RESUMEN
Pioglitazone is in the class of compounds known as the thiazolidinediones and is used to treat type 2 diabetes mellitus. The first in its class compound, troglitazone, was withdrawn from the U.S. market in 2000 due to a high incidence of hepatotoxicity and drug-induced liver failure. Reactive ring-opened products of troglitazone have been identified and evidence suggests that these reactive intermediates might be a potential cause of hepatotoxicity. The present work shows that pioglitazone has a reactive ring-opened product which was trapped by glutathione and positively identified by high performance liquid chromatography with tandem mass spectrometry accurate mass measurements. The novel thiazolidinedione ring-opened products of pioglitazone were identified in rat and human liver microsomes and in freshly isolated rat but not human hepatocytes.
Asunto(s)
Hepatocitos/metabolismo , Microsomas Hepáticos/metabolismo , Tiazolidinedionas/metabolismo , Animales , Hepatocitos/química , Humanos , Masculino , Microsomas Hepáticos/química , Pioglitazona , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Tiazolidinedionas/análisis , Tiazolidinedionas/químicaRESUMEN
PURPOSE: Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO-LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes. Similar modifications occurring in vivo in diabetes may contribute to retinopathy. The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL. METHODS: Minimal in vitro modification of LDL (3 days, 37 degrees C) was achieved with glucose (0, 50 mM), under antioxidant conditions (for N-LDL, G-LDL), or under mild oxidant conditions (for MO-, GO-LDL) in the presence/absence of aminoguanidine (0, 1, 10, 100 microM). Glucose and aminoguanidine were then removed by dialysis. Confluent bovine retinal capillary endothelial cells (n = 13) and pericytes (n = 14) were exposed to LDL (100 mg/l) for 3 days, with and without aminoguanidine (100 microM) in media. Cell counts were determined by hemocytometer. RESULTS: A decrease in cell counts after exposure to modified compared with N-LDL was confirmed (P < 0.001) but was significantly mitigated if LDL had been modified in the presence of aminoguanidine (P < 0.001). Aminoguanidine was as effective at 1 microM as at the higher concentrations. Aminoguanidine (100 microM) present in culture media conferred no additional protection, and showed slight evidence of toxicity. Aminoguanidine present during LDL modification had no effect on measured glycation or oxidation products, or on LDL oxidizability. CONCLUSIONS: Very low concentrations of aminoguanidine mitigate toxicity of LDL exposed to stresses that simulate the diabetic environment. This action may contribute to the beneficial effects of aminoguanidine observed in experimental diabetic retinopathy.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Guanidinas/farmacología , Lipoproteínas LDL/toxicidad , Pericitos/efectos de los fármacos , Vasos Retinianos/efectos de los fármacos , Animales , Capilares/citología , Capilares/efectos de los fármacos , Bovinos , Recuento de Células , Células Cultivadas , Endotelio Vascular/citología , Glicosilación , Oxidación-Reducción , Pericitos/citología , Vasos Retinianos/citologíaRESUMEN
The glycoxidation products Nepsilon-(carboxymethyl)lysine and pentosidine increase in skin collagen with age and at an accelerated rate in diabetes. Their age-adjusted concentrations in skin collagen are correlated with the severity of diabetic complications. To determine the relative roles of increased glycation and/or oxidation in the accelerated formation of glycoxidation products in diabetes, we measured levels of amino acid oxidation products, distinct from glycoxidative modifications of amino acids, as independent indicators of oxidative stress and damage to collagen in aging and diabetes. We show that ortho-tyrosine and methionine sulfoxide are formed in concert with Nepsilon-(carboxymethyl)lysine and pentosidine during glycoxidation of collagen in vitro, and that they also increase with age in human skin collagen. The age-adjusted levels of these oxidized amino acids in collagen was the same in diabetic and nondiabetic subjects, arguing that diabetes per se does not cause an increase in oxidative stress or damage to extracellular matrix proteins. These results provide evidence for an age-dependent increase in oxidative damage to collagen and support previous conclusions that the increase in glycoxidation products in skin collagen in diabetes can be explained by the increase in glycemia alone, without invoking a generalized, diabetes-dependent increase in oxidative stress.
