Melanin quantitation from human erythrocytes; interference by haeme derivatives

Precipitates were obtained by 6 N HCl hydrolysis of human erythrocytes and subsequent extractions with ethanol-ether 1:1 and with tetrahydrofuran. The mean quantity of these precipitates (n = 16) was 16.60 +/- 1.60 (standard deviation) mg/ml, (15.09 +/- 1.45 mg/g) and from saline washed erythrocyte samples (n = 8) 16.65 +/- 0.73 mg/ml, (15.14 +/- 0.67 mg/g). A large part of these precipitates (about 74%) is associated with haemoglobin (in average 12.34 mg/ml). Melanins account for the difference (16.60-12.34) = 4.26 mg/ml, approximately 8.7% of haemoglobin-free erythrocyte solids. Precipitates from red cells, and from haemoglobin produced similar UV-VIS and IR spectra. The precipitates from haemoglobin are mainly derivatives of haeme (about 97%); the remaining approximately 3% are melanins from globin. The total melanins are about 1.3% of haeme-free solids of erythrocytes. Precipitation from the erythrocytes with 6 N HCl was also achieved in practically complete argon atmosphere, and similar quantities were obtained as those in air with the same UV-VIS and IR spectra. Since granules or solid particles are not found in the cytoplasm of normal human erythrocytes, we conclude that soluble melanins are present. Small amounts of melanins can be present in the membranes as well, since the precursors of melanins: norepinephrine, epinephrine are present in these membranes.