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Freedom from drug interference in new immunoassays for urinary catecholamines and metanephrines.

Pheochromocytoma is a tumor arising from neural crest tissue that secretes catecholamines directly into the circulation, producing episodes of hypertension, severe headaches, and sweating (1, 2). The measurement of free epinephrine (fE),[3] free norepinephrine (fNE), total (i.e., free plus conjugated) metanephrine (tM), and total normetanephrine (tNM) in urine are, when used in combination, considered the most sensitive and specific biochemical test for the detection of this tumor (3-5). Plasma fE and fNE measurements suffer from problems of sample timing and stability (4). Recently, the measurement of plasma free metanephrine and free normetanephrine has been advocated as being more clinically sensitive than urinary free catecholamines and metanephrines (tM plus tNM together) (6). However, these tests still need to be evaluated against individual urinary tM and tNM excretion before claims for the superiority of these difficult assays can be upheld.

HPLC is the most commonly used technique for measuring fE, fNE, tM, and tNM in urine. Methods, however, vary between laboratories (2), with some procedures being prone to interference (7-9), often by antihypertensive agents (e.g., angiotensin-converting enzyme inhibitors, (3-Mockers, calcium channel Mockers, and diuretics) that patients being screened for pheochromocytoma are likely to be taking. For HPLC, interferents usually manifest as peaks coeluting, partially or totally, with the peaks of interest. The former are easily detected by inspection of the chromatogram. Total coelution is less easy to detect and can never be ruled out unless the method of detection relies on primary chemical properties of the analyte of interest (e.g., mass spectroscopy). Interference can also occur in the sample preparation method before chromatography (10).

Over the past 15 years, attempts have been made to develop immunoassays for the catecholamines and metanephrines (11-13). However, problems in raising antibodies against epinephrine and norepinephrine and their 3-O-methylated metabolites, metanephrine and normetanephrine, because of their small molecular size and the high susceptibility of the catecholamines to oxidation (14,15) have prevented such assays from becoming available until recently (16). These new immunochemical methods should be easier to use than HPLC techniques and will be more accessible to laboratories, at the same time hopefully maintaining or improving assay sensitivity and specificity. However, the detection of interference in immunoassays is more difficult than for HPLC because no visual hardcopy of the measured end product is produced; a number is simply generated. It is therefore vital to assess the specificity these immunoassays. This is achieved by testing the reaction of the antibody with molecules structurally related to the intended analyte (17). However, cross-reactants with the antibody may be removed during the extraction procedure and separation stages of the immunoassay method. Thus, it is usually the method cross-reactivity that is evaluated. For the immunoassays used in this study, method cross-reactivity to related biogenic amines was established by the manufacturers for all four immunoassays (fE, fNE, tM, and tNM), with a maximum cross-reactivity of 3%.

The aim of this study was to determine whether drugs commonly prescribed to subjects with symptoms of pheochromocytoma interfere with the measurement of urinary free catecholamines and total metanephrines by these new immunoassays. We took urine samples from patients on a variety of medications (angiotensin-converting enzyme inhibitors, [beta]-Mockers, calcium channel Mockers, diuretics, statins, [alpha]-methyldopa, and a-receptor antagonists). By statistically comparing fE, fNE, tM, and tNM excretion in these patients with that in a nonmedicated population, we were able to examine potential interference from genuine drug metabolites, such as may occur during the use of these immunoassays in the clinical field. In addition, to further elucidate the specificity of the immunoassays, we compared results with those from an established HPLC method (18) that in our laboratory has been shown to suffer little from visually detectable interferences. To aid result interpretation, the precision, accuracy, linearity, and sensitivity of each immunoassay were determined. We used the RIA rather than the ELISA versions of the methods because of the ready availability of a gamma counter in our laboratory. The antigen-antibody system is identical in both types of assay and should therefore be similarly affected by any interferences.

Materials and Methods

CHEMICALS

All chemicals were analytical grade, unless otherwise stated, and solvents were filtered through 0.2 [micro]m nylon filters before use. All RIA reagents were provided by the manufacturers.

