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D-Alanine, a rare enantiomer of alanine, can potentially alleviate the worsening of viral infections and maintain circadian rhythm. This study aimed to analyze the kinetics of D-Alanine upon oral intake. Five healthy volunteers were administered D-Alanine as a single oral dose at 11,236 or 33,708 µmoL (1-3 g). Upon intake of the lower dose, the plasma level of D-Alanine reached its peak concentration of 588.4 ± 40.9 µM with a peak time of 0.60 ± 0.06 h. The compartment model estimated the clearance of D-Alanine at 12.5 ± 0.3 L/h, or 208 ± 5 mL/min, distribution volume of 8.3 ± 0.7 L and half-life of 0.46 ± 0.04 h, suggesting a rapid clearance of D-Alanine. The peak concentration and area under the curve increased proportionally upon intake of the higher dose, while the clearance, distribution volume and half-life did not. The urinary ratio of D-Alanine per sum of D- and L-Alanine reached its peak of nearly 100%, followed by a slow decline. The peak time of the urinary ratio was 1.15 ± 0.15 h, showing a time lag of blood to urine excretion. Fractional excretion, a ratio of the clearance of a substance per a standard molecule in kidney, of D-Alanine increased from 14.0 ± 5.8% to 64.5 ± 10.3%; the latter corresponded to the urinary clearance of D-Alanine as about 77 mL/min for an adult, with a peak time of 1.90 ± 0.56 h. D-Alanine was quickly absorbed and appeared in blood, followed by urinary excretion. This kinetic analysis increases our fundamental knowledge of the oral intake of D-Alanine for the chronic dosing. Trial number: #UMIN000050865. Date of registration: 2023/6/30., (© 2024. The Author(s).)

The aim of this study was to assess the pharmacokinetics of the existing remdesivir intravenous formulation (100 mg dose) against the newly developed oral formulation (20 mg dose) for remdesivir and its active nucleoside metabolite (GS-441524) in beagle dogs followed by healthy human volunteers. A quantification method for remdesivir and its active nucleoside metabolite (GS-441524) in beagle dog and human plasma has been developed and validated using liquid chromatography coupled to triple quadrupole mass spectrometry detection. The analytical methods for beagle dogs and human differ in the calibration curve range, plasma matrix, processing volume, reconstitution volume and injection volume; however all other parameters were same in both methods. A simple protein precipitation extraction was carried out using acetonitrile containing the internal standard remdesivir D5. Remdesivir and GS-441524 were separated on an Endurus C-18P, 100 × 4.6 mm, 3 μm column and detected using a mass spectrometer with electrospray ionization in positive ion mode. The ion transitions used were m/z 603.1 → m/z 200.0 for remdesivir, m/z 292.0 → m/z 202.2 for GS-441524 and m/z 608.2 → m/z 205.1 for remdesivir D5. The calibration curve results were linear in beagle dog plasma (2.0-2,000.8 ng/ml range for remdesivir and 2.0-1,500.4 ng/ml for GS-441524) and human plasma (30.0-4,503.9 ng/ml range for remdesivir and 2.0-200.4 ng/ml for GS-441524). The recovery was >90% in beagle dog and human plasma. These methods were successfully used to determine the pharmacokinetic parameters of the intravenous injection and subcutaneous tablets dosage forms in beagle dogs and healthy humans., (© 2024 John Wiley & Sons, Ltd.)

Aims: Many transgender and gender diverse (TGD) individuals have expressed concerns about the potential for oral pre-exposure prophylaxis to affect hormonal concentrations achieved from taking gender-affirming hormone therapy (GAHT). The purpose of this study was to understand the bidirectional effects between hormone and intraerythrocytic tenofovir diphosphate concentrations when switching from tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) to tenofovir alafenamide/emtricitabine (TAF/FTC) in TGD users/nonusers of GAHT., Methods: The study evaluated stored blood samples and dried blood spot cards from TGD adults without HIV who took ≥12 weeks of TDF/FTC and then switched to ≥12 weeks of TAF/FTC for pre-exposure prophylaxis., Results: Thirty-nine individuals met the study inclusion criteria. Regardless of sex assigned at birth and the use of GAHT, there were no significant differences in hormone concentrations when individuals taking GAHT were taking TDF/FTC and then switched to TAF/FTC. Further, there was no significant difference in intraerythrocytic tenofovir diphosphate concentrations between users and nonusers of GAHT., Conclusion: There are no bidirectional effects between hormone and intraerythocytic tenofovir diphosphate concentrations when switching from TDF/FTC to TAF/FTC in TGD users/nonusers of GAHT., (© 2024 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

A green and simple UPLC method was developed and optimized, adopting a factorial design for simultaneous determination of oseltamivir phosphate and remdesivir with dexamethasone as a co-administered drug in human plasma and using daclatasvir dihydrochloride as an internal standard within 5 min. The separation was established on UPLC column BEH C 18 1.7 μm (2.1 × 100.0 mm) connected to UPLC pre-column BEH 1.7 μm (2.1 × 5.0 mm) at 50 °C with an injection volume of 10 μL. The photodiode array detector (PDA) was set at three wavelengths of 220, 315, and 245 nm for oseltamivir phosphate, the internal standard, and both dexamethasone and remdesivir, respectively. The mobile phase consisted of methanol and ammonium acetate solution (40 mM) adjusted to pH 4 in a ratio of 61.5:38.5 (v/v) with a flow rate of 0.25 mL min -1 . The calibration curves were linear over 500.0-5000.0 ng mL -1 for oseltamivir phosphate, over 10.0-500.0 ng mL -1 and 500.0-5000.0 ng mL -1 for dexamethasone, and over 20.0-500 ng mL -1 and 500.0-5000.0 ng mL -1 for remdesivir. The Gibbs free energy and Van't Hoff plots were used to investigate the effect of column oven temperatures on retention times. Fluoride-EDTA anticoagulant showed inhibition activity on the esterase enzyme in plasma. The proposed method was validated according to the M10 ICH, FDA, and EMA's bioanalytical guidelines. According to Eco-score, GAPI, and AGREE criteria, the proposed method was considered acceptable green., (© 2024. The Author(s).)

Aim: To evaluate insulin and glucagon sensitivity in Han Chinese women with and without gestational diabetes mellitus (GDM)., Methods: In total, 81 women with GDM and 81 age-matched healthy controls were evaluated with a 75 g oral glucose tolerance test (OGTT) at gestational weeks 24-28. Plasma glucose concentrations were measured at fasting and 1 h and 2 h post-OGTT. Fasting plasma insulin, glucagon and amino acids were also measured. Insulin and glucagon sensitivity were assessed by the homeostatic model assessment of insulin resistance (HOMA-IR) and glucagon-alanine index, respectively., Results: As expected, plasma glucose concentrations were higher at fasting and 1 h and 2 h post-OGTT in GDM participants (p < .001 each). Both the HOMA-IR and the glucagon-alanine index were higher in GDM participants. There was a weak positive correlation between HOMA-IR and glucagon-alanine index (r = 0.24, p = .0024). Combining the HOMA-IR and the glucagon-alanine index yielded better capacity (area under the curve = 0.878) than either alone (area under the curve = 0.828 for HOMA-IR and 0.751 for glucagon-alanine index, respectively) in differentiating GDM from healthy participants. While the majority of GDM participants (64%) exhibited both reduced insulin and glucagon sensitivity, a third of them presented either reduced insulin (20%) or glucagon (14%) sensitivity alone. HOMA-IR and glucagon-alanine index correlated differentially with fasting glucose, triglycerides, low-density lipoprotein cholesterol, sum of amino acids and hepatic steatosis index., Conclusions: Impairments of both insulin and glucagon sensitivity occur frequently in Chinese women with GDM, which may, individually or together, drive metabolic derangements in GDM. These observations provide new insights into the pathophysiology of GDM and support the need to target insulin or glucagon resistance, or both, in the management of GDM., (© 2024 John Wiley & Sons Ltd.)

