Association between Moraxella keratitis and advanced glycation end products.

Diabetes mellitus is recognized as a major predisposing factor for Moraxella keratitis. However, how diabetes mellitus contributes to Moraxella keratitis remains unclear. In this study, we examined Moraxella keratitis; based on the findings, we investigated the impact of advanced glycation end products (AGEs) deposition in the cornea of individuals with diabetic mellitus on the adhesion of Moraxella isolates to the cornea. A retrospective analysis of 27 culture-proven cases of Moraxella keratitis at Ehime University Hospital (March 2006 to February 2022) was performed. Moraxella isolates were identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among the patients, 30.4% had diabetes mellitus and 22.2% had the predominant ocular condition of using steroid eye drops. The species identified were Moraxella nonliquefaciens in 59.3% and Moraxella lacunata in 40.7% of patients. To investigate the underlying mechanisms, we assessed the effects of M. nonliquefaciens adherence to simian virus 40-immortalized human corneal epithelial cells (HCECs) with or without AGEs. The results demonstrated the number of M. nonliquefaciens adhering to HCECs was significantly increased by adding AGEs compared with that in controls (p < 0.01). Furthermore, in the corneas of streptozotocin-induced diabetic C57BL/6 mice treated with or without pyridoxamine, an AGE inhibitor, the number of M. nonliquefaciens adhering to the corneas of diabetic mice was significantly reduced by pyridoxamine treatment (p < 0.05). In conclusion, the development of Moraxella keratitis may be significantly influenced by the deposition of AGEs on the corneal epithelium of patients with diabetes mellitus.

Pyridoxamine Attenuates Doxorubicin-Induced Cardiomyopathy without Affecting Its Antitumor Effect on Rat Mammary Tumor Cells.

Doxorubicin (DOX) is commonly used in cancer treatment but associated with cardiotoxicity. Pyridoxamine (PM), a vitamin B6 derivative, could be a cardioprotectant. This study investigated the effect of PM on DOX cardiotoxicity and DOX antitumor effectiveness. Sprague Dawley rats were treated intravenously with DOX (2 mg/kg/week) or saline over eight weeks. Two other groups received PM via oral intake (1 g/L in water bottles) next to DOX or saline. Echocardiography was performed after eight weeks. PM treatment significantly attenuated the DOX-induced reduction in left ventricular ejection fraction (72 ± 2% vs. 58 ± 3% in DOX; p < 0.001) and increase in left ventricular end-systolic volume (0.24 ± 0.02 µL/cm2 vs. 0.38 ± 0.03 µL/cm2 in DOX; p < 0.0001). Additionally, LA7 tumor cells were exposed to DOX, PM, or DOX and PM for 24 h, 48 h, and 72 h. Cell viability, proliferation, cytotoxicity, and apoptosis were assessed. DOX significantly reduced LA7 cell viability and proliferation (p < 0.0001) and increased cytotoxicity (p < 0.05) and cleaved caspase-3 (p < 0.001). Concomitant PM treatment did not alter the DOX effect on LA7 cells. In conclusion, PM attenuated DOX-induced cardiomyopathy in vivo without affecting the antitumor effect of DOX in vitro, highlighting PM as a promising cardioprotectant for DOX-induced cardiotoxicity.

Pyridoxamine protects human granulosa cells against advanced glycation end-products-induced steroidogenesis disturbances.

BACKGROUND: Ovarian advanced glycation end-products (AGEs) accumulation is associated with ovarian granulosa cells (GCs) dysfunction. Vitamin B6 derivatives positively affected reproduction. The current study was conducted to elucidate the AGEs effects on human luteinized mural GCs steroidogenesis in the presence or absence of pyridoxamine (PM). METHODS AND RESULTS: Isolated GCs of 50 healthy women were divided into four parts and treated with media alone (Control), PM alone, or human glycated albumin (HGA) with/without PM. Main steroidogenic enzymes and hormones were assessed by qRT-PCR and ELISA. The AGE receptor (RAGE) protein was also determined using Western blotting. The non-toxic concentration of HGA increased the expression of RAGE, StAR, 3ß-HSD, and 17ß-HSD (P < 0.0001 for all) but decreased the expression of CYP19A1 at mRNA levels. The increased RAGE protein expression was also confirmed by western blot analysis. These effects resulted in declined estradiol (E2), slightly, and a sharp rise in progesterone (P4) and testosterone (T) levels, respectively. PM, on its own, ameliorated the HGA-altered enzyme expression and, thereby, corrected the aberrant levels of E2, P4, and T. These effects are likely mediated by regulating the RAGE gene and protein expression. CONCLUSION: This study indicates that hormonal dysfunctions induced by the AGEs-RAGE axis in luteinized GCs are likely rectified by PM treatment. This effect is likely acquired by reduced expression of RAGE. A better understanding of how AGEs and PM interact in ovarian physiology and pathology may lead to more targeted therapy for treating ovarian dysfunction.

