Cysteinyl Glycine Research Articles

Urinary Thioproline and Thioprolinyl Glycine as Specific Biomarkers of Formaldehyde Exposure in Humans.

Assessment of personal formaldehyde (FA) exposure is most commonly carried out using formate as a biomarker, as it is the major product from FA metabolism. However, formate could also have originated from the metabolism of other endogenous and exogenous substances or from dietary intake, which may give rise to overestimated results with regard to FA exposure. We have developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with an isotope-dilution method for rigorous quantitation of two major urinary FA conjugation products: thioproline (SPro) and thioprolinyl glycine (SPro-Gly), formed in the reaction between FA and endogenous cysteine or cysteinyl glycine, respectively, as marker molecules to assess personal FA exposure. Using this newly developed method, we measured the FA exposure levels in cigarette smokers, occupants of a chemistry research laboratory and typical domestic household, and visitors to a Chinese temple with a Pearson correlation coefficient greater than 0.94, showing a strong linear correlation between urinary adduct levels and the airborne FA level. It is believed that quantitation of urinary SPro and SPro-Gly may represent a noninvasive, interference-free method for assessing personal FA exposure.

Impact of homocysteine on acute ischemic stroke severity: possible role of aminothiols redox status

BackgroundAcute ischemic stroke (AIS) is one of the most common cerebrovascular diseases which accompanied by a disruption of aminothiols homeostasis. To explore the relationship of aminothiols with neurologic impairment severity, we investigated four aminothiols, homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CG) and glutathione (GSH) in plasma and its influence on ischemic stroke severity in AIS patients.MethodsA total of 150 clinical samples from AIS patients were selected for our study. The concentrations of free reduced Hcy (Hcy), own oxidized Hcy (HHcy), free reduced Cys (Cys), own oxidized Cys (cysteine, Cyss), free reduced CG (CG) and free reduced GSH (GSH) were measured by our previously developed hollow fiber centrifugal ultrafiltration (HFCF-UF) method coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The concentration ratio of Hcy to HHcy (Hcy/HHcy), Cys to Cyss (Cys/Cyss) were also calculated. The neurologic impairment severity of AIS was evaluated using National Institutes of Health Stroke Scale (NIHSS). The Spearman correlation coefficient and logistic regression analysis was used to estimate and perform the correlation between Hcy, HHcy, Cys, Cyss, CG, GSH, Hcy/HHcy, Cys/Cyss and total Hcy with NIHSS score.ResultsThe reduced Hcy and Hcy/HHcy was both negatively correlated with NIHSS score in AIS patients with P = 0.008, r=-0.215 and P = 0.002, r=-0.249, respectively. There was no significant correlation of Cys, CG, GSH, HHcy, Cyss, Cys/Cyss and total Hcy with NIHSS score in AIS patients with P value > 0.05.ConclusionsThe reduced Hcy and Hcy/HHcy, not total Hcy concentration should be used to evaluate neurologic impairment severity of AIS patient.

Intravenous Glutathione: A Promising Therapy for the Alcoholic Liver Disease

Alcoholic liver disease (ALD) and sepsis are life-threatening conditions marked by severe oxidative stress. Chronic alcohol use triggers oxidative stress and inflammation that damages liver cells. Glutathione (GSH), a tripeptide consisting of gamma glutamyl cysteinyl glycine possesses a thiol group and participates in oxidation reduction reactions, acting as a principal cellular scavenger of free radicles. GSH is present in large concentrations in the liver, and its endogenous levels are depleted in ALD, exacerbating the condition. Intravenous GSH supplementation has shown promising results in improving liver function and reducing fibrosis markers in ALD patients. Intravenous GSH treatment has demonstrated the potential to reduce oxidative injury and mortality rates in patients with sepsis in the intensive care unit. The versatility of GSH in mitigating oxidative stress, inflammation, and tissue damage and proven safety and tolerability profile make it a valuable adjunct to current treatments. Further research is imperative to comprehensively unravel the therapeutic benefits of GSH injection adjunct to standard of care in patients with ALD and sepsis. Keywords: Glutathione, Alcoholic liver disease, Sepsis, Antioxidants, Intensive care unit, Intravenous

Evaluation of the Drug-Induced Liver Injury Potential of Saxagliptin through Reactive Metabolite Identification in Rats.

