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Pheochromocytoma: pitfalls
in the biochemical evaluation
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Expert Rev. Endocrinol. Metab. 9(2), 123–135 (2014)
Georgiana A Dobri1,
Emmanuel Bravo2,
Amir H Hamrahian*1
1
Department of Endocrinology and
Metabolism, Cleveland Clinic
Foundation, Cleveland, OH, USA
2
Department of Hypertension and
Nephrology, Cleveland Clinic
Foundation, Cleveland, OH, USA
*Author for correspondence:
Tel.: +1 216 445 6709
Fax: +1 216 445 1656
[email protected]
The current work-up of a patient suspected to have a pheochromocytoma starts with the
measurement of plasma or urine metanephrines. Notably, up to a quarter of these patients
will have a false positive result. When the plasma or urine metanephrines are less than the
4-fold upper limit of normal, clinicians struggle between the fear of missing a potentially fatal
condition and ordering costly follow up tests. In many cases, ordering unnecessary imaging
studies may only increase the level of patient anxiety. This article will review various
physiologic factors, pathologic conditions and medications that may influence the levels of
catecholamines and their metabolites yielding false positive or false negative results. Acquiring
familiarity with these conditions as well as interfering medications will equip clinicians with
better interpretation skills of the biochemical tests.
KEYWORDS: catecholamines • dopamine • false negative • false positive • metanephrines • pheochromocytoma
Pheochromocytoma, a neuroendocrine tumor
arising from chromaffin cells, is characterized by
excessive catecholamine production. About
85% of the tumors arise from chromaffin cells
in the adrenal medulla – termed pheochromocytoma – and the rest from extra-adrenal chromaffin cells – termed paraganglioma. Based on
the origin of the chromaffin tissue, paragangliomas are divided into two groups: parasympathetic paragangliomas (most commonly along
the cranial nerves; for example, glomus tumors,
chemodectoma and carotid body tumor) and
sympathetic paragangliomas (sympathetic ganglia in the abdomen, less commonly in the pelvis, mediastinum and neck) [1].
Depending on the type of catecholamines
produced (epinephrine [E], norepinephrine
[NE] or dopamine [DA]) and individual’s
response to the catecholamines, pheochromocytomas have a highly variable clinical picture,
most commonly presenting with headaches,
sweating, palpitations or hypertension (HTN).
Of note, up to 20% of the patients have normal blood pressure and 10–15% have few or
no symptoms at the time of the evaluation –
termed the silent pheochromocytomas, making
the diagnosis even more challenging [2–4].
Of all the hypertensive patients tested for
pheochromocytoma, less than 1% have a
catecholamine-producing tumor. On the other
hand, looking at autopsy studies, an undiagnosed
pheochromocytoma was found in 0.05–0.3% of
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10.1586/17446651.2014.887985
the cases. Surgery was the precipitating cause of
death in about 25% of these patients [5–7]. Pheochromocytoma is an uncommon disorder, frequently looked for and rarely diagnosed.
As missing a pheochromocytoma can have
serious and potentially fatal consequences, the
reference intervals for the plasma and urine
catecholamines and metanephrines should ideally be set to provide optimal diagnostic sensitivity. As one might expect, the result is
decreased test specificity. Therefore, clinicians
are faced with a number of potential falsepositive results – in one study, 22% of the
patients tested for pheochromocytoma had at
least one false-positive result [8].
A proper interpretation of the diagnostic
test results is influenced by the knowledge and
understanding of catecholamine secretion,
metabolism and excretion, since any situation
that may affect any one of these steps may
lead to abnormal biochemical testing. We will
review various physiological factors, pathological conditions and medications that might
influence the levels of catecholamines and their
metabolites, resulting in false-positive or falsenegative results.
The word metanephrine in its plural form –
‘metanephrines’ – is generally used as a term
encompassing both metabolites of E and NE
(e.g., plasma metanephrines, urine metanephrines). When used to denote the direct
metabolite of E, it is usually in singular
2014 Informa UK Ltd
ISSN 1744-6651
123
Review
Dobri, Bravo & Hamrahian
The most important enzymes in catecholamine metabolism are monoamine
oxidase (MAO), COMT and sulfotransferTH
ase (FIGURE 1). The presence of high-affinity
DOPA
COMT isoenzyme in pheochromocytoma
leads to local metabolism to metanephrines
L-AADC
of a large proportion of the catecholamines
COMT
MAO
produced by the tumor before release into
Dopamine
Methoxytyramine
HVA
circulation. On the other hand, in a state
of sympathetic overactivity, the NE proSULT
DBH
NMN-SO4
Normetanephrine
COMT
duced by the neurons is released in the
circulation or preferentially processed to
Norepinephrine
MAO
MAO
3,4-dihydroxyphenylglycol by MAO.
COMT
Accordingly, there is a substantial dissoVMA
PNMT
ciation between the more elevated plasma
COMT
catecholamines compared with metaMAO
MAO
nephrines, which may help to distinguish
hypernoradrenergic HTN from pheoEpinephrine
SULT
chromocytoma [10]. In addition, even if
MN-SO
4
Metanephrine
COMT
the catecholamines are produced episodically in the tumor, they are stored in
Figure 1. A simplified overview of catecholamine synthesis and metabolism
granules from where they continuously
excluding a number of intermediary compounds.
leak and get metabolized by COMT;
COMT: Catechol-O-methyltransferase; DBH: Dopamine b-hydroxylase;
DHPG: 3,4 dihydroxyphenylglycol; DOPA: 3,4-dihydroxyphenylalanine; HVA: Homovanillic
therefore, the metanephrines may be
acid; L-AADC: L-aromatic amino acid decarboxylase; MAO: Monoamine oxidase;
measured at any time regardless of the
MN-SO4: Sulfated metanephrine; NMN-SO4: Sulfated normetanephrine;
presence or absence of symptoms. SimiPNMT: Phenylethanolamine N-methyltransferase; SULT: Sulphotranferase; TH: Tyrosine
larly, methoxytyramine – the COMT
hydroxylase; VMA: Vanillylmandelic acid.
metabolite of DA is a better marker for
detection of DA-producing tumors than
form, ‘metanephrine’, and in this review, it will be used in measurement of DA alone [11].
its abbreviated form, MN. The direct metabolite of NE,
NE is derived from multiple sources including adrenal and
‘normetanephrine’, in this review, will be referred to as NMN. extra-adrenal chromaffin cells, neurons, mesenteric organs and
diet [12]. E is secreted mainly from the adrenal; thus, elevations
in the E and MN levels are much more specific for the presCatecholamine synthesis & metabolism
Catecholamine synthesis begins from the amino acid tyrosine; ence of pheochromocytoma compared with elevations in NE or
some of the tyrosine is formed from phenylalanine, but most of NMN levels.
