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July 2009

Clinical potential of advanced glycation end-product inhibitors in diabetes mellitus.

Non-enzymatic accumulation of advanced glycation end-products (AGE) is to some extent a physiologic consequence of tissue aging. On the other hand, circulating AGE and tissue deposits mark the course of diabetes mellitus as well as a variety of other vascular or degenerative diseases. AGE generation is paralleled by oxidative damage and lipid peroxidation within target tissue, with features of inflammation through the involvement of monocytes/macrophages expressing receptors for glycated macromolecules. Over the past 15 years, a wealth of data concerning the pharmacology of AGE have been gathered through animal and human investigations, targeting their likely contribution to the progression of diabetic and non-diabetic vascular damage. Several agents have been shown to interfere with the formation of AGE or AGE precursors, bind to tissue receptors, or promote breakdown of deposits. The first and most studied inhibitor, aminoguanidine, has shown extensive beneficial effects in experimental models of diabetic vascular damage, recently entering phase I-III clinical investigation. Newer anti-AGE agents include pyridoxamine and the so-called ‘amadorins’, cross-link breakers, AGE binders and receptor antagonists.

Am J Cardiovasc Drugs. 2003;3(5):315-20

An in vitro approach to the chronological aging of skin by glycation of the collagen: the biological effect of glycation on the reconstructed skin model.

Glycation is a slow, nonenzymatic reaction that takes place between free amino groups in proteins primarily from lysine and a reducing sugar such as glucose or ribose. In skin, this reaction creates new residues or formations of cross-links (advanced glycation end products, AGEs) in the extracellular matrix of the dermis. The formation of these bridges between dermal molecules is supposed to be responsible for loss of elasticity or other properties of the dermis observed during aging. Glycation may therefore play an important role in chronologic aging. In order to examine this hypothesis, we have developed a reconstructed skin model made of a modified dermal compartment that is a fibroblast-contracted collagen lattice prepared with preglycated collagen. The presence of AGEs (glycoxidation products) in the skin equivalents was evidenced using specific antibodies against carboxymethyllysine (CML). Several changes were observed after collagen glycation: (1) fibroblast shape and distribution (vimentin staining) were modified; (2) extracellular matrix molecules and the dermal-epidermal junction zone seemed to be enhanced (procollagen I and III, collagen IV and VII stainings); (3) stainings for beta1 and alpha6 integrins were also increased in the epidermal cell layer; and (4) collagenase activity was increased. To verify the biological effect of glycation, we used the well-known glycation inhibitor aminoguanidine. After aminoguanidine treatment, we found a low CML amount and decreased distribution of markers previously overexpressed in glycated skin constructs. These in vitro findings were at least in part related to aging in vivo and demonstrate an actual effect of glycation in skin aging.

Ann N Y Acad Sci. 2005 Jun;1043:529-32

Novel inhibitors of glycation and AGE formation.

Accelerated formation of advanced glycation/lipoxidation and endproducts (AGEs/ALEs) has been implicated in the pathogenesis of various diabetic complications. Several natural and synthetic compounds have been proposed and tested as inhibitors of AGE/ALE formation. We have previously reported the therapeutic effects of several new AGE/ALE inhibitors on the prevention of nephropathy and dyslipidemia in streptozotocin (STZ)-induced diabetic rats. In this study, we investigated the effects of various concentrations of a compound, LR-90, on the progression of renal disease and its effects on AGE and receptor for AGE (RAGE) protein expression on the kidneys of diabetic STZ-rats. Diabetic male Sprague-Dawley rats were treated with or without LR-90 (0, 5, 20, 25, and 50 mg/l of drinking water). After 32 weeks, body weight, glycemic status, renal function, and plasma lipids were measured. Kidney histopathology and AGE/ALE accumulation and RAGE protein expression in tissues were also determined. In vitro studies were also performed to determine the possible mechanism of action of LR-90 in inhibiting AGE formation and AGE-protein cross-linking. LR-90 protected the diabetic kidneys by inhibiting the increase in urinary albumin-to-creatinine ratio and ameliorated hyperlipidemia in diabetic rats in a concentration-dependent fashion without any effects on hyperglycemia. LR-90 treatment also reduced kidney AGE/ALE accumulation and RAGE protein expression in a concentration-dependent manner. In vitro, LR-90 exhibited general antioxidant properties by inhibiting metal-catalyzed reactions and reactive oxygen species (OH radical) and reactive carbonyl species (methlyglyoxal, glyoxal) generations without any effect on pyridoxal 5’ phosphate. The compound also prevents AGE-protein cross-linking reactions. These findings demonstrate the bioefficacy of LR-90 in treating nephropathy and hyperlipidemia in diabetic animals by inhibiting AGE accumulation, RAGE protein expression, and protein oxidation in the diabetic kidney. Additionally, our study suggests that LR-90 may be useful also to delay the onset and progression of diabetic atherosclerosis as the compound can inhibit the expression of RAGE and inflammation-related pathology, as well as prevent lipid peroxidation reactions.

