676 Humans, Clinical

The Value of Immunoassays for Metanephrines in the

Biochemical Diagnosis of Pheochromocytomas

Authors

N. Unger1, T. Deutschbein1, M. K. Walz2, K. Mann1, S. Petersenn1

Aliations

Key words

Abstract
&

received 18.12.2008
accepted 24.04.2009
Bibliography
DOI 10.1055/s-0029-1224133
Published online:
June 25, 2009
Horm Metab Res 2009;
41: 676679
Georg Thieme Verlag KG
Stuttgart New York
ISSN 0018-5043
Correspondence
S. Petersenn, MD
Division of Endocrinology,
Medical Center
University of Duisburg-Essen
Hufelandstrae 55
45122 Essen
Germany
Tel.: + 49/201/723 28 54
Fax: + 49/201/723 59 76
stephan.petersenn@uni-due.de

An undiagnosed pheochromocytoma may result

in life-threatening consequences. The diagnosis of pheochromocytoma is based on the overproduction of catecholamines. Highly sensitive
biochemical assays are essential to avoid falsenegative results. Determinations of 24-h urinary
epinephrine and norepinephrine levels are established diagnostic tools. However, they may be
falsely negative in patients with a biochemicallysilent or periodically-secreting pheochromocytoma. Metanephrines, which are metabolites of
catecholamines, have been suggested as an alternative diagnostic tool. Urinary metanephrines
are determined by high-pressure liquid chro-

Introduction
&
Pheochromocytomas are tumours arising from
chroman cells of the adrenal medulla. The
overproduction of catecholamines may result in
severe hypertension, either sustained or periodic,
as well as headache, sweating, and tachycardia. In
patients with these typical signs and symptoms,
screening for pheochromocytoma should be performed. Furthermore, with an increase in incidentally discovered adrenal masses by advanced
imaging techniques, reliable biochemical parameters are mandatory to distinguish pheochromocytomas from other hormonally active or
inactive adrenal adenomas. An undiagnosed
pheochromocytoma may result in life-threatening consequences. The diagnosis is based on the
overproduction of catecholamines. Highly sensitive biochemical assays are essential to avoid
false-negative results. Determinations of 24-h
urinary epinephrine and norepinephrine levels

Unger N et al. Immunoassays for Metanephrines Horm Metab Res 2009; 41: 676679

matography (HPLC) in an increasing number of

laboratories, whereas plasma metanephrines
measured by HPLC are available in specialised
centres only. The dierent HPLC methods may be
cost- and time-intensive. Immunoassays such as
radio- or enzyme-immunoassays may be alternative procedures. Measurement of metanephrines
instead of catecholamines by either technique
improved the diagnostic accuracy for the diagnosis of pheochromocytomas. Determination
of plasma free metanephrines demonstrated a
higher accuracy than their urinary counterparts.
The use of immunoassays may be an alternative to the laborious HPLC, although the method
needs to be evaluated in more detail.

are established diagnostic tools. However, they

may be falsely negative in patients with a biochemically-silent or periodically-secreting pheochromocytoma, and falsely elevated in patients
with panic disorder or congestive heart failure
[1]. Metanephrines, which are metabolites of catecholamines, have been suggested as an alternative diagnostic tool [24]. An explanation for the
potential superiority of metanephrines compared with catecholamines was provided by a
recent study [5]. Pheochromocytomas contain
high amounts of catechol-O-methyltransferase
(COMT), an enzyme that metabolises catecholamines into free metanephrines. Rapid metabolism of catecholamines into metanephrines
within the tumour may result in a continued high
secretion of the metabolites, but in an episodic
and low secretion of the parent amines. In this
article, the value of the currently available assays
for plasma and urinary metanephrines and their
pitfalls are discussed.