Kidneys do not excrete free metanephrines or catecholamines
it is of dietary origin (FIGURE 1). The rate-limiting step is the conversion of tyrosine to 3,4-dihydroxyphenylalanine by tyrosine efficiently, until these are further sulfate conjugated or metabolized
hydroxylase. 3,4-dihydroxyphenylalanine is converted to DA, to vanillylmandelic acid (VMA) or homovanillic acid. The clearwhich is further converted to NE. NMN, MN and methoxytyr- ance of conjugated metanephrines is totally dependent on the
amine are the catechol-O-methyltransferase (COMT)-methylated kidney function, and accordingly their half-life is much longer than
free metanephrines, which is little affected by renal function.
products of NE, E and DA, respectively.
Chromaffin cells, mainly of adrenal origin, harbor phenylethanolamine N-methyltransferase (PNMT) in the cytoplasm, which Catecholamine receptors & action
converts the NE leaking from the storage granules into E. In the Generically classified as a- and b-receptors, 2-a, 3-b and
storage vesicles, NE and E are bound to a protein called chromogra- 5 DA adrenoreceptors, with their organ-specific roles have been
nin A. PNMT expression is dependent upon high levels of onsite described [13–16]. They are widely distributed throughout the
cortisol. This feature explains why E is mainly secreted from the body (eye, heart, arteries and veins, muscle, hepatomesenteric
adrenal medulla and why adrenal pheochromocytomas produce E, system, lungs, kidney, bladder, uterus, sex organs, skin, fat
frequently in addition to NE, while extra-adrenal tumors and cells) but for the purpose of this review, we will only discuss
metastasis (which lack local cortisol synthesis and PNMT) produce the effects most relevant to the presentation and diagnosis
NE and DA. Rarely, paragangliomas may be associated with of pheochromocytoma.
increased E and its metabolites, up to twofold upper limit of normal
a-1 adrenoreceptors are postsynaptic receptors located in the
range (ULN) in the authors’ experience; however, the NE/E and vascular smooth muscle and are responsible for vascular conNMN/MN ratios are considerably increased in such patients [9].
striction and HTN. a-2 receptors are present at presynaptic
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Tyrosine
124
Expert Rev. Endocrinol. Metab. 9(2), (2014)
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Pheochromocytoma: pitfalls in diagnosis
level inhibiting secretion of NE from the neuron through a
negative feedback loop. They have minor contribution to the
vascular smooth muscle constriction compared with a-1
receptors activation.
b-1 adrenoreceptors increase renin secretion by the kidney
contributing to some extent to HTN; they have major actions
in the heart with positive inotropic and chronotropic effects,
making the b-1 receptor anatagonists medications of choice in
controlling the pheochromocytoma-associated tachyarrhythmias.
b-2 adrenoreceptor stimulation causes bronchodilatation, glycogenolysis, vasodilatation and increased NE release from neurons. E has more potent effects on b-2 adrenoreceptors, which
provides an explanation for hypotension and hyperglycemia as
a presentation of predominantly E secreting pheochromocytomas. b-3 receptors activation promotes lipolysis.
DA-1 receptors are present in vessels and kidneys and promote vasodilatation, diuresis and natriuresis. DA receptors
2 through 5 have various roles at the brain level. DA, at physiological concentrations, has minor effects on a and b receptors,
but as the levels increase (like in DA secreting pheochromocytomas), cross-reaction can cause vasoconstriction and elevated
blood pressure [17,18]. The majority of patients with rare, purely
DA-producing pheochromocytomas are normotensive, but may
develop hypotension if a-blockade is used, secondary to unopposed stimulation of DA receptors [11,19].
Biochemical evaluation of pheochromocytoma
From a diagnostic standpoint, the most important metabolites
are methoxytyramine for DA and MN, NMN for E and NE,
respectively. For screening purposes, plasma and urine measurements of catecholamines and urine VMA have fallen out of
favor because of lower sensitivity and specificity compared with
the metanephrines.
In plasma, the commonly used commercial assays measure free
MN and NMN levels. The measured 24-h urine MN and
NMN levels are mostly made up of sulfated compounds with
small amounts of free metanephrines. Currently available assays
measure the free form of catecholamines in plasma and urine.
Ideally, the biochemical investigation is pursued first, and a confirmed diagnosis is followed by imaging studies for localization.
In some cases, when it is challenging to confirm the biochemical
diagnosis (repeated metanephrines elevation <4-times the ULN)
[20,21], imaging can be used as an aid for the diagnosis [22]. There
is a lack of consensus on the best initial diagnostic test with clear
differences among institutions in their choice [2,23]. While both
plasma and urine metanephrines represent reasonable options for
initial biochemical evaluation in patients suspected to have pheochromocytoma, the authors prefer plasma metanephrines since
almost invariably normal levels rule out a diagnosis of pheochromocytoma and because of the ease of collection [24]. In our experience, when the normotensive reference range is used for plasma
and urine metanephrines, their diagnostic specificity is comparable or in favor of plasma metanephrines [20].
