ADVANCED GLYCATION END-PRODUCTS OF THE MAILLARD REACTION IN AORTIC PEPSIN-INSOLUBLE AND PEPSIN-SOLUBLE COLLAGEN FROM DIABETIC RATS

Recent immunohistological studies using antibodies against advanced gl ycation end products (AGEs) have demonstrated the presence of AGEs in several tissues. By an enzyme-linked immunosorbent assay using the mon oclonal anti-AGE antibody, the present study aimed to determine AGEs i n pepsin-insoluble collagen (PIG) as web as in pepsin-soluble collagen (PSC) from the aortas of streptozotocin (STZ)-induced diabetic rats ( at 4, 16, and 28 weeks after STZ injection) and those of age-matched c ontrol rats. Addition of EDTA to the immunoassay buffer has led us to successful determination of AGEs in the aortic PIC samples with follow ing results: 1) in diabetic rats, there was a time-related increase in the AGE contents at 28 weeks (n = 9, 226.4 +/- 13.5 ng/mg collagen [m ean +/- SE]), compared with that at 4 and 16 weeks (n = 6, 79.6 +/- 9. 5 ng/mg collagen, and n = 8, 149.4 +/- 30.9 ng/mg collagen at 4 and 16 weeks, respectively; both P < 0.05, between 4 and 16 weeks and 28 wee ks); 2) after 28 weeks of diabetes, the AGE contents in PIC of aortas were significantly higher in diabetic rats than in controls (n = 9, 22 6.4 +/- 13.5 ng/mg collagen vs. n = 8, 129.6 +/- 14.9 ng/mg collagen, P < 0.01, diabetic vs. control); and 3) the level of the AGE content w as strongly correlated with the PIC/total collagen (TC) ratio (n = 45, r = 0.698, P = 0.0001). By treating the samples of PSC with alkaline solution, the AGE content of PSC was also determined. In the PSC fract ion, the AGE levels in the diabetic rats tended to increase with time and to be higher than those of control rats at 28 weeks although these changes were not statistically significant (diabetic: n = 4, 19.4 +/- 9.7; n = 6, 22.3 +/- 6.2; n = 6, 39.6 +/- 10.8; control: n = 4, 19.7 +/- 9.8; n = 6, 22.9 +/- 7.3; n = 7, 30.7 +/- 7.2; at 4, 16, and 28 we eks, respectively). Compared with the AGE levels of PSC, those of PIC were about four to seven times and four to five times higher in diabet ic and control rats, respectively (PIG versus PSC in diabetic or contr ol rats, all P < 0.001, at 4, 16, and 28 weeks, respectively). These f indings provide the first immuno-chemical evidence that AGE adducts ar e present in the materials extracted sequentially by pepsin and collag enase and that these adducts in PIC accumulated as a function of the i ncrease in the aortic PIC/TC ratio.