A liver injury was recently reported for saxagliptin, which is a dipeptidyl peptidase-4 (DPP-4) inhibitor. However, the underlying mechanisms of saxagliptin-induced liver injury remain unknown. This study aimed to evaluate whether saxagliptin, a potent and selective DPP-4 inhibitor that is globally used for treating type 2 diabetes mellitus, binds to the nucleophiles in vitro. Four DPP-4 inhibitors, including vildagliptin, were evaluated for comparison. Only saxagliptin and vildagliptin, which both contain a cyanopyrrolidine group, quickly reacted with L-cysteine to enzyme-independently produce thiazolinic acid metabolites. This saxagliptin-cysteine adduct was also found in saxagliptin-administered male Sprague-Dawley rats. In addition, this study newly identified cysteinyl glycine conjugates of saxagliptin and 5-hydroxysaxagliptin. The observed metabolic pathways were hydroxylation and conjugation with cysteine, glutathione, sulfate, and glucuronide. In summary, we determined four new thiazoline-containing thiol metabolites (cysteine and cysteinylglycine conjugates of saxagliptin and 5-hydroxysaxagliptin) in saxagliptin-administered male rats. Our results reveal that saxagliptin can covalently bind to the thiol groups of cysteine residues of endogenous proteins in vivo, indicating the potential for saxagliptin to cause drug-induced liver injury.

REVERSED PHASE HPLC DETERMINATION OF TOTAL HOMOCYSTEINE, CYSTEINE, CYSTEINYL GLYCINE, GLUTATHIONE IN PLASMA OF EPILEPTIC PATIENTS

The fast and accurate reversed-phase HPLC method was predicated on qualitative and quantitative estimation of the total concentrations of the essential thiols in the plasma of adults volunteers compared with epileptic patients, which is l-cysteine (Cys) total homocysteine (tHys), cysteinyl glycine (Cys-Gly), glutathione (GSH) and cysteamine (Cstm), respectively. The plasma samples of (50) epileptic patients were classified into three groups; the non-anti-epileptic drug consisted of 20 patients (12 male, eight female) who were mainly diagnosed with idiopathic generalized epilepsy. The mean age was 34 ± 13 (range 19–48) years. The second group consisted of 30 patients receiving two types of anti-epileptic drugs, 18 patients (12 male and six female) receiving Carbamazepine, and 12 patients (7 males and five female) receiving phenytoin. The mean age of the patients was 32 ± 11 Compared with 31 healthy volunteers (20 male and 11 female). The mean age of the controls was 31 ± 9 (range 26–49 years). The mean duration of treatment of patients receiving the anti-epileptic drug was six months). All measurement was done using pre-column derivatization with bromobimane, and the derivative of each standard mixture were baseline separated on C18-DB (50 x 4.6 mm ID) column, 3μm particle size. The regression coefficients for the separated and standard deviation SD within-run ranged from 0.09 to 8.40 μmol/L, and between-run ranged from 0.15to 9.16 μmol/L; the analytical procedure gave good linearity in the range between 1.25 to 20 µmole/ l, the detection limit was 0.1 µmole /L for all the thiol groups. Analytical recovery was 96.9–107.4 %; the mean concentration of plasma cysteine and total homocysteine was slightly higher in males than females; it was 221± 75 for adult males and 190± 44 μmol/L for adult females, while t-homocysteine was (10.55 ±2.45 vs. 9.79 ± 1.88 μmol/L, the results observed that the value of cysteine and homocysteine were significantly higher in epileptic patients using Carbamazepine and phenytoin than in healthy volunteers. Mean values for glutathione were lower, while cysteinyl glycine showed no significant difference in healthy and epileptic patients and no sex- and age-dependent.

