Direct liquid chromatography-mass spectrometry method for the detection of glutathione S-transferase isozymes and investigation of their expression in response to dietary flavone.
The cytosolic GSTs were measured directly in tissue homogenates using HPLC interfaced via ESI to a mass spectrometer (LC/MS). Total ion chromatograms were generated and filtered for ion currents corresponding to m/z ratio characteristic of individual GST isozymes. Direct LC/MS has a high degree of precision (8%) and low instrument detection limits (50-100 ng) and offers the advantage of monitoring GST expression at the protein level. In this study we describe the sub-chronic effect of feeding flavone (2500 mg/kg diet) on the expression of mGSTA3, mGSTP1, mGSTM1, and mGSTM2 in male and female mice. Additionally, we tentatively identify mGSTO and its up-regulation by flavone; a result that will facilitate the study of this novel enzyme. Flavone induced mGSTM1 and mGSTP1 in a gender and isozyme specific manner yet had no appreciable effect on the expression of mGSTA3. Male animals (day 5) displayed a 8-fold increase in mGSTM1 and a 2.6-fold increase in mGSTP1 whereas female animals displayed a 5- and 3-fold increase in mGSTM1 and mGSTP1, respectively. The mGSTM2 was detected only in flavone-fed animals, indicating an up-regulation of this isozyme by flavone. Results obtained using direct LC/MS compare favorably to the specific activity of individual isozymes (P = 0.19), and are comparable to GST levels determined using affinity chromatography followed by LC/MS (P = 0.79).
Department of Food Science and Technology, Cruess Hall, University of California, One Shields Ave, Davis, CA 95616, USA.
Stephanie A
SA
Burns
Yun-Jeong
YJ
Hong
Alyson E
AE
Mitchell
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004032:Diet; D005260:Female; D047309:Flavones / Q000008:administration & dosage / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000032:analysis*; D007527:Isoenzymes / Q000032:analysis*; D008297:Male; D051379:Mice; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D047309:Flavones; D007527:Isoenzymes; D005982:Glutathione Transferase