ascorbic-acid and pimagedine

The treatment of diabetes mellitus aftermaths became one of medicine's most significant therapeutical and financial issues in the XXI century. Most of which are related to protein glycation and oxidative stress caused by long lasting periods of hyperglycemia. Thus, even within a venerable one, searching for new drugs, displaying anti-glycation and anti-oxidative properties seem useful as an additive therapy of diabetes. In this paper, we assessed the anti-glycating properties of phloroglucinol, a drug discovered in the XIX century and still used in many countries for its antispasmodic action. Herewith, we present its effect on protein glycation, glycoxidation, and oxidative damage in an albumin glycation/oxidation model and HepG2 cells treated with high glucose concentrations. The phloroglucinol showed the strongest and the widest protective effect within all analyzed antiglycating (aminoguanidine, pioglitazone) and anti-oxidative (vitamin C, GSH) agents. To the very best of our knowledge, this is the first study showing the properties of phloroglucinol in vitro what once is proven in other models might deepen its clinical applications.

Topics: Albumins; Antioxidants; Ascorbic Acid; Glucose; Glycation End Products, Advanced; Guanidines; Hep G2 Cells; Hepatocytes; Humans; Inflammation; Nitrosative Stress; Oxidative Stress; Phloroglucinol; Pioglitazone; Reactive Oxygen Species

We explored the association between the activities of antioxidant enzymes and their metallic cofactors in rats treated with cisplatin. The antioxidant effects of aminoguanidine, and a combination of vitamins E and C were investigated. Plasma platin was significantly lower than liver and kidney. Cisplatin treatment caused significant increase in plasma Se-glutathione peroxidase activity. Activities of Se-glutathione peroxidase, glutathione S-transferase, catalase and Cu,Zn-superoxide dismutase have been found to be significantly decreased in liver and kidney compared to controls. Zn levels in these organs were diminished upon cisplatin treatment, while levels of Cu were unaffected. Interestingly, levels of iron, the cofactor of catalase, were found to be significantly increased in liver and kidney. Intervention with aminoguanidine or vitamins was generally prevented cisplatin-caused changes in the activity of enzymes and in the tissue levels of cofactor metals. These observations suggest that relation between activities of enzymes and levels of cofactor metals is multifactorial.

Topics: Animals; Antineoplastic Agents; Antioxidants; Ascorbic Acid; Catalase; Cisplatin; Coenzymes; Enzymes; Glutathione Peroxidase; Glutathione Transferase; Guanidines; Kidney; Liver; Male; Metals; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Vitamin E

Arsenic enhances the genotoxicity of other carcinogenic agents such as ultraviolet radiation and benzo[a]pyrene. Recent reports suggest that inhibition of DNA repair is an important aspect of arsenic cocarcinogenesis, and DNA repair proteins such as poly(ADP ribose) polymerase (PARP)-1 are direct molecular targets of arsenic. Although arsenic has been shown to generate reactive oxygen/nitrogen species (ROS/RNS), little is known about the role of arsenic-induced ROS/RNS in the mechanism underlying arsenic inhibition of DNA repair. We report herein that arsenite-generated ROS/RNS inhibits PARP-1 activity in cells. Cellular exposure to arsenite, as well as hydrogen peroxide and NONOate (nitric oxide donor), decreased PARP-1 zinc content, enzymatic activity, and PARP-1 DNA binding. Furthermore, the effects of arsenite on PARP-1 activity, DNA binding, and zinc content were partially reversed by the antioxidant ascorbic acid, catalase, and the NOS inhibitor, aminoguanidine. Most importantly, arsenite incubation with purified PARP-1 protein in vitro did not alter PARP-1 activity or DNA-binding ability, whereas hydrogen peroxide or NONOate retained PARP-1 inhibitory activity. These results strongly suggest that cellular generation of ROS/RNS plays an important role in arsenite inhibition of PARP-1 activity, leading to the loss of PARP-1 DNA-binding ability and enzymatic activity.

Topics: Arsenites; Ascorbic Acid; Cells, Cultured; DNA; Guanidines; Humans; Hydrogen Peroxide; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Reactive Nitrogen Species; Reactive Oxygen Species; Zinc; Zinc Fingers

Density functional theory calculations [CPCM/UM06/6-31+G(d,p)] were used to elucidate the structures and relative stability of Fe(III) complexes with various ligands that inhibit the formation of advanced glycation end products (AGEs) or iron overloaded disease (viz. aminoguanidine, pyridoxamine, LR-74, Amadori compounds, and ascorbic acid). EDTA was used as the free energy reference ligand. The distorted neutral octahedral complex containing one iron atom and three molecules of pyridoxamine [Fe(PM)(3)] was found to be the most stable. The stability of the complexes decreases in the following chelate sequence: pyridoxamine, Amadori complex, aminoguanidine, LR inhibitor, and ascorbic acid.

