Analytical and Bioanalytical Chemistry

, Volume 409, Issue 24, pp 5735–5745 | Cite as

Profiling of cardiolipins and their hydroperoxides in HepG2 cells by LC/MS

  • Zhen Chen
  • Yue Wu
  • Yi-Shing Ma
  • Yuu Kobayashi
  • Yao-Yao Zhao
  • Yusuke Miura
  • Hitoshi Chiba
  • Shu-Ping HuiEmail author
Research Paper


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.

Graphical abstract

Illustration represents cardiolipin hydroperoxide structure and its content increasing in AAPH-treated HepG2 cells by LC/MS analysis


Cardiolipin Lipid hydroperoxides CL-OOH Molecular species Mitochondria LC-HR-MS/MS 



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.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interests.

Supplementary material

216_2017_515_MOESM1_ESM.pdf (509 kb)
ESM 1 (PDF 509 kb)


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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Zhen Chen
    • 1
  • Yue Wu
    • 1
  • Yi-Shing Ma
    • 1
  • Yuu Kobayashi
    • 1
  • Yao-Yao Zhao
    • 1
  • Yusuke Miura
    • 1
  • Hitoshi Chiba
    • 1
  • Shu-Ping Hui
    • 1
    Email author
  1. 1.Faculty of Health SciencesHokkaido UniversitySapporoJapan

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