Simultaneous Analysis of the Majority of Low-Molecular-Weight, Redox-Active Compounds from Mitochondria

Abstract

Studies of the interaction between oxidative stress and mitochondrial dysfunction are complicated by analytical limitations, especially the need to assess multiple parameters in relatively small samples. We have addressed this problem by developing a methodology for the simultaneous analysis of the majority of low-molecular-weight, redox-active compounds from mitochondria using HPLC separations followed by coulometric array detection. The method described should also be applicable for the study of redox-active compounds in other subcellular organelles as well as in intact cells and tissues. The protocol described enables simultaneous measurement of antioxidants (e.g., tocopherols, ascorbate, lipoates, uric acid, and glutathione), markers of oxidative stress (e.g.,o-tyrosine,m-tyrosine, nitrotyrosine, dityrosine, glutathione disulfide, and 8-hydroxydeoxyguanosine) as well as other metabolites (e.g., purines and indoles). In all, ca. 600 redox active compounds can be detected, most with a limit of detection of ∼5 pg on column. Results, including analytical parameters, from a study of liver mitochondria from control and diabetic rats are presented to demonstrate utility of this methodology.