Upper Reference Limits for Urinary Catecholamines, Metanephrines and 3-Methoxy-4-Hydroxymandelic Acid in Hypertensive Patients

Assessment of the excretion rates of catecholamines (CAT) and their metabolites in urine remains the mainstay of initial biochemical investigations and follow-up of tumours of neural crest origin, which commonly produce high levels of parent catecholamines. These lesions include pheochromocytoma (in adults), neuroblastoma (in children) and ganglioneuroma (in young adults). In most patients, these potentially lethal but curable lesions are manifested by hypertension, which provides the clue to the diagnosis. The reference intervals for urinary CAT and their metabolites are usually based on “normal” or normotensive patients (1). Literature on the reference values for adult hypertensive in-patients without pheochromocytoma as the relevant reference population is scarce (2–6). Moreover, the values are differently expressed: as urinary excretion per 24 hours (24-h) or as excretion per urinary creatinine, to allow for possible incomplete 24-h collections and permit comparison with the results of timed excretion. To overcome these problems, we estimated the upper reference limits (URL) for urinary free epinephrine (E), norepinephrine (NE) and 3-methoxy-4-hydroxymandelic acid (VMA), and for total (free and conjugated) metanephrine (M) and normetanephrine (NM) in adult hypertensive patients treated in the University Medical Centre Ljubljana. The study population comprised patients with sustained hypertension, for whom determination of CAT and their metabolites was ordered as part of routine investigations for the exclusion of pheochromocytoma. Four patients were excluded from the study because of incomplete urine collection (daily urine creatinine <5.0 mmol for men and <3.0 mmol for women) and two because of significant drug interference (dopamine infusion). We evaluated the estimated URL values by comparing them with the results of other studies. Urine samples (24-h) from 318 treated hypertensive adult in-patients aged 20–64 years (180 women and 138 men) were collected into polyethylene containers with 10 ml of 6.8 mol/l HCl. An aliquot of each sample was stored at 4 °C until analysis within 1 week. All measurements were performed by isocratic high performance liquid chromatography (HPLC) with electrochemical detector Model 1640 (Bio-Rad Diagnostic Group, Hercules, California, USA) and original sets of reagents including Lyphochek® controls (Bio-Rad Laboratories GmbH, Munich, Germany). Initial clean-up was performed by ion exchange chromatography, followed by ion pair chromatography on a reversedphase column. The methodological principles described in the manufacturers’ instruction manuals were followed. Creatinine in urine was measured by automated Jaffe endpoint reaction on an Alcyon 300 analyser (Abbott Laboratories, Abbott Park, IL 60064, USA). The average intra-assay and inter-assay precision, expressed as the coefficient of variation (CV), was below 3.0% for creatinine and below 10.0% for all the other measured parameters. All statistical analyses, including distributions, medians and URL with the confidence limits, were performed with the Statistica for Windows software (StatSoft Inc., Version 5.0, Tulsa, OK 74104, USA). The CAT and metabolite values for each sex, expressed per day and in relation to urinary creatinine, showed a nonGaussian distribution (positive skewness). There were statistically significant differences between men and women (p<0.001). In contrast to other studies, we found no age-dependence for any of the metabolites when expressed per urinary creatinine (p>0.2), and all excretion values, including epinephrine, per urinary creatinine were higher in women (6). In accordance with these findings, we calculated the URL values as the 97.5th percentile with 90% confidence intervals (CI) based on the rank number limits. The values were determined separately for each sex, irrespective of age (Table 1). The calculations were performed by the nonparametric method. As shown in Table 1, our URL values for 24-h urinary excretion of CAT and their metabolites are very similar for both sexes, but they differ considerably when expressed in relation to creatinine excretion. The results, with the exception of NE, are appreciably below the values determined in a study of overnight urinary excretion (2). The estimation of reference limits for CAT and their metabolites, including the evaluation of biological variability, need to be improved because the current procedures are too long, complex and expensive. This is the *E-mail of the corresponding author: milan.skitek@mf.uni-lj.si Letter to the Editor