Asunto(s)
Colágeno/metabolismo , Diabetes Mellitus/metabolismo , Metionina/análogos & derivados , Piel/metabolismo , Tirosina/metabolismo , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Arginina/análogos & derivados , Arginina/metabolismo , Niño , Preescolar , Glucosa/farmacología , Humanos , Lactante , Recién Nacido , Lisina/análogos & derivados , Lisina/metabolismo , Metionina/metabolismo , Persona de Mediana Edad , Estrés Oxidativo , Factores de TiempoRESUMEN
The amount of advanced glycation end-products (AGE) in tissue proteins increases in diabetes mellitus, and the concentration of a subclass of AGEs, known as glycoxidation products, also increases with chronological age in proteins. The rate of accumulation of glycoxidation products is accelerated in diabetes and age-adjusted concentrations of two glycoxidation products, N epsilon-(carboxymethyl)lysine (CML) and pentosidine, correlate with the severity of complication in diabetic patients. Although AGEs and glycoxidation products are implicated in the development of diabetic complications, these compounds are present at only trace concentrations in tissue proteins and account for only a fraction of the chemical modifications in AGE proteins prepared in vitro. The future of the AGE hypothesis depends on the chemical characterization of a significant fraction of the total AGEs in tissue proteins, a quantitative assessment of their effects on protein structure and function, and an assessment of their role as mediators of biological responses. In this manuscript we describe recent work leading to characterization of new AGEs and glycoxidation products. These compounds include: (1) the imidazolone adduct formed by reaction of 3-deoxyglucosone with arginine residues in protein; (2) N epsilon-(carboxyethyl)lysine, an analogue of CML formed on reaction of methylglyoxal with lysine; (3) glyoxal-lysine dimer; and (4) methyl-glyoxal-lysine dimer, which are imidazolium crosslinks formed by reaction of glyoxal or methylglyoxal with lysine residues in protein. The presence of 3-deoxyglucosone, methylglyoxal and glyoxal in vivo and the formation of the above AGEs in model carbonyl-amine reaction systems suggests that these AGEs are also formed in vivo and contribute to tissue damage resulting from the Maillard reaction.
Asunto(s)
Productos Finales de Glicación Avanzada/metabolismo , Reacción de Maillard , Proteínas/metabolismo , Envejecimiento/metabolismo , Arginina/análogos & derivados , Arginina/metabolismo , Biomarcadores , Complicaciones de la Diabetes , Diabetes Mellitus/metabolismo , Productos Finales de Glicación Avanzada/química , Glicosilación , Humanos , Lisina/análogos & derivados , Lisina/metabolismo , Estructura Molecular , Norleucina/análogos & derivados , Norleucina/metabolismo , Oxidación-Reducción , Pirroles/metabolismoRESUMEN
Glycoxidation products (GOPs), such as N epsilon-(carboxymethyl)lysine (CML) and pentosidine, are formed during reaction of glucose with protein under oxidative conditions in vitro. It is uncertain whether these GOPs are derived from oxidation of Amadori adducts on protein or from oxidation of glucose or intermediates formed prior to the Amadori rearrangement. To address this question, we reacted collagen with 250 mM glucose in 200 mM phosphate buffer, pH 7.4, under antioxidative conditions, yielding a protein rich in Amadori adducts, but with only traces of GOPs. This "preglycated" collagen was then exposed to [13C6]glucose under oxidative conditions, producing both natural and [13C2]-CML. At 200 mM phosphate buffer, [13C2]-CML was the major product, even at low (5 mM) [13C6]glucose concentration, indicating a limited role for Amadori compounds in formation of CML in high phosphate. The relative yields of natural and [13C2]-CML varied with phosphate concentration, becoming similar at more physiological (10 mM) phosphate. We conclude that during glycation of proteins at high phosphate concentrations in vitro, GOPs are formed primarily by oxidation of free glucose or rapidly-formed intermediates preceding the Amadori rearrangement, such as carbinolamine or Schiff base adducts. In contrast, at lower phosphate and glucose concentrations in vivo, the Amadori adduct may be the more significant precursor of GOPs. The fact that glycoxidation reactions proceed by multiple routes must be considered in the development of therapeutic approaches for inhibiting the Maillard reaction in diabetes.
