Indirect Antioxidant Effects of the Nitrite Anion: Focus on Xanthine Oxidase

One electron reduction of nitrite (NO₂⁻) has been determined to be a significant, noncanonical source of nitric oxide (NO) with molybdopterin enzymes being identified as critical to this process. Of the molybdopterin enzymes identified as NO₂⁻ reductases, xanthine oxidoreductase (XOR) is the most extensively studied. Paradoxically, XOR generates oxidants and thus can contribute to oxidative stress under inflammatory conditions when the oxidase form (XO) of XOR is abundant. However, under similar inflammatory conditions XO has been associated with NO generation, especially when NO₂⁻ levels are elevated which begs the question: if reaction of nitrite with XO consumes electrons, then does it subsequently reduce oxidant generation? To address this question, electron paramagnetic resonance (EPR) was used, under controlled O₂ tensions, to assess superoxide (O₂^·−) generation by endothelial-bound XO plus xanthine and the resultant impact of introducing NO₂⁻. Nitrite diminished XO-derived O₂^·− under hypoxia (1% O₂) whereas at 21% O₂, it had no impact. To confirm these results and discount contributions from the reaction of NO with O₂^·−, molecular O₂ consumption was assessed. The presence of NO₂⁻ decreased the rate of XO/xanthine-dependent O₂ consumption in a concentration-dependent manner with greater impact under hypoxic conditions (1% O₂) compared to 21% O₂. In a more biologic setting, NO₂⁻ also diminished XO-dependent H₂O₂ formation in murine liver homogenates supplemented with xanthine. Interestingly, nitrate (NO₃⁻) did not alter XO-dependent O₂ consumption at either 21% or 1% O₂; yet it did slightly impact nitrite-mediated effects when present at 2:1 ratio vs. NO₂⁻. When combined, these data: 1) show a significant indirect antioxidant function for NO₂⁻ by decreasing oxidant generation from XO, 2) demonstrate that both XO-derived H₂O₂ and O₂^·− production are diminished by the presence of NO₂⁻ and 3) incentivize further exploration of the difference between XO reaction with NO₂⁻ vs. NO₃⁻.