Topics: Ascorbic Acid; Coordination Complexes; Edetic Acid; Ferric Compounds; Glycation End Products, Advanced; Guanidines; Kinetics; Ligands; Molecular Structure; Propionates; Pyridoxamine; Quantum Theory; Quinolines; Thermodynamics

The aim of this article is to investigate the effects of Aminoguanidine and vitamin C (VitC) on type IV collagen in diabetic nephropathy rats. Diabetic nephropathy rats were induced by intraperitoneal injection of STZ. Rats were randomly divided into five groups: normal control group (n = 10), diabetes group (n = 10), aminoguanidine group (n = 10), VitC group (n = 10), aminoguanidine and VitC group (n = 10). After 16 weeks, the general conditions, blood gloucose, glycosylated hemoglobin, blood urea nitrogen, serum creatinine, serum type IV collagen, urinary albumin excretion rate, and creatinine clearance rate were detected, type IV collagen protein was determined by immunohistochemical analysis as well as the expression of collagen type IVα1 mRNA were determined by in situ hybridization analysis in the kidneys of each group. The results were (1) diabetes mellitus and renal lesions occurred in the diabetes group, aminoguanidine group, VitC group, VitC and aminoguanidine group; (2) aminoguanidine and VitC improved the general conditions of diabetic nephropathy rats, decreased blood urea nitrogen, serum creatinine, and urinary albumin excretion rate as well as increased creatinine clearance rate. The expressions of collagen type IV were significantly down-regulated in treatment groups in contrast to the diabetes group. Aminoguanidine and VitC protect renal lesions in diabetic nephropathy, respectively, by inhibiting expression of type IV collagen, while aminoguanidine and VitC have a synergistic effect on them.

Topics: Animals; Ascorbic Acid; Collagen Type IV; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Drug Synergism; Glycated Hemoglobin; Guanidines; Immunohistochemistry; Kidney; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger

In this study, the antioxidant effect of aminoguanidine on nephrotoxicity of a single dose of cisplatin is investigated and compared with the effects of well-known antioxidants vitamin C and E combination. Tubular damage and perivascular inflammation were observed in kidney samples of the cisplatin-administered groups. Aminoguanidine and vitamin C-E combination are found to be capable of preventing these effects of cisplatin. Liver tissues of all groups were intact. Cisplatin-induced oxidative stress was evidenced by significant decrease in glutathione and significant increase in malondialdehyde levels in kidney samples. Antioxidants with cisplatin decreased malondialdehyde levels. Antioxidants with cisplatin prevented the decrease in liver glutathione levels. The nephrotoxicity was confirmed biochemically by significant elevation of serum urea and creatinine levels. Both vitamin C-E combination and aminoguanidine prevented the increase in serum urea levels according to the cisplatin group.

Topics: Animals; Antineoplastic Agents; Antioxidants; Ascorbic Acid; Chromatography, High Pressure Liquid; Cisplatin; Enzyme Inhibitors; Glutathione; Guanidines; Kidney Diseases; Male; Malondialdehyde; Oxidative Stress; Rats; Rats, Sprague-Dawley; Urea; Vitamin E

Two experiments were conducted to evaluate the effects of arginine, vitamin E (VE), and vitamin C (VC) on cardiopulmonary performance and ascites parameters of broilers reared under a cold environmental temperature. One-day-old male broilers were fed a basal corn-soybean meal diet (control, 1.2% arginine and 40 IU of VE), or the basal diet supplemented with 1% arginine and either 200 IU vitamin E (AE), 500 mg of vitamin C (AC), or a combination of VE and VC at the same amounts (AEC) per kilogram of feed. Pulmonary arterial pressure (PAP) and mean arterial pressure were recorded in clinically healthy, anesthetized birds (28 to 42 d old) before and after an epinephrine (Epi) challenge (0.5 mg/kg of BW, i.v.), an aminoguanidine hemisulfate challenge (100 mg/kg of BW, i.v.), and an N-nitro-l-arginine methyl ester challenge (50 mg/kg of BW, i.v.) at 20-min intervals. Data were analyzed by repeated measures ANOVA, and the Student Newman-Keuls test was used to separate means within groups. The PAP increased 30 s after the Epi challenge in all birds, but the peak PAP was lower in the AEC group than in all the other groups, whereas birds in the AE and AC groups had lower PAP peaks than did the control group. After 120 s of challenge, the PAP was lower in AEC birds compared with the other birds. The PAP returned to pre-Epi amounts within 300 s in all groups. The PAP was increased (P < 0.05) within 60 s after the aminoguanidine hemisulfate and N-nitro-l-arginine methyl ester challenges in all groups, but no differences were found among groups. The mean arterial pressure responses did not differ among groups. Plasma NO was greater in the AEC group than in all the other groups before and after the Epi challenge. These results showed that Epi elicited lower amplitude PAP and less prolonged increases in PAP in birds from the AEC group, and this may have been related to the increased vasodilation attributable to NO production. The AEC may have had complementary effects against oxidative stress, protecting the endothelium and preserving NO function.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Arginine; Ascorbic Acid; Blood Pressure; Chickens; Diet; Drug Therapy, Combination; Epinephrine; Guanidines; Male; Nitric Oxide; Vitamin E

