Department of Chemistry, Xuzhou Normal University, Xuzhou, PR China.
Electrophoresis. 2011 Nov;32(21):3025-33. doi: 10.1002/elps.201100165. Epub 2011 Oct 18.
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.
心磷脂是一种线粒体特异性磷脂，已知其与许多线粒体功能密切相关。因此，心磷脂的定量测定为评估线粒体含量和功能提供了有价值的方面。本研究旨在开发一种简单可靠的方法，用于直接分析心磷脂，特别是用于评估 HepG2 细胞的线粒体数量。所提出的方法基于在线 10-N-壬基吖啶橙（NAO）与心磷脂的相互作用，使用 CE 与 LIF 检测。由 10% H(2)O-40%甲醇-50%ACN（均为 v/v）组成的水-有机溶剂系统，含有 20 μM NAO，与单独的 NAO 相比，可在 2 分钟内提供较短的分析时间，并在 525nm 处对 NAO-心磷脂复合物产生明确的荧光增强。在最佳条件下，在心磷脂浓度为 0.1-200 μM 范围内建立了峰面积与浓度之间的校准曲线，相关系数为 0.9955。检测限为 9nM。该方法成功应用于 HepG2 细胞线粒体中心磷脂的分析。开发了一种估计每个细胞线粒体数量的新生化方法，并与所提出的方法一起用于每个细胞心磷脂的测量和之前报道的心磷脂/每个线粒体的测量。
