One-step fabrication of dipeptide-based bifunctional polymer for individual enrichment of glycopeptides and phosphopeptides from serum.

A dipeptide-based bifunctional material immobilized with Ti4+ (denoted as APE-MBA-VPA-Ti4+) was developed using precipitation polymerization. This polymer combines hydrophilic interaction liquid chromatography (HILIC) and immobilized metal affinity chromatography (IMAC) enrichment strategies, allowing for the individual and simultaneous enrichment of glycopeptides and phosphopeptides. It demonstrated high sensitivity (0.1 fmol µL-1 for glycopeptides, 0.005 fmol µL-1 for phosphopeptides), strong selectivity (molar ratio HRP: BSA = 1:1000, ß-casein: BSA = 1:2500), consistent reusability (10 cycles) and satisfactory recovery rate (93.5 ± 1.8 % for glycopeptides, 91.6 ± 0.6 % for phosphopeptides) in the individual enrichment. Utilizing nano LC-MS/MS technology, the serum of liver cancer patients was analyzed after enrichment individually, resulting in the successful capture of 333 glycopeptides covering 262 glycosylation sites, corresponding to 131 glycoproteins, as well as 67 phosphopeptides covering 57 phosphorylation sites, related to 48 phosphoproteins. In comparison, the serum of normal healthy individuals yielded a total of 283 glycopeptides covering 244 glycosylation sites corresponding to 126 glycoproteins, as well as 66 phosphopeptides covering 56 phosphorylation sites related to 37 phosphoproteins. Label-free quantification identified 10 differentially expressed glycoproteins and 8 differentially expressed phosphoproteins in the serum of liver cancer patients. Among them, glycoproteins (HP, BCHE, AGT, C3, and PROC) and phosphoproteins (ZYX, GOLM1, GP1BB, CLU, and TNXB) showed upregulation and displayed potential as biomarkers for liver cancer.

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

BACKGROUND: Acute 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. METHODS: A 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. RESULTS: The 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. CONCLUSIONS: The reduced Hcy and Hcy/HHcy, not total Hcy concentration should be used to evaluate neurologic impairment severity of AIS patient.

Plasma metabolome and cytokine profile reveal glycylproline modulating antibody fading in convalescent COVID-19 patients.

The COVID-19 pandemic has incurred tremendous costs worldwide and is still threatening public health in the "new normal." The association between neutralizing antibody levels and metabolic alterations in convalescent patients with COVID-19 is still poorly understood. In the present work, we conducted absolutely quantitative profiling to compare the plasma cytokines and metabolome of ordinary convalescent patients with antibodies (CA), convalescents with rapidly faded antibodies (CO), and healthy subjects. As a result, we identified that cytokines such as M-CSF and IL-12p40 and plasma metabolites such as glycylproline (gly-pro) and long-chain acylcarnitines could be associated with antibody fading in COVID-19 convalescent patients. Following feature selection, we built machine-learning-based classification models using 17 features (six cytokines and 11 metabolites). Overall accuracies of more than 90% were attained in at least six machine-learning models. Of note, the dipeptide gly-pro, a product of enzymatic peptide cleavage catalyzed by dipeptidyl peptidase 4 (DPP4), strongly accumulated in CO individuals compared with the CA group. Furthermore, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination experiments in healthy mice demonstrated that supplementation of gly-pro down-regulates SARS-CoV-2-specific receptor-binding domain antibody levels and suppresses immune responses, whereas the DPP4 inhibitor sitagliptin can counteract the inhibitory effects of gly-pro upon SARS-CoV-2 vaccination. Our findings not only reveal the important role of gly-pro in the immune responses to SARS-CoV-2 infection but also indicate a possible mechanism underlying the beneficial outcomes of treatment with DPP4 inhibitors in convalescent COVID-19 patients, shedding light on therapeutic and vaccination strategies against COVID-19.

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.

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.

Humoral response to neurofilaments and dipeptide repeats in ALS progression.