The glycation reaction involves a series of non-enzymatic reactions between the carbonyl group on reducing sugars and the amino group on proteins leading to the formation of advanced glycation end-products (AGEs), which are acknowledged to be involved in the pathogenesis of diabetic and aging-related complications. Consequently, the development of AGE inhibitors is considered to have therapeutic potential in patients with diabetes or age-related diseases. The preliminary results showed that a methanol extract (PAE) of Plantago asiatica, which is traditionally used as a folk medicine in Asian countries to treat fever, cough, wound etc., had strong glycation inhibitory activity. The effects of the extract on AGE fluorescence were dose-dependent, reaching 41% inhibition at 0.1 microg/mL of extract. The purified principle from PAE was identified as plantamajoside. As well as antioxidant activities, in vitro glycation inhibitory activities with 10 and 25 mm plantamajoside were higher than those with 10 and 25 mm aminoguanidine. The results demonstrate that PAE and plantamajoside had significant effects on in vitro AGE formation, and the glycation inhibitory activity and antioxidant activity of plantamajoside were comparable to those obtained using millimolar concentrations of the standard antiglycation agent aminoguanidine, and the antioxidant ascorbate, respectively.

Topics: Antioxidants; Ascorbic Acid; Biphenyl Compounds; Catechols; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Glucosides; Glycation End Products, Advanced; Guanidines; Magnetic Resonance Spectroscopy; Methanol; Phenols; Picrates; Plant Extracts; Plantago; Polyphenols

Amadori compounds act as precursors in the formation of advanced glycation end products (AGEs) by non-enzymatic protein glycation, which are involved in ensuing protein damage. Pyridoxamine is a potent drug against protein glycation, and can act on several pathways in the glycation process. Nevertheless, the pyridoxamine inhibition action on Amadori compounds oxidation is still unclear. In this work, we have studied the Schiff base formation between pyridoxamine and various Amadori models at pH 7.4 at 37 degrees C in the presence of NaCNBH(3). We detected an adduct formation, which suggests that pyridoxamine reacts with the carbonyl group in Amadori compounds. The significance of this mechanism is tested by comparison of the obtained kinetics rate constants with that obtained for 4-(aminomethyl)-pyridine, a structural analogue of pyridoxamine without post-Amadori action. We also study the chelating effect of pyridoxamine on metal ions. We have determined the complexation equilibrium constants between pyridoxamine, N-(1-deoxy-d-fructos-1-yl)-l-tryptophan, aminoguanidine, and ascorbic acid in the presence of Zn(2+). The results show that the strong stability of pyridoxamine complexes is the key in its post-Amadori inhibition action. On the other hand results explain the lack of inhibition of aminoguanidine (a glycation inhibitor) in the post-Amadori reactions.

Topics: Amino Acids; Ascorbic Acid; Chelating Agents; Chromatography, High Pressure Liquid; Free Radical Scavengers; Fructose; Glucose; Glycation End Products, Advanced; Glycosylation; Guanidines; Kinetics; Ligands; Magnetic Resonance Spectroscopy; Molecular Structure; Organometallic Compounds; Oxidation-Reduction; Pyridoxamine; Schiff Bases; Stereoisomerism; Time Factors; Tryptophan; Zinc

