Capillary electrophoresis with LIF detection for assessment of mitochondrial number based on the cardiolipin content

Abstract

Cardiolipin is a mitochondria-specific phospholipid known to be intimately involved with numerous mitochondrial functions. Accordingly, quantitative determination of cardiolipin provides a valuable aspect for assessing mitochondrial content and function. The current study was undertaken to develop a simple and reliable method for direct analysis of cardiolipin with particular application for the assessment of mitochondrial number of HepG2 cells. The method presented is based on the online 10-N-nonyl acridine orange (NAO) interaction with cardiolipin using CE with LIF detection. An aqueous-organic solvent system composed of 10% H 2O-40% methanol-50% ACN (all v/v) containing 20μM NAO provides both short analysis time within 2min and a definite fluorescence enhancement at 525nm for NAO-cardiolipin complex as compared with NAO alone. Under the optimum condition, a calibration curve between the peak area and the concentration of cardiolipin was established in the range of 0.1-200μM with a correlation coefficient of 0.9955. The detection limit is 9nM. The proposed method was successfully applied to the analysis of cardiolipin in mitochondria from HepG2 cells. A new biochemical method estimating the mitochondrial number per cell was developed and used together with the proposed method for cardiolipin per cell measurement and cardiolipin per mitochondrion reported before. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.