Abstract
Nonezymatic glucosylation of plasma protein was determined with tiobarbituric acid (Fluckiger and Winterhalter). Plasma glucose should be removed out for avoiding cross-reaction with the thiobarbituric acid. Glucose-free plasma was hydrolyzed after 4.5 hour-boiling and deproteinzed with 40% trichloracetic acid. Supernatant was incubated with 0.05 M thiobarbituric acid at 40° for 40 min. The reaction product was absorbed at 443nm with a spectrophotometer. Hydroxymethylfurfural (HMF) was used as a standard and glucosylated plasma protein was expressed as nmol HMF/mg protein. Plasma protein was measured with the method of protein dye binding (Bradford). Glucosylated hemoglobin (HbA1) was determined with agar gel electrophoresis (Lionel).
In 83 diabetic patients newly diagnosed glucosylated plasma protein was 1.58±0.5nmol HMF/mg protein which was significantly higher than that in healthy subjects or impaired glucose tolerance (IGT). Hb A1 in diabetic patients was 10.86±2.4%. There is significant correlation between Hb A1 and fasting blood glucose or Hb A1 and 2 hour-blood glucose during 75 g GTT.
In 96 diabetic patients having been treated with sulphonylurea or insulin the average value of blood glucose measured frequently at the past three months was significantly correlated with Hb A1 (r=0.77, P<0.001).
Glucosylated plasma protein also was significantly correlated with the average value of blood glucose measured at the past three months in the diabetic patients (r=0.45, P<0.05). Because a turnover of plasma protein is shorter than the life span of blood red cells, the past three monthaverage value of blood glucose is correlated more tightly with Hb A1 than glucosylated plasma protein.
Blood red cells of healthy subjects were washed by physiological saline, and glucose solution was added to them at each concentration of glucose, 100, 300 and 500mg/100ml. The mixture was incubated at 37° and glucosylation of hemoglobin began 24 hours after the incubation with glucose as well as glucosylation of protein.
In 13 diabetic patients blood glucose, Hb A1 and glucosylated plasma protein were determined every week for 4 weeks after the beginning of treatment. At 4 weeks the blood glucose decreased by 48% but Hb A1 and glucosylated plasma protein slightly decreased by 18% and 29%, respectively. Improvement of Hb A1 is very slow as compared with blood glucose.
The measurement of Hb A1 is very useful for evaluation of diabetic control. However, in anemia and abnormal hemoglobinemia such as thalassemia, Hb A1 value is erroneous. On the other hand glucosylated plasma protein reflects on the condition of diabetes at the past 1 or 2 weeks. When the glucosylated plasma protein is measured with thiobarbituric acid, because TBA reacts to glucose, it is necessary to exclude glucose from plasma with dialysis.
Based on our findings we suggest that Hb A1 or glucosylated plasma protein should be measured monthly at least. Glucosylated plasma protein may be an adequate index of diabetic control as well as Hb A1. Any increase in glucosylated plasma protein may indicate impairment of diabetic control. Further study is necessary to prove the worth of detecting early diabetic microangiopathy by measurement of glucosylated protein.