The Role of Vitamin B6 in Peripheral Neuropathy: A Systematic Review.

INTRODUCTION: Vitamin B6 is a water-soluble vitamin that is naturally present in many foods and is accessible in many dietary supplements. The three natural forms are pyridoxine, pyridoxal, and pyridoxamine. Both vitamin B6 deficiency and high B6 intake have been described as risk factors for developing peripheral neuropathy (PN). The aim of this systematic review is to characterize and comprehensively describe B6-related PN. METHOD: A systematic, computer-based search was conducted using the PubMed database. Twenty articles were included in this review. RESULTS: Higher vitamin B6 levels, which usually occur following the taking of nutritional supplements, may lead to the development of a predominantly, if not exclusively, sensory neuropathy of the axonal type. After pyridoxine discontinuation, such patients subjectively report improved symptoms. However, although low vitamin B6 levels can be seen in patients suffering from peripheral neuropathy of various etiologies, there is no firm evidence that low B6 levels have a direct causal relationship with PN. Many studies suggest subjective improvement of neuropathy symptoms in patients suffering from PN of various etiologies after receiving B6 supplementation; however, no data about B6 administration as a monotherapy exist, only as part of a combination treatment, usually with other vitamins. Therefore, the potential therapeutic role of B6 cannot be confirmed to date. Supplementation with vitamin B6, even as part of a nutritional multivitamin supplement, has not been proven harmful at permitted daily doses in patients who already suffer from PN. CONCLUSION: Current scientific evidence supports a neurotoxic role of B6 at high levels. Although some studies suggest that low B6 is also a potential risk factor, further studies in this area are needed.

Mechanism of D-Cycloserine Inhibition of D-Amino Acid Transaminase from Haliscomenobacter hydrossis.

D-cycloserine inhibits pyridoxal-5'-phosphate (PLP)-dependent enzymes. Inhibition effect depend on organization of the active site and mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of the enzyme similarly to the substrate (amino acid), and this interaction is predominantly reversible. Several products of the interaction of PLP with D-cycloserine are known. For some enzymes formation of a stable aromatic product - hydroxyisoxazole-pyridoxamine-5'-phosphate at certain pH - leads to irreversible inhibition. The aim of this work was to study the mechanism of D-cycloserine inhibition of the PLP-dependent D-amino acid transaminase from Haliscomenobacter hydrossis. Spectral methods revealed several products of interaction of D-cycloserine with PLP in the active site of transaminase: oxime between PLP and ß-aminooxy-D-alanine, ketimine between pyridoxamine-5'-phosphate and cyclic form of D-cycloserine, and pyridoxamine-5'-phosphate. Formation of hydroxyisoxazole-pyridoxamine-5'-phosphate was not observed. 3D structure of the complex with D-cycloserine was obtained using X-ray diffraction analysis. In the active site of transaminase, a ketimine adduct between pyridoxamine-5'-phosphate and D-cycloserine in the cyclic form was found. Ketimine occupied two positions interacting with different active site residues via hydrogen bonds. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and activity of the inhibited transaminase from H. hydrossis could be restored by adding excess of keto substrate or excess of cofactor. The obtained results confirm reversibility of the inhibition by D-cycloserine and interconversion of various adducts of D-cycloserine and PLP.

Vitamin B6-Based Biomimetic Asymmetric Catalysis.