Despite its effectiveness, combination antiretroviral treatment (cART) has a limited effect on HIV DNA reservoir, which establishes early during primary HIV infection (PHI) and is maintained by latency, homeostatic T-cells proliferation, and residual replication. This limited effect can be associated with low drug exposure in lymphoid tissues and/or suboptimal adherence to antiretroviral drugs (ARVs). The aim of this study was to assess ARV concentrations in plasma, peripheral blood mononuclear cells (PBMCs) and lymph nodes (LNs), and their association to HIV RNA and HIV DNA decay during PHI. Participants were randomised to receive standard doses of darunavir/cobicistat (Arm I), dolutegravir (Arm II) or both (Arm III), with a backbone of tenofovir alafenamide and emtricitabine. Total HIV DNA was measured using digital-droplet PCR in PBMCs at baseline, 12 and 48 weeks. Drug concentrations in plasma and PBMCs were determined at 2, 12 and 48 weeks (LNs at 12 weeks) by UHPLC-MS/MS. Seventy-two participants were enrolled, mostly male (n=68), with a median age of 34 years and variable Fiebig stages (V-VI 57.7%, I-II 23.9%, and III-IV 18.3%). Twenty-six patients were assigned to Arm I, 27 to Arm II and 19 to Arm III. After 48 weeks, most patients had undetectable viremia, with minor differences in HIV RNA decay between arms. Patients with Fiebig I-II showed faster HIV RNA and HIV DNA decay. Intracellular tissue penetration was high for nucleoside analogues and low-moderate for darunavir and dolutegravir. Only tenofovir diphosphate concentrations in PBMCs showed correlation with HIV DNA decay. Overall, these results indicate that the timing of treatment initiation and intracellular tenofovir penetration are primary and secondary factors, respectively, affecting HIV reservoir., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Sepsis is characterized by a metabolic disorder of amino acid occurs in the early stage; however, the profile of serum amino acids and their alterations associated with the onset of sepsis remain unclear. Thus, our objective is to identify the specific kinds of amino acids as diagnostic biomarkers in pediatric patients with sepsis. Serum samples were collected from patients with sepsis admitted to the pediatric intensive care unit (PICU) between January 2019 and December 2019 on the 1 st , 3 rd and 7 th day following admission. Demographic and laboratory variables were also retrieved from the medical records specified times. Serum amino acid concentrations were detected by UPLC-MS/MS system. PLS-DA (VIP > 1.0) and Kruskal-Wallis test (p < 0.05) were employed to identify potential biomarkers. Spearman's rank correlation analysis was conducted to find the potential association between amino acid levels and clinical features. The diagnostic utility for pediatric sepsis was assessed using receiver operating characteristic (ROC) curve analysis. Most of amino acid contents in serum were significantly decreased in patients with sepsis, but approached normal levels by the seventh day post-diagnosis. Threonine (THR), lysine (LYS), valine (VAL) and alanine (ALA) emerged as potential biomarkers related for sepsis occurrence, though they were not associated with PELOD/PELOD-2 scores. Moreover, alterations in serum THR, LYS and ALA were linked to complications of brain injury, and serum ALA levels were also related to sepsis-associated acute kidney injury. Further analysis revealed that ALA was significantly correlated with the Glasgow score, serum lactate and glucose levels, C-reactive protein (CRP), and other indicators for liver or kidney dysfunction. Notably, the area under the ROC curve (AUC) for ALA in distinguishing sepsis from healthy controls was 0.977 (95% CI: 0.925-1.000). The serum amino acid profile of children with sepsis is significantly altered compared to that of healthy controls. Notably, ALA shows promise as a potential biomarker for the early diagnosis in septic children., (© 2024. The Author(s).)

A novel, highly sensitive and eco-friendly micellar-mediated spectrofluorimetric method was developed and validated for the determination of the novel antiparkinsonian drug safinamide mesylate in the presence of its related precursor impurity, 4-hydroxybenzaldehyde. The proposed approach relies on increasing the inherent fluorescence emission at 296 nm of safinamide, by forming hydrogen bonds between the mentioned drug and sodium dodecyl sulfate in the micellar system using 0.1 N HCl as a solvent, following excitation at 226 nm. A thorough investigation was conducted into the experimental factors affecting spectrofluorimetric behavior of the studied drug. A linearity plot of safinamide over the concentration range of 10.0-1000.0 ng/mL against the relative fluorescence intensities was established. The proposed method demonstrated excellent sensitivity down to the nano-gram level with detection and quantitation limits of 1.91 and 5.79 ng/mL, respectively. The studied drug was effectively determined in Parkimedine ® Tablets. Furthermore, the proposed method allows for ultrasensitive quantification of safinamide in spiked human plasma, with satisfactory percentage recovery (98.97-102.28%). Additionally, the greenness assessment using the advanced green certificate classification approach, the complementary green analytical procedure index (Complex-GAPI), and the analytical GREEness metric approach (AGREE), along with the practicality check using the Blue Applicability Grade Index in addition to the all-inclusive overall whiteness evaluation using the RGB-12 model were carried out. The outcomes demonstrated the effectiveness and whiteness of the proposed technique. Clearly, the suggested approach has the advantages of being simple, requiring no pretreatment steps, and relying solely on direct measuring procedures., (© 2024. The Author(s).)

Estimation of the postmortem interval (PMI), especially the early PMI, plays a key role in forensic practice. Although several studies based on metabolomics approaches have presented significant findings for PMI estimation, most did not examine the effects of ambient temperature. In this study, gas chromatography-mass spectrometry (GC‒MS)‒based metabolomics was adopted to explore the changes in metabolites in the cardiac blood of suffocated rats at various ambient temperatures (5 °C, 15 °C, 25 °C, and 35 °C) from 0 to 24 h after death. Isoleucine, alanine, proline, valine, glycerol, glycerol phosphate, xanthine, and hypoxanthine were found to contribute to PMI in all temperature groups. Hypoxanthine and isoleucine were chosen to establish estimation models (equations) with an interpolation function using PMI as the dependent variable (f (x, y) ), relative intensity as the independent variable x, and temperature as the independent variable y. Thereafter, these two models were validated with predictive samples and shown to have potential predictive ability. The findings indicate that isoleucine, alanine, proline, valine, glycerol, glycerol phosphate, xanthine, and hypoxanthine may be significant for PMI estimation at various ambient temperatures. Furthermore, a method to determine PMI based on ambient temperature and PMI-related metabolites was explored, which may provide a basis for future studies and practical applications., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Introduction: The DOLAM trial revealed that switching from triple antiretroviral therapy (three-drug regimen; 3DR) to dolutegravir plus lamivudine (two-drug regimen; 2DR) was virologically non-inferior to continuing 3DR after 48 weeks of follow-up. Weight increased with 2DR relative to 3DR but it did not impact on metabolic parameters., Methods: Multiomics plasma profile was performed to gain further insight into whether this therapy switch might affect specific biological pathways. DOLAM (EudraCT 201500027435) is a Phase 4, randomized, open-label, non-inferiority trial in which virologically suppressed persons with HIV treated with 3DR were assigned (1:1) to switch to 2DR or to continue 3DR for 48 weeks. Untargeted proteomics, metabolomics and lipidomics analyses were performed at baseline and at 48 weeks. Univariate and multivariate analyses were performed to identify changes in key molecules between both therapy arms., Results: Switching from 3DR to 2DR showed a multiomic impact on circulating plasma concentration of N-acetylmuramoyl-L-alanine amidase (Q96PD5), insulin-like growth factor-binding protein 3 (A6XND0), alanine and triglyceride (TG) (48:0). Correlation analyses identified an association among the up-regulation of these four molecules in persons treated with 2DR., Conclusions: Untargeted multiomics profiling studies identified molecular changes potentially associated with inflammation immune pathways, and with lipid and glucose metabolism. Although these changes could be associated with potential metabolic or cardiovascular consequences, their clinical significance remains uncertain. Further work is needed to confirm these findings and to assess their long-term clinical consequences., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The coronavirus-2 has led to a global pandemic of COVID-19 with an outbreak of severe acute respiratory syndrome leading to worldwide quarantine measures and a rise in death rates. The objective of this study is to propose a green, sensitive, and selective densitometric method to simultaneously quantify remdesivir (REM) in the presence of the co-administered drug linezolid (LNZ) and rivaroxaban (RIV) in spiked human plasma. TLC silica gel aluminum plates 60 F254 were used as the stationary phase, and the mobile phase was composed of dichloromethane (DCM): acetone (8.5:1.5, v/v) with densitometric detection at 254 nm. Well-resolved peaks have been observed with retardation factors (R f ) of 0.23, 0.53, and 0.72 for REM, LNZ, and RIV, respectively. A validation study was conducted according to ICH Q2 (R1) Guidelines. The method was rectilinear over the concentration ranges of 0.2-5.5 μg/band, 0.2-4.5 μg/band and 0.1-3.0 μg/band for REM, LNZ and RIV, respectively. The sensitivities of REM, LIN, and RIV were outstanding, with quantitation limits of 128.8, 50.5, and 55.8 ng/band, respectively. The approach has shown outstanding recoveries ranging from 98.3 to 101.2% when applied to pharmaceutical formulations and spiked human plasma. The method's greenness was assessed using Analytical Eco-scale, GAPI, and AGREE metrics., (© 2024. The Author(s).)

