In vitro glycation of human serum albumin by dihydroxyacetone and dihydroxyacetone phosphate

Amino groups in proteins can non-enzymatically react with reducing sugars to generate a structurally diverse group of compounds referred to as advanced glycation end products (AGEs). The in vivo formation of AGEs contributes to some of the complications of diabetes including atherosclerosis, cataract formation, and renal failure. The formation of AGEs is dependent on both sugar and protein concentrations. Increases in temperature, pH, and exposure time of sugars to the proteins also play a significant role in the rate of AGE formation. This study focuses on the use of a combination of analytical techniques to study the in vitro AGE formation of HSA with dihydroxyacetone phosphate (DHAP), a ketose generated during glycolysis, and its dephosphorylated analog, dihydroxy acetone (DHA), commonly used as a browning reagent in skin tanning preparations. The extent of AGE formation was affected by DHAP and DHA concentrations and by the duration of HSA exposure to these glycating agents. Increases in temperature and pH sped the glycation process and enhanced the formation of the AGEs of HSA. MALDI-TOF mass spectroscopic data provided a reliable result to evaluate the extent of the AGE formation.     

The in vitro inhibition effect of 2 nm gold nanoparticles on non-enzymatic glycation of human serum albumin

The reaction of amino groups of protein and the carbonyl groups of reducing sugar molecules, non-enzymatically induce a series of chemical reactions that form a heterogeneous group of compounds known as advanced glycation end products (AGEs). The accumulation of AGEs is associated with various disease conditions that include complications in diabetes, Alzheimer's disease and aging. The current study monitored the extent of non-enzymatic glycation of human serum albumin (HSA) in order to estimate the formation of HSA related AGEs in the presence of 2 nm gold nanoparticles. The rate of glycation was evaluated using several analytical methods. Physiological concentrations of HSA and glyceraldehyde mixtures, incubated with various concentrations of negatively charged 2 nm gold nanoparticles, resulted in a lower reaction rate than mixtures without 2GNP. Moreover, increasing concentrations of gold nanoparticles exhibited a pronounced reduction in AGE formation. High performance liquid chromatography, UV-visible spectroscopy and circular dichroism analytical methods provide reliable techniques for evaluating AGE formation of HSA adducts.     

Identification of AGE-precursors and AGE formation in glycation-induced BSA peptides

The glycation of BSA leads to protein/peptide modifications that result in the formation of AGEs. AGEs react with the amino groups of N-terminal amino acid residues, particularly arginine and lysine residues. Enhanced AGE formation exists in the blood and tissues of diabetics, as well as in aging and other disorders. The Identification of AGEs is of great importance. Mass spectrometry has been applied to identify and structurally elucidate AGEs. Here, we report on the identification of AGE- peptides and AGE-precursors based on relative mass changes as a result of specific AGE formation. HPLC-ESIMS, ESI-MS/MS, and the Mascot database were used. The relative mass changes due to the specific type of AGE formation were added to the identified peptides followed by a manual search of the glycated samples, which resulted in the identification of seven peptides for the formation of five AGEs, namely CML, pyrraline, imidazolone A, imidazolone B, and AFGP. Four glycated peptides (FPK, ECCDKPLLEK, IETMR, and HLVDEPQNLIK) were identified in the formation of AGE-precursors.     

Characterization of advanced glycation end products: mass changes in correlation to side chain modifications

Advanced glycation end products (AGEs) that arise from the reaction of sugars with protein side chains are supposed to be involved in the pathogenesis of several diseases; therefore, the effects of AGEs on cells are the objective of numerous investigations. Because AGE modifications are an extremely heterogeneous group of side chain modifications, the exact characterization of an AGE-modified protein is impossible. To gain a deeper understanding about AGE formation kinetics and structures, AGEs can be characterized with respect to the degree of modification, specific side chain modifications, absorbance and fluorescence characteristics, and changes in the protein structure and molecular weight. For this study, human serum albumin (HSA)-AGEs derived from different concentrations of glucose, methyl glyoxal, and glyoxylic acid were used. The molecular mass of the obtained AGEs was determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mass data were compared with earlier results concerning the degree of lysine and arginine side chain modifications and AGE-specific fluorescence and absorbance data. The molecular masses were found to gradually increase with increasing concentrations of the individual modifier without reaching a plateau. The mass increase correlates very well with the AGE-specific absorbance at 360 nm and with the degree of side chain modifications. The mass spectrometric data prove, for the first time, that an increasing absorbance at 360 nm is directly correlated to a mass increase during the AGE formation process.     

