- Email: [email protected]
Biochemical diagnosis of pheochromocytoma and paraganglioma
Annales d’Endocrinologie 70 (2009) 161–165
Biochemical diagnosis of pheochromocytoma and paraganglioma Diagnostic biochimique du phéochromocytome et paragangliome J.W.M. Lenders a,b a
Department of Medicine, Division of General Internal Medicine, Radboud University Nijmegen Medical Center, Geert Grooteplein Zuid, 8, 6525GA, Nijmegen, The Netherlands b Department of Internal Medicine III, University Hospital Carl Gustav Carus, Dresden, Germany Available online 17 March 2009
1. Introduction Pheochromocytomas and paragangliomas are catecholamine-producing neuroendocrine tumors arising from chromaffin cells in the adrenal glands and in the ganglia. The catecholamines secreted by these tumors are usually responsible for signs and symptoms like hypertension, headache, palpitations, excessive sweating and pallor [1]. The archetypical patient in whom this tumor is considered is the hypertensive patient with paroxysmal symptoms. Although many patients are tested, its incidence is extremely rare, being 50–75 new patients per year in the dutch population of approximately 16 million inhabitants. Despite improved diagnostic test methods, the diagnosis is missed in a considerable number of patients [2,3]. The spectrum of disorders for which testing for pheochromocytoma is necessary includes symptomatic patients suspected for having primary or recurrent pheochromocytoma, patients with an incidentaloma, patients with therapy-resistant hypertension and patients with a hereditary predisposition for pheochromocytoma like von Hippel Lindau (VHL), multiple endocrine neoplasia (MEN-2), neurofibromatosis (NFI) and succinate dehydrogenase B, D and C mutations. For the diagnosis of pheochromocytoma one has to demonstrate that there is an excessive production of catecholamines and/or metabolites of catecholamines in plasma or urine. There is a variety of different biochemical tests available and for initial testing a test with a maximal negative predictive value is mandatory since a missed diagnosis due to a false-negative test result can have catastrophic consequences for the patient. Before discussing the diagnostic pros and cons of the different tests, first the metabolism of catecholamines will be discussed because knowledge of the different metabolites may help to understand their different diagnostic characteristics. E-mail address: [email protected]. 0003-4266/$ – see front matter © 2009 Published by Elsevier Masson SAS. doi:10.1016/j.ando.2009.02.008
2. Metabolism and measurements of catecholamines or metabolites Deamination within sympathetic nerves by monoamine oxidase (MAO) represents the major route for metabolism of catecholamines and is more important than extraneuronal metabolism [4]. The principal deaminated metabolite of norepinephrine is 3,4-dihydroxyphenylglycol (DHPG). DHPG is O-methylated by catechol-O methyltransferase (COMT) to 3-methoxy-4-hydroxyphenylglycol (MHPG). In contrast to MAO, COMT is located exclusively in extraneuronal tissues. MHPG is also formed by the combined actions of COMT and MAO on the norepinephrine released by sympathetic nerves that escapes neuronal uptake or the norepinephrine and epinephrine secreted directly into the bloodstream by the adrenal medulla. An alternative metabolic pathway is the conversion of catecholamines into metanephrines within adrenal medullary or pheochromocytoma tumor cells. This is due to the presence of COMT in adrenal medullary chromaffin cells and pheochromocytoma tumor cells [5,6]. Over 90% of circulating metanephrine and up to 40% of normetanephrine are formed within adrenal medullary cells from catecholamines leaking into the cytoplasm from vesicular storage granules. Less than 10% of circulating metanephrine is derived from metabolism of epinephrine after release by the adrenals into the circulation. In patients with pheochromocytoma, more than 94% of the elevated plasma levels of normetanephrine or metanephrine are derived from metabolism of catecholamines within tumor cells, a process that occurs continuously and independently of any fluctuations in catecholamine secretion [6]. Despite substantial intra-adrenal production of metanephrines, plasma concentrations of normetanephrine, and particularly metanephrine, are relatively insensitive markers of sympathoadrenal activation which is in contrast to increased norepinephrine release by
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sympathetic nerves or increased epinephrine release from the adrenal medulla [6,7]. So, comparisons of plasma concentrations of catecholamines and metanephrines can be helpful in distinguishing false-positive from true-positive biochemical test results for pheochromocytoma. All catecholamines and their metabolites (except vanillyl mandelic acid, VMA) are converted by monoamine-preferring sulphotransferase (SULT1A3) into sulphate conjugates which are excreted in the urine. It is important to realize that the urinary metanephrines are different metabolites from the free metanephrines measured in plasma. The free (unconjugated) metanephrines in plasma are formed by COMT in adrenal or tumor tissues and these are partially conjugated to sulphate-conjugated metanephrines which have much longer plasma half-lives than the free metanephrines. These conjugated metanephrines represent the principal form which is eliminated in the urine and therefore measurements of urinary metanephrines mainly reflect sulphate-conjugated metanephrines. The major end-product of norepinephrine and epinephrine metabolism, VMA, is produced mainly