Cell Biochem Biophys. 2007;48(2-3):147-57

Inhibition of crystallin ascorbylation by nucleophilic compounds in the hSVCT2 mouse model of lenticular aging.

PURPOSE: Senile cataracts are associated with oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. This process is partially explained by advanced glycation end products (AGEs) from ascorbic acid (ASA), as the authors unequivocally demonstrated in an hSVCT2 transgenic mouse. The authors present the first pharmacologic intervention study against ascorbylation in these mice. METHODS: Five groups of mice from 2 to 9 months of age (10 mice/group) were fed a diet containing 0.1% (wt/wt) aminoguanidine, pyridoxamine, penicillamine, and nucleophilic compounds NC-I and NC-II. AGEs were determined in crystallin digests using high-performance liquid chromatography, liquid chromatography-mass spectrometry, or gas chromatography-mass spectrometry. Lens protein extract was incubated in vitro with ASA or dehydroascorbic acid. RESULTS: The ASA level increased approximately 10-fold in all groups and was unaffected by treatment. AGEs were increased several-fold in transgenic compared with control lenses. Body weight, food intake, lenticular glutathione, and glycated lysine level were unaltered. In vitro, all compounds inhibited AGE formation. In vivo, NC-I and NC-II significantly decreased protein fluorescence at lambda(ex)335/(em)385 (P = 0.045, P = 0.017, respectively) and lambda(ex)370/(em)440 (P = 0.029, P = 0.007, respectively). Other inhibitors had no effect. After 7 months, only NC-I and NC-II induced a 50% reduction in pentosidine (P = NS for NC-I; P = 0.035 for NC-II). NC-I also decreased carboxymethyllysine (P = 0.032) and carboxyethyllysine (P = NS). Fluorescent cross-link K2P was decreased by NC-I, NC-II, aminoguanidine, and pyridoxamine (P = NS). CONCLUSIONS: Pharmacologically blocking protein ascorbylation with absorbable guanidino compounds is feasible and may represent a new strategy for the delay of age-related nuclear sclerosis of the lens.

Invest Ophthalmol Vis Sci. 2008 Nov;49(11):4945-52

Preventing cell death induced by carbonyl stress, oxidative stress or mitochondrial toxins with vitamin B anti-AGE agents.

Carbonyls generated by autoxidation of carbohydrates or lipid peroxidation have been implicated in advanced glycation end product (AGE) formation in tissues adversely affected by diabetes complications. Tissue AGE and associated pathology have been decreased by vitamin B(1)/B(6) in trials involving diabetic animal models. To understand the molecular cytoprotective mechanisms involved, the effects of B(1)/B(6) vitamers against cytotoxicity induced by AGE/advanced lipid end product (ALE) carbonyl precursors (glyoxal/acrolein) have been compared to cytotoxicity induced by oxidative stress (hydroperoxide) or mitochondrial toxins (cyanide/copper). Thiamin was found to be best at preventing cell death induced by carbonyl stress and mitochondrial toxins but not oxidative stress cell death suggesting that thiamin pyrophosphate restored pyruvate and alpha-ketoglutarate dehydrogenases inhibited by mitochondrial toxicity. However, B(6) vitamers were most effective at preventing oxidative stress or lipid peroxidation cytotoxicity suggesting that pyridoxal or pyridoxal phosphate were antioxidants and/or Fe/Cu chelators. A therapeutic vitamin cocktail could provide maximal prevention against carbonyl stress toxicity associated with diabetic complications.

Mol Nutr Food Res. 2008 Mar;52(3):379-85

Renoprotective effects of the AGE-inhibitor pyridoxamine in experimental chronic allograft nephropathy in rats.

