Plasmalogens: Free Radical Reactivity and Identification of Trans Isomers Relevant to Biological Membranes

Abstract

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.

Keywords: cis-trans isomerization; erythrocyte membrane; fatty acids; free radicals; lipidomics; plasmalogen analysis; trans plasmalogens.

Figure 1

(a) General structure of plasmalogens with a fatty acid in position sn-2 and a vinyl ether-containing hydrocarbon chain in position sn-1. The natural structure has a cis-vinyl ether function. The trans-vinyl ether containing plasmalogen is a synthetically modified lipid. (b) 1-(1Z-octadecenyl)-2-arachidonoyl-sn-glycero-3-phosphocholine (acronym: C18 plasm 20:4-PC) with all cis double bonds. (c) Mechanism of thiyl radical catalyzed cis-trans double bond isomerization of unsaturated fatty acid moieties in lipids depicted as consecutive addition-elimination process. (d) The four monotrans isomers of arachidonic acid (mtAra) structures, as product mix obtained from the thiyl radical-catalyzed isomerization of Ara.

Figure 2

NMR spectra of the commercially available cis plasmalogen and the corresponding mixture of cis/ trans plasmalogen obtained after 4 min of UV photolysis; (a) ¹H NMR and (b) ¹³C NMR regions relative to vinyl ether group; the red and blue lines give the H-C correlations of the OCH=CH group in cis configuration; red and blue dashed lines give the H-C correlations of the trans OCH=CH group; (c) region of bis-allylic and allylic proton signals; the red circle highlights the new signals at 2.78 and 1.97 ppm; (d) ¹³C NMR spectral region relative to C15 resonance of cis plasmalogen and the corresponding cis/trans plasmalogen isomers, where red dashed lines indicate the mtAra isomers: 14-trans (130.90 ppm), 11-trans (130.51 ppm), 8-trans (130.26 ppm) and 5-trans (130.21 ppm) isomers [32].

Figure 3

Follow-up of the isomerization of C18 plasm-20:4 PC (0.125 mM) performed in deuterated benzene in presence of 2-mercaptoethanol (0.5 equiv.) after 1, 2.5, and 4 min of photo-irradiation: (A) trans vinyl ether formation quantitatively determined by ¹H NMR, using tetramethylsilane (TMS) molarity (7.4 mM) as a reference, and mt Ara isomers obtained by quantitative GC analysis after transesterification of the reaction mixture at each time point; (B) graphical representation of the values reported in Table A for the plasmalogen photoirradiation with the formation of trans isomers and decrease of the starting cis material. See details in Table S4.

Figure 4

Proposed mechanism for the thiyl radical catalyzed cis-trans isomerization of vinyl ether moiety in plasmalogens.

Figure 5

The isomerization and peroxidation processes occurring under Fenton-like conditions to double bond and PUFA moieties, respectively, in the presence of thiol RSH. The double bond can belong to the vinyl ether function or the unsaturated fatty acid chain.