Ritov Vladimir B, Menshikova Elizabeth V, Kelley David E
Department of Medicine, Division of Endocrinology and Metabolism, School of Medicine, University of Pittsburgh, 3459 Fifth Avenue, MUH N809 Pittsburgh, PA 15213-3236, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Feb 2;831(1-2):63-71. doi: 10.1016/j.jchromb.2005.11.031. Epub 2005 Dec 6.
Cardiolipin is a phospholipid that is specific to the inner mitochondrial membrane and essential for numerous mitochondrial functions. Accordingly, a quantitative assay for cardiolipin can be a valuable aspect of assessing mitochondrial content and functional capacity. The current study was undertaken to develop a simple and reliable method for direct analysis of the major molecular species of cardiolipin and with particular application for analysis of human skeletal muscle. The method that is presented is based on derivatization of cardiolipin in a total lipid extract with 1-pyrenyldiazomethane (PDAM), to form stable, fluorescent 1-pyrenylmethyl esters. The derivatization reaction takes 30 min on ice in a two-phase system (chloroform:methanol:H(2)O:H(2)SO(4)) containing 0.5-1.0mM PDAM and detergent. The contents of the major cardiolipin species in the derivatization mixture can be estimated by HPLC separation with fluorescent detection during a 20 min run on a reverse phase column and with HPLC grade ethanol/0.5mM H(3)PO(4) as the mobile phase. The recovery is about 80%. The method is specific and sensitive with quantitation limits of 0.5-1 pmol cardiolipin. The response of the fluorescence detector (peak area) is linear across a range 5-40 pmol. The assay is linear over the range between 0.3 and 3.0mg of tissue (R(2)=0.998). The assay provides good reproducibility and accuracy (within 5-10%).
心磷脂是一种线粒体内膜特有的磷脂，对众多线粒体功能至关重要。因此，心磷脂的定量测定可能是评估线粒体含量和功能能力的一个重要方面。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的主要分子种类，并特别应用于人类骨骼肌的分析。所提出的方法基于用1-芘基重氮甲烷（PDAM）对总脂质提取物中的心磷脂进行衍生化，以形成稳定的荧光1-芘基甲酯。衍生化反应在含有0.5 - 1.0 mM PDAM和去污剂的两相系统（氯仿：甲醇：水：硫酸）中于冰上进行30分钟。衍生化混合物中主要心磷脂种类的含量可通过在反相柱上进行20分钟运行并以HPLC级乙醇/0.5 mM磷酸作为流动相的HPLC分离和荧光检测来估计。回收率约为80%。该方法具有特异性和敏感性，定量限为0.5 - 1 pmol心磷脂。荧光检测器的响应（峰面积）在5 - 40 pmol范围内呈线性。该测定在0.3至3.0 mg组织范围内呈线性（R² = 0.998）。该测定具有良好的重现性和准确性（在5 - 10%以内）。
