Measurement artifacts in the dithiothreitol (DTT) oxidative potential assay caused by interactions between aqueous...

Area of Science:

• Environmental Science
• Toxicology
• Analytical Chemistry

Background:

• Metals in particulate matter (PM) are suspected to increase toxicity by catalyzing reactive oxygen species (ROS).
• Acellular assays, like the dithiothreitol (DTT) assay, measure the oxidative potential (OP) of PM, often using phosphate buffers.

Purpose of the Study:

• To investigate how metal precipitation affects the oxidative potential (OP) measurements in the DTT assay.
• To determine the influence of varying phosphate concentrations on metal precipitation and subsequent OP readings.

Main Methods:

• Characterized metal precipitation in DTT assays using ambient PM from Baltimore, MD, and NIST SRM-1648a.
• Analyzed the impact of aqueous metal concentrations, ionic strength, and phosphate concentrations on precipitation.
• Assessed OP responses of the DTT assay under different phosphate buffer concentrations.

Main Results:

• Metal precipitation was observed to be dependent on aqueous metal concentrations, ionic strength, and phosphate levels.
• Differences in metal precipitation led to varied OP responses in the DTT assay across different phosphate concentrations.
• These effects were consistent across all analyzed PM samples.

Conclusions:

• Direct comparison of DTT assay results obtained using different phosphate buffer concentrations is unreliable.
• Metal precipitation in phosphate buffers poses a significant challenge for accurately inferring PM toxicity.
• Findings have implications for other assays relying on phosphate buffers for pH control in toxicity assessments.