Glycation and the Maillard Reaction In Vitro: Implications for Diabetes Mellitus

Ahmed, Nessar (1992). Glycation and the Maillard Reaction In Vitro: Implications for Diabetes Mellitus. PhD thesis The Open University.



A colorimetric microassay for protein glycation based on the periodate method has been developed. This microassay has improved sensitivity, speed and yield of chromophore enabling it to be used on 0.1 mg aliquots of intact protein. However this assay is unsuitable for collagen and so a second microassay based on the TBA method was developed. This TBA microassay has an improved yield of HMF, is less cumbersome, requires less protein than earlier versions, and is suitable as a rapid assay for glycated collagen.

Fructation induces AGE formation considerably faster than glucation in vitro probably because of the highly reactive fructose-derived aldehydic AP. However glycation assays designed for the glucose-AP (a ketose) were found to underestimate the true extent of fructation. For this reason a microassay based on the DNPH method was developed to quantify fructation.

Comparative rates of protein fluorescence generated by different sugars were identical to their comparative rates of SB formation but not crosslinking. Using model proteins and different sugars, this study has revealed that fluorescent-AGE and crosslinked-AGE may be formed by independent pathways ie some proteins are more susceptible to crosslinked-AGE and vice versa. Phosphate, pH and calcium have been found to increase glucose and fructose-derived fluorescent-AGE in BSA.

Contrary to current dogma, no evidence for an accelerated rise in AGE levels in reincubated proteins after removal of free sugars was found. Only a small increase in fluorescence of reincubated glucated-BSA but not fructated-BSA was detectable. These studies suggest that free sugars have a significant role in fluorescence generation and indeed using chemically modified proteins, evidence has been presented for a reaction between the AP and free sugars to form fluorescent products. This secondary glycation is more pronounced for fructose and explains in part the higher fluorescence generated by fructated proteins.

Comparative studies between inhibitors with different modes of action have revealed that aminoguanidine is the most effective at reducing fluorescence and crosslinking of proteins in vitro on a molar basis. This is probably because the compound has multiple sites of action and more than one amino group. Phenylenediamine reacts with 3-DG preventing formation of AGE and was found to have a greater inhibitory effect on fructose compared to glucose-derived AGE.

In this study, no evidence was found for sugar-induced protein fragmentation in vitro unless transition metals were included. Furthermore binding of the transition metal to protein appears to be important for significant fragmentation to occur and in vivo this autoxidative glycation may be restricted to the limited number of copper binding proteins only.

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