PO0026 Utilizing Purifiedβ-Glucuronidase and Arylsulfatase to Accurately Quantitate Metabolites in Human Urine
來源:IMCS|作者:Pongkwan (Nikki) Sitasuwan*, Margarita Marinova, L. Andrew Lee|發布時間: 87天前|40 次瀏覽|分享到:
Several metabolites are known to be glucuronidated and sulfated in the human body. Monitoring the aglycones with the gluruco-nidated and sulfated metabolites creates six additional masses to monitor on tandem mass spectrometry, which can limit the total number of metabolites being monitored. In addition, the conjugated standards are very expensive and often not available. Herein,we report the use of purifiedβ-glucuronidase (IMCSzyme?) and recombinant arylsulfatase that effectively cleave glucuronidesand sulfates at neutral pH with no detectable CYP activity or esterase activity. The initial studies explored urinary corticosteroidmetabolites on tandem mass spectrometry before and after cleaving glucuronides and sulfates with the purified enzymes. Among corticosteroids, glucuronidated allo-tetrahydrocortisol (aTHF) has been reported to be recalcitrant substrate for enzymatic decon-jugation. We demonstrate rapid hydrolysis within 10 minutes, while the reported 4 hour incubation using crude snail enzyme is in-adequate for recovering the higher levels of a THF glucuronide (1,2).
The study and quantitation of metabolites can give insightsto health state and human diseases. This poster focus-es on cortisol metabolism as an example. Cortisol is quan-titatively the major glucocorti-coid product of the adrenalcortex. The deficiency of ad-renal steroid excretion isfound in Addison’s disease leading to hypotension, and on the other hand the over-production is found in Cush-ing’s syndrome leading to hy-pertension (3). Quantitative measurement of cortisol in both serum and urine are widely done by immunoas-says, however the limitations