RIA METHODS FOR THE DETERMINATION OF FREE URINARY CATECHOLAMINES AND TOTAL METANEPHRINES

The RIA kits were manufactured by Immuno Biological Laboratories, Hamburg, Germany, and supplied by Immunodiagnostic Systems Ltd. (IDS), Tyne and Wear, UK. The AMICYL-Test[TM] KATCOMBI kit contained all reagents, including calibrators and controls, for the measurement of fE and fNE. The AMICYL-Test METCOMBI kit contained all reagents for the measurement of tM and tNM.

The concentration of free catecholamines and total metanephrines in each urine was determined using competitive immunoassay with 1~I-labeled antigen and polyethylene glycol-enhanced precipitation of the antibody-antigen complex. The primary antibody for all assays was raised against an acylated derivative of either metanephrine or normetanephrine. Therefore, not only did all samples require acylation, but for the catecholamine immunoassays, enzyme-catalyzed methylation to convert fE and fNE into free metanephrine and normetanephrine was also needed. Methylation was preceded by extraction of the catecholamines from the sample matrix (and removal of metanephrines) by borate affinity gels. No extraction step was required for the tM and tNM assays.

ANALYTICAL PROCEDURE FOR FREE CATECHOLAMINES (fE AND fNE)

Extraction. Catecholamines in samples, calibrators, and controls were adsorbed onto borate-coated wells of a macrotiter plate. After incubation (30 min at room temperature) and washing (twice with doubly distilled water), catecholamines were eluted from the gel by shaking with 300 [micro]L of 0.05 mol/L HCI.

Methylation and acylation. The extracted catecholamines were aliquoted to duplicate tubes coated with Bolton-Hunter acylation reagent (16), and the supplied methylation reagent (rat liver catechol-O-methyltransferase with S-adenosylmethionine as the coenzyme) was added. All tubes were vortex-mixed and incubated in a water bath at 37[degrees]C for 60 min.

RIA. After incubation, 50 [micro]L of the appropriate labeled antibody (i.e., the [I.sup.125]-derivative of acyl-M or acyl-NM) was added to all tubes, followed by 50 [micro]L of antiserum. Samples were vortex-mixed, centrifuged (4508 for 1 min at 4[degrees]C), and incubated overnight at either room temperature (fE assay) or 2-8[degrees]C (fNE assay). The following day, precipitating antiserum was added to all tubes, and after further vortex-mixing and a 15-min incubation period at room temperature, the resulting precipitate was pelleted by centrifugation (30008 for 15 min at 4[degrees]C). The supernatant was removed, and the amount of radioactivity in each tube was determined.

Calculation of results. A calibration curve of the percentage of binding of label against log concentration of the calibrator was fitted to the data for the calibrators using the spline-function program in the Wallac Multicalc computer software (19). The fE or fNE concentrations in subsequent samples were determined from these calibration curves.

ANALYTICAL PROCEDURE FOR TOTAL METANEPHRINES

For the measurement of total (i.e., both conjugated and unconjugated) metanephrine and normetanephrine, sulfate and glucuronate conjugates were firstly acid hydrolyzed such that all metanephrines were in their free form before acylation.

Acid hydrolysis. Samples were heated for 20 min at 90[degrees]C with 0.1 mol/L hydrochloric acid. After cooling, 50 [micro]L of each sample was diluted 20-fold with assay buffer and vortex-mixed before acylation.

Acylation. Samples were added to duplicate coated acylation tubes, vortex-mixed, and incubated for at least 15 min at room temperature.

RIA. The appropriate labeled antibody (i.e., 50 [micro]L of [I.sup.125]-derivative of acyl-M or acyl-NM) was added to the acylated samples, followed by an equal volume of antiserum. Samples were vortex-mixed, centrifuged (4508 for 1 min at 4[degrees]C), and incubated overnight at 2-8[degrees]C. The following day, precipitating antiserum was added to all tubes, and after vortex-mixing and incubation (15 min at room temperature), the resulting precipitate was pelleted by centrifugation (3000g for 15 min at 4[degrees]C). The amount of radioactivity in each tube was determined. The concentrations of tM and tNM in each sample were calculated by comparison with a calibration curve as described for the catecholamine assay.