in the liver by oxidation of MHPG catalyzed by alcohol dehydrogenase and excreted in the urine. The considerable derivation of most of the produced VMA from the sympathoneuronal pathway of catecholamine metabolism (through DHPG and MHPG) is the major explanation for the poor diagnostic sensitivity of the urinary VMA test for pheochromocytoma [8–10]. Therefore, normal urinary excretion of VMA does not reliably exclude pheochromocytoma. The biochemical tests to document catecholamine excess in patients suspected for having pheochromocytoma include measurements of urinary and plasma catecholamines, urinary and plasma metanephrines. The utility of measuring either plasma or urinary catecholamines for diagnosis of pheochromocytoma is well documented [8,11]. High-performance liquid chromatography (HPLC) with electrochemical or fluorometric detection (ECD, FD) or liquid chromatography coupled with mass-spectroscopy (LC-MS/MS) which provide higher analytical specificity than HPLC methods, are currently the methods of choice for measuring catecholamines. HPLC and LC-MS/MS assays allow separate measurement of urinary normetanephrine and metanephrine, termed “fractionated” metanephrines. Immunological methods like RIA are not yet reliable enough for use in clinical practice. 3. Biochemical effects of heterogeneity of pheochromocytoma Pheochromocytomas are heterogeneous tumors in terms of clinical signs and symptoms and biochemical characteristics. It is important to take this into account when performing biochemical testing. Most but not all patients with a pheochromocytoma are symptomatic with hypertension and paroxysms of perspiration, palpitations and headache as the most prevalent symptoms. It is clear that these patients have the highest chance to demonstrate increased plasma or urinary levels of catecholamines. However, some patients have only minor or no symptoms at all and in these patients biochemical tests for catecholamines
may be negative. This may be due to the fact that the tumor is still small and or is biochemically not very active. An additional possibility is that plasma catecholamine levels are normal between episodes of paroxysmal increased secretion of catecholamines. In particular this might be the case when patients with a hereditary predisposition are screened for excess production of catecholamines because the tumors in these, usually asymptomatic patients, are small. As a consequence, plasma or urinary catecholamines and metanephrines show smaller increases in cases of hereditary than in sporadic pheochromocytoma. Yet, since the free metanephrines are produced continuously within pheochromocytoma tumor cells and independently of catecholamine release, biochemical tests of these metabolites are much more sensitive and less fallible to miss the tumor [10,12]. In both hereditary and sporadic pheochromocytoma, increases in plasma free metanephrines above the upper reference limits are larger than those of catecholamines. Consequently, there are less false-negative results for the measurements of metanephrines than for those of catecholamines. This pattern is particularly evident for epinephrine-producing pheochromocytomas where there are larger and more consistent increases in plasma free metanephrine than of its precursor, epinephrine [10]. The underlying type of mutation in patients with a hereditary predisposition also determines the biochemical phenotype since these mutations might have different effects on the genetic make-up of the enzymes that are involved in the synthesis and metabolism of catecholamines. MEN-2 tumors can be detected more easily by increased levels of plasma metanephrine in contrast to VHL tumors which are more easily detected by plasma normetanephrine. In addition, pheochromocytomas in MEN-2 patients have an adrenergic phenotype and produce both epinephrine and norepinephrine, whereas tumors in VHL patients have a distinctly noradrenergic phenotype and produce almost exclusively norepinephrine. Furthermore, pheochromocytomas in VHL patients produce smaller amounts of catecholamines and are less likely to produce signs and symptoms than tumors in MEN-2 patients [13]. Mutation-dependent differences in expression of genes may also explain progression from benign to malignant pheochromocytoma, which are often characterized by a dedifferentiated state. Norepinephrine is usually the leading catecholamine that is produced but more importantly metastatic pheochromocytomas are often characterized by high tissue, plasma and urinary levels of dihydroxyphenylalanine and dopamine, the immediate precursors of norepinephrine [14–16]. 4. Initial biochemical testing To ensure that a negative test result in a tested patient indeed excludes the tumor, an initial biochemical test with a maximal negative predictive value is needed. The obvious reason for this requirement is that missing the diagnosis can have fatal consequences for the patient. Translated in test chartacteristics this means that a test with a nearly maximal sensitivity is needed. Currently there are two tests that fulfill this requirement: measurement of plasma free metanephrines and measurement
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Table 1 Data from 7 studies using plasma free metanephrines. Study
Pheo-patients (n)
Non-pheo-patients (n)
URL (NMN)
URL (MN)
Sensitivity (%)
Specificity (%)
NIH Sterberk Vienna Essen Mayo Cleveland New Castle Ut
214 25 17 24 56 32 11
644 1235 14 126 445 – 114
0.61 0.61 0.66 0.69 0.90 0.90 0.90
0.31 0.31 0.31 0.19 0.50 0.50 0.55
99 100 100 96 96 97 100
89 97 100 79 85 – 91
URL: upper reference limit (nmol/L).
