Pheochromocytomas, although a rare cause of hypertension, are dangerous tumors that require consideration in large numbers of patients. The resulting low prevalence of these tumors among the tested population and the inadequate sensitivity and specificity of commonly used biochemical tests make diagnosis of pheochromocytoma difficult and time-consuming.
As outlined in two articles in this issue of the Journal (1)(2), measurements of plasma concentrations of metanephrines provide a promising, new, highly sensitive test for diagnosis of pheochromocytoma. A negative test result for plasma metanephrines means that a pheochromocytoma is highly unlikely so that no other tests are necessary (3)(4). Thus, measurements of plasma metanephrines provide a particularly good initial diagnostic test for exclusion of pheochromocytoma.
The diagnostic superiority of plasma metanephrines over plasma or urinary catecholamines and urinary vanillylmandelic acid is clear (3)(4)(5)(6). What remains unclear, as illustrated in the two articles in this issue of the Journal (1)(2), is whether measurements of plasma free (unconjugated) metanephrines or total metanephrines provide the better diagnostic test, and how these tests differ. Also unclear is how these tests differ from measurements of urinary metanephrines.
Part of the confusion about tests of urinary total or fractionated metanephrines and plasma free, unconjugated, or total metanephrines stems from the unfortunate and confusing terminology used to distinguish these analytes. The term “metanephrines” describes two catecholamine metabolites: normetanephrine, the O-methylated metabolite of norepinephrine, and metanephrine, the O-methylated metabolite of epinephrine (Fig. 1 ). The term urinary “total” metanephrines was coined, based on historical precedents, to describe both normetanephrine and metanephrine measured together as a single concentration by early spectrophotometric assays.
Diagram illustrating the pathways of metabolism of catecholamines to free and sulfate-conjugated metanephrines.
PNMT, phenylethanolamine N-methyltransferase; COMT, catechol-O-methyltransferase; SULT1A3, monoamine-preferring sulfotransferase.
Spectrophotometric assays of urinary total metanephrines have been superseded by HPLC assays that allow separate measurement of normetanephrine and metanephrine, termed “fractionated” metanephrines. Despite the superiority of HPLC assays of plasma or urinary fractionated metanephrines over urinary total metanephrines (3)(4)(5)(7)(8), demand for the latter test persists. The delay of laboratories and clinicians to move to the better tests may partly reflect the confusing nature of the terminology used to distinguish the various assays.
Adding to the confusion in terminology is the fact that the “free” or “unconjugated” metanephrines measured by Roden et al. (1) are different metabolites from the metanephrines commonly measured in urine or the plasma total metanephrines measured by Grouzmann et al. (2). Moreover, assays of plasma total metanephrines reflect fractionated measurements of normetanephrine and metanephrine; thus, these assays are not analogous to combined measurement of normetanephrine and metanephrine in assays of urinary total metanephrines.
Plasma total metanephrines and urinary fractionated or total metanephrines are determined after urine or plasma samples are subjected to acid hydrolysis or enzymatic deconjugation with sulfatase. This liberates free metanephrines from the sulfate-conjugated metabolites. Plasma concentrations of total metanephrines (i.e., free plus conjugated metanephrines) are 20- to 30-fold higher than free metanephrines and therefore largely reflect conjugated metanephrines, metabolites different from the free metanephrines. In assays of urinary metanephrines, the difference is even larger, with free metanephrines representing a small proportion (<3%) of the total measured as free plus conjugated metanephrines.
Sulfate-conjugated metanephrines and catecholamines are formed from the free amines by the actions of a specific sulfotransferase isoenzyme, monoamine-preferring sulfotransferase (SULT1A3) (9). The presence of this sulfotransferase isoenzyme, and thus formation of sulfate-conjugated metanephrines, in adrenal medullary chromaffin cells or pheochromocytoma tumor cells has not been established. Monoamine-preferring sulfotransferase is, however, found in high concentrations in the gastrointestinal tract, which is the source of most of the sulfate-conjugated catecholamines produced in the body (10)(11). Therefore, in contrast to the considerable amounts of free metanephrines formed in adrenal medullary or pheochromocytoma tumor tissue, it seems likely that sulfate-conjugated metanephrines are formed predominantly in gastrointestinal tissue.
