Oxidative lipidomics of apoptosis: quantitative assessment of phospholipid hydroperoxides in cells and tissues

Abstract

Oxidized phospholipids play essential roles in execution of mitochondrial stage of apoptosis and clearance of apoptotic cells. The identification and quantification of oxidized phospholipids generated during apoptosis can be successfully achieved by oxidative lipidomics. With this approach, diverse molecular species of phospholipids and their hydroperoxides are identified and characterized by soft-ionization mass-spectrometry techniques such as electrospray ionization (ESI). Quantitative assessment of lipid hydroperoxides is performed by fluorescence HPLC-based protocol. The protocol is based on separation of phospholipids using two-dimensional-high-performance thin-layer chromatography (2-D-HPTLC). Phospholipids are hydrolyzed using phospholipase A(2). The fatty acid hydroperoxides (FA-OOH) released is quantified by a fluorometric assay using Amplex red reagent and microperoxidase-11 (MP-11). Detection limit of this protocol is 1-2 pmol of lipid hydroperoxides. Lipid arrays vs. oxidized lipid arrays can be performed by comparing the abundance of phospholipids with the abundance of oxidized phospholipids. Using oxidative lipidomics approach we show that the pattern of phospholipid oxidation during apoptosis is nonrandom and does not follow their abundance in several types of cells undergoing apoptosis and a variety of disease states. This has important implications for evaluation of apoptosis in vivo. The anionic phospholipids, cardiolipin (CL) and phosphatidylserine (PS), are the preferred peroxidation substrates.

Fig. 21.1.

Typical ESI mass spectra of molecular species of PE (A), PI (B), PC (C), and CL (D) obtained from rat brain cortical P2 fraction. Phospholipids separated by 2D-HPTLC were subjected to MS analysis by direct infusion into mass spectrometer. Typical MS/MS fragmentation experiments for the major molecular species of PE, PI, PC, and CL are shown on panels Ab, Bb, Cb, and Db, respectively. Note that MS spectra for PE, PI, CL were recorded in negative mode and for PC in positive mode.

Fig. 21.2.

Typical negative-ion ESI mass spectra of PS isolated from rat brain cortex (A – control and B – after CCI), (C) – midfrontal gyrus from human with Alzheimer’s disease patients and (D) – from mouse gut after total body irradiation. Identification of individual oxidized molecular species of PS containing C_22:6+OOH. Tandem MS-MS experiments confirmed the structures of non-oxidized and oxidized PS species (panels A, b; B, b).

Fig. 21.3.

Determination of FA-OOH using Amplex red protocol. Formation of resorufin from Amplex red in the presence of MP-11 and FA-OOH (a). Typical separation of the resorufin by HPLC (b). Insert shows fluorescent response of resorufin vs concentration of MP-11 in the presence (closed circus) and in the absence (opened circus) of FA-OOH (9S HpODE). Fluorescent response of resorufin vs concentration of resorufin or 9S-HpODE (c). Typical negative-ion ESI-MS of arachidonic acid (d), after its oxidation by cyt c/H₂O₂ (e), and oxidized arachidonic acid after reaction catalyzed by MP-11 in the presence of Amlex red (f). Quantification of arachidonic acid hydroperoxides catalyzed by cyt c/H₂O₂ (g). Reaction conditions: Arachidonic acid (2 mM) was incubated with or without cyt c (40 μM), H₂O₂ (2 × 500 μM), 100 μM DTPA in 50 mM phosphate buffer pH 7.4 at 37°C in for 30 min. Amount of arachidonic acid hydroperoxides was determined by Amlex red/MP-11 system (68.4 ± 5.9 LOOH pmol/nmol FA).

Fig. 21.4.

Comparison of the abundance of major PL classes with their oxidation products. Profiles of phospholipids and PL-OOH in: (1) rat brain cortical P2 fractions after CCI, (2) human brain midfrontal gyrus with Alzheimer’s disease, and (3) mouse gut after total body irradiation. Phospholipid content is expressed as percent of total phospholipids and shown in green-blue scale. Phospholipid hydroperoxides are presented as percent of phospholipids (pmol PL-OOH per nmol of phospholipid) and shown in yellow-brown scale. In a variety of apoptotic cells and the disease processes investigated, CL and PS underwent most robust oxidation compared with other phospholipids (PE, PI, and PC). One hundred percent corresponds to 110 ± 20 pmol; 71 ± 32 pmol, and 79 ± 20 pmol of PL-OOH per nmol of phospholipid in rat brain cortex, human brain (midfrontal gyrus), and mouse gut, respectively.