Structure of a synthetic glucose derived advanced glycation end product that is immunologically cross-reactive with its naturally occurring counterparts

Glucose reacts nonenzymatically with the amino groups of proteins to form stable, cross-linking adducts called advanced glycation end products or AGEs. While several lines of evidence have established that AGEs accumulate in tissues and contribute to the pathological sequelae of diabetes and aging, the identity of the major cross-link(s) that forms in vivo has remained enigmatic. This has been considered to be due to the labile nature and to the low fluorescence properties of this cross-link, despite the fact that fluorescence has been generally associated with AGE formation in vivo. Accordingly, the few AGE adducts that have been isolated thus far from proteins in vivo or from model systems in vitro have been found to account for only a fraction of the glucose-derived cross-links that occur in tissues. This situation has been further underscored by the development of a well-characterized class of antibodies that recognize in vivo AGEs but which fail to react with the structurally defined AGEs that have been identified to date. This particular class of anti-AGE antibodies has proven valuable in the quantification of AGE-modified forms of hemoglobin, low-density lipoprotein (LDL), and beta-amyloid peptide, and can provide prognostic information on the course of certain diabetic complications. To obtain insight into the structure of this immunoreactive, AGE adduct, we used an anti-AGE antibody (RU) as a probe to isolate novel AGE(s) that formed within a reaction mixture of glucose and the model glycation substrate, N(alpha)-CBZ-Arg-Lys. HPLC purification of an immunoreactive fraction that accumulated in this preparation showed the presence of a compound that was determined by (1)H NMR and electrospray ionization mass spectrometry (ESI-MS) to be a stable, imidazole-based cross-link (termed arginine-lysine imidazole or ALI). The properties of ALI, immunoreactivity, acid-lability, nonfluorescence, and inhibition of formation by aminoguanidine, suggest that ALI is likely to typify an important class of the AGE cross-links that form in vivo.     

In Vitro Study on Structural Alteration of Myoglobin by Methylglyoxal

Methylglyoxal (MG), a reactive α-oxoaldehyde, reacts with proteins to form irreversible advanced glycation end products (AGEs) following Maillard-like reaction. MG-induced AGE (MAGE) formation may be significant, particularly in diabetic condition with increased level of MG. Although myoglobin (Mb) is known to react with sugars to form AGEs, its interaction with MG is not known. Here we have studied interaction of Mb with MG. After in vitro reaction between Mb and MG at 25 °C for 7 days, the unchanged Mb and modified Mb (MG-Mb) were separated by ion exchange chromatography. Compared to Mb, MG-Mb exhibited higher electrophoretic mobility in native polyacrylamide gel electrophoresis, increased absorbance around 280 nm and more α-helical content, indicating structural changes of the modified protein. As shown by MALDI-mass spectrometry, MG converted Lys-16 and Lys-133 to carboxyethyllysine in MG-Mb. MAGE thus formed in MG-Mb may be associated with its enhanced mobility in native gel due to neutralization of positive charges and the observed structural changes in comparison with Mb.     

Increased glyoxalase I levels inhibit accumulation of oxidative stress and an advanced glycation end product in mouse mesangial cells cultured in high glucose

Chronic high glucose levels lead to the formation of advanced glycation end-products (AGEs) as well as AGE precursors, such as methylglyoxal (MG) and glyoxal, via non-enzymatic glycation reactions in patients with diabetic mellitus. Glyoxalase 1 (GLO-1) detoxifies reactive dicarbonyls that form AGEs. To investigate the interaction between AGEs and GLO-1 in mesangial cells (MCs) under diabetic conditions, AGE levels and markers of oxidative stress were measured in GLO-1-overexpressing MCs (GLO-1-MCs) cultured in high glucose. Furthermore, we also examined levels of high glucose-induced apoptosis in GLO-1-MCs. In glomerular MCs, high glucose levels increased the formation of both MG and argpyrimidine (an MG-derived adduct) as well as GLO-1 expression. GLO-1-MCs had lower intracellular levels of MG accumulation, 8-hydroxy-deoxyguanosine (an oxidative DNA damage marker), 4-hydroxyl-2-nonenal (a lipid peroxidation product), and nitrosylated protein (a marker of oxidative-nitrosative stress) compared to control cells. Expression of mitochondrial oxidative phosphorylation complexes I, II, and III was also decreased in GLO-1-MCs. Furthermore, fewer GLO-1-MCs showed evidence of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling assay, and activation of both poly (ADP-ribose) polymerase 1 cleavage and caspase-3 was lower in GLO-1-MCs than in control cells cultured in high glucose. These results suggest that GLO-1 plays a role in high glucose-mediated signaling by reducing MG accumulation and oxidative stress in diabetes mellitus.     

