Graeme Eisenhofer Ph.D. and Karel Pacak M.D. Ph.D.
National Institutes of Health, Bethesda, Maryland
Pheochromocytomas are endocrine tumors that are characterized by production of certain chemicals (catecholamines) normally produced by the endocrine cells (chromaffin cells) from which the tumors are derived. There are two important catecholamines produced by these chromaffin cells: norepinephrine and epinephrine. Norepinephrine, or noradrenaline as it is also known, is a catecholamine produced and released as a neurotransmitter by certain nerves in the brain (central nervous system noradrenergic nerves) and also in the body (sympathetic nerves). Sympathetic nerves innervate the blood vessels and the heart to control blood pressure and cardiac function. Norepinephrine is also formed in the adrenal medulla, the central part of the adrenal gland, but here it is further metabolized to another catecholamine called epinephrine or adrenaline. The epinephrine produced and released by the adrenal gland functions as a hormone; it acts principally at sites distant from its site of release from the adrenal medulla. This contrasts with the norepinephrine released by nerves which acts to control the function of the cells at the sites in the immediate proximity of the nerve endings from which the transmitter is released. It is the elevations of one or both norepinephrine or epinephrine in the bloodstream that cause the distinctive but variable symptoms of pheochromocytoma.
Diagnosis of pheochromocytoma typically requires confirmation by several tests, perhaps the most important being biochemical evidence of excessive catecholamine production by the tumor. This is usually achieved from measurements of catecholamines and certain catecholamine metabolites in urine or plasma (Table 1). However, the catecholamines, norepinephrine and epinephrine, are also produced by sympathetic nerves and the adrenal medulla and are thus not specific to pheochromocytomas. Therefore, high levels of catecholamines and their metabolites may be produced by a variety of conditions or disease states involving increased release of catecholamines from sympathetic nerves or the adrenal medulla (1). Sometimes pheochromocytomas may be “silent”; that is they may not produce catecholamines in amounts sufficient to produce a positive biochemical test result or the associated typical clinical signs and symptoms. Also, many pheochromocytomas secrete catecholamines episodically; between episodes, plasma concentrations or urinary excretion of catecholamines may be normal. Thus, tests of plasma or urinary catecholamines and urinary metabolites of catecholamines do not always reliably exclude or confirm the presence of a tumor (2-6). A more recently developed biochemical test involving measurements of plasma free normetanephrine and metanephrine, respective metabolites of norepinephrine and epinephrine, offers advantages over other tests for diagnosis of pheochromocytoma (5).
_____________________________________________________________ TABLE 2. BIOCHEMICAL TESTS FOR DIAGNOSIS OF PHEOCHROMOCYTOMA _____________________________________________________________ Biochemical Test and Normal Reference Range (lower and upper reference limits of normal)* _____________________________________________________________ 1. Urine Catecholamines (measured by HPLC) Norepinephrine (15-80 micrograms/day) Epinephrine (0-20 micrograms/day) 2. Urine Deconjugated Fractionated Metanephrines (measured by HPLC) Normetanephrine-sulfate (44-540 micrograms/day) Metanephrine-sulfate (26-230 micrograms/day) 3. Urine Deconjugated Total Metanephrines (measured by Spectrofluorimetry) (0-1.2 milligrams/day) (Sum of free plus sulfate conjugated metanephrine & normetanephrine) 4. Urine VMA (measured by Spectrofluorimetry) (0-7.9 milligrams/day) 5. Plasma Catecholamines (measured by HPLC) Norepinephrine (80-498 picograms/milliliter) Epinephrine (4-83 picograms/milliliter) 6. Plasma Free Metanephrines (measured by HPLC) Normetanephrine (18-112 picograms/milliliter) Metanephrine (12-61 picograms/milliliter) 7. Plasma Deconjugated Metanephrines (measured by HPLC) Normetanephrine-sulfate (610-3170 picograms/milliliter) Metanephrine-sulfate (316-1706 picograms/milliliter) _____________________________________________________________ * Reference ranges indicate lower and upper reference limits of a normal population commonly estimated from the 95% confidence intervals. Reference ranges may vary from laboratory to laboratory. A milligram is 1/1000 th (10 to the -3) of a gram; A microgram is 1/1,000,0000 th (10 to the -6) of a gram. A picogram is 1/1,000,000,000,000 (10 to the -12) of a gram.
