title: Assessing bioenergetic function in response to oxidative stress by metabolic profiling.
abstract: It is now clear that mitochondria are an important target for oxidative stress in a broad range of pathologies, including cardiovascular disease, diabetes, neurodegeneration, and cancer. Methods for assessing the impact of reactive species on isolated mitochondria are well established but constrained by the need for large amounts of material to prepare intact mitochondria for polarographic measurements. With the availability of high-resolution polarography and fluorescence techniques for the measurement of oxygen concentration in solution, measurements of mitochondrial function in intact cells can be made. Recently, the development of extracellular flux methods to monitor changes in oxygen concentration and pH in cultures of adherent cells in multiple-sample wells simultaneously has greatly enhanced the ability to measure bioenergetic function in response to oxidative stress. Here we describe these methods in detail using representative cell types from renal, cardiovascular, nervous, and tumorigenic model systems while illustrating the application of three protocols to analyze the bioenergetic response of cells to oxidative stress.
authors.affiliation: Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Dranka
authors.forename: Gloria A
authors.initials: GA
authors.lastname: Benavides
authors.forename: Anne R
authors.initials: AR
authors.lastname: Diers
authors.forename: Samantha
authors.initials: S
authors.lastname: Giordano
authors.forename: Blake R
authors.initials: BR
authors.lastname: Zelickson
authors.forename: Colin
authors.initials: C
authors.lastname: Reily
authors.forename: Luyun
authors.initials: L
authors.lastname: Zou
authors.forename: John C
authors.initials: JC
authors.lastname: Chatham
authors.forename: Bradford G
authors.initials: BG
authors.lastname: Hill
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhang
authors.forename: Aimee
authors.initials: A
authors.lastname: Landar
authors.forename: Victor M
authors.initials: VM
authors.lastname: Darley-Usmar
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004734:Energy Metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000378:metabolism*; D018384:Oxidative Stress; D010100:Oxygen / Q000032:analysis / Q000378:metabolism
chemical_list: D010100:Oxygen