Peptide Mapping of Type IV Collagen Modified Minimally by Methylglyoxal in Vitro

Diabetes Obesity Metabolism

2006

315

234

Glycation is a major cause of spontaneous damage to proteins in physiological systems. This is exacerbated in diabetes as a consequence of the increase in glucose and other saccharides derivatives in plasma and at the sites of vascular complications. Protein damage by the formation of early glycation adducts is limited to lysine side chain and N-terminal amino groups whereas later stage adducts, advanced glycation endproducts (AGEs), modify these and also arginine and cysteine residues. Metabolic dysfunction in vascular cells leads to the increased formation of methylglyoxal which adds disproportionately to the glycation damage in hyperglycaemia. AGE-modified proteins undergo cellular proteolysis leading to the formation and urinary excretion of glycation free adducts. AGEs may potentiate the development of diabetic complications by activation of cell responses by AGE-modified proteins interacting with specific cell surface receptors, activation of cell responses by AGE free adducts, impairment of protein-protein and enzyme-substrate interactions by AGE residue formation, and increasing resistance to proteolysis of extracellular matrix proteins. The formation of AGEs is suppressed by intensive glycaemic control, and may in future be suppressed by thiamine and pyridoxamine supplementation, and several other pharmacological agents. Increasing expression of enzymes of the enzymatic defence against glycation provides a novel and potentially effective future therapeutic strategy to suppress protein glycation.

Section: Future Prospectsmentioning

confidence: 99%

Advanced glycation endproducts: what is their relevance to diabetic complications?

Ahmed

Diabetes Obesity Metabolism

2006

315

234

“…Steps must be taken to avoid oxidative degradation of the adduct residues of proteins during enzymatic hydrolysis; the addition of the antioxidant thymol and incubation under nitrogen or argon are typical methods (11,13). After an initial step with pepsin (replaced by collagenase for analysis of collagen) under acidic conditions (14), antibiotics are included in the enzymatic digest to prevent bacterial growth in the amino acid solution being produced (13). These procedures yield acceptable analyte stabilities and exhaustive proteolysis, and then proceed to near-completion for proteins modified only minimally by glycation, oxidation, and nitration.…”

Section: Measurement Of Glycated Oxidized and Nitrated Proteins And Related Free Adducts By Stable Isotopic Dilution Analysismentioning

confidence: 99%

Quantitation of Markers of Protein Damage by Glycation, Oxidation, and Nitration in Peritoneal Dialysis

Rabbani

2009

Perit Dial Int

Self Cite

Proteolysis products of proteins damaged by glycation, oxidation, and nitration—glycated, oxidized, and nitrated amino acids (glycation, oxidation, and nitration free adducts)—are waste products normally excreted in urine and cleared in peritoneal dialysate. Glucose degradation products in peritoneal dialysis (PD) fluids may increase protein damage, giving rise to increased protein glycation, oxidation, and nitration adduct residues of proteins and increased flux of glycation, oxidation, and nitration free adducts. Increased protein damage has been linked to mortality in end-stage renal disease. Reliable quantitation of markers for adducts of protein glycation, oxidation, and nitration is required for mechanistic studies and for morbidity and mortality risk analysis in PD patients. We review the available analytical techniques for such quantitation. Stable isotopic dilution analysis with tandem mass spectrometry is the “gold standard.” This method needs to be applied further in the study of PD and to validate other techniques so that the effect of PD on the metabolism and clearance of damaged proteins and related products can be quantified, and so that best-practice fluid management can be established to minimize cardiovascular risk.

“…Oxidative degradation of protein adduct residues during enzymatic hydrolysis is suppressed by addition of the antioxidant thymol and incubation under nitrogen or carbon monoxide (the latter to exclude oxygen and inactivate heme in hemoglobin digests) (18,29). After an initial step with pepsin under acidic conditions (replaced by collagenase for analysis of collagen) (30), antibiotics are included in the enzymatic digest to prevent bacterial growth in the amino acid solution being produced (29). These procedures give acceptable analyte stabilities and exhaustive proteolysis that proceed to near completion for proteins minimally modified by glycation, oxidation, and nitration.…”

Section: Quantitation Of Glycation Adducts In Uremia — the Advantages Of Liquid Chromatography With Tandem Mass Spectrometric (Lc-ms/ms) mentioning

confidence: 99%

Measurement of Protein Glycation, Glycated Peptides, and Glycation Free Adducts

2005

Perit Dial Int

Self Cite

Protein glycation adducts, early glycation adducts, such as N∊-fructosyl-lysine, and advanced glycation end products (AGEs) are uremic toxins. Glycation adducts are found in plasma and tissue proteins (glycation adduct residues), in peptides (glycation adduct peptide residues), and glycated amino acids (glycation free adducts). The latter two analyte groups arise from proteolysis of glycated proteins and glycation of peptides and amino acids. Quantitation of glycation adducts in uremia is difficult because of the presence of many different AGEs at low concentrations in different forms in the presence of many potential interferences. Application of liquid chromatography with tandem mass spectrometric (LC-MS/MS) detection to plasma, urine, and dialysate samples of uremic patients has provided a comprehensive and quantitative analysis of glycation adducts in uremia. Glycation free adducts accumulate markedly in the plasma of uremic patients and are eliminated in the peritoneal dialysate. Multiple glycation adducts, and also protein oxidation and nitration adducts, may be quantified concurrently. Glycation free adducts are the major form of glycation adduct eliminated in dialysate. LC-MS/MS may now be used to quantify concentrations, extents of protein modification, clearances, and excretion rates of glycation adducts in uremia.