In this study, we investigate the protective effects of eugenosedin-A on p38 mitogen-activated protein kinase (MAPK), inflammatory nitric oxide (NO) and cyclooxygenase-2 (COX-2) pathways in a rat model of endotoxin shock. Rats were pretreated with eugenosedin-A, trazodone, yohimbine (1 mg kg(-1), i.v.), aminoguanidine or ascorbic acid (15 mg kg(-1), i.v.) 30 min before endotoxin challenge. Endotoxaemia was induced by a single i.v. injection of lipopolysaccharide (LPS, 10 mg kg(-1)). In rats not treated with eugenosedin-A, LPS increased plasma concentrations of NO and prostaglandin E(2) (PGE(2)), and levels of p38 MAPK, inducible NO synthase (iNOS) and COX-2 proteins in the liver, lung, aorta and lymphocytes. In the pre-treated rats, eugenosedin-A not only inhibited the LPS-induced NO and PGE(2) levels but also attenuated the LPS-induced increase in p38 MAPK and iNOS levels in the liver, aorta and lymphocytes. Eugenosedin-A also reduced LPS-induced COX-2 proteins in the aorta and lymphocytes. Likewise, aminoguanidine, ascorbic acid, yohimbine and trazodone were also found to decrease NO and PGE(2) concentrations after endotoxin challenge. While aminoguanidine and ascorbic acid also attenuated the LPS-induced increase in p38 MAPK, iNOS and COX-2 proteins in the aorta and lymphocytes, trazodone and yohimbine inhibited only the increase in p38 MAPK, iNOS and COX-2 proteins in lymphocytes. Finally, eugenosedin-A (10(-10)-10(-8) M) significantly inhibited the biphasic response induced by hydrogen peroxide (10(-6)-3 x 10(-5) M) in rat denudated aorta. Taken together, the results of this study indicate that eugenosedin-A, as well as ascorbic acid, can attenuate free-radical-mediated aortic contraction and relaxation. It may therefore be able to reduce the damage caused by septic shock by inhibiting formation of p38 MAPK, iNOS, COX-2 and free radicals.

Topics: Adrenergic alpha-Antagonists; Animals; Antioxidants; Aorta, Thoracic; Ascorbic Acid; Blotting, Western; Cyclooxygenase 2; Endotoxemia; Guanidines; Hydrogen Peroxide; In Vitro Techniques; Lipopolysaccharides; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide Synthase Type II; Organ Specificity; p38 Mitogen-Activated Protein Kinases; Piperazines; Rats; Rats, Wistar; Serotonin Antagonists; Trazodone; Up-Regulation; Yohimbine

The water-soluble and cell permeable nitroxide derivative 4-hydroxy tempol (TPL) has been shown to reduce or ameliorate oxidative stress-induced dysfunction and damage in vascular endothelial cells. We studied the effects of TPL on glucose transport and metabolism in bovine aortic endothelial (VEC) and smooth muscle cells (VSMC) under normal and high glucose conditions. Normally, these cells operate an autoregulatory protective mechanism that limits the rate of glucose transport under hyperglycemic conditions by decreasing the cell content of their typical glucose transporter GLUT-1 mRNA and protein as well as its plasma membrane abundance. TPL augmented the rate of glucose transport both under normo- and hyperglycemic conditions by increasing GLUT-1 mRNA and protein content and its plasma membrane abundance in both types of cells, leading to an increased flux of glucose into the cells. These effects were found related to ROS-generating and oxidant activities of TPL and to a decreased rate of mitochondrial ATP production under both normo- and hyperglycemic conditions. Since impaired mitochondrial functions, and in particular decreased rate of ATP production, augment the expression of GLUT-1 protein and glucose transport and metabolism, we suggest that the stimulatory effects of TPL in vascular cells results from its unfavorable interactions in the mitochondrion. It is therefore suggested that effects of TPL in cells of cardiovascular system be evaluated in parallel to its adverse effects on glucose and energy metabolism.

Topics: Acetylcysteine; Adenosine Triphosphate; Animals; Antioxidants; Ascorbic Acid; Biological Transport; Cattle; Cell Membrane; Chromans; Cyclic N-Oxides; Endothelium, Vascular; Glucose; Glucose Transporter Type 1; Guanidines; Mitochondria; Monosaccharide Transport Proteins; Myocytes, Smooth Muscle; RNA, Messenger; Spin Labels