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plasma metanephrines

urinary metanephrines

pheochromocytoma

HPLC

immunoassays

Department of Endocrinology and Division of Laboratory Research, Medical Center, University of Duisburg-Essen, Essen,
Germany
2
Department of Surgery and Center of Minimally Invasive Surgery, Kliniken Essen-Mitte, Essen, Germany

Humans, Clinical

677

Table 1 Comparison of dierent thresholds in various studies for the measurement of plasma free metanephrine and normetanephrine for the diagnosis of
pheochromocytoma
Reference

Method

Plasma free parameters

Threshold (pg/ml)

Sens

Spec

Eisenhofer et al. [14]

HPLC

Sawka et al. [9]

HPLC

Raber et al. [10]

HPLC

Unger et al. [11]

RIA

Gao et al. [12]

EIA

Lenz et al. [13]

RIA

MN
NMN
MN
NMN
MN
NMN
MN
NMN
MN
NMN
MN
NMN

61
112
99
165
60
121
38
126
92
135
100
170

53 %
96 %
50 %
87 %
71 %
100 %
71 %
92 %
30 %
83 %
n.a.
n.a.

95 %
92 %
94 %
94 %
100 %
100 %
79 %
96 %
94 %
92 %
n.a.
n.a.

MN: metanephrine; NMN: normetanephrine; Sens: sensitivity;

10000

1000

100

10

EH

NF
M

CP
A

AP
A

Ph
eo

EH

NF
M

CP
A

AP
A

Ph
eo

Normetanephrines in plasma (pg/ml)

High-pressure liquid chromatographic (HPLC) procedures have

been established for the determination of plasma free metanephrines (metanephrine and normetanephrine) (Table 1) [6]. In a
large study by Lenders et al., plasma free metanephrines demonstrated a higher sensitivity than plasma or urinary catecholamines [7]. Sensitivity was slightly higher for sporadic than for
hereditary pheochromocytoma (99 vs. 97 %), while the specificity was lower in comparison (82 vs. 96 %). When considering
the combination of plasma metanephrine and normetanephrine
for the diagnosis of pheochromocytoma, Sawka et al. and another
study by Lenders et al. revealed a sensitivity of 97 and 100 %,
respectively, with a lower specificity of 85 % [8, 9]. Raber et al.
studied patients with pheochromocytoma and compared them
with 14 patients suering from other adrenal tumours [10]. In
that study, normal values for plasma catecholamines and free
metanephrines were adapted from the previous study by Lenders et al [8]. The authors demonstrated a sensitivity and specificity of 100 % each for plasma normetanephrine.
Due to the cost- and time-intensive liquid chromatography technique, the method is limited to specialised centres. Immunoassays may be an alternative process. We have previously
reported on the diagnostic value of plasma free and urinary
metanephrines measured by radioimmunoassay (RIA) for the
diagnosis of pheochromocytoma [11]. We compared catecholamines and metanephrines in plasma and urine in patients
with adrenal tumours. Receiver operating characteristics (ROC)
analysis was employed to provide thresholds with an optimal
balance between sensitivity and specificity. Scattergrams of
plasma metanephrine and normetanephrine are shown in
Fig. 1. Plasma normetanephrine was the best single parame
ter for the diagnosis of pheochromocytomas. A threshold of
126 pg/ml for plasma normetanephrine demonstrated a sensitivity of 92 % and specificity of 96 %, while a threshold of 38 pg/
ml for plasma free metanephrine revealed a sensitivity of 71 %
with a specificity of 79 % (Table 1). However, optimal thresholds
may not reflect clinical requirements: it may be more important
to truly exclude a potentially lethal pheochromocytoma. The use
of a threshold with high clinical sensitivity ( 95 %) results in a
reduced number of false-negative test results, with the trade-o
of low specificity representing a high number of false-positive
test results. Those patients falsely classified as having a pheo-

Metanephrines in plasma (pg/ml)

Biochemical Detection Methods for Plasma

Metanephrines
&

10000

1000

100

10

Fig. 1 Plasma metanephrine (a) and normetanephrine (b) measured by

RIA in patients with pheochromocytoma (PHAEO), aldosterone-producing
(APA) and cortisol-producing (CPA) adrenal adenoma, nonfunctioning
adrenal mass (NFM), essential hypertension (EH), and in healthy
volunteers (C). The horizontal bars demonstrate the threshold for plasma
metanephrine and normetanephrine, respectively.

