摘要
Human HbA is nonenzymatically glycated at several sites. Approximately half of glycated Hb is formed by the addition of glucose to the amino-terminal valine of the Hb βchains, this species is called HbA1c.Most studies examining the effects of various agents on Hb glycation have focused on HbA1c formation in vitro.However,approximately hal f of Hb glycation in vivo also occurs at other sites,i.e.epsilon amino groups on lysines.Our purpose,therefore,was to develop a model for testing the effects of several parameters and /or chemicals on glycohemoglobin formation which would be representative of the st.uation in vivo in terms of glycation sites.Hb was glycated by several methods:① Drying of whole blood on filter paper ② Incubation of erythrocyte hemolysates in various buffers ③Incubation of intact erythrocytes in plasma and cell culture medium. Results show that drying of Hb on filter paper caused rapid .Hb glycation which could be measured by affinity chromatography; the measured glycated Hb more than doubled in less than one week. However,measurement of HbA1c formation by immunoassay showed minimal glycation at the βchain N-terminal valine from filter paper elutes(HbA1c increased by less than 2% in one week).Similarly, incubation of hemolysates showed minimal formation of HbA1c compared to other glycation products. However,incubation of intact erythtocytes in either plasma or culture medium showed formation of HbA1c and iotal glycated Hb in proportions similar to that formed in vivo. We conclude that in vitro conditions will affect the Hb glycation site and that incubation of intact erythrocytes provides the most representative model for the study of Hb glycation.
Human HbA is nonenzymatically glycated at several sites. Approximately half of glycated Hb is formed by the addition of glucose to the amino-terminal valine of the Hb βchains, this species is called HbA1c.Most studies examining the effects of various agents on Hb glycation have focused on HbA1c formation in vitro.However,approximately hal f of Hb glycation in vivo also occurs at other sites,i.e.epsilon amino groups on lysines.Our purpose,therefore,was to develop a model for testing the effects of several parameters and /or chemicals on glycohemoglobin formation which would be representative of the st.uation in vivo in terms of glycation sites.Hb was glycated by several methods:① Drying of whole blood on filter paper ② Incubation of erythrocyte hemolysates in various buffers ③Incubation of intact erythrocytes in plasma and cell culture medium. Results show that drying of Hb on filter paper caused rapid .Hb glycation which could be measured by affinity chromatography; the measured glycated Hb more than doubled in less than one week. However,measurement of HbA1c formation by immunoassay showed minimal glycation at the βchain N-terminal valine from filter paper elutes(HbA1c increased by less than 2% in one week).Similarly, incubation of hemolysates showed minimal formation of HbA1c compared to other glycation products. However,incubation of intact erythtocytes in either plasma or culture medium showed formation of HbA1c and iotal glycated Hb in proportions similar to that formed in vivo. We conclude that in vitro conditions will affect the Hb glycation site and that incubation of intact erythrocytes provides the most representative model for the study of Hb glycation.
