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 (MT-CL) and other trans CL isomers (T-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 the 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 the reduction in unilamellar liposome diameter, and accompanied the well-known process of oxidative consumption induced by Fenton reagents. These results highlight a potential new molecular modification pathway of mitochondrial lipids with wide applications to membrane functions and biological consequences.

Plasmalogens are membrane phospholipids with two fatty acid hydrocarbon chains linked to L-glycerol, one containing a characteristic cis-vinyl ether function and the other one being a polyunsaturated fatty acid (PUFA) residue linked through an acyl function. All double bonds in these structures display the cis geometrical configuration due to desaturase enzymatic activity and they are known to be involved in the peroxidation process, whereas the reactivity through cis-trans double bond isomerization has not yet been identified. Using 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine (C18 plasm-20:4 PC) as a representative molecule, we showed that the cis-trans isomerization can occur at both plasmalogen unsaturated moieties, and the product has characteristic analytical signatures useful for omics applications. Using plasmalogen-containing liposomes and red blood cell (RBC) ghosts under biomimetic Fenton-like conditions, in the presence or absence of thiols, peroxidation, and isomerization processes were found to occur with different reaction outcomes due to the particular liposome compositions. These results allow gaining a full scenario of plasmalogen reactivity under free radical conditions. Moreover, clarification of the plasmalogen reactivity under acidic and alkaline conditions was carried out, identifying the best protocol for RBC membrane fatty acid analysis due to their plasmalogen content of 15–20%. These results are important for lipidomic applications and for achieving a full scenario of radical stress in living organisms.