The capillary-isotachophoretic analysis of reactants and products of glucuronidation. A new method for the assay of UDPGlucuronosyltransferase.
UDPglucuronosyltransferase competes with a nucleotide pyrophosphatase for UDPglucuronate as a substrate. Both enzyme systems are located in the microsomal fraction of mammalian liver homogenates, and the pyrophosphatase is present in all but the purest preparations of UDP-glucuronosyltransferase, since solubilisation procedures yield both activities in the 100 000 x g supernatant. In glucuronidation studies, it is important to be able to ascertain the degree of hydrolysis of the substrate UDPglucuronate due to the pyrophosphatase activity. A new method is reported using analytical capillary isotachophoresis whereby reactants and products of both glucuronidation and hydrolysis can be assayed simultaneously. The example presented in this paper is the glucuronidation of paracetamol (4-acetamidophenol) an important phenolic drug of toxicological interest. Rabbit liver microsomes glucuronidate this substance much more slowly than other phenolic compounds, so that hydrolysis of the donor molecule UDPglucuronic acid becomes of primary importance. Furthermore, the ionic mobility of 4-acetamidophenyl glucuronoside is much less than the other constitutents of assay mixtures under the analytical conditions employed, so that a well-defined separation from other reaction species is afforded.
D000082:Acetaminophen / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000378:metabolism; D006868:Hydrolysis; D008658:Inactivation, Metabolic; D008722:Methods; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D011817:Rabbits; D013379:Substrate Specificity
D000082:Acetaminophen; D014453:Glucuronosyltransferase; D011755:Pyrophosphatases