We recently reported that PL-AG, a Schiff base of pyridoxal and aminoguanidine, was more effective than aminoguanidine (AG), a well-known anti-diabetic-complication compound, in preventing nephropathy in diabetic mice and presented brief data indicating the antioxidant activity of the adduct. In the present study, we additionally investigated the inhibitory activity of PL-AG in comparison with that of AG against in vitro and in vivo oxidation. PL-AG was more potent than AG and reference compounds such as pyridoxal and pyridoxamine in any of the five antioxidant activities examined in vitro, i.e., hydrogen peroxide-scavenging, hydroxyl radical-scavenging, superoxide radical-scavenging, ascorbic acid-autoxidation inhibitory, and low-density lipoprotein (LDL)-oxidation inhibitory activities, the last two of which were assessed in the presence of Cu(2+). Unlike AG, PL-AG did not show the pro-oxidant activity. The inhibitory activity of PL-AG against lipid peroxidation in diabetic rats was higher than that of AG, for example, the amounts of malondialdehyde in erythrocytes (nmol/g hemoglobin; mean +/- SD) in normal, untreated diabetic, AG-treated diabetic, and PL-AG-treated diabetic rats were 3.53 +/- 0.35, 4.99 +/- 0.23, 4.65 +/- 0.45, and 4.06 +/- 0.35, respectively. A fluorescent substance different from PL-AG was found in the plasma and urine of rats treated with PL-AG. The chemical structure of this substance, i.e., oxidized PL-AG, was determined by a combination of nuclear magnetic resonance, mass, and infrared spectrometry. AG dramatically decreased the pyridoxal phosphate level in the diabetic rat liver, whereas PL-AG only moderately affected it. Our results indicate that the antioxidant activity of PL-AG is due to its chelation with transition metal ions and to scavenging of reactive oxygen species. They also suggest that PL-AG is more promising for the treatment of diabetic complications than AG.

Topics: Animals; Antioxidants; Ascorbic Acid; Aspartate Aminotransferases; Benzoates; Blood Glucose; Body Weight; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Diabetes Mellitus; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Erythrocytes; Fluorescent Dyes; Guanidines; Hydrogen Peroxide; Ions; Lipid Peroxidation; Lipoproteins, LDL; Liver; Magnetic Resonance Spectroscopy; Male; Malondialdehyde; Mass Spectrometry; Mice; Microsomes, Liver; Models, Chemical; Oxidants; Oxygen; Pyridoxal; Rats; Rats, Wistar; Reactive Oxygen Species; Spectrophotometry; Spectrophotometry, Infrared; Superoxides; Time Factors

Endogenously produced dicarbonyls, such as methylglyoxal (MG), are involved in advanced glycation end-product formation and thus linked to the pathophysiology of diabetic chronic complications. While the search for synthetic new antiglycation agents continues, little attention has been paid to putative antiglycation agents in natural compounds. Given the link between glycation and oxidation, in this work, we study the effects of methylglyoxal on two model systems; plasminogen and antithrombin III (AT III), then we set out to unravel a possible antiglycation effect for extracts of the flavonoid-rich common herbal species Achyrocline satureoides (AS) and Ilex paraguariensis (IP). Using SAR-PRO-ARG-pNA as a specific thrombin substrate, we show that incubation of plasma with MG decreases heparin activation of AT III by up to a 70%, in a dose-dependent manner. A parallel dose-dependent decrease in plasminogen activity reaching more than 50% was shown using D-BUT-CHT-lys-pNA as a plasmin-specific substrate. Extracts of AS and IP display a dose dependent inhibition of the action of the dicarbonyl, already significant at a 1/100 dilution of the herbal infusions. The inhibition was comparable to that obtained by using millimolar concentrations of known AGE inhibitors such as aminoguanidine and carnosine as well as micromolar concentrations of the antioxidant ascorbic acid. We believe our system of whole plasma glycation over 16 h with micromolar concentrations of MG, coupled with the measurement of activities of plasminogen and AT III by specific substrates provides a straightforward, practical method for monitoring the action of putative antiglycation agents. If predictably milder glycated forms of AT III and plasminogen were to be secreted in vivo, the loss of activities shown here could act synergistically to generate hyperthrombicity.

Topics: Achyrocline; Antioxidants; Antithrombin III; Ascorbic Acid; Carnosine; Dose-Response Relationship, Drug; Guanidines; Ilex paraguariensis; Kinetics; Models, Biological; Plant Extracts; Plasminogen; Pyruvaldehyde

Glycation is a process whereby sugar molecules form a covalent adduct with protein amino groups. In this study, we used ascorbic acid (AsA) as a glycating agent and purified cucumber (Cucumis sativus L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a model protein in chloroplast tissues, and examined effects of glycation on the activity and susceptibility of Rubisco to proteases. Glycation proceeded via two phases during incubation with AsA and Rubisco in vitro at physiological conditions (10 mM AsA, pH 7.5, 25 degrees C in the presence of atmospheric oxygen). At the early stage of glycation (phase 1), the amount of AsA attaching to Rubisco increased at an almost linear rate (0.5-0.7 mol AsA incorporated (mol Rubisco)(-1) d(-1)). By Western blotting using monoclonal antibodies recognizing glycation adducts, a major glycation adduct, N( epsilon )-(carboxymethyl)lysine was detected. At the late stage of glycation (phase 2), incorporation of AsA reached saturation, and a glycation adduct, pentosidine mediating intramolecular cross-linking, was detected corresponding to formation of high molecular weight aggregates cross-linked between subunits. Glycation led to a decrease in Rubisco activity (half-life about 7-8 d). Furthermore, glycated Rubisco of phase 2 drastically increased protease susceptibility in contrast to unchanged susceptibility of glycated Rubisco of phase 1 compared to that of native Rubisco. Results obtained here suggest that AsA is possibly an important factor in the loss of activity and turnover of Rubisco.

