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

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

Institute for Infectious Diseases, University of Berne, Berne 3010

Corresponding Author: 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 $gamma$ -tocopherol, the major form of vitamin E in the North American diet, may play an important role in these diseases. $gamma$ -Tocopherol scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro- $gamma$ -tocopherol (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, and $alpha$ - and $gamma$ -tocopherol 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 $gamma$ -tocopherol. 24 hours after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to $gamma$ -tocopherol or corrected for total neutral lipids, while $alpha$ - and $gamma$ -tocopherol levels remained unchanged. These results demonstrate that nitration of $gamma$ -tocopherol 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 $gamma$ -tocopherol.

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