Remdesivir (RDV) is the first drug approved by the US Food and Drug Administration (FDA) for the treatment of coronavirus disease 2019 (COVID-19) in certain patients requiring hospitalization. As a nucleoside analogue prodrug, RDV undergoes intracellular multistep activation to form its pharmacologically active species, GS-443902, which is not detectable in the plasma. A question arises that whether the observed plasma exposure of RDV and its metabolites would correlate with or be informative about the exposure of GS-443902 in tissues. A whole body physiologically-based pharmacokinetic (PBPK) modeling and simulation approach was utilized to elucidate the disposition mechanism of RDV and its metabolites in the lungs and liver and explore the relationship between plasma and tissue pharmacokinetics (PK) of RDV and its metabolites in healthy subjects. In addition, the potential alteration of plasma and tissue PK of RDV and its metabolites in patients with organ dysfunction was explored. Our simulation results indicated that intracellular exposure of GS-443902 was decreased in the liver and increased in the lungs in subjects with hepatic impairment relative to the subjects with normal liver function. In subjects with severe renal impairment, the exposure of GS-443902 in the liver was slightly increased, whereas the lung exposure of GS-443902 was not impacted. These predictions along with the organ impairment study results may be used to support decision making regarding the RDV dosage adjustment in these patient subgroups. The modeling exercise illustrated the potential of whole body PBPK modeling to aid in decision making for nucleotide analogue prodrugs, particularly when the active metabolite exposure in the target tissues is not available., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

In pasture-based systems, there are nutritional and climatic challenges exacerbated across lactation; thus, dairy cows require an enhanced adaptive capacity compared with cows in confined systems. We aimed to evaluate the effect of lactation stage (21 vs. 180 days in milk, DIM) and Holstein genetic strain (North American Holstein, NAH, n = 8; New Zealand Holstein, NZH, n = 8) on metabolic adaptations of grazing dairy cows through plasma metabolomic profiling and its association with classical metabolites. Although 67 metabolites were affected (FDR < 0.05) by DIM, no metabolite was observed to differ between genetic strains while only alanine was affected (FDR = 0.02) by the interaction between genetic strain and DIM. However, complementary tools for time-series analysis (ASCA analysis, MEBA ranking) indicated that alanine and the branched-chain amino acids (BCAA) differed between genetic strains in a lactation-stage dependent manner. Indeed, NZH cows had lower (P-Tukey < 0.05) plasma concentrations of leucine, isoleucine and valine than NAH cows at 21 DIM, probably signaling for greater insulin sensitivity. Metabolic pathway analysis also revealed that, independently of genetic strains, AA metabolism might be structurally involved in homeorhetic changes as 40% (19/46) of metabolic pathways differentially expressed (FDR < 0.05) between 21 and 180 DIM belonged to AA metabolism., (© 2021. The Author(s).)

Remdesivir is a nucleotide analog prodrug that has received much attention since the outbreak of the COVID-19 pandemic in December 2019. GS-441524 (Nuc) is the active metabolite of remdesivir and plays a pivotal role in the clinical treatment of COVID-19. Here, a robust HPLC-MS/MS method was developed to determine Nuc concentrations in rat plasma samples after a one-step protein precipitation process. Chromatographic separation was accomplished on Waters XBrige C 18 column (50 × 2.1 mm, 3.5 μm) under gradient elution conditions. Multiple reaction monitoring transitions in electrospray positive ion mode were m/z 292.2 → 163.2 for Nuc and 237.1 → 194.1 for the internal standard (carbamazepine). The quantitative analysis method was fully validated in line with the United States Food and Drug Administration guidelines. The linearity, accuracy and precision, matrix effect, recovery, and stability results met the requirements of the guidelines. Uncertainty of measurement and incurred sample reanalysis were analyzed to further ensure the robustness and reproducibility of the method. This optimized method was successfully applied in a rat pharmacokinetics study of remdesivir (intravenously administration, 5 mg kg -1 ). The method can act as a basis for further pharmacokinetic and clinical efficacy investigations in patients with COVID-19. Graphical abstract., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Background and Objectives: Metabolomics facilitates the discovery of biomarkers and potential therapeutic targets for CKD progression., Design, Setting, Participants, & Measurements: We evaluated an untargeted metabolomics quantification of stored plasma samples from 645 Chronic Kidney Disease in Children (CKiD) participants. Metabolites were standardized and logarithmically transformed. Cox proportional hazards regression examined the association between 825 nondrug metabolites and progression to the composite outcome of KRT or 50% reduction of eGFR, adjusting for age, sex, race, body mass index, hypertension, glomerular versus nonglomerular diagnosis, proteinuria, and baseline eGFR. Stratified analyses were performed within subgroups of glomerular/nonglomerular diagnosis and baseline eGFR., Results: Baseline characteristics were 391 (61%) male; median age 12 years; median eGFR 54 ml/min per 1.73 m 2 ; 448 (69%) nonglomerular diagnosis. Over a median follow-up of 4.8 years, 209 (32%) participants developed the composite outcome. Unique association signals were identified in subgroups of baseline eGFR. Among participants with baseline eGFR ≥60 ml/min per 1.73 m 2 , two-fold higher levels of seven metabolites were significantly associated with higher hazards of KRT/halving of eGFR events: three involved in purine and pyrimidine metabolism (N6-carbamoylthreonyladenosine, hazard ratio, 16; 95% confidence interval, 4 to 60; 5,6-dihydrouridine, hazard ratio, 17; 95% confidence interval, 5 to 55; pseudouridine, hazard ratio, 39; 95% confidence interval, 8 to 200); two amino acids, C-glycosyltryptophan, hazard ratio, 24; 95% confidence interval 6 to 95 and lanthionine, hazard ratio, 3; 95% confidence interval, 2 to 5; the tricarboxylic acid cycle intermediate 2-methylcitrate/homocitrate, hazard ratio, 4; 95% confidence interval, 2 to 7; and gulonate, hazard ratio, 10; 95% confidence interval, 3 to 29. Among those with baseline eGFR <60 ml/min per 1.73 m 2 , a higher level of tetrahydrocortisol sulfate was associated with lower risk of progression (hazard ratio, 0.8; 95% confidence interval, 0.7 to 0.9)., Conclusions: Untargeted plasma metabolomic profiling facilitated discovery of novel metabolite associations with CKD progression in children that were independent of established clinical predictors and highlight the role of select biologic pathways., (Copyright © 2021 by the American Society of Nephrology.)

