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Quantification of unconjugated metanephrines in human plasma without interference by acetaminophen.

Pheochromocytomas are tumors of chromaffin cells most frequently originating from the adrenal medulla and represent a rare cause of secondary hypertension attributable to excessive production of norepinephrine (NE)[3] and/or epinephrine (E) (1). Because almost every other afflicted patient has no or only episodic hypertension and not all suffer from classic clinical symptoms (1), highly sensitive tests are mandatory for the diagnosis of the disease (2).

Standard biochemical testing, such as the measurement of urinary vanilly1mandelic acid and catecholamine excretion or of plasma catecholamine concentrations, can be influenced by external factors such as posture (3), stress (3, 4), or difficulty in obtaining complete 24-h urine collections (2,5). Quantification of plasma concentrations of the metanephrines normetanephrine (NMN) and metanephrine (MN), the O-methylated extraneuronal metabolites of NE and E, respectively, may overcome these limitations. Measuring total, i.e., both unconjugated and conjugated metanephrines, increases the sensitivity (6) but is less accurate than free, i.e., unconjugated, metanephrines for the detection of pheochromocytoma (7). This could well relate to the preparation procedures, which require acid hydrolysis or enzymatic deconjugation by sulfatase before measurement of total metanephrines. Unconjugated plasma NMN and MN also identify pheochromocytoma in subjects with familial predisposition for the tumor (8) and patients with adrenomedullary hypofunction (9). These studies used HPLC with electrochemical detection to quantify plasma metanephrines (10-12). The method of Lenders et al. (11) provides a two- to fourfold greater sensitivity, but still suffers from interference by acetaminophen, a frequently prescribed analgesic drug that may be also used to treat headache of hypertensive patients.

The present study describes a simple HPLC method for the determination of unconjugated NMN and MN in human plasma that (a) has been shown to offer high diagnostic accuracy compared with plasma catecholamines for the detection of pheochromocytoma (3) and (b) is not affected by simultaneous acetaminophen medication.

Materials and Methods

SUBJECTS

Patients were recruited from the Hypertension and Diabetes Outpatient Services of the Department of Internal Medicine III and from inpatients of the Department of Surgery at the University of Vienna Medical School. The anthropometric data of patients with or without hypertension (defined by systolic blood pressure >135 mmHg or diastolic blood pressure >85 mmHg) are shown in Table 1. Because kidney failure may affect the plasma metanephrine concentration (13), only patients with serum creatinine within the reference interval were included. None was taking acetaminophen, which was previously shown to interfere with the plasma NMN assay (5).

The study was performed in accordance with the current revision of the Helsinki Declaration of 1975 and the guidelines for Good Clinical Practice.

PROTOCOLS

Blood sampling was performed on patients in sitting or lying position, which do not affect unconjugated metanephrines (3). Heart rate and blood pressure were recorded simultaneously. Venous blood (5 mL) was collected in chilled heparin-containing tubes through an intravenous cannula in the forearm. The blood samples were immediately placed in ice-water and centrifuged (4 [degrees]C at 14008) within 15 min; the supernatant was stored at -80 [degrees]C until assayed. In addition, three 24-h urine samples (of which the highest concentrations are given) for the determination of NE and E were obtained within 2-4 weeks before or after blood sampling.

To examine the reported interference of acetaminophen with the determination of plasma NMN, five healthy volunteers were admitted to the Clinical Research Unit. After an overnight fast, a plastic catheter was introduced into a forearm vein, and blood was drawn as described above after 15 min (baseline). The subjects then ingested acetaminophen at an antipyretic effective dose of 500 mg (Mexalen[R]; Merckle), and blood samples were collected 60 and 120 min later.

DETERMINATION OF PLASMA UNCONJUGATED METANEPHRINES

The assay described here was recently used to determine plasma concentrations of unconjugated MN and NMN in patients with adrenal tumors before and after surgery (3). The assay represents the modification and optimization of a previously described method (11).

Sample extraction. All reagents, water, and methanol used for the extraction procedures were HPLC grade. The ion-exchange matrix of the solid-phase cation-exchange column (ICT ISOLUTE; 500 mg, 6 mL) was activated by washing three times with 5 mL of ammoniac methanol (7.5 mL of concentrated ammonia, 67.5 mL of water, 25 mL of methanol), once with 2.5 mL of potassium hydroxide in methanol (1 g/L), and once with 2.5 mL of water.

