Incomplete Hydrolysis of Curcumin Conjugates by β-Glucuronidase: Detection of Complex Conjugates in Plasma.

Scope The diphenol curcumin from turmeric is rapidly metabolized into phase II conjugates following oral administration, resulting in negligible plasma concentration of the free compound, which is considered the bioactive form. Total plasma concentration of curcumin is often quantified after treatment with beta-glucuronidase to hydrolyze curcumin-glucuronide, the most abundant conjugate in vivo. The efficiency of enzymatic hydrolysis has not been tested. Methods and results Using liquid chromatography-mass spectrometry (LC-MS) analyses the efficiency of beta-glucuronidase and sulfatase from Helix pomatia is compared to hydrolyze curcumin conjugates in human and mouse plasma after oral administration of turmeric. Both beta-glucuronidase and sulfatase completely hydrolyze curcumin-glucuronide. Unexpectedly, beta-glucuronidase hydrolysis is incomplete, affording a large amount of curcumin-sulfate, whereas sulfatase hydrolyzed both glucuronide and sulfate conjugates. With sulfatase, the concentration of free curcumin is doubled in human and increased in mouse plasma compared to beta-glucuronidase treatment. Incomplete hydrolysis by beta-glucuronidase suggests the presence of mixed glucuronide-sulfate conjugates. LC-MS based searches detect diglucuronide, disulfate, and mixed sulfate-glucuronide and sulfate-diglucuronide conjugates in plasma that likely contribute to the increase of free curcumin upon sulfatase treatment. Conclusion beta-Glucuronidase incompletely hydrolyzes complex sulfate-containing conjugates that appear to be major metabolites, resulting in an underestimation of the total plasma concentration of curcumin.