Naila Ahmed

Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry.

Glycation of proteins forms fructosamines and advanced glycation endproducts. Glycation adducts may be risk markers and risk factors of disease development. We measured the concentrations of the early glycation adduct fructosyl-lysine and 12 advanced glycation endproducts by liquid chromatography with tandem mass spectrometric detection. Underivatized analytes were detected free in physiological fluids and in enzymic hydrolysates of cellular and extracellular proteins. Hydroimidazolones were the most important glycation biomarkers quantitatively; monolysyl adducts (N(epsilon)-carboxymethyl-lysine and N(epsilon)-1-carboxyethyl-lysine) were found in moderate amounts, and bis(lysyl)imidazolium cross-links and pentosidine in lowest amounts. Quantitative screening showed high levels of advanced glycation endproducts in cellular protein and moderate levels in protein of blood plasma. Glycation adduct accumulation in tissues depended on the particular adduct and tissue type. Low levels of free advanced glycation endproducts were found in blood plasma and levels were 10-100-fold higher in urine. Advanced glycation endproduct residues were increased in blood plasma and at sites of vascular complications development in experimental diabetes; renal glomeruli, retina and peripheral nerve. In clinical uraemia, the concentrations of plasma protein advanced glycation endproduct residues increased 1-7-fold and free adduct concentrations increased up to 50-fold. Comprehensive screening of glycation adducts revealed the relative and quantitative importance of alpha-oxoaldehyde-derived advanced glycation endproducts in physiological modification of proteins-particularly hydroimidazolones, the efficient renal clearance of free adducts, and the marked increases of glycation adducts in diabetes and uraemia-particularly free advanced glycation endproducts in uraemia. Increased levels of these advanced glycation endproducts were associated with vascular complications in diabetes and uraemia.

Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans

Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C. elegans: progressive mitochondrial protein modification by the glycolysis‐derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase‐1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase‐1 mRNA. The decrease in enzymatic activity promotes accumulation of MG‐derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase‐1 expression. Over‐expression of the C. elegans glyoxalase‐1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C. elegans lifespan. In contrast, knock‐down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C. elegans lifespan.

Increased Dicarbonyl Metabolism in Endothelial Cells in Hyperglycemia Induces Anoikis and Impairs Angiogenesis by RGD and GFOGER Motif Modification

Chronic vascular disease in diabetes is associated with disruption of extracellular matrix (ECM) interactions with adherent endothelial cells, compromising cell survival and impairing vasculature structure. Loss of functional contact with integrins activates anoikis and impairs angiogenesis. The metabolic dysfunction underlying this vascular damage and disruption is unclear. Here, we show that increased modification of vascular basement membrane type IV collagen by methylglyoxal, a dicarbonyl glycating agent with increased formation in hyperglycemia, formed arginine-derived hydroimidazolone residues at hotspot modification sites in RGD and GFOGER integrin-binding sites of collagen, causing endothelial cell detachment, anoikis, and inhibition of angiogenesis. Endothelial cells incubated in model hyperglycemia in vitro and experimental diabetes in vivo produced the same modifications of vascular collagen, inducing similar responses. Pharmacological scavenging of methylglyoxal prevented anoikis and maintained angiogenesis, and inhibition of methylglyoxal metabolism with a cell permeable glyoxalase I inhibitor provoked these responses in normoglycemia. Thus, increased formation of methylglyoxal and ECM glycation in hyperglycemia impairs endothelial cell survival and angiogenesis and likely contributes to similar vascular dysfunction in diabetes.

Degradation products of proteins damaged by glycation, oxidation and nitration in clinical type 1 diabetes

