Measurement Artifacts in the Dithiothreitol (DTT) Oxidative Potential Assay Caused by Precipitation of Aqueous Transition Metals
JAYASHREE YALAMANCHILI, Christopher Hennigan, Brian Reed,
University of Maryland, Baltimore County Abstract Number: 13
Working Group: Health-Related Aerosols
AbstractTransition metals in particulate matter (PM) are hypothesized to have enhanced toxicity based on their ability to catalyze reactive oxygen species (ROS) generation. Acellular assays are widely used to measure the oxidative potential (OP) of various species – including transition metals – in PM. Many OP assays, including the dithiothreitol (DTT) and ascorbic acid (AA) assays, use a phosphate buffer matrix to simulate human body conditions (pH 7.4 and 37 °C). In this work, we conducted experiments to characterize metal precipitation in the DTT assay using aqueous extracts of a well-studied PM standard (NIST SRM-1648a, urban particulate matter). Precipitation was detected using laser particle light scattering as well as microscopic analysis (SEM and TEM) of filtered solids coupled with chemical analysis of dissolved metals to quantify metal removal. Our combination of measurements confirmed rapid metal precipitation in the DTT assay. Metal phase transitions from the aqueous to solid form for Fe, Mn, Al, Ca, Pb, Mg, Zn, Cu, and V were detected and likely occur through a variety of mechanisms, including co-precipitation and adsorption. We observed an increase in particle formation with increased metal concentrations. The effect of the metal phase (solid vs. aqueous) on the OP measured by the DTT assay was characterized using the urban PM: there was a significant difference in the OP measured due to the metal phase. Finally, the effects of the phosphate buffer concentration on metal precipitation and the measured OP in the DTT assay were investigated. The buffer concentration was directly proportional to the measured OP for Fe and Mn, but inversely proportional to Cu, indicating that the DTT assay is likely sensitive to the type and amount of solids in solution. The implications of these results for other acellular OP assays and PM toxicity are discussed.