Free radical-mediated formation of trans-cardiolipin isomers, analytical approaches for lipidomics and consequences for the structural organization of membranes

Free radical-mediated processes, such as peroxidation, isomerization and hydrogenation affecting fatty acid integrity and biological functions have a trans-disciplinary relevance. Cardiolipins [CL, (1,3-diphosphatidyl-sn-glycerol)] and tetra-linoleoyl-CL are complex phospholipids, exclusively present in the Inner Mitochondrial Membrane (IMM) lipids where they maintain membrane integrity and regulate enzyme functionalities. Peroxidation pathways and fatty acid remodeling are known causes of mitochondrial disfunctions and pathologies, including cancer. Free radical-mediated isomerization with the change of the cis CL into geometrical trans isomers is an unknown process with possible consequences on the supramolecular membrane lipid organization. Here, the formation of mono-trans CL and all-trans CL is reported using CL from bovine heart mitochondria and thiyl radicals generated by UV-photolysis from 2-mercaptoethanol. Analytical approaches for CL isomer separation and identification via 1H/13C NMR are provided, together with chemical study of CL derivatization to fatty acid methyl esters (FAME), useful for lipidomics and metabolomics research. Kinetics information of the radical chain isomerization process was obtained using γ-irradiation conditions. The CL isomerization affected the structural organization of membranes, as tested by reduction of unilamellar liposome diameter, and accompanied the well-known process of oxidative consumption induced by Fenton reagents. These results highlight a new molecular modification pathway of mitochondrial lipids with wide applications to membrane functions and biological consequences.