Characterization of glutathione-specific gamma glutamyl cyclotransferase (ChaC) in Bombyx mori.

Glutathione (GSH) contributes to redox maintenance and detoxification of various xenobiotic and endogenous substances. γ-glutamyl cyclotransferase (ChaC) is involved in GSH degradation. However, the molecular mechanism underlying GSH degradation in silkworms (Bombyx mori) remains unknown. Silkworms are lepidopteran insects that are considered to be an agricultural pest model. We aimed to examine the metabolic mechanism underlying GSH degradation mediated by B. mori ChaC and successfully identified a novel ChaC gene in silkworms (herein, bmChaC). The amino acid sequence and phylogenetic tree revealed that bmChaC was closely related to mammalian ChaC2. We overexpressed recombinant bmChaC in Escherichia coli, and the purified bmChaC showed specific activity toward GSH. Additionally, we examined the degradation of GSH to 5-oxoproline and cysteinyl glycine via liquid chromatography-tandem mass spectrometry. Quantitative real-time polymerase chain reactionrevealed that bmChaC mRNA expression was observed in various tissues. Our results suggest that bmChaC participates in tissue protection via GSH homeostasis. This study provides new insights into the activities of ChaC and the underlying molecular mechanisms that can aid the development of insecticides to control agricultural pests.

Untargeted Metabolomics by Ultra-High-Performance Liquid Chromatography Coupled with Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry Analysis Identifies a Specific Metabolomic Profile in Patients with Early Chronic Kidney Disease

Chronic kidney disease (CKD) has emerged as one of the most progressive diseases with increased mortality and morbidity. Metabolomics offers new insights into CKD pathogenesis and the discovery of new biomarkers for the early diagnosis of CKD. The aim of this cross-sectional study was to assess metabolomic profiling of serum and urine samples obtained from CKD patients. Untargeted metabolomics followed by multivariate and univariate analysis of blood and urine samples from 88 patients with CKD, staged by estimated glomerular filtration rate (eGFR), and 20 healthy control subjects was performed using ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry. Serum levels of Oleoyl glycine, alpha-lipoic acid, Propylthiouracil, and L-cysteine correlated directly with eGFR. Negative correlations were observed between serum 5-Hydroxyindoleacetic acid, Phenylalanine, Pyridoxamine, Cysteinyl glycine, Propenoylcarnitine, Uridine, and All-trans retinoic acid levels and eGFR. In urine samples, the majority of molecules were increased in patients with advanced CKD as compared with early CKD patients and controls. Amino acids, antioxidants, uremic toxins, acylcarnitines, and tryptophane metabolites were found in all CKD stages. Their dual variations in serum and urine may explain their impact on both glomerular and tubular structures, even in the early stages of CKD. Patients with CKD display a specific metabolomic profile. Since this paper represents a pilot study, future research is needed to confirm our findings that metabolites can serve as indicators of early CKD.

A simple and accurate HFCF-UF method for the analysis of homocysteine, cysteine, cysteinyl-glycine, and glutathione in human blood

The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study.

Long-term effect of parental selenium supplementation on the one-carbon metabolism in rainbow trout (Oncorhynchus mykiss) fry exposed to hypoxic stress.

