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Articles |
1
Central Laboratory for Clinical Chemistry, University Hospital, P.O. Box 30.001, 9700 RB Groningen, The Netherlands.
2
Repromed GmbH, Flughafenstraße 52A, D-22335 Hamburg,
Germany.
a Author for correspondence. Fax 31-50-3612290.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: gas chromatography • mass spectrometry • pheochromocytoma
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pheochromocytomas usually produce free catecholamines, which very likely before entering the circulation are already converted into metanephrines. Eisenhofer et al. (4) showed that after intravenous infusion of 1 H-labeled catecholamine precursors, steady-state plasma concentrations of 1 H-labeled free metanephrines were <6% of steady-state concentrations of the precursor amines, whereas in pheochromocytoma patients plasma concentrations of free metanephrines were ~50% of their precursor amines (1). Moreover, Eisenhofer et al. (5) demonstrated that >90% of circulating free M is formed by metabolism of adrenaline within the adrenals and thus <10% is formed by metabolism of adrenaline after its release into the circulation. Also, formation of metanephrines within pheochromocytoma tissue is supported by findings of high concentrations of metanephrines within tumor tissue (6)(7) and a high concentration of free NM in the plasma draining a tumor (8). Deactivation of circulating 3-O-methylated catecholamines followed by sulfoconjugation and excretion suggests high specificity and sensitivity for urinary total metanephrines, compared with oxidatively deaminated catecholamine metabolites. In addition, unlike urinary total catecholamines, urinary total M concentrations are not significantly influenced by diet (9).
For the reasons stated above, one of the most sensitive and specific methods for the clinical chemical diagnosis and follow-up of patients with pheochromocytoma remains the assay of urinary metanephrines, provided that their analysis is carried out by modern chromatographic techniques (3)(10)(11). As urinary M and NM are in majority present as sulfoconjugates (12), a deconjugation (hydrolysis) step to determine each of them as the sum of their free and conjugated fractions generally precedes their analysis. The determination of urinary M and NM separately has been reported to be useful to distinguish between tumors located in the adrenal medulla (secreting mainly adrenaline, to be converted into M) and extraadrenal tumors (secreting mainly noradrenaline, to be converted into NM) (13). Moreover, measuring total metanephrines (sum of M and NM) is less sensitive than measuring M and NM separately (14).
Although chromatographic techniques such as HPLC, gas chromatography (GC), and GC-mass spectrometry (MS) allow the measurement of urinary metanephrines with good precision and accuracy (3), the necessary know-how and the time-consuming analytical procedures (and the restricted availability of some types of equipment, e.g., GC-MS) impede an intensive use of these methods, resulting in relatively few laboratories actually carrying out these assays. In recent years more laboratories introduced M assays, since commercial HPLC equipment along with reagents in kit form as "turn-key instrumentation" became available. Nevertheless, in past years, development of immunoassays for these analytes was also attempted. Thus, papers describing RIA techniques for urinary M and NM were published some years ago (15)(16). Again, the use of radioactive substances constitutes a disadvantage, not offering an attractive substitute for existing chromatographic methods. Recently, however, enzyme immunoassay kits, based on microtiter plate technology, for the quantitative determination of urinary M and NM have become commercially available. These kits for the first time offer an opportunity to replace chromatographic techniques for methods accessible to common routine clinical laboratories. The principles on which these kits are based will be described here.
We have evaluated these M and NM kits by assaying several urine samples obtained from healthy persons and patients with and without pheochromocytoma by applying the ELISA methods as well as a GC-MS method previously developed in our laboratory, which was recently published (17).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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elisa kits for determination of metanephrine and
normetanephrine
These new kits are available either as RIA or ELISA versions.
Although according to the supplier the performance of the RIA and ELISA
kits are comparable, we prefer the ELISA version, as no handling of
radioactive substances is required. Thus only the ELISA kits were
evaluated in this study. The kits are based on the following principle.