One of the fundamental goals of chemists is to develop highly efficient methods for producing optically active compounds, given their wide range of applications in chemistry, pharmaceutical industry, chemical biology, and material science. Biomimetic asymmetric catalysis, which imitates the structures and functions of enzymes, has emerged as an extremely attractive strategy for producing chiral compounds. This field has drawn tremendous research interest and has led to various protocols for constructing complex molecular scaffolds. The Vitamin B6 family, including pyridoxal, pyridoxamine, pyridoxine, and the corresponding phosphorylated derivatives, serves as the cofactors to catalyze more than 200 enzymatic functions, accounting for ∼4% of all enzyme activities. Although significant progress has been made in simulating the biological roles of vitamin B6 during the past several decades, its extraordinary catalytic power has not yet been successfully applied into asymmetric synthesis. In recent years, our group has been devoted to developing vitamin B6-based biomimetic asymmetric catalysis using chiral pyridoxals/pyridoxamines as catalysts. We are particularly interested in mimicking the processes of enzymatic transamination and biological aldol reaction of glycine, respectively, developing asymmetric biomimetic transamination and carbonyl catalysis enabled α-C-H transformation of primary amines. Using a chiral α,α-diarylprolinol-derived pyridoxal as the catalyst, we reported the first chiral pyridoxal catalyzed asymmetric transamination of α-keto acids in 2015. A significant breakthrough in biomimetic transamination was achieved by using an axially chiral biaryl pyridoxamine catalyst that bears a lateral amine side arm. The amine side arm acts as an intramolecular base, accelerating the transamination and proving highly effective for transamination of α-keto acids and α-keto amides. In addition, we discovered the catalytic power of chiral pyridoxals as carbonyl catalysts for asymmetric biomimetic Mannich/aldol reactions of glycinates. These chiral pyridoxals also enabled more α-C-H conversions of glycinates, such as asymmetric 1,4-addition toward α,ß-unsaturated esters and asymmetric α-allylation with Morita-Baylis-Hillman acetates. Moreover, carbonyl catalysis can be further applied to highly challenging primary amines with inert α-C-H bonds, such as propargylamines and benzylamines, which represents a powerful strategy for direct asymmetric α-C-H functionalization of various primary amines without protection of the NH2 group. These biomimetic/bioinspired transformations provide efficient new protocols for the synthesis of chiral amines. Herein, we summarize our recent efforts on the development of the vitamin B6-based biomimetic asymmetric catalysis.

Pyridoxamine prevents increased atherosclerosis by intermittent methylglyoxal spikes in the aortic arches of ApoE-/- mice.

Methylglyoxal (MGO) is a reactive glucose metabolite linked to diabetic cardiovascular disease (CVD). MGO levels surge during intermittent hyperglycemia. We hypothesize that these MGO spikes contribute to atherosclerosis, and that pyridoxamine as a MGO quencher prevents this injury. To study this, we intravenously injected normoglycemic 8-week old male C57Bl6 ApoE-/- mice with normal saline (NS, n = 10) or 25 µg MGO for 10 consecutive weeks (MGOiv, n = 11) with or without 1 g/L pyridoxamine (MGOiv+PD, n = 11) in the drinking water. We measured circulating immune cells by flow cytometry. We quantified aortic arch lesion area in aortic roots after Sudan-black staining. We quantified the expression of inflammatory genes in the aorta by qPCR. Intermittent MGO spikes weekly increased atherosclerotic burden in the arch 1.8-fold (NS: 0.9 ± 0.1 vs 1.6 ± 0.2 %), and this was prevented by pyridoxamine (0.8 ± 0.1 %). MGOiv spikes increased circulating neutrophils and monocytes (2-fold relative to NS) and the expression of ICAM (3-fold), RAGE (5-fold), S100A9 (2-fold) and MCP1 (2-fold). All these changes were attenuated by pyridoxamine. This study suggests that MGO spikes damages the vasculature independently of plasma glucose levels. Pyridoxamine and potentially other approaches to reduce MGO may prevent excess cardiovascular risk in diabetes.

Pyridoxamine reduces methylglyoxal and markers of glycation and endothelial dysfunction, but does not improve insulin sensitivity or vascular function in abdominally obese individuals: A randomized double-blind placebo-controlled trial.

AIM: To investigate the effects of pyridoxamine (PM), a B6 vitamer and dicarbonyl scavenger, on glycation and a large panel of metabolic and vascular measurements in a randomized double-blind placebo-controlled trial in abdominally obese individuals. MATERIALS AND METHODS: Individuals (54% female; mean age 50 years; mean body mass index 32 kg/m2 ) were randomized to an 8-week intervention with either placebo (n = 36), 25 mg PM (n = 36) or 200 mg PM (n = 36). We assessed insulin sensitivity, ß-cell function, insulin-mediated microvascular recruitment, skin microvascular function, flow-mediated dilation, and plasma inflammation and endothelial function markers. PM metabolites, dicarbonyls and advanced glycation endproducts (AGEs) were measured using ultra-performance liquid chromatography tandem mass spectrometry. Treatment effects were evaluated by one-way ANCOVA. RESULTS: In the high PM dose group, we found a reduction of plasma methylglyoxal (MGO) and protein-bound Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), as compared to placebo. We found a reduction of the endothelial dysfunction marker soluble vascular cell adhesion molecule-1 (sVCAM-1) in the low and high PM dose group and of soluble intercellular adhesion molecule-1 (sICAM-1) in the high PM dose, as compared to placebo. We found no treatment effects on insulin sensitivity, vascular function or other functional outcome measurements. CONCLUSIONS: This study shows that PM is metabolically active and reduces MGO, AGEs, sVCAM-1 and sICAM-1, but does not affect insulin sensitivity and vascular function in abdominally obese individuals. The reduction in adhesion markers is promising because these are important in the pathogenesis of endothelial damage and atherosclerosis.