This study evaluated how different forms of selenium (Se)supplementation into rainbow trout broodstock diets modified the one-carbon metabolism of the progeny after the beginning of exogenous feeding and followed by hypoxia challenge. The progeny of three groups of rainbow trout broodstock fed either a control diet (Se level: 0·3 µg/g) or a diet supplemented with inorganic sodium selenite (Se level: 0·6 µg/g) or organic hydroxy-selenomethionine (Se level: 0·6 µg/g) was cross-fed with diets of similar Se composition for 11 weeks. Offspring were sampled either before or after being subjected to an acute hypoxic stress (1·7 mg/l dissolved oxygen) for 30 min. In normoxic fry, parental Se supplementation allowed higher glutathione levels compared with fry originating from parents fed the control diet. Parental hydroxy-selenomethionine treatment also increased cysteine and cysteinyl-glycine concentrations in fry. Dietary Se supplementation decreased glutamate-cysteine ligase (cgl) mRNA levels. Hydroxy-selenomethionine feeding also lowered the levels of some essential free amino acids in muscle tissue. Supplementation of organic Se to parents and fry reduced betaine-homocysteine S-methyltransferase (bhmt) expression in fry. The hypoxic stress decreased whole-body homocysteine, cysteine, cysteinyl-glycine and glutathione levels. Together with the higher mRNA levels of cystathionine beta-synthase (cbs), a transsulphuration enzyme, this suggests that under hypoxia, glutathione synthesis through transsulphuration might have been impaired by depletion of a glutathione precursor. In stressed fry, S-adenosylmethionine levels were significantly decreased, but S-adenosylhomocysteine remained stable. Decreased bhmt and adenosylmethionine decarboxylase 1a (amd1a) mRNA levels in stressed fry suggest a nutritional programming by parental Se also on methionine metabolism of rainbow trout.

Association of Low Molecular Weight Plasma Aminothiols with the Severity of Coronavirus Disease 2019.

Objective Aminothiols (glutathione (GSH), cysteinylglycine (CG)) may play an important role in the pathogenesis of coronavirus disease 2019 (COVID-19), but the possible association of these indicators with the severity of COVID-19 has not yet been investigated. Methods The total content (t) and reduced forms (r) of aminothiols were determined in patients with COVID-19 (n = 59) on admission. Lung injury was characterized by computed tomography (CT) findings in accordance with the CT0-4 classification. Results Low tGSH level was associated with the risk of severe COVID-19 (tGSH ≤ 1.5 μM, mild vs. moderate/severe: risk ratio (RR) = 3.09, p = 0.007) and degree of lung damage (tGSH ≤ 1.8 μM, CT < 2 vs. CT ≥ 2: RR = 2.14, p = 0.0094). The rGSH level showed a negative association with D-dimer levels (ρ = −0.599, p = 0.014). Low rCG level was also associated with the risk of lung damage (rCG ≤ 1.3 μM, CT < 2 vs. CT ≥ 2: RR = 2.28, p = 0.001). Levels of rCG (ρ = −0.339, p = 0.012) and especially tCG (ρ = −0.551, p = 0.004) were negatively associated with platelet count. In addition, a significant relationship was found between the advanced oxidation protein product level and tGSH in patients with moderate or severe but not in patients with mild COVID-19. Conclusion Thus, tGSH and rCG can be seen as potential markers for the risk of severe COVID-19. GSH appears to be an important factor to oxidative damage prevention as infection progresses. This suggests the potential clinical efficacy of correcting glutathione metabolism as an adjunct therapy for COVID-19.

The Therapeutic Potential of Glutathione Supplement: A Review of Clinical Trials

Glutathione (GSH) is a tripeptide (γ-glutamyl cysteinyl glycine) involved in a variety of biological processes indispensable to sustain life and the most abundant free radical scavenger synthesized endogenously in humans. There are adverse health consequences from glutathione deficiency. The present mini –review aims to provide an extensive overview to glutathione supplement therapeutic effects in human subjects.

Sequestration and Elimination of Toxic Aldehydes.

