Oxidized phospholipids as biomarkers of tissue and cell damage with a focus on cardiolipin

Abstract

Oxidized phospholipid species are important, biologically relevant, lipid signaling molecules that usually exist in low abundance in biological tissues. Along with their inherent stability issues, these oxidized lipids present themselves as a challenge in their detection and identification. Often times, oxidized lipid species can co-chromatograph with non-oxidized species making the detection of the former extremely difficult, even with the use of mass spectrometry. In this study, a normal-phase and reverse-phase two dimensional HPLC-mass spectrometric system was applied to separate oxidized phospholipids from their non-oxidized counterparts, allowing unambiguous detection in a total lipid extract. We have utilized bovine heart cardiolipin as well as commercially available tetralinoleoyl cardiolipin oxidized with cytochrome c (cyt c) and hydrogen peroxide as well as with lipoxygenase to test the separation power of the system. Our findings indicate that oxidized species of not only cardiolipin, but other phospholipid species, can be effectively separated from their non-oxidized counterparts in this two dimensional system. We utilized three types of biological tissues and oxidative insults, namely rotenone treatment of lymphocytes to induce mitochondrial damage and cell death, pulmonary inhalation exposure to single walled carbon nanotubes, as well as total body irradiation, in order to identify cardiolipin oxidation products, critical to the cell damage/cell death pathways in these tissues following cellular stress/injury. Our results indicate that selective cardiolipin (CL) oxidation is a result of a non-random free radical process. In addition, we assessed the ability of the system to identify CL oxidation products in the brain, a tissue known for its extreme complexity and diversity of CL species. The ability of the two-dimensional HPLC-mass spectrometric system to detect and characterize oxidized lipid products will allow new studies to be formulated to probe the answers to biologically important questions with regard to oxidative lipidomics and cellular insult.

Molecular species composition of plant cardiolipin determined by liquid chromatography mass spectrometry

“…4, 5). The elution of CL molecular species from the reverse phase column depended on the acyl chain length and degree of unsaturation as previously described (31,33,50,51). CL molecular species with shorter acyl chains or with a higher degree of unsaturation were eluted earlier (Figs.…”

“…2B, C). Previously, CL was enriched from animal lipids before quantification by MS using silica/normal phase chromatography (32,33,51) (Table 3).…”

Challenges in Separations of Proteins and Small Biomolecules and the Role of Modern Mass Spectroscopy Tools for Solving Them, as Well as Bypassing Them, in Structural Analytical Studies of Complex Biomolecular Mixtures

“…In Figure 11, the first dimension chromatogram (normal-phase chromatography) is shown. Although various classes of lipids and phospholipids (ceramide/glycolipids, phosphatidylglycerols, cardiolipins, phosphatidylinositols, phosphatidylethanolamines, phosphatidylserines, phosphatidylcholines, phosphatidic acids and sphingomyelins were efficiently separated, individual lipids within those classes were either poorly resolved or not separated at all [60]. Thus, a second dimension, using reverse-phase chromatography was employed, as shown in Figure 12.…”

“…MALDI MS/MS approach, using diagnostic precursor ion and neutral loss scans was shown to be highly efficient for analysis of biological lipid mixtures [3]. LC-MS approach has been applied as well [60]. Although LC-MS methodology has many advantages for analysis of complex biological mixtures, MALDI MS is advantageous for detailed MS/MS structural analyses.…”

“…In a study shown in Figures 11 and 12 [60], 2D chromatography (normal-phase+reverse-phase chromatography) in combination with MS was employed. In Figure 11, the first dimension chromatogram (normal-phase chromatography) is shown.…”