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Journal of Biochemistry Advance Access originally published online on July 23, 2007
Journal of Biochemistry 2007 142(1):73-78; doi:10.1093/jb/mvm114
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© 2007 The Japanese Biochemical Society.

Identification of Radicals Formed in the Reaction Mixtures of Rat Liver Microsomes with ADP, Fe3+ and NADPH Using HPLC–EPR and HPLC–EPR–MS

Katsuyuki Minakata1, Etsuo Okuno2, Masayuki Nakamura1 and Hideo Iwahashi1,*

1Department of Chemistry, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan; and 2Department of Clinical Nutrition, Kyusyu Nutrition Welfare University, 5-1-1 Shimoitoudu Kokurakita-ku Kitakyusyu, Fukuoka 803-8511, Japan

*To whom correspondence should be addressed. Tel/Fax: 073 441 0772, 073 441 0772, Email: chem1{at}wakayama-med.ac.jp

Received March 15, 2007; Accepted April 24, 2007


  Abstract

The reaction of rat liver microsomes with Fe3+, ADP and NADPH was examined using EPR, HPLC–EPR and HPLC–EPR–MS combined use of spin trapping technique. A prominent EPR spectrum ({alpha}N = 1.58 mT and {alpha}Hß = 0.26 mT) was observed in the complete reaction mixture. The EPR spectrum was hardly observed for the complete reaction mixture without rat liver microsomes. The radicals appear to be derived from microsomal components. The EPR spectrum was also hardly observed in the absence of Fe3+. Addition of some iron chelators such as EDTA, citrate and ADP resulted in the dramatic change in the EPR intensity. Iron ions seem to be essential for this reaction. For the complete reaction mixture with boiled microsomes, a weak EPR spectrum was observed, suggesting that enzymes participate in the reaction. Five peaks were separated on the HPLC–EPR elution profile of the complete reaction mixture of rat liver microsomes with ADP, Fe3+ and NADPH. The retention times of the peaks 1 to 5 were 19.4, 22.5, 27.3, 29.8 and 31.4 min, respectively. To identify the radical adducts, HPLC–EPR–MS analyses were performed for the three prominent peaks. The HPLC–EPR–MS analyses showed that a new radical adduct, 4-POBN/1-hydroxypentyl radical, in addition to 4-POBN/ethyl radical adducts, forms in a reaction mixture of rat liver microsomes with ADP, Fe3+ and NADPH.

Key Words: ethyl radical, iron ion, lipid peroxidation, 1-hydroxypentyl radical, P450, spin trapping

Abbreviations: 4-POBN, {alpha}-(4-Pyridyl-1-oxide)-N-tert-butylnitrone


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