For adsorption of metanephrines, 1 mL of plasma containing the internal standard, 4-hydroxy-3-methoxybenzylamine (HMBA; 1000 pg in 100 [micro]L of 0.1 mol/L acetic acid), and 400 [micro]L of 0.1 mol/L acetic acid were dissolved with water (final volume, 6 mL). Pooled plasma (control plasma) and pooled plasma supplemented with 1000 pg each of NMN and MN were treated identically. Columns were washed once with 6 mL of 10 mmol/L acetic acid-methanol (9:1 by volume), once with 5 mL of water, once with 5 mL of 10 mmol/L ammonium phosphate (pH 7.8), and twice with 5 mL of water. The metanephrines were eluted with one 3.5-mL volume of ammoniac methanol, and the eluate was concentrated by vacuum centrifugation and lyophilized. Samples were stored at -80 [degrees]C until further use.

Chromatography. The mobile phase consisted of 0.85 mmol/L octane sulfonate, 0.16 mmol/L EDTA, 0.55 mol/L sodium dihydrogen phosphate, and 45 mL/L acetonitrile; the pH was adjusted with 85% o-phosphoric acid to 3.3 (all reagents were HPLC grade). Following extraction, the lyophilized material was reconstituted in 125 [micro]L of mobile phase, and 50 [micro]L was injected (Rheodyne 7125 Injector). The mobile phase was pumped through the chromatographic system at a flow rate of 1.25 mL/min. Liquid chromatography was performed on a HPLC column (Waters-Spherisorb; S5 ODS2, 4.6 X 250 mm; 5-[micro]m particle size; Bischoff Chromatography) using the ESA 580 HPLC with two additional pulse dampers (Environmental Sciences Associates). For quantification of MN and NMN, we used the Coulochem II electrochemical detector equipped with a Model 5021 conditioning cell and Model 5014A microdialysis cell (Environmental Sciences Associates). Potentials for electrochemical detection were as follows: +0.4 V for the conditioning cell; +0.1 V for the first electrode of the microdialysis cell; -0.065 V for the second electrode of the microdialysis cell. The signal of the latter electrode was used for analysis. Data were transferred and analyzed using a software package (Perkin-Elmer-Nelson 900 integration interface and integration software; Perkin-Elmer Nelson Systems) on an IBM PS2 personal computer.

[FIGURE 1 OMITTED]

PLASMA CATECHOLAMINES

Plasma catecholamines were measured by reversed-phase HPLC using plasma catecholamine extraction tubes (Environmental Sciences Associates) for the isolation procedure (14). Inter- and intraassay CVs were <5% for both compounds (3).

URINE CATECHOLAMINES

Urine samples were collected in plastic flasks containing 10 mL of 8 mol/L HCI. After extraction by ion-exchange columns and separation by HPLC, catecholamines were measured by electrochemical detection (Pharmacia LKB) using reagent sets from Chromsystem (3).

ASSAY VALIDATION

Intraassay CVs were assessed from multiple (n = 20) measurements of NMN and MN in pooled plasma (target concentrations, -700 pmol/L for NMN and -200 pmol/L for MN) within one extraction procedure. Interassay CVs were calculated from repeated measurements of NMN and MN in a different pooled plasma (target concentrations, ~400 pmol/L for NMN and ~200 pmol/L for MN) after extraction on different days. The limit of detection of the assay was assessed from the peak heights of NMN or MN equaling three times the basal oscillation (signal-to-noise ratio, 3:1).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

STATISTICAL ANALYSIS

All data are presented as means [+ or -] SD unless stated otherwise. Correlation between variables was described by the Pearson rank correlation. P <0.05 was considered statistically significant.

Results

PERFORMANCE OF THE METANEPHRINE ASSAY

A typical chromatogram of a blood sample obtained from a healthy man under resting conditions is presented in Fig. 1A. For identification and quantification of metanephrines, 1000 pg each of NMN and MN was added to the same plasma sample (1 mL) before extraction (Fig. 1B). Linearity of the assays for MN and NMN was observed after addition of defined amounts (0, 100, 200, 500, 1000, and 2000 pg) of MN (0, 547, 1095, 2737, 5473, and 10 947 fmol) and NMN (0, 508,1017, 2542, 5084, and 10 168 fmol), which were added to triplets of pooled plasma before standard analysis (Fig. 2).

Minor differences in the recoveries of NMN and MN (90-105%) compared with the internal standard HMBA between different series of extraction were corrected by use of control plasma and supplemented control plasma.

Intraassay CVs were 4.7% for NMN (plasma NMN, 671 [+ or -] 32 pmol/L) and 7.0% for MN (plasma MN, 153 [+ or -] 11 pmol/L). The interassay CV was 12% for both NMN (plasma NMN, 437 [+ or -] 54 pmol/L) and MN (plasma MN, 210 [+ or -] 25 pmol/L). The limits of detection were 11 fmol for NMN and 17 fmol for MN. The limits of quantification in plasma extracts were 66 pmol/L for NMN and 44 pmol/L for MN.