QUALITY CONTROL

Included in each batch of assays were four quality-control samples, prepared by mixing urines from patients not receiving any medications and pheochromocytoma patients to cover the entire analytical range of the assays. These samples were stored as aliquots at -20[degrees]C.

ASSESSMENT OF ANALYTICAL VARIABLES

Accuracy. The accuracy of each immunoassay was assessed by comparing RIA results with those from the HPLC method, using a paired two-tailed t-test, Pearson correlation analysis, linear regression, and difference plots (20, 21).

Linearity. Three urine samples were serially diluted with either pooled urine containing low concentrations of analytes or 0.05 mol/L HCI.

Detection limit. The detection limit was defined as the concentration of analyte corresponding the an activity 3 SD above the response of the zero calibrator (n = 8) for each analyte (22, 23).

Precision. Intraassay precision was determined from the results of duplicate analysis of samples from patients. The interassay precision was calculated from analysis of the quality-control samples included in every assay (22, 23).

HPLC MEASUREMENT OF FREE CATECHOLAMINES AND TOTAL METANEPHRINES

The HPLC measurement of urinary fE, fNE, tM, and tNM was a modification of the method described by Green et al. (18). To analyze total metanephrines, urine samples were first acid hydrolyzed (2 mol/L HCl at 100[degrees]C for 20 min) to convert conjugated metanephrines into their free form before HPLC. A Gilson automated ASTED sample processor (Anachem) allowed the automated online extraction of analytes from urine by strong cation exchange. The HPLC mobile phase (0.125 mol/L diammonium hydrogen orthophosphate, pH 3.3, 4.4 mmol/L heptane sulfonic acid, 2 mmol/L EDTA, and 2 mL/L methanol) eluted the catecholamines and metanephrines from the ion exchanger directly onto the analytical column (150 X 46 mm, packed with 5 [micro]m particles of Spherisorb OD52). Detection was electrochemical (redox mode), using a coulometric detector with a dual analytical cell (ESA Analytical). Quality-control material was prepared from commercial preparations (Bio-Rad) and urine samples from patients with pheochromocytoma.

INVESTIGATION OF POTENTIAL IMMUNOASSAY INTERFERENCES

Urine samples (24-h or random) were collected from patients on a variety of medications (angiotensin-converting enzyme inhibitors, [beta]-Mockers, calcium channel Mockers, diuretics, statins, a-methyldopa, and a-receptor antagonists). All samples were acidified with HCl and stored at -20[degrees]C. Urine pH was determined before analysis; samples with pH >3 were discarded because of potential catecholamine instability (14,15).

[FIGURE 1 OMITTED]

Urine samples were grouped according to the specific type of drug being taken by the patient. Subjects in more than one group were not included in subsequent analysis. We used one-way ANOVA to assess the significance of any differences in the concentrations of free catecholamines and total metanephrines between the different drug types. Patients not on medication were included as a control group (no analytical interference in the immunoassay). However, it is possible that drug interference may have been negated by physiological effects of the drug in question on catecholamine metabolism. To investigate this possibility, the analysis was repeated using data from an HPLC method that has no known analytical interference (Reed P, Kane J, Weinkove C, unpublished laboratory observations). Significance based on these results would, therefore, suggest physiological effects of the drug in question on catecholamine metabolism.

STATISTICAL ANALYSIS

All statistical analysis was performed using Astute, Ver. 1.51 (DDU Software).

Results

ASSESSMENT OF ANALYTICAL VARIABLES

Accuracy. No significant difference was found between the mean concentrations of catecholamines measured by immunoassay and by HPLC (Table 1). For tM, the immunoassay gave a mean concentration significantly higher than the HPLC method (P <0.001; n = 211); no such difference was found for tNM (Table 1). Correlation coefficients showed acceptable agreement between the two methods, with values of 0.921 (fE), 0.808 (fNE), 0.822 (tM), and 0.896 (tNM). However, the regression lines for the catecholamine immunoassays were significantly different from 1.00 (P <0.01), with values of 0.842 and 0.627 for fE and fNE, respectively (Fig. 1). Difference plots comparing RIA and HPLC results showed no concentration-dependent bias (Figs. 1 and 2).

