Cardiolipin (CL) exists as crucial functional phospholipid in mitochondria. The oxidation of CL is concerned with mitochondrial dysfunction and various diseases. As main oxidation products, CL hydroperoxide (CL-OOH) plays a key role in intermediating oxidative reaction. Thus, direct analysis of CL-OOH is of great interest. In the present study, CL and CL-OOH profiles were analyzed in oxidized HepG2 cell lipid via HPLC-Orbitrap MS/MS. Furthermore, the contents of individual molecular species were compared between intact and AAPH-oxidized HepG2 cells. In total, 46 CL and 18 CL-OOH were identified from oxidized cell lipids, while 21 CL and 9 CL-OOH were detected in AAPH-treated cells. Most CL depleted significantly after AAPH inducement, with percentages varying from 8.3% (CL70:7) to 73.7% (CL72:4), depending on fatty acyl composition. While almost all the CL-OOH remarkably increased, among them 68:6-, 72:6-, and 72:7-OOHs were only detected in AAPH-treated cells. CL68:5- and CL68:4-OOH were the most abundant species, while CL70:5-OOH among all the species expressed the highest oxidation percentage of the corresponding CL. Our results showed practical separation, identification, and semi-quantitation of CL-OOH species, which could contribute to approaches to lipidomic analysis of CL and CL-OOH, as well as tracing biomarkers in mitochondrial oxidative stress diagnosis.
Cardiolipin Lipid hydroperoxides CL-OOH Molecular species Mitochondria LC-HR-MS/MS
This is a preview of subscription content, log in to check access.
This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, by the Regional Innovation Strategy Support Program, Sapporo Health Innovation “Smart-H”, of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
Zhang M, Mileykovskaya E, Dowhan W. Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria. J Biol Chem. 2005;280:29403–8. doi:10.1074/jbc.M504955200.CrossRefGoogle Scholar
Samhan-Arias AK, Ji J, Demidova OM, Sparvero LJ, Feng W, Tyurin V, et al. Oxidized phospholipids as biomarkers of tissue and cell damage with a focus on cardiolipin. Biochim Biophys Acta Biomembr. 2012;1818:2413–23. doi:10.1016/j.bbamem.2012.03.014.CrossRefGoogle Scholar
Wortmann SB, Vaz FM, Gardeitchik T, Vissers LELM, Renkema GH, Schuurs-Hoeijmakers JHM, et al. Mutations in the phospholipid remodeling gene SERAC1 impair mitochondrial function and intracellular cholesterol trafficking and cause dystonia and deafness. Nat Genet. 2012;44:797–802. doi:10.1038/ng.2325.CrossRefGoogle Scholar
Tyurina YY, Tyurin VA, Kapralova VI, Wasserloos K, Mosher M, Epperly MW, et al. Oxidative lipidomics of γ-radiation-induced lung injury: mass spectrometric characterization of cardiolipin and phosphatidylserine peroxidation. Radiat Res. 2011;175:610–21. doi:10.1667/RR2297.1.CrossRefGoogle Scholar
Hui SP, Taguchi Y, Takeda S, Ohkawa F, Sakurai T, Yamaki S, et al. Quantitative determination of phosphatidylcholine hydroperoxides during copper oxidation of LDL and HDL by liquid chromatography/mass spectrometry. Anal Bioanal Chem. 2012;403:1831–40. doi:10.1007/s00216-012-5833-x.CrossRefGoogle Scholar
Shrestha R, Hui S-P, Sakurai T, Yagi A, Takahashi Y, Takeda S, et al. Identification of molecular species of cholesteryl ester hydroperoxides in very low-density and intermediate-density lipoproteins. Ann Clin Biochem. 2014;51:662–71. doi:10.1177/0004563213516093.CrossRefGoogle Scholar
Hui S-P, Sakurai T, Takeda S, Jin S, Fuda H, Kurosawa T, et al. Analysis of triacylglycerol hydroperoxides in human lipoproteins by Orbitrap mass spectrometer. Anal Bioanal Chem. 2013;405:4981–7. doi:10.1007/s00216-013-6903-4.CrossRefGoogle Scholar
Ji J, Kline AE, Amoscato A, Samhan-Arias AK, Sparvero LJ, Tyurin VA, et al. Lipidomics identifies cardiolipin oxidation as a mitochondrial target for redox therapy of brain injury. Nat Neurosci. 2012;15:1407–13. doi:10.1038/nn.3195.CrossRefGoogle Scholar
Zhong H, Lu J, Xia L, Zhu M, Yin H. Formation of electrophilic oxidation products from mitochondrial cardiolipin in vitro and in vivo in the context of apoptosis and atherosclerosis. Redox Biol. 2014;2:878–83. doi:10.1016/j.redox.2014.04.003.CrossRefGoogle Scholar
Hui SP, Sakurai T, Ohkawa F, Furumaki H, Jin S, Fuda H, et al. Detection and characterization of cholesteryl ester hydroperoxides in oxidized LDL and oxidized HDL by use of an Orbitrap mass spectrometer. Anal Bioanal Chem. 2012;404:101–12. doi:10.1007/s00216-012-6118-0.CrossRefGoogle Scholar
Suzuki E, Sano A, Kuriki T, Miki T. Improved separation and determination of phospholipids in animal tissues employing solid phase extraction. Biol Pharm Bull. 1997;20:299–303. doi:10.1248/bpb.20.299.CrossRefGoogle Scholar
Fauland A, Trötzmüller M, Eberl A, Afiuni-Zadeh S, Köfeler H, Guo X, et al. An improved SPE method for fractionation and identification of phospholipids. J Sep Sci. 2013;36:744–51. doi:10.1002/jssc.201200708.CrossRefGoogle Scholar
MacIel E, Domingues P, Domingues MRM. Liquid chromatography/tandem mass spectrometry analysis of long-chain oxidation products of cardiolipin induced by the hydroxyl radical. Rapid Commun Mass Spectrom. 2011;25:316–26. doi:10.1002/rcm.4866.CrossRefGoogle Scholar
Bird SS, Marur VR, Sniatynski MJ, Greenberg HK, Kristal BS (2011) Lipidomics profiling by high-resolution LC-MS and high-energy collisional dissociation fragmentation: focus on characterization of mitochondrial cardiolipins and monolysocardiolipins. Anal Chem 83:940–949. doi: 10.1021/ac102598u
Minkler PE, Hoppel CL. Separation and characterization of cardiolipin molecular species by reverse-phase ion pair high-performance liquid chromatography-mass spectrometry. J Lipid Res. 2010;51:856–65. doi:10.1194/jlr.D002857.CrossRefGoogle Scholar
Sullivan EM, Fix A, Crouch MJ, Sparagna GC, Zeczycki TN, Brown DA, et al. Murine diet-induced obesity remodels cardiac and liver mitochondrial phospholipid acyl chains with differential effects on respiratory enzyme activity. J Nutr Biochem. 2017;45:94–103. doi:10.1016/j.jnutbio.2017.04.004.CrossRefGoogle Scholar
Zhong H, Xiao M, Zarkovic K, Zhu M, Sa R, Lu J, et al. Mitochondrial control of apoptosis through modulation of cardiolipin oxidation in hepatocellular carcinoma: a novel link between oxidative stress and cancer. Free Radic Biol Med. 2017;102:67–76. doi:10.1016/j.freeradbiomed.2016.10.494.CrossRefGoogle Scholar
Ostrander DB, Sparagna GC, Amoscato AA, McMillin JB, Dowhan W. Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis. J Biol Chem. 2001;276:38061–7. doi:10.1074/jbc.M107067200.CrossRefGoogle Scholar