In a landmark paper by Lenders et al., the investigators analyzed the characteristics of various biochemical tests for
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investigation of pheochromocytoma [20]. The authors showed a
99% sensitivity, 89% specificity for plasma fractionated metanephrines and 97% sensitivity, 69% specificity for urine fractionated metanephrines, rendering plasma fractionated metanephrines
as the best test for initial biochemical evaluation. Plasma
and urine catecholamines as well as VMA had low sensitivities
64–86% making these inadequate screening tools [25–27]. Moreover, using multiple tests may not increase the diagnostic accuracy but leave more room for false-positive results [2,20].
Some investigators recommend the use of plasma metanephrines for screening patients with a high suspicion for pheochromocytoma and urine metanephrines in low-risk patients [28]. In their
study, different population reference ranges for plasma and urine
metanephrines were used. For the 24-h urine metanephrines, the
upper range of a hypertensive population was used, artificially
rendering the test more specific; in contrast, for the plasma metanephrines, the upper range of a normotensive population was
used making the test more sensitive but less specific.
Chromogranin A is not a reliable screening test for pheochromocytoma but has been proposed as an add-on test to clarify
intermediate elevations of plasma fractionated metanephrines
and catecholamines [29–31]. In patients with elevation in plasma
metanephrines, <fourfold the ULN and not taking protonpump inhibitors, since these are associated with elevated levels,
a Chromogranin A >270 ng/ml (normal up to 225 ng/ml)
had a 87% sensitivity and 89% specificity in diagnosing
pheochromocytoma [30].
Concomitant measurement of catecholamines may be useful
in interpretation of the metanephrines in some circumstances.
A ratio of MN over E and NMN over NE has been used by
some investigators to differentiate intermediate results in
patients suspected to have pheochromocytoma. Eisenhofer et al.
showed that all patients without pheochromocytoma had MN/
E <4.2 and NMN/NE <0.52, but these ratios were useful in
30 and 15% of the patients, respectively, to make the diagnosis
of pheochromocytoma [25].
Lenders et al. showed that all patients without pheochromocytoma had plasma NMN <3.6 and MN <3.9-fold ULN and that
80% of the patients with pheochromocytoma were diagnosed by
these cutoffs [20]. Only a few case reports with false-positive MN
and NMN levels >fourfold ULN have been reported [8].
False-positive results
A number of physiological and pathological conditions can
increase the levels of catecholamines and their metabolites
(TABLE 1). Therefore, caution should be taken when interpreting
the results particularly in patients with levels in the indeterminate range for the diagnosis of pheochromocytoma (NM and
NMN <fourfold ULN).
Age adjustments
Eisenhofer et al. looked at the optimal reference intervals for
metanephrines based on sex and age, by examining blood samples from 1226 subjects aged 5–84 years, including 535 patients
in whom pheochromocytoma was ruled out, were examined.
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Dobri, Bravo & Hamrahian
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Table 1. Confounding variables associated with false-positive metanephrines.
Variable
Effect on metanephrines levels
Biochemical pattern
Intervention
Ref.
Age
Increase in plasma
NMN with age
Twofold increase in plasma
NMN from childhood to
60 years old
Age-specific ranges
Posture – sitting
versus supine
Increase in plasma MN,
NMN in seated position
Increase in plasma MN, NMN
by up to 30%
Blood draw in supine position
after 30 min rest
Exercise
Increase in plasma MN,
NMN with the degree
of activity
Increase in plasma MN, NMN
up to two- to threefold
Avoid exercise on the day of
blood draw
High catecholamine
diet
Increase in NMN and
methoxytyramine
Increase in urine NMN twofold
and methoxytyramine threefold
Avoid high catecholamine
foods for 24 h
[40,41]
Renal impairment
MN and NMN plasma levels increase
with the degree of impairment
and 24-h urine levels become
unreliable
Increase in plasma
NMN <fourfold ULN, increase
in plasma MN <twofold ULN
Avoid measuring urine
metanephrines. Plasma MN
is the least affected
[10,45]
HTN
Increase in plasma and urine
metanephrines levels with
the degree of HTN
Increase in plasma MN, NMN
up to 50%
Use different ranges for
hypertensive population
Stroke/ICH
Increase in urine MN, NMN
Increase in urine MN,
NMN >twofold ULN
Avoid biochemical evaluation
within one week of the event
[59]
Decompensated
congestive heart
failure
Increase in plasma NMN
Increase in plasma NMN
– two- to fourfold
Stabilize underlying disease (if
possible) and repeat testing.
Plasma MN not affected
[8,55]
OSA
Increase in urine NMN
Increase in urine NMN by 30%
Treat OSA and repeat levels
[32,33]
[20,35–38]
[35,38,39]
[46–50]
[56–58]
DA: Dopamine; E: Epinephrine; MN: Metanephrine; NE: Norepinephrine; NMN: Normetanephrine; OSA: Obstructive sleep apnea; ULN: Upper limit of normal.
The data were further validated in 3888 patients evaluated for
pheochromocytoma including 558 in whom pheochromocytoma was confirmed. The study showed a consistent increase in
NMN with aging; a weaker correlation was described in MN
levels [32]. Upper cutoffs for reference intervals showed
age-associated increases with almost doubling in the upper
limits, from 0.588 nmol/l in adults 18–30 years of age to
1.047 nmol/l in adults over 60 years. Based on these data, the
investigators developed a curvilinear model for age-adjusted
upper cutoffs for NMN, which significantly increased specificity (from 88.3 to 96%) with virtually no change in the diagnostic sensitivity (from 93.9 to 93.6%).
Similar findings were reported by Sawka et al., who measured
plasma metanephrines in a total of 501 subjects (including
56 patients with pheochromocytoma). The proposed age-adjusted
MN score would decrease the false-positive rate from 16.3 to
3%, keeping the sensitivity at 100% [33]. The study shows that
adjustment for age in the interpretation of results of plasma
metanephrines significantly decreases false positives in patients
being evaluated for sporadic pheochromocytoma.
