Ion-exchange microchromatography and thiobarbituric acid colorimetry for the measurement of canine glycated hemoglobins.

Abstract

Nonenzymatic glycation of hemoglobin is a slow, continuous, and irreversible process which takes place during the whole lifespan of the erythrocyte. When hemolytic diseases are ruled out, the levels of glycated hemoglobins reflect the time-averaged serum glucose concentration for the preceding weeks. Canine hemoglobin also binds physiologically to intraerythrocytic glucose to form a glycated fraction which provides information on the animal's long-term glycemic status. This study describes an overall evaluation of ion-exchange microchromatography and thiobarbituric acid (TBA) colorimetry for the measurement of canine glycated hemoglobins. The intra- and inter-assay coefficients of variation (CVs) found were less than 5% in normal and diabetic canine samples, and both assays proved linear over the analytical range tested, which was wide enough to include the expected clinical values. Under our laboratory's conditions, the reference range for HbA(1) was 5.82 +/- 0.62% and for HbA(1)c was 2.35 -/+ 0.47%. Sample stability was lower using the ion-exchange procedure, with increases in HbA(1) observed after 4 days in whole blood and hemolysates stored at room temperature, after 12 days in whole blood stored at 4 degrees C, and after 7 days in hemolysates stored at 4 degrees C and -20 degrees C. In the case of TBA colorimetry, whole blood was stable for at least 21 days at room temperature and at 4 degrees C, and hemolysates were stable for 18 days at room temperature, at least 21 days at 4 degrees C, and up to 3 months at -20 degrees C.