BACKGROUND: Advanced glycation end products (AGEs) are involved in diabetic nephropathy (DN). The AGE formation inhibitor pyridoxamine (PM) is renoprotective in DN and in normoglycaemic obese Zucker rats. In chronic allograft nephropathy (CAN), renal AGE accumulation occurs as well. METHODS: To investigate whether inhibition of AGE formation is renoprotective in CAN, we studied the Fisher 344 to Lewis (F-L) allograft rat model of experimental CAN. Fisher to Fisher (F-F) isografts served as controls. Proteinuria, renal function and renal histology of untreated transplanted rats (F-L n = 8, F-F n = 8) were compared to rats receiving PM 2 g/l in drinking water for 20 weeks starting at transplantation (F-L n = 5, F-F n = 10). All rats received cyclosporin A (1.5 mg/kg/day) for 10 days after transplantation to prevent early acute rejection. RESULTS: Compared to untreated allografts, PM significantly decreased proteinuria (76 +/- 18 vs 29 +/- 3 mg/day), serum creatinine (130 +/- 12 vs 98 +/- 5 micromol/l), focal glomerulosclerosis (116 +/- 27 vs 16 +/- 5 AU), glomerular macrophage influx (5.6 +/- 0.6 vs 3.3 +/- 1.0), interstitial fibrosis (132 +/- 24 vs 76 +/- 2 AU) and interstitial macrophage influx (47.0 +/- 8.7 vs 15.4 +/- 5.0. Moreover, PM significantly ameliorated tubular accumulation of pentosidine, compared to untreated allografts (2.5 +/- 0.6 vs 0.3 +/- 0.3, all p < 0.05). In the isograft controls, these values did not differ between untreated and PM treated rats. CONCLUSION: PM exerts renoprotective effects and decreases renal pentosidine accumulation in experimental CAN, suggesting a detrimental role for renal AGE accumulation in the pathogenesis of renal damage in this non-diabetic model. These results indicate that inhibition of AGE formation might be a useful adjunct therapy to attenuate CAN.

Nephrol Dial Transplant. 2008 Feb;23(2):518-24

Could oxidative stress associate with age products in cataractogenesis?

BACKGROUND: Oxidative stress has been reported to contribute to aging and cataract formation in the lens. The aim was to determine the association of oxidative stress with advanced glycation end products (AGEs) in elderly diabetic and non-diabetic patients with cataract. METHODS: In the present study, malondialdehyde, vitamin E, serum AGEs, and glycemic control were investigated. The study included 156 subjects. Out of them, 30 were normal elderly subjects, 31 were elderly diabetic patients without cataract, 33 were elderly diabetic patients with cataract, 32 were elderly non-diabetic with cataract, and 30 were normal young subjects. The patients were selected on clinical grounds from Eye Ward, Jinnah Postgraduate Medical Centre, Karachi, Pakistan. RESULTS: Positive significant correlation was observed between s-AGEs and malondialdehyde in elderly diabetic and non-diabetic patients with cataract. Negative significant correlation was observed between s-AGEs and vitamin E in elderly diabetic and non-diabetic patients with cataract. However, the malondialdehyde and serum AGEs were found to be significantly increased (p < 0.001) in elderly diabetic and non-diabetic patients with and without cataract compared with elderly control subjects. In contrast to all four senile groups, the serum AGEs was significantly lower (p < 0.001) in young control subjects. Serum vitamin E was found to be significantly decreased (p < 0.001) in elderly diabetic patients with and without cataract compared with elderly control subjects. Fasting blood glucose, HbA(1C) and serum fructosamine levels were significantly increased (p < 0.001) in elderly diabetic patients with and without cataract compared with non-diabetic elderly patients with cataract and elderly control subjects. CONCLUSIONS: This study revealed that increased AGEs were associated with oxidative stress in the elderly groups. AGE, as a result of oxidative stress, might have a role in cataract formation, which, in diabetic patients, occurs vigorously as compared with non-diabetic cataract patients.

Curr Eye Res. 2008 Aug;33(8):669-75

DNA damage during glycation of lysine by methylglyoxal: assessment of vitamins in preventing damage.

Amino acids react with methylglyoxal to form advanced glycation end products. This reaction is known to produce free radicals. In this study, cleavage to plasmid DNA was induced by the glycation of lysine with methylglyoxal in the presence of iron(III). This system was found to produce superoxide as well as hydroxyl radicals. The abilities of various vitamins to prevent damage to plasmid DNA were evaluated. Pyridoxal-5-phosphate showed maximum protection, while pyridoxamine showed no protection. The protective abilities could be directly correlated to inhibition of production of hydroxyl and superoxide radicals. Pyridoxal-5-phosphate exhibited low radical scavenging ability as evaluated by its TEAC, but showed maximum protection probably by interfering in free radical production. Pyridoxamine did not inhibit free radical production. Thiamine and thiamine pyrophosphate, both showed protective effects albeit to different extents. Tetrahydrofolic acid showed better antioxidant activity than folic acid but was found to damage DNA by itself probably by superoxide generation.

