Induced Oxidations in Blood. Hemoglobin Destruction by Methylene Blue in Lactic Acid Peroxidation

Abstract

We have previously reported experiments 1 which showed that the observations of Barron and Harrop 2 , 3 that methylene blue added to blood increases the rate of oxygen consumption and decreases lactic acid production, may, in part, be accounted for by the oxidation of formed lactic acid. The incubation of washed dog erythrocytes in a solution containing added dl-lactate, in the presence but not in the absence of methylene blue, results in a disappearance of lactate.

Further study indicates that the lactic acid is oxidized to pyruvic acid, apparently quantitatively. That this oxidation is not mediated by the lactic “dehydrogenase” of the Wieland school is evident from the fact that molecular O₂ alone is incapable of effecting it; the dye is necessary. Experiments of Harned (unpublished) in this laboratory, indicating intermediate peroxide formation when leucomethylene blue undergoes oxidation, suggest that the mechanism by which the lactic acid is oxidized may be one of peroxidation. We find, however, that lactic acid is not affected by the hypothetical methylene blue peroxide alone, since shaking leucomethylene blue and buffered sodium lactate with O₂ fails to touch the lactate. Evidently another cell constituent is also essential.

A series of experiments with oxygenated dog erythrocytes (substantially free from sugar) in phosphate buffers containing added dl-lactate were carried out with the following determinations at intervals during 2 to 6 hours: O₂ content and capacity, CO₂ content, lactic acid and pyruvic acid. The following experiment illustrates the results obtained.

Experiment 128. Dog erythrocytes separated from serum and leucocytes by centrifugation, incubated 1.5 hours at 37°C, suspended in isotonic NaCl and phosphate buffer (pH 7.4) containing sodium dl-lactate and 0.005% methylene blue. Incubated with gentle shaking at 37°C. in closed filled tubes containing glass beads.