OBJECTIVE: To appraise the utility as biomarkers of blood antibodies and immune complexes to neurofilaments and dipeptide repeat proteins, the products of translation of the most common genetic mutation in amyotrophic lateral sclerosis (ALS). METHODS: Antibodies and immune complexes against neurofilament light, medium, heavy chains as well as poly-(GP)-(GR) dipeptide repeats were measured in blood samples from the ALS Biomarkers (n = 107) and the phenotype-genotype biomarker (n = 129) studies and in 140 healthy controls. Target analyte levels were studied longitudinally in 37 ALS cases. Participants were stratified according to the rate of disease progression estimated before and after baseline and C9orf72 genetic status. Survival and longitudinal analyses were undertaken with reference to matched neurofilament protein expression. RESULTS: Compared to healthy controls, total neurofilament proteins and antibodies, neurofilament light immune complexes (p < 0.0001), and neurofilament heavy antibodies (p = 0.0061) were significantly elevated in ALS, patients with faster progressing disease (p < 0.0001) and in ALS cases with a C9orf72 mutation (p < 0.0003). Blood neurofilament light protein discriminated better ALS from healthy controls (AUC: 0.92; p < 0.0001) and faster from slower progressing ALS (AUC: 0.86; p < 0.0001) compared to heavy-chain antibodies and light-chain immune complexes (AUC: 0.79; p < 0.0001 and AUC: 0.74; p < 0.0001). Lower neurofilament heavy antibodies were associated with longer survival (Log-rank Chi-square: 7.39; p = 0.0065). Increasing levels of antibodies and immune complexes between time points were observed in faster progressing ALS. CONCLUSIONS: We report a distinctive humoral response characterized by raising antibodies against neurofilaments and dipeptide repeats in faster progressing and C9orf72 genetic mutation carriers ALS patients. We confirm the significance of plasma neurofilament proteins in the clinical stratification of ALS.

Plasma metabolites were associated with spatial working memory in major depressive disorder.

ABSTRACT: Major depressive disorder (MDD) is a common disease with both affective and cognitive disorders. Alterations in metabolic systems of MDD patients have been reported, but the underlying mechanisms still remains unclear. We sought to identify abnormal metabolites in MDD by metabolomics and to explore the association between differential metabolites and neurocognitive dysfunction.Plasma samples from 53 MDD patients and 83 sex-, gender-, BMI-matched healthy controls (HCs) were collected. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was then used to detect metabolites in those samples. Two different algorithms were applied to identify differential metabolites in 2 groups. Of the 136 participants, 35 MDD patients and 48 HCs had completed spatial working memory test. Spearman rank correlation coefficient was applied to explore the relationship between differential metabolites and working memory in these 2 groups.The top 5 metabolites which were found in sparse partial least squares-discriminant analysis (sPLS-DA) model and random forest (RF) model were the same, and significant difference was found in 3 metabolites between MDD and HCs, namely, gamma-glutamyl leucine, leucine-enkephalin, and valeric acid. In addition, MDD patients had higher scores in spatial working memory (SWM) between errors and total errors than HCs. Valeric acid was positively correlated with working memory in MDD group.Gamma-glutamyl leucine, leucine-enkephalin, and valeric acid were preliminarily proven to be decreased in MDD patients. In addition, MDD patients performed worse in working memory than HCs. Dysfunction in working memory of MDD individuals was associated with valeric acid.

Effect of the Natural Sweetener Xylitol on Gut Hormone Secretion and Gastric Emptying in Humans: A Pilot Dose-Ranging Study.

Sugar consumption is associated with a whole range of negative health effects and should be reduced and the natural sweetener xylitol might be helpful in achieving this goal. The present study was conducted as a randomized, placebo-controlled, double-blind, cross-over trial. Twelve healthy, lean volunteers received intragastric solutions with 7, 17 or 35 g xylitol or tap water on four separate days. We examined effects on: gut hormones, glucose, insulin, glucagon, uric acid, lipid profile, as well as gastric emptying rates, appetite-related sensations and gastrointestinal symptoms. We found: (i) a dose-dependent stimulation of cholecystokinin (CCK), active glucagon-like peptide-1 (aGLP-1), peptide tyrosine tyrosine (PYY)-release, and decelerated gastric emptying rates, (ii) a dose-dependent increase in blood glucose and insulin, (iii) no effect on motilin, glucagon, or glucose-dependent insulinotropic peptide (GIP)-release, (iv) no effect on blood lipids, but a rise in uric acid, and (v) increased bowel sounds as only side effects. In conclusion, low doses of xylitol stimulate the secretion of gut hormones and induce a deceleration in gastric emptying rates. There is no effect on blood lipids and only little effect on plasma glucose and insulin. This combination of properties (low-glycemic sweetener which stimulates satiation hormone release) makes xylitol an attractive candidate for sugar replacement.

Role of Magnetic Resonance Spectroscopy in Evaluation of Cerebral Metabolic Status Before and After Carotid Endarterectomy/Thromboendarterectomy and Carotid Artery Stenting in Patients with Asymptomatic Critical Internal Carotid Artery Stenosis.

