It is widely accepted that the Maillard reaction, specifically the development of Advanced Glycation End-products (AGEs) in vivo, is linked to the pathogenesis of diabetic secondary complications. The same diseases that occur in old age are similarly thought to develop as a function of the accumulation of AGEs. This thesis presents an in vitro investigation into glycation by fructose (fructation) and discusses the potential for in vivo fructation. The contribution of in vivo fructation to degenerative diseases is unknown, largely because assays for glycation underestimate, or do not detect, fructation (Ahmed N& Furth A. J. 1992, Clin. Chem. X8,1301-1303). In vitro radiolabelling illustrates that incorporation of fructose into protein, through the Maillard reaction, is more swift than glucose. The development of AGE-fluorescence reflects the incorporation of the two sugars, i. e. higher upon fructation. A novel colorimetric assay for glycation (including fructation), based on the reaction of 2,4-dinitrophenylhydrazine (DNPH) with protein-bound carbonyl groups, is presented. The DNPH assay detects compounds that are intermediates in post-Amadori, or post-Heyns reactions, and precede development of fluorescence. The production of the protein-bound carbonyl intermediates is largely dependent on the presence of lipid and free metal. The same group of compounds are probably the principle site of action of aminoguanidine, which blocks the production of AGEs. An in vitro model is presented, to show how protein glycated in one part of the body, can long after maintenance of euglycaemia, become covalently cross-linked to a second protein. Lysozyme was glycated with low concentrations of fructose, or using short periods of fructation, and the free fructose removed. The fructated lysozyme was subsequently incubated in sugar-free buffer with native ßlactoglobulin and produced a 32kD heterodimer of the two covalently cross-linked proteins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:295258 |
| Date | January 1995 |
| Creators | Liggins, Jason |
| Publisher | Open University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://oro.open.ac.uk/57550/ |
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