Determination and Clinical Significance of Glycosylated Hemoglobins

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A new chromatographic method for the determination of glycosylated hemoglobin was developed in our laboratory. In this method, IEX-530CM (Tôyôsôda Co.) was used instead of Bio Rex 70 cation exchange resin (Bio Rad Inc.) and gradient elution was performed with 30mM sodium phosphate, pH6.50 buffer and 30mM sodium phosphate containing 0.2M NaCl, pH6.50 buffer. Blood samples were obtained by venipuncture and hemolysates were prepared by the method of Goldstein13). Hemoglobins of the blood sample were divided into about sixteen fractions within twentyeight minutes by our new method. Characteristic of each hemoglobin fraction was examined before and after the pretreatment of normal and diabetic erythrocytes in vivo and in vitro. Four major labile glycosylated hemoglobins were found in blood red cell, namely, L-GHb4, L-GHb7, L-GHb9 and L-GHb12. And stable glycosylated hemoglobin (St-GHb8) was separated from the labile glycosylated hemoglobin (L-GHb7), which were commented as two chromatographically indistinguishable forms by Goldstein et al. The Hb6 fraction was identified as HbF by its properties against alkaline conditions and by its chromatographical kinetics. More than one percent of HbF occured in the samples from normal and diabetic subjects. The Hb10 fraction was found to increase fairly high after the incubation of erythrocytes with saline at 37° for 6hrs. Comparing the fraction of hemoglobin divided by McDonald et al.21) with our fractions, A1a1 corresponded to Hb1, A1a2 to Hb2, A1b to Hb3, L-GH4 and Hb5, A1c to HbF, L-GHb7, St-GHb8, L-GHb9, Hb10 and Hb11, A0 to Hb12-Hb16, respectively. Fluctuations in A1c and A1 were due to variable amounts of components, which we know here were L-GHb4, HbF, L-GHb7 and L-GHb9. Recently, removal of labile fractions by dialysis of hemolysate or by saline inculation of red cells was recommended. But with these procedure Hb10 increases fairly high, as mentioned above, and is not separated from A1 or A1c by the conventional methods. For the correct interpretation of glycosylated hemoglobins, it is necessary to mind these factors which flucturate and complicate the observed values or to measure the stable glycosylated hemoglobin (St-GHb8) directly. Otherwise the obtained results will not be accurate indicators of longterm blood glucose control.
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Determination and Clinical Significance of Glycosylated Hemoglobins

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