Topics: Ascorbic Acid; Blotting, Western; Carbon Radioisotopes; Chymotrypsin; Endopeptidases; Enzyme Stability; Glutathione; Glycation End Products, Advanced; Guanidines; Immunochemistry; Lysine; Ribulose-Bisphosphate Carboxylase

The toxicity of the beta-amyloid (Abeta) peptide of Alzheimer's disease may relate to its polymerisation state (i.e. fibril content). We have shown previously that plasma lipoproteins, particularly when oxidised, greatly enhance Abeta polymerisation. In the present study the nature of the interactions between both native and oxidised lipoproteins and Abeta1-40 was investigated employing various chemical treatments. The addition of ascorbic acid or the vitamin E analogue, trolox, to lipoprotein/Abeta coincubations failed to inhibit Abeta fibrillogenesis, as did the treatment of lipoproteins with the aldehyde reductant, sodium borohydride. The putative lipid peroxide-derived aldehyde scavenger, aminoguanidine, however, inhibited Abeta-oxidised lipoprotein-potentiated polymerisation, but in a manner consistent with an antioxidant action for the drug. Lipoprotein treatment with the reactive aldehyde 4-hydroxy-2-trans-nonenal enhanced Abeta polymerisation in a concentration-dependent fashion. Incubation of Abeta with lipoprotein fractions from which the apoprotein components had been removed resulted in extents of polymerisation comparable to those observed with Abeta alone. These data indicate that the apoprotein components of plasma lipoproteins play a key role in promoting Abeta polymerisation, possibly via interactions with aldehydes.

Topics: Aldehydes; Alzheimer Disease; Amyloid beta-Peptides; Antioxidants; Apolipoproteins; Ascorbic Acid; Biopolymers; Borohydrides; Chromans; Guanidines; Humans; Kinetics; Lipoproteins; Oxidation-Reduction; Peptide Fragments

Antioxidants and antioxidant enzymes are known to protect against cell death induced by reactive oxygen species. However, apart from directly quenching free radicals, little is known about the effect of antioxidants on hormone-activated second messenger systems. We previously found that antioxidants such as 17-beta estradiol and resveratrol activate membrane-bound guanylate cyclase GC-A, the receptor for atrial natriuretic factor (ANF), in PC12 cells. It is possible that other antioxidants may also activate membrane-bound guanylate cyclase GC-A. The aim of this study was to determine if dithiothreitol (DTT), vitamin C, and vitamin E activate membrane-bound guanylate cyclase GC-A in PC12 cells. The results showed that both DTT and vitamin C increased cGMP levels in PC12 cells, whereas vitamin E had no effect. DTT and vitamin C inhibited membrane-bound guanylate cyclase activity stimulated by ANF in PC12 cells. In contrast, DTT and vitamin C had no effect on soluble guanylate cyclase activity stimulated by substance P. Furthermore, NO synthase inhibitors L-NAME and aminoguanidine did not affect DTT- and vitamin C-stimulated guanylate cyclase activity. The results indicate that DTT and vitamin C, but not vitamin E, activate membrane-bound guanylate cyclase GC-A in PC12 cells.

Topics: Animals; Antioxidants; Ascorbic Acid; Atrial Natriuretic Factor; Cell Membrane; Dithiothreitol; Enzyme Activators; Enzyme Inhibitors; Guanidines; Guanylate Cyclase; NG-Nitroarginine Methyl Ester; PC12 Cells; Rats; Substance P; Vitamin E