Understanding the utility and limitations of biochemical tests for diagnosis of pheochromocytoma can benefit from an understanding of catecholamine release and metabolism under normal conditions as well as in the many disease states associated with elevated catecholamine release. Central to this is a basic understanding of the pathways of catecholamine metabolism. Both norepinephrine and epinephrine are metabolized by a multiplicity of pathways catalyzed by an array of enzymes, resulting in a considerable number of different metabolites, only some of which are routinely used for diagnosis of pheochromocytoma (Figure 1).
The first step in catecholamine metabolism involves the actions either one of two enzymes:  monoamine oxidase, an enzyme that removes the amine part of the catecholamine; and  catechol-O-methyltransferase, an enzyme that adds a methyl group to form normetanephrine from norepinephrine and metanephrine from epinephrine (Figure 1). The actions of monoamine oxidase on norepinephrine and epinephrine results in formation of a single metabolite called 3,4-dihydroxyphenylglycol, or as it is commonly abbreviated, DHPG. Removal of the amine to form the metabolite, 3,4-dihydroxymandelic acid (DHMA), is not a favored pathway (7-9). DHPG is further metabolized by catechol-O-methyltransferase to form 3-methoxy-4-hydroxyphenylglycol (MHPG). This metabolite is also produced to a limited extent by the actions of monoamine oxidase on both normetanephrine and metanephrine (10,11).
In humans, 3-methoxy-4-hydroxymandelic acid – more commonly known as vanillylmandelic acid (VMA) – is the principal end-product of norepinephrine and epinephrine metabolism, produced largely from MHPG and to a lesser extent from normetanephrine and metanephrine (12-14) (Figure 1). VMA is present in urine and plasma at very high concentrations (Table 1), which like the conjugated metanephrines, makes measurement of VMA relatively simple. It is this that has made measurements of urinary VMA a time honored, though not necessarily sensitive, biochemical test for diagnosis of pheochromocytoma.
With the exception of VMA, all the catecholamines and their metabolites are metabolized by a further enzyme that adds a sulfate group to the molecules. These sulfate conjugates, as they are called, represent other major end-products of catecholamine metabolism that are typically present in plasma and urine at higher concentrations than the free compounds. In particular, the sulfate conjugates of the normetanephrine and metanephrine are present in plasma and urine in concentrations more than 25-fold higher than those of the free compounds. Assays of urine metanephrines, typically used for diagnosis of pheochromocytoma, employ a deconjugation step so that sulfate-conjugated metanephrines comprise the bulk of these measurements. Thus, these assays are largely measurements of different metabolites (i.e., sulfate- conjugated derivatives) from those of the free metanephrines. The latter are present in plasma and urine at much lower and harder to detect concentrations than their sulfate-conjugated derivatives.
Although knowledge of the pathways of catecholamine metabolism is useful, what is perhaps more important to an understanding of the utility of catecholamines and their metabolites in the diagnosis of pheochromocytoma is an appreciation of how catecholamines are metabolized differently within nerves and other cells, before and after their entry into the bloodstream and among various organs and tissues, including chromaffin cells and pheochromocytoma tumor cells. Pheochromocytomas differ from sympathetic nerves or central nervous system noradrenergic nerves but are similar to adrenal medullary cells in that they secrete catecholamines directly into the bloodstream. In contrast, the norepinephrine released from sympathetic nerves acts and is metabolized locally so that only a small proportion escapes local removal and metabolism to diffuse into the bloodstream (15-18)(Figure 2).
Pheochromocytomas, differ from the adrenal medulla in that the tumors mainly secrete norepinephrine, whereas the predominant catecholamine secreted by the adrenal medulla is epinephrine. It is norepinephrine that is therefore more consistently elevated in patients with pheochromocytoma, although a significant but much smaller proportion may also show elevations in plasma or urinary epinephrine (19). Pheochromocytomas causing elevations in only epinephrine are generally rather uncommon. Increases in epinephrine either occuring alone or in combination with norepinephrine are, however, quite common in pheochromocytomas associated with multiple endocrine neoplasia type 2 (20,21).
