Capillary electrophoresis with LIF detection for assessment of mitochondrial number based on the cardiolipin content.
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.
Department of Chemistry, Xuzhou Normal University, Xuzhou, PR China. wenfengzhao2002@gmail.com
Wenfeng
W
Zhao
Qidan
Q
Chen
Ruige
R
Wu
Hong
H
Wu
Yingsing
Y
Fung
Waisum
W
O
D000097:Acetonitriles / Q000737:chemistry; D000609:Aminoacridines / Q000737:chemistry; D015415:Biomarkers / Q000032:analysis / Q000737:chemistry; D002308:Cardiolipins / Q000032:analysis* / Q000737:chemistry; D003584:Cytological Techniques; D019075:Electrophoresis, Capillary / Q000379:methods*; D056945:Hep G2 Cells; D006801:Humans; D016014:Linear Models; D000432:Methanol / Q000737:chemistry; D008928:Mitochondria / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000097:Acetonitriles; D000609:Aminoacridines; D015415:Biomarkers; D002308:Cardiolipins; C041691:10-N-nonylacridinium orange; D000432:Methanol; C032159:acetonitrile