Aims/hypothesisHyperglycaemia in diabetes is associated with increased glycation, oxidative stress and nitrosative stress. Proteins modified consequently contain glycation, oxidation and nitration adduct residues, and undergo cellular proteolysis with release of corresponding free adducts. These free adducts leak into blood plasma for eventual renal excretion. The aim of this study was to perform a comprehensive quantitative analysis of protein glycation, oxidation and nitration adduct residues in plasma protein and haemoglobin as well as of free adducts in plasma and urine to quantify increased protein damage and flux of proteolytic degradation products in diabetes.MethodsType 1 diabetic patients (n=21) and normal healthy control subjects (n=12) were studied. Venous blood samples, with heparin anticoagulant, and 24-h urine samples were taken. Samples were analysed for protein glycation, oxidation and nitration adducts by a quantitative comprehensive screening method using liquid chromatography with triple quadrupole mass spectrometric detection.ResultsIn type 1 diabetic patients, the concentrations of protein glycation, oxidation and nitration adduct residues increased up to three-fold in plasma protein and up to one-fold in haemoglobin, except for decreases in pentosidine and 3-nitrotyrosine residues in haemoglobin when compared with normal control subjects. In contrast, the concentrations of protein glycation and oxidation free adducts increased up to ten-fold in blood plasma, and urinary excretion increased up to 15-fold in diabetic patients.Conclusions/interpretationWe conclude that there are profound increases in proteolytic products of glycated and oxidised proteins in diabetic patients, concurrent with much lower increases in protein glycation and oxidation adduct residues.

Methylglyoxal administration induces diabetes-like microvascular changes and perturbs the healing process of cutaneous wounds.

Increased formation of MG (methylglyoxal) and related protein glycation in diabetes has been linked to the development of diabetic vascular complications. Diabetes is also associated with impaired wound healing. In the present study, we investigated if prolonged exposure of rats to MG (50-75 mg/kg of body weight) induced impairment of wound healing and diabetes-like vascular damage. MG treatment arrested growth, increased serum creatinine, induced hypercholesterolaemia (all P < 0.05) and impaired vasodilation (P < 0.01) compared with saline controls. Degenerative changes in cutaneous microvessels with loss of endothelial cells, basement membrane thickening and luminal occlusion were also detected. Acute granulation appeared immature (P < 0.01) and was associated with an impaired infiltration of regenerative cells with reduced proliferative rates (P < 0.01). Immunohistochemical staining indicated the presence of AGEs (advanced glycation end-products) in vascular structures, cutaneous tissue and peripheral nerve fibres. Expression of RAGE (receptor for AGEs) appeared to be increased in the cutaneous vasculature. There were also pro-inflammatory and profibrotic responses, including increased IL-1beta (interleukin-1beta) expression in intact epidermis, TNF-alpha (tumour necrosis factor-alpha) in regions of angiogenesis, CTGF (connective tissue growth factor) in medial layers of arteries, and TGF-beta (transforming growth factor-beta) in glomerular tufts, tubular epithelial cells and interstitial endothelial cells. We conclude that exposure to increased MG in vivo is associated with the onset of microvascular damage and other diabetes-like complications within a normoglycaemic context.

Profound Mishandling of Protein Glycation Degradation Products in Uremia and Dialysis

The aim of this study was to define the severe deficits of protein glycation adduct clearance in chronic renal failure and elimination in peritoneal dialysis (PD) and hemodialysis (HD) therapy using a liquid chromatography-triple quadrupole mass spectrometric detection method. Physiologic proteolysis of proteins damaged by glycation, oxidation, and nitration forms protein glycation, oxidation, and nitration free adducts that are released into plasma for urinary excretion. Inefficient elimination of these free adducts in uremia may lead to their accumulation. Patients with mild uremic chronic renal failure had plasma glycation free adduct concentrations increased up to five-fold associated with a decline in renal clearance. In patients with ESRD, plasma glycation free adducts were increased up to 18-fold on PD and up to 40-fold on HD. Glycation free adduct concentrations in peritoneal dialysate increased over 2- to 12-h dwell time, exceeding the plasma levels markedly. Plasma glycation free adducts equilibrated rapidly with dialysate of HD patients, with both plasma and dialysate concentrations decreasing during a 4-h dialysis session. It is concluded that there are severe deficits of protein glycation free adduct clearance in chronic renal failure and in ESRD on PD and HD therapy.

Methylglyoxal modification of mSin3A links glycolysis to angiopoietin-2 transcription.

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here, we report that in retinal Müller cells, increased glycolytic flux causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc transferase to an mSin3A-Sp3 complex, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding of the repressor complex to a glucose-responsive GC box in the angiopoietin-2 promoter, resulting in increased Ang-2 expression. A similar mechanism involving methylglyoxal-modification of other coregulator proteins may play a role in the pathobiology of a variety of conditions associated with changes in methylglyoxal concentration, including cancer and diabetic vascular disease.