Posture, rest & exercise
Several studies have shown that levels of plasma metanephrines
in a seated position are about 30% higher compared with sampling after 30 min of supine rest [34,35]. The 20–30 min period
was initially derived from the time needed to achieve a steady
126
state for concentration of catecholamines (4–5 half-lives of
about 5 min). This time frame was later verified by showing a
decrease in plasma MN and NMN levels in the first 30 min
after transition from a seated to a supine position and insignificant change beyond that [36]. The circulatory clearances and
plasma half-lives of free MN and NMN are similar to those of
their catecholamine precursors [35,37,38].
It would seem reasonable to expect that diagnostic performance would be similar if one used the upper cutoff range for
plasma metanephrines matched to the phlebotomy technique
used. However, a recent study by Därr et al. showed that there
was an unacceptable drop in diagnostic sensitivity for plasma
metanephrines from 100 to 77.6% when comparing blood
draws in fasting rested supine position (using supine cutoffs) to
blood draws in nonfasting seated position (using seated cutoffs)
with no significant change in diagnostic specificity [36].
On the other hand, in order to avoid the possibility of a
false-positive test with a random blood draw, we would suggest
repeating the test after 30 min of supine rest, if the initial levels
were elevated [34].
Physical activity significantly increases plasma catecholamines
and metanephrines by 60–230% depending on the degree of activity. Levels do not return to normal after 15 min of rest in a seated
position. Therefore, it is important to avoid exercise prior to the
blood sampling as 15–30 min of rest cannot counteract the rise in
the metanephrines. DA is not affected by exercise [35,38,39].
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Diet & fasting state
Dietary catecholamines are metabolized by the monoaminepreferring sulfotransferases present in the gut and get absorbed
into the blood stream as conjugated catecholamines and metanephrines. Foods with high catecholamine content include
nuts, fruits – especially bananas, vegetables like potatoes, tomatoes, beans and cheeses [40]. Since a substantial proportion of
the total metanephrines (conjugated and free) is derived from
the diet, the measurement of free metanephrines is more accurate for the diagnosis of pheochromocytoma.
De Jong et al. studied the influence of a change from a
poor to a rich catecholamine diet and they found no appreciable change in plasma free NMN, but up to twofold elevation in urine total NMN. There was no significant change in
either plasma or urine MN levels. Methoxytyramine in both
plasma and urine was up to threefold elevated after a rich
catechol diet. This diet has resulted in a modest effect of up
to a 1.5-fold increase in 24-h urine free DA and NE but a
negligible influence on E [41]. These observations may be
explained by the content of DA and NE compared with E in
food. A 24-h restriction of catecholamine-rich foods is recommended when measuring 24-h urine catecholamines or
metanephrines; no specific diet is necessary when measuring
plasma metanephrines [40,41].
A study of 180 healthy subjects showed that drinking two
cups of coffee resulted in a 20% increase in plasma NMN,
with no change in the MN levels [35]. High salt diets may
decrease plasma NE, NMN by about 10% [42] in both normotensive and hypertensive subjects, with low salt diets having the
opposite effect [43]. These fluctuations seem to be small in
absolute number and likely clinically insignificant. The high
salt diet did not have a statistically significant effect on plasma
E and MN levels [42,43].
Smoking
A small study demonstrated a 1.5- to 3-fold increase from
baseline in plasma NE and E levels, but no data on the magnitude of the increase above the ULN or the effect on plasma
or urine metanephrines were provided [44]. Notably, we do
not know if nicotine withdrawal would generate a sympathetic response possibly leading to an even more generous
catecholamine response. Some reference labs recommend
refraining from smoking for 4 h prior to blood draw for
plasma catecholamines.
Renal impairment
The sulfate-conjugated MN and NMN are excreted by the
kidney, and renal impairment makes their plasma and urine
levels inaccurate in reflecting the true rate of production. As
the urine metanephrines measured by the current assays are
mostly sulfate conjugated, their measurement in the 24-h
urine would be unreliable for evaluation of patients suspected
to have pheochromocytoma [10].
Measurement of free plasma metanephrines is the best biochemical test for evaluation of patients with renal insufficiency
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suspected of having pheochromocytoma, since their clearance is
considerably less affected by renal function [10]. In a study comparing free plasma metanephrines in patients with variable
degrees of renal insufficiency, including end-stage renal disease
requiring hemodialysis, to patients with essential HTN and
normotensive volunteers, the mean plasma free metanephrines
level was elevated up to twofold ULN in the majority of the
former group. The liquid chromatography with electrochemical
detection HPLC assay was used to measure the plasma metanephrines [45]. The upper 95% CI for plasma free NMN and
MN levels were <three- and twofold the ULN compared with
the hypertensive control group and <four- and twofold the
ULN compared with the normotensive group, respectively. In
patients on hemodialysis, only one out of four patients showed
increased plasma metanephrines. However, the investigators
could not reliably measure plasma catecholamines and metanephrines in about 30% of patients on dialysis because of interfering substances present in the blood. The use of liquid
chromatography with tandem mass spectrometry (LC–MS/MS)
is expected to minimize the analytical problems in patients
with renal insufficiency.
Hypertension
The majority of studies examining catecholamine levels in
patients with HTN have reported up to 25–50% higher plasma
and urine NE levels than in normotensive controls. E is also
higher in patients with HTN compared with controls but to a
lesser degree compared with NE. Catecholamine levels increase
with the degree of HTN [46]. Urine metanephrines are also higher
in patients with HTN; however, the effect on plasma metanephrines is small with at least one study showing no increase in hypertensive patients [47–49]. Some reference labs report different
reference ranges for plasma and urine metanephrines in patients
with HTN (up to 50% higher), which would decrease the falsepositive results at the expense of a slight decrease in diagnostic
sensitivity [50].
