Abstract Title
Use of Liquid Chromatography Mass Spectrometry for Measurement of Microsomal UGT Activity
Additional Funding Sources
The project described was supported by National Science Foundation (NSF) award number OIA-1757324 from the NSF Idaho EPSCoR Program. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NSF. Additional support was received from the M.J. Murdock Charitable Trust, Instrument Grant No. 201914150:05/28/2020, “Liquid Chromatography Mass Spectrometer,” and NSF RII Track-2 FEC Award No. OIA-1826801, “Genomes Underlying Toxin Tolerance (GUTT).
Abstract
Sagebrush (Artemisia tridentate) produces a large number of toxic chemicals to protect itself from herbivores.4,5 Nevertheless, it is a major part of the diet of sage-grouse (Centrocercus urophasianus) to the extent that over the winter, sagebrush constitutes around 90% of the sage grouse’s diet.5 In order to understand how sage-grouse can be proficient at metabolizing sagebrush toxic compounds, we designed a protocol to analyze the UDP-glucuronosyltransferase (UGT) enzymes activity in vitro using mice microsomes. UGT enzymes add a glucuronic acid group to substances through a condensation reaction.1,2,3 4-methylumbelliferone2 was used as a substrate for the enzyme. Liquid chromatography mass spectrometry (LCMS) was used to demonstrate that the amount of 4-methylumbelliferone decreased over time, and a product, 4-methylumbelliferone glucuronide appeared correspondingly. This protocol will be used to investigate the transformation of sagebrush compounds by sage-grouse liver UGTs.
Use of Liquid Chromatography Mass Spectrometry for Measurement of Microsomal UGT Activity
Sagebrush (Artemisia tridentate) produces a large number of toxic chemicals to protect itself from herbivores.4,5 Nevertheless, it is a major part of the diet of sage-grouse (Centrocercus urophasianus) to the extent that over the winter, sagebrush constitutes around 90% of the sage grouse’s diet.5 In order to understand how sage-grouse can be proficient at metabolizing sagebrush toxic compounds, we designed a protocol to analyze the UDP-glucuronosyltransferase (UGT) enzymes activity in vitro using mice microsomes. UGT enzymes add a glucuronic acid group to substances through a condensation reaction.1,2,3 4-methylumbelliferone2 was used as a substrate for the enzyme. Liquid chromatography mass spectrometry (LCMS) was used to demonstrate that the amount of 4-methylumbelliferone decreased over time, and a product, 4-methylumbelliferone glucuronide appeared correspondingly. This protocol will be used to investigate the transformation of sagebrush compounds by sage-grouse liver UGTs.