Background: Recent studies highlight the role of metabolites in immune diseases, but it remains unknown how much of this effect is driven by genetic and non-genetic host factors., Result: We systematically investigate circulating metabolites in a cohort of 500 healthy subjects (500FG) in whom immune function and activity are deeply measured and whose genetics are profiled. Our data reveal that several major metabolic pathways, including the alanine/glutamate pathway and the arachidonic acid pathway, have a strong impact on cytokine production in response to ex vivo stimulation. We also examine the genetic regulation of metabolites associated with immune phenotypes through genome-wide association analysis and identify 29 significant loci, including eight novel independent loci. Of these, one locus (rs174584-FADS2) associated with arachidonic acid metabolism is causally associated with Crohn's disease, suggesting it is a potential therapeutic target., Conclusion: This study provides a comprehensive map of the integration between the blood metabolome and immune phenotypes, reveals novel genetic factors that regulate blood metabolite concentrations, and proposes an integrative approach for identifying new disease treatment targets.

Vascular calcification (VC) is a risk factor for cardiovascular events and mortality in chronic kidney disease (CKD). Several components influence the occurrence of VC, among which inflammation. A novel uremic toxin, lanthionine, was shown to increase intracellular calcium in endothelial cells and may have a role in VC. A group of CKD patients was selected and divided into patients with a glomerular filtration rate (GFR) of <45 mL/min/1.73 m 2 and ≥45 mL/min/1.73 m 2 . Total Calcium Score (TCS), based on the Agatston score, was assessed as circulating lanthionine and a panel of different cytokines. A hemodialysis patient group was also considered. Lanthionine was elevated in CKD patients, and levels increased significantly in hemodialysis patients with respect to the two CKD groups; in addition, lanthionine increased along with the increase in TCS, starting from one up to three. Interleukin IL-6, IL-8, and Eotaxin were significantly increased in patients with GFR < 45 mL/min/1.73 m 2 with respect to those with GFR ≥ 45 mL/min/1.73 m 2 . IL-1b, IL-7, IL-8, IL-12, Eotaxin, and VEGF increased in calcified patients with respect to the non-calcified. IL-8 and Eotaxin were elevated both in the low GFR group and in the calcified group. We propose that lanthionine, but also IL-8 and Eotaxin, in particular, are a key feature of VC of CKD, with possible marker significance.

Remdesivir, formerly GS-5734, has recently become the first antiviral drug approved by the U.S. Food and Drug Administration (FDA) to treat COVID-19, the disease caused by SARS-CoV-2. Therapeutic dosing and pharmacokinetic studies require a simple, sensitive, and selective validated assay to quantify drug concentrations in clinical samples. Therefore, we developed a rapid and sensitive LC-MS/MS assay for the quantification of remdesivir in human plasma with its deuterium-labeled analog, remdesivir- 2 H5, as the internal standard. Chromatographic separation was achieved on a Phenomenex® Synergi™ HPLC Fusion-RP (100 × 2 mm, 4 μm) column by gradient elution. Excellent accuracy and precision (<5.2% within-run variations and. <9.8% between-run variations) were obtained over the range of 0.5-5000 ng/mL. The assay met the FDA Bioanalytical Guidelines for selectivity and specificity, and low inter-matrix lot variability (<2.7%) was observed for extraction efficiency (77%) and matrix effect (123%) studies. Further, stability tests showed that the analyte does not degrade under working conditions, nor during freezing and thawing processes., (Published by Elsevier B.V.)

Both antiviral treatment with remdesivir and hemoadsorption using a CytoSorb ® adsorption device are applied in the treatment of severe COVID-19. The CytoSorb ® adsorber consists of porous polymer beads that adsorb a broad range of molecules, including cytokines but also several therapeutic drugs. In this study, we evaluated whether remdesivir and its main active metabolite GS-441524 would be adsorbed by CytoSorb ® . Serum containing remdesivir or GS-441524 was circulated in a custom-made system containing a CytoSorb ® device. Concentrations of remdesivir and GS-441524 before and after the adsorber were analyzed by liquid chromatography-tandem mass spectrometry. Measurements of remdesivir in the outgoing tube after the adsorber indicated almost complete removal of remdesivir by the device. In the reservoir, concentration of remdesivir showed an exponential decay and was not longer detectable after 60 mins. GS-441524 showed a similar exponential decay but, unlike remdesivir, it reached an adsorption-desorption equilibrium at ~48 µg/L. Remdesivir and its main active metabolite GS-441524 are rapidly eliminated from the perfusate by the CytoSorb ® adsorber device in vitro. This should be considered in patients for whom both therapies are indicated, and simultaneous application should be avoided. In general, plasma levels of therapeutic drugs should be closely monitored under concurrent CytoSorb ® therapy., (© 2021 The Authors. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Background: The present COVID-19 pandemic has prompted worldwide repurposing of drugs. The aim of the present work was to develop and validate a two-dimensional isotope-dilution liquid chromatrography tandem mass spectrometry (ID-LC-MS/MS) method for accurate quantification of remdesivir and its active metabolite GS-441524, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in serum; drugs that have gained attention for repurposing in the treatment of COVID-19., Methods: Following protein precipitation, samples were separated with a two-dimensional ultra-high performance liquid chromatography (2D-UHPLC) setup, consisting of an online solid phase extraction (SPE) coupled to an analytical column. For quantification, stable isotope-labelled analogues were used as internal standards for all analytes. The method was validated on the basis of the European Medicines Agency bioanalytical method validation protocol., Results: Detuning of lopinavir and ritonavir allowed simultaneous quantification of all analytes with different concentration ranges and sensitivity with a uniform injection volume of 5 μL. The method provided robust validation results with inaccuracy and imprecision values of ≤ 9.59 % and ≤ 11.1 % for all quality controls., Conclusion: The presented method is suitable for accurate and simultaneous quantification of remdesivir, its metabolite GS-441525, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in human serum. The quantitative assay may be an efficient tool for the therapeutic drug monitoring of these potential drug candidates in COVID-19 patients in order to increase treatment efficacy and safety., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Remdesivir (RDV) is a phosphoramidate prodrug designed to have activity against a broad spectrum of viruses. Following IV administration, RDV is rapidly distributed into cells and tissues and simultaneously metabolized into GS-441524 and GS-704277 in plasma. LC-MS/MS methods were validated for determination of the 3 analytes in human plasma that involved two key aspects to guarantee their precision, accuracy and robustness. First, instability issues of the analytes were overcome by diluted formic acid (FA) treatment of the plasma samples. Secondly, a separate injection for each analyte was performed with different ESI modes and organic gradients to achieve sensitivity and minimize carryover. Chromatographic separation was achieved on an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 μm) with a run time of 3.4 min. The calibration ranges were 4-4000, 2-2000, and 2-2000 ng/mL, respectively for RDV, GS-441524 and GS-704277. The intraday and interday precision (%CV) across validation runs at 3 QC levels for all 3 analytes was less than 6.6%, and the accuracy was within ±11.5%. The long-term storage stability in FA-treated plasma was established to be 392, 392 and 257 days at -70 °C, respectively for RDV, GS-441524 and GS-704277. The validated method was successfully applied in COVID-19 related clinical studies., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aims/hypothesis: Many individuals who develop type 2 diabetes also display increased glucagon levels (hyperglucagonaemia), which we have previously found to be associated with the metabolic syndrome. The concept of a liver-alpha cell axis provides a possible link between hyperglucagonaemia and elevated liver fat content, a typical finding in the metabolic syndrome. However, this association has only been studied in individuals with non-alcoholic fatty liver disease. Hence, we searched for a link between the liver and the alpha cells in individuals with non-steatotic levels of liver fat content. We hypothesised that the glucagon-alanine index, an indicator of the functional integrity of the liver-alpha cell axis, would associate with liver fat and insulin resistance in our cohort of women with low levels of liver fat., Methods: We analysed data from 79 individuals participating in the Prediction, Prevention and Subclassification of Type 2 Diabetes (PPSDiab) study, a prospective observational study of young women at low to high risk for the development of type 2 diabetes. Liver fat content was determined by MRI. Insulin resistance was calculated as HOMA-IR. We conducted Spearman correlation analyses of liver fat content and HOMA-IR with the glucagon-alanine index (the product of fasting plasma levels of glucagon and alanine). The prediction of the glucagon-alanine index by liver fat or HOMA-IR was tested in multivariate linear regression analyses in the whole cohort as well as after stratification for liver fat content ≤0.5% (n = 39) or >0.5% (n = 40)., Results: The glucagon-alanine index significantly correlated with liver fat and HOMA-IR in the entire cohort (ρ = 0.484, p < 0.001 and ρ = 0.417, p < 0.001, respectively). These associations resulted from significant correlations in participants with a liver fat content >0.5% (liver fat, ρ = 0.550, p < 0.001; HOMA-IR, ρ = 0.429, p = 0.006). In linear regression analyses, the association of the glucagon-alanine index with liver fat remained significant after adjustment for age and HOMA-IR in all participants and in those with liver fat >0.5% (β = 0.246, p = 0.0.23 and β = 0.430, p = 0.007, respectively) but not in participants with liver fat ≤0.5% (β = -0.184, p = 0.286)., Conclusions/interpretation: We reproduced the previously reported association of liver fat content and HOMA-IR with the glucagon-alanine index in an independent study cohort of young women with low to high risk for type 2 diabetes. Furthermore, our data indicates an insulin-resistance-independent association of liver fat content with the glucagon-alanine index. In summary, our study supports the concept that even lower levels of liver fat (from 0.5%) are connected to relative hyperglucagonaemia, reflecting an imminent impairment of the liver-alpha cell axis.