BACKGROUND The relative efficacy of carotid endarterectomy (CEA)/thromboendarterectomy (TEA) and carotid artery stenting (CAS) already has been compared in randomized controlled trials and a meta-analysis, but only limited data exist describing the status of cerebral metabolism before and after these interventions. The aim of the present study was to compare metabolic changes before and after treatment of carotid stenosis and assess their potential clinical implications.   MATERIAL AND METHODS Patients with asymptomatic unilateral critical internal CAS were imaged with proton 3T magnetic resonance spectroscopy (H-MRS) because the technique is more sensitive than regular magnetic resonance imaging for detection of the early signs of ischemic events. Abnormal metabolite ratios detected with H-MRS may precede actual morphological changes associated with hypoperfusion as well as reperfusion changes. Ipsilateral and contralateral middle cerebral artery vascular territories were both evaluated before and after vascular intervention. H-MRS was performed within 24 h before and after surgery. Correlations in the metabolic data from H-MRS for N-acetylaspartic acid (NAA)+N-acetylaspartylglutamate, creatinine (Cr)+phosphocreatinine, and phosphocholine+glycerophosphocholine (Cho) were sought. RESULTS H-MRS voxels from 11 subjects were analyzed. Values for dCho/CrI, dCho/CrC and Cho/Naal (P<0.001) were significantly higher ipsilaterally than contralaterally. Ratios for dNaa/ChoC and Cho/NaaC were significantly higher on the non-operated side (P<0.001). CONCLUSIONS H-MRS may be helpful for assessment of patients with CAS, particularly because unlike other modalities, it reveals postoperative changes in metabolic brain status. Initial results indicate the important role of perioperative neuroprotective treatment.

Anethole Dithiolethione Increases Glutathione in Kidney by Inhibiting γ-Glutamyltranspeptidase: Biochemical Interpretation and Pharmacological Consequences.

AIMS: Anethole dithiolethione (ADT) is a marketed drug to treat xerostomia. Its mechanism of action is still unknown, but several preclinical studies indicate that it is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Here, we investigated the molecular mechanisms behind these effects. RESULTS: Oral treatment of rats confirmed the GSH enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ-glutamyltranspeptidase (γ-GT) activity of the different tissues. In vitro and ex vivo experiments with tubular renal cells and isolated perfused rat kidney showed that the cellular uptake of intact GSH was correlated with the extracellular concentrations of GSH. CONCLUSION: s. The prominent in vivopharmacological effect of ADT was a marked increase of GSH concentration in the kidney and a decrease of some systemic and renal biomarkers of oxidative stress. In particular, by inhibition of γ-GT activity, it decreased the production cysteinylglycine, a thiol that has prooxidant effects as the consequence of its autooxidation. The activity of ADT as GSH enhancer in both the circulation and the kidney was long-lasting. All these characteristics make ADT a promising drug to protect the kidney, and in particular proximal tubule cells, from xenobiotic-induced damage.

Pre-symptomatic Caspase-1 inhibitor delays cognitive decline in a mouse model of Alzheimer disease and aging.

Early therapeutic interventions are essential to prevent Alzheimer Disease (AD). The association of several inflammation-related genetic markers with AD and the early activation of pro-inflammatory pathways in AD suggest inflammation as a plausible therapeutic target. Inflammatory Caspase-1 has a significant impact on AD-like pathophysiology and Caspase-1 inhibitor, VX-765, reverses cognitive deficits in AD mouse models. Here, a one-month pre-symptomatic treatment of Swedish/Indiana mutant amyloid precursor protein (APPSw/Ind) J20 and wild-type mice with VX-765 delays both APPSw/Ind- and age-induced episodic and spatial memory deficits. VX-765 delays inflammation without considerably affecting soluble and aggregated amyloid beta peptide (Aß) levels. Episodic memory scores correlate negatively with microglial activation. These results suggest that Caspase-1-mediated inflammation occurs early in the disease and raise hope that VX-765, a previously Food and Drug Administration-approved drug for human CNS clinical trials, may be a useful drug to prevent the onset of cognitive deficits and brain inflammation in AD.

Sucralose Consumption over 2 Weeks in Healthy Subjects Does Not Modify Fasting Plasma Concentrations of Appetite-Regulating Hormones: A Randomized Clinical Trial.