It is well-known that aldehydes resulting from the in vivo oxidation of primary alcohols are toxic. Here, we experimentally demonstrate in rat models that the dipeptide cysteinylglycine (CG), formed in vivo from its oxidized product, cystinyl-bis-glycine (CbG), will sequester acetaldehyde and isoamyl aldehyde, two model aldehydes resulting from the oxidation of ethanol and isoamyl alcohol, respectively, and excrete them in urine as their respective conjugation products with CG. These data suggest that a whole series of toxic aldehydes can be sequestered and detoxified by CG and may prevent the flushing syndrome exhibited by individuals with a defective enzyme that converts acetaldehyde to acetate. The data also suggest the possibility of alleviating the hangover syndrome we believe to be caused by aldehydes, such as isoamyl aldehyde derived from short, branched-chain alcohols, present as congeners in certain alcoholic beverages. The sequestration of other toxic agents, such as cyanide, that can react with CG can also be envisioned.

The dynamics of cellular and humoral immunity during the treatment of pneumonia in patients on a background of acute leukemia

Pneumonia is often life threatening infectious complication on a back ground of software treatment in patients with acute lymphoblastic leukemia (ALL). The existing consensus documents on the treatment of pneumonia in patients with impaired immunity focuses on antibac terial therapy, at the same time, questions about the need for correc tion of the immune status of patients remain poorly understood. A promising direction is the use of glutamyl cysteinyl glycine sodium in the treatment of pneumonia in patients with OL to enhance the activ ity of the immune system and improve the anti infective protection. The aim of the study : was the analysis of indicators of cellular and humoral immunity in the process of applying immunocorrigirutee drug glutamyl cysteinyl glycine sodium (Glutoxim) in patients with pneu monia, which arise on the background of ALL optimization approach es in the treatment of pneumonia in patients with impaired immunity. Materials and methods . Study group of 87 patients with pneumonia on the background of ALL that took place a software based treatment of hematological centre «SE City Multidisciplinary Clinical Hospital No. 4» (str) for 2014 2015, the average age of patients was from 28 to 39 years old. ALL diagnoses of pneumonia was verified in accordance with generally accepted clinical and morphological criteria. Patients from the main study group (n=57) was additionally appointed glu tamyl cysteinyl glycine sodium to 2 ml of 3% (60 mg), N 10, in the morning, intravenous, total dose of 600 mg. All patients investigated indicators immunogram: T lymphocytes, b lymphocytes and indicators subpopulation composition (CD3+, CD4+, CD8+, CD19+, CD19 , CD16+, CD56+). The study was conducted using flow through laser cytofluometry (BeckmanCoulter, USA) and calculated the derived indices. Indicators of immunogram in patients of study groups were evaluated in the dynamics of treatment. Statistical processing was per formed using packages of applied programs «Excel» and «Statistic 10». Results . The analysis of indicators of cellular and humoral immunity of patients with pneumonia accompanied by АLL in the process of applying glutamyl cysteinyl glycin sodium showed significant activation of phagocytosis and anti infective protection of the results of increasing the relative amount of T helpers (CD4 +), PC cells, increased immunoregu latory index, FA and FC. The results of the dynamics of humoral immu nity also confirm the positive influence of the use of glutamyl cysteinyl glycine sodium on the immune reactivity of patients with reliable change indices of IgA and IgM, which are responsible for neutralizing infectious agents, bacterial toxins and indirectly activate phagocytosis. Conclusion . The use of immunomodulatory therapy with the drug glu tamyl cysteinyl glycine sodium (Glutoxim) in patients with pneumo nia on the background of ALL leads to improved clinical manifesta tions of the disease, reduces the duration of ABT improves the quality of life and leads to earlier return (average 3 4 days) to the program treatment АLL. The results of the research prove the possibility of optimizing the treatment of pneumonia by the using of immunomodu latory therapy glutamyl cysteinyl glycine sodium (Glutoxim) patients with pneumonia on a background of АLL.