For studies on the interference of acetaminophen with plasma NMN, one tablet of Mexalen containing 500 mg of acetaminophen was suspended in 100 mL of water-methanol (4:1 by volume). Aliquots of the supernatant (100 [micro]L each) were added as an external acetaminophen standard to aliquots of baseline plasma sample before extraction and analyzed by HPLC (Fig. 3A). In addition, blood samples were drawn from healthy volunteers after ingestion of 500 mg of Mexalen. Under our experimental conditions, no interference of the drug with the NMN and MN peaks was found after 1 and 2 h. The relative retention factors for NMN and MN were 0.67 and 1.21 compared with acetaminophen (relative retention factor = 1.00) as shown in a typical chromatogram (Fig. 3B).

Chromatograms of one typical patient with histologically confirmed pheochromocytoma showed increased concentrations of NMN (20 500 pmol/L) and MN (12 200 pmol/L; Fig. 4A) and the expected decrease to reference values (361 and 71 pmol/L, respectively) after surgical removal of the tumor (Fig. 4B).

[FIGURE 4 OMITTED]

PATIENT DATA

Anthropometric data and concentrations of catecholamines and metanephrines of the total study population as well as of defined subgroups are summarized in Table 1. Thirty-seven (77%) of the subjects were hypertensive. Serum creatinine was slightly but significantly higher (P <0.05) in male than in female subjects. Plasma NMN concentrations were higher (P <0.01) in male than in female subjects.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Plasma NMN and MN were positively correlated with the respective plasma (r = 0.52 and 0.49; P <0.01 for both) and urinary catecholamines, NE (r = 0.31; P = 0.04) and E (r = 0.38; P = 0.03; Fig. 5). Plasma NMN was weakly but significantly (P = 0.02) related to age, which did not hold true for plasma MN and catecholamines (Fig. 6). Plasma NE was positively correlated with systolic (r = 0.19; P = 0.04) and diastolic (r = 0.20; P = 0.04) blood pressure, whereas plasma E was correlated with diastolic (r = 0.30; P = 0.03) but not with systolic blood pressure (r = 0.15; P = 0.09). Both plasma NMN and MN were unrelated to systolic or diastolic blood pressure.

Discussion

The described assay for the determination of unconjugated plasma metanephrines is based on the method described by Lenders et al. (11). The latter method was modified by the use of (a) different solid-phase extraction columns, (b) a different chromatographic system (column material, mobile phase), and (c) a coulometric detector with a different analytical cell, which allowed improved selective setting of oxidizing and reducing potentials.

Both methods allow detection of pheochromocytomas with higher sensitivity and specificity compared with the measurement of plasma catecholamines (3, 7,8). Baseline concentrations of NMN and MN under resting conditions were in good agreement with the previous method (11) and some radioenzymatic techniques (15,16), but different from other methods (10, 17,18). The greater sensitivities of both newer methods and the resulting lower limits of detection most likely explain these differences from previous assays. The intra- and interassay CVs for NMN were comparable to those reported by Lenders et al. (11). The mean values for our control subjects were slightly higher than the values reported in that study (11), but were very similar to the values obtained in other studies by the same group (4, 7-9,12) as well as those in our recent report (3).

Although otherwise comparable, the major advantage of the present assay compared with the other established HPLC method (11) resides in its ability to clearly separate the NMN peak from that of the analgesic and antipyretic drug acetaminophen. Because bilateral diffuse headache is typical for essential hypertension but also belongs to the classical symptom triad of pheochromocytoma (1), patients at risk of pheochromocytoma can be expected to use acetaminophen (as Tylenol[R] in the US or as Mexalen in Europe). Thus, application of the present method for the quantification of plasma NMN does not require patients to stop taking acetaminophen. Minor differences in the sample extraction procedures, particularly the binding pH of the sample and the cation-exchange column, which are also critical for the purification of the metanephrines (19), could have led to lower recoveries of acetaminophen and/or its metabolites. Slightly different chromatographic conditions, such as column material and mobile phase, gave retention factors for both NMN and MN relative to acetaminophen that enabled clear separation of the peaks of these compounds.