Both obesity and sarcopenia are frequently associated in ageing, and together may promote the progression of related conditions such as diabetes and frailty. However, little is known about the pathophysiological mechanisms underpinning this association. Here we show that systemic alanine metabolism is linked to glycaemic control. We find that expression of alanine aminotransferases is increased in the liver in mice with obesity and diabetes, as well as in humans with type 2 diabetes. Hepatocyte-selective silencing of both alanine aminotransferase enzymes in mice with obesity and diabetes retards hyperglycaemia and reverses skeletal muscle atrophy through restoration of skeletal muscle protein synthesis. Mechanistically, liver alanine catabolism driven by chronic glucocorticoid and glucagon signalling promotes hyperglycaemia and skeletal muscle wasting. We further provide evidence for amino acid-induced metabolic cross-talk between the liver and skeletal muscle in ex vivo experiments. Taken together, we reveal a metabolic inter-tissue cross-talk that links skeletal muscle atrophy and hyperglycaemia in type 2 diabetes.

Objective: To validate our previously identified candidate metabolites, and to assess the ability of these metabolites to predict hypoxic-ischemic encephalopathy (HIE) both individually and combined with clinical data., Study Design: Term neonates with signs of perinatal asphyxia, with and without HIE, and matched controls were recruited prospectively at birth from 2 large maternity units. Umbilical cord blood was collected for later batch metabolomic analysis by mass spectroscopy along with clinical details. The optimum selection of clinical and metabolites features with the ability to predict the development of HIE was determined using logistic regression modelling and machine learning techniques. Outcome of HIE was determined by clinical Sarnat grading and confirmed by electroencephalogram grade at 24 hours., Results: Fifteen of 27 candidate metabolites showed significant alteration in infants with perinatal asphyxia or HIE when compared with matched controls. Metabolomic data predicted the development of HIE with an area under the curve of 0.67 (95% CI, 0.62-0.71). Lactic acid and alanine were the primary metabolite predictors for the development of HIE, and when combined with clinical data, gave an area under the curve of 0.96 (95% CI, 0.92-0.95)., Conclusions: By combining clinical and metabolic data, accurate identification of infants who will develop HIE is possible shortly after birth, allowing early initiation of therapeutic hypothermia., (Copyright © 2020. Published by Elsevier Inc.)

Mitochondrial disorders represent a large collection of rare syndromes that are difficult to manage both because we do not fully understand biochemical pathogenesis and because we currently lack facile markers of severity. The m.3243A>G variant is the most common heteroplasmic mitochondrial DNA mutation and underlies a spectrum of diseases, notably mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). To identify robust circulating markers of m.3243A>G disease, we first performed discovery proteomics, targeted metabolomics, and untargeted metabolomics on plasma from a deeply phenotyped cohort (102 patients, 32 controls). In a validation phase, we measured concentrations of prioritized metabolites in an independent cohort using distinct methods. We validated 20 analytes (1 protein, 19 metabolites) that distinguish patients with MELAS from controls. The collection includes classic (lactate, alanine) and more recently identified (GDF-15, α-hydroxybutyrate) mitochondrial markers. By mining untargeted mass-spectra we uncovered 3 less well-studied metabolite families: N-lactoyl-amino acids, β-hydroxy acylcarnitines, and β-hydroxy fatty acids. Many of these 20 analytes correlate strongly with established measures of severity, including Karnofsky status, and mechanistically, nearly all markers are attributable to an elevated NADH/NAD+ ratio, or NADH-reductive stress. Our work defines a panel of organelle function tests related to NADH-reductive stress that should enable classification and monitoring of mitochondrial disease.

The world is in the midst of a pandemic caused by a novel coronavirus and is desperately searching for possible treatments. The antiviral remdesivir has shown some effectiveness against SARS-CoV-2 in vitro and in a recent animal study. We use data from a study of remdesivir in rhesus macaques to fit a viral kinetics model in an effort to determine the most appropriate mathematical descripton of the effect of remdesivir. We find statistically significant differences in the viral decay rate and use this to inform a possible mathematical formulation of the effect of remdesivir. Unfortunately, this model formulation suggests that the application of remdesivir will lengthen SARS-CoV-2 infections, putting into question its potential clinical benefit., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Currently, type 2 diabetes mellitus (T2DM) and obesity are major global public health issues, and their prevalence in the United Arab Emirates (UAE) are among the highest in the world. In 2019, The UAE diabetes national prevalence was 15.4%. In recent years there has been a considerable investigation of predictive biomarkers associated with these conditions. This study analysed fasting (8 h) blood samples from an obese, normoglycemic cohort and an obese, T2DM cohort of UAE nationals, employing clinical chemistry analysis, 1D 1 H NMR and mass spectroscopy (FIA-MS/MS and LC-MS/MS) techniques. The novel findings reported for the first time in a UAE population revealed significant differences in a number of metabolites in the T2DM cohort. Metabolic fingerprints identified by NMR included BCAAs, trimethylamine N-oxide, β-hydroxybutyrate, trimethyl uric acid, and alanine. A targeted MS approach showed significant differences in lysophosphatidylcholines, phosphatidylcholines, acylcarnitine, amino acids and sphingomyelins; Lyso.PC.a.C18.0, PC.ae.C34.2, C3.DC..C4.OH, glutamine and SM.C16.1, being the most significant metabolites. Pearson's correlation studies showed associations between these metabolites and the clinical chemistry parameters across both cohorts. This report identified differences in metabolites in response to T2DM in agreement with many published population studies. This contributes to the global search for a bank of metabolite biomarkers that can predict the advent of T2DM and give insight to its pathogenic mechanisms.

Systemic metabolic changes after renal transplantation reflect the key processes that are related to graft accommodation. In order to describe and better understand these changes, the 1 HNMR based metabolomics approach was used. The changes of 47 metabolites in the serum samples of 19 individuals were interpreted over time with respect to their levels prior to transplantation. Considering the specific repeated measures design of the experiments, data analysis was mainly focused on the multiple analyses of variance (ANOVA) methods such as ANOVA simultaneous component analysis and ANOVA-target projection. We also propose here the combined use of ANOVA and classification and regression trees (ANOVA-CART) under the assumption that a small set of metabolites the binary splits on which may better describe the graft accommodation processes over time. This assumption is very important for developing a medical protocol for evaluating a patient's health state. The results showed that besides creatinine, which is routinely used to monitor renal activity, the changes in levels of hippurate, mannitol and alanine may be associated with the changes in renal function during the post-transplantation recovery period. Specifically, the level of hippurate (or histidine) is more sensitive to any short-term changes in renal activity than creatinine.