Amino Acids. 2007 Nov;33(4):615-21

Aminophospholipid glycation and its inhibitor screening system: a new role of pyridoxal 5’-phosphate as the inhibitor.

Peroxidized phospholipid-mediated cytotoxity is involved in the pathophysiology of a number of diseases [i.e., the abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) found in the plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, or Amadori-PE), because Amadori-PE causes oxidative stress. However, lipid glycation inhibitor has not been discovered yet because of the lack of a lipid glycation model useful for inhibitor screening. We optimized and developed a lipid glycation model considering various reaction conditions (glucose concentration, temperature, buffer type, and pH) between PE and glucose. Using the developed model, various protein glycation inhibitors (aminoguanidine, pyridoxamine, and carnosine), antioxidants (ascorbic acid, alpha-tocopherol, quercetin, and rutin), and other food compounds (L-lysine, L-cysteine, pyridoxine, pyridoxal, and pyridoxal 5’-phosphate) were evaluated for their antiglycative properties. Pyridoxal 5’-phosphate and pyridoxal (vitamin B(6) derivatives) were the most effective antiglycative compounds. These pyridoxals could easily be condensed with PE before the glucose/PE reaction occurred. Because PE-pyridoxal 5’-phosphate adduct was detectable in human red blood cells and the increased plasma Amadori-PE concentration in streptozotocin-induced diabetic rats was decreased by dietary supplementation of pyridoxal 5’-phosphate, it is likely that pyridoxal 5’-phosphate acts as a lipid glycation inhibitor in vivo, which possibly contributes to diabetes prevention.

J Lipid Res. 2006 May;47(5):964-74

Availability of vitamin B6 and pantothenate in an average American diet in man.

The availability of vitamin B6 and pantothenate in an average American diet was assessed healthy male volunteers. The subjects received two types of diets, both nutritionally equivalent to the average American diet: period 1 (35 days), semipurified formula diet (low in both vitamins) with daily supplements of 1.1 mg pyridoxine and 8.2 mg pantothenate; period 2 (35 days), natural food sources, providing 2.3 mg vitamin B6 and 11.5 mg pantothenate/day; period 3 (21 days), formula diet, providing 2.7 mg pyridoxine and 8.2 mg pantothenate/day. Daily protein intake was 96 g throughout the study. Vitamins in food and urine samples were determined microbiologically and plasma pyridoxal phosphate by a tyrosine apodecarboxylase radioassay method. Compared to the availability of the pure vitamins as 100%, the availability of vitamin B6 ranged from 61 to 81% with a mean of 71% using plasma pyridoxal phosphate data, and ranged from 73 to 92% with a mean of 79% according to urinary vitamin B6 data. Availability of pantothenate ranged from 40 to 61% with a mean of 50%, according to urinary pantothenate data. The average American diet used in our study contained 1.7 and 5.8 mg/day of available vitamin B6 and pantothenate, respectively.

Am J Clin Nutr. 1981 Jul;34(7):1328-37

Effects of pyridoxal-5’-phosphate (MC-1) in patients undergoing high-risk coronary artery bypass surgery: results of the MEND-CABG randomized study.

OBJECTIVE: Coronary artery bypass graft surgery remains associated with significant postoperative cardiovascular morbidity and mortality in high-risk patients. MC-1 (pyridoxal-5’-phosphate monohydrate) inhibits purinergic receptors and intracellular influx of Ca2+, thereby reducing cellular injury during experimental ischemia and reperfusion. The MEND-CABG trial tested the hypothesis that MC-1 reduces cardiovascular morbidity and mortality after coronary artery bypass graft. METHODS: In a phase 2, double-blinded, placebo-controlled study, 901 patients scheduled for coronary artery bypass graft surgery with cardiopulmonary bypass and at high risk for subsequent cardiac or neurologic complications were randomly assigned to receive oral MC-1 (250 mg or 750 mg/d once daily) or placebo beginning 3 to 10 hours prior to surgery and continued to postoperative day 30. RESULTS: At 30 days, MC-1 250 mg (compared with placebo) reduced the composite of death, nonfatal cerebral infarction, and nonfatal myocardial infarction by 14.0% (P = .3124) with peak creatinine kinase-myocardial band > or =50 ng/mL (prespecified primary end point); 32.3% (P = .0349) with peak creatinine kinase-myocardial band > or =70 ng/mL; and 37.2% (P = .0283) with peak creatinine kinase-myocardial band > or =100 ng/mL. Myocardial infarctions with peak creatinine kinase-myocardial band> or =100 ng/mL were reduced by 47.2% in the MC-1 250-mg group versus placebo (P = .0083). Greater efficacy was demonstrated with 250 mg than with the 750-mg dose of MC-1. CONCLUSIONS: In high-risk patients undergoing coronary artery bypass graft, treatment with MC-1 did not significantly affect the prespecified primary end point but was associated with a significant reduction in perioperative myocardial infarction with creatinine kinase-myocardial band > or =100 ng/mL. A larger, well-powered trial is needed to evaluate the cardioprotective effects of MC-1.

