Enzyme and acid deconjugation of plasma sulfated metanephrines

Abstract

Background: Total (i.e. free + sulfated) metanephrines in plasma is a biomarker for the diagnosis of pheochromocytoma/paraganglioma. Sulfated metanephrines must be completely deconjugated by perchloric acid hydrolysis or sulfatase treatment prior to analytical measurement to enable quantification by current techniques. In this report, we compare the yield and efficiency of both methods. Methods: The deconjugation rate of synthetic sulfated metanephrines (normetanephrine (S-NMN), metanephrine (S-MN) and methoxytyramine (S-MT)) spiked in charcoal-stripped plasma was determined by boiling perchloric acid and compared to sulfatase treatment. Total plasma metanephrines (MN, NMN and MT) were also determined in patient samples by both methods. Results: The complete deconjugation of sulfated metanephrines is achieved after 30 min incubation with 0.1 M boiling perchloric acid or upon sulfatase treatment. Ten minutes of acid hydrolysis (gold-standard) leads to a 30% underestimation of metanephrine concentrations. The enzyme hydrolysis is time and amount of sulfatase dependent. The rate of hydrolysis is analyte-dependent (MTN N NMN N MN), although it must contain at least 0.8 U/ml of sample. The Deming regression curves comparing acid versus enzyme hydrolysis on patient samples assessed that both methods gave similar unbiased concentrations. Conclusion: Enzyme and acid treatments are equivalent and efficient for removing sulfate from metanephrines as long as the optimal protocol is used for each method. However, the gold standard method for acid hydrolysis at 10 min established more than 20 years ago was not satisfactory regarding the hydrolysis of metanephrines in plasma.

Catecholamines and their O-methylated metabolites in vitreous humor in hypothermia cases

“…The term metanephrines describes two catecholamine metabolites: normetanephrine (the O-methylated [14]. The term urinary ''total'' metanephrines was coined to describe both normetanephrine and metanephrine measured together as a single concentration by early spectrophotometry assays.…”

“…This enzyme is located in high concentrations in the gastrointestinal tract, the source of most sulfateconjugated catecholamines produced in the body. The sulfate conjugation of metanephrines gives rise to hydrosoluble metabolites, considered as end point products eventually eliminated in urine [14][15][16].…”

High concentration of plasma methoxytyramine: dopamine-producing tumour or Parkinson’s disease therapy?

“…Total MNs and MT were desulphated using perchloric acid method or treated with sulphatase as described in a previous study. 10 Total and free MNs and MT were extracted on µElution plate and quantified by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). 11,12 The validation of the method to quantify free MNs and MT was described by Peaston and colleagues and calculated CV inter-assay from three different concentrations and 20 different assays was reported to be lower than 13% for MT.…”

“…The method used for free and total MT quantification is identical after the deconjugation treatment applied to the sulphated MNs and MT. 10…”

Laboratory Evaluation of Pheochromocytoma and Paraganglioma

“…Importantly, efficiency of the acid hydrolysis conversion of sulfate conjugated to free metanephrines de-pends on pH, temperature, and time of incubation (21,22 ). This would not be a problem if calibrators and QC samples recapitulated the proportions of free and conjugated metabolites in patient samples.…”