Plasma-aminothiols status and inverse correlation of total homocysteine with B-vitamins in arsenic exposed population of West Bengal, India

ABSTRACTChronic arsenic toxicity is a serious environmental health problem across the world. Bangladesh and India (particularly the state of West Bengal) are the worst affected countries with such problem. The present study reports plasma-aminothiols (p-aminothiols) like L-cysteine (L-Cys), cysteinyl glycine (Cys-gly), total homocysteine (t-Hcy) and glutathione (GSH) status, and the inverse relationship of t-Hcy with B-vitamins (B1, B6, B9 and B12) in arsenic exposed population of West Bengal, India. Reverse phase HPLC was used to measure p-aminothiols and serum B-vitamins in different arsenic exposed population. Arsenic in drinking water and urine were measured by flow injection analysis system – Atomic Absorption Spectrometry (FIAS-AAS) and Transversely heated graphite atomizer (THGA-AAS) techniques, respectively. Water arsenic exposure was >50 µg/L in 50% population, of which majority (33.58%) belong to the range of >50–500 µg/L and more than 8% were even >1000 µg/L. Urine arsenic (µg/g creatinine) levels increased with arsenic exposure. The variability among p-aminothiols was also observed with higher exposure to arsenic in drinking water. A significant difference between exposed and control population was noticed for plasma L-Cys. The difference of B-vitamins between the population exposed to <50 and >50 µg/L arsenic in drinking water was also found to be significant. B9 and B12 deficiency with increased consumption of arsenic in water corroborates the anemic conditions commonly observed among arsenic exposed population. The aminothiol status indicated oxidative stress in exposed population. This study demonstrated progressive increase in plasma t-Hcy as well as inverse relationships of serum B-vitamins with increased water arsenic concentration.

The therapeutic effect of medlar polysaccharide on blood fat metabolism of rats with nonalcoholic fatty liver disease

Objective To explore the effect of medlar polysaccharide(MP) on blood fat metabolism of rat with nonalcoholic fatty liver disease(NAFLD). Methods 40 SD rats were randomly divided into a blank control group(n=7), and a NAFLD model group (n=33). The control group was treated with normal forage, and the control group was treated with high-fat forage. After 8 weeks, 30 NAFLD models were successfully established and divided into three groups randomly: a LBP intervention group (treatment group, n=10) was perfused with 60 mg/(kg•d)MP, a simvastatin intervention group(control group, n=10) was perfused with 5mg/(kg•d) simvastatin, and a model group(model group, n=10) was perfused with the same volume of water along with high fat diet at the same time. After 8 weeks, triglycerides, total cholesterol, ALT, AST, leptin, and adiponectin in the plasma in rat were test. Malondialdehyde, Superoxide Dismutase and Gamma glutamyl cysteinyl glycine were also detected in the liver tissue. Results After 8 weeks, the contents of TG (0.94 ± 0.09 mmol/L vs.1.19 ± 0.13 mmol/L), TC (2.15 ± 0.20 mmol/L vs. 3.52 ± 0.29 mmol/L), ALT (41.75 ± 4.14 U/L vs. 55.34 ± 5.38 U/L), AST (129.61 ± 12.07 U/L vs. 164.96 ± 15.49 U/L), LP (235.69 ± 23.52 pg/ml vs. 284.01 ± 29.43 pg/ml) were decreased, the contents of ADP (35.47 ± 3.09 µg/L vs. 26.31 ± 2.58 µg/L) was increased in serum of the treatment group compared with the model group (P<0.05 or P<0.01)；the contents of SOD (92.40 ± 8.89 U/mg vs. 61.60 ± 5.89 U/mg), GSH (22.25 ± 2.21 mg/g vs. 15.50 ± 1.36 mg/g)were increased, and the contents of MDA (0.84 ± 0.08 nmol/mg vs. 1.07 ± 0.09 nmol/mg) was decreased in liver tissue of the treatment group compared with the model group (P<0.05 or P<0.01). HE stain showed that the treatment group improved the injury. Conclusions Medlar polysaccharide(MP) can inhibit lipid peroxidation by adjusting lipid metabolism. Key words: Nonalcoholic fatty liver disease; Medlar polysaccharidel superoxidase dismutase; Malondialdehyde; Adiponectin; Leptin