Since norepinephrine is the predominant catecholamine secreted by pheochromocytomas, an understanding of its metabolism after release and production within sympathetic nerves, as compared with after release directly into the bloodstream by a pheochromocytoma, is particularly important. Because monoamine oxidase is the only catecholamine-metabolizing enzyme present in noradrenergic or sympathetic nerves, the norepinephrine metabolized within these nerves is all converted to DHPG (22-24) (Figure 2). As a consequence, the DHPG appearing in plasma is almost exclusively produced in sympathetic nerves, whereas the additional presence of catechol-O-methyltransferase in extraneuronal cells means that normetanephrine is exclusively produced from norepinephrine in extraneuronal cells, such as smooth muscle cells or liver cells (17,23). Much of the DHPG formed in nerves is metabolized further to 3-methoxy-4-hydroxyphenylglcol (MHPG) by catechol-O-methyltransferase in extraneuronal cells (10,23). Thus, in contrast to DHPG and normetanephrine, MHPG reflects both neuronal metabolism of norepinephrine to DHPG and extraneuronal metabolism of DHPG and normetanephrine, but is mainly derived from the DHPG produced initially in nerves (10).
Comparison of removal and metabolism of catecholamines by neuronal and extraneuronal cells has indicated that the former cells are far more important than the latter for inactivation of neuronally released norepinephrine (11,17,25). This means that most of the norepinephrine produced and released by nerves is metabolized within the nerves themselves (Figure 2). Most of this is from metabolism of norepinephrine that leaks from stores of the transmitter into the neuronal cytoplasm where the enzyme monoamine oxidase is located.
The above considerations combined with the series nature of neuronal and extraneuronal removal and metabolism (25) explain why very little of the DHPG in plasma (less than 1%) is derived from neuronal metabolism of norepinephrine released directly into the bloodstream (Figure 2). Thus, release of norepinephrine from a pheochromocytoma directly into the bloodstream causes only small increases in DHPG compared with release of norepinephrine from nerves (24,26). Hence, patients with pheochromocytoma and high norepinephrine levels often have normal or only slightly elevated plasma concentrations of DHPG (26,27). Therefore, findings of a high norepinephrine combined with a normal DHPG provide supportive evidence that an increased plasma concentration of norepinephrine is not due to excessive release from sympathetic nerves and might rather reflect a tumor (26,28,29). While it cannot be ignored that both an increased norepinephrine and DHPG could reflect an unusual tumor that also produces DHPG, this pattern is more typical of a state of increased release of norepinephrine by sympathetic nerves.
In contrast to the greater importance of neuronal over extraneuronal pathways for metabolism of neuronally released norepinephrine, the reverse is the situation for metabolism of circulating norepinephrine (Figure 2). In humans only 20-30% of the norepinephrine in the bloodstream is removed by and metabolized in nerves, whereas this proportion is 90% and more for that produced and released by nerves (11,30,31). This in part reflects the series nature of neuronal and extraneuronal removal and metabolizing mechanisms operating between sites of neuronal release within tissues and the bloodstream, but is also influenced by organs such as the liver that play an important role in the removal of circulating catecholamines by uptake into and metabolism by extraneuronal cells (14,32). As a result, about 20% of circulating levels of the extraneuronal metabolite, normetanephrine, are produced from circulating norepinephrine (32,33), a relatively high amount compared to the less than 1% for the neuronal metabolite, DHPG. Since pheochromocytomas secrete catecholamines directly into the bloodstream, extraneuronal production of normetanephrine and metanephrine from circulating catecholamines provides one reason why the metanephrines are better markers for a tumor than other metabolites that are largely derived from neuronal metabolism.
VMA, the major end-product of norepinephrine and epinephrine metabolism, is produced almost exclusively from the removal and metabolism by the liver of catecholamines and their metabolites that circulate in the bloodstream (14) (Figure 2). This is because the enzyme responsible for formation of VMA from MHPG, alcohol dehydrogenase, is localized to the liver (34,35). The substantial production of VMA from circulating DHPG and MHPG, most of which is derived from neuronal norepinephrine metabolism, explains why VMA is a relatively insensitive marker for pheochromocytoma compared with the precursors norepinephrine, epinephrine, normetanephrine and metanephrine (36-40).
