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 -.

Human and rodent red blood cells do not demonstrate xanthine oxidase activity or XO-catalyzed nitrite reduction to NO.

A number of molybdopterin enzymes, including xanthine oxidoreductase (XOR), aldehyde oxidase (AO), sulfite oxidase (SO), and mitochondrial amidoxime reducing component (mARC), have been identified as nitrate and nitrite reductases. Of these enzymes, XOR has been the most extensively studied and reported to be a substantive source of nitric oxide (NO) under inflammatory/hypoxic conditions that limit the catalytic activity of the canonical NOS pathway. It has also been postulated that XOR nitrite reductase activity extends to red blood cell (RBCs) membranes where it has been immunohistochemically identified. These findings, when combined with countervailing reports of XOR activity in RBCs, incentivized our current study to critically evaluate XOR protein abundance/enzymatic activity in/on RBCs from human, mouse, and rat sources. Using various protein concentrations of RBC homogenates for both human and rodent samples, neither XOR protein nor enzymatic activity (xanthine → uric acid) was detectable. In addition, potential loading of RBC-associated glycosaminoglycans (GAGs) by exposing RBC preparations to purified XO before washing did not solicit detectable enzymatic activity (xanthine → uric acid) or alter NO generation profiles. To ensure these observations extended to absence of XOR-mediated contributions to overall RBC-associated nitrite reduction, we examined the nitrite reductase activity of washed and lysed RBC preparations via enhanced chemiluminescence in the presence or absence of the XOR-specific inhibitor febuxostat (Uloric®). Neither addition of inhibitor nor the presence of the XOR substrate xanthine significantly altered the rates of nitrite reduction to NO by RBC preparations from either human or rodent sources confirming the absence of XO enzymatic activity. Furthermore, examination of the influence of the age (young cells vs. old cells) of human RBCs on XO activity also failed to demonstrate detectable XO protein. Combined, these data suggest: 1) that XO does not contribute to nitrite reduction in/on human and rodent erythrocytes, 2) care should be taken to validate immuno-detectable XO by demonstrating enzymatic activity, and 3) XO does not associate with human erythrocytic glycosaminoglycans or participate in nonspecific binding.

Xanthine Oxidase Drives Hemolysis and Vascular Malfunction in Sickle Cell Disease.

OBJECTIVE: Chronic hemolysis is a hallmark of sickle cell disease (SCD) and a driver of vasculopathy; however, the mechanisms contributing to hemolysis remain incompletely understood. Although XO (xanthine oxidase) activity has been shown to be elevated in SCD, its role remains unknown. XO binds endothelium and generates oxidants as a byproduct of hypoxanthine and xanthine catabolism. We hypothesized that XO inhibition decreases oxidant production leading to less hemolysis. Approach and Results: Wild-type mice were bone marrow transplanted with control (AA) or sickle (SS) Townes bone marrow. After 12 weeks, mice were treated with 10 mg/kg per day of febuxostat (Uloric), Food and Drug Administration-approved XO inhibitor, for 10 weeks. Hematologic analysis demonstrated increased hematocrit, cellular hemoglobin, and red blood cells, with no change in reticulocyte percentage. Significant decreases in cell-free hemoglobin and increases in haptoglobin suggest XO inhibition decreased hemolysis. Myographic studies demonstrated improved pulmonary vascular dilation and blunted constriction, indicating improved pulmonary vasoreactivity, whereas pulmonary pressure and cardiac function were unaffected. The role of hepatic XO in SCD was evaluated by bone marrow transplanting hepatocyte-specific XO knockout mice with SS Townes bone marrow. However, hepatocyte-specific XO knockout, which results in >50% diminution in circulating XO, did not affect hemolysis levels or vascular function, suggesting hepatocyte-derived elevation of circulating XO is not the driver of hemolysis in SCD. CONCLUSIONS: Ten weeks of febuxostat treatment significantly decreased hemolysis and improved pulmonary vasoreactivity in a mouse model of SCD. Although hepatic XO accounts for >50% of circulating XO, it is not the source of XO driving hemolysis in SCD.