A Simplified Colorimetric Assay of Nonenzymatic Glycosylation of Human low Density Lipoproteins in Normal and Diabetes Mellitus

ABSTRACT

A simplified colorimetric method for measurement of the levels of glycosylation of proteins was developed by a modification of an existing method. Employing this method, the extent of nonenzymatic glycosylation of apolipoprotein B subspecies(B-100, B-74, B-26), LDL, VLDL and total serum proteins in human plasma obtained from patients with diabetes mellitus and control subjects was compared. Plasma LDL (1.019 < d < 1.063) and VLDL(d < 1.006) were separated using the sequential ultracentrifugation method, and the subspecies of apolipoprotein B were isolated by extracting them from polyacrylamide gels after they were separated by preparative SDS-polyacrylamide gel electrophoresis. Increases in the level of glycosylation of serum proteins, LDL, VLDL, and apo B subspecies obtained from diabetic patients were observed. Among them, the increases of glycosylated LDL and apo B-26 were most significant (p < .001). Also, good correlations were found between glycosylations of apo B-26 and LDL (r=.88), and glycosylation of LDL and LDL cholesterol level(r=.79). The results also showed an excellent correlation between levels of HbA1c and glycosylated apo B-26(r=.93).