The clonidine suppression test, initially described by Bravo et al. in
1981, revealed that in hypertensive patients without pheochromocytoma, NE levels decreased to within the normal range 3 h after
an oral dose of 0.3 mg clonidine. Another criterion used for a normal response is a fall in plasma NE of >50% from baseline. This
has a better sensitivity but higher rate of false-positive results [51–54].
The clonidine suppression test is based on the central action of clonidine on a-2 adrenoreceptors located at presynaptic level, which
when activated would inhibit the secretion of NE from the neuronal level. The test may be used to distinguish between patients with
pheochromocytoma from hypertensive patients suspected to have
sympathetic overdrive, particularly in those with elevation of NE
and NMN in the two- to fourfold ULN range.
Investigators from the NIH re-evaluated the clonidine suppression test [25]. Using the criteria of normalization and a
decrease by >40% in the plasma NMN levels, the test yielded a
96% sensitivity and 100% specificity. Using normalization of
NE and a 50% decrease in its level yielded 67% sensitivity and
98% specificity in the same cohort. Because clonidine acts at the
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Dobri, Bravo & Hamrahian
neuronal synaptic level and not in the adrenal, the test cannot be
used to evaluate patients with elevated E and MN levels.
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Medical conditions
About a third of the false-positive results in patients evaluated
for pheochromocytoma are due to physiological variations in
catecholamines [8]. The increased sympathetic tone in response
to anxiety, pain, alcohol or clonidine withdrawal, acidosis,
hypotension, infection or bleeding can result in elevated plasma
and urine catecholamines and metanephrines. The dissociation
between the high degree of elevation in the NE compared with
the NMN level and minimal or no elevation in the E and MN
may serve as clues to the underlying sympathetic overdrive.
Not uncommonly, the treatment of the underlying condition
will result in normalization of elevated catecholamine and MN
levels. It becomes particularly challenging to decide if conditions like stroke, myocardial infarction and decompensated
heart failure are causes for false-positive results or complications
of a pheochromocytoma crisis [8,55].
In population-based studies, an association between sleepdisordered breathing and MN and NMN levels, independent
of major confounding factors, has been reported [56–58]. In a
number of case reports, the elevated NE in patients with
obstructive sleep apnea returned to normal after treatment [56].
In a small prospective study, patients with intracerebral hemorrhage (ICH), four out of six patients had elevated 24-h urine
catecholamines and/or metanephrines above twofold ULN [59].
The levels peaked between the third and sixth day after the
event were proportional to the ICH size and continued to
decrease but were still elevated 1 month after with subsequent
normalization [59]. The authors recommended that screening
for pheochromocytoma to be avoided in the first week after an
ICH event and preferably deferred for at least 1 month.
Elevated NE levels are described in patients with decompensated cirrhosis with four-times higher levels than controls.
Increased secretion secondary to lower effective arterial blood
volume rather than a decrease in clearance has been postulated
as the underlying etiology, since the elevated NE levels were
suppressible by central blood volume expansion [60].
In a study by Eisenhofer et al., that included 35 patients
with congestive heart failure, there was a two- to fourfold
increase in plasma NE and NMN compared with controls.
While there also was an increase in E levels, MN levels were
not affected [48]. In another study of patients with pulmonary
HTN of noncardiac origin, circulating NE levels were twotimes higher than controls [61].
Medications
Levels of catecholamines and their metabolites may be influenced by a number of medications. Knowledge of the potential
mechanisms of drug interference is essential in patients being
evaluated for pheochromocytoma (TABLE 2). The drug interference
with the assay is now a rare event if modern mass spectroscopy
assays are used. Overall, medication interaction may account
for about 20% of false-positive results [8].
128
Medications: pharmacological effect
Tricyclic antidepressants (e.g., amitriptyline, doxepin, imipramine) block the neuronal reuptake of NE, increasing its escape
into the circulation. Accordingly, the neuronal MAO action is
bypassed, resulting in higher amounts of COMT metabolite
NMN. This class of drugs does not affect E and MN levels.
These medications may also increase blood pressure, further
contributing to a false impression of pheochromocytoma
[25,62,63]. The increase in plasma or urine NMN in patients taking this class of medication is usually less than fourfold
ULN [25].
Although tricyclic antidepressants are known to increase
NE/NMN levels, newer classes of antidepressants which inhibit
NE neuronal reuptake are potentially associated with falsepositive results. Clinicians need to inquire about the use of
serotonin NE reuptake inhibitors (venlafaxine, duloxetine),
selective serotonin reuptake inhibitors (SSRIs: fluoxetine, sertraline, citalopram, escitalopram) and NE DA reuptake inhibitors
(bupropion). Neary et al. reported a patient with plasma
NMN > fourfolds ULN while on venlafaxine, which returned
to normal 2 weeks after discontinuation of the drug [64]. Some
of the atypical antipsychotics, such as quetiapine, may be associated with two- to three-times elevations in urine and plasma
NMN through blocking NE reuptake and increasing NE secretion via presynaptic a-2 receptors block [65].
Use of recreational drugs such as cocaine which inhibits NE
reuptake, amphetamines, agents used for ADHD such as methylphenidate and antiobesity agents like phentermine, which stimulate the release of catecholamines may account for false-positive
results [66]. Sympathomimetics (ephedrine, pseudoephedrine)
increase the production of catecholamines along with their
metabolites, MN and NMN [25]. Withdrawal from sedative drugs
like benzodiazepines, opioids, clonidine and alcohol increases
sympathetic drive, which may also lead to false-positive results.
MAO inhibitors (tranylcypromine, phenelzine, selegiline)
decrease the MAO conversion of NE and E, resulting in preferential metabolism through the COMT pathway, thereby
increasing the MN and NMN levels (FIGURE 1).
