Advanced glycation end products induce crosslinking of collagen in vitro.
We have investigated the effect of advanced glycation end products (AGEs) on the crosslinking of collagen. The potential pathological significance of AGEs and the altered metabolism of ascorbic acid (ASA) in diabetes have prompted us to investigate the role of ASA in the crosslinking and advanced glycation of collagen. Rat tail tendons were incubated with ASA and dehydroascorbic acid (DHA) under physiological conditions of temperature and pH, and the crosslinking and the level of AGEs were analyzed. Analysis of crosslinking was conducted by pepsin solubility and cyanogen bromide digestion. Level of AGEs was estimated by enzyme-linked immunosorbent assay (ELISA) using antibodies raised against AGE-ribonuclease. It was noted that ASA and DHA induced crosslinking of collagen and stimulated the formation of AGEs. It was also noted that these pathways were dependent on oxidative conditions. Similarly incubation of collagen with AGEs, prepared by the in vitro incubation of bovine serum albumin (BSA) with glucose, also resulted in increased crosslinking. The extent of crosslinking was dependent on the duration of incubation. The novel finding of this study, which is in contrast to the earlier reports on glucose-induced crosslinking of collagen, was that AGEs-induced crosslinking of collagen was not inhibited by radical scavengers and the metal chelator. EDTA, whereas glucose-induced crosslinking of collagen was almost completely prevented by free radical scavengers. The increased fluorescence intensity observed in collagen incubated with AGEs was also not prevented by radical scavengers. Estimation of AGEs by ELISA revealed an increased accumulation of AGEs in collagen incubated with AGE-BSA. The inhibitory effect of aminoguanidine and aspirin on AGEs-induced modification of collagen, strongly suggests that the amino-carbonyl interaction between AGEs and collagen may play a key role in the crosslinking process. The results obtained in this study indicate that soluble AGEs can directly induce crosslinking of collagen and this process is independent of oxidative conditions. From these results it may be hypothesized that glucose, under oxidative conditions, reacts with proteins to form potentially reactive end products called AGEs. These AGEs, once formed, could induce crosslinking of collagen even in the absence of both glucose and oxygen.
Sajithlal
G B
GB
Department of Biochemistry, Central Leather Research Institute, Adyar, Chennai, India.
Chithra
P
P
Chandrakasan
G
G
D000818:Animals; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003488:Cyanogen Bromide; D003683:Dehydroascorbic Acid / Q000737:chemistry / Q000494:pharmacology; D003920:Diabetes Mellitus / Q000378:metabolism; D016166:Free Radical Scavengers; D005947:Glucose / Q000737:chemistry / Q000494:pharmacology; D017127:Glycation End Products, Advanced / Q000737:chemistry* / Q000378:metabolism; D010434:Pepsin A; D010449:Peptide Mapping; D051381:Rats; D013710:Tendons / Q000378:metabolism
D003432:Cross-Linking Reagents; D016166:Free Radical Scavengers; D017127:Glycation End Products, Advanced; D003094:Collagen; D010434:Pepsin A; D005947:Glucose; D003488:Cyanogen Bromide; D003683:Dehydroascorbic Acid