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Capillary electrophoresis with LIF detection for assessment of mitochondrial number based on the cardiolipin content
Author(s) -
Zhao Wenfeng,
Chen Qidan,
Wu Ruige,
Wu Hong,
Fung Yingsing,
O Waisum
Publication year - 2011
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100165
Subject(s) - cardiolipin , acridine orange , mitochondrion , chemistry , chromatography , phospholipid , detection limit , capillary electrophoresis , analytical chemistry (journal) , biochemistry , membrane , apoptosis
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 2 O–40% methanol–50% ACN (all v/v) containing 20 μM NAO provides both short analysis time within 2 min and a definite fluorescence enhancement at 525 nm 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 0.1–200 μM with a correlation coefficient of 0.9955. The detection limit is 9 nM. 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.