The advanced glycation end-product (AGE) hypothesis proposes that accelerated chemical modification of proteins by glucose during hyperglycemia contributes to the pathogenesis of diabetic complications. The two most commonly measured AGEs, N(epsilon)-(carboxymethyl)lysine and pentosidine, are glycoxidation products, formed from glucose by sequential glycation and autoxidation reactions. Although several compounds have been developed as AGE inhibitors and are being tested in animal models of diabetes and in clinical trials, the mechanism of action of these inhibitors is poorly understood. In general, they are thought to function as nucleophilic traps for reactive carbonyl intermediates in the formation of AGEs; however alternative mechanisms of actions, such as chelation, have not been rigorously examined. To distinguish between the carbonyl trapping and antioxidant activity of AGE inhibitors, we have measured the chelating activity of the inhibitors by determining the concentration required for 50% inhibition of the rate of copper-catalyzed autoxidation of ascorbic acid in phosphate buffer. All AGE inhibitors studied were chelators of copper, as measured by inhibition of metal-catalyzed autoxidation of ascorbate. Apparent binding constants for copper ranged from approximately 2 mm for aminoguanidine and pyridoxamine, to 10-100 microm for carnosine, phenazinediamine, OPB-9195 and tenilsetam. The AGE-breakers, phenacylthiazolium and phenacyldimethylthiazolium bromide, and their hydrolysis products, were among the most potent inhibitors of ascorbate oxidation. We conclude that, at millimolar concentrations of AGE inhibitors used in many in vitro studies, inhibition of AGE formation results primarily from the chelating or antioxidant activity of the AGE inhibitors, rather than their carbonyl trapping activity. Further, at therapeutic concentrations, the chelating activity of AGE inhibitors and AGE-breakers may contribute to their inhibition of AGE formation and protection against development of diabetic complications.

Topics: Amines; Antioxidants; Ascorbic Acid; Bromides; Chelating Agents; Copper; Diabetes Complications; Diabetes Mellitus; Enzyme Inhibitors; Glycation End Products, Advanced; Guanidines; Oxidation-Reduction; Pyridoxamine; Serum Albumin, Bovine

Characteristics of cellular insulin glycation were examined in the pancreatic B-cell line, BRIN-BD11. The extent of insulin glycation increased stepwise during 72 h of culture at 5.6-33.3 mmol/l glucose, attaining levels up to 27%. Glycation of insulin at 33.3 mmol/l glucose was rapid, reaching maximal values within 2 h, and not readily reversible during 2 to 24 h of subsequent exposure to 5.6 mmol/l glucose. Glycated insulin was readily secreted by BRIN-BD11 cells upon active stimulation with glucose and other secretagogues. Cellular insulin glycation was decreased by 66-80% by inhibitors of protein glycation, vitamin C, aminoguanidine or acetylsalicylic acid. Modulation of insulin-secretory activity of BRIN-BD11 cells by co-culture at high glucose with diazoxide. L-alanine or glibenclamide indicated that long-term stimulation of secretion was associated with a decrease in the extent of insulin glycation. Glycation of insulin in vitro was substantially less extensive than in BRIN-BD11 cells, although glucose-6-phosphate and glyceraldehyde-3-phosphate were 1.4- to 2.0-fold more reactive than glucose per se. These observations indicate that insulin is readily glycated and secreted from insulin-secreting cells under hyperglycaemic conditions in culture.

Topics: Ascorbic Acid; Aspirin; Cell Line; Diazoxide; Enzyme Inhibitors; Glucose; Glyburide; Glycosylation; Guanidines; Humans; Hypoglycemic Agents; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Stimulation, Chemical; Time Factors

Topics: Alanine; Animals; Ascorbic Acid; Aspirin; Cell Fusion; Cell Line; Chromatography, Affinity; Glucose; Glycosylation; Guanidines; Insulin; Insulin Secretion; Islets of Langerhans; Potassium; Rats

The ageing of connective tissues involves modifications of collagen, which are currently generating much interest amongst protein researchers. Protein glycation, a non-enzymic reaction involving sugar, appears to play a role in the evolution of age-related physical changes and diabetic complications-retinopathy, neuropathy, renal failure and atherosclerosis. Our studies show that the glycation of human corneal and scleral collagen produces increases in the collagen intermolecular spacing-these increases are similar to those we previously reported on the ageing of collagen in these tissues. The present investigation employs X-ray diffraction to look at the structural effects of various substances that are believed in inhibit protein glycation. Aspirin-like compounds and certain vitamins successfully prevented the sugar-induced molecular changes from occurring in corneal and scleral collagen, suggesting that such compounds could have a useful role in this aspect of ageing.

Topics: Acetaminophen; Aged; Aging; Anti-Inflammatory Agents, Non-Steroidal; Ascorbic Acid; Aspirin; Cellular Senescence; Chelating Agents; Collagen; Cornea; Culture Techniques; Enzyme Inhibitors; Glycosylation; Guanidines; Humans; Ibuprofen; Pentetic Acid; Sclera; Vitamin E; Vitamins