Since the metanephrines as such do not possess sufficient immunogenic
properties, making it difficult to raise specific antibodies against
them, use is being made of their N-acetylated derivatives. By reaction
with Bolton–Hunter reagent [3-(p-hydroxyphenyl)propionic
acid N-hydroxysuccinimide ester], both metanephrines are
readily converted to their N-acylhemisuccinates, with excellent
immunogenic properties (see Fig. 1
). Specific antibodies against these derivatives can be produced
in rabbits by coupling the latter to bovine serum albumin as hapten,
whereas as tracers (in the ELISA version) these hemisuccinates coupled
to biotin are used. In short, the procedure is as follows. Urinary M
(or NM) is first converted to its N-acyl derivative by means of the
Bolton–Hunter reagent and then the product transferred to microtiter
plates coated with goat anti-rabbit IgG. After addition of a fixed
amount of the appropriate tracer and (rabbit) antibody, subsequent
incubation overnight, and washing, anti-biotin–alkaline phosphatase
conjugate is added. After incubation for some time, plates are washed,
followed by addition of alkaline phosphatase substrate
(p-nitrophenylphosphate) and measurement of the released
p-nitrophenol spectrophotometrically after a fixed
incubation time. Maximal enzyme activity is observed when no acylated M
(or NM) is present in urine, because in that case all tracer molecules
(acylated M or NM coupled to biotin) are bound to the walls of the
microtiter plate, which consists of a coating of M-antibody (the goat
anti-rabbit IgG:M rabbit-antibody complex) after the first incubation
step. On the other hand acylated M (or NM) molecules, if present in
urine, compete with tracer molecules in binding to the antibody,
resulting in less binding of biotin-containing tracer to the wall and
consequently less binding of anti-biotin–alkaline phosphatase
conjugate after the final incubation, leading to reduced enzyme
activity. Thus, lowest activity is reached at highest urinary M
concentration.
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The test kits were supplied by Immuno Biological Laboratories (IBL), Hamburg, Germany [cat. no. RE 591 71 (AMICYL-TestTM normetanephrine-ELISA) and cat. no. RE 591 81 (AMICYL-TestTM metanephrine-ELISA]. They contain all necessary materials and reagents (in total 16 components, among which is one microtiter plate) to carry out 96 assays of either M or NM, including calibration samples. The contents of the kits can be divided in such a way that three separate runs can be performed, each comprising 32 determinations, equivalent to 4 microtiter strips of 8 wells each. Both kits are practically similar in composition and differ only with respect to the tracer (M-biotin or NM-biotin), antiserum (specific for M or NM), and calibrator solutions (M or NM). Included in all kits is a detailed prescription of the analytical procedure to be followed. The kits should be stored in the dark at 2–8 °C. To carry out tests, only appropiate pipettes, a vortex-type mixer, a heating block (set at 90 °C), a temperature-controlled water bath (37 °C), a microtiter washer, and a microtiter plate reader, able to measure at 405 nm, are further required.
analytical procedure followed for elisa kits
For a detailed description of the analytical procedure (both for M
and NM) we refer to the booklets (edited in both English and German)
supplied by the manufacturer and included in each test kit. Here we
present a shortened version describing the different analytical steps
in less detail.
Hydrolysis and derivatization
. Since urinary
metanephrines are predominantly present as sulfated conjugates, a
hydrolysis step is necessary, unless one wants to determine free
(unconjugated) metanephrines. In reagent tubes, 200 µL of 0.1 mol/L
HCl solution was added to 50 µL of patient urines, 7 calibrators
(present in the kits), and 2 control samples, and all mixtures were
heated for 1 h at 90 °C. After cooling, the acylation reaction
(with Bolton–Hunter reagent) was carried out for 30 min at 37 °C.
Incubation of acylated samples with tracer and antibody
.
Twenty microliters of the acylated samples was pipetted in duplicate
into the appropiate wells, followed by addition of 50 µL of M (or
NM)-biotin tracer solution and 50 µL of antiserum solution (specific
for M or NM), and allowed to stand at 2–8 °C overnight (at least
for 14 h).