Evaluation of coronary function in female rats with severe type 1 diabetes: Effects of combined treatment with insulin and pyridoxamine.

BACKGROUND: This study aimed to evaluate the coronary function, myocardium, and epicardial adipose tissue (EAT) in female rats with severe type 1 diabetes and the effects of combined treatment with insulin and pyridoxamine (AGEs inhibitor). METHODS: Female Wistar rats were divided into groups: control (CTR, n = 13), type 1 diabetes (DM1, n = 12), type 1 diabetes treated with insulin (DM1 + INS, n = 11), and type 1 diabetes treated with insulin and pyridoxamine (DM1 + INS + PDX, n = 14). The vascular responsiveness was performed in the septal coronary artery and the protein expressions of AGE, RAGE, GPER, NF-kB was evaluated in the left ventricle (LV), as well as the reactive oxygen species (ROS) was measured in LV and in EAT. We analyzed plasma levels of glucose, estradiol, Nε-carboxymethylisine (CML), thiobarbituric acid reactive substances (TBARS), catalase (CAT), and superoxide dismutase (SOD). RESULTS: The maximal responses to ACh were reduced in the DM1 compared with the CTR group, accompanied by an increase in circulating glucose, CML, and TBARS. Additionally, the expression of NF-kB in LV and generation of ROS in the presence of MnTMPyP (SOD mimetic) were increased in the DM1 group compared with CTR. Only the combined treatment was effective for fully re-establish ACh relaxation response, NF-kB protein expression, ROS generation, and increased SOD activity in the DM1 + INS + PDX group. CONCLUSION: The reduction of the endothelium-dependent relaxation response in the septal coronary artery of female rats with severe type 1 diabetes was normalized with the combined treatment with insulin and pyridoxamine, associated with reduced inflammation and oxidative stress in the myocardium and increased circulating antioxidant activity.

Chemical Modifications of Pyridoxine for Biological Applications: An Overview.

Pyridoxine and its derivatives, pyridoxamine, and pyridoxal have been recognized for more than 70 years and are known for regulating cellular biology and metabolism. During the past few decades, the anti-oxidant and anti-inflammatory properties of pyridoxine and its vitamers were explored. However, an interesting turnabout was observed in pyridoxine chemical modification in the last two decades. The various important pathophysiological aspects of pyridoxine and its derivatives on several cellular systems have been discovered by researchers. Recent findings have shown that many diseases, like cancer, diabetes, hypertension, tuberculosis, epilepsy, and neurodegenerative diseases are linked to the alteration of pyridoxine. Herein, our main focus is to review the importance of pyridoxine and its derivatives obtained by various chemical modifications, in various disease areas and to recognize important directions for future research.

Scavenging dicarbonyls with 5'-O-pentyl-pyridoxamine increases HDL net cholesterol efflux capacity and attenuates atherosclerosis and insulin resistance.

