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

Abstract

AbstractCardiolipin 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.