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Journal of Lipid Research, Vol. 43, 1978-1985, November 2002
Copyright © 2002 by Lipid Research, Inc.

Methods

Analysis of plasma tocopherols , , and 5-nitro- in rats with inflammation by HPLC coulometric detection

Stephan Christen^1,*,, Qing Jiang^*,, Mark K. Shigenaga^*, and Bruce N. Ames^*,

^* Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609
Institute for Infectious Diseases, University of Berne, CH-3010 Berne, Switzerland

¹ To whom correspondence should be addressed. e-mail: stephan.christen{at}ifik.unibe.ch

Reactive nitrogen oxide species (RNOS) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that -tocopherol (T), the major form of vitamin E in the North American diet, may play an important role in these diseases. T scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-T (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, T, and T in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing 4% of T. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to T or corrected for total neutral lipids, while - and T levels remained unchanged. These results demonstrate that nitration of T is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase.

The present HPLC method should be helpful in clarifying the precise physiological role of T.

Abbreviations: T, -tocopherol; BHT, butylated hydroxytoluene; Ch18:1, cholesteryl oleate; Ch18:2, cholesteryl linoleate; Ch20:4, cholesteryl arachidonate; FC, free cholesterol; T, -tocopherol; NGT, 5-nitro--tocopherol; TC, total cholesterol; TG, triglycerides; TNL, total neutral lipids

Supplementary key words reactive nitrogen oxide species • nitration • vitamin E

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