Comparison of modification sites formed on human serum albumin at various stages of glycation

Abstract

Background-Many of the complications encountered during diabetes can be linked to the nonenzymatic glycation of proteins, including human serum albumin (HSA). However, there is little information regarding how the glycation pattern of HSA changes as the total extent of glycation is varied. The goal of this study was to identify and conduct a semi-quantitative comparison of the glycation products on HSA that are produced in the presence of various levels of glycation.Methods-Three glycated HSA samples were prepared in vitro by incubating physiological concentrations of HSA with 15 mmol/l glucose for 2 or 5 weeks, or with 30 mmol/l glucose for 4 weeks. These samples were then digested and examined by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the glycation products that were formed.Results-It was found that the glycation pattern of HSA changed with its overall extent of total glycation. Many modifications including previously-reported primary glycation sites (e.g., K199, K281, and the N-terminus) were consistently found in the tested samples. Lysines 199 and 281, as well as arginine 428, contained the most consistently identified and abundant glycation products. Lysines 93, 276, 286, 414, 439, and 524/525, as well as the N-terminus and arginines 98, 197, and 521, were also found to be modified at various degrees of HSA glycation.Conclusions-The glycation pattern of HSA was found to vary with different levels of total glycation and included modifications at the 2 major drug binding sites on this protein. This result suggests that different modified forms of HSA, both in terms of the total extent of glycation and glycation pattern, may be found at various stages of diabetes. The clinical implication of these results is that the binding of HSA to some drug may be altered at various stages of diabetes as the extent of glycation and types of modifications in this protein are varied.

Mass Spectrometric Characterization of Circulating Covalent Protein Adducts Derived from a Drug Acyl Glucuronide Metabolite: Multiple Albumin Adductions in Diclofenac Patients

“…This seemingly novel adduct is attributed tentatively to a somewhat rare, nonenzymic, posttranslational modification occurring in vivo by the slow reaction of HSA with glucuronic acid in plasma (Mazzuchin et al, 1971;Smith et al, 1990). In a separate study using similar analytical methods, we found glucosylated Lys525 (Barnaby et al, 2011) in vivo (data not shown). Neither glucuronylated Lys525 nor any of the N-acylations and glycations associable with either diclofenac AG or reactive thioester metabolites of diclofenac were found on HSA isolated from the three control subjects.…”

“…Lys414, in HSA site 2, diclofenac's high-affinity binding site (Chamouard et al, 1985), is modified by the cyclic imide but not by diclofenac AG or benzylpenicillin. Lys199 and Lys414, but neither Lys195 nor Lys432, are glycated spontaneously by glucose (Barnaby et al, 2011) via the same pathway of reversible imine formation and slow, cumulative, Amadori rearrangement. Diclofenac AG glycations were identified notwithstanding incubations did not contain cyanoborohydride, which reduces imines and enhances binding of AG, including diclofenac AG (Kenny et al, 2004), to HSA (Smith et al, 1990).…”

Mitochondria Function in Diabetes – From Health to Pathology – New Perspectives for Treatment of Diabetes-Driven Disorders

“…1). The total amount of such accumulated products is known to be dependent on the type of sugar that is causing the glycation, the incubation time and sugar concentration, as well as the type protein that is being modified (Barnaby et al, 2011). The first product of Maillard reaction is a simple glycosylamine, which readily undergoes the Amadori rearrangement to produce 1-amino-1-deoxy-2-ketoses.…”

Aqueous extract of some indigenous medicinal plants inhibits glycation at multiple stages and protects erythrocytes from oxidative damage–an in vitro study

“…Albumin is one of the main circulating proteins which undergoes glycation due to its abundance in serum (35 g/l), long half-life (21 days) and presence of multiple glycation sites (Barnaby et al 2011). Glycation induced modifications have a detrimental impact on albumin structure as its-1) conformation changes, (Rondeau and Bourdon 2011) 2) free thiols gets oxidized to disulfide linkages or thiol radicals (S-S or S, respectively) (Bourdon et al 1999), 3) initially globular albumin refolds into amyloid fibrils comprising cross-β structure (the β aggregates specifically bind to amyloid specific dyes-Congo red and thioflavin T (Th T) (Bouma et al 2003).…”