OBJECTIVE: Oxidative stress contributes to the development of insulin resistance (IR) and atherosclerosis. Peroxidation of lipids produces reactive dicarbonyls such as Isolevuglandins (IsoLG) and malondialdehyde (MDA) that covalently bind plasma/cellular proteins, phospholipids, and DNA leading to altered function and toxicity. We examined whether scavenging reactive dicarbonyls with 5'-O-pentyl-pyridoxamine (PPM) protects against the development of IR and atherosclerosis in Ldlr-/- mice. METHODS: Male or female Ldlr-/- mice were fed a western diet (WD) for 16 weeks and treated with PPM versus vehicle alone. Plaque extent, dicarbonyl-lysyl adducts, efferocytosis, apoptosis, macrophage inflammation, and necrotic area were measured. Plasma MDA-LDL adducts and the in vivo and in vitro effects of PPM on the ability of HDL to reduce macrophage cholesterol were measured. Blood Ly6Chi monocytes and ex vivo 5-ethynyl-2'-deoxyuridine (EdU) incorporation into bone marrow CD11b+ monocytes and CD34+ hematopoietic stem and progenitor cells (HSPC) were also examined. IR was examined by measuring fasting glucose/insulin levels and tolerance to insulin/glucose challenge. RESULTS: PPM reduced the proximal aortic atherosclerosis by 48% and by 46% in female and male Ldlr-/- mice, respectively. PPM also decreased IR and hepatic fat and inflammation in male Ldlr-/- mice. Importantly, PPM decreased plasma MDA-LDL adducts and prevented the accumulation of plaque MDA- and IsoLG-lysyl adducts in Ldlr-/- mice. In addition, PPM increased the net cholesterol efflux capacity of HDL from Ldlr-/- mice and prevented both the in vitro impairment of HDL net cholesterol efflux capacity and apoAI crosslinking by MPO generated hypochlorous acid. Moreover, PPM decreased features of plaque instability including decreased proinflammatory M1-like macrophages, IL-1ß expression, myeloperoxidase, apoptosis, and necrotic core. In contrast, PPM increased M2-like macrophages, Tregs, fibrous cap thickness, and efferocytosis. Furthermore, PPM reduced inflammatory monocytosis as evidenced by decreased blood Ly6Chi monocytes and proliferation of bone marrow monocytes and HSPC from Ldlr-/- mice. CONCLUSIONS: PPM has pleotropic atheroprotective effects in a murine model of familial hypercholesterolemia, supporting the therapeutic potential of reactive dicarbonyl scavenging in the treatment of IR and atherosclerotic cardiovascular disease.

Pyridoxamine treatment ameliorates large artery stiffening and cerebral artery endothelial dysfunction in old mice.

Age-related increases in large artery stiffness are associated with cerebrovascular dysfunction and cognitive impairment. Pyridoxamine treatment prevents large artery stiffening with advancing age, but the effects of pyridoxamine treatment on the cerebral vasculature or cognition is unknown. The purpose of this study was to investigate the effects of pyridoxamine on blood pressure, large artery stiffness, cerebral artery function, and cognitive function in old mice. Old male C57BL/6 mice consumed either pyridoxamine (2 g/L) or vehicle control in drinking water for ∼7.5 months and were compared with young male C57BL/6 mice. From pre- to post-treatment, systolic blood pressure increased in old control mice, but was maintained in pyridoxamine treated mice. Large artery stiffness decreased in pyridoxamine-treated mice but was unaffected in control mice. Pyridoxamine-treated mice had greater cerebral artery endothelium-dependent dilation compared with old control mice, and not different from young mice. Old control mice had impaired cognitive function; however, pyridoxamine only partially preserved cognitive function in old mice. In summary, pyridoxamine treatment in old mice prevented age-related increases in blood pressure, reduced large artery stiffness, preserved cerebral artery endothelial function, and partially preserved cognitive function. Taken together, these results suggest that pyridoxamine treatment may limit vascular aging.

Modeling studies on the role of vitamins B1 (thiamin), B3 (nicotinamide), B6 (pyridoxamine), and caffeine as potential leads for the drug design against COVID-19.

In response to the COVID-19 pandemic, and the lack of effective and safe antivirals against it, we adopted a new approach in which food supplements with vital antiviral characteristics, low toxicity, and fast excretion have been targeted. The structures and chemical properties of the food supplements were compared to the promising antivirals against SARS-COV-2. Our goal was to exploit the food supplements to mimic the topical antivirals' functions but circumventing their severe side effects, which has limited the necessary dosage needed to exhibit the desired antiviral activity. On this line, after a comparative structural analysis of the chemicals mentioned above, and investigation of their potential mechanisms of action, we selected caffeine and some compounds of the vitamin B family and further applied molecular modeling techniques to evaluate their interactions with the RDB domain of the Spike protein of SARS-CoV-2 (SC2Spike) and its corresponding binding site on human ACE-2 (HssACE2). Our results pointed to vitamins B1 and B6 in the neutral form as potential binders to the HssACE2 RDB binding pocket that might be able to impair the SARS-CoV-2 mechanism of cell invasion, qualifying as potential leads for experimental investigation against COVID-19.

Validated UPLC-MS/MS method for the analysis of vitamin B6 pyridoxal 5́-phosphate, pyridoxal, pyridoxine, pyridoxamine, and pyridoxic acid in human cerebrospinal fluid.