Kinetic study on the aromatic nucleophilic substitution reaction of 3,6‐dichloro‐1,2,4,5‐tetrazine by biothiols

The aromatic nucleophilic substitution reaction of 3,6‐dichloro‐1,2,4,5‐tetrazine (DCT) with a series of biothiols RSH: (cysteine, homocysteine, cysteinyl–glycine, N‐acetylcysteine, and glutathione) is subjected to a kinetic investigation. The reactions are studied by following spectrophotometrically the disappearance of DCT at 370 nm. In the case of an excess of N‐acetylcysteine and glutathione, clean pseudo first‐order rate constants (kobs1) are found. However, for cysteine, homocysteine and cysteinyl–glycine, two consecutive reactions are observed. The first one is the nucleophilic aromatic substitution of the chlorine by the sulfhydryl group of these biothiols (RSH) and the second one is the intramolecular and intermolecular nucleophilic aromatic substitutions of their alkylthio with the amine group of RSH to give the di‐substituted compound. Therefore, in these cases, two pseudo first‐order rate constants (kobs1 and kobs2, respectively) are found under biothiol excess. Plots of kobs1 versus free thiol concentration at constant pH are linear, with the slope (kN) independent of pH (from 6.8 to 7.4). The kinetic data analysis (Brønsted‐type plot and activation parameters) is consistent with an addition–elimination mechanism with the nucleophilic attack as the rate‐determining step. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Kidney donors and kidney transplants have abnormal aminothiol redox status, and are at increased risk of oxidative stress and reduced redox buffer capacity

ObjectiveLiving kidney donors have been part of a successful kidney transplant programme in Norway for almost 50years. Glomerular filtration rates (GFRs) have tended to remain stable at about 70% of pre-donation levels. Plasma total homocysteine (Hcy) has an inverse relationship to kidney function, and previous reports indicate elevated levels of Hcy in kidney donors. We wanted to examine the most important plasma aminothiols in kidney donors, i.e. Hcy, cysteine (Cys) and cysteinylglycine (CG) with their redox species. The aminothiol redox-system appears to be an integral part of the extracellular antioxidant defence system in the body. Design and methodsPlasma concentrations of total Hcy were obtained in 82 previous kidney donors, 82 healthy controls and 26 kidney transplants with stable and good kidney function. In a subset of 30 kidney donors, 30 matched controls and 12 kidney transplants plasma samples were analysed for Hcy, Cys, CG and their redox species. There were no differences between groups for B-vitamin status. ResultsKidney donors and kidney transplants had elevated plasma concentrations of total Hcy, Cys and CG. The plasma levels of reduced Hcy species were high — with a high reduced/oxidized ratio. The plasma levels of reduced Cys species were low — with a low reduced/oxidized ratio. ConclusionsPrevious kidney donors have abnormal plasma aminothiol redox status. The present findings indicate that donors may have increased risk of oxidative stress with low redox buffer capacity and disturbed cellular redox-dependent signalling pathways. Similar observations were made in the kidney transplants.

Levodopa-related cysteinyl–glycine and cysteine reduction with and without catechol-O-methyltransferase inhibition in Parkinson’s disease patients

Oxidative stress is influenced by the thiol homeostasis, which regulates the redox milieu via glutathione. Components of glutathione metabolism are cysteine and cysteinyl-glycine. Both substrates decay following levodopa application or dopamine-related oxidative stress. Objective was to investigate the impact of an acute levodopa application with and without catechol-O-methyltransferase inhibitor on cysteine- and cysteinyl-glycine plasma levels. On two investigation days, 13 patients with Parkinson's disease took one retarded release 200-mg levodopa/50mg carbidopa-containing tablet or one 150-mg levodopa/50-mg carbidopa/200-mg entacapone formulation under standardized conditions. Levodopa, 3-O-methyldopa, cysteine and cysteinyl-glycine were measured at baseline, 80 and 140min following levodopa administration. Cysteine and cysteinyl-glycine similarly decreased, levodopa was nearly equal during both conditions. Entacapone lowered 3-O-methyldopa. Cysteine decay may be due to an elevated glutathione generation, which consumes cysteine. Cysteinyl-glycine decrease results from the alternative glutathione transformation to its oxidized form glutathione dissulfide after free radical scavenging.

