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Journal of Lipid Research, Vol. 50, 2064-2071, October 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Determination of leukotriene A₄ stabilization by S100A8/A9 proteins using mass spectrometry

Christopher L. Rector and Robert C. Murphy¹

Department of Pharmacology, Mail Stop 8303, University of Colorado Denver, Aurora, CO 80045

¹ To whom correspondence should be addressed. e-mail: Robert.Murphy{at}ucdenver.edu

Leukotriene A₄ (LTA₄) is the precursor for the formation of bioactive leukotrienes, but is highly susceptible to nonenzymatic hydrolysis. Although it is chemically reactive, LTA₄ participates in the process of transcellular metabolism, which requires the transfer of LTA₄ from one cell to another for the production of additional leukotrienes. Due to the susceptibility of LTA₄ to hydrolysis, various methods have been used to measure the half-life of LTA₄ in the presence of different proteins in efforts to understand how it is transported between cells. In this work, a new liquid chromatography mass spectrometry technique was developed to improve upon these previous assays that analyzed LTA₄ directly. The new technique derivatizes LTA₄ to stable compounds for analysis and removes the potential for sample decomposition between analytical runs. This assay was used in measuring the capabilities of the S100A8/A9 protein complex isolated from human neutrophils to stabilize LTA₄. It was determined that the S100A8/A9 protein complex protects LTA₄ from hydrolysis in a Ca²⁺ dependent manner and increases LTA₄ half-life to in excess of 35 and 5 min at 4°C and 37°C, respectively.

Supplementary key words transcellular metabolism • leukotriene biosynthesis • S100 protein

Abbreviations: CID, collision induced dissociation; diHETE, dihydroxyeicosatetraenoic acid; 5,6-(EtO,OH)-ETE, 5,6-ethoxy-hydroxyeicosatetraenoic acid; FPLC, fast-protein liquid chromatography; 5-HPETE, 5S-hydroperoxy-eicosa-6E,8Z,11Z,14Z-tetraenoic acid; 5-LO, 5-lipoxygenase; LTA₄, 5S-trans-5,6-oxido-eicosa-7E,9E,11Z,14Z-tetraenoic acid, leukotriene A₄; LTB₄, 5S, 12R-dihydroxy-eicosa-6Z,8E,10E,14Z-tetraenoic acid, leukotriene B₄; LTC₄, 5S-hydroxy, 6R-(S-glutathionyl)-eicosa-7E,9E,11Z,14Z-tetraenoic acid, leukotriene C₄; 5-OH, 12-EtO-LTB₄, 5-hydroxy, 12-ethoxy-⁶-trans-LTB₄; 5-oxoETE, 5-oxo-eicosa-6E,8Z,11Z,14Z-tetraenoic acid; PMN, polymorphonuclear leukocyte; RP-HPLC, reversed phase high performance liquid chromatography; RP/LC/MS, reversed phase liquid chromatography mass spectrometry; SPE, solid phase extraction

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