Measurement of methemoglobin formation from oxyhemoglobin a real-time, continuous assay of nitric oxide release by human polymorphonuclear leukocytes

Abstract

We have evaluated the spectrophotometric measurement (at 401 vs. 411 nm) of nitric oxide (NO)-dependent methemoglobin formation from oxygemoglobin in order to assess NO release from human polymorphonuclear neutrophil leukocytes (PMN). S-nitroso- d,l-acetyl-penicillamine (SNAP, 25–200 μM), a donor of NO, induced a dose-dependent methemoglobin formation. Furthermore, when PMN were activated with N-formyl-methionylleucyl-phenylalanine or phorbol myristate acetate in the presence of superoxide dismutase (SOD) and catalase, methemoglobin formation ensued. The amount of methemoglobin formed was dependent on the amounts of oxyhemoglobin and stimulus used, and the number of PMN in the assay. The NO synthase (NOS) inhibitors N G -monomethyl- l-arginine or nitro- l-arginine methyl ester did not affect methemoglobin generation from oxyhemoglobin induced by SNAP but inhibited that mediated by activated PMN with IC 50 values of 250 μM and 340 μM, respectively. The substrate for NO formation from NOS, l-arginine in concentrations up to 1 mM did not significantly influence the methemoglobin formation either induced bu SNAP or activated PMN. Exclusion of SOD did not affect SNAP-dependent oxidation of oxyhemoglobin. Exclusion of SOD from the cell-containing system attenuated methemoglobin formation, and if catalase was also excluded the repsonse was further reduced. Finally, PMN from a patient with X-linked chronic granulomatous disease, unable to produce superoxide anions, showed a similar production of methemoglobin from HbO 2 as did healthy PMN, activated with the respective agonists. We conclude that spectrophotometric measurement of methemoglobin formation from oxyhemoglobin in the presence of SOD and catalase is a suitable method for the measurement of NO release from PMN, with the benefits of a real-time, continuous assay.