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5-Lipoxygenase (5-LO) converts arachidonic acid to 5-hydroperoxy-trans-6,8,11,14-eicosatetraenoic acid (5-HpETE). The modulation of 5-LO activity would be beneficial in the treatment of inflammatory conditions. Trimellitic anhydride (TMAA) is a cyclooxygenase (COX)-2 inhibitor that is much more selective than the popular COX-2 inhibitor, aspirin. In this paper, we have used this cyclization reaction and high-resolution liquid chromatography with tandem mass spectrometry to explore the utility of TMAA as a tool for identifying inhibitors of 5-LO. After incubating human neutrophils with 50 microM arachidonic acid and 100 microM TMAA, levels of 5-HpETE in the presence and absence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) were determined. TMAA was found to inhibit fMLP-stimulated 5-LO activity in a dose- and time-dependent manner. Studies of thromboxane B2 (TXB2) production in the presence of arachidonic acid showed that TMAA at 100 microM is a weak inhibitor of 5-LO (82% inhibition) when compared to a more potent inhibitor, nordihydroguaiaretic acid (NDGA, 61% inhibition). Stable isotope labeling studies with trideuterated arachidonic acid confirmed that TMAA inhibited 5-LO activity and that 5-LO was the primary target of this trimellitic anhydride inhibitor of 5-LO. [

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