Incubation with enzyme conjugate
. After three washing
cycles, 150 µL of enzyme conjugate solution (alkaline
phosphatase–anti-biotin conjugate) was pipetted into the wells and the
plate was shaken for 2 h at room temperature.
Enzyme reaction.
After three washing cycles 200 µL of
substrate solution (p-nitrophenylphosphate) was pipetted
into each well, followed by incubation for 20 min at room temperature
while shaking. The reaction was stopped by addition of 50 µL of a 1
mol/L NaOH + 0.25 mol/L EDTA solution. Within 60 min extinction
coefficients were measured in the wells by means of a microtiter plate
reader at a wavelength of 405 nm.
Calculation of results.
Calculations were performed with
the computer program "Easy Fit" distributed by SLT/Tecan
Labinstruments, Grödig, Austria, which converts the absorbance
readings acquired by the microtiter plate reader into concentrations
(µmol/L) of M or NM by taking into account absorbance values of seven
calibrators (ranging from 0 to 12.7 µmol/L for M and from 0 to 40.9
µmol/L for NM). Both for the calibrators and for the patient samples
the mean of the obtained duplicate absorbance readings was taken and
absorbance as percentage of the absorbance value of the zero calibrator
was calculated. To obtain a calibration curve, the absorbance
percentages (y-values) of the calibrators were plotted
against the logarithms of the concentrations of the calibrators
(x-axis, see Fig. 2
). From these calibration curves, concentrations, and the
calculated absorbance percentages of the patient samples, M or NM
concentrations in these samples were derived.
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The microtiter equipment (washer and reader) used in the present investigation was from Dynatech Labs., Chantilly, VA. The reader (type MR 400) was linked to an IBM-type AT compatible computer.
cross-reactivity measurements in elisa kits
For these measurements, calibrator solutions in water of M or NM
(depending on the investigated ELISA kit) were used, to which a known
amount of calibrator, containing the potentially cross-reactive
substance to be investigated, was added. The following procedure was
used. A 10 000-fold concentration excess of this substance, in
comparison with calibration M or NM concentrations needed for a 50%
reduction in absorbance, was added in the application of the test
procedure instead of urine to see whether or not it could achieve a
>50% reduction in absorbance. If so, a 3000-fold concentration excess
was added and again checked, to determine if a >50% absorbance
reduction could be observed. Likewise, if necessary, a 1000-, 300-, or
100-fold concentration was tried. In this way cross-reactivity could be
estimated: An observed 50% absorbance reduction occurring at an added
100-fold concentration excess is equivalent to a cross-reactivity of
1%. No drugs commonly used in treating hypertensive or other patients
suspected of having a pheochromocytoma were investigated, because of
the observed low cross-reactivity percentages of the tried endogenous
compounds with high structural resemblance to metanephrines. Therefore
significant cross-reactivity of such substances is highly improbable.
gc-ms measurements
For the assessment of urinary metanephrines with isotope dilution
MS our previously described procedure (17) was used.
Essentially the procedure was as follows: To a 300-µL urine sample,
deuterated analogs of 3-methoxytyramine, M, and NM were added as
internal standards. After evaporation to dryness in a stream of
nitrogen, 300 µL of a derivatization mixture consisting of
acetonitrile, dimethylformamide, and pentafluoropropionic anhydride was
added.