J Thorac Cardiovasc Surg. 2007 Jun;133(6):1604-11

Protective effect of pyridoxal-5-phosphate (MC-1) on perioperative myocardial infarction is independent of aortic cross clamp time: results from the MEND-CABG trial.

AIM: Aortic cross-clamp time remains a significant marker of mortality and morbidity after coronary artery bypass graft (CABG) surgery. Pyridoxal-5-phosphate (MC-1), blocking purinergic receptors and intracellular influx of calcium, was shown to decrease the incidence of perioperative myocardial infarction in the prospective, randomized, double-blinded MC-1 to Eliminate Necrosis and Damage in CABG (MEND-CABG) clinical trial. METHODS: We studied the relationship between treatment with MC-1 and aortic cross-clamping relative to the incidence of cardiovascular (CV) death and myocardial infarction (MI) in the trial that enrolled 901 high-risk patients undergoing CABG with cardiopulmonary bypass. Patients were randomized to receive either placebo, MC-1 250 mg/day or MC-1 750 mg/day starting 3-10 h before CABG and continued for 30 days after surgery. Serial creatine kinase-myocardial band (CK-MB) determinations, ECGs and clinical evaluations were performed. RESULTS: Cross-clamping time increased the event rate of death and MI with an odds ratio (95% confidence interval) of 1.67 (1.17-2.37, P=0.0044). Treatment with MC-1 decreased the rate of events (P=0.0073) with odds ratios of 0.52 (0.31-0.88 for MC-1 250 mg/day versus placebo) and 0.48 (0.29-0.82 for MC-1 750 mg/day versus placebo). There was no interaction between cross-clamp time and treatment (P=0.61) on the occurrence of the combined endpoint. CONCLUSION: MC-1 decreased the incidence of CV death and MI (CK-MB >or=100 ng/mL) during the first 90 days after CABG in the MEND-CABG trial. Although longer aortic clamping time increased the risk of cardiovascular events, the protective effect of MC-1 was independent of ischemic time during CABG.

J Cardiovasc Surg (Torino). 2008 Apr;49(2):249-53

Pyridoxal phosphate and hepatocyte growth factor prevent dialysate-induced peritoneal damage.

Glucose-based peritoneal dialysate (PD) is responsible for increased accumulation of advanced glycation end products (AGE) in the peritoneum of continuous ambulatory peritoneal dialysis patients. Pyridoxal 5’-phosphate (PLP), a derivative of vitamin B(6), protects proteins from glycation. Hepatocyte growth factor (HGF) heals damaged tissues in a reciprocal manner against TGF-beta1. First, with the use of gas chromatography-mass spectrometry, whether PLP traps 3-deoxyglucosone (3DG), a major glucose degradation product in PD, was determined. Then, whether rat peritoneal tissue damages induced by intraperitoneal administration of glucose-based PD is ameliorated by PLP or HGF was examined. In vitro incubation with PLP markedly decreased concentration of 3DG in a dose-dependent manner, demonstrating the 3DG-trapping effect of PLP. The peritoneum of PD-treated rats was significantly thickened compared with that of physiologic saline-treated rats. Both PLP and HGF prevented the thickening of rat peritoneum induced by PD and ameliorated accumulation of AGE and expression of TGF-beta1, vascular endothelial growth factor, and type 1 collagen and a number of blood vessels. Furthermore, expression of HGF was significantly increased in the peritoneum of PLP-treated rats compared with that of PD-treated rats. In conclusion, PLP shows 3DG-trapping effect. PLP and HGF prevented peritoneal thickening; accumulation of AGE; expression of TGF-beta1, vascular endothelial growth factor, and type 1 collagen; and neoangiogenesis in rat peritoneum induced by PD.

J Am Soc Nephrol. 2005 Jan;16(1):144-50

Pyridoxamine protects proteins from functional damage by 3-deoxyglucosone: mechanism of action of pyridoxamine.

