Abstract
The advanced glycation end-product (AGE) hypothesis proposes that accelerated chemical modification of proteins by glucose during hyperglycemia contributes to the pathogenesis of diabetic complications. The two most commonly measured AGEs, N(epsilon)-(carboxymethyl)lysine and pentosidine, are glycoxidation products, formed from glucose by sequential glycation and autoxidation reactions. Although several compounds have been developed as AGE inhibitors and are being tested in animal models of diabetes and in clinical trials, the mechanism of action of these inhibitors is poorly understood. In general, they are thought to function as nucleophilic traps for reactive carbonyl intermediates in the formation of AGEs; however alternative mechanisms of actions, such as chelation, have not been rigorously examined. To distinguish between the carbonyl trapping and antioxidant activity of AGE inhibitors, we have measured the chelating activity of the inhibitors by determining the concentration required for 50% inhibition of the rate of copper-catalyzed autoxidation of ascorbic acid in phosphate buffer. All AGE inhibitors studied were chelators of copper, as measured by inhibition of metal-catalyzed autoxidation of ascorbate. Apparent binding constants for copper ranged from approximately 2 mm for aminoguanidine and pyridoxamine, to 10-100 microm for carnosine, phenazinediamine, OPB-9195 and tenilsetam. The AGE-breakers, phenacylthiazolium and phenacyldimethylthiazolium bromide, and their hydrolysis products, were among the most potent inhibitors of ascorbate oxidation. We conclude that, at millimolar concentrations of AGE inhibitors used in many in vitro studies, inhibition of AGE formation results primarily from the chelating or antioxidant activity of the AGE inhibitors, rather than their carbonyl trapping activity. Further, at therapeutic concentrations, the chelating activity of AGE inhibitors and AGE-breakers may contribute to their inhibition of AGE formation and protection against development of diabetic complications.
Highlights
MethodsReagents—Unless otherwise noted, all chemicals were purchased from Sigma/Aldrich Chemical Co. Monobasic and dibasic sodium phosphate salts were purchased from EM Science (Gibbstown, NJ); heptafluorobutyric acid (HFBA) from Acros (Pittsburgh, PA), and nitric acid and HPLC grade acetonitrile from Fisher Scientific (Pittsburgh, PA)
Summary—We have shown that AGE inhibitors have significant copper chelating activity
While we have not studied other metals in detail, similar inhibitory effects were observable with phosphate buffers containing a mixture of trace metal ions
Summary
Reagents—Unless otherwise noted, all chemicals were purchased from Sigma/Aldrich Chemical Co. Monobasic and dibasic sodium phosphate salts were purchased from EM Science (Gibbstown, NJ); heptafluorobutyric acid (HFBA) from Acros (Pittsburgh, PA), and nitric acid and HPLC grade acetonitrile from Fisher Scientific (Pittsburgh, PA). Phenacylthiazolium bromide (PTB) and phenacyldimethylthiazolium bromide (PMTB) were synthesized according to the methods of Vasan et al (13) and Wolfenbuttel et al (14), respectively, and purified to homogeneity by reverse-phase HPLC. 2,3Diaminophenzine (DAP) was synthesized according to Soulis et al (15). PTB, PMTB, and DAP were homogeneous by reverse phase-HPLC analysis and by electrospray ionization liquid chromatography-mass spectrometry. All water used in these experiments was distilled and deionized (15–18 M⍀) using a mixed bed ion-exchanger with activated charcoal polisher.
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