of urinary fractionated metanephrines. Seven studies in 379 patients with a pheochromocytoma and more than 2500 patients without a pheochromocytoma have established a sensitivity of plasma free metanephrines of about 98% with a specificity ranging from 85–100% (Table 1). The consequent high negative predictive value, even at high pretest probabilities, indicates that the presence of a pheochromocytoma is extremely unlikely when these tests are negative (Table 2). Only in a few small hereditary pheochromocytomas this test was false-negative. Although the sensitivity of urinary excretion of fractionated metanephrines is similar to that of plasma free metanephrines, the specificity is lower. Measuring urinary excretion of metanephrines carries the disadvantage of inconveniency for the patient and also the possibility of incomplete urine sampling. Other tests such as urinary catecholamines, total metanephrines and VMA lack sufficient sensitivity to exclude the presence of a pheochromocytoma. In general, sensitivities are higher in sporadic than in hereditary pheochromocytoma while specificities are higher in hereditary than in sporadic pheochromocytomas and this applies to all tests. This is not unexpected since sporadic patients are usually tested because of symptoms in contrast to patients with a hereditary pheochromocytoma who are tested because of the hereditary predisposition and not because of symptoms. One could argue that combining different tests has a more accurate diagnostic yield. This is however not the case if one compares plasma free metanephrines with combinations of other tests since plasma free metanephrines still supersede the receiver operating characteristic (ROC) curves of the combined tests. In addition, combining several tests is fraught with an increased likelihood of a false-positive result requiring additional time and effort for appropriate follow-up. The diagnostic characteristics of biochemical tests should be put into the perspective of the expected prevalence rate or pretest probability of a pheochromocytoma in the patient to be tested. In a population of hypertensive patients where the prevalence is low, it is likely that the number of false-positive Table 2 The negative and positive predictive values of plasma free metanephrines at three different prevalence rate (pretest probability). Prevalence (%) Sensitivity (%) Negative predictive value (%) Specificity (%) Positive predictive value (%)
1 98 99.9 85 6
30 98 99 85 74
80 98 91 85 96
test results will exceed the number of true-positive test results. However, from the data of the NIH study it can be calculated that all patients with a pheochromocytoma (i.e., the truepositive test results) have elevations in plasma normetanephrine above 2.20 nmol/L or of metanephrine above 1.20 nmol/L. These values are nearly four times the upper reference limits of 0.61 nmol/L for normetanephrine and 0.31 nmol/L for metanephrine. In these patients, the extent of increase in plasma normetanephrine or metanephrine indicates with high probability the presence of a pheochromocytoma and the tumor should be located by imaging studies. However, in patients with only slightly elevated plasma concentrations of free metanephrines (less than four times the upper reference limits), only some have a pheochromocytoma but most have not. In this group, several possible explanations for this false-positive test result should be considered. 5. False-positive biochemical test results Analytical interference and increased plasma or urinary levels of catecholamines and their metabolites secondary to medications and physiological or pathological conditions represent the leading causes of false-positive test results. One typical example of a false-positive test result for plasma free metanephrines is the position of the patient during blood sampling. To minimize any influence of sympathoadrenal activation in the sitting position, one can sample blood using an indwelling intravenous canula with patients recumbent for at least 20 minutes before sampling (like is necessary for catecholamines). Since in daily patient care this is a laborious approach for many clinics, blood samples can be taken in the sitting position provided that the upper reference limits are used as established for the supine position (i.e. 0.61 nmol/L for normetanephrine and 0.31 nmol/L for metanephrine). Although this approach will increase the number of false-positive test results by 16% as opposed to drawing a sample after at least 20 minutes of supine rest, the sensitivity will remain unaltered [17]. Despite the need to repeat sampling if plasma metanephrines are elevated in the sitting position, this approach is cost-effective. Analytical interference with drugs and dietary constituents can also lead to false-positive test results. Assays for plasma or urinary catecholamines or metanephrines like LC-MS/MS and XLC-MS/MS are hardly prone to analytical interference [18]. Yet, for the time being, it is reasonable that all blood samples for these assays be collected after an overnight fast and after
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refraining from caffeinated beverages. Pathological conditions like renal or liver failure can lead to false-positive test results or render chromatographic recordings difficult to interpret due to circulating interfering metabolic substances. Drug-induced elevations of plasma catecholamines and metanephrines represent a major cause of false-positive test results [1]. This involves indirect interference which occurs during use of tricyclic antidepressants (by inhibition of neuronal uptake of norepinephrine by sympathetic nerves), ␣-adrenoceptor blocking therapy (by baroreflex-mediated sympathetic activation) or phenoxybenzamine (by reversal of presynaptic alpha2 -adrenoceptor inhibitory actions on norepinephrine release from sympathetic nerves). Several, in particular cardiovascular conditions are accompanied by sympathoadrenal activation like heart failure and renovascular hypertension. Although normetanephrine and particularly metanephrine are less sensitive to sympathoadrenal activation than the parent amines, the levels of these metabolites can be increased by the same stimuli or clinical conditions that increase plasma catecholamines. Finally, patients with renal failure are prone to false-positive test results [19]. Twenty-four hour urinary testing is difficult to interpret in these patients since urine collections may be impossible. In addition, impaired renal function results in dramatic increases in plasma concentrations of VMA and sulfate-conjugated metanephrines, rendering these tests invalid [20,21]. In contrast, since the circulatory clearance of plasma catecholamines and free metanephrines is largely independent of renal function, measurements of these analytes in plasma represent the most appropriate tests for diagnosis of pheochromocytoma in renal failure [21]. 