Lining Qi 1 Neil D. Danielson 1 Qiang Dai 2 Ray M. Lee 2 1 Chemistry and Biochemistry Department, Miami University, Oxford, OH, USA 2 Huntsman Cancer Institute at the University of Utah, Salt Lake City, UT, USA Capillary electrophoresis of cardiolipin with on-line dye interaction and spectrophotometric detection Cardiolipin is an important phospholipid present in the mitochondrial inner membrane. It plays a key function in mitochondrial respiration by interacting with many enzymes or cofactors related to oxidative phosphorylation complexes. We have determined the concentration of cardiolipin using on-line 10-N-nonyl acridine orange (NAO) dye inter- action capillary electrophoresis (CE) and spectrophotometric detection with a sample throughput of 3 min. In addition to the presence of 0.1 mM NAO, the background elec- trolyte (BGE) composition has been set at 80% methanol-10% acetonitrile-10% H 2 O (all v/v) to provide both good solubility and the maximum absorbance enhancement at 497 nm for the NAO-cardiolipin complex as compared to NAO alone. Sample con- sumption for each injection is about 57 nL. A calibration curve is established from 0.5 mM to 0.1 mM with R 2 = 0.9912 with a detection limit of 0.05 mM for cardiolipin. In a blind study, actual mitochondrial cell membrane samples in the mL range before or after UV light exposure were analyzed using the CE method. Cardiolipin concentra- tion decreased in the different parts of the membrane sample upon UV photolysis of the cells. Support for the theory that UV light can induce cardiolipin translocation from the inner membrane (IM) to the outer membrane (OM) was indicated by a significant percentage increase of cardiolipin (as measured by the cardiolipin in the OM as com- pared to the sum total in the OM and IM) from 30.7 6 2.4% before UV light photolysis to 38.3 6 2.2% after UV irradiation. Keywords: Apoptosis / Capillary electrophoresis / Cardiolipin / Mitochondria / 10-N-Nonylacridine orange / Spectrophotometric detection DOI 10.1002/elps.200305448 1 Introduction Mitochondria have a central role in regulation of pro- grammed cell death, or apoptosis [1]. Mitochondrial defects happen in a wide variety of degenerative dis- eases, ageing, and cancer. Phospholipids are distributed asymmetrically in cell membranes [2]. Cardiolipin (diphos- phatidylglycerol) is often used as a biochemical marker for mitochondria [3–5]. It is the most acidic of eubacterial and mitochondrial membrane phospholipids and it is the only anionic phospholipid present in significant quantities in the inner mitochondrial membrane (IM). The asym- metrical distribution of cardiolipin is controlled by trans- bilayer movements and it can be perturbed, transiently or permanently, by some events [6–8], such as apoptosis [9, 10]. Phospholipid scramblase (PLS) is a family of enzymes, which transfer phospholipids bidirectionally be- tween two compartments [11, 12]. The cardiolipin from beef heart, first isolated in 1941, contains four fatty acid chains and two phosphate head groups [13]. Cardiolipin phosphoryl groups are the most extended and rigidly fixed; the ester carbonyl groups and the free hydroxyl group of the charged – phos- phoryl-CH 2 -CH(OH)-CH 2 -phosphoryl – backbone stabilize the intra- and intermolecular, bidimensional, hydrogen- bonded network that contains the water molecules of the hydration layer [14, 15]. Cardiolipin fatty acyl satura- tion modulates surface conduction of protons for the membrane systems [16]. Cardiolipin plays a key function in regenerating enzymatic activity from phospholipid- depleted preparations of Complexes I, III and IV of the mitochondrial respiratory chain [17]. It can suffice in the case of cytochrome oxidase [18] or work with other phos- pholipids in the case of Complex I or II. Recently, cardio- lipin as a proton trap for oxidative phosphorylation has been reviewed [19]. Phospholipids are difficult to detect due to the lack of conjugated double bonds and unreactive aliphatic func- tional groups in the structure. Classical methods to quantify phospholipids require extraction and subse- quently separation by thin-layer chromatography with colorimetric determination of inorganic phosphate [3]. Liquid chromatography (LC) in the normal phase mode Correspondence: Dr. Neil D. Danielson, Department of Chemis- try and Biochemistry, Miami University, Oxford, OH 45056, USA E-mail: danielnd@muohio.edu Fax: +513-529-5715 Abbreviations: IM, inner membrane; MeOH, methanol; NAO, 10-N-nonyl acridine orange 1680 Electrophoresis 2003, 24, 1680–1686  2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/1005–1680 $17.501.50/0