The dithiothreitol (DTT) assay is widely used to measure the oxidative potential of particulate matter. Results are typically presented in mass-normalized units (e.g., pmols DTT lost per minute per microgram PM) to allow for comparison among samples. Use of this unit assumes that the mass-normalized DTT response is constant and independent of the mass concentration of PM added to the DTT assay. However, based on previous work that identified non-linear DTT responses for copper and manganese, this basic assumption (that the mass-normalized DTT response is independent of the concentration of PM added to the assay) should not be true for samples where Cu and Mn contribute significantly to the DTT signal. To test this we measured the DTT response at multiple PM concentrations for eight ambient particulate samples collected at two locations in California. The results confirm that for samples with significant contributions from Cu and Mn, the mass-normalized DTT response can strongly depend on the concentration of PM added to the assay, varying by up to an order of magnitude for PM concentrations between 2 and 34 μg mL-1. This mass dependence confounds useful interpretation of DTT assay data in samples with significant contributions from Cu and Mn, requiring additional quality control steps to check for this bias. To minimize this problem, we discuss two methods to correct the mass-normalized DTT result and we apply those methods to our samples. We find that it is possible to correct the mass-normalized DTT result, although the correction methods have some drawbacks and add uncertainty to DTT analyses. More broadly, other DTT-active species might also have non-linear concentration-responses in the assay and cause a bias. In addition, the same problem of Cu- and Mn-mediated bias in mass-normalized DTT results might affect other measures of acellular redox activity in PM and needs to be addressed.
Keywords: Dithiothreitol; copper; manganese; oxidative potential; particulate matter; quinones.