6. Follow-up biochemical testing If all potential causes of false-positive test results have been taken into account and excluded as far as possible, still some patients may disclose slightly elevated plasma levels of metanephrines. Additional follow-up biochemical tests can include any of the standard tests used for diagnosis of pheochromocytoma but should include repeat measurements of plasma free metanephrines or urinary fractionated metanephrines. Since metanephrines are continuously produced by a pheochromocytoma, normal plasma metanephrines in a second test do exclude a pheochromocytoma, regardless of whether the first test was positive. Plasma catecholamine measurements are specifically helpful when blood sampling is possible during an episode of paroxysmal signs or symptoms. The results of this sample should then be compared to the result of a sample taken when the patient is asymptomatic. A further step for follow-up testing in case of a positive test result can be provided by the clonidine test. This test is not useful when plasma concentrations of catecholamines or metanephrines are normal because it is likely that clonidine will decrease plasma catecholamines or metanephrines, regardless whether the patient has a tumor or not [22,23]. This test is particularly useful for distinguishing elevated levels of plasma norepinephrine or normetanephrine due to increased release from sympathetic nerves from high levels due to release
from a pheochromocytoma. Measuring plasma normetanephrine instead of norepinephrine improves the sensitivity from 67 to 96% while the specificity is similar with respectively 98 and 100%. An abnormal response for plasma normetanephrine, defined as a decrease in plasma normetanephrine of less than 40% and a plasma normetanephrine level of >0.61 nmol/L after clonidine, strongly indicate the presence of a pheochromocytoma [24]. Appropriate execution and interpretation of the above described tests in combination with a thorough assessment of clinical history, symptoms and signs is sufficient to refute the diagnosis of pheochromocytoma in most patients. In some patients however, unnecessary imaging studies can not be avoided since there is not one ideal biochemical test that definitely can exclude or confirm a pheochromocytoma. Repeated testing or more involved testing (e.g., novel imaging approaches, vena caval blood sampling) might be helpful in these patients. 7. Conclusions Biochemical testing for pheochromocytoma requires a maximal sensitive test for initial screening while for confirmation a very specific test should be used. Urinary fractionated metanephrines or plasma free metanephrines are the best tests for initial screening. Plasma free metanephrines can be measured in a blood sample drawn in the fasting state and in the sitting position without preceding rest, provided that the upper reference limits are used that are established form studies in which the samples were drawn after supine rest with an intravenous indwelling canula. In case of a slightly increased test result, the test should be repeated after rest in the recumbent position. This test has not the disadvantage of 24 hour urine collection and can be done at any time the patient comes in. For follow-up testing, urinary 24 hour testing for fractionated metanephrines can be done or plasma free metanephrines measurement can be repeated. Finally, a clonidine suppression test offers the possibility to distinguish elevated levels of plasma norepinephrine or normetanephrine due to increased release from sympathetic nerves from high levels due to release from a pheochromocytoma. This diagnostic approach limits the unnecessary use of expensive imaging modalities, thus resulting in considerable savings in cost without missing patients with a pheochromocytoma. References [1] Lenders JW, Eisenhofer G, Mannelli M, Pacak K. Pheochromocytoma. Lancet 2005;366:665–75. [2] Lo CY, Lam KY, Wat MS, et al. Adrenal pheochromocytoma remains a frequently overlooked diagnosis. Am J Surg 2001;179:212–5. [3] Yu R, Nissen NN, Chopra P, Dhall D, et al. Diagnosis and treatment of pheochromocytoma in an academic hospital from 1997 to 2007. Am J Med 2009;122:85–95. [4] Eisenhofer G, Huynh T-T, Hiroi M, Pacak K. Understanding catecholamine metabolism as a guide to the biochemical diagnosis of pheochromocytoma. Rev End Metab Dis 2001;2:297–311. [5] Eisenhofer G, Rundqvist B, Aneman A, et al. Regional release and removal of catecholamines and extraneuronal metabolism to metanephrines. J Clin Endocrinol Metab 1995;80:3009–17.
J.W.M. Lenders / Annales d’Endocrinologie 70 (2009) 161–165 [6] Eisenhofer G, Keiser H, Friberg P, et al. Plasma metanephrines are markers of pheochromocytoma produced by catechol-O-methyltransferase within tumors. J Clin Endocrinol Metab 1998;83:2175–85. [7] Eisenhofer G, Friberg P, Pacak K, et al. Plasma metadrenalines: do they provide useful information about sympatho-adrenal function and catecholamine metabolism? Clin Sci 1995;88:533–42. [8] Bravo EL, Tarazi RC, Gifford RW, Stewart BH. Circulating and urinary catecholamines in pheochromocytoma. Diagnostic and pathophysiologic implications. N Engl J Med 1979;301:682–6. [9] Peaston RT, Lai L. Biochemical detection of phaechromocytoma: should we still be measuring urinary HMMA? J Clin Pathol 1993;46: 734–7. [10] Lenders JWM, Pacak K, Walther MM, et al. Biochemical diagnosis of pheochromocytoma: which test is best? JAMA 2002;287:1427–34. [11] Duncan MW, Compton P, Lazarus L, Smythe GA. Measurement of norepinephrine and 3,4-dihydroxyphenylglycol in urine and plasma for the diagnosis of pheochromocytoma. N Engl J Med 1988;319: 136–42. [12] Eisenhofer G, Lenders JW, Linehan WM, et al. Plasma normetanephrine and metanephrine for detecting pheochromocytoma in von HippelLindau disease and multiple endocrine neoplasia type 2. N Engl J Med 1999;340:1872–9. [13] Eisenhofer G, Walther MM, Huynh TT, et al. Pheochromocytomas in von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2 display distinct biochemical and clinical phenotypes. J Clin Endocrinol Metab 2001;86:1999–2008. [14] Goldstein DS, Stull R, Eisenhofer G, et al. Plasma 3,4dihydroxyphenylalanine (dopa) and catecholamines in neuroblastoma or pheochromocytoma. Ann Intern Med 1986;105:887–8.