COMT inhibitors used as adjuvants in Parkinson’s disease
increase DA, E and NE. The same biochemical pattern is
found with the use of Levodopa and a Methyldopa as both
drugs are competitive substrates with catecholamines for the
COMT enzyme. Use of levodopa results in small increases
in E, NE, MN and NMN usually below the ULN, but can
cause marked DA and methoxytyramine elevations of up to
20- and 150-fold, respectively [67–69]. Elevation of urine catecholamines of up to 10-times the ULN has been described
with the use of Methyldopa [70].
a-blockers are medications of choice in controlling blood
pressure in patients with pheochromocytoma during their preoperative preparation. Phenoxybenzamine, a noncompetitive
a-1 and 2 adrenoreceptor blocker, is an important cause for
false-positive results [25]. It is usually associated with <fourfold
ULN elevation in plasma and urine NMN. The drug has no
significant influence on the E or MN levels [25].
Expert Rev. Endocrinol. Metab. 9(2), (2014)
Pheochromocytoma: pitfalls in diagnosis
Review
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Table 2. Medication interaction with metanephrines levels through pharmacologic effect.
Medication
Site of action
Biochemical pattern
Ref.
a-1 and -2 blockers (phenoxybenzamine)
Baroreflex mediated sympathetic
activation
Increase in NE release through
presynaptic a-2 receptor inhibition
Increase in plasma and urine NMN up
to two- to threefold ULN
[25]
Amphetamines, methylphenidate,
phentermine
Sympathetic stimulation
Increase in MN, NMN
[66]
Antidepressants
Tricyclics – (amitriptyline, doxepin,
imipramine)
Serotonin NE reuptake inhibitors – SNRIs
(venlafaxine, duloxetine)
Selective serotonin reuptake
inhibitors – SSRIs (fluoxetine, sertraline,
citalopram)
NE DA reuptake inhibitors – NDRIs
(bupropion)
Block neuronal uptake of NE
Increase in plasma and urine NMN up
to fourfold ULN. Rarely may result in
levels >fourfold ULN
b-blockers (atenolol, metoprolol,
propranolol)
Mechanism uncertain
Increase in plasma MN – mild
Levodopa
Competitive substrate for COMT
Minimal increase in plasma and urine
MN/NMN within normal range,
increase in plasma and urine
methoxytyramine 149-fold
MAO inhibitors (tranylcypromine,
phenelzine, selegiline)
MAO inhibition (enzyme implicated in
both neuronal and extraneuronal
catecholamine metabolism)
Increase in plasma and urine MN, NMN
[10,55]
Pseudoephedrine
Sympathetic stimulation
Increase in plasma and urine MN, NMN
[25,55]
Quetiapine
a-blocker
Block neuronal uptake of NE
Increase in plasma and urine NMN
two- to threefold
[65]
Withdrawal – alcohol, benzodiazepines,
clonidine, opioids
Sympathetic stimulation
Increase in plasma and urine MN, NMN
[55]
[25,62–64]
[25]
[67–69]
COMT: Catechol-O-methyltransferase; DA: Dopamine; E: Epinephrine; MAO: Monoamine oxidase; MN: Metanephrine; NE: Norepinephrine; NMN: Normetanephrine;
ULN: Upper limit of normal.
As a class, b-adrenoreceptor blockers, including the combined a- and b-adrenoreceptor blockers such as labetolol, were
found to be the most frequent cause for false-positive results in
plasma MN (12% of the patients taking b-blockers), but the
magnitude of the elevation was not reported [25]. In the same
report, this class of drugs was not associated with an increased
frequency of false-positive results for plasma catecholamines
and NMN. In agreement with the conclusion made by the
study authors, we do not feel that stopping these drugs is justified unless there are equivocal results.
Calcium channel blockers and a1-adrenoreceptor blocking
drugs may be associated with mild elevations in NE levels via
reflex sympathetic activation in response to the drop in blood
pressure with minimal influence on E or metanephrines. These
classes represent alternative options to control blood pressure in
patients evaluated for pheochromocytoma. Angiotensinconverting enzyme inhibitors, angiotensin receptor blockers and
diuretics seem to have little influence on causing false-positive
results [25].
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The use of D2 receptor antagonists such as metoclopramide
may result in a catecholamine increase via presynaptic release of
NE from the secondary to a decrease in the DA inhibitory system [71]. Intravenous metoclopramide given to patients with
essential HTN increased plasma catecholamine levels <twofold
ULN [72].
Medications: assay interference
Spectrophotometry has been used in the past for measurement
of catecholamines and metanephrines. This assay was prone to
false-positive results due to multiple medication interactions.
HPLC method currently used for plasma and urine catecholamine measurement underwent considerable improvement and
most of the medication–assay interactions have been addressed;
when the issue cannot be resolved, the interference is usually
reported.
For the measurement of metanephrines, most of the reference labs in the USA use the LC–MS/MS method, which is
significantly less prone to drug interference [73–75].
129
Review
Dobri, Bravo & Hamrahian
Elevated plasma NMN
>4-fold ULN
<4-fold ULN
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PHEO very likely
Repeat levels after:
– Stopping interfering medications and
stabilizing interfering diseases, if possible
– Avoiding exercise on the day of testing
– Drawing blood supine after 30 min rest
Normal
<2-fold ULN
PHEO unlikely
Functional
PHEO ruled out
Follow up and repeat levels
including 24-h urinary
metanephrines. May consider
clonidine suppression test or
imaging in selected cases
Imaging after
medication
interference ruled out
2–4-fold ULN
Clonidine
suppression test
Normal
Abnormal
did not vary; NMN levels did not show a
gender difference [32,35]. A retrospective
study of 24-h urine fractionated metanephrines showed significantly higher
urine MN and NMN values men versus
women in the Korean population. The
authors proposed gender-specific cutoffs
to improve diagnostic sensitivity and
specificity [82]. However, further studies
are needed to determine if these findings
can be reproduced and extended to
other races.
A small increase in plasma free NMN
has been seen in parallel with the BMI,
but the increase was not sufficient to alter
cutoff values. Accordingly, a gender and
BMI-adjusted reference range does not
seem to be warranted [35].
