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

Abstract

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 β‐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 β‐glucuronidase and sulfatase from Helix pomatia is compared to hydrolyze curcumin conjugates in human and mouse plasma after oral administration of turmeric. Both β‐glucuronidase and sulfatase completely hydrolyze curcumin‐glucuronide. Unexpectedly, β‐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 β‐glucuronidase treatment. Incomplete hydrolysis by β‐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 β‐Glucuronidase incompletely hydrolyzes complex sulfate‐containing conjugates that appear to be major metabolites, resulting in an underestimation of the total plasma concentration of curcumin.

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“…Also, as NC plasma concentration decreased, the NC content in tissues increased and it was shown that the distribution of NC in tissues is higher than C. It has been shown that C can be metabolized in the kidney by the β-glucuronidase and sulfatase enzymes. 32 , 33 Therefore, complete clearance from plasma 72 h after the last dose of NC and C in the present study indicates complete urinary C and NC excretion.…”

Polar Phenol Detection in Plasma and Serum: Insights on Sample Pre-Treatment for LC/MS Analysis and Application on the Serum of Corinthian Currant-Fed Rats

“…As the suitable conjugate standards of polar phenolics are rarely commercially available for analysis, the majority of studies on their bioavailability repeatedly treat biofluid samples with glucuronidase/sulfatase enzymes for the subsequent quantification of the released aglycones [ 38 ]. To date, a few studies have questioned the efficacy of enzymatic hydrolysis for the reliable quantification of glucuronidated and sulfated metabolites of polar phenolics in biofluids [ 24 , 29 , 39 ]. According to Quifer-Rada et al [ 29 ], enzymatic hydrolysis negatively affected the recovery of the precursor and free-form polar phenolics present in human urine samples.…”

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“…are often pre-treated with deconjugating enzymes to liberate the aglycone prior to pharmacokinetic analyses (51,58). This practice, however, is not always well documented or characterized, nor are the clinical implications of low in vivo, bioactive aglycones always considered (60,67).…”

“…Indeed, in the case of curcuminoids, these conjugates can persist in the circulation for over 24 h (e.g., 10% of administered curcuminoids, independent of dose) ( 61 ), due in part to enterohepatic recirculation ( Figure 4A ) ( 60 – 65 ). For this reason, and because glucuronide conjugates are difficult to analyze ( 66 ), serum samples for curcuminoids and other botanicals are often pre-treated with deconjugating enzymes to liberate the aglycone prior to pharmacokinetic analyses ( 51 , 58 ). This practice, however, is not always well documented or characterized, nor are the clinical implications of low in vivo , bioactive aglycones always considered ( 60 , 67 ).…”

“…This practice, however, is not always well documented or characterized, nor are the clinical implications of low in vivo , bioactive aglycones always considered ( 60 , 67 ). For example, data from our laboratories indicate that the common practice of glucuronidase hydrolysis can underestimate curcumin exposure due to incomplete hydrolysis of significant quantities of sulfated or higher order conjugates and suggest the use of sulfatase instead of glucuronidase since the former enzyme achieves a more complete hydrolysis of conjugates ( 58 ). Some have questioned the clinical relevance of such measures as conjugates typically lack bioactivity ( 68 ).…”