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[15] Proye C, Fossati P, Fontaine P, et al. Dopamine-secreting pheochromocytoma: an unrecognized entity? Classification of pheochromocytomas according to their type of secretion. Surgery 1986;100:1154–62. [16] John H, Ziegler WH, Hauri D, Jaeger P. Pheochromocytomas: can malignant potential be predicted? Urology 1999;53:679–83. [17] Lenders JW, Willemsen JJ, Eisenhofer G, Ross HA, Pacak K, Timmers HJ, et al. Is supine rest necessary before blood sampling for plasma metanephrines? Clin Chem 2007;53:352–4. [18] de Jong WH, Graham KS, van der Molen JC, Links TP, Morris MR, Ross HA, et al. Plasma free metanephrine measurement using automated online solid-phase extraction HPLC tandem mass spectrometry. Clin Chem 2007;53:1684–93. [19] Stumvoll M, Radjaipour M, Seif F. Diagnostic considerations in pheochromocytoma and chronic hemodialysis: case report and review of the literature. Am J Nephrol 1995;15:147–51. [20] Mornex R, Peyrin L, Pagliari R, Cottet-Emard JM. Measurement of plasma methoxyamines for the diagnosis of pheochromocytoma. Horm Res 1991;36:220–6. [21] Eisenhofer G, Huysmans F, Pacak K, Walther MM, Sweep FC, Lenders JW. Plasma metanephrines in renal failure. Kidney Int 2005;67:668–77. [22] Bravo EL, Gifford Jr RW. Current concepts. Pheochromocytoma: diagnosis, localization and management. N Engl J Med 1984;311:1298–303. [23] Grossman E, Goldstein DS, Hoffman A, Keiser HR. Glucagon and clonidine testing in the diagnosis of pheochromocytoma. Hypertension 1991;17:733–41. [24] Eisenhofer G, Goldstein DS, Walther MM, Friberg P, Lenders JW, Keiser HR, et al. Biochemical diagnosis of pheochromocytoma: how to distinguish true- from false-positive test results. J Clin Endocrinol Metab 2003;88:2656–66.
Biochemical diagnosis of pheochromocytoma and paraganglioma Diagnostic biochimique du phéochromocytome et paragangliome J.W.M. Lenders a,b a
Department of Medicine, Division of General Internal Medicine, Radboud University Nijmegen Medical Center, Geert Grooteplein Zuid, 8, 6525GA, Nijmegen, The Netherlands b Department of Internal Medicine III, University Hospital Carl Gustav Carus, Dresden, Germany Available online 17 March 2009
1. Introduction Pheochromocytomas and paragangliomas are catecholamine-producing neuroendocrine tumors arising from chromaffin cells in the adrenal glands and in the ganglia. The catecholamines secreted by these tumors are usually responsible for signs and symptoms like hypertension, headache, palpitations, excessive sweating and pallor [1]. The archetypical patient in whom this tumor is considered is the hypertensive patient with paroxysmal symptoms. Although many patients are tested, its incidence is extremely rare, being 50–75 new patients per year in the dutch population of approximately 16 million inhabitants. Despite improved diagnostic test methods, the diagnosis is missed in a considerable number of patients [2,3]. The spectrum of disorders for which testing for pheochromocytoma is necessary includes symptomatic patients suspected for having primary or recurrent pheochromocytoma, patients with an incidentaloma, patients with therapy-resistant hypertension and patients with a hereditary predisposition for pheochromocytoma like von Hippel Lindau (VHL), multiple endocrine neoplasia (MEN-2), neurofibromatosis (NFI) and succinate dehydrogenase B, D and C mutations. For the diagnosis of pheochromocytoma one has to demonstrate that there is an excessive production of catecholamines and/or metabolites of catecholamines in plasma or urine. There is a variety of different biochemical tests available and for initial testing a test with a maximal negative predictive value is mandatory since a missed diagnosis due to a false-negative test result can have catastrophic consequences for the patient. Before discussing the diagnostic pros and cons of the different tests, first the metabolism of catecholamines will be discussed because knowledge of the different metabolites may help to understand their different diagnostic characteristics. E-mail address: [email protected]. 0003-4266/$ – see front matter © 2009 Published by Elsevier Masson SAS. doi:10.1016/j.ando.2009.02.008
2. Metabolism and measurements of catecholamines or metabolites Deamination within sympathetic nerves by monoamine oxidase (MAO) represents the major route for metabolism of catecholamines and is more important than extraneuronal metabolism [4]. The principal deaminated metabolite of norepinephrine is 3,4-dihydroxyphenylglycol (DHPG). DHPG is O-methylated by catechol-O methyltransferase (COMT) to 3-methoxy-4-hydroxyphenylglycol (MHPG). In contrast to MAO, COMT is located exclusively in extraneuronal tissues. MHPG is also formed by the combined actions of COMT and MAO on the norepinephrine released by sympathetic nerves that escapes neuronal uptake or the norepinephrine and epinephrine secreted directly into the bloodstream by the adrenal medulla. An alternative metabolic pathway is the conversion of catecholamines into metanephrines within adrenal medullary or pheochromocytoma tumor cells. This is due to the presence of COMT in adrenal medullary chromaffin cells and pheochromocytoma tumor cells [5,6]. Over 90% of circulating metanephrine and up to 40% of normetanephrine are formed within adrenal medullary cells from catecholamines leaking into the cytoplasm from vesicular storage granules. Less than 10% of circulating metanephrine is derived from metabolism of epinephrine after release by the adrenals into the circulation. In patients with pheochromocytoma, more than 94% of the elevated plasma levels of normetanephrine or metanephrine are derived from metabolism of catecholamines within tumor cells, a process that occurs continuously and independently of any fluctuations in catecholamine secretion [6]. Despite substantial intra-adrenal production of metanephrines, plasma concentrations of normetanephrine, and particularly metanephrine, are relatively insensitive markers of sympathoadrenal activation which is in contrast to increased norepinephrine release by
