Enzyme and Acid Deconjugation of Plasma Sulfated Metanephrines

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: 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: 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 amount of sulfatase required is analyte-dependent (MT>>NMN>MN) though it must contain at least 0.8 mU/ml of sample. The Deming regression curves comparing acid vs 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 hydrolysis of metanephrines in plasma.