Linearity. Assays were linear up to 1.5 and 2.0 [micro]mol/L for fE and fNE, respectively. For the metanephrines, linearity was observed up to 4.9 [micro]mol/L for tM and 14.7 [micro]mol/L for tNM. Linearity was not affected by the diluent matrix (urine or 0.1 mol/L hydrochloric acid).

Detection limit. For fE and fNE, the detection limits were 0.009 and 0.027 [micro]mol/L, respectively; for tM and tNM, the detection limits were 0.119 and 0.346 [micro]mol/L, respectively.

Precision. For the fE immunoassay, the intraassay CVs were 12-22% (0.021-0.65 [micro]mol/L); the intraassay CVs for fNE were 4.6-14% (0.071-2.36 [micro]mol/L) (Table 2). The interassay CVs were 11-19% for fE (0.036-0.37 [micro]mol/L) and 10-25% for fNE (0.39-1.40 [micro]mol/L; Table 2). The intraassay CVs were 6.9-27% (0.15-5.07 [micro]mol/L) for tM and 9.6-12% for tNM (0.49-16.4 [micro]mol/L; Table 3). The interassay CVs were 10-22% for tM (0.23-3.73 [micro]mol/L) and 5.8-16% for tNM (0.62-13.9 [micro]mol/L; Table 3).

INVESTIGATION FOR POTENTIAL IMMUNOASSAY INTERFERENCES

By one-way ANOVA, no significant differences were found between the concentrations of free catecholamines or total metanephrines in urines from patients taking the medications investigated and the control group on no medication. The individual P values were 0.649 (fE), 0.221 (fNE), 0.149 (tM), and 0.170 (tNM; Tables 4 and 5). Additionally, no significant differences were found when the analysis was repeated using the data obtained by HPLC (P = 0.796 for fE, 0.246 for fNE; 0.259 for tM, and 0.251 for tNM).

[FIGURE 2 OMITTED]

Discussion

ASSESSMENT OF ASSAY PARAMETERS

Accuracy. The tNM immunoassay showed good agreement with HPLC using the paired two-tailed t-test (Table 1). However, a significant positive bias was observed for the tM immunoassay (P <0.001; n = 211). Higher metanephrine concentrations were also observed by Worthers et al. (24), who compared the ELISA metanephrine assay with measurements by gas chromatography with mass spectroscopy. These differences may be attributable to variations in the calibration of the tM immunoassay. For the catecholamine assays, although the mean concentrations of fE and fNE agreed well with HPLC, the slopes of the regression lines comparing HPLC and RIA results were significantly different from unity. This may be attributable to the few high results exerting a disproportionately strong influence on the regression line. In addition, for fE, most of our samples contained only low analyte concentrations, near the detection limit of the HPLC method (0.02 [micro]mol/L); discrepancies here may have been caused by errors in the HPLC measurement.

Precision. This study involved a more rigorous assessment of the precision of the immunoassays than previous publications (13, 24).

The interassay CVs for tM and tNM from this study were similar to those reported by Worthers et al. (24), who used the ELISA version of the immunoassays. They found interassay CVs of 10% and 14% (mean metanephrine concentrations, 0.37 and 1.54 [micro]mol/L) and 9% and 13% (mean normetanephrine concentrations, 1.09 and 4.73 [micro]mol/L). Their results were based on the analysis of two samples 10 times by only two kits; in our study, four samples were analyzed up to 10 times by eight different kits.

For the catecholamine immunoassays, few other studies have reported precision data. Manz et al. (16) found interassay CVs of 8% and 13% (13.8 and 3.38 [micro]mol/L, respectively) for fE, and 9% and 16% (33.2 and 8.74 [micro]mol/L, respectively) for fNE. These compared well with our CVs (Table 2) despite the much lower analyte concentrations used in this study.

The HPLC method showed better precision than the immunoassays, with between-batch CVs of 1-8% for both catecholamines and metanephrines. This is in agreement with published precision data for the measurement of these analytes by other HPLC methods (25-27). The better precision of HPLC may reflect the more automated nature of the technique compared with the immunoassays, suggesting that in this study, precision may improve with the use of automated work stations. In addition, a new version of the catecholamine kit is now available. Preliminary data from our laboratory have shown a marked improvement in interassay CV (2.1-7.4% for fE at 0.0210.65 [micro]mol/L, and 1.8-6.4% for fNE at 0.071-2.36 [micro]mol/ L). Although insufficient assays were performed to assess interassay precision, it may be predicted that this will improve similarly.