Vitamin B6 and its metabolites play a crucial role in the development and interaction of brain metabolism. Following diagnostic improvements additional inherited disorders in vitamin B6 metabolism have been identified, most of them leading to a severe epileptic disorder accompanied by progressive neurological deficits including intellectual disability and microcephaly. Since early treatment can improve the outcome, fast and reliable detection of metabolic biomarkers is important. Therefore, the analysis of vitamin B6 metabolites has become increasingly important, but is, however, still challenging and limited to a few specialized laboratories. Until today, vitamin B6 metabolites are measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using trichloroacetic acid for protein precipitation. In this work, we present the development and validation of a new, accurate and reliable method for analysis and quantification of the vitamin B6 vitamers pyridoxal 5́-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and pyridoxic acid (PA) in human CSF samples using acetonitrile for protein precipitation. The method is based on ultra-performance liquid chromatography-tandem mass spectrometry using electrospray ionization (UPLC-ESI-MS/MS). The calibration was performed in surrogate matrix Ringer solution and metabolites were quantified by their corresponding isotopically labelled internal standards. A protein precipitation by acetonitrile was applied greatly improving chromatographic separation of the metabolites in a 4.7 min chromatographic run. The method was validated following the European Medical Agency (EMA) and Food and Drug Administration (FDA) guidelines for bioanalytical method validation. The metabolites were quantified from 5 to 200 nmol/L with a seven-point calibration curve and minimum coefficient of regression of 0.99. The validation was performed with quality control samples at four concentration levels with surrogate matrix ringer solution and pooled CSF material. Within- and inter-day accuracy and precision in Ringer solution were within 85.4 % (PLP) and 114.5 % (PM) and from 2.6 % (PA) to 16.5 % (PLP). Within- and inter-day accuracy and precision in pooled CSF material were within 90.5 % (PN) and 120.1 % (PL) and from 1.7 % (PA) to 19.0 % (PM). The method was tested by measuring of 158 CSF samples to determine reference ranges. The B6 vitamers PLP and PL were determined in all CSF samples above 5 nmol/L while PN, PM and PA showed concentrations below or near LOQ. Probable supplementation of PLP was detected in eight CSF samples, which revealed high concentrations of PM, PN, PL, or PA, whereas PLP was in the reference range or slightly elevated. The method is suitable for the application within a routine diagnostic laboratory.

Metabolic rewiring revealed by cell-specific rate analyses from nontargeted exometabolomics during simultaneous consumption of glucose and lactic acid in a CHO fed-batch process.

Previously, we reported, based on an untargeted metabolomics, carnitine derivatives are part of a mechanism to overcome impaired mitochondrial functioning triggered by an acyl-group overflow in CHO cells. In this study, we analyzed the cell-specific rates of 24 selected metabolites using two metrics: correlation coefficients and root-mean-square deviations (RMSDs) between glucose-fed versus glucose/lactic acid-fed cultures. The time-course profiles of acetylcarnitine, adipoylcarnitine, glutarylcarnitine, glutamate, and succinate exhibited significant negative correlations between the two culture conditions. Based on RMSDs, seven carnitine derivatives, 3-hydroxy-methyl-glutarate, mevalonate, pyridoxamine-5-phosphate, succinate, and glycine were substantially different. The analyses from the two metrics reveal a distinctive rearrangement of rates from the following metabolic pathways: (i) high secretion rates of carnitines as part of the acyl-group removal, (ii) low secretion rates of succinate, related to the tricarboxylic acid cycle and the electron-transport chain, (iii) low secretion rates of pyridoxamine-5-phosphate - a co-factor for amino acid catabolism, transaminations, and transsulfuration, and (iv) increases in the consumption rates of glutamate and glycine, both used to produce glutathione. The rewiring in rates observed upon feeding lactic acid is best explained by the activation of pathways supporting homeostasis of acyl-groups and antioxidant synthesis, which are required for continuous proper functioning of oxidative phosphorylation.

Inhibition of advanced glycation end product formation and serum protein infiltration in bioprosthetic heart valve leaflets: Investigations of anti-glycation agents and anticalcification interactions with ethanol pretreatment.

