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Journal of Lipid Research, Vol. 46, 2514-2524, November 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Methods

Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes

Kiyotaka Nakagawa^*, Jeong-Ho Oak^*, Ohki Higuchi^*, Tsuyoshi Tsuzuki^*, Shinichi Oikawa, Haruhisa Otani, Masatoshi Mune, Hua Cai^** and Teruo Miyazawa^1,*

^* Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
Department of Medicine, Nippon Medical School, Tokyo 113-8603, Japan
Department of Internal Medicine, Wakayama Medical College, Wakayama 641-8509, Japan
^** Section of Cardiology, Department of Medicine, Division of Biological Sciences and Pritzker School of Medicine, University of Chicago, Chicago, IL 60637

Published, JLR Papers in Press, September 8, 2005. DOI 10.1194/jlr.D500025-JLR200

¹ To whom correspondence should be addressed. e-mail: miyazawa{at}biochem.tohoku.ac.jp

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (Amadori-PE; deoxy-D-fructosyl phosphatidylethanolamine), because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo is still unclear. Consequently, we developed an analysis method of Amadori-PE using a quadrupole/linear ion-trap mass spectrometer, the Applied Biosystems QTRAP. In positive ion mode, collision-induced dissociation of Amadori-PE produced a well-characterized diglyceride ion ([M+H–303]⁺) permitting neutral loss scanning and multiple reaction monitoring (MRM). When lipid extract from diabetic plasma was infused directly into the QTRAP, Amadori-PE molecular species could be screened out by neutral loss scanning. Interfacing liquid chromatography with QTRAP mass spectrometry enabled the separation and determination of predominant plasma Amadori-PE species with sensitivity of 0.1 pmol/injection in MRM. The plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15–0.29 mol% in diabetic patients. Furthermore, plasma Amadori-PE levels were positively correlated with PCOOH (a maker for oxidative stress).

These results show the involvement between lipid glycation and lipid peroxidation in diabetes pathogenesis.

Abbreviations: AGE, advanced glycation end product; Amadori-PE, Amadori-glycated phosphatidylethanolamine; Hb_A1c, hemoglobin A1c; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRM, multiple reaction monitoring; PCOOH, phosphatidylcholine hydroperoxide; PE, phosphatidylethanolamine; PEOOH phosphatidylethanolamine hydroperoxide; PS, phosphatidylserine; QqLIT, hybrid quadrupole/linear ion trap

Supplementary key words glycation • QTRAP mass spectrometer • lipid peroxidation

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