BACKGROUND: The effect of nonnutritive sweeteners on appetite is controversial. Some studies have found changes in certain appetite control hormones with sucralose intake that may be through interaction with sweet taste receptors located in the intestine. OBJECTIVE: The aim of this study was to evaluate whether sucralose consumption could produce changes in fasting plasma concentrations of appetite-regulating hormones, including glucagon-like peptide 1, ghrelin, peptide tyrosine tyrosine, and leptin, and secondarily in insulin resistance. DESIGN: A 2-week parallel randomized clinical trial with an additional visit conducted 1 week after dosing termination. PARTICIPANTS/SETTING: Sixty healthy, normal-weight individuals, without habitual consumption of nonnutritive sweeteners were recruited from July 2015 to March 2017 in Mexico City. INTERVENTION: Daily sucralose consumption at 15% of the acceptable daily intake by using commercial sachets added to food. The control group followed the same protocol without an intervention. MAIN OUTCOMES MEASURED: Fasting concentrations of appetite regulating hormones before and after the intervention. Fasting glucose and insulin concentrations were measured to assess insulin resistance as a secondary outcome. STATISTICAL ANALYSIS PERFORMED: Basal and final concentrations were compared using Wilcoxon matched-pairs test and Mann-Whitney U test for analysis between groups. Repeated measures analysis of variance was used to evaluate changes in the homeostasis model assessment of insulin resistance. RESULTS: Sucralose was not associated with changes in any of the hormones measured. One week postintervention, an incremental change (P=0.04) in the homeostasis model assessment of insulin resistance was found in the intervention group. CONCLUSIONS: Sucralose intake is not associated with changes in fasting concentrations of glucagon-like peptide 1, ghrelin, peptide tyrosine tyrosine, or leptin. An increase in the homeostasis model assessment of insulin resistance observed only at 1 week postdosing is of unknown clinical significance, if any.

Influence of Dietary Supplementation for Hyperhomocysteinemia Treatments.

Hyperhomocysteinemia is recognized as risk factor for cardiovascular and age-associated diseases. Folic acid supplementation efficiently lowers plasma homocysteine (Hcy) levels, but high intake may negatively affect health because of unnatural levels of unmetabolized folic acid in the systemic circulation. Oxoproline (Oxo) provides by glutamic acid production an increase of intracellular folic acid trapping. Aim of this study was to compare the efficacy of three supplementation protocols: (1) traditional therapy (5-methyl-tetrahydrofolate: 15 mg/day); (2) 5 mL/day of Oxo with 300 µg folic acid (oxifolic); (3) 5 mL/day of Oxo alone (magnesio+) in a 90 days randomized trial on thirty-two moderate hyperhomocysteinemic (18.6 ± 2.4 µmol.L-1) patients (age 48 ± 14 yrs). Thiols: cysteine (Cys), cysteinylglycine (Cys-Gly) and glutathione levels were assessed too. Every supplementation induced significant (p range <0.05-0.0001) reductions of Hcy level and Cys concentration after the three protocols adopted. Otherwise glutathione concentration significantly increased after oxifolic (p < 0.01) and traditional (p < 0.05) supplementation. The integration of Oxo resulted an interesting alternative to traditional therapy because absence or minimal number of folates in the integrator eliminates any chance of excess that can constitute a long-term risk.

LC-MS/MS Analysis of Thiol-Containing Amino Acids in Exosomal Fraction of Serum.

It has been suggested that thiol-containing amino acids could be used as biomarkers for diseases associated with oxidative stress. We investigated the thiol-containing amino acids, homocysteine (Hcy), cysteine (Cys), glutathione (GSH) and γ-glutamylcysteine (γ-GluCys), in commercial human serum by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after precolumn derivatization with 4-fluoro-7-sulfobenzofurazan. This method was applied to determine the composition of thiol-containing amino acids in exosomes prepared from the serum. Hcy, Cys, GSH and γ-GluCys could be detected in the exosomal fraction, and the ratio of each thiol-containing amino acid was similar to those in the corresponding native serum. Cys (94.76%) was most enriched in the exosomal fraction, followed by GSH (2.97%), γ-GluCys (1.59%) and Hcy (0.68%). These findings suggest that thiol-containing amino acids, Hcy, Cys, GSH and γ-GluCys, are included in exosomes in human serum.

Physiological responses to maximal eating in men.