High-dose thiamine therapy counters dyslipidemia and advanced glycation of plasma protein in streptozotocin-induced diabetic rats.

Abstract: The streptozotocin‐induced (STZ) diabetic rat experimental model of diabetes on insulin maintenance therapy exhibits dyslipidemia, mild thiamine deficiency, and increased plasma protein advanced glycation end products (AGEs). The reversal of thiamine deficiency by high‐dose thiamine and S‐benzoylthiamine monophosphate (benfotiamine) prevented the development of incipient nephropathy. Recently, we reported that high‐dose thiamine (but not benfotiamine) countered diabetic dyslipidemia. To understand further the differences between the effects of thiamine and benfotiamine therapy, we quantified the levels of the AGEs in plasma protein. We found hydroimidazolone AGE residues derived from glyoxal and methylglyoxal, G‐H1 and MG‐H1, were increased 115% and 68% in STZ diabetic rats, with respect to normal controls, and were normalized by both thiamine and benfotiamine; whereas N‐carboxymethyl‐lysine (CML) and N‐carboxyethyl‐lysine (CEL) residues were increased 74% and 118% in STZ diabetic rats and were normalized by thiamine only. The lack of effect of benfotiamine on plasma CML and CEL residue concentrations suggests there may be important precursors of plasma protein CML and CEL residues other than glyoxal and methylglyoxal. These are probably lipid‐derived aldehydes.

Peptide mapping of human serum albumin modified minimally by methylglyoxal in vitro and in vivo.

Abstract: Methylglyoxal is a potent glycating agent and important precursor of advanced glycation end products (AGEs) in physiological systems. Unlike glucose, methylglyoxal is predominantly an arginine‐directed glycating agent. Methylglyoxal reacts with proteins to form mainly the arginine‐derived hydroimidazolone AGE, Nδ‐(5‐hydro‐5‐methyl‐4‐imidazolon‐2‐yl)‐ornithine (MG‐H1), argpyrimidine, the lysine‐derived AGEs, Nε‐(1‐carboxyethyl)lysine (CEL), and methylglyoxal‐derived lysine dimer (MOLD). Sites within proteins susceptible to modification by methylglyoxal have not been identified. Here we show that modification of human serum albumin by methylglyoxal forms mainly hydroimidazolone MG‐H1 residues. The location of MG‐H1 residues was identified by mass spectrometric peptide mapping. This method identified a hot spot of hydroimidazolone formation at Arg‐410, with other minor MG‐H1 modifications at Arg‐114, Arg‐186, Arg‐218, and Arg‐428. Other extracellular and intracellular proteins are modified by methylglyoxal in physiological systems. Modification of arginine residues by methylglyoxal may be particularly damaging because arginine residues have a high frequency of occurrence in ligand and substrate recognition sites in receptor and enzyme active sites.

Increased Protein Glycation in Cirrhosis and Therapeutic Strategies to Prevent It

Abstract: Glycation of liver proteins by reactive aldehydes formed from the metabolism of ethanol and lipid peroxidation has been implicated in the development of both alcoholic and nonalcoholic liver cirrhosis. Modified proteins are targeted to the proteasome for proteolysis. Release of glycation‐free adducts into the circulation may provide a diagnostic “signature” of hepatic protein damage. We quantitatively screened protein glycation, oxidation, and nitrosation adduct residues and free adducts in portal, hepatic, and peripheral venous blood plasma of cirrhotic patients; we also screened the hepatic and peripheral venous blood plasma of control subjects by liquid chromatography‐mass spectrometry. There was a remarkable 14‐16‐fold increase of glyoxal‐derived, hydroimidazolone‐free adduct in portal and hepatic venous plasma of cirrhotic patients with respect to normal controls. There was only a twofold increase of glycation adduct residues in plasma proteins in cirrhotic patients, which was attributed mainly to decreased albumin turnover. Therapeutic strategies to decrease dicarbonyl compounds may be beneficial, such as dicarbonyl scavengers, glutathione repleting agents, and high‐dose thiamine therapy.