Abstract
The inhibition of post-Amadori advanced glycation end product (AGE) formation by three different classes of AGE inhibitors, carbonyl group traps, chelators, and radical-trapping antioxidants, challenge the current paradigms that: 1) AGE inhibitors will not increase the formation of any AGE product, 2) transition metal ions are required for oxidative formation of AGE, and 3) screening AGE inhibitors only in systems containing transition metal ions represents a valid estimate of potential in vivo mechanisms. This work also introduces a novel multifunctional AGE inhibitor, 6-dimethylaminopyridoxamine (dmaPM), designed to function as a combined carbonyl trap, metal ion chelator, and radical-trapping antioxidant. Other AGE inhibitors including pyridoxamine, aminoguanidine, o-phenylenediamine, dipyridoxylamine, and diethylenetriaminepentaacetic acid were also examined. The results during uninterrupted and interrupted ribose glycations show: 1) an unexpected increase in the yield of pentosidine in the presence of radical-trapping phenolic antioxidants such as Trolox and dmaPM, 2) significant formation of Nepsilon-carboxymethyllysine (CML) in the presence of strong chelators and phenolic antioxidants, which implies that there must be nonradical routes to CML, 3) prevention of intermolecular cross-links with radical-trapping inhibitors, and 4) that dmaPM shows excellent inhibition of AGE. Glucose glycations reveal the expected inhibition of pentosidine and CML with all compounds tested, but in a buffer free of trace metal ions the yield of CML in the presence of radical-trapping antioxidants was between the metal ion-free and metal ion-containing controls. Protein molecular weight analyses support the conclusion that Amadori decomposition pathways are constrained in the presence of metal ion chelators and radical traps.
Highlights
The inhibition of post-Amadori advanced glycation end product (AGE) formation by three different classes of advanced glycation end products (AGEs) inhibitors, carbonyl group traps, chelators, and radical-trapping antioxidants, challenge the current paradigms that: 1) AGE inhibitors will not increase the formation of any AGE product, 2) transition metal ions are required for oxidative formation of AGE, and 3) screening AGE inhibitors only in systems containing transition metal ions represents a valid estimate of potential in vivo mechanisms
A pyridoxine (PN) derivative (6-dimethylaminopyridoxine, dmaPN) was tested to study the importance of the nucleophilic amine group during glycation reactions. Both dmaPM and dmaPN were found to be much better radical-trapping antioxidants than PM or PN [59], as evaluated by Impact of Antioxidants on Post-Amadori Glycoxidation following the peroxyl radical-induced quenching of allophycocyanin fluorescence [37, 38]. This new carbonyl group trapping and radical-trapping AGE inhibitor, dmaPM, and a number of other AGE inhibitors were tested in metal ion-containing and metal ion-free (ϪM buffer) experiments
Our findings show a phenomenal impact of radicaltrapping antioxidants on post-Amadori glycoxidation and challenge the current paradigms that: 1) AGE inhibitors will not increase the formation of any AGE product, 2) transition metal ions are required for oxidative formation of AGE, and 3) screening AGE inhibitors only in systems containing transition metal ions represents a valid estimate of potential in vivo mechanisms
Summary
The inhibition of post-Amadori advanced glycation end product (AGE) formation by three different classes of AGE inhibitors, carbonyl group traps, chelators, and radical-trapping antioxidants, challenge the current paradigms that: 1) AGE inhibitors will not increase the formation of any AGE product, 2) transition metal ions are required for oxidative formation of AGE, and 3) screening AGE inhibitors only in systems containing transition metal ions represents a valid estimate of potential in vivo mechanisms. Nonenzymatic protein glycation by reducing sugars, such as glucose or ribose, is a complicated cascade of condensations, rearrangements, fragmentations, and oxidative modifications that lead to a plethora of compounds collectively called advanced glycation end products (AGEs)1 [1]. The “classical” or Hodge pathway begins with reversible formation of a Schiff base aldimine adduct that undergoes rearrangement to a relatively irreversible ketoamine Amadori product (see supplemental material). Interrupted glycation methods have been developed by Booth et al [1] that allow Amadori-rich proteins to be isolated This method allows more detailed studies to be carried out, which may provide insight into the specific mechanisms involved in post-Amadori formation of AGEs. Generally, glycation alters protein physicochemical properties by forming a variety of cross-links, browning (nonfluorescent AGE), and fluorescent AGE products. CML, N⑀-carboxymethyllysine; diPM, dipyridoxylamine trihydrochloride; dmaPM, 6-dimethylaminopyridoxamine dihydrochloride; dmaPN, 6-dimethylaminopyridoxine hydrochloride (6-dimethylaminopyridoxol-HCl); DTPA, diethylenetriaminepentaacetic acid; MW, molecular weight; o-PD, ortho-phenylenediamine; PM, pyridoxamine dihydrochloride; PN, pyridoxine hydrochloride (pyridoxol); RI, refractive index; SEC, size exclusion chromatography; Tx, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid); HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight
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