Effect of pyridoxamine on chemical modification of proteins by carbonyls in diabetic rats: characterization of a major product from the reaction of pyridoxamine and methylglyoxal

Advanced glycation end products (AGEs) from the Maillard reaction contribute to protein aging and the pathogenesis of age- and diabetes-associated complications. The alpha-dicarbonyl compound methylglyoxal (MG) is an important intermediate in AGE synthesis. Recent studies suggest that pyridoxamine inhibits formation of advanced glycation and lipoxidation products. We wanted to determine if pyridoxamine could inhibit MG-mediated Maillard reactions and thereby prevent AGE formation. When lens proteins were incubated with MG at 37 degrees C, pH 7.4, we found that pyridoxamine inhibits formation of methylglyoxal-derived AGEs concentration dependently. Pyridoxamine reduces MG levels in red blood cells and plasma and blocks formation of methylglyoxal-lysine dimer in plasma proteins from diabetic rats and it prevents pentosidine (an AGE derived from sugars) from forming in plasma proteins. Pyridoxamine also decreases formation of protein carbonyls and thiobarbituric-acid-reactive substances in plasma proteins from diabetic rats. Pyridoxamine treatment did not restore erythrocyte glutathione (which was reduced by almost half) in diabetic animals, but it enhanced erythrocyte glyoxalase I activity. We isolated a major product of the reaction between MG and pyridoxamine and identified it as methylglyoxal-pyridoxamine dimer. Our studies show that pyridoxamine reduces oxidative stress and AGE formation. We suspect that a direct interaction of pyridoxamine with MG partly accounts for AGE inhibition.     

Glucose Modification of Human Serum Albumin: A Structural Study

Structural changes associated with the exposure of human serum albumin (HSA) to glucose with or without the presence of Cu (II) have been characterized using a bank of methods for structural analysis including circular dichroism (CD), amino acid analysis (AAA), fluorescence measurements, SDS-PAGE, and boronate binding (which is a measure of Amadori product formation). We show that in the short-term (10 d) incubation mixtures, HSA is resistant to Cu (II)-mediated oxidative damage and that the early products of glycation of HSA had minimal effects on the folded structure. Amino acid analysis showed that there was no formation of advanced glycation endproducts (AGE), which can be measured by loss of lysine. This remained the case in longer term incubation of HSA (56 d) in the hyperglycemic concentration range (5–25 mM glucose) despite increased levels of Amadori product (60% boronate binding) and the formation of glycophore (Excitation 350, Emission 425). At high, nonphysiological concentrations (100 mM and 500 mM) of glucose, glycophore formation increased and 3 and 11 mol Lysine-glucose adduct/mol HSA were converted to AGE, respectively. This was accompanied by increased damage to tryptophan and protein-protein crosslinking but only minor tertiary structural change. In the presence of Cu (II), however, AGE formation was accompanied by extensive damage to histidine and tryptophan side chains, main chain fragmentation, and loss of both secondary and tertiary structure. Thus, changes in structure appear to be the result of oxidation as opposed to glycation, per se. © 1997 Elsevier Science Inc.     

Capillary electrophoretic analysis of advanced glycation endproducts formed from the reaction of reducing sugars with the amino group of glucosamine

Glucosamine (GlcN) is an amino sugar sold over-the-counter and is widely used as a dietary supplement to relieve symptoms of osteoarthritis. It is not known whether it is the GlcN alone or one of its many possible nonenzymatic glycation products that is responsible for this effect. The current study demonstrates that reducing sugars form advanced glycation endproducts (AGEs) with GlcN and, as a result, decrease GlcN autocondensation by reducing the availability of the GlcN amino group. Capillary electrophoresis (CE) was used to analyze the in vitro Maillard reaction of GlcN with glyceraldehyde (GA), glucose (Glc), and fructose (Fru) as well as their inhibition of GlcN autocondensation under physiological conditions. Formation of AGEs was monitored by UV and fluorescence spectroscopy. Major components were separated by CE using a bare capillary and UV detection at 214 nm. AGE species were separated by HPLC and were complementary to the CE results. The effects of sugar concentration and incubation time on the AGE profile are also reported for each of the GlcN reducing sugar model systems. A simple and rapid CE method was developed to analyze the AGE formation in this initial report of the reaction of reducing sugars with the amino group of GlcN.     