Bioprosthetic heart valves (BHV) fabricated from heterograft tissue, such as glutaraldehyde pretreated bovine pericardium (BP), are the most frequently used heart valve replacements. BHV durability is limited by structural valve degeneration (SVD), mechanistically associated with calcification, advanced glycation end products (AGE), and serum protein infiltration. We investigated the hypothesis that anti-AGE agents, Aminoguanidine, Pyridoxamine [PYR], and N-Acetylcysteine could mitigate AGE-serum protein SVD mechanisms in vitro and in vivo, and that these agents could mitigate calcification or demonstrate anti-calcification interactions with BP pretreatment with ethanol. In vitro, each of these agents significantly inhibited AGE-serum protein infiltration in BP. However, in 28-day rat subdermal BP implants only orally administered PYR demonstrated significant inhibition of AGE and serum protein uptake. Furthermore, BP PYR preincubation of BP mitigated AGE-serum protein SVD mechanisms in vitro, and demonstrated mitigation of both AGE-serum protein uptake and reduced calcification in vivo in 28-day rat subdermal BP explants. Inhibition of BP calcification as well as inhibition of AGE-serum protein infiltration was observed in 28-day rat subdermal BP explants pretreated with ethanol followed by PYR preincubation. In conclusion, AGE-serum protein and calcification SVD pathophysiology are significantly mitigated by both PYR oral therapy and PYR and ethanol pretreatment of BP.

Exploitation of a rod-shaped, acid-labile curcumin-loaded polymeric nanogel system in the treatment of systemic inflammation.

Curcumin is proven to have potent anti-inflammatory activity, but its low water solubility and rapid degradation in physiological conditions limit its clinical use, particularly in intravenous drug delivery. In this study, we fabricated rod-shaped, acid-labile nanogels, using high biosafe and biocompatible polymers, for intravenous application in systemic inflammation treatment. The constituent polymers of the nanogels were prepared via the conjugation of vitamin B6 derivatives, including pyridoxal and pyridoxamine, onto poly(glutamate) with ester bonds. The aldehyde groups of the pyridoxal and amine groups of the pyridoxamine on the polymers enable crosslinking using a Schiff base during the solvent evaporation procedure for the preparation of the rod-shaped nanogels. Our study is the first to introduce this linkage, which is generated from two vitamin B6 derivatives into a nanogel system. It is also the first to fabricate a rod-shaped nanogel system via simple solvent evaporation. Under acidic conditions, such as those encountered in the endosomes and lysosomes within inflammatory macrophage cells spread in the whole body, imine bonds are cleaved and release payloads. The nanogel polymers were successfully synthesized and characterized, and the formation and disappearance of the Schiff base under neutral and acidic conditions were also confirmed using Fourier transform infrared spectroscopy. Following curcumin encapsulation, the long, rod-shaped nanogels were able to rapidly internalize into macrophage cells in static or adhere to cells under the flows, release their payloads in the acid milieus, and, thus, mitigate curcumin degradation. Consequently, curcumin-loaded, rod-shaped nanogels displayed exceptional anti-inflammatory activity both in vitro and in vivo, by efficiently inhibiting pro-inflammatory mediator secretion. These results demonstrate the feasibility of our acid-labile, rod-shaped nanogels for the treatment of systemic inflammation.

Comparative Study to Evaluate the Effect of Low-Protein Diet Supplementation with Taurine and N-Acetylcysteine, N-Acetylcysteine and Pyridoxamine Dihydrochloride in Preventing the Progression of Chronic Renal Failure in Patients with Non-Diabetic Kidney Disease.