Normally at least 90% of metanephrine and up to 40% of normetanephrine are formed from metabolism of epinephrine and norepinephrine within the adrenals before release of these catecholamines into the circulation (32,33). This makes the adrenal medulla the single largest source of both normetanephrine and metanephrine in the body, exceeding the contribution of the liver (32). This helps explain why so little of these metabolites (6% for metanephrine and 20% for normetanephrine) are formed from metabolism of catecholamines after release into the bloodstream (33). Both adrenal medullary and pheochromocytoma tumor cells contain high quantities of catechol-O-methyltransferase (41), the enzyme that is responsible for O-methylation of catecholamines to form normetanephrine and metanephrine (42). The catechol-O-methyltransferase is localized within chromaffin cells so it would appear that the normetanephrine and metanephrine from these sources are derived from catecholamines leaking from stores into the chromaffin cell cytoplasm. In patients with pheochromocytoma, over 94% of the elevated plasma concentrations of normetanephrine or metanephrine are derived from metabolism of catecholamines by the catechol-O-methyltransferase within pheochromocytoma tumor cells and not by actions of extra-adrenal catechol-O-methyltransferase on catecholamines released by tumors into the circulation (41). This means that production of normetanephrine and metanephrine is an ongoing process within pheochromocytoma tumor cells, independent of catecholamine release. This explains why plasma concentrations of normetanephrine and metanephrine are relatively insensitive markers of increased norepinephrine release by nerves or increased epinephrine release from the adrenals or of a paroxysmal attack associated with large increases in catecholamine release from a pheochromocytoma (41). However, this also means that even when tumors are not secreting catecholamines into the bloodstream they are nevertheless constantly metabolizing catecholamines to normetanephrine and/or metanephrine.
The sensitivity of a test for diagnosis of pheochromocytoma is indicated by the proportion of patients with a tumor who have a positive test result (i.e., an elevated plasma concentration or urinary output). A sensitivity of one hundred percent indicates that in all cases of patients who have a tumor the test will be positive. Put another way, a high sensitivity of 100% indicates that a normal test result reliably excludes the presence of a tumor in all cases where the result is normal. Importantly, it must be realized that a high sensitivity of 100% does not necessarilly prove the existence of a pheochromocytoma in all patients who have a positive test result. This is another matter of the specificity of the test (see below).
A problem with use of plasma or urinary catecholamines for diagnosis of pheochromocytoma is that some tumors are quiescent and may not secrete large amounts of catecholamines while other tumors appear to secrete catecholamines episodically. Thus, plasma levels and urinary outputs of catecholamines are normal in some patients with pheochromocytoma and the presence of a pheochromocytoma cannot be reliably excluded using measurements of plasma or urinary catecholamine concentrations (2-6,37,43-45). In contrast, the metanephrines (either normetanephrine or metanephrine or both) are constantly produced by the actions of catechol-O-methyltransferase on catecholamines leaking from stores within tumor cells and therefore show much more consistent increases above normal in patients with pheochromocytoma than plasma catecholamines (5,41). This means that measurements of plasma normetanephrine and metanephrine reliably excludes the presence of all but the smallest of pheochromocytomas. Where excluded, no other tests are necessary. This means that measurements of plasma metanephrines avoid a missed diagnosis and minimize the need to run multiple diagnostic tests to exclude the presence of a tumor. It also means that a person with normal plasma concentrations of normetanephrine and metanephrine can be fairly confident of not having a pheochromocytoma.
Since, the free metanephrines are formed extraneuronally, and to a large extent within chromaffin tissues (e.g., adrenal medulla and pheochromocytomas), these metabolites are also more sensitive markers for a pheochromocytoma than the other catecholamine metabolites that are derived mainly from neuronal sources.
Urinary metanephrines are commonly measured after acid hydrolysis and thus largely represent sulfate-conjugated normetanephrine and metanephrine. A substantial amount of the normetanephrine-sulfate is derived from sulfate conjugation of normetanephrine produced in parts of the body other than the adrenal medulla or pheochromocytoma tumor chromaffin tissue. Therefore the sulfate-conjugated normetanephrine, as commonly measured in urine, is a less sensitive marker of pheochromocytoma than the free normetanephrine measured in plasma. Nevertheless, measurements of urinary deconjugated normetanephrine and metanephrine, performed by modern HPLC methods, do provide a reasonably sensitive biochemical test for diagnosis of pheochromocytoma; these measurements may be more sensitive than measurements of catecholamines (6,46).
Measurements of the combined sum of urinary outputs of normetanephrine and metanephrine in sulfate-conjugated plus free form (commonly known as urinary total metanephrines), as measured by out-dated spectrofluorometric methods, are not sensitive tests of a pheochromocytoma and have limited value in the initial work-up of a patient suspected of having a pheochromocytoma. The low sensitivity of urinary VMA also makes this test less than satisfactory for the initial biochemical diagnosis of a pheochromocytoma.
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Pheochromocytoma Org – Page Revised 02/10/05