Methemoglobin production by nitric oxide in fresh sheep blood

As nitric oxide (NO) inhalation is used therapeutically, we studied the production of methemoglobin (metHb) by NO in fresh adult sheep blood. NO solutions were prepared by bubbling a 10% NO-90% N₂ gas mixture in phosphate-buffered saline (pH = 7.41 at 20°C) for at least 60 min. Fresh blood samples were obtained from catheterized femoral arteries or veins just prior to mixing with NO solution. Measurements of metHb were made at times 0, 30 sec, 2 min and 5 min after mixing of NO-containing buffer and blood using a Radiometer OSM-3 hemoximeter. Mixing was performed using two syringes connected via a stopcock. The reaction of NO with blood occurred rapidly after mixing since data values for each of the time points after 30 sec were unchanged for all mixtures. The mixing volume ratio of NO-containing buffer to blood was either 1:1 (protocol A) for comparisons of arterial vs venous blood, or the ratios were randomized (protocol B) to investigate effects of Hb oxygenation. Protocol A elicited only slight increases of metHb in arterial and venous blood which did not differ significantly. In protocol B, an increase of metHb was associated with a relative decline of deoxyhemoglobin (deoxyHb). A higher molar ratio of NO/deoxyHb yielded greater amounts of metHb. Therefore, in fresh sheep blood, deoxyHb is converted to metHb in the presence of NO. This reaction is not affected by the presence of oxyhemoglobin.