Free metanephrine

The basis for the high diagnostic efficacy of plasma free metanephrines is explained by several factors (1) plasma free metanephrines are produced by metabolism of catecholamines within pheochromocytomas, a process that occurs continuously and independently of variations in catecholamine release by tumors (2) normally only small amounts of metanephrines are produced in the body, and these are relatively unresponsive to sympathoadrenal activation compared with the parent amines and (3) VMA and the metanephrines commonly measured in urine are different metabofites from the free metanephrines measured in plasma, and are produced in different parts of the body by metabolic processes not directly related to the tumor itself." ... 

After the potential confounding influence of medications or other causes of false-positive results have been eliminated, some consideration should be given to the choice of additional biochemical tests and patterns of results necessary for more firmly establishing or refuting the diagnosis of a pheochromocytoma. When initial testing yields elevations in plasma normetanephrine, metanephrine, or both amines, this may be corroborated by a similar pattern of results after additional measurements of urinary normetanephrine and metanephrine. Conversely, when initial testing yields positive results for urinary fractionated metanephrines, additional measurements of plasma free metanephrines are useful. 

Patterns of increases in plasma free metanephrines and catecholamines can also be useful for confirming pheo-chromocytomas in patients in whom initial tests of free metanephrines are positive but msufficiently elevated for a firm diagnosis. More specifically, patients with a pheochromocytoma usually have larger relative increases in metanephrines than of the parent catecholammes, whereas patients with false-positive results caused by sympathoadrenal activation usually have larger increases in catecholamines than metanephrines. 

An HPLC method for the more difficult measurement of plasma free metanephrines was first described in 1993. This method, like those involving measurements of plasma or urinary deconjugated metanephrines, requires a preana-lytical cation-exchange extraction and purification step. The low plasma concentrations of free metanephrines present several technical challenges. In particular, low levels of interfering substances, such as acetaminophen, tend to be more troublesome to measurements of plasma concentrations of the free metabolites than to the higher decon-... 

Weise M, Merke DP, Pacak K, Walther MM, Eisenhofer G. Utility of plasma free metanephrines for detecting childhood pheochromocytoma. 

G. Eisenhofer, J.W.M. Lenders, D.S. Goldstein, M. Mannelli, G. Csako, M.M. Walther, F.M. Brouwers and K. Pacak, Pheochromocytoma catecholamine phenotypes and prediction of tumor size and location by use of plasma free metanephrines, Clin. Chem., 51, 735-744 (2005). 

S.A. Lagerstedt, D.J. O Kane and R.J. Singh, Measurement of plasma free metanephrine and nometanephrine by liquid chromatography-tandem mass spectrometry for diagnosis of pheochromocytoma, Clin. Chem., 50, 603-611 (2004). 

Precipitation "protein crash" Ease, speed Requires centrifugation, no analyte preconcentration Plasma-free metanephrines by LC-MS/MS [3]... 

Marney LC, Laha TJ, Baird GS, Rainey PM, Hoofnagle AN. Isopropanol protein precipitation for the analysis of plasma free metanephrines by liquid chromatography— tandem mass spectrometry. Clin Chem 2008 54 1729—32. 

The high diagnostic sensitivity of measurements of plasma free or urinary fractionated normetanephrine and metanephrine makes these tests the most suitable choice for the initial work up of a patient with a suspected pheochromocytoma. Negative results by these tests virtually exclude a pheochromocytoma, whereas negative results by other tests do not. Exceptions include small or microscopic ([Pg.1047]

Additional markers of catecholamine overproduction have been employed to improve the biochemical detection of neuroblastomas. Free dopamine may be abnormal in urine from neuroblastoma patients with VMA and HVA excretion. Combined testing for VMA, HVA, and dopamine may therefore improve tumor detection, and in 1993 an international consensus report on neuroblastoma diagnosis added dopamine to the Hst of acceptable measurements to document the adrenergic nature of the tumor. Plasma measurements of dopamine and L-dopa, the amino acid precursor of dopamine, may also have clinical value and allow the alternate use of plasma. Measurement of methylated metabolites, especially normetanephrine, has also been explored. When urinary normetanephrine, metanephrine, methoxytyra-mine, dopamine, norepinephrine, VMA, and HVA were measured, clinical sensitivity for detection of neuroblastomas was 97% to 100% when results of normetanephrine testing were coupled either with VMA in the infants or with HVA in children greater than age 1. Even with an extended panel of catecholamines and metabolite measurements, a low incidence of nonsecreting tumors continues to be identified and should be considered in the interpretation of a negative test result. 

Interpretation of a biochemical test result as normal or abnormal depends on availability of valid reference intervals (see Chapter 16). For tests of a single analyte, such as VMA, it can be expected that at least 2.5% of patients without pheochromocytomas will have values for the analyte above the upper reference limit and 2.5% below the lower reference limit. Up to a 5% incidence of false-positive results might be expected for tests of pairs of analytes, such as norepinephrine and epinephrine in tests of urinary or plasma catecholamines or normetanephrine and metanephrine in tests of plasma free or urinary fractionated metanephrines. False-positive rates usually, however, tend to be higher than expected this is likely due to reduced control over sampling conditions and sources of interference or differences in clinical characteristics of reference and patient populations. 

The free 0-methylated amine metabolites are present in plasma at picomolar concentrations that have made their accurate measurement technically difficult. Measurements of plasma metanephrines therefore represent relatively recent developments. The.first method enabling accurate measurement of plasma free normetanephrine involved a radioenzymatic assay in which normetanephrine was converted to H-iabeled metanephrine using preparations of the enzyme phenylethanolamine-N-methyltransferase, incubated with H-methyi-labeled S-adenosylmethionine. This method, however, did not allow measurements of metanephrine or methoxytyramine, and therefore had limited clinical utility. 

The first HPLC methods for measuring plasma metanephrines in the early 1990s featured an acid-hydrolysis step similar to that used for routine measurements of urinary metanephrines. These measurements of plasma deconjugated (free plus conjugated) metanephrines indicated promise for diagnosis of pheochromocytomas. Very high levels of the deconjugated metabolites were also found in patients with renal failure. 

TABLE 29 8 Reference Intervals for Plasma Free and Deconjugated Metanephrines in Normotensive and Hypertensive Adults and in Normotensive Children... 

Buu NT, Angers M, Chevalier D, Kuchel O. A new method for the simultaneous analysis of free and sulfoconjugated normetanephrine, metanephrine, and 3-methoxytyramine in human urine by HPLC with electrochemical detection. J Lab Clin Med 1984 104 425-32. 

These challenges observed for both electiochemical and fluorimetric modes of detection prompted the need for a more sensitive, selective and maintenance-free HPLC assay for routine, rapid, simultaneous measurements of catecholamines and metanephrines in urine. 

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