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Identifying and applying a highly selective probe to simultaneously determine the O-glucuronidation activity of human UGT1A3 and UGT1A4.

Jiang L, Liang SC, Wang C, Ge GB, Huo XK, Qi XY, Deng S, Liu KX, Ma XC - Sci Rep (2015)

Bottom Line: Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway.However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme.DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Key Laboratory of Pharmacokinetic and Drug Transport of Liaoning, Academy of Integrative Medicine, Dalian Medical University, Dalian, 116044, China.

ABSTRACT
Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway. However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme. In this paper, desacetylcinobufagin (DACB) 3-O- and 16-O-glucuronidation were found to be isoform-specific probe reactions for UGT1A4 and UGT1A3, respectively. DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

No MeSH data available.


3β- (A) and 16β-O-glucuronidation (B) in 12 individual HLMs and organ microsomes including the kidney and intestine from different genders.
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f6: 3β- (A) and 16β-O-glucuronidation (B) in 12 individual HLMs and organ microsomes including the kidney and intestine from different genders.

Mentions: To widely apply DACB in measuring the real O-glucuronidation bioactivities of UGT1A3 and UGT1A4 in various biological samples, we had established a sensitive method using LC-MS/MS (Fig. S8). After optimizing the pH values of various incubation systems, we found that pH values in the range of 7–9 were best for determining the bioactivities of UGT1A3 and UGT1A4 (Fig. S9). In using DACB as a probe, significant O-glucuronidation bioactivity differences between UGT1A3 and UGT1A4 in individual human livers (Fig. 6) were elucidated for the first time. And we also revealed that the activity of UGT1A4 in mediating O- and N-glucuronidation had a positive correlation in HLMs (Fig. S10), which is important for further clarifying the stereochemical structure and catalytic mechanism of UGT1A4. Additionally, animal species differences for UGT1A3 and UGT1A4 were analyzed using DACB. The UGT metabolic profiles indicate that the 16-O-glucuronidation of DACB can occur in all species except RtLMs, whereas 3-O- glucuronidation occurs in DLMs, MLMs, MsLMs, RLMs and TLMs. Kinetic studies using DACB as a probe for UGT1A3 and UGT1A4 were performed to compare differences between species (Table S2). For DACB-3-O-glucuronidation (M-1) mediated by UGT1A4, PLMs, RLMs and MLMs exhibited Michaelis–Menten kinetics, whereas DLMs showed biphasic kinetics (Fig. S11). According to the CLint values, the activities of UGT1A4 in different animal species were order as Rabbit > Monkey > Mouse > Dog > Pig for M-1 formation. Similarly, the formation of DACB-16-O-glucuronidation (M-2) was selectively mediated by UGT1A3 following Michaelis–Menten kinetics in PLMs, DLMs and MLMs, while substrate inhibition kinetics was displayed in RLMs (Fig. S12). Additionally, the CLint activities of UGT1A3 were MLM > RLM > PLM > DLM (Table S2). These results for the UGT1A3 and UGT1A4 activities, as measuring by using DACB as a selective probe, may provide important guidance for the rational selection of model animals in preclinical studies of new drugs.


Identifying and applying a highly selective probe to simultaneously determine the O-glucuronidation activity of human UGT1A3 and UGT1A4.

Jiang L, Liang SC, Wang C, Ge GB, Huo XK, Qi XY, Deng S, Liu KX, Ma XC - Sci Rep (2015)

3β- (A) and 16β-O-glucuronidation (B) in 12 individual HLMs and organ microsomes including the kidney and intestine from different genders.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401096&req=5

f6: 3β- (A) and 16β-O-glucuronidation (B) in 12 individual HLMs and organ microsomes including the kidney and intestine from different genders.
Mentions: To widely apply DACB in measuring the real O-glucuronidation bioactivities of UGT1A3 and UGT1A4 in various biological samples, we had established a sensitive method using LC-MS/MS (Fig. S8). After optimizing the pH values of various incubation systems, we found that pH values in the range of 7–9 were best for determining the bioactivities of UGT1A3 and UGT1A4 (Fig. S9). In using DACB as a probe, significant O-glucuronidation bioactivity differences between UGT1A3 and UGT1A4 in individual human livers (Fig. 6) were elucidated for the first time. And we also revealed that the activity of UGT1A4 in mediating O- and N-glucuronidation had a positive correlation in HLMs (Fig. S10), which is important for further clarifying the stereochemical structure and catalytic mechanism of UGT1A4. Additionally, animal species differences for UGT1A3 and UGT1A4 were analyzed using DACB. The UGT metabolic profiles indicate that the 16-O-glucuronidation of DACB can occur in all species except RtLMs, whereas 3-O- glucuronidation occurs in DLMs, MLMs, MsLMs, RLMs and TLMs. Kinetic studies using DACB as a probe for UGT1A3 and UGT1A4 were performed to compare differences between species (Table S2). For DACB-3-O-glucuronidation (M-1) mediated by UGT1A4, PLMs, RLMs and MLMs exhibited Michaelis–Menten kinetics, whereas DLMs showed biphasic kinetics (Fig. S11). According to the CLint values, the activities of UGT1A4 in different animal species were order as Rabbit > Monkey > Mouse > Dog > Pig for M-1 formation. Similarly, the formation of DACB-16-O-glucuronidation (M-2) was selectively mediated by UGT1A3 following Michaelis–Menten kinetics in PLMs, DLMs and MLMs, while substrate inhibition kinetics was displayed in RLMs (Fig. S12). Additionally, the CLint activities of UGT1A3 were MLM > RLM > PLM > DLM (Table S2). These results for the UGT1A3 and UGT1A4 activities, as measuring by using DACB as a selective probe, may provide important guidance for the rational selection of model animals in preclinical studies of new drugs.

Bottom Line: Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway.However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme.DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Key Laboratory of Pharmacokinetic and Drug Transport of Liaoning, Academy of Integrative Medicine, Dalian Medical University, Dalian, 116044, China.

ABSTRACT
Glucuronidation mediated by uridine 5'-diphospho (UDP)-glucuronosyltransferase is an important detoxification pathway. However, identifying a selective probe of UDP- glucuronosyltransferase is complicated because of the significant overlapping substrate specificity displayed by the enzyme. In this paper, desacetylcinobufagin (DACB) 3-O- and 16-O-glucuronidation were found to be isoform-specific probe reactions for UGT1A4 and UGT1A3, respectively. DACB was well characterized as a probe for simultaneously determining the catalytic activities of O-glucuronidation mediated by UGT1A3 and UGT1A4 from various enzyme sources, through a sensitive analysis method.

No MeSH data available.