Study of degradation pathways of Amadori compounds obtained by glycation of opioid pentapeptide and related smaller fragments: Stability, reactions, and spectroscopic properties

Abstract

Reactions between biological amines and reducing sugars (the Maillard reaction) are among the most important of the chemical and oxidative changes occurring in biological systems that contribute to the formation of a complex family of rearranged and dehydrated covalent adducts that have been implicated in the pathogenesis of human diseases. In this study, chemistry of the Maillard reactions was studied in four model systems containing fructosamines (Amadori compounds) obtained from the endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu), leucine-enkephalin methyl ester, structurally related tripeptide (Tyr-Gly-Gly), or from amino acid (Tyr). The degradation of model compounds as well as their ability to develop Maillard fluorescence was investigated under oxidative conditions in methanol and phosphate buffer pH 7.4 at two different temperatures (37 and 70 degrees C). At 37 degrees C, glycated leucine-enkephalin degraded slowly in methanol (t(1/2) approximately 13 days) and phosphate buffer (t(1/2) approximately 9 days), producing a parent peptide compound as a major product throughout a three-week incubation period. Whereas fluorescence slowly increased over time at 37 degrees C, incubations off all studied Amadori compounds at 70 degrees C resulted in a rapid appearance of a brown color and sharp increase in AGE (advanced glycation end products)-associated fluorescence (excitation 320 nm/emmision 420 nm) as well as in distinctly higher amounts of fragmentation products. The obtained data indicated that the shorter the peptide chain the more degradation products were formed. These studies have also helped to identify a new chemical transformation of the peptide backbone in the Maillard reaction that lead to beta-scission of N-terminal tyrosine side chain and p-hydroxybenzaldehyde formation under both aqueous and nonaqueous conditions.

Oxidative degradation of N ε-fructosylamine-substituted peptides in heated aqueous systems

“…For example, 5 % or more of N-(1-deoxy-d-fructos-1-yl)glycine remained when incubated at 90-100 °C for 1.5 h (Staempfli et al 1994;Davidek et al 2002;. N-terminally glycated peptides were studied only up to 70 °C, but showed an even slower degradation behavior (Jakas and Horvat 2003), which could be attributed to either the lower temperature and the lower concentration of catalytically active phosphate buffer or their generally higher stability. Thus, our studies indicate a substantially lower stability of ε-N-Amadori modifications at the peptide level.…”

“…On the other hand, protein models are too complex and difficult to interpret (Wells-Knecht et al 1995). Thus, peptides represent a good compromise (Jakas and Horvat 2003). Few years ago, we have established comprehensive methods to synthesize and purify glycated peptides (Frolov et al 2006;Frolov and Hoffmann 2008), providing a convenient basis for Amadori degradation studies.…”

Free Radical Processes in Non-enzymatic Browning of Glucose and Lysine: Influence of Temperature and Unsaturated Lipids

“…Fluorescent compounds formed in vivo have been used as indicators of AGEs-modification of proteins, but emission characteristics depend on the type of protein adduct formed. [17][18][19] Lipid oxidation products could further enhance AGEs formation in food processed especially at high temperatures because lipid oxidation produces similar reaction intermediates to those formed during sugar degradation, leading to formation of AGEs. However, such protein modifications are also affected by the changes in the hydrophobic and hydrophilic balance and the molecular mobility induced by the presences of lipids, as recently described for milk powder with varying lipid contents.…”

Characterization of Site-Specific Glycation During Process Development of a Human Therapeutic Monoclonal Antibody

“…The Schiff base intermediate is reversible but it rapidly converts through Amadori rearrangement into a more stable ketoamine adduct. This reaction is also commonly referred to as a Maillard reaction after the name of the inventor 21,22. As glucose is the major source of energy in the cell culture media, small amounts of glycation in mAbs during cell culture can be expected 23–25.…”

“…This reaction is also commonly referred to as a Maillard reaction after the name of the inventor. 21,22 As glucose is the major source of energy in the cell culture media, small amounts of glycation in mAbs during cell culture can be expected. [23][24][25] The majority of this glycation is distributed over greater than 30 lysine residues.…”