Pyridoxamine (PM) is a promising drug candidate for treatment of diabetic nephropathy. The therapeutic effect of PM has been demonstrated in multiple animal models of diabetes and in phase II clinical trials. However, the mechanism of PM therapeutic action is poorly understood. One potential mechanism is scavenging of pathogenic reactive carbonyl species (RCS) found to be elevated in diabetes. We have suggested previously that the pathogenicity of RCS methylglyoxal (MGO) may be due to modification of critical arginine residues in matrix proteins and interference with renal cell-matrix interactions. We have also shown that this MGO effect can be inhibited by PM (Pedchenko et al. (2005) Diabetes 54, 2952-2960). These findings raised the questions of whether the effect is specific to MGO, whether other structurally different physiological RCS can act via the same mechanism, and whether their action is amenable to PM protection. In the present study, we have shown that the important physiological RCS 3-deoxyglucosone (3-DG) can damage protein functionality, including the ability of collagen IV to interact with glomerular mesangial cells. We have also demonstrated that PM can protect against 3-DG-induced protein damage via a novel mechanism that includes transient adduction of 3-DG by PM followed by irreversible PM-mediated oxidative cleavage of 3-DG. Our results suggest that, in diabetic nephropathy, the therapeutic effect of PM is achieved, in part, via protection of renal cell-matrix interactions from damage by a variety of RCS. Our data emphasize the potential importance of the contribution by 3-DG, along with other more reactive RCS, to this pathogenic mechanism.

Biochemistry. 2008 Jan 22;47(3):997-1006

A post-Amadori inhibitor pyridoxamine also inhibits chemical modification of proteins by scavenging carbonyl intermediates of carbohydrate and lipid degradation.

Reactive carbonyl compounds are formed during autoxidation of carbohydrates and peroxidation of lipids. These compounds are intermediates in the formation of advanced glycation end products (AGE) and advanced lipoxidation end products (ALE) in tissue proteins during aging and in chronic disease. We studied the reaction of carbonyl compounds glyoxal (GO) and glycolaldehyde (GLA) with pyridoxamine (PM), a potent post-Amadori inhibitor of AGE formation in vitro and of development of renal and retinal pathology in diabetic animals. PM reacted rapidly with GO and GLA in neutral, aqueous buffer, forming a Schiff base intermediate that cyclized to a hemiaminal adduct by intramolecular reaction with the phenolic hydroxyl group of PM. This bicyclic intermediate dimerized to form a five-ring compound with a central piperazine ring, which was characterized by electrospray ionization-liquid chromatography/mass spectrometry, NMR, and x-ray crystallography. PM also inhibited the modification of lysine residues and loss of enzymatic activity of RNase in the presence of GO and GLA and inhibited formation of the AGE/ALE N(epsilon)-(carboxymethyl)lysine during reaction of GO and GLA with bovine serum albumin. Our data suggest that the AGE/ALE inhibitory activity and the therapeutic effects of PM observed in diabetic animal models depend, at least in part, on its ability to trap reactive carbonyl intermediates in AGE/ALE formation, thereby inhibiting the chemical modification of tissue proteins.

J Biol Chem. 2002 Feb 1;277(5):3397-403

Oral vitamin B12 supplementation reduces plasma total homocysteine concentration in women in India.

People in India have a high prevalence of low vitamin B12 status and high plasma total homocysteine (tHcy) concentrations. In a proof of principle trial, we studied the effect of oral vitamin B12 (500 microg) and/or 100 g cooked green leafy vegetables (GLV) every alternate day in a 2x2 factorial design over a 6-week period. Forty-two non-pregnant vegetarian women (age 20-50 years) were randomly allocated to four study groups. Clinical measurements were made at the beginning and at the end of the study, and blood samples were collected before, and 2 and 6 weeks after commencement of intervention. Forty women completed the trial. Twenty-six women had low vitamin B12 status (<150 pmol/L) and 24 had hyperhomocysteinemia (>15 micromol/L). GLV supplementation did not alter plasma folate or tHcy. Vitamin B12 supplementation increased plasma vitamin B12 concentration (125 to 215 pmol/L, p <0.05) and reduced tHcy concentration (18.0 to 13.0 micromol/L, p <0.05) within first 2 weeks, both of which remained stable for the next 4 weeks. Plasma vitamin B12 and tHcy concentrations did not change in those who did not receive vitamin B12, and there was no change in plasma folate concentration in any of the groups. Blood haemoglobin concentration increased marginally within first two weeks in those women who received vitamin B12 (by 3 g/L, p <0.05) and the number of women with macrocytosis decreased from 2 to zero. There was no change in vibration sensory threshold during the period of the study. High-dose per oral vitamin B12 supplementation significantly reduced plasma tHcy within 2 weeks but did not achieve normal plasma tHcy concentration even after 6 weeks. People in India have a high prevalence of low vitamin B12 status and high plasma total homocysteine (tHcy) concentrations.

