Analysis of mitochondria by capillary electrophoresis: Cardiolipin levels decrease in response to carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone

Abstract

Cardiolipin is an important phospholipid present in the inner membrane of mitochondria. It plays a critical role in adenosine triphosphate (ATP) synthesis mediated by oxidative phosphorylation. Exposure of HepG2 cells to carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) caused the inhibition of ATP synthesis and the depolarization of mitochondria. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) analysis of fluorescent mitochondrion-selective probe 10-N-nonyl acridine orange (NAO) labeled mitochondria was employed to in situ estimate the cardiolipin levels under FCCP-induced de-energization of mitochondria. NAO, stoichiometriclly bound to cardiolipin at a 1:1 or 2:1 molar ratio (NAO/cardiolipin), emitted green and red fluorescence, respectively. Green fluorescence was chosen for cardiolipin content analysis because it was more intense than red fluorescence. A significant decrease in the cardiolipin content, up to 11% of the control, was evident when the ATP content and mitochondrial membrane potential (MMP) correspondingly decreased. These related findings suggested that CE-LIF may provide a sensitive strategy to determine cardiolipin content in response to exposure to chemical uncouplers. This reinforces the hypothesis that alterations in ATP synthesis and MMP have a close association with cardiolipin content, which correlated tightly with mitochondrial membrane assembly and activity. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.