A simple, precise, rapid and accurate reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for analysis of safinamide mesylate (SAF) in presence of its basic degradate, and co-administered drugs levodopa and ondansetron. The mobile phase consisted of acetonitrile and 20 mM potassium dihyrogen orthophosphate buffer having pH = 5 (40: 60 v/v). Quantification was achieved with ultraviolet detector at 226 nm. The linear range was 0.5-10 μg/mL with mean recovery ± SD of 99.72 ± 1.59. The peak purity of SAF in pharmaceutical preparation spiked with its degradate and co-administered drugs revealed symmetry factor (999.8) within the calculated threshold (>998.1). The suggested method was validated in compliance with the International Conference on Harmonization (ICH) guidelines and statistically compared with the manufacturer HPLC method with no significant difference in terms of accuracy and precision. The assay method was successfully used to estimate SAF in tablets with good percentage recoveries. The high sensitivity (lower than Cmax of the drug 0.65 μg/mL) of the proposed HPLC method enabled the determination of SAF in presence of its basic degradate and co-administered drug, ondansetron in human plasma with acceptable accuracy. The suggested HPLC method could be used in Quality Control (QC) lab for analysis of the studied drug in pharmaceutical preparation., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Remdesivir has received significant attention for its potential application in the treatment of COVID-19, caused by SARS-CoV-2. Remdesivir has already been tested for Ebola virus disease treatment and found to have activity against SARS and MERS coronaviruses. The remdesivir core contains GS-441524, which interferes with RNA-dependent RNA polymerases alone. In non-human primates, following IV administration, remdesivir is rapidly distributed into PBMCs and converted within 2 h to the active nucleoside triphosphate form, while GS-441524 is detectable in plasma for up to 24 h. Nevertheless, remdesivir pharmacokinetics and pharmacodynamics in humans are still unexplored, highlighting the need for a precise analytical method for remdesivir and GS-441524 quantification., Objectives: The validation of a reliable UHPLC-MS/MS method for remdesivir and GS-441524 quantification in human plasma., Methods: Remdesivir and GS-441524 standards and quality controls were prepared in plasma from healthy donors. Sample preparation consisted of protein precipitation, followed by dilution and injection into the QSight 220 UHPLC-MS/MS system. Chromatographic separation was obtained through an Acquity HSS T3 1.8 μm, 2.1 × 50 mm column, with a gradient of water and acetonitrile with 0.05% formic acid. The method was validated using EMA and FDA guidelines., Results: Analyte stability has been evaluated and described in detail. The method successfully fulfilled the validation process and it was demonstrated that, when possible, sample thermal inactivation could be a good choice in order to improve biosafety., Conclusions: This method represents a useful tool for studying remdesivir and GS-441524 clinical pharmacokinetics, particularly during the current COVID-19 outbreak., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: A method based on liquid chromatography coupled to triple quadrupole mass spectrometry detection using 50 µL of plasma was developed and fully validated for quantification of remdesivir and its active metabolites GS-441524., Methods: A simple protein precipitation was carried out using 75 µL of methanol containing the internal standard (IS) remdesivir-13C6 and 5 µL ZnSO4 1 M. After separation on Kinetex® 2.6 µm Polar C18 100A LC column (100 × 2.1 mm i.d.), both compounds were detected by a mass spectrometer with electrospray ionization in positive mode. The ion transitions used were m/z 603.3 → m/z 200.0 and m/z 229.0 for remdesivir, m/z 292.2 → m/z 173.1 and m/z 147.1 for GS-441524 and m/z 609.3 → m/z 206.0 for remdesivir-13C6., Results: Calibration curves were linear in the 1-5000 μg/L range for remdesivir and 5-2500 for GS-441524, with limit of detection set at 0.5 and 2 μg/L and limit of quantification at 1 and 5 μg/L, respectively. Precisions evaluated at 2.5, 400 and 4000 μg/L for remdesivir and 12.5, 125, 2000 μg/L for GS-441524 were lower than 14.7% and accuracy was in the [89.6-110.2%] range. A slight matrix effect was observed, compensated by IS. Higher stability of remdesivir and metabolite was observed on NaF-plasma. After 200 mg IV single administration, remdesivir concentration decrease rapidly with a half-life less than 1 h while GS-441524 appeared rapidly and decreased slowly until H24 with a half-life around 12 h., Conclusions: This method would be useful for therapeutic drug monitoring of these compounds in Covid-19 pandemic.

Rebamipide is used widely in East Asia for the treatment of gastric ulcers, acute gastritis, and exacerbated chronic gastritis. The objective of this study was to investigate the pharmacokinetic (PK) properties of rebamipide following single oral administration in rats and dogs. Eleven rats and dogs received single oral administrations of rebamipide (35 mg/kg and 100 mg, respectively). Blood samples were collected according to the assigned schedule, and the plasma concentration of rebamipide was determined using liquid chromatography-tandem mass spectrometry. A double-peak phenomenon was observed in the PK profile of rebamipide in rats. In contrast, rebamipide showed a conventional PK profile without double peaks in dogs. The half-life of rebamipide in rats (12.85 ± 7.86 h) was longer than that in dogs (5.62 ± 2.24 h), and the apparent total clearance (Cl t /F) of rebamipide in rats (3.32 ± 1.18 L/h) was lower than that in dogs (105.01 ± 42.37 L/h). Simple allometric approaches showed that the correlation between body weight and Cl t /F (R 2 = 0.9287) among rats, dogs, and humans appeared satisfactory. This finding will help not only in understanding the pharmacology of rebamipide but also in establishing a strategy for in vivo evaluation of novel rebamipide formulations., (© 2020 John Wiley & Sons, Ltd.)

Remdesivir is one of the most promising drugs to treat COVID-19 based on the following facts: remdesivir has a broad-spectrum antiviral mechanism of action; it demonstrated in vitro activity against SARS-CoV-2 and in vivo efficacy in animal models against the similar coronavirus MERS-CoV; its safety profile has been tested in Ebola patients and in compassionate use in COVID-19 patients. Currently, remdesivir is being investigated in ten randomized controlled trials against COVID-19. The dose regimen of remdesivir is an IV loading dose of 200 mg on day 1 followed by daily IV maintenance doses of 100 mg for 5-9 days. Based on our data analysis, however, remdesivir with IV administration alone is unlikely to achieve excellent clinical efficacy. This analysis is based on the following observations: plasma exposures of remdesivir and its active metabolite are unlikely to be correlated with its clinical efficacy; remdesivir and its active metabolites are unlikely to be adequate in the lung to kill the SARS-CoV-2 virus. Even if remdesivir demonstrates benefits in the current randomized controlled trials, its efficacy may be limited. We suggest that a combination of an IV and pulmonary delivery dose regimen should be studied immediately to realize a potentially more effective antiviral therapy against COVID-19. Graphical abstract.

