Dithiothreitol (DTT) concentration effect and its implications on the applicability of DTT assay to evaluate the oxidative potential of atmospheric aerosol samples

Abstract

The cell-free dithiothreitol (DTT) assay is widely used and the DTT consumption rate is interpreted to assess the oxidative potential (OP). Most researchers use an experimental procedure developed by Cho et al. (2005) while some adopt a procedure by Li et al. (2009). The key difference between the two procedures is the initial DTT concentration, 100 μM used in the former and 20 μM in the latter, raising an unaddressed issue of comparability. We examine in this work this issue using metal-free humic-like substance (HULIS) samples isolated from ambient aerosol and two metals (i.e. copper and manganese). We found that higher initial DTT concentrations led to higher DTT consumption rates for both HULIS and metals. For HULIS, the increase in DTT consumption rate was proportional to the initial DTT concentration (i.e., roughly by 5-fold), allowing correction of the concentration effect and direct comparison of results from the two protocols. However, the proportionality did not hold for the metals or metal-organic mixtures. The increase was much lower than the proportionality of 5 and metal concentration-dependent, specifically, 1.2–1.3 for Cu and from negligible to 2.0 for Mn. For six water extracts of ambient aerosol samples, in which HULIS and metals co-exist, the proportionality ranged from 1.3 to 2.2. This deviation from a linear dependence on initial DTT concentration, plausibly due to metal-DTT binding, impedes assessing and comparing OP of metals and metal-organic mixtures using different implementations of the DTT assay. Considering the different antioxidants concentrations in real human lung fluid, this work raises caution about using the DTT assay to assess metal-containing mixtures, such as ambient aerosol samples.