Asia Pac J Clin Nutr. 2007;16(1):103-9

Plasma total homocysteine level and bone mineral density: the Hordaland Homocysteine Study.

BACKGROUND: Plasma total homocysteine (tHcy) has been associated with hip fracture but not directly with bone mineral density (BMD). We examined the association of hip BMD with levels of plasma tHcy, folate, and vitamin B12 and the methylenetetrahydrofolate reductase (MTHFR) 677C—>T and 1298A—>C polymorphisms. METHODS: Bone mineral density was measured between 1997 and 2000 in 2,268 men and 3,070 women, aged 47 to 50 and 71 to 75 years, from the Hordaland Homocysteine Study cohort. Low BMD was defined as BMD in the lowest quintile for each sex and age group. Linear, logistic, and generalized additive regression models were used. RESULTS: Plasma levels of tHcy were inversely related to BMD among middle-aged and elderly women (P<.001) but not among men. The multiple adjusted odds ratio for low BMD among subjects with high (>or=15 micromol/L [>or=2.02 mg/L]) compared with low (<9 micromol/L [<1.22 mg/L]) tHcy level was 1.96 (95% confidence interval, 1.40-2.75) for women and was not significant for men. Additional adjustments for plasma folate level or intake of calcium and vitamin D did not substantially alter the results. Plasma folate level was associated with BMD in women only. We observed no association between BMD and vitamin B12 level or the MTHFR polymorphisms. CONCLUSIONS: Elevated tHcy and low folate levels were associated with reduced BMD in women but not in men. These findings suggest that tHcy may be a potential modifiable risk factor for osteoporosis in women.

Arch Intern Med. 2006 Jan 9;166(1):88-94

Chronic kidney disease, prevalence of premature cardiovascular disease, and relationship to short-term mortality.

BACKGROUND: Chronic kidney disease (CKD) is recognized as an independent cardiovascular disease (CVD) risk state, particularly in the elderly, and has been defined by levels of estimated glomerular filtration rate (eGFR) and markers of kidney damage. The relationship between CKD and CVD in younger and middle-aged adults has not been fully explored. METHODS: Community volunteers completed surveys regarding past medical events and underwent blood pressure and laboratory testing. Chronic kidney disease was defined as an eGFR <60 mL x min(-1) x 1.73 m(-2) or urine albumin-creatinine ratio (ACR) > or =30 mg/g. Premature CVD was defined as self-reported myocardial infarction or stroke at <55 years of age in men and <65 years of age in women. Mortality was ascertained by linkage to national data systems. RESULTS: Of 31,417 participants, the mean age was 45.1 +/- 11.2 years, 75.5% were female, 36.8% African American, and 21.6% had diabetes. A total of 20.6% were found to have CKD, with the ACR and eGFR being the dominant positive screening tests in the younger and older age deciles, respectively. The prevalences of premature myocardial infarction (MI), stroke, or death, and the composite were 5.3%, 4.7%, 0.8%, 9.2%, and 2.5%, 2.2%, 0.2%, 4.2% for those with and without CKD, respectively (P < .0001 for composite). Multivariable analysis found CKD (OR 1.44, 95% CI 1.27-1.63), age (OR 1.05 [per year], 95% CI 1.04-1.06), hypertension (OR 1.61, 95% CI 1.40-1.84), diabetes (OR 2.03, 95% CI 1.79-2.29), smoking (OR 1.91, 95% CI 1.66-2.21), and less than high school education (OR 1.59, 95% CI 1.37-1.85) as the most significantly associated factors for premature CVD or death (all P < .0001). Survival analysis found those with premature MI or stroke and CKD had the poorest short-term survival over the next 3 years after screening. CONCLUSIONS: Chronic kidney disease is an independent predictor of MI, stroke, and death among men and women younger than age 55 and 65 years, respectively. These data suggest the biologic changes that occur with kidney failure promote CVD at an accelerated rate that cannot be fully explained by conventional risk factors or older age. Screening for CKD by using both the ACR and eGFR can identify younger and middle-aged individuals at high risk for premature CVD and near-term death.

Am Heart J. 2008 Aug;156(2):277-83

Effects of pyridoxamine in combined phase 2 studies of patients with type 1 and type 2 diabetes and overt nephropathy.