Several lines of evidence support a role for oxidized low density lipoprotein (LDL) in the genesis of the atherosclerotic lesion. Hence, the effect of compounds with antioxidant properties on LDL oxidation assumes great significance. Ascorbate, a potent water-soluble chain-breaking antioxidant, has been shown to inhibit LDL oxidation. Aminoguanidine (AMG) is a pharmacological inhibitor of advanced non-enzymatic glycosylation. Recently it has been suggested that aminoguanidine might have an inhibitory effect on LDL oxidation, but total lipid peroxidation assayed by conjugated diene formation was not inhibited. Thus, in this study, we compared the effect of aminoguanidine with ascorbate to obtain a better appreciation of the effect of AMG on Cu(2+)-catalyzed LDL oxidation. Oxidative modification of LDL was monitored by assaying intermediates and end products of lipid peroxidation, conjugated dienes (CD), lipid peroxides (LPO), and relative electrophoretic mobility (REM). Apolipoprotein B-100 modification (increased fluorescence, fragmentation on SDS-PAGE, and 125I-labeled LDL degradation by human macrophages) was also measured. Ascorbate (100 microM) inhibited LDL oxidation by > 95%, as evidenced by all of the selected indices. Aminoguanidine (20 mM) substantially decreased thiobarbituric acid-reactive substances (TBARS) activity and lipid peroxide formation, but only partially prevented the increase of REM (-55%), apoB fluorescence (-39%), and degradation by macrophages (-54%). Unlike ascorbate, AMG failed to preserve alpha-tocopherol in LDL, prevent apoB-100 fragmentation, or inhibit conjugated diene formation during LDL oxidation. Furthermore, incubation of AMG with already oxidized LDL resulted in a significant decrease in TBARS activity and LPO, and 26.9% decrease in the REM of LDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Apolipoprotein B-100; Apolipoproteins B; Ascorbic Acid; Copper; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Guanidines; Humans; Kinetics; Lipid Peroxidation; Lipoproteins, LDL; Macrophages; Oxidation-Reduction; Thiobarbituric Acid Reactive Substances

A simple, quantitative assay has been established to determine the glycation-dependent crosslinking ability of any sugar by measuring the incorporation of [14C]-lysine into protein. The assay was shown to be both sugar-dependent and protein-dependent and was completely inhibited by sodium cyanoborohydride, 2-aminoguanidine, and semicarbazide. A typical 1.0-ml reaction mixture contained 5 mg lysozyme, 20 mumol threose, and 5 microCi of [14C]lysine and exhibited an incorporation of 8 x 10(5) cpm (1.6 nmol of lysine) after 7 days of incubation. A comparison of the crosslinking ability of a variety of sugars showed glyceraldehyde and dihydroxyacetone to be twice as active as erythrose and threose and eight times more reactive than ribose. Little or no crosslinking could be demonstrated with three different hexoses as well as their phosphorylated derivatives. The dicarbonyl sugars 3-deoxyglucosone and xylosone were at least as effective as ribose in crosslinking, as were the oxidation products of ascorbic acid. Several amine-containing compounds were tested as inhibitors of crosslinking; however, 2-aminoguanidine was the most effective. The rate of synthesis of Lys-Lys, Lys-Arg, and Lys-His crosslinks was determined by measuring the incorporation of [14C]lysine into specific amino acid homopolymers. The relative incorporation was polylysine > polyarginine > polyhistidine with threose, but polyarginine > polyhistidine > polylysine with dehydroascorbic acid, suggesting a different crosslinking mechanism for these two compounds.

Topics: Amino Acids; Ascorbic Acid; Carbohydrate Metabolism; Carbon Radioisotopes; Cross-Linking Reagents; Glycosylation; Guanidines; Kinetics; Lysine; Oxidation-Reduction; Proteins

Nonenzymatic glycosylation of serum albumin was studied in the presence of naturally occurring metabolites, pyridoxal, pyridoxal phosphate and ascorbate/dehydroascorbate, and a hydrazine compound, aminoguanidine. Pyridoxal, pyridoxal phosphate, ascorbate and dehydroascorbate, at concentrations of 0.1 mM or greater, significantly inhibited the nonenzymatic glycosylation of albumin. Aminoguanidine was the most potent inhibitor of nonenzymatic glycosylation and 54% or 85% inhibition occurred when 5 or 50 mM aminoguanidine, respectively, was present in the incubation mixture containing 20 mM glucose. A major effect of aminoguanidine was to lower the free glucose concentration in the incubation mixture by a direct reaction with glucose as judged by thin layer chromatography. The present studies suggest that vital metabolites such as pyridoxal phosphate and ascorbate may be potentially important in controlling glucose-induced nonenzymatic glycosylation of proteins. Pyridoxal phosphate forms a Schiff base with proteins as does glucose and therefore may be a preferable drug, over aminoguanidine which is a hydrazine, for inhibiting the effects of glucose-induced nonenzymatic glycosylation.

Topics: Ascorbic Acid; Glycosylation; Guanidines; Kinetics; Pyridoxal Phosphate; Serum Albumin, Bovine