Immunochemical detection of advanced glycosylation end products in vivo.

Reducing sugars react with protein amino groups to form a diverse group of protein-bound moieties with fluorescent and cross-linking properties. These compounds, called advanced glycosylation end products (AGEs), have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. Although several AGEs have been identified on the basis of de novo synthesis and tissue isolation procedures, the measurement of AGE compounds in vivo has remained difficult. As an approach to the study of AGE formation in vivo, we prepared polyclonal antiserum to an AGE epitope(s) which forms in vitro after incubation of glucose with ribonuclease (RNase). This antiserum proved suitable for the detection of AGEs which form in vivo. Both diabetic tissue and serum known to contain elevated levels of AGEs readily competed for antibody binding. Cross-reactivity studies revealed the presence of a common AGE epitope(s) which forms after the incubation of diverse proteins with glucose. Cross-reactive epitopes also formed with glucose 6-phosphate or fructose. These data suggest that tissue AGEs which form in vivo appear to contain a common immunological epitope which cross-reacts with AGEs prepared in vitro, supporting the concept that immunologically similar AGE structures form from the incubation of sugars with different proteins (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332). None of the known AGEs, such as 4-furanyl-2-furoyl-1H-imidazole, 1-alkyl-2-formyl-3,4-diglycosylpyrrole, pyrraline, carboxymethyllysine, or pentosidine, were found to compete for binding to anti-AGE antibody. These data further suggest that the dominant AGE epitope which forms from the reaction of glucose with proteins under native conditions is immunologically distinct from the structurally defined AGEs described to date.     

Monitoring nonenzymatic glycation of human immunoglobulin G by methylglyoxal and glyoxal: A spectroscopic study

The accumulation of dicarbonyl compounds, methylglyoxal (MG) and glyoxal (G), has been observed in diabetic conditions. They are formed from nonoxidative mechanisms in anaerobic glycolysis and lipid peroxidation, and they act as advanced glycation endproduct (AGE) precursors. The objective of this study was to monitor and characterize the AGE formation of human immunoglobulin G (hIgG) by MG and G using ultraviolet (UV) and fluorescence spectroscopy, circular dichroism (CD), and matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS). hIgG was incubated over time with MG and G at different concentrations. Formation of AGE was monitored by UV and fluorescence spectroscopy. The effect of AGE formation on secondary structure of hIgG was studied by CD. Comparison of AGE profile for MG and G was performed by MALDI–MS. Both MG and G formed AGE, with MG being nearly twice as reactive as G. The combination of these techniques is a convenient method for evaluating and characterizing the AGE proteins.     

Nonenzymatic glycation of DNA nucleosides with reducing sugars

Reducing sugars can react with the free amino groups of proteins to form a heterogeneous group of compounds known as advanced glycation endproducts (AGEs) or Maillard reaction products. The objective of this investigation was to monitor the nonenzymatic glycation of DNA nucleosides and to characterize the formation of nucleoside AGEs using capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), UV fluorescence spectroscopy, and mass spectrometry. Deoxyguanosine, deoxyadenosine, deoxythymidine, and deoxycytidine were used as the model nucleosides and were incubated over time with glucose, galactose, or glyceraldehyde. Under increasing concentrations and time, deoxyguanosine exhibited the highest rate of glycation with glyceraldehyde. Deoxyadenosine and deoxycytidine exhibited comparable reactivity with glyceraldehyde and no appreciable reactivity with galactose or glucose. No reactivity was observed between deoxythymidine and the sugars. A combination of CE, HPLC, UV fluorescence spectroscopy, and mass spectrometry provided a convenient method for characterizing nucleoside AGEs and for monitoring the physical factors that influence the formation of sugar adducts of DNA nucleosides.     

