Abstract
To elucidate the role of enzymatic lipid peroxidation in disease pathogenesis and in food deterioration, we recently achieved stereoselective analysis of phosphatidylcholine hydroperoxide (PCOOH) possessing 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-9Z,11E-HPODE) using HPLC-MS/MS with a CHIRALPAK OP (+) column. Because enzymatic oxidation progresses concurrently with auto-oxidation, we need to distinguish them further. Here, we attempted such an analysis. First, we used lipoxygenase, linoleic acid, and lysophosphatidylcholine (LPC) to synthesize the enzymatic oxidation product 13(S)-9Z,11E-HPODE PC, and the auto-oxidation products 13(RS)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC, which were used as standards to test the ability of various columns to separate the enzymatic oxidation product from auto-oxidation products. Separation was achieved by connecting in series two columns with different properties: CHIRALPAK OP (+) and CHIRALPAK IB-3. The CHIRALPAK OP (+) column separated 13(R)-9Z,11E-HPODE PC and 13(S)-9Z,11E-HPODE PC, whereas CHIRALPAK IB-3 enabled separation of 13(S)-9Z,11E-HPODE PC and 13(RS)-9E,11E-HPODE PC. The results for the analysis of both enzymatically oxidized and auto-oxidized lecithin (an important phospholipid mixture in vivo and in food) indicate that our method would be useful for distinguishing enzymatic oxidation and auto-oxidation reactions. Such information will be invaluable for elucidating the involvement of PCOOH in disease pathogenesis and in food deterioration.
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Abbreviations
- 16:0 LPC:
-
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine
- 16:0/18:2 PC:
-
1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine
- C18:
-
octadecylsilane
- CD3OD:
-
methanol-d4
- CSP:
-
chiral stationary phase
- DCC:
-
dicyclohexylcarbodiimide
- DMAP:
-
4-dimethylaminopyridine
- HPETE:
-
hydroperoxyeicosatetraenoic acid
- HPODE:
-
hydroperoxyoctadecadienoic acid
- LA:
-
linoleic acid
- LOX:
-
lipoxygenase
- LPC:
-
lysophosphatidylcholine
- MxP:
-
2-methoxypropene
- NMR:
-
nuclear magnetic resonance
- PC:
-
phosphatidylcholine
- PCOOH:
-
phosphatidylcholine hydroperoxide
- PPTS:
-
pyridinium p-toluenesulfonate
- SRM:
-
selected reaction monitoring.
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Acknowledgments
This work was supported by Grant-in-Aid for Scientific Research (B) Grant Number 15H04497.
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Ito, J., Nakagawa, K., Kato, S. et al. A novel chiral stationary phase HPLC-MS/MS method to discriminate between enzymatic oxidation and auto-oxidation of phosphatidylcholine. Anal Bioanal Chem 408, 7785–7793 (2016). https://doi.org/10.1007/s00216-016-9882-4
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DOI: https://doi.org/10.1007/s00216-016-9882-4