The data demonstrate that unconjugated plasma metanephrines not only correlate with the respective plasma concentrations of their parent compounds, as shown previously (4), but also with urinary excretion of the corresponding catecholamines. Nevertheless, NMN and MN are less sensitive to stimulation by insulin and glucagon (12), a change to upright posture (3), mental challenge (4), and intraoperative stress (3). These findings along with the good correlation of tumor size and volume with plasma metanephrine concentrations, which most likely results from intratumoral metabolism of catecholamines, are considered to explain the improved diagnostic efficacy for the detection of pheochromocytomas (3,12).

With regard to their proposed role as tumor markers, it was of interest to evaluate plasma NMN and MN concentrations in a control population. The positive correlation between age and plasma NMN but not MN is in agreement with previous results obtained in a slightly younger population [mean age, ~40 years vs ~50 years (4)]. Such correlation was not detectable when total plasma metanephrines were measured, which could be attributable to the markedly lower intraassay ([less than or equal to] 7.0% each) and interassay imprecision ([less than or equal to] 12.5% each) of that HPLC method (19). The positive relationship between plasma catecholamines and systolic as well as diastolic blood pressure is in agreement with sympathetic regulation of blood pressure (20).

In conclusion, this sensitive metanephrine assay is not affected by simultaneous acetaminophen medication and reveals a correlation of metanephrines with plasma and urinary catecholamines and age but not with blood pressure.

This study was supported by a grant (Jubilaumsfonds, 0NB 6768) from the Austrian National Bank to S.G. and M.R.

References

(1.) Manger WM, Gifford RW Jr. Pheochromocytoma: a clinical overview. In: Swales JD, ed. Textbook of hypertension. Oxford: Blackwell Scientific Publications, 1994:941pp.

(2.) Rosano TG, Swift TA, Hayes LW. Advances in catecholamine and metabolite measurements for the diagnosis of pheochromocytoma. Clin Chem 1991;37:1854-67.

(3.) Raber W, Raffesberg W, Bischof M, Scheuba C, Niederle B, Gasic S, et al. Diagnostic efficacy of unconjugated plasma metanephrines for the detection of pheochromocytoma. Arch Intern Med 2000;160:2959-63.

(4.) Eisenhofer G, Friberg P, Pacak K, Goldstein DS, Murphy DL, Tsigos C, et al. Plasma metadrenalines: do they provide useful information about sympatho-adrenal function and catecholamine metabolism? Clin Sci 1995;88:533-42.

(5.) Bravo EL. Plasma or urinary metanephrines for the diagnosis of pheochromocytoma? That is the question. Ann Intern Med 1996; 125:331-2.

(6.) Mornex R, Peyrin L, Pagliari R, Cottet-Emard JM. Measurement of plasma methoxyamines for the diagnosis of pheochromocytoma. Horm Res 1991;36:220-6.

(7.) Lenders JW, Keiser HR, Goldstein DS, Willemsen JJ, Friberg P, Jacobs MC, et al. Plasma metanephrines in the diagnosis of pheochromocytoma. Ann Intern Med 1995;123:101-9.

(8.) Eisenhofer G, Lenders JW, Linehan WM, Walther MM, Goldstein DS, Keiser HR. Plasma normetanephrine and metanephrine for detecting pheochromocytoma in von Hippel-Lindau disease and multiple endocrine neoplasia type 2. N Engl J Med 1999;340: 1872-9.

(9.) Merke DP, Chrousos GP, Eisenhofer G, Weise M, Keil MF, Rogol AD, et al. Adrenomedullary dysplasia and hypofunction in patients with classic 21-hydroxylase deficiency. N Engl J Med 2000;343: 1362-8.

(10.) Pagliari R, Cottet-Emard JM, Peyrin L. Determination of free and conjugated normetanephrine and metanephrine in human plasma by high-performance liquid chromatography with electrochemical detection. J Chromatogr 1991;563:23-6.

(11.) Lenders JWM, Eisenhofer G, Armando I, Keiser HR, Goldstein DS, Kopin IJ. Determination of metanephrines by liquid chromatography with electrochemical detection. Clin Chem 1993;39:97-103.

(12.) Eisenhofer G, Keiser H, Friberg P, Mezey E, Huynh TT, Hiremagalur B, et al. Plasma metanephrines are markers of pheochromocytoma produced by catechol-O-methyltransferase within tumors. J Clin Endocrinol Metab 1998;83:2175-85.

(13.) Marini M, Fathi M, Vallotton M. Determination of serum metanephrines in the diagnosis of pheochromocytoma. Ann Endocrinol (Paris) 1994;54:337-42.

(14.) Musso NR, Vergassola C, Pende A, Lotti G. Reversed-phase HPLC separation of plasma norepinephrine, epinephrine and dopamine with three-electrode coulometric detection. Clin Chem 1989;35: 1975-7.