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sympathetic nerves or increased epinephrine release from the adrenal medulla [6,7]. So, comparisons of plasma concentrations of catecholamines and metanephrines can be helpful in distinguishing false-positive from true-positive biochemical test results for pheochromocytoma. All catecholamines and their metabolites (except vanillyl mandelic acid, VMA) are converted by monoamine-preferring sulphotransferase (SULT1A3) into sulphate conjugates which are excreted in the urine. It is important to realize that the urinary metanephrines are different metabolites from the free metanephrines measured in plasma. The free (unconjugated) metanephrines in plasma are formed by COMT in adrenal or tumor tissues and these are partially conjugated to sulphate-conjugated metanephrines which have much longer plasma half-lives than the free metanephrines. These conjugated metanephrines represent the principal form which is eliminated in the urine and therefore measurements of urinary metanephrines mainly reflect sulphate-conjugated metanephrines. The major end-product of norepinephrine and epinephrine metabolism, VMA, is produced mainly in the liver by oxidation of MHPG catalyzed by alcohol dehydrogenase and excreted in the urine. The considerable derivation of most of the produced VMA from the sympathoneuronal pathway of catecholamine metabolism (through DHPG and MHPG) is the major explanation for the poor diagnostic sensitivity of the urinary VMA test for pheochromocytoma [8–10]. Therefore, normal urinary excretion of VMA does not reliably exclude pheochromocytoma. The biochemical tests to document catecholamine excess in patients suspected for having pheochromocytoma include measurements of urinary and plasma catecholamines, urinary and plasma metanephrines. The utility of measuring either plasma or urinary catecholamines for diagnosis of pheochromocytoma is well documented [8,11]. High-performance liquid chromatography (HPLC) with electrochemical or fluorometric detection (ECD, FD) or liquid chromatography coupled with mass-spectroscopy (LC-MS/MS) which provide higher analytical specificity than HPLC methods, are currently the methods of choice for measuring catecholamines. HPLC and LC-MS/MS assays allow separate measurement of urinary normetanephrine and metanephrine, termed “fractionated” metanephrines. Immunological methods like RIA are not yet reliable enough for use in clinical practice. 3. Biochemical effects of heterogeneity of pheochromocytoma Pheochromocytomas are heterogeneous tumors in terms of clinical signs and symptoms and biochemical characteristics. It is important to take this into account when performing biochemical testing. Most but not all patients with a pheochromocytoma are symptomatic with hypertension and paroxysms of perspiration, palpitations and headache as the most prevalent symptoms. It is clear that these patients have the highest chance to demonstrate increased plasma or urinary levels of catecholamines. However, some patients have only minor or no symptoms at all and in these patients biochemical tests for catecholamines
may be negative. This may be due to the fact that the tumor is still small and or is biochemically not very active. An additional possibility is that plasma catecholamine levels are normal between episodes of paroxysmal increased secretion of catecholamines. In particular this might be the case when patients with a hereditary predisposition are screened for excess production of catecholamines because the tumors in these, usually asymptomatic patients, are small. As a consequence, plasma or urinary catecholamines and metanephrines show smaller increases in cases of hereditary than in sporadic pheochromocytoma. Yet, since the free metanephrines are produced continuously within pheochromocytoma tumor cells and independently of catecholamine release, biochemical tests of these metabolites are much more sensitive and less fallible to miss the tumor [10,12]. In both hereditary and sporadic pheochromocytoma, increases in plasma free metanephrines above the upper reference limits are larger than those of catecholamines. Consequently, there are less false-negative results for the measurements of metanephrines than for those of catecholamines. This pattern is particularly evident for epinephrine-producing pheochromocytomas where there are larger and more consistent increases in plasma free metanephrine than of its precursor, epinephrine [10]. The underlying type of mutation in patients with a hereditary predisposition also determines the biochemical phenotype since these mutations might have different effects on the genetic make-up of the enzymes that are involved in the synthesis and metabolism of catecholamines. MEN-2 tumors can be detected more easily by increased levels of plasma metanephrine in contrast to VHL tumors which are more easily detected by plasma normetanephrine. In addition, pheochromocytomas in MEN-2 patients have an adrenergic phenotype and produce both epinephrine and norepinephrine, whereas tumors in VHL patients have a distinctly noradrenergic phenotype and produce almost exclusively norepinephrine. Furthermore, pheochromocytomas in VHL patients produce smaller amounts of catecholamines and are less likely to produce signs and symptoms than tumors in MEN-2 patients [13]. Mutation-dependent differences in expression of genes may also explain progression from benign to malignant pheochromocytoma, which are often characterized by a dedifferentiated state. Norepinephrine is usually the leading catecholamine that is produced but more importantly metastatic pheochromocytomas are often characterized by high tissue, plasma and urinary levels of dihydroxyphenylalanine and dopamine, the immediate precursors of norepinephrine [14–16]. 4. Initial biochemical testing To ensure that a negative test result in a tested patient indeed excludes the tumor, an initial biochemical test with a maximal negative predictive value is needed. The obvious reason for this requirement is that missing the diagnosis can have fatal consequences for the patient. Translated in test chartacteristics this means that a test with a nearly maximal sensitivity is needed. Currently there are two tests that fulfill this requirement: measurement of plasma free metanephrines and measurement
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Table 1 Data from 7 studies using plasma free metanephrines. Study
Pheo-patients (n)
Non-pheo-patients (n)
URL (NMN)
URL (MN)
Sensitivity (%)
Specificity (%)
NIH Sterberk Vienna Essen Mayo Cleveland New Castle Ut
214 25 17 24 56 32 11
644 1235 14 126 445 – 114
0.61 0.61 0.66 0.69 0.90 0.90 0.90
0.31 0.31 0.31 0.19 0.50 0.50 0.55
99 100 100 96 96 97 100
89 97 100 79 85 – 91
URL: upper reference limit (nmol/L).