Mixtures were derivatized at 80 °C for 15 min. After cooling, samples were extracted and washed with a mixture of heptane and water. Heptane layers were evaporated to dryness and redissolved in 50 µL of ethyl acetate:pentafluoropropionic anhydride (250:1, by vol). Volumes of 1–5 µL were injected into a gas chromatograph–mass spectrometer combination. Samples were monitored in the ammonia–chemical ionization mode. Quantification was done by use of calibration curves.
sample collection and preservation
Urine samples were collected from 47 apparently healthy persons
(ages 3–27 years, median 8.8) and 10 patients previously diagnosed as
having pheochromocytoma [on the basis of high urinary vanillylmandelic
acid (VMA) excretion]. Some patient urine samples were collected after
surgical treatment. Samples were collected in 2-L brown polypropylene
bottles (Sarsted, Nuembrecht, Germany) containing ~250 mg each of
Na2S2O5 and EDTA as
preservatives. Samples were acidified to pH 4 with acetic acid before
freezing at -20 °C. Urinary creatinine concentrations used to
quantify excretion in terms of creatinine were measured by a picric
acid method on a SMA-2 analyzer (Bayer, Tarrytown, NY). For clinical
purposes, concentration values (originally obtained in nmol/L or
µmol/L) were either expressed in nmol (or µmol) excreted in 24
h (when 24-h urine portions were collected) or in µmol/mol creatinine
(when no 24-h portions were collected).
statistics
Results of comparisons between measurements by ELISA and GC-MS
were obtained by method comparison statistics according to Passing and
Bablok (18) [correlation coefficients calculated on the
basis of usual least-squares (Pearson) linear regression analysis].
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Linearity.
Three different pathological urine samples
were assayed undiluted and after twofold, fourfold, eightfold, 16-fold,
and 32-fold dilution. The results, both for M and NM in µmol/L, are
shown in Table 1
, indicating a satisfactory linearity of both assays.
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Recovery measurements.
Four different urine samples in
the case of NM and three in the case of M (all samples from control
persons) were determined as such and after supplementing with various
amounts of NM and M respectively. From the obtained concentrations,
recovery percentages were calculated. For NM, recovery ranged from 91%
to 117% and for M from 74% to 120%.
Cross-reactivity.
Several compounds with chemical
structures related to M and NM were investigated as to their possible
interference in the two test kits. As seen in Table 2
, both test kits showed minimal cross-reactivity with other
substances.
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comparison of elisa with gc-ms
To check the accuracy of the ELISA test kits by comparison with
GC-MS, two series of urine samples, one comprising samples of a group
of 47 apparently healthy control children or young adults, and another
comprising samples of a group of 10 adult patients from whom urine was
collected because of previously established pheochromocytoma, were
investigated. Results obtained by applying the ELISA and GC-MS
procedure were compared and graphically displayed in Fig. 3
, in which the two upper panels comprise only the results for
the controls and the two lower panels the combined results of controls
and patients. The obtained parameters from the statistical analyses are
given in Table 3
. In the control group (n = 47) the M concentrations
obtained from the ELISA method showed a tendency to be slightly higher
than those obtained from GC-MS, whereas the reverse held for the NM
results; correlation coefficients were 0.911 and 0.928, respectively.
The combined results of patients and controls (n = 56) showed
correlation coefficients of 0.993 for M and of 0.988 for NM; the slopes
of the linear regression curves were 0.984 and 0.988, respectively.
From Fig. 3
and Table 3
one can conclude that an acceptable correlation
between both methods exists. Passing–Bablok regression analysis,
applied to the results obtained from the control group and also the
combined control/patient group, further revealed that in the regression
lines no significant deviation from zero for intercept and from 1.00
for slope could be observed, both for M and NM (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Also, no significant interference between each of the two
metanephrines (small cross-reactivity of NM in the M test kit and vice
versa) has been observed. Reproducibility data, linearity data (Table 1
), and recovery data taken together indicate that the precision of an
obtained test result for both urinary metanephrines is in the order of
10–20%, which, although less than experienced for chromatographic
techniques, is in principle sufficient to discriminate between normal
and pathological states. ELISA data demonstrate a good accordance with
GC-MS data, for concentrations both in the normal and pathological
ranges.
To establish whether or not a determined concentration of urinary M and
(or) NM indicates the presence of pheochromocytoma, the amount of
metanephrines present in a 24-h urine collection or expressed as
µmol/mol creatinine (in that case no 24-h collection is necessary)
must be calculated and compared with the respective reference values.
