A short-duration colorimetric method based on phenol-sulfuric acid reaction for the estimation of glucosylhemoglobin

Hemolysates were treated with HCl (0.18 M)-acetone solution to remove heme and the globin precipitated was washed with acetone. It was dissolved in 1 ml of 0.05 M Tris-HCl, pH 7.0, subjected to heat treatment for 10 min at 100 degrees C to remove traces of acetone, and treated with 0.05 ml of 80% phenol and 3 ml of H2SO4. The color was measured at 480 nm. Glucosylhemoglobin values in control subjects and diabetics were respectively 0.286 +/- 0.051 and 0.513 +/- 0.081 mole hexose/mole hemoglobin. The increase in diabetics was highly significant (P less than 0.001). A good correlation (r = 0.85) between fasting blood sugar values and glucosylhemoglobin level was observed. When globin solution was subjected to 4 hr hydrolysis with HCl-oxalic acid (2 and 1 mole/liter) solution prior to phenol-sulfuric acid reaction, estimated glucosylhemoglobin values increased to 0.720 +/- 0.083 in control subjects and 1.036 +/- 0.115 in diabetics. The possible reasons for this increase are discussed.