Asunto(s)
Colágeno/química , Glucosa/química , Animales , Arginina/análogos & derivados , Arginina/química , Isótopos de Carbono , Glicosilación , Lisina/análogos & derivados , Lisina/química , Oxidación-Reducción , Fosfatos/farmacología , RatasRESUMEN
To investigate the role of modified low-density lipoproteins (LDL) in the pathogenesis of diabetic retinopathy, we studied the cytotoxicity of normal and mildly modified human LDL to bovine retinal capillary endothelial cells and pericytes in vitro. Pooled LDL was incubated (in phosphate-buffered saline-EDTA, 3 days, 37 degrees C) under 1) nitrogen with additional chelating agents and 2) air, to prepare normal and minimally oxidized LDL, respectively. Similar conditions, but with the addition of 50 mM D-glucose, were used to prepare glycated and glycoxidized LDL. None of the LDL preparations was recognized by the macrophage scavenger receptor, confirming limited modification. Retinal capillary endothelial cells and pericytes were grown to confluence and then exposed for 2 or 3 days to serum-free medium (1% albumin) supplemented with normal or modified LDL (100 mg/l) or to serum-free medium alone. Cytotoxicity was assessed by cell counting (live and total cells) and by cell protein determination. Compared with normal LDL, modified LDL were cytotoxic to both cell types at both time points, causing highly significant decreases in live and total cell counts (P < 0.001) (analysis of variance). Reductions in cell protein also were significant for pericytes at day 3 (P = 0.016) and of borderline significance for endothelial cells at day 2 (P = 0.05) and day 3 (P = 0.063). Cytotoxicity increased as follows: normal < glycated < or = minimally oxidized < glycoxidized LDL. We conclude that, in diabetes, mild modification of LDL resulting from separate or combined processes of glycation and oxidation may contribute to chronic retinal capillary injury and thus to the development of diabetic retinopathy.
Asunto(s)
Capilares/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Lipoproteínas LDL/toxicidad , Proteínas de la Membrana , Músculo Liso Vascular/efectos de los fármacos , Receptores de Lipoproteína , Vasos Retinianos/efectos de los fármacos , Análisis de Varianza , Animales , Capilares/citología , Bovinos , Supervivencia Celular/efectos de los fármacos , Endotelio Vascular/citología , Productos Finales de Glicación Avanzada , Humanos , Lipoproteínas LDL/sangre , Lipoproteínas LDL/aislamiento & purificación , Músculo Liso Vascular/citología , Receptores Inmunológicos/metabolismo , Receptores de LDL/metabolismo , Receptores Depuradores , Vasos Retinianos/citología , Receptores Depuradores de Clase B , Sustancias Reactivas al Ácido Tiobarbitúrico/análisisRESUMEN
To evaluate their usefulness as chemical indicators of cumulative oxidative damage to proteins, we studied the kinetics and extent of formation of ortho-tyrosine (o-Tyr), dityrosine (DT), and dityrosine-like fluorescence (Ex = 317 nm, Em = 407 nm) in the model proteins RNase and lysozyme exposed to radiolytic and metal-catalyzed (H2O2/Cu2+) oxidation (MCO). Although there were protein-dependent differences, o-Tyr, DT, and fluorescence increased coordinately during oxidation of the proteins in both oxidation systems. The contribution of DT to total dityrosine-like fluorescence in oxidized proteins varied from 2-100%, depending on the protein, type of oxidation, and extent of oxidative damage. In proteins exposed to MCO, DT typically accounted for > 50% of the fluorescence at DT wavelengths. These studies indicate that o-Tyr and DT should be useful chemical markers of cumulative exposure of proteins to MCO in vitro and in vivo.
Asunto(s)
Proteínas/química , Tirosina/análogos & derivados , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Peróxido de Hidrógeno/farmacología , Isomerismo , Cinética , Muramidasa/química , Muramidasa/efectos de los fármacos , Muramidasa/efectos de la radiación , Oxidación-Reducción , Proteínas/efectos de los fármacos , Proteínas/efectos de la radiación , Ribonucleasas/química , Ribonucleasas/efectos de los fármacos , Ribonucleasas/efectos de la radiaciónRESUMEN
The concentrations of ortho-tyrosine (o-Tyr) and dityrosine (DT) were measured in noncataractous human lenses in order to assess the role of protein oxidation reactions in the aging of lens proteins. The measurements were conducted by selected ion monitoring-gas chromatography/mass spectrometry using deuterium-labeled internal standards, which provided both high sensitivity and specificity for the quantitation of o-Tyr and DT. Between ages 1 and 78 years, the o-Tyr concentration in lens proteins varied from 0.3 to 0.9 mmol of o-Tyr/mol of Phe (n = 19), while DT ranged from 1 to 3 mumol of DT/mol of Tyr (n = 30). There were no significant changes in levels of o-Tyr with lens age. There was a statistically significant, but only slight, increase in DT in lens proteins with age (approximately 33% increases between ages 1 and 78, r = 0.5, p < 0.01). At the same time, total protein fluorescence, measured at DT wavelengths (Ex = 317 nm, Em = 407 nm), increased 11-fold between ages 1 and 78 and correlated strongly with age (r = 0.82, p < 0.0001). Although the fluorescence maxima of lens proteins were similar to those of DT, DT accounted for less than 1% of the DT-like fluorescence in lens protein at all ages. These observations indicate that oxidation of Phe and Tyr plays a limited role in the normal aging of lens proteins in vivo.