This study investigated metabolic, endocrine, appetite and mood responses to a maximal eating occasion in fourteen men (mean: age 28 (sd 5) years, body mass 77·2 (sd 6·6) kg and BMI 24·2 (sd 2·2) kg/m2) who completed two trials in a randomised crossover design. On each occasion, participants ate a homogenous mixed-macronutrient meal (pizza). On one occasion, they ate until 'comfortably full' (ad libitum) and on the other, until they 'could not eat another bite' (maximal). Mean energy intake was double in the maximal (13 024 (95 % CI 10 964, 15 084) kJ; 3113 (95 % CI 2620, 3605) kcal) compared with the ad libitum trial (6627 (95 % CI 5708, 7547) kJ; 1584 (95 % CI 1364, 1804) kcal). Serum insulin incremental AUC (iAUC) increased approximately 1·5-fold in the maximal compared with ad libitum trial (mean: ad libitum 43·8 (95 % CI 28·3, 59·3) nmol/l × 240 min and maximal 67·7 (95 % CI 47·0, 88·5) nmol/l × 240 min, P < 0·01), but glucose iAUC did not differ between trials (ad libitum 94·3 (95 % CI 30·3, 158·2) mmol/l × 240 min and maximal 126·5 (95 % CI 76·9, 176·0) mmol/l × 240 min, P = 0·19). TAG iAUC was approximately 1·5-fold greater in the maximal v. ad libitum trial (ad libitum 98·6 (95 % CI 69·9, 127·2) mmol/l × 240 min and maximal 146·4 (95 % CI 88·6, 204·1) mmol/l × 240 min, P < 0·01). Total glucagon-like peptide-1, glucose-dependent insulinotropic peptide and peptide tyrosine-tyrosine iAUC were greater in the maximal compared with ad libitum trial (P < 0·05). Total ghrelin concentrations decreased to a similar extent, but AUC was slightly lower in the maximal v. ad libitum trial (P = 0·02). There were marked differences on appetite and mood between trials, most notably maximal eating caused a prolonged increase in lethargy. Healthy men have the capacity to eat twice the energy content required to achieve comfortable fullness at a single meal. Postprandial glycaemia is well regulated following initial overeating, with elevated postprandial insulinaemia probably contributing.

Identification of the urine and serum metabolomics signature of gout.

OBJECTIVE: Gout is the most common inflammatory arthritis and the worldwide incidence is increasing. By revealing the metabolic alterations in serum and urine of gout patients, the first aim of our study was to discover novel molecular biomarkers allowing for early diagnosis. We also aimed to investigate the underlying pathogenic pathways. METHODS: Serum and urine samples from gout patients (n = 30) and age-matched healthy controls (n = 30) were analysed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) to screen the differential metabolites and construct a diagnostic model. Next, the model was verified and optimized in the second validation cohort (n = 100). The pathways were illustrated to understand the underlying pathogenesis of gout. RESULTS: In general, serum metabolomics demonstrated a clearer distinction than urine metabolomics. In the discovery cohort, 40 differential serum metabolites were identified that could distinguish gout patients from healthy controls. Among them, eight serum metabolites were verified in the validation cohort. Through regression analysis, the final model consisted of three serum metabolites-pyroglutamic acid, 2-methylbutyryl carnitine and Phe-Phe-that presented optimal diagnostic power. The three proposed metabolites produced an area under the curve of 0.956 (95% CI 0.911, 1.000). Additionally, the proposed metabolic pathways were primarily involved in purine metabolism, branched-chain amino acids (BCAAs) metabolism, the tricarboxylic acid cycle, synthesis and degradation of ketone bodies, bile secretion and arachidonic acid metabolism. CONCLUSION: The metabolomics signatures could serve as an efficient tool for early diagnosis and provide novel insights into the pathogenesis of gout.

γ-Glutamylcysteine synthetase and γ-glutamyl transferase as differential enzymatic sources of γ-glutamylpeptides in mice.