chromocytoma may then undergo a cost- and time-intensive follow-up to detect the suspected tumour. Depending on the
clinical requirements, we provided thresholds with either high
sensitivity or high specificity, when using an immunoassay
(Table 2). A recent study using an enzyme immunoassay (EIA)
confirmed a higher sensitivity for plasma free normetanephrine

Unger N et al. Immunoassays for Metanephrines Horm Metab Res 2009; 41: 676679

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Spec: specificity; n.a.: not applicable

678 Humans, Clinical

Plasma

Threshold

Spec

(for sens > 95 %)

Metanephrine
Normetanephrine

21 pg/ml
(sens 80 %)
86 pg/ml

Threshold

Sens

(for spec > 95 %)

50 %

92 pg/ml

54 %

78 %

126 pg/ml

92 %

For plasma metanephrine, a sensitivity of 95 % was not available. Thus, the thresh-

old with the highest sensitivity is provided (sensitivity in brackets)

Metanephrines in urine (g/24h)

10000

1000

Biochemical Detection Methods for Urinary

Metanephrines
&

100

10

EH

NF
M

CP
A

AP
A

Ph
eo

EH

NF
M

CP
A

AP
A

Ph
eo

Normetanephrines in urine (g/24h)

cific for plasma free metanephrine and normetanephrine. A

potential cross-reactivity with various blood components such
as other catecholamines was excluded. Especially, caeic and
ferula acid did not demonstrate a significant interference with
the RIA. The results of the RIA were compared with results
obtained by tandem mass spectrometry (LC-MS/MS), revealing a
good correlation for metanephrine (r2 = 0.985) and normetanephrine (r2 = 0.975). Unfortunately, a comparison of immunoassays with the well-established HPLC method is still lacking.
Whereas the RIA-threshold of 126 pg/ml for normetanephrine
in Unger et al. [11] is quite similar to those of the previous studies using HPLC (range: 112165 pg/ml), a threshold of 38 pg/ml
for metanephrine is rather low compared with thresholds
between 60 pg/ml and 99 pg/ml in HPLC studies [9, 10, 14]. However, when adjusting the threshold for a similar specificity as in
the other studies, the value of 92 pg/ml is quite comparable
(Table 2).

10000

1000

100

10

Fig. 2 Urinary metanephrine (a) and normetanephrine (b) measured by

RIA in patients with pheochromocytoma (PHAEO), aldosterone-producing
(APA) and cortisol-producing (CPA) adrenal adenoma, nonfunctioning
adrenal mass (NFM), essential hypertension (EH), and in healthy
volunteers (C). The horizontal bars demonstrate the threshold for urinary
metanephrine and normetanephrine, respectively.

than for metanephrine, with a comparable specificity (Table 1)

[12]. Gao et al. analysed 30 patients with pheochromocytomas:
the thresholds used in that study were calculated (mean + 2 SD)
from the investigation of 36 patients with essential hypertension and 15 patients with excluded pheochromocytomas. This
study might be limited due to the small total number of controls
(n = 51). Furthermore, Lenz et al. investigated a dierent RIA to
ours [13]. An upper reference range of 170 pg/ml for plasma
normetanephrine and 100 pg/ml for plasma metanephrine was
found, when analysing 178 healthy volunteers. Hypertensive
patients demonstrated slightly, but statistically not significant
higher levels compared to controls. The assays were highly spe-