IDENTIFICATION OF POTENTIAL INTERFERENCES

Immunoassay detection systems rely on the biological specificity of antigen/antibody reactions. Therefore, substances with similar structures to the catecholamines and/or metanephrines (e.g., [alpha]-methyldopa and [beta]-blockers) may interfere in these assays. No such interference was observed in this study. No significant difference was found in fE and fNE concentrations (P = 0.649 and 0.221, respectively) or tM and tNM concentrations (P = 0.149 and 0.170, respectively) in urines samples from subjects in the different drug groups and those on no drugs. A similar lack of interference in catecholamine and metanephrine immunoassays has been reported previously (11,16, 24). However, these studies examined only compounds structurally related to the catecholamines rather than the clinically used drugs examined in this study. In addition, in previous studies urine samples were supplemented with the parent compound; therefore, interference by metabolites was not investigated.

In addition to affecting antigen/antibody recognition, drugs and/or their metabolites could have interfered with other steps in the immunoassays, namely acylation, extraction with boronic acid, and methylation, with the last two steps applying only to the catecholamine immunoassay. It has been reported that the speed of the Bolton-Hunter acylation reaction depends on sample pH, with the reaction being slow at too low a pH, and denaturation of the acylation reagent occurring most rapidly at alkaline pH (28). This pH factor, although not examined in this study, is unlikely to have any significant effect on the RIAs because sample acidity should be sufficient to prevent denaturation of the acylation reagent, and the length of the acylation period was sufficient to allow for any pH-related variations in acylation rate.

With the additional extraction and methylation steps in the catecholamine immunoassay, it may be expected that these assays would be more prone to interference. Borate extraction has been shown to be better than other techniques because this material binds only compounds with cis-diol groups, as opposed to a requirement simply for diol groups in, for example, alumina extraction (10, 27, 29). This, coupled with the high specificity of immunological reactions, may be sufficient to prevent interferences at this stage of the analysis. Both calcium and an unidentified "inhibiting factor" have been shown to inhibit catecholamine-O-methyltransferase (30, 31). However, because only 25-[micro]L samples of urine were used in the catecholamine immunoassay, these inhibitors are unlikely to have any significant effect.

In summary, overall these new immunoassays compared well with HPLC in the measurement of fE, fNE, tM, and tNM, and none of the drugs examined in this study showed significant interference. Therefore, in combination, they may provide a viable, accessible alternative to HPLC for the initial screening of subjects with suspected pheochromocytoma.

We would like to thank Immunodiagnostics Ltd. and Immuno Biological Laboratories Ltd for the immunoassays kits used in this study.

References

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JULIE WASSELL, [1] PAUL REED, [2] JOHN KANE, [2] and CYRIL WEINKOVE [2]

[1] Department of Biochemistry, Wythenshawe Hospital, Southmoor Rd., Manchester M23 9LT, UK. [2] Department of Biochemistry, Hope Hospital, Stott Lane, Manchester M6 SHD, UK.

[3] Nonstandard abbreviafions: fE, free epinephrine; fNE, free norepinephrine; tM, total metanephrine; and tNM, total normetanephrine.

* Author for correspondence. Fax 0161-291-2125; e-mail juliewassell@hotmail.com.

Received August 18, 1999; accepted October 4, 1999.
Table 1. Comparison of results obtained by RIA and HPLC.

 fE fNE tM tNM

HPLC mean, [micro]mol/L 0.117 0.234 0.482 0.768
RIA mean, [micro]mol/L 0.116 0.219 0.576 0.796
n 128 198 211 190
p[a] 0.926 0.104 <0.001 0.917

[a] Two-tailed t-test of paired samples.