L-Arginine inhibits in vitro nonenzymatic glycation and advanced glycosylated end product formation of human serum albumin

Summary L-Arginine (Arg) has a structure similar to that of aminoguanidine (AG) and may inhibit glycation and advanced glycosylated end product (AGE) formation. Human serum albumin (HSA) (100mg/ml) was incubated for 2 weeks with glucose (200mM) at 37°C or with glucose and equimolar concentrations of Arg, N--acetyl Arg, or AG with or without 25mM diethylenetriaminepentaacetic acid (DTPA). In the absence of DTPA, electrospray ionization mass spectrometry showed a 70% reduction of covalently bound glucose in the presence of Arg and a 30% reduction with AG. Digestibility by trypsin of HSA incubated with glucose and Arg was similar to that of HSA incubated alone. This suggests less covalent modification of HSA in the presence of Arg as compared with the absence of Arg. When incubations contained DTPA, autoradiography showed less¹⁴C labeling of HSA subunits in the presence of Arg and AG. When the-amino group of Arg was blocked with an acetyl group, labeling was similar to that of HSA incubated with glucose, suggesting involvement of the-amino group in the inhibition. Fluorescence of HSA at ex₃₇₀ and em₄₄₀ was reduced with Arg, but AG was more effective than Arg. These results suggest that Arg, like AG, can inhibit glycation and AGE formation.Presented in part at the FASEB meeting, Atlanta, GA, 1991.     

Below the radar: advanced glycation end products that detour "around the side". Is HbA1c not an accurate enough predictor of long term progression and glycaemic control in diabetes?

Advanced glycation is the irreversible attachment of reducing sugars onto the free amino groups of proteins. Its physiological roles are thought to include the identification of senescent proteins and hence there is a time dependent accumulation of advanced glycation end products (AGEs). AGE labelled proteins are catabolised by cells into low molecular weight peptides and amino acids and excreted primarily via the kidneys. This process appears to be tightly controlled by AGE clearance receptor complexes containing AGE-R1, AGE-R2 and AGE-R3 and scavenger receptors such as CD36, SR-AII and SR-BI. Conditions such as diabetes, however, which have a metabolic overload of reducing sugars, rapidly accelerate AGE formation. In addition, advanced glycation is facilitated by oxidative stress and renal disease even in the absence of increases in reducing sugar concentrations. As part of our western diet, we also ingest AGEs of which approximately 50-80% are absorbed, catabolised and excreted over a period of two days. As AGE levels rise during diabetes, interruption of normal function occurs via three distinct mechanisms, namely AGE induced cross-linking of extracellular matrices, stiffening elastic fibres, disturbing cellular adhesion and preventing turnover. The second is by intracellular formation of AGEs, which causes generalised cellular dysfunction. The third is via the chronic activation of specific receptors such as RAGE, the receptor for advanced glycation end products, which produces excesses in inflammatory molecule production. Due to the range of dysfunction produced by the accumulation of AGEs in diabetes, there is a growing need for early recognition and intervention in this process.     

The anti-apoptotic activity of albumin for endothelium is inhibited by advanced glycation end products restricting intramolecular movement

Human serum albumin (HSA) inhibits endothelial apoptosis in a highly specific manner. CNBr fragmentation greatly increases the effectiveness of this activity, suggesting that this type of protection is mediated by a partially cryptic albumin domain which is transiently exposed by intramolecular movement. Advanced glycation end-product (AGE) formation in HSA greatly reduces its intra-molecular movement. This study aimed to determine if this inhibits the anti-apoptotic activity of HSA, and if such inactivation could be reversed by CNBr fragmentation. HSA-AGE was prepared by incubating HSA with glucose, and assessed using the fructosamine assay, mass spectrometry, SDS-PAGE and fluorometry. Low levels of AGE in the HSA had little effect upon its anti-apoptotic activity, but when the levels of AGE were high and the intra-molecular movement was reduced, endothelial cell survival was also found to be reduced to levels equivalent to those in cultures without HSA or serum (p > 0.001). Survival was restored by the inclusion of native HSA, despite the presence of HSA with high levels of AGE. Also, CNBr fragmentation of otherwise inactive HSA-AGE restored the anti-apoptotic activity for endothelium. Apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and fluorescence-activated cell sorting analysis, and there was no evidence for direct toxicity in the HSA-AGE preparations. The results are consistent with the proposed role of intra-molecular movement in exposing the anti-apoptotic domain in HSA for endothelium. The levels of AGE formation required to inhibit the anti-apoptotic activity of HSA exceeded those reported for diabetes. Nonetheless, the data from this study seems to be the first example of reduced protein function due to AGE-restricted intra-molecular movement.     