When considering urinary metanephrines for the diagnosis of

pheochromocytoma, previous studies relied mostly on the
measurement of urinary total metanephrines, with low sensitivities between 65 % and 89 % and specificities from 89 % to 95 %
[7, 8, 14, 15]. In contrast, the determination of fractionated
metanephrines measured by HPLC in previous studies resulted
in higher sensitivities, from 88 % to 97 %, but impaired specificities, from 69 % to 85 % [7, 15]. We previously reported on the
value of determination of urinary fractionated metanephrines
by radioimmunoassay [11]. Thresholds were calculated by ROC
analysis. Scattergrams of plasma metanephrine and normetane Fig. 2. We provided a threshold of
phrine are shown in
111 g/d (sensitivity 80 %, specificity 83 %) for urinary metanephrine and 437 g/d (sensitivity 93 %, specificity 87 %) for urinary normetanephrine. In our study, urinary fractionated
metanephrines demonstrated a slightly lower sensitivity and
specificity than their plasma counterparts. Urinary metanephrines largely reflect sulfate-conjugated metabolites, which are
formed in gastrointestinal tissue. Thus, they are not related only
to pheochromocytoma, which may explain the reduced accuracy compared with plasma metanephrines [16, 17].

Influencing Variables
&
Eisenhofer et al. demonstrated the possible influence of drugs
on the levels of catecholamines and their metabolites [14]. Nonselective alpha-blockers such as phenoxybenzamine and tricyclic antidepressants may be associated with higher plasma and
urinary levels of norepinephrine and normetanephrine, but not
epinephrine or metanephrine. Beta-blockers, to a much lower
extent, may be responsible for elevated levels of plasma metanephrines and urinary catecholamines and metanephrines. This
rather physiological influence of antihypertensive drugs is independent of the assay used to determine metanephrines. However, a direct interference of antihypertensive and other drugs
on the levels of metanephrines was demonstrated for the HPLC
system [1820]. In contrast, Wassell et al. investigated the interference of beta-blockers, alpha-blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, diuretics,

Unger N et al. Immunoassays for Metanephrines Horm Metab Res 2009; 41: 676679

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Table 2 Thresholds with high sensitivity ( 95 %) and specificity ( 95 %) for

the diagnosis of pheochromocytoma as determined by RIA [11] a

alpha-methyldopa, and statins in an RIA for urinary total

metanephrines [21]. They revealed no significant influence of
these drugs on the RIA. Lenz et al. investigated the impact of
haemoglobin, bilirubin, and triglycerides on their RIA for plasma
free metanephrines by adding these blood components to the
blood samples [13]. It revealed no significant interference with
the RIA. The same authors also tested the influence of the use of
heparin- and EDTA-tubes on the measurement of plasma free
metanephrines in the RIA applied [13]. They found a good correlation of the probes for metanephrine (r = 0.985) and normetanephrine (r = 0.992).
Lenders et al. investigated the influence of the patients position
[22]. The study revealed that levels of plasma normetanephrine
and metanephrine were 30 and 27 %, respectively, higher in
patients in a sitting position than those in patients in a supine
position. Use of the upper limit of normal derived from samples
taken in supine rest resulted in a profound loss of specificity (91
vs. 75 %) for patients investigated in seated position. In order to
preserve high accuracy, the authors therefore proposed that
thresholds should be established using samples taken only from
patients in the supine position.
Biochemical activity may dier between sporadic and various
genetically-determined pheochromocytomas. Lenders et al.
found biochemical parameters to be higher in sporadic compared to hereditary pheochromocytomas [7]. Eisenhofer et al.
found that patients with von HippelLindau syndrome almost
exclusively had high plasma levels of only normetanephrine,
whereas patients with multiple endocrine neoplasia type 2
demonstrated high levels of plasma metanephrine [23]. Therefore, adjusted thresholds may be required for patients with
hereditary syndromes, and the biochemical profile may provide
a hint as to the underlying disease.

Conclusion
&
The measurement of metanephrines instead of catecholamines
improves the diagnostic accuracy for the diagnosis of pheochromocytomas. The determination of plasma free metanephrines
demonstrates a higher accuracy than their urinary counterparts.
The use of immunoassays may be an alternative to the laborious
technique of HPLC, although the method needs to be evaluated
in more detail.

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Humans, Clinical