Table 2. Precision of the measurement of free catecholamines by RIA.

 fE

Intraassay
Mean, [micro]mol/L 0.021 0.065 0.21 0.65
CV, % 19 12 12 22
n 112 85 90 37
Interassay
Mean, [micro]mol/L 0.036 0.25 0.29 0.37
CV, % 19 11 19 17
n 8 8 8 8

 fNE

Intraassay
Mean, [micro]mol/L 0.071 0.24 0.71 2.36
CV, % 13 8.2 4.6 14
n 88 178 115 32
Interassay
Mean, [micro]mol/L 0.39 0.51 0.65 1.4
CV, % 13 10 10 25
n 10 10 10 10

Table 3. Precision of the measurement of total metanephrines by RIA.

 tM

Intraassay
Mean, [micro]mol/L 0.15 0.51 1.52 5.07
CV, % 27 8.3 6.9 7.6
n 103 265 133 57
Interassay
Mean, [micro]mol/L 0.23 0.43 0.73 3.73
CV, % 22 11 10 12
n 10 10 10 10

 tNM

Intraassay 0.49 1.64 4.91 16.4
Mean, [micro]mol/L 12 10 9.6 10
CV, % 99 43 84 31
n
Interassay 0.62 3.56 8.07 13.9
Mean, [micro]mol/L 5.8 16 12 9.5
CV, % 10 10 10 10
n

Table 4. Comparison of mean urine free catecholamine concentrations
determined by RIA in patients taking specific medications.

Drug fE[1]

 Mean, [micro]mol/L SD (n)

Control (no drugs) 0.052 0.042 (14)
Amlodipine 0.076 0.060 (15)
Atenolol 0.032 0.026 (11)
Bendrofluazide 0.031 0.026 (10)
Bumetanide -[c] -
Captopril - -
Doxazosin - -
Enalapril 0.076 0.17 (16)
Frusemide 0.061 0.053 (11)
Methyldopa - -
Nifedipine 0.036 0.043 (10)
Statins - -

Drug fNE[b]

 Mean, [micro]mol/L SD (n)

Control (no drugs) 0.247 0.154 (25)
Amlodipine 0.24 0.192 (32)
Atenolol 0.157 0.097 (24)
Bendrofluazide 0.176 0.132 (15)
Bumetanide - -
Captopril 0.137 0.138 (8)
Doxazosin 0.146 0.097 (9)
Enalapril 0.211 0.168 (24)
Frusemide 0.249 0.245 (18)
Methyldopa - -
Nifedipine 0.168 0.139 (23)
Statins 0.183 0.279 (8)

[a] P = 0.649.

[b] P = 0.221.

[c] -, insufficient number of urines above the assay detection
limit for further analysis.

Table 5. Comparison of mean urine total metanephrine concentrations
determined by RIA in patients taking specific medications.

Drug tM[a]

 Mean, SD (n)
 [micro]mol/L

Control (no drugs) 0.581 0.321 (21)
Amlodipine 0.636 0.407 (32)
Atenolol 0.451 0.258 (33)
Bendrofluazide 0.439 0.227 (20)
Bumetanide 0.487 0.308 (12)
Captopril 0.435 0.251 (10)
Doxazosin 0.484 0.293 (11)
Enalapril 0.616 0.385 (28)
Frusemide 0.767 0.960 (23)
Methyldopa 0.505 0.172 (11)
Nifedipine 0.410 0.212 (27)
Statins 0.662 0.554 (8)

Drug tNM[b]

 Mean, SD (n)
 [micro]mol/L

Control (no drugs) 1.19 0.526 (19)
Amlodipine 1.47 1.05 (32)
Atenolol 1.07 0.742 (34)
Bendrofluazide 0.893 0.643 (21)
Bumetanide 0.98 0.713 (11)
Captopril 1.06 0.567 (10)
Doxazosin 0.991 0.538 (11)
Enalapril 1.35 1.11 (26)
Frusemide 1.61 1.37 (20)
Methyldopa 1.05 0.944 (11)
Nifedipine 0.978 0.438 (26)
Statins 1.70 2.24 (7)

[a] P = 0.149.

[b] P = 0.170.
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Title Annotation:Endocrinology and Metabolism
Author:Wassell, Julie; Reed, Paul; Kane, John; Weinkove, Cyril
Publication:Clinical Chemistry
Date:Dec 1, 1999
Words:4774
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