(15.) Kobayashi K, DeQuattro V, Bernheimer J, Kolloch R, Miano L. Plasma normetanephrine: a biochemical marker of sympathetic nerve function in man. Life Sci 1980;26:1821-6.

(16.) Foti A, Adachi M, DeQuattro V. The relationships of free to conjugated normetanephrine in plasma and spinal fluid of hypertensive patients. J Clin Endocrinol Metab 1982;55:81-6.

(17.) Ishimitsu T, Hirose S. Simultaneous assay of 3,4-dihydroxyphenylalanine catecholamines O-methylated metabolites in human plasma using high-performance liquid chromatography. J Chromatogr 1985;337:239-48.

(18.) Nohta H, Yamaguchi E, Ohkura Y. Measurement of catecholamines, their precursors and metabolites in human urine and plasma by solid-phase extraction followed by high-performance liquid. J Chromatogr 1989;493:15-26.

(19.) Pallant A, Mathian B, Prost L, Theodore C, Patricot MC. Determination of plasma methoxyamines. Clin Chem Lab Med 2000;38: 513-7.

(20.) Floras JS. Epinephrine and the genesis of hypertension. Hypertension 1992;19:1-18.

MICHAEL RODEN, [1] * WOLFGANG RAFFESBERG, [1] WOLFGANG RABER, [1] ELISABETH BERNROIDER, [1] BRUNO NIEDERLE, [2] WERNER WALDHAUSL, [1] and SLOBODAN GASIC [1]

[1] Division of Endocrinology and Metabolism, Department of Medicine III, and [2] Department of Surgery, University of Vienna Medical School, A-1090 Vienna, Austria.

[3] Nonstandard abbreviations: NE, norepinephrine; E, epinephrine; NMN, normetanephrine; MN, metanephrine; and HMBA, 4-hydroxy-3-methoxybenzylamine.

* Address correspondence to this author at: Division of Endocrinology and Metabolism, Department of Internal Medicine III, University of Vienna Medical School, Wahringer Gurtel 18-20, A-1090 Vienna, Austria. Fax 43-1-404007790; e-mail michael.roden@akh-wien.ac.at.

Received January 25, 2001; accepted March 12, 2001.
Table 1. Anthropometric characteristics and laboratory data in the
total group and in selected subgroups. (a)

 Blood pressure,
 mmHg
 Age,
 n years Systolic Diastolic

Total 48 49 [+ or -] 14 148 [+ or -] 27 96 [+ or -] 15
Male 23 49 [+ or -] 16 145 [+ or -] 21 97 [+ or -] 14
Female 25 50 [+ or -] 12 151 [+ or -] 31 95 [+ or -] 17
Normotensive 11 51 [+ or -] 19 118 [+ or -] 14 75 [+ or -] 7
Hypertensive 37 48 [+ or -] 13 157 [+ or -] 22 102 [+ or -] 11

 Plasma metanephrines,
 pmol/L
 S-Creatinine,
 [mu] mol/L NMN MN

Total 82 [+ or -] 11 296 [+ or -] 134 108 [+ or -] 60
Male 87 [+ or -] 11 361 [+ or -] 138 131 [+ or -] 69
Female 77 [+ or -] 9 237 [+ or -] 101 86 [+ or -] 41
Normotensive 88 [+ or -] 17 267 [+ or -] 157 108 [+ or -] 83
Hypertensive 80 [+ or -] 9 302 [+ or -] 130 108 [+ or -] 52

 Plasma catecholamines,
 pmol/L

 NE E

Total 1606 [+ or -] 691 189 [+ or -] 128
Male 1703 [+ or -] 716 206 [+ or -] 101
Female 1525 [+ or -] 679 175 [+ or -] 150
Normotensive 1104 [+ or -] 658 132 [+ or -] 82
Hypertensive 1704 [+ or -] 680 201 [+ or -] 139

 Urinary catecholamines,
 nmol/24 h

 NE E

Total 397 [+ or -] 271 45 [+ or -] 35
Male 484 [+ or -] 310 56 [+ or -] 41
Female 299 [+ or -] 181 33 [+ or -] 22
Normotensive 282 [+ or -] 145 41 [+ or -] 25
Hypertensive 414 [+ or -] 288 46 [+ or -] 37

(a) Data are presented as means [+ or -] SD.
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Title Annotation:Endocrinology and Metabolism
Author:Roden, Michael; Raffesberg, Wolfgang; Raber, Wolfgang; Bernroider, Elisabeth; Niederle, Bruno; Waldh
Publication:Clinical Chemistry
Date:Jun 1, 2001
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