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Diphenylamine: an unusual antioxidant
T Sugihara1, G Rao, R P Hebbel
1Department of Medicine, University of Minnesota Medical School, Minneapolis 55455.
Free Radical Biology & Medicine
|April 1, 1993
Summary
Diphenylamine (DPA) surprisingly promotes lipid peroxidation and red blood cell (RBC) potassium leak, despite inhibiting some markers. This highlights the unreliability of thiobarbituric acid reactive substances (TBARS) assays for assessing peroxidation.
Area of Science:
- Biochemistry
- Cell Biology
- Oxidative Stress Research
Background:
- Diphenylamine (DPA) is commonly used as an antioxidant in lipid peroxidation studies.
- Red blood cell (RBC) membranes are susceptible to peroxidative damage.
- Thiobarbituric acid reactive substances (TBARS) are a common, but potentially misleading, indicator of lipid peroxidation.
Purpose of the Study:
- To investigate the actual effect of Diphenylamine (DPA) on lipid peroxidation and associated red blood cell (RBC) membrane integrity.
- To evaluate the reliability of TBARS assays in the presence of DPA.
- To explore the mechanism behind DPA's influence on peroxidation.
Main Methods:
- Utilized peroxidizing RBC membranes to assess DPA's impact.
- Measured lipid hydroperoxide (LOOH) formation, oxygen consumption, and TBARS generation.
- Quantified potassium (K) ion leakage from RBCs.
- Examined DPA's effect on cyclooxygenase-dependent arachidonate metabolites.
- Assessed DPA's ability to inhibit peroxyl radical-induced phycoerythrin fluorescence destruction.
- Studied DPA analogues to identify the functional group responsible for its effects.
Main Results:
- DPA promoted lipid hydroperoxide (LOOH) formation and oxygen consumption.
- DPA inhibited TBARS generation, creating a discrepancy in peroxidation assessment.
- DPA increased RBC K leak, exacerbating peroxidative stress effects.
- DPA abolished cyclooxygenase-dependent arachidonate conversion products.
- DPA demonstrated significant protection against peroxyl radical damage to phycoerythrin fluorescence, comparable to BHT.
- The secondary amine function of DPA was identified as crucial for its observed effects, suggesting a pro-oxidant mechanism via radical formation and redox cycling.
Conclusions:
- Diphenylamine (DPA) acts as a pro-oxidant in RBC membranes, promoting lipid peroxidation and potassium leak, contrary to its assumed antioxidant role.
- Relying solely on TBARS measurements can be misleading when assessing peroxidation in the presence of compounds like DPA.
- The secondary amine group in DPA is responsible for its pro-oxidant activity, likely through redox cycling and radical generation.