Free-Radical-Mediated Formation of Trans-Cardiolipin Isomers, Analytical Approaches for Lipidomics and Consequences of the Structural Organization of Membranes

Abstract

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.

Keywords: Fenton reaction; cardiolipin; cis-trans isomerization; free radicals; linoleic acid; liposome dimension; γ-irradiation.

Figure 1

Structure of tetra-linoleoyl cardiolipin.

Figure 2

(a) Thiyl radical-catalyzed isomerization of monounsaturated fatty acid moiety L_Z to L_E; (b) Cis−trans isomerization of linoleic moiety (L_ZZ) catalyzed by thiyl radicals.

Figure 3

NMR spectral regions of natural cardiolipin (CL) and its reaction crude in the first minutes of UV photolysis containing mono-trans CL (MT-CL, see Figure S4 for the structures): (a) ¹H NMR region relative to bis-allylic and allylic proton signals; (b) ¹³C NMR region relative to alkene carbon atom signals.

Figure 4

Dose profile of cis−trans isomerization of linoleoyl residue (L_ZZ corresponding to 1.32 mM) of CL (0.35 mM) catalyzed by thiyl radicals generated by γ-radiolysis of HO(CH₂)₂SH (0.5 equiv.) in the presence of 0.33 equiv. of methyl oleate as control in N₂O-saturated i-PrOH at 22 °C. Profiles of L_ZZ (green ▲), L_ZE + L_EZ (red ■) and L_EE (blue ●), and the sum of all geometrical isomers (purple ▼).

Figure 5

γ-Radiolysis of 0.5 mM POPC/CL liposomes (3:1) in the presence of 0.5 mM HO(CH₂)₂SH as a function of the irradiation dose. The concentration (mM) of L_ZZ (green ▼), L_ZE (grey ■), L_EZ (red ▲) and L_EE (blue ●), and the sum of all geometrical isomers (purple ◆).