Melanie Glauser1, Michael Metrailler1, Sandrine Gerber-Lemaire2, Catherine Centeno1, Caroline Seghezzi1, Marielle Dunand1, Karim Abid1, Adeline Herren1, Eric Grouzmann3. 1. Centre Hospitalier Universitaire Vaudois, Service de Biomédecine, Hôpital Beaumont, Av de Beaumont 29, CH-1011 Lausanne, Switzerland. 2. Institute of Chemistry and Chemical Engineering, Ecole Polytechnique Fédérale de Lausanne, Batochime (Bat BCH), CH-1015 Lausanne, Switzerland. 3. Service de Biomédecine, Centre Hospitalier Universitaire Vaudois, Hôpital Nestlé 5th floor, office 5017, Av Pierre Decker, 1011 Lausanne, CH-1011 Lausanne, Switzerland. Electronic address: Eric.Grouzmann@chuv.ch.
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 0.1M 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 (MT>>NMN>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.
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 0.1M 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 (MT>>NMN>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.
Authors: Foteini Christou; Edward Pivin; Alban Denys; Karim A Abid; Tobias Zingg; Maurice Matter; Antoinette Pechère-Bertschi; Marc Maillard; Eric Grouzmann; Gregoire Wuerzner Journal: Front Endocrinol (Lausanne) Date: 2022-02-24 Impact factor: 5.555