Acetoacetate promotes the formation of fluorescent advanced glycation end products (AGEs)

Acetoacetate (AA) is an important ketone body, which produces reactive oxygen species (ROS). Advanced glycation end products (AGEs) are defined as final products of glycation process whose production is influenced by the levels of ROS. The accumulation of AGEs in the body contributes to pathogenesis of many diseases including complications of diabetes, and Alzheimer’s and Parkinson’s disease. Here, we evaluated the impact of AA on production of AGEs upon incubation of human serum albumin (HSA) with glucose. The effect of AA on the AGEs formation of HSA was studied under physiological conditions after incubation with glucose for 35 days. The physical techniques including circular dichroism (CD) and fluorescence spectroscopy were used to assess the impact of AA on formation and structural changes of glycated HSA (GHSA). Our results indicated that the secondary and tertiary structural changes of GHSA were increased in the presence of AA. The fluorescence intensity measurements of AGEs also showed an increase in AGEs formation. Acetoacetate has an activator effect in formation of AGEs through ROS production. The presence of AA may result in enhanced glycation in the presence of glucose and severity of complications associated with accumulation of AGEs.     

The pro-oxidant role of methylglyoxal in mesenteric artery smooth muscle cells

Methylglyoxal (MG), a highly reactive metabolite of glucose, causes non-enzymatic glycation of proteins to form irreversible advanced glycation endproducts (AGEs). The present study investigated whether methylglyoxal induced oxidative stress and activated nuclear factor kappa B (NF-kappaB) in freshly isolated and cultured smooth muscle cells (SMCs) from rat mesenteric artery. The treatment of cells with MG (50 or 100 micromol/L) induced a significant increase in AGE formation and oxidation of DCF. MG-enhanced generation of AGEs and the oxidation of DCF was markedly inhibited by antioxidant n-acetylcysteine (NAC, 600 micromol/L). MG at a concentration of 100 micromol/L increased the heme-oxygenase-1 expression in these cells. Moreover, MG activated NF-kappaB p65, indicated by an increased immuno cytochemistry stain for NF-kappaB p65 located in the nucleus after the treatment of mesenteric artery SMCs with MG. MG-induced activation of NF-kappaB p65 was inhibited by NAC. In summary, MG significantly increases oxidative stress and activates NF-kappaB p65 in mesenteric artery SMCs. The pro-oxidant role of methylglyoxal may contribute to various pathological changes of SMCs from resistance arteries.     

Pyridoxamine traps intermediates in lipid peroxidation reactions in vivo: evidence on the role of lipids in chemical modification of protein and development of diabetic complications

Maillard or browning reactions between reducing sugars and protein lead to formation of advanced glycation end products (AGEs) and are thought to contribute to the pathogenesis of diabetic complications. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) inhibit both the formation of AGEs and development of complications in animal models of diabetes. PM also inhibits the chemical modification of protein by advanced lipoxidation end products (ALEs) during lipid peroxidation reactions in vitro. We show here that several PM adducts, formed in incubations of PM with linoleate and arachidonate in vitro, are also excreted in the urine of PM-treated animals. The PM adducts N-nonanedioyl-PM (derived from linoleate), N-pentanedioyl-PM, N-pyrrolo-PM, and N-(2-formyl)-pyrrolo-PM (derived from arachidonate), and N-formyl-PM and N-hexanoyl-PM (derived from both fatty acids) were quantified by liquid chromatography-mass spectrometry analysis of rat urine. Levels of these adducts were increased 5-10-fold in the urine of PM-treated diabetic and hyperlipidemic rats, compared with control animals. We conclude that the PM functions, at least in part, by trapping intermediates in AGE/ALE formation and propose a mechanism for PM inhibition of AGE/ALE formation involving cleavage of alpha-dicarbonyl intermediates in glycoxidation and lipoxidation reactions. We also conclude that ALEs derived from polyunsaturated fatty acids are increased in diabetes and hyperlipidemia and may contribute to development of long term renal and vascular pathology in these diseases.