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Abstract Herein we report an improved method to separate cardiolipin (Ptd^sub 2^Gro) from tissue total lipid extracts using a biphasic solvent system combined with high performance liquid chromatography. This method uses a normal phase silica column and two mobile phases: mobile phase A that was n-hexane:2-propanol (3:2 by vol) and mobile phase B that was n-hexane:2-propanol:water (56.7:37.8:5.5 by vol). The initial solvent conditions were 95% A and 5% B, with a flow rate of 1.5 mL/min. The samples were from non-derivatized aliquots of liver, heart, or brain lipid extracts. The peak corresponding to Ptd^sub 2^Gro appeared at 31 min, was well defined and did not overlap with neighboring peaks. The adjacent peak corresponded to ethanolamine glycerophospholipids and the remaining phospholipids were eluted in a single peak. The identity of the phospholipids separated by this method was verified by thin layer chromatography (TLC) and fatty acid analysis, which confirmed that the Ptd^sub 2^Gro was well resolved from other phospholipids. This method is useful to separate and quantify Ptd^sub 2^Gro from small tissue samples thereby avoiding the variability associated with TLC methods.
Keywords High Performance liquid Chromatography Cardiolipin - Phospholipids - Brain - Liver - Heart Separation
Abbreviations
ARA Arachidonic acid
DHA Docosahexaenoic acid
EtnGp1 Ethanolamine glycerophospholipids
FAME Fatty acid methyl esters
GLC Gas liquid chromatography
HPLC High performance liquid chromatography
TLC Thin layer chromatography
Ptd^sub 2^Gro Cardiolipin
Introduction
Cardiolipin (Ptd^sub 2^Gro) is a unique phospholipid that contains three glycerol molecules, two phosphate groups, and four fatty acids. Ptd^sub 2^Gro is synthesized in mitochondria where it represents 12% of the total mitochondrial phospholipids [I]. In rat heart and liver, it represents approximately 7% of total phospholipids, while the brain contains substantially less Ptd^sub 2^Gro, where it represents only 2.5% of the total brain phospholipids [2]. While Ptd^sub 2^Gro is found in bacteria, in mammals it is found almost exclusively in the inner mitochondrial membrane and at intermembrane contact sites [3, 4]. Ptd^sub 2^Gro is essential for the optimal function of numerous mitochondrial enzymes involved in energy metabolism, such as cytochrome c oxidase. It binds tightly to the cytochrome c oxidase complex and this interaction is required for efficient electron transport activity [5]. In addition, alterations in the content or structure of Ptd^sub 2^Gro have been observed...