Non-enzymatic glycosylation of plasma lipoproteins in vitro

Human very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoproteins (HDL2 and HDL3) were glycosylated in vitro by incubation with high concentrations of glucose and glucose-6-phosphate. Glycosylated lipoproteins showed enhanced mobility on agarose electrophoresis when compared to control lipoproteins, samples treated with glucose-6-phosphate being more strongly affected. Extent of glycosylation of LDL was determined using thiobarbituric acid, LDL incubated with glucose exhibiting degrees of glycosylation 15-117% in excess of control. LDL incubated with glucose-6-phosphate however, gave values similar to those obtained with control incubations. Amino acid analysis of protein hydrolysates prepared from VLDL and LDL fractions incubated with glucose revealed reductions of up to 53% in the levels of free lysine when compared to control lipoprotein samples. The appearance of 2 novel peaks, probably corresponding to glucosyllysine, was also observed. The effects seen with glucose-6-phosphate however, were not as marked as expected. None of the other amino acids measured were decreased. These data show that plasma lipoproteins can be glycosylated in vitro and that an indication of the degree of glycosylation (by glucose) may be obtainable using thiobarbituric acid. Amino acid analysis revealed that the probable binding site for glucose on the apoproteins are the lysine residues. Although glucose-6-phosphate also binds to lysine residues the effects produced by this agent on plasma lipoproteins may also involve other mechanisms requiring further investigation.