of urinary fractionated metanephrines. Seven studies in 379 patients with a pheochromocytoma and more than 2500 patients without a pheochromocytoma have established a sensitivity of plasma free metanephrines of about 98% with a specificity ranging from 85–100% (Table 1). The consequent high negative predictive value, even at high pretest probabilities, indicates that the presence of a pheochromocytoma is extremely unlikely when these tests are negative (Table 2). Only in a few small hereditary pheochromocytomas this test was false-negative. Although the sensitivity of urinary excretion of fractionated metanephrines is similar to that of plasma free metanephrines, the specificity is lower. Measuring urinary excretion of metanephrines carries the disadvantage of inconveniency for the patient and also the possibility of incomplete urine sampling. Other tests such as urinary catecholamines, total metanephrines and VMA lack sufficient sensitivity to exclude the presence of a pheochromocytoma. In general, sensitivities are higher in sporadic than in hereditary pheochromocytoma while specificities are higher in hereditary than in sporadic pheochromocytomas and this applies to all tests. This is not unexpected since sporadic patients are usually tested because of symptoms in contrast to patients with a hereditary pheochromocytoma who are tested because of the hereditary predisposition and not because of symptoms. One could argue that combining different tests has a more accurate diagnostic yield. This is however not the case if one compares plasma free metanephrines with combinations of other tests since plasma free metanephrines still supersede the receiver operating characteristic (ROC) curves of the combined tests. In addition, combining several tests is fraught with an increased likelihood of a false-positive result requiring additional time and effort for appropriate follow-up. The diagnostic characteristics of biochemical tests should be put into the perspective of the expected prevalence rate or pretest probability of a pheochromocytoma in the patient to be tested. In a population of hypertensive patients where the prevalence is low, it is likely that the number of false-positive Table 2 The negative and positive predictive values of plasma free metanephrines at three different prevalence rate (pretest probability). Prevalence (%) Sensitivity (%) Negative predictive value (%) Specificity (%) Positive predictive value (%)
1 98 99.9 85 6
30 98 99 85 74
80 98 91 85 96
test results will exceed the number of true-positive test results. However, from the data of the NIH study it can be calculated that all patients with a pheochromocytoma (i.e., the truepositive test results) have elevations in plasma normetanephrine above 2.20 nmol/L or of metanephrine above 1.20 nmol/L. These values are nearly four times the upper reference limits of 0.61 nmol/L for normetanephrine and 0.31 nmol/L for metanephrine. In these patients, the extent of increase in plasma normetanephrine or metanephrine indicates with high probability the presence of a pheochromocytoma and the tumor should be located by imaging studies. However, in patients with only slightly elevated plasma concentrations of free metanephrines (less than four times the upper reference limits), only some have a pheochromocytoma but most have not. In this group, several possible explanations for this false-positive test result should be considered. 5. False-positive biochemical test results Analytical interference and increased plasma or urinary levels of catecholamines and their metabolites secondary to medications and physiological or pathological conditions represent the leading causes of false-positive test results. One typical example of a false-positive test result for plasma free metanephrines is the position of the patient during blood sampling. To minimize any influence of sympathoadrenal activation in the sitting position, one can sample blood using an indwelling intravenous canula with patients recumbent for at least 20 minutes before sampling (like is necessary for catecholamines). Since in daily patient care this is a laborious approach for many clinics, blood samples can be taken in the sitting position provided that the upper reference limits are used as established for the supine position (i.e. 0.61 nmol/L for normetanephrine and 0.31 nmol/L for metanephrine). Although this approach will increase the number of false-positive test results by 16% as opposed to drawing a sample after at least 20 minutes of supine rest, the sensitivity will remain unaltered [17]. Despite the need to repeat sampling if plasma metanephrines are elevated in the sitting position, this approach is cost-effective. Analytical interference with drugs and dietary constituents can also lead to false-positive test results. Assays for plasma or urinary catecholamines or metanephrines like LC-MS/MS and XLC-MS/MS are hardly prone to analytical interference [18]. Yet, for the time being, it is reasonable that all blood samples for these assays be collected after an overnight fast and after