Cysteinyl‐glycine reduces mucosal pro‐inflammatory cytokine response to fMLP in a piglet model of intestinal atrophy

PepT1 is a small intestinal (SI) di/tripeptide transporter also capable of transporting bacterial peptides. We measured the mucosal cytokine response to a pro‐inflammatory peptide (fMLP) when delivered with cysteinyl‐glycine (CG); CG competes for PepT1‐mediated uptake and has anti‐inflammatory potential. Pigs (n=6, 10 d) received parenteral nutrition (PN) for 4 d to induce SI atrophy; littermates (n=6) remained with the sow (S). Subsequently, five 10 cm loops of the distal SI were isolated and perfused for 3 h with one of: 1) 5 mM cysteine + 5 mM glycine (5 mM cys + gly) 2) 5 mM CG 3) 10 μM fMLP 4) 5 mM cys + gly + 10 μM fMLP 5) 5 mM CG + 10 μM fMLP. In both S and PN, SI exposed to fMLP had higher mucosal TNF‐α and IFN‐γ compared to unexposed loops (p&lt;0.001), while IFN‐γ was higher in PN compared to S pigs (p &lt; 0.01). CG + fMLP treatment lowered the IFN‐γ response in both S and PN (p &lt;0.05), but neither group responded significantly to free cys + gly. Interestingly, free cys + gly reduced the TNF‐α response in S pigs (p&lt;0.001), but not in the PN group. CG + fMLP lowered TNF‐α compared to fMLP alone in both groups (p&lt;0.001) and in S pigs the response was significantly more abated than with cys + gly (p &lt; 0.001). Thus, CG was more effective than free cys + gly at attenuating a bacterial peptide‐induced cytokine response particularly in the injured SI; this may be due to impaired free amino acid uptake or effective competitive inhibition of fMLP uptake. (NSERC)

Human organic anion transporter OAT1 is not responsible for glutathione transport but mediates transport of glutamate derivatives

Due to their clearance function, the kidneys are exposed to high concentrations of oxidants and potentially toxic substances. To maintain cellular integrity, renal cells have to be protected by sufficient concentrations of the antioxidant glutathione (GSH). We tested whether GSH or its precursors are taken up by human organic anion transporters 1 (OAT1) and 3 (OAT3) stably expressed in HEK293 cells. GSH did not inhibit uptake of p-aminohippurate (PAH) or of estrone sulfate (ES) in OAT3-transfected HEK293 cells. In OAT1-transfected cells, GSH reduced the uptake of PAH marginally. Among the GSH constituent amino acids, glutamate, cysteine, and glycine, only glutamate inhibited OAT1, but labeled glutamate was not taken up by a probenecid-inhibitable transport system. Thus OAT1 binds glutamate but is unable to translocate it. The GSH precursor dipeptide, cysteinyl glycine (cysgly), and the glutamate derivative N-acetyl glutamate (NAG), inhibited uptake of PAH when present in the medium and trans-stimulated uptake of PAH from the intracellular side, indicating that they are hitherto unrecognized transported substrates of OAT1. N-acetyl aspartate weakly interacted with OAT1, but aspartate did not. NAG inhibited also OAT3, albeit with much lower affinity compared with OAT1, and glutamate did not interact with OAT3 at all. Taken together, human OAT3 and OAT1 cannot be involved in renal GSH extraction from the blood. However, OAT1 could support intracellular GSH synthesis by taking up cysteinyl glycine.