Objectives: The differential diagnosis of seronegative rheumatoid arthritis (negRA) and psoriasis arthritis (PsA) is often difficult due to the similarity of symptoms and the unavailability of reliable clinical markers. Since chronic inflammation induces major changes in the serum metabolome and lipidome, we tested whether differences in serum metabolites and lipids could aid in improving the differential diagnosis of these diseases., Methods: Sera from negRA and PsA patients with established diagnosis were collected to build a biomarker-discovery cohort and a blinded validation cohort. Samples were analysed by proton nuclear magnetic resonance. Metabolite concentrations were calculated from the spectra and used to select the variables to build a multivariate diagnostic model., Results: Univariate analysis demonstrated differences in serological concentrations of amino acids: alanine, threonine, leucine, phenylalanine and valine; organic compounds: acetate, creatine, lactate and choline; and lipid ratios L3/L1, L5/L1 and L6/L1, but yielded area under the curve (AUC) values lower than 70%, indicating poor specificity and sensitivity. A multivariate diagnostic model that included age, gender, the concentrations of alanine, succinate and creatine phosphate and the lipid ratios L2/L1, L5/L1 and L6/L1 improved the sensitivity and specificity of the diagnosis with an AUC of 84.5%. Using this biomarker model, 71% of patients from a blinded validation cohort were correctly classified., Conclusions: PsA and negRA have distinct serum metabolomic and lipidomic signatures that can be used as biomarkers to discriminate between them. After validation in larger multiethnic cohorts this diagnostic model may become a valuable tool for a definite diagnosis of negRA or PsA patients., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Mild cognitive impairment (MCI) is a transition phase between healthy individuals and Alzheimer's disease (AD). Therefore, diagnosis of MCI at early stage will help to delay or prevent its progression to disease. In the present study, we aim to identify the metabolic biomarkers, which can help in the diagnosis of MCI. We have screened 2000 elderly individuals from north India, out of which 200 were identified as MCI. We continued our study on 10 MCI individuals who regularly participated in the follow-up. The age and gender matched 10 healthy individuals were taken as control. These control and MCI individuals were subjected to neuropsychological examination such as Hindi mental state examination (HMSE) and Montreal cognitive assessment (MOCA) followed by 1 H Nuclear Magnetic Resonance (NMR) analysis. Remarkable changes were noted between control and MCI individuals at metabolic level. In silico study showed the involvement of eight metabolites in MCI. We found higher level of lactate, N-acetyl aspartate, histidine and lower level of formate, choline, alanine, creatinine and glucose in blood plasma of MCI individuals compared to control. Further, In silico study showed that choline might be directly associated with MCI or AD. Such In silico study with quantitative metabolite analysis of plasma could be used as diagnostic biomarkers for the identification of MCI., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The present study aimed to characterize the relationships between plasma lactate, plasma alanine, monocarboxylate transporter (MCT) polymorphisms, and indices of diabetes in patients with type 2 diabetes (T2D) in Japan. Eighty-three patients with T2D were prospectively enrolled. The gluconeogenesis and glycogenolysis are enhanced and uptake of glucose is decreased in the T2D liver. Since the liver plays an important role in maintaining glucose metabolism, we examined the relationships between liver enzymes and indices of diabetes. Some studies have reported that MCT1 (SLC16A1) polymorphism causes metabolic diseases. In addition, a high frequency of MCT1 polymorphism was reported in a healthy Japanese population. However, little is known about the relationships between T2D and MCT polymorphisms. Plasma l-lactate concentration positively correlated with indices of diabetes (fasting plasma glucose [FPG] and hemoglobin A1c [HbA1c]) and with the liver enzymes alanine aminotransferase (ALT) and gamma-glutamyl transpeptidase (γ-GTP). MCT1 polymorphisms were associated with all of these markers. We identified no significant correlations between d-lactate or alanine concentrations and any of these markers, but a significant association was observed between l-lactate, a marker of oxidative capacity, and indices of diabetes. We conclude that plasma l-lactate concentration may represent a predictor of the progression or severity of T2D., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2019. Published by Elsevier Ltd.)

Background: D-amino acids have been recognized as bioactive substances in humans. d-Serine and D-alanine are co-agonists of N-methyl-d-aspartate receptors. Glutamate has been suggested to be involved in the pathophysiology of Alzheimer's disease (AD). This study aimed to explore the roles of amino acids, particularly D-amino acids, in cognitive decline among patients with AD or mild cognitive impairment (MCI)., Methods: We enrolled 144 patients: 20 amnestic MCI, 85 mild AD, 25 moderate AD, and 14 severe AD. Serum levels of amino acids were measured by high performance liquid chromatography and confirmed by D-amino acid oxidase assay. The cognitive function was mainly evaluated by Alzheimer's Disease Assessment Scale - Cognitive Subscale (ADAS-cog)., Results: ADAS-cog total scores were positively correlated with d-serine (r = 0.186, p = 0.026) and D-/Total- serine ratio (r = 0.191, p = 0.022). ADAS-cog behavior scores were negatively correlated with D-glutamate (r = -0.177, p = 0.034) and L-glutamate (r = -0.250, p = 0.003), but positively correlated with D-alanine (r = 0.236, p = 0.005) and D-/Total- alanine ratio (r = 0.252, p = 0.002). Among the 11 tasks of ADAS-cog, D-glutamate and d-serine were correlated with different items respectively, noteworthily in the opposite direction., Conclusion: This is the first study suggesting that D-amino acids in blood may be correlated with ADAS-cog in different items and in the opposite direction. Lower D-glutamate and higher D-alanine levels may predict more behavioral symptoms. In summary, D-glutamate, d-serine and D-alanine play different and characteristic roles in AD. Further longitudinal studies are warranted to elucidate the function and interaction of D-amino acids in specific cognitive domains as well as various phases of dementia., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The identification of metabolites in complex biological matrices is a challenging task in 1D 1 H-NMR-based metabolomics studies. Statistical total correlation spectroscopy (STOCSY) has emerged for aiding the structural elucidation by revealing the peaks that present a high correlation to a driver peak of interest (which would likely belong to the same molecule). However, in these studies, the signals from metabolites are normally present as a mixture of overlapping resonances, limiting the performance of STOCSY. As an alternative to avoid the overlap issue, 2D 1 H homonuclear J-resolved (JRES) spectra were projected, in their usual tilted and symmetrized processed form, and STOCSY was applied on these 1D projections (p-JRES-STOCSY). Nonetheless, this approach suffers in cases where the signals are very close. In addition, STOCSY was applied to the whole JRES spectra (also tilted) to identify correlated multiplets, although the overlap issue in itself was not addressed directly and the subsequent search in databases is complicated in cases of higher order coupling. With these limitations in mind, in the present work, we propose a new methodology based on the application of STOCSY on a set of nontilted JRES spectra, detecting peaks that would overlap in 1D spectra of the same sample set. Correlation comparison analysis for peak overlap detection (COCOA-POD) is able to reconstruct projected 1D STOCSY traces that result in more suitable database queries, as all peaks are summed at their f2 resonances instead of the resonance corresponding to the multiplet center in the tilted JRES spectra. (The peak dispersion and resolution enhancement gained are not sacrificed by the projection.) Besides improving database queries with better peak lists obtained from the projections of the 2D STOCSY analysis, the overlap region is examined, and the multiplet itself is analyzed from the correlation trace at 45° to obtain a cleaner multiplet profile, free from contributions from uncorrelated neighboring peaks.

The potential health benefit of dietary fiber has attracted considerable attention in recent decades. In this study, the effects of modified dietary fibers (MDF) derived from okara on body composition, fat distribution, serum metabolomic parameters, and fatty acid profiles in mice fed high-fat diets (HFD) were evaluated by nuclear magnetic resonance (NMR)-based metabolic approach. HFD-induced C57BL mice were fed with a diet containing 100 g/kg MDF for 12 weeks. Compared with control mice, MDF-fed mice exhibited less fat and lower body weights, altered serum metabolomic profiles, and distinct fatty acid profiles. The levels of choline, phosphatidylcholine, glycerophosphorylcholine, glucose, lysine, scyllo-inositol, and glutamate for MDF group were higher than those for both CONT and HFD groups. A remarkable reduction of total cholesterol, total triglycerides, ω-6 fatty acids, alanine, citrate, creatine, or succinate was also observable for MDF group compared with HFD group. These findings demonstrated that the intake of MDF derived from okara clearly ameliorated some of the HFD-induced adverse metabolic effects and prevented adipose tissue accumulation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat ('good freezer') and Black-Red Andaluza ('bad freezer') showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages., Competing Interests: The authors have declared that no competing interests exist.