As to which M and NM concentrations (in µmol/24 h or in µmol/mol
creatinine) should be considered abnormal and thus may be indicative of
pheochromocytoma, no general agreement exists, as they depend more or
less on the analytical methods used and the choice of the reference
group. In Table 4
some upper limits of reference values for M and NM expressed in
µmol/24 h are given, derived from the cited review papers, the GC-MS
method used by us, and the values reported for the present ELISA kits.
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Taking into account that the GC-MS and the ELISA method were concordant
in identifying the pathological urinary samples (see Fig. 3
, lower
panels), we are justified in stating that the ELISA test kits can be
successfully applied in the detection of pheochromocytoma patients. The
analytical procedures are relatively simple and require only low-cost
instrumentation (microtiter plate washer and reader) generally
available in the clinical laboratory. Therefore the present ELISA
methods for determining urinary metanephrines can be considered
acceptable alternatives for chromatographic methods, allowing any
clinical laboratory to extend with ease their arsenal of laboratory
tests with a procedure for detection of pheochromocytoma.
For those laboratories that would like to determine plasma metanephrines or urinary free (unconjugated) metanephrines, the described test kits do not possess the required sensitivity, since concentrations <1 nmol/L must be measured with sufficient accuracy, which is below the detection limits of these kits. As argued above, there is no valid reason to prefer such determinations over those of urinary total metanephrines and, therefore, clinically there is no need for the availability of such sensitive M or NM test kits.
One may speculate that in the near future, apart from catecholamines, other diagnostically important biogenic amines, e.g., serotonin, histamine, and their metabolites in body fluids, will be determined likewise by simple immunoassay, becoming the method of choice for the analysis of these molecules. However, one should realize that, although easier to perform, with immunoassays each analyte (e.g., M and NM) must be determined with separate kits, which might lead to higher costs and in some cases might consume more analytical time in comparison with chromatographic methods, in which several analytes can be quantified together within one assay run (e.g., M and NM).
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  B Havekes, A A van der Klaauw, M M Weiss, J C Jansen, A G L van der Mey, A H J T Vriends, B A Bonsing, J A Romijn, and E P M Corssmit Pheochromocytomas and extra-adrenal paragangliomas detected by screening in patients with SDHD-associated head-and-neck paragangliomas Endocr. Relat. Cancer, June 1, 2009; 16(2): 527 - 536. [Abstract] [Full Text] [PDF]
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 Q. Pu, Y. Chang, C. Zhang, Y. Cai, and A. Hassid Chronic insulin treatment suppresses PTP1B function, induces increased PDGF signaling, and amplifies neointima formation in the balloon-injured rat artery Am J Physiol Heart Circ Physiol, January 1, 2009; 296(1): H132 - H139. [Abstract] [Full Text] [PDF]
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 M. M. Kushnir, F. M. Urry, E. L. Frank, W. L. Roberts, and B. Shushan Analysis of Catecholamines in Urine by Positive-Ion Electrospray Tandem Mass Spectrometry Clin. Chem., February 1, 2002; 48(2): 323 - 331. [Abstract] [Full Text] [PDF]
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D. K. Crockett, E. L. Frank, and W. L. Roberts Rapid Analysis of Metanephrine and Normetanephrine in Urine by Gas Chromatography-Mass Spectrometry Clin. Chem., February 1, 2002; 48(2): 332 - 337. [Abstract] [Full Text] [PDF]
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J. Wassell, P. Reed, J. Kane, and C. Weinkove Freedom from Drug Interference in New Immunoassays for Urinary Catecholamines and Metanephrines Clin. Chem., December 1, 1999; 45(12): 2216 - 2223. [Abstract] [Full Text] [PDF]
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G. A. Smythe, M. W. Duncan, J. Grassi, and P. Pradelles Immunoassay of Catecholamines and Metabolites • Two of the authors of the article referred to reply: Clin. Chem., October 1, 1997; 43(10): 2011 - 2012. [Full Text]
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,
control data.