BACKGROUND/AIMS: Treat-ments of diabetic nephropathy (DN) delay the onset of end-stage renal disease. We report the results of safety/tolerability studies in patients with overt nephropathy and type 1/type 2 diabetes treated with pyridoxamine, a broad inhibitor of advanced glycation. METHODS: The two 24-week studies were multicenter Phase 2 trials in patients under standard-of-care. In PYR-206, patients were randomized 1:1 and had baseline serum creatinine (bSCr) <or=2.0 mg/dl. In PYR-205/207, randomization was 2:1 and bSCr was <or=2.0 for PYR-205 and >or=2.0 but <or=3.5 mg/dl for PYR-207. Treated patients (122 active, 90 placebo) received 50 mg pyridoxamine twice daily in PYR-206; PYR-205/207 patients were escalated to 250 mg twice daily. RESULTS: Adverse events were balanced between the groups (p = NS). Slight imbalances, mainly in the PYR-205/207 groups, were noted in deaths (from diverse causes, p = NS) and serious adverse events (p = 0.05) that were attributed to pre-existing conditions. In a merged data set, pyridoxamine significantly reduced the change from baseline in serum creatinine (p < 0.03). In patients similar to the RENAAL/IDNT studies (bSCr >or=1.3 mg/dl, type 2 diabetes), a treatment effect was observed on the rise in serum creatinine (p = 0.007). No differences in urinary albumin excretion were seen. Urinary TGF-beta1 also tended to decrease with pyridoxamine (p = 0.049) as did the CML and CEL AGEs. CONCLUSION: These data provide a foundation for further evaluation of this AGE inhibitor in DN.

Am J Nephrol. 2007;27(6):605-14

High-dose vitamin B6 decreases homocysteine serum levels in patients with schizophrenia and schizoaffective disorders: a preliminary study.

Vitamin B6 plays an essential role in the normal functioning of the central nervous system. Normal homocysteine (Hcy) serum level is maintained by remethylation of Hcy to methionine by enzymes that require folic acid and vitamin B12 and by catabolism to cysteine by a vitamin B6-dependent enzyme. These findings may be consistent with the hypothesis that the vitamin B6 status may influence plasma Hcy levels. The aims of this preliminary study were (1) to determine whether a correlation exists between Hcy and vitamin B6 levels in patients with schizophrenia and schizoaffective disorders and (2) to investigate whether treatment with high-dose vitamin B6 may reduce Hcy levels in these patients. METHODS: In this preliminary study, we enrolled 11 patients with schizophrenia or schizoaffective disorders (7 men and 4 women; mean age +/- SD, 50 +/- 12 years) receiving high doses of vitamin B6 treatment (1200 mg/d) for 12 weeks. Blood samples for the assessment of pyridoxal-5-phosphate and Hcy serum levels were obtained at baseline and after 12 weeks of treatment. RESULTS: Age was significantly positively correlated with Hcy levels at baseline (r = 0.392, P = 0.004). All other parameters, including diagnosis, disease duration, and pyridoxal-5-phosphate serum level, were not correlated with Hcy serum levels at baseline. After vitamin B6 treatment, Hcy serum levels significantly decreased (14.2 +/- 3.4 vs. 11.8 +/- 2.0 micromol/L, respectively, t = 2.679, P = 0.023); this decrease being statistically significant in men but not in women. CONCLUSIONS: High doses of vitamin B6 lead to a decrease in Hcy serum level in male patients with schizophrenia or schizoaffective disorder.

Clin Neuropharmacol. 2007 Jan-Feb;30(1):13-7

AGES in brain ageing: AGE-inhibitors as neuroprotective and anti-dementia drugs?

In Alzheimer’s disease, age-related cellular changes such as compromised energy production and increased radical formation are worsened by the presence of AGEs as additional, AD specific stress factors. Intracellular AGEs (most likely derived from methylglyoxal) crosslink cytoskeletal proteins and render them insoluble. These aggregates inhibit cellular functions including transport processes and contribute to neuronal dysfunction and death. Extracellular AGEs, which accumulate in ageing tissue (but most prominently on long-lived protein deposits like the senile plaques) exert chronic oxidative stress on neurons. In addition, they activate glial cells to produce free radicals (superoxide and NO) and neurotoxic cytokines such as TNF-alpha. Drugs, which inhibit the formation of AGEs by specific chemical mechanisms (AGE-inhibitors), including aminoguanidine, carnosine, tenilsetam, OPB-9195 and pyridoxamine, attenuate the development of (AGE-mediated) diabetic complications. Assuming that ‘carbonyl stress’ contributes significantly to the progression of Alzheimer’s disease, AGE-inhibitors might also become interesting novel therapeutic drugs for treatment of AD.

Biogerontology. 2001;2(1):19-34