What do the above differences mean in terms of the performance of the various tests of plasma and urinary metanephrines for diagnosis of pheochromocytoma? Because sulfate-conjugated metanephrines are derived entirely from the free metanephrines, it might be expected that plasma free and total metanephrines and urinary metanephrines would correlate closely and that the various tests should provide similar diagnostic sensitivity and specificity. However, a larger and more variable contribution of the norepinephrine produced in gastrointestinal tissues to sulfate-conjugated than to free normetanephrine might also be expected to compromise the diagnostic sensitivity and specificity of measurements of the conjugated compared with the free metabolite.
Differences in circulatory clearance and elimination of free and sulfate-conjugated metanephrines might also affect diagnostic sensitivity and specificity. The free metanephrines are cleared rapidly from the circulation by the same extraneuronal monoamine transporters that are responsible for the rapid circulatory clearance of catecholamines. The free metanephrines are then metabolized by monoamine oxidase to 3-methoxy-4-hydroxyphenylglyol or by monoamine-preferring sulfotransferase to sulfate-conjugated metanephrines. As metabolic end-products, the sulfate-conjugated metanephrines are cleared slowly from the circulation, with clearance dependent almost entirely on elimination by the kidneys. Thus, plasma concentrations of total metanephrines are grossly increased in patients with renal failure, and impaired kidney function is well known to limit the diagnostic utility of these metabolites (12)(13)(14). In contrast, because the kidneys contribute little to the circulatory clearance of free metanephrines, impaired renal function would be expected to have little effect on these metabolites.
Grouzmann et al. (2) argue that the slow circulatory clearance of sulfate-conjugated metanephrines should make measurements of total metanephrines superior to measurements of catecholamines or of the free metanephrines for diagnosis of pheochromocytoma. Presumably, this is because a slow circulatory clearance would minimize any wide variations in plasma concentrations of total metanephrines that might accompany intermittent catecholamine release by a tumor. However, because free metanephrines are produced continuously within pheochromocytoma tumor cells and independently of catecholamine release (15), it is unclear how a slower circulatory clearance of conjugated than of free metanephrines would make the total metanephrines better markers of a pheochromocytoma than the free metanephrines.
Because the circulatory clearance of an analyte inversely determines the resulting plasma concentration, the slower clearance of sulfate-conjugated than of free metanephrines does produce higher plasma concentrations of sulfate-conjugated than of free metanephrines. This in turn minimizes the requirement for high analytical sensitivity, providing an advantage for measurements of total metanephrines. Nevertheless, as shown by Roden et al. (1), technical advances are now making it possible to very accurately measure the low concentrations of free metanephrines in plasma without interference from compounds such as acetaminophen.
It remains to be determined which test, plasma free or total metanephrines, offers the better method for diagnosis of pheochromocytoma. In the interim, it is important to consider that the free metanephrines measured in plasma by some laboratories represent metabolites largely different from the metanephrines commonly measured in urine or the total metanephrines measured in plasma by other laboratories.
References
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Eisenhofer G, Aneman A, Hooper D, Rundqvist B, Friberg P. Mesenteric organ production, hepatic metabolism, and renal elimination of norepinephrine and its metabolites in humans.
Eisenhofer G, Coughtrie MW, Goldstein DS. Dopamine sulphate: an enigma resolved.
Marini M, Fathi M, Vallotton M. Determination of serum metanephrines in the diagnosis of pheochromocytoma.
Pallant A, Mathian B, Prost L, Theodore C, Patricot MC. Determination of plasma methoxyamines.
