MSACL 2016 EU Abstract

Reference Ranges for Free Urine Metanephrines Measured by Tandem Mass Spectrometry

Malcolm Whiting (Presenter)
Flinders University

Bio: Bio: Malcolm Whiting is Head of the Pharmacology Unit of Chemical Pathology Directorate in SA Pathology, and Senior Lecturer in Medical Biochemistry at Flinders University in South Australia. He has worked in many areas of clinical chemistry including proteins, amino acids, lipids, biogenic amines and drugs. His qualifications include a Ph.D. in biochemistry from Adelaide University and MAACB from the Australasian Association of Clinical Biochemists. His main analytical interest is in chromatography and tandem mass spectrometry, and he has set up isotope-dilution assays for a number of analytes of clinical interest. Publications include over 80 research articles, reviews and book chapters in a wide range of medical and biochemical journals.

Authorship: Malcolm Whiting1,2, Kate Coleman1,
1Chemical Pathology, SA Pathology, Adelaide, South Australia 5042 & 2Medical Biochemistry, School of Medicine, Flinders University, South Australia 5042

Short Abstract

Phaeochromocytoma and paraganglioma (PPGL) are characterised by increased secretion of catecholamines and their metabolites. The biochemical test that is recommended by the Endocrine Society to diagnose and monitor these neuroendocrine tumours is either total metanephrines in urine or free metanephrines in plasma. However, free metanephrines in urine may prove to be the most convenient and best performing test for PPGL diagnosis in the future. In this study, we have used our LC-MSMS method for their measurement in the clinical laboratory to derive gender-specific reference ranges for free metanephrines for both spot urines and 24-hour urine collections.

Long Abstract

The measurement of the urinary excretion of biogenic amines is used for the biochemical diagnosis of phaeochromocytoma and paraganglioma (PPGL), and for monitoring their treatment. Guidelines from the Endocrine Society recommend that metanephrines in plasma or urine should be determined in preference to urine free catecholamines. With urine metanephrine testing, an acid hydrolysis step is commonly used to convert conjugated (sulphated) metanephrines to free metanephrines prior to the estimation of total urine normetanephrine, metanephrine and 3-methoxytyramine. The proportion of urine metanephrines that are free is small and is around 20% of total metanephrines, but their concentrations are easily measured by LC-MSMS. In this study, we have used our previously-developed LC-MSMS profiling method for simultaneous measurement of free catecholamines and free metanephrines in urine to derive gender-specific reference ranges for free metanephrines in both spot urines and 24-hour urine collections.

An Agilent 1290-6460 LC-MSMS was tuned to measure ions from noradrenaline, adrenaline, dopamine, normetanephrine, metanephrine, and 3-methoxytyramine (3-MT). Deuterated internal standards were added to 100 µL urine, calibrator and QC materials, and the equilibrated samples purified on Agilent Plexa solid-phase extraction (SPE) Versa-Plate cartridges. Amines were bound to the SPE support as boronate-complexes, and eluted with dilute formic acid for simultaneous LC-MSMS analysis1. The MRM method monitored both catecholamines and metanephrines after positive electrospray ionization. Separation of all analytes was achieved on a Kinetex F5 column (100 x 3 mm) with a methanol gradient and run time of 6.2 min. Between-run imprecision of commercial QC material gave CV between 1.5 and 5.9% for all analytes.

Early-morning non-acidified spot urines (Group 1) were collected from 198 adult patients (age range 23 – 91 y; urine creatinine 1.5 - 30.8 mmol/L), after overnight rest for measurement of urine albumin as part of renal function testing. They were analyzed for free metanephrines and reference ranges determined as 95th percentiles of normal distributions of log-transformed data using an Analyse-It statistics package. Mean concentrations calculated as µmol/mol creatinine were significantly higher in females than in males, with upper limits of normal (ULN) of 31, 20 and 42 (females) and 20, 14, and 28 (males) for free normetanephrine, metanephrine and 3-MT respectively. Urine concentrations of free metanephrines were also higher in adults over 60 compared to under 60 years old, but this difference did not reach statistical significance in this group.

24-Hour urine collections (Group 2) were obtained in acid-containers from 76 adult patients (age range 18 – 93 y), as is current laboratory practice for biochemical investigation of possible PPGL. They were also analysed for free metanephrines and catecholamines by LC-MSMS, and included in this study if found to have normal excretion of catecholamines. In group 2, free metanephrine outputs expressed as nmol/day were similar or higher in males compared to females, with ULNs being 320, 220 and 440 (males) and 340, 160, and 390 (females) for free normetanephrine, metanephrine and 3-MT respectively.

Urine creatinine was also measured for the 24-hour acidified urines to allow free metanephrine concentrations to be expressed as µmol/mol creatinine in Group 2, and converted to fractions of the gender-matched ULN that were obtained from overnight non-acidified spot (Group 1) urines. The distributions of these concentrations of free metanephrines expressed as fractions of ULN were similar to values obtained from the same patients using ULNs calculated from nmol/day. The reference ranges in µmol/mol creatinine that were obtained for spot urines in this study therefore should be useful to exclude PPGL in patients who have collected either an early-morning spot urine or a 24-hour collection. Clearly, a change in urine specimen collection practice from 24-hour samples to unacidified early-morning spot samples, and measurement of free rather than total metanephrines, will benefit both patients and the clinical laboratory, but further studies with large groups of patients with and without PPGL are required to clinically validate this approach.


References & Acknowledgements:

1 Whiting (2009) Ann Clin Biochem 46: 129-36.


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