Use of leuco-dyes in the quantitative colorimetric microdetermination of hemoglobin and other heme compounds

Sensitivity, stability, and specificity of the color-producing reaction of hydrogen peroxide with bezidine, leuco-malachite green, or o-dianisidine were tested in numerous systems containing hemoglobin and other hemoproteins. Use of urea or low temperature (to minus 12 degrees C), or both, was highly beneficial, especially with leuco-malachite green, for which the color reaction was stable, after about 15 min, for longer than 24 h, with a colorless bank. Absorbance was 0.3 at a final hemoglobin concentration of 0.27 mg/liter. Nonspecific color produced by substances such as FeCl-3 and ascorbic acid was completely eliminated. Of the three leuco-dyes studied, only benzidine yielded a completely linear calibration curve, but its relative instability and reported carcinogenicity are serious disadvantages. No system tested eliminated completely the known inhibition by plasma of the peroxidase activity of these leuco dyes.