An (1)O2 route to γ-hydroxyalkenal phospholipids by vitamin E-induced fragmentation of hydroperoxydiene-derived endoperoxides

Abstract

Biologically active phospholipids that incorporate an oxidatively truncated acyl chain terminated by a γ-hydroxyalkenal are generated in vivo. The γ-hydroxyalkenal moiety protrudes from lipid bilayers like whiskers that serve as ligands for the scavenger receptor CD36, fostering endocytosis, e.g., of oxidatively damaged photoreceptor cell outer segments by retinal pigmented endothelial cells. They also covalently modify proteins generating carboxyalkyl pyrroles incorporating the ε-amino group of protein lysyl residues. We postulated that γ-hydroxyalkenals could be generated, e.g., in the eye, through fragmentation of hydroperoxy endoperoxides produced in the retina through reactions of singlet molecular oxygen with polyunsaturated phospholipids. Since phospholipid esters are far more abundant in the retina than free fatty acids, we examined the influence of a membrane environment on the fate of hydroperoxy endoperoxides. We now report that linoleate hydroperoxy endoperoxides in thin films and their phospholipid esters in biomimetic membranes fragment to γ-hydroxyalkenals, and fragmentation is stoichiometrically induced by vitamin E. The product distribution from fragmentation of the free acid in the homogeneous environment of a thin film is remarkably different from that from the corresponding phospholipid in a membrane. In the membrane, further oxidation of the initially formed γ-hydroxyalkenal to a butenolide is disfavored. A conformational preference for the γ-hydroxyalkenal, to protrude from the membrane into the aqueous phase, may protect it from oxidation induced by lipid hydroperoxides that remain buried in the lipophilic membrane core.

Figure 1

Oxidative cleavage of polyunsaturated fatty acyl (PUFA) phosphatidylcholines generates γ-hydroxyalkenal phosphatidylcholines that react with proteins to deliver carboxyalkyl pyrroles.

Figure 2

Singlet molecular oxygen reaction pathways to γ-hydroxyalkenals. Further oxidation produces butenolides in competition with adduction to proteins to generate carboxyalkyl pyrroles.

Figure 3

Postulated fragmentation products from 13-HP-Endo.

Figure 4

Structurally specific synthesis of a 13-hydroperoxyendoperoxide.

Figure 5

Synthesis of the butenolide ODFO.

Figure 6

Negative ESI-MS/MS spectrum of KODA (A) and ODFO (B), and HPLC chromatogram of KODA and ODFO (1:1 mixture) eluting with methanol/water (C) and acetonitrile/water (D). The chromatogram was monitored by LC-MS in the negative ion mode with SRM of appropriate mass transitions as noted.

Figure 7

Time courses for the generation of HODA, ODFO, KODA and FOA during the thermal decomposition of 13-HP-Endo in the absence (open symbols) or presence (solid symbols) of α-tocopherol (Vit E). Error bars are deviation from average of duplicate runs within less than 24 h.

Figure 8

Formation of HODA and ODFO from 13-HP-Endo with various amounts of α-tocopherol in the absence and presence of the chelating agent diethylene triamine pentaacetic acid (DTPA). Error bars are deviation from average of duplicate runs within less than 24 h.

Figure 9

CML-bound redox-active metal ions may catalyze reductive fragmentation of hydroperoxy endoperoxides to γ-hydroxyalkenals.

Figure 10

A possible redox metal ion catalyzed reductive homolysis of 13-HP-Endo by α-tocopherol.

Figure 11

Formation of HODA and ODFO from 13-HP-Endo in the presence of various amounts of α-tocopherol or γ-tocopherol.

Figure 12

Synthesis of a hydroperoxy endoperoxide phosphocholine ester 13-HP-Endo-PC.

Figure 13

Formation of HODA-PC and ODFO-PC from 13-HP-Endo-PC in small unilamellar vesicles, in PBS buffer (50 mM), with one equivalent of α-tocopherol (Vit E) in PBS buffer (50 mM), or with one equivalent of α-tocopherol in PBS buffer (50 mM) with DTPA (100 μM).

Figure 14

Fragmentation of 13-HP-Endo-PC in unilamellar vesicles. Octanol/water partition coefficients (Log P) for α-tocopherol and the methyl esters of the oxidized PUFAs, calculated by ChemDraw^®, are shown as a measure of relative hydrophobicity.

Figure 15

Formation of HODA-PC and ODFO-PC from 13-HP-Endo-PC in methanol and buffer homogeneous solution and in small unilamellar vesicles.

Figure 16

Possible mechanism that links elevated carboxymethyllysine (CML) accumulation with the generation of carboxyethylpyrroles (CEPs) in the retina of AMD eyes.

Chart 1