Effect of sodium azide on hydrogen peroxide production by zymosan-activated human neutrophils

Stimulated neutrophils (PMNs) produce large quantities of superoxide anion, which is the precursor for hydrogen peroxide (H₂O₂). We developed a new fluorimetric assay to measure the H₂O₂ released by zymosan A-activated PMNs utilizing the oxidation ofp-hydroxyphenylacetic acid by H₂O₂ to its fluorescent dimer in the presence of horseradish peroxidase. Zymosan-activated PMNs isolated from nine healthy volunteers and 20 patients with acute hypoxemic respiratory failure (AHRF) released after 90 min 2.3±0.3 and 2.4±1.3 nmol H₂O₂/10⁶ PMNs, respectively. Inhibition of the heme enzymes by 1.0 mM sodium azide (NaN₃) increased the H₂O₂ production to 21.6±4.4 nmol H₂O₂/10⁶ PMNs in the control group (P<0.001), and to 22.5±14.7 nmol H₂O₂/10⁶ PMNs in patients with AHRF (P<0.001). Incubation temperature, room temperature or 37‡C, did not change the total amount of H₂O₂ produced after 90 min by zymosan-activated PMN. Addition of NaN₃ improved both the sensitivity and reproducibility of the measurement of H₂O₂ and allowed detection of H₂O₂ released by PMNs with coefficients of variation of less than 5% at PMN concentrations as low as 0.1×10⁶ cells/ml. The amount of H₂O₂ released by activated PMNs did not distinguish healthy controls from patients with AHRF.