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refraining from caffeinated beverages. Pathological conditions like renal or liver failure can lead to false-positive test results or render chromatographic recordings difficult to interpret due to circulating interfering metabolic substances. Drug-induced elevations of plasma catecholamines and metanephrines represent a major cause of false-positive test results [1]. This involves indirect interference which occurs during use of tricyclic antidepressants (by inhibition of neuronal uptake of norepinephrine by sympathetic nerves), ␣-adrenoceptor blocking therapy (by baroreflex-mediated sympathetic activation) or phenoxybenzamine (by reversal of presynaptic alpha2 -adrenoceptor inhibitory actions on norepinephrine release from sympathetic nerves). Several, in particular cardiovascular conditions are accompanied by sympathoadrenal activation like heart failure and renovascular hypertension. Although normetanephrine and particularly metanephrine are less sensitive to sympathoadrenal activation than the parent amines, the levels of these metabolites can be increased by the same stimuli or clinical conditions that increase plasma catecholamines. Finally, patients with renal failure are prone to false-positive test results [19]. Twenty-four hour urinary testing is difficult to interpret in these patients since urine collections may be impossible. In addition, impaired renal function results in dramatic increases in plasma concentrations of VMA and sulfate-conjugated metanephrines, rendering these tests invalid [20,21]. In contrast, since the circulatory clearance of plasma catecholamines and free metanephrines is largely independent of renal function, measurements of these analytes in plasma represent the most appropriate tests for diagnosis of pheochromocytoma in renal failure [21]. 6. Follow-up biochemical testing If all potential causes of false-positive test results have been taken into account and excluded as far as possible, still some patients may disclose slightly elevated plasma levels of metanephrines. Additional follow-up biochemical tests can include any of the standard tests used for diagnosis of pheochromocytoma but should include repeat measurements of plasma free metanephrines or urinary fractionated metanephrines. Since metanephrines are continuously produced by a pheochromocytoma, normal plasma metanephrines in a second test do exclude a pheochromocytoma, regardless of whether the first test was positive. Plasma catecholamine measurements are specifically helpful when blood sampling is possible during an episode of paroxysmal signs or symptoms. The results of this sample should then be compared to the result of a sample taken when the patient is asymptomatic. A further step for follow-up testing in case of a positive test result can be provided by the clonidine test. This test is not useful when plasma concentrations of catecholamines or metanephrines are normal because it is likely that clonidine will decrease plasma catecholamines or metanephrines, regardless whether the patient has a tumor or not [22,23]. This test is particularly useful for distinguishing elevated levels of plasma norepinephrine or normetanephrine due to increased release from sympathetic nerves from high levels due to release
from a pheochromocytoma. Measuring plasma normetanephrine instead of norepinephrine improves the sensitivity from 67 to 96% while the specificity is similar with respectively 98 and 100%. An abnormal response for plasma normetanephrine, defined as a decrease in plasma normetanephrine of less than 40% and a plasma normetanephrine level of >0.61 nmol/L after clonidine, strongly indicate the presence of a pheochromocytoma [24]. Appropriate execution and interpretation of the above described tests in combination with a thorough assessment of clinical history, symptoms and signs is sufficient to refute the diagnosis of pheochromocytoma in most patients. In some patients however, unnecessary imaging studies can not be avoided since there is not one ideal biochemical test that definitely can exclude or confirm a pheochromocytoma. Repeated testing or more involved testing (e.g., novel imaging approaches, vena caval blood sampling) might be helpful in these patients. 7. Conclusions Biochemical testing for pheochromocytoma requires a maximal sensitive test for initial screening while for confirmation a very specific test should be used. Urinary fractionated metanephrines or plasma free metanephrines are the best tests for initial screening. Plasma free metanephrines can be measured in a blood sample drawn in the fasting state and in the sitting position without preceding rest, provided that the upper reference limits are used that are established form studies in which the samples were drawn after supine rest with an intravenous indwelling canula. In case of a slightly increased test result, the test should be repeated after rest in the recumbent position. This test has not the disadvantage of 24 hour urine collection and can be done at any time the patient comes in. For follow-up testing, urinary 24 hour testing for fractionated metanephrines can be done or plasma free metanephrines measurement can be repeated. Finally, a clonidine suppression test offers the possibility to distinguish elevated levels of plasma norepinephrine or normetanephrine due to increased release from sympathetic nerves from high levels due to release from a pheochromocytoma. This diagnostic approach limits the unnecessary use of expensive imaging modalities, thus resulting in considerable savings in cost without missing patients with a pheochromocytoma. References [1] Lenders JW, Eisenhofer G, Mannelli M, Pacak K. Pheochromocytoma. Lancet 2005;366:665–75. [2] Lo CY, Lam KY, Wat MS, et al. Adrenal pheochromocytoma remains a frequently overlooked diagnosis. Am J Surg 2001;179:212–5. [3] Yu R, Nissen NN, Chopra P, Dhall D, et al. Diagnosis and treatment of pheochromocytoma in an academic hospital from 1997 to 2007. Am J Med 2009;122:85–95. [4] Eisenhofer G, Huynh T-T, Hiroi M, Pacak K. Understanding catecholamine metabolism as a guide to the biochemical diagnosis of pheochromocytoma. Rev End Metab Dis 2001;2:297–311. [5] Eisenhofer G, Rundqvist B, Aneman A, et al. Regional release and removal of catecholamines and extraneuronal metabolism to metanephrines. J Clin Endocrinol Metab 1995;80:3009–17.
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