Time of day & venipuncture
Catecholamines and their metabolites
have no diurnal variation and therefore
Follow-up and repeat levels Imaging
sampling could be made at any time durif high clinical suspicion
ing the day. However, urine and plasma
catecholamines are lower overnight comFigure 2. Approach to elevated plasma normetanephrine in patients suspected
pared with waking hours, which may be
to have sporadic pheochromocytoma.
related to activity and posture [35,83,84].
NM: Normetanephrine; PHEO: Pheochromocytoma; ULN: Upper limit of normal range.
No spike in plasma MN and NMN levDA, originating from exogenous compounds (e.g., levo- els after venipuncture has been reported [35].
dopa, carbidopa), may be measured along with endogenous
DA. Clinicians should be aware of this interference Menstrual cycle
when measuring catecholamine levels in a patient using such There is no relationship of metanephrines with phases of the
medications.
menstrual cycle, although a moderate, transient increase in
Caffeic acid is a catechol found in coffee, including the plasma NMN has been shown just before and after the ovuladecaffeinated ones, and interferes with plasma catecholamine tory LH peak. This finding is likely of no diagnostic signifiassays, producing falsely high levels of E and DA. Refraining cance in the work-up of reproductive age women suspected to
from caffeinated products, including decaffeinated coffee, is rec- have pheochromocytoma [35].
ommended for 24 h prior to testing [76]. It is not clear if current generation HPLC assays have overcome this interference as False-negative results
newer studies have not been performed.
With a few exceptions, current assays for plasma and urine
The HPLC assay is rarely used for the measurement of metanephrines provide a very high sensitivity in diagnosis of
metanephrines in the USA. Not only is the LC–MS/MS assay patients suspected to have pheochromocytoma.
associated with a far lesser problem with drug interference, but
Small, asymptomatic tumors or early recurrences may proalso is more cost-effective since its far simpler sample prepara- duce very low levels of E, NE and go undetected. Such
tion allows for a higher sample throughput. Acetamino- patients usually have tumors less than 2 cm in size and are
phen [20,77], methenamine (urine antiseptic) [78], mesalamine normotensive. Rarely, histologically proven larger pheochro(anti-inflammatory agent used to treat inflammatory bowel dis- mocytomas may be associated with a normal biochemical
ease) [79], buspirone [80] and sotalol [81] have been reported to work-up including plasma and urine metanephrines. In these
cause false-positive results, using the HPLC assay.
cases, it appears that such pheochromocytomas possess ineffective catecholamine biosynthesis, yielding silent clinical and
biochemical presentations [25,28].
Other conditions associated with negligible increase in
catecholamines & metanephrines
DA-producing pheochromocytomas, which usually present
BMI & gender
as paragangliomas, may be missed either because only E, NE
A few studies have shown that plasma MN levels were and their metabolites are measured or because the tumor has
marginally higher in men than women, but reference intervals a very active COMT isoenzyme that quickly metabolizes DA
PHEO unlikely
130
PHEO likely
Expert Rev. Endocrinol. Metab. 9(2), (2014)
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Pheochromocytoma: pitfalls in diagnosis
to methoxytyramine. This is why methoxytyramine is
thought to be a better marker for detection of DA-producing
tumors than measurement of DA alone [11,28]. However, currently, measurement of methoxytyramine is limited to
research labs. HTN is usually not a feature of DA-producing
paragangliomas and indeed such tumors may be associated
with hypotension [11].
Patients with familial pheochromocytoma syndromes (Von
Hippel Lindau, multiple endocrine neoplasia, neurofibromatosis, paraganglioma), who are screened at early stages of their
disease, may have negative biochemical work-up similar to
those with a small or microscopic tumor burden [20,28].
Recurrence in patients with metastatic pheochromocytoma
with documented negative biochemical work-up has been
reported in a few patients with extra-adrenal pheochromocytoma, without a clear explanation for the finding [20]. The current authors have also come across a similar case. One may
speculate that the metastases might have suffered histological
changes, rendering the tissue ineffective in catecholamines production or release.
It is important to note that plasma and urine MN concentrations decline following adrenalectomy. This recognition may
be particularly important in detecting early recurrences in a
patient with pheochromocytoma postadrenalectomy and can
account for false-negative results [85]. In contrast, the NMN
levels increase up to twofold the ULN after adrenalectomy, a
finding that should not be confused with recurrent disease. The
rise in NMN levels persists at least for 5 years after adrenalectomy and possibly lifelong. This is tentatively explained by
increased sympathetic NE production to compensate for the
decrease in E or previous a and b receptor desensitization by
high circulating levels. These findings might warrant adjusted
reference ranges for MN and NMN in patients with adrenalectomy to further minimize the false-positive and -negative
results.
Assay interference has rarely been reported as a cause of
false-negative results in the past, when the measurement of
total urine metanephrines was done by spectrophotometry [86].
Spilker et al. reported readings below control levels when
studying the effect of theophylline and propranolol on the
measurement of total urine metanephrines, using a similar
assay [86].
Expert commentary
Pheochromocytoma and paraganglioma are rare disorders,
frequently looked for, rarely found and when missed they can
be associated with fatal consequences. The biochemical diagnosis of pheochromocytoma may at times be challenging as
catecholamine secretion and their metabolic pathway can be
influenced by a number of medications, underlying diseases
or in response to exercise and daily stressors. Reaching a correct diagnosis can be enhanced by increasing awareness of
such interactions.
We believe that the measurement of plasma fractionated
metanephrines is the best test for excluding or confirming a
informahealthcare.com
Review
diagnosis of pheochromocytoma, and we recommend it as
the screening test of choice because of the ease of collection
and its excellent diagnostic sensitivity. Measurement of 24-h
urine fractionated metanephrines may be used as an alternative. In our experience, when normotensive reference ranges
are used for both plasma and urine metanephrines, the diagnostic specificity of plasma metanephrines is similar to or
better than urine metanephrines.
The sensitivity and specificity of plasma and urine metanephrines for evaluation of patients suspected to have pheochromocytoma are highly influenced by the cutoff values used.