Some γ-glutamylpeptides in blood plasma are putative biomarkers for pathological conditions of the liver. γ-Glutamyltransferase (GGT) and γ-glutamylcysteine synthetase (γ-GCS) are two such potential enzymes that are responsible for the production of γ-glutamylpeptides. GGT produces γ-glutamylpeptides by transferring the γ-glutamyl moiety from glutathione to an amino acid or a peptide. γ-GCS normally catalyzes the production of γ-glutamylcysteine from glutamate and cysteine in the glutathione-synthesizing reaction, but other amino acids can also serve as an acceptor of a γ-glutamyl group, thus resulting in the formation of a variety of γ-glutamylpeptides. Based on liquid chromatography-mass spectrometry analyses, we observed differences in the distribution of γ-glutamylpeptides between the liver and kidney and were able to measure the activities of γ-GCS as well as the GGT reactions by quantifying the resulting γ-glutamylpeptides. The enzymatic characterization of γ-GCS in liver homogenates indicated that several γ-glutamylpeptides including γ-glutamyltaurine are actually produced. Cys showed the lowest Km value (0.06 mM) while other amino acids had much higher Km values (ranging from 21 to 1800 mM). The moderate Km values for these amino acids suggest that they were not the preferred amino acids in this conversion but were utilized as acceptor substrates for the production of the corresponding γ-glutamylpeptides by the γ-GCS reaction under Cys-deficient conditions. Thus, the production of these γ-glutamylpeptides by γ-GCS is directly correlated with a low Cys content, suggesting that their measurement in blood plasma could be useful for predicting the presymptomatic disease state of the liver with a defect in GSH redox balance.

Bioanalysis of EGFRm inhibitor osimertinib, and its glutathione cycle- and desmethyl metabolites by liquid chromatography-tandem mass spectrometry.

Osimertinib is a "third-generation'' oral, irreversible, tyrosine kinase inhibitor. It is used in the treatment of non-small cellular lung carcinoma and spares wild-type EGFR. Due to its reactive nature, osimertinib is, in addition to oxidative routes, metabolized through GSH coupling and subsequent further metabolism of these conjugates. The extent of the non-oxidative metabolism of osimertinib is unknown, and methods to quantify this metabolic route have not been reported yet. To gain insight into this metabolic route, a sensitive bioanalytical assay was developed for osimertinib, the active desmethyl metabolite AZ5104, and the thio-metabolites osimertinibs glutathione, cysteinylglycine, and cysteine conjugates was developed. The ease of synthesis of these metabolites was a key-part in the development of this assay. This was done through simple one-step synthesis and subsequent LC-purification. The compounds were characterized by NMR and high-resolution mass spectrometry. Sample preparation was done by a simple protein crash with acetonitrile containing the stable isotopically labeled internal standards for osimertinib and the thio-metabolites, partial evaporation of solvents, and reconstitution in eluent, followed by UHPLC-MS/MS quantification. The assay was successfully validated in a 2-2000 nM calibration range for all compounds except the glutathione metabolite, where the LLOQ was set at 6 nM due to low accuracy at 2 nM. Limited stability was observed for osimertinib, AZ5104, and the glutathione metabolite. The clinical applicability of the assay was demonstrated in samples of patients treated with 80 mg osimertinib once daily, containing all investigated compounds at detectable and quantifiable levels.

Comparative Preclinical Pharmacokinetics and Bioavailability of Antidepressant GSB-106 Tablet Form.

Pharmacokinetics and relative bioavailability of antidepressant GSB-106 (hexamethylendiamide bis-(N-monosuccinyl-L-seryl-L-lysine) were determined in rabbits after single oral administration of the pharmaceutical substance and tablet mass. GSB-106 concentrations in the blood plasma were determined by HPLC-mass spectrometry. Relative bioavailability of GSB-106 tablet form was 160.79±24.33%.

Transport Versus Hydrolysis: Reassessing Intestinal Assimilation of Di- and Tripeptides by LC-MS/MS Analysis.

SCOPE: The role of PEPT1 in the uptake of intact peptides as compared to hydrolysis prior to uptake of their constituents is unknown. Here, dipeptides, tripeptides, and amino acids are quantified to study the fate of selected peptides in different intestinal models. METHODS AND RESULTS: An LC-MS/MS-based method is applied for the simultaneous assessment of rates of hydrolysis and transport of a peptide panel in Caco-2 transwell cell culture, in vitro and in vivo in mice expressing or lacking PEPT1, and in hydrolysis studies in vitro using human intestinal samples. It is shown that susceptibility to hydrolysis of peptides at the brush border membrane or within epithelial cells is practically identical in all tested models and strictly structure-dependent. Peptides with high luminal disappearance show substantial hydrolysis and low basolateral appearance, while peptides with low disappearance show strong PEPT1 dependency and high basolateral appearance in intact form in Caco-2 transwell culture. CONCLUSION: Hydrolysis and transport of intact peptides are highly variable and structure-dependent. For peptides possessing less polar N-terminal residues, hydrolysis usually dominates over transport via PEPT1. For other peptides with high intrinsic hydrolysis resistance, including anserine, carnosine, ɣ-glutamyl-dipeptides, and aminocephalosporins, PEPT1 is the main determinant for appearance in peripheral blood.