Chronic Kidney Disease(CKD) has multifactorial etiology and there are lots of grey zone in understanding its complex pathophysiology. There is no silver bullet for optimal care of CKD. Oxidative stress being well understood and considered as an important common progressive factor for CKD of different etiology. Several research studies focused on reducing oxidative stress and have shown diverse outcomes. In this randomized, open-label, three arms, controlled, single center study we evaluated the role of N acetylcysteine which is a direct scavenger of free radical, in combination with taurine and pyridoxamine in retarding the progression of non-diabetic kidney disease. METHODS: 69 non-dialysis, non-diabetic patients diagnosed with chronic renal failure with GFR more than 15 ml/min/1.73m2 and less than 60ml/min/1.73m2 receiving standard of care were enrolled in the study, of which 22 were in the placebo arm, 23 treated with NT (500 mg Taurine + 150 mg NAC) arm and 24 in the NP (300mg NAC+ 50mg pyridoxamine di-hydrochloride) arm. The subjects in the treatment arm received the study drug twice a day along with low protein (0.6gm protein per Kg body weight) isocaloric diet with 25-30 Kcal/Kg/D and were evaluated monthly up to 6 months. Change in eGFR accorss 3 groups over 6 months were compared. RESULT: Mean age of the subjects was 57 ± 13 years of 56.25% were male and 43.75% were female. 69 patients completed the study. The Empirical Distribution Function (EDF) of NP group was dominant over control and NT group indicating a positive effect of NT on non-diabetic CKD at 10% level of significance. In the subgroup analysis a significant effect was observed in the cases of patients receiving NP with baseline eGFR more than 45 ml/min. The mean increase in eGFR readings over six months was 8.15 units higher in the NP group than in the control group. The two-sided p-values of the t-test, the Wilcoxon test and the Kolmogorov-Smirnov test were 0.0496, 0.0316 and 0.0354, respectively. Thus, all the three tests reject the hypothesis of identical changes in eGFR at the 5% level. In subjects with bicarbonate more than 22 mg/dl, the mean increase in eGFR over six months was 10.86 units higher in the NP group than in the control group indicating NP has a positive effect on increasing eGFR over 6 months, in patients without the presence of any metabolic acidosis. The two-sided p-vales of the t-test, the Wilcoxon test and the Kolmogorov-Smirnov test were 0.0325, 0.0205 and 0.1495, respectively. Thus, two of the three tests reject the hypothesis of identical changes in eGFR at the 5% level which clearly indicates that NP had better efficacy than other groups. CONCLUSION: N-acetyl cysteine along with pyridoxine may be a useful intervention along with a low protein diet in retarding progression of CKD in the nondiabetic population in early CKD.

Increased fermentable carbohydrate intake alters colonic mucus barrier function through glycation processes and increased mast cell counts.

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder for which dietary interventions can be a useful treatment. In recent years, the low-FODMAP approach is gaining traction in this regard. The fermentation of these non-absorbed carbohydrates by the gut microbiota can generate toxic glycating metabolites, such as methylglyoxal. These metabolites can have harmful effects by their role in the generation of advanced glycation end products (AGEs), which activates Receptor for AGEs (AGER). Mast cells can be stimulated by AGEs and play a role in IBS. We have treated mice with lactose or fructo-oligosaccharides (FOS), with or without co-administration of pyridoxamine and investigated the colonic mucus barrier. We have found that an increased intake of lactose and fructo-oligosaccharides induces a dysregulation of the colonic mucus barrier, increasing mucus discharge in empty colon, while increasing variability and decreasing average thickness mucus layer covering the fecal pellet. Changes were correlated with increased mast cell counts, pointing to a role for the crosstalk between these and goblet cells. Additionally, AGE levels in colonic epithelium were increased by treatment with the selected fermentable carbohydrates. Observed effects were prevented by co-treatment with anti-glycation agent pyridoxamine, implicating glycation processes in the negative impact of fermentable carbohydrate ingestion. This study shows that excessive intake of fermentable carbohydrates can cause colonic mucus barrier dysregulation in mice, by a process that involves glycating agents and increased mucosal mast cell counts.

Cycloserine enantiomers inhibit PLP-dependent cysteine desulfurase SufS via distinct mechanisms.

The cysteine desulfurase SufS is a pyridoxal-5'-phosphate-dependent enzyme and is essential for the SUF system, which participates in iron-sulfur cluster biosynthesis. Inhibition of SufS in the SUF system by D-cycloserine (DCS) in Plasmodium falciparum apicoplast has recently been reported, indicating that SufS could be a target for malaria therapeutics. However, the mechanistic details underlying the inhibition of SufS by DCS have not yet been clarified. Moreover, inhibition of SufS by the other enantiomer, L-cycloserine (LCS), has not been investigated. Herein, we investigated the structure-based inhibition mechanisms of SufS by DCS and LCS using Bacillus subtilis SufS, whose catalytic mechanism has been well characterized in comparison to that of the P. falciparum SufS. Surprisingly, DCS- and LCS-mediated inhibitions of SufS occur via distinct mechanisms resulting in pyridoxamine-5'-phosphate (PMP) in DCS-mediated inhibition and PMP-3-hydroxyisoxazole adduct (PMP-isoxazole) in LCS-mediated inhibition. Biochemical and structural evaluation of SufS variants identified conserved His and Arg residues at the active site as the key determinants of the distinct inhibition mechanisms. The importance of structural elements involved in DCS and LCS-mediated inhibitions of SufS provides valuable insights for the structure-based design of new drugs targeting SufS. DATABASE: Structural data are available in PDB database under the accession numbers 6KFY, 7CEO, 7CEP, 7CEQ, 7CER, 7CES, 7CET, 7CEU, 7E6A, 7E6B, 7E6C, 7E6D, 7E6E, and 7E6F.