Therefore, we recommend the use of a large reference population of normotensive and hypertensive controls to establish validated reference ranges for patients with and without HTN,
and ultimately age-specific reference ranges for each of these
two groups.
Previous studies have shown that with the exception of few
circumstances such as the use of tricyclic antidepressants or
decompensated heart failure, plasma fractionated metanephrines
more than fourfold the ULN establishes a diagnosis of pheochromocytoma, thus proceeding with imaging would be the
reasonable next step.
In the case of elevated fractionated plasma metanephrines
of < fourfold the ULN (indeterminate range), the first task is to
review the medication list, ask about illicits, identify possible
pharmacological interferences and, if safe, to stop the respective
medications and repeat the testing.
Any elevation in the plasma MN above the normal range
should be considered suspicious for an underlying pheochromocytoma and if confirmed on repeat testing should be
pursued by adrenal imaging. This approach is based on a
low frequency of false-positive results for plasma MN
(<5%) [25,28].
FIGURE 2 depicts our approach to patients with elevated
plasma NMN who are suspected to have a sporadic pheochromocytoma. Family members of an index patient with
hereditary pheochromocytoma are usually identified at an
earlier stage of their disease. Accordingly, in these cases,
imaging may be considered even in the presence of only
mildly elevated metanephrines, a recommendation that may
not conform to the algorithm (FIGURE 2).
Most patients with plasma NMN <twofold ULN do not
have pheochromocytoma. Concomitant medical conditions that
increase sympathetic tone or decrease catecholamine clearance
should be closely evaluated. Drawing the blood sample in a
supine position after 30 min rest may help to resolve some
falsely elevated results. If not resolved, especially in the absence
of suggestive symptoms, such patients may be followed at intervals and have their levels repeated. In selected cases with persistently elevated levels, a clonidine suppression test or imaging
may be considered.
The main challenge lies in patients with plasma NMN levels
that are between two- to fourfold the ULN. If the review of
the medical history and concomitant medications does not
point toward an identifiable cause and the repeated levels
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Dobri, Bravo & Hamrahian
continue to be in the indeterminate range, the use of clonidine
suppression test is a reasonable next step in evaluation. If the
NMN levels are not appropriately suppressed by clonidine,
then proceeding with imaging to clarify the diagnosis is our
recommended approach.
Normal plasma or urine metanephrines almost always rule
out an underlying functional pheochromocytoma. Falsenegative results may rarely be seen in adrenal pheochromocytomas <2 cm in size, DA-producing pheochromocytomas,
patients with familial pheochromocytoma syndromes who may
have microscopic or small tumors, early recurrence or small
residual tumor tissue after surgical resection of pheochromocytomas and in patients with metastatic disease after resection of
the primary tumor.
The pathways of tumorigenesis will be further investigated. Additional molecular and genetic markers and possibly circulating tumor cells will be discovered that will be
implemented in the evaluation of patients suspected to have
pheochromocytoma. Such markers would provide a more
sensitive screening test for familial pheochromocytoma syndromes and detect recurrences much earlier with a decrease
in false-negative results. At the same time, these may play
an important role in investigation of patients with elevated
plasma and urine metanephrines in the indeterminate
range and could help clinicians to distinguish among
comorbidities that may be secondary to an underlying
pheochromocytoma.
Acknowledgements
Five-year view
MS assay will replace other commercial assays including the
HPLC methods. The use of MS will eliminate or minimize
drug interference with the assay analytical method. There will
be a broader use of normotensive and hypertensive reference
ranges for plasma and urine fractionated metanephrines with
introduction of age-specific normative ranges for each group
resulting in a decrease in the number of false-positive results.
The more specific reference ranges may also be defined
for patients following unilateral or bilateral adrenalectomy. The
use of methoxytyramine assays in the diagnosis of DA-secreting
pheochromocytomas may become commercially available.
We are indebted to C Faiman for reviewing the manuscript and for his
thoughtful suggestions.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with
any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.
This includes employment, consultancies, honoraria, stock ownership
or options, expert testimony, grants or patents received or pending, or
royalties.
No writing assistance was utilized in the production of this
manuscript.
Key issues
• Up to a quarter of patients evaluated for pheochromocytoma will have at least one false-positive biochemical test.
• We recommend plasma fractionated metanephrines (metanephrines) for screening purposes.
• metanephrines are continuously made out of catecholamines stored in the pheochromocytoma vesicular stores; therefore, biochemical
evaluation of patients suspected to have pheochromocytoma may be done at any time irrespective of the presence or absence
of symptoms.
• Plasma metanephrines >fourfold the upper limit of normal range (ULN) usually establishes the diagnosis.
• The majority of false-positive results are related to the plasma or urine normetanephrine (NMN) fraction; any degree of elevation in the
plasma or urine MN should be carefully evaluated and not ignored as false positive.
• Most patients with plasma or urine NMN <twofold the ULN do not have pheochromocytoma; such patients should have their levels
repeated in intervals.
• Patients with plasma NMN level between two- to fourfold the ULN need close monitoring and further evaluation to rule out an
underlying pheochromocytoma.
• Review the medication list for possible interference and if a drug interaction is suspected, then consider stopping it and repeating the
levels afterward; familiarity with the assay used and its potential drug interference profile is critical.
• Drugs most frequently associated with false-positive results are tricyclic antidepressants and phenoxybenzamine.
• Evaluation for underlying medical conditions including renal impairment and repeating the blood draws in the supine position after
30 min of rest may help to eliminate some of the false-positive results.
• Rare false-negative results in pheochromocytoma may be seen in patients with tumors <2 cm in size, dopamine-secreting tumors, early
stage familial pheochromocytoma syndromes, early recurrence, small residual tumor tissue after surgical resection and in patients with
metastatic pheochromocytoma after resection of the primary tumor.
132
Expert Rev. Endocrinol. Metab. 9(2), (2014)
Pheochromocytoma: pitfalls in diagnosis
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