L-Pyroglutamic acid succinimidyl ester (L-PGA-OSu) and its isotopic variant (L-PGA[d 5 ]-OSu) were synthesized and used as the chiral labeling reagents for the enantioseparation of amino acids by reversed-phase UPLC-ESI-MS/MS. The enantiomers of amino acids were labeled with the reagents at 60 °C for 10 min in an alkaline medium. The resulting diastereomers were well separated by the reversed-phase chromatography using an ODS column, packed with small particles (1.7 μm) (Rs = 1.95-8.05). A highly sensitive detection at a low-fmol level (0.5-3.2 fmol) was obtained from the selected reaction monitoring (SRM) chromatograms. An isotope labeling strategy using light and heavy variants for the differential analysis of the DL-amino acids in different sample groups is also presented in this paper. The ratios of D/L-alanine in different yogurt products were successfully determined by the proposed method. The D/L ratios were almost comparable to those obtained from only using light reagent (i.e., L-PGA-OSu). Therefore, the proposed strategy seems to be useful for the differential analysis of DL-amino acids, not only in food products but also in biological samples.

In the context of the vascular effects of hydrogen sulfide (H 2 S), it is known that this gaseous endogenous biological modulator of inflammation, oxidative stress, etc. is a potent vasodilator. Chronic renal failure, a common disease affecting the aging population, is characterized by low levels of H 2 S in plasma and tissues, which could mediate their typical hypertensive pattern, along with other abnormalities. Lanthionine and homolanthionine, natural non-proteinogenic amino acids, are formed as side products of H 2 S production. Also in consideration of the intrinsic difficulties in H 2 S measuring, these compounds have been proposed as reliable and stable markers of H 2 S synthesis. However, in the setting of chronic renal failure patients on hemodialysis, they represent typical retention products (without ruling out the possibility of an increased intestinal synthesis) and prospective novel uremic toxins. Here, a method utilizing liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring ion mode has been developed and evaluated for the determination of these key H 2 S metabolites in plasma, by using a triple quadrupole mass spectrometer.

Wilson's disease (WD) is caused by mutations in the ATP7B gene responsible for a toxic copper overload mainly in the liver and the central nervous system. Phenotypic heterogeneity may challenge the diagnostic confirmation. Exchangeable copper (CuEXC) has recently been proposed as a new marker of WD, and its ratio to the total serum copper (Cus), Relative Exchangeable Copper (REC = CuEXC/Cus), as a diagnostic marker. This study aimed to investigate whether this could be confirmed in Atp7b -/- mice, an engineered WD animal model. Atp7b -/- (n = 137) and wild type (WT; n = 101) mice were investigated under the same conditions at 6-8, 20, 39, or 50 weeks of age. Twenty-four Atp7b -/- mice received D-penicillamine treatment from 39 to 50 weeks of age. Serum and liver [histology and intrahepatic copper (IHCu)] data were evaluated. In the WT group, all serum and liver data were normal. Atp7b -/- livers developed a chronic injury from isolated moderate inflammation (6-8 weeks: 16/33 = 48%) to inflammatory fibrosis with cirrhosis (50 weeks: 25/25 = 100% and 16/25 = 64% respectively). Cus and CuEXC increased until week 39, whereas IHCu and REC were stable with increasing age and much higher than in WT mice (mean ± SD: 669 ± 269 vs. 13 ± 3 μg/g dry liver and 39 ± 12 vs. 11 ± 3%, respectively). A threshold value of 20% for REC provided a diagnostic sensitivity and specificity of 100%, regardless of sex, age, or the use of D-penicillamine. Eleven weeks of 100 mg/kg D-penicillamine reduced liver fibrosis (p = 0.001), IHCu (p = 0.026) and CuEXC (p = 0.175). In conclusion, this study confirms REC as a WD diagnostic marker in a mouse model of chronic liver disease caused by copper overload. Further studies are needed to assess the usefulness of CuEXC to monitor the evolution of WD, particularly during treatment., (Copyright © 2018. Published by Elsevier GmbH.)

Docosahexaenoic acid (DHA, 22:6n-3) must be consumed in the diet or synthesized from n-3 polyunsaturated fatty acid (PUFA) precursors. However, the effect of dietary DHA on the metabolic pathway is not fully understood. Presently, 21-day-old Long Evans rats were weaned onto one of four dietary protocols: 1) 8 weeks of 2% ALA (ALA), 2) 6 weeks ALA followed by 2 weeks of 2% ALA + 2% DHA (DHA), 3) 4 weeks ALA followed by 4 weeks DHA and 4) 8 weeks of DHA. After the feeding period, 2 H 5 -ALA and 13 C 20 -eicosapentaenoic acid (EPA, 20:5n-3) were co-infused and blood was collected over 3 h for determination of whole-body synthesis-secretion kinetics. The synthesis-secretion coefficient (ml/min, means ± SEM) for EPA (0.238±0.104 vs. 0.021±0.001) and DPAn-3 (0.194±0.060 vs. 0.020±0.008) synthesis from plasma unesterified ALA, and DPAn-3 from plasma unesterified EPA (2.04±0.89 vs. 0.163±0.025) were higher (P<.05) after 2 weeks compared to 8 weeks of DHA feeding. The daily synthesis-secretion rate (nmol/d) of DHA from EPA was highest after 4 weeks of DHA feeding (843±409) compared to no DHA (70±22). Liver gene expression of ELOVL2 and FADS2 were lower (P<.05) after 4 vs. 8 weeks of DHA. Higher synthesis-secretion kinetics after 2 and 4 weeks of DHA feeding suggests an increased throughput of the PUFA metabolic pathway. Furthermore, these findings may lead to novel dietary strategies to maximize DHA levels while minimizing dietary requirements., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: 1-Deoxysphingolipids (1-deoxySLs) are atypical sphingolipids. They are formed during sphingolipid de novo synthesis by the enzyme serine palmitoyltransferase, due to the alternate use of alanine over its canonical substrate serine. Pathologically elevated 1-deoxySL are involved in several neurological and metabolic disorders. The objective of this study was to investigate the role of 1-deoxySL in glycogen storage disease type I (GSDI)., Methods: In this prospective, longitudinal observational study (median follow-up 1.8y), the plasma 1-deoxySL profile was analyzed in 15 adult GSDI patients (12 GSDIa, 3 GSDIb), and 31 healthy controls, along with standard parameters for monitoring GSDI., Results: 1-Deoxysphinganine (1-deoxySA) concentrations were elevated in GSDI compared to controls (191 ± 129 vs 35 ± 14 nmol/l, p < 0.0001). Concordant with the mechanism of 1-deoxySL synthesis, plasma alanine was higher (625 ± 182 vs 398 ± 90 μmol/l, p < 0.0001), while serine was lower in GSDI than in controls (88 ± 22 vs 110 ± 18 μmol/l. p < 0.001). Accordingly, serine, alanine and triglycerides were determinants of 1-deoxySA in the longitudinal analysis of GSDIa. 1-deoxySA concentrations correlated with the occurrence of low blood glucose (area under the curve below 4 mmol/l) in continuous glucose monitoring. The 1-deoxySL profile in GSDIb was distinct from GSDIa, with a different ratio of saturated to unsaturated 1-deoxySL., Conclusion: In addition to the known abnormalities of lipoproteins, GSDI patients also have a disturbed sphingolipid metabolism with elevated plasma 1-deoxySL concentrations. 1-DeoxySA relates to the occurrence of low blood glucose, and may constitute a potential new biomarker for assessing metabolic control. GSDIa and Ib have distinct 1-deoxySL profiles indicating that both GSD subtypes have diverse phenotypes regarding lipid metabolism., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: To explore the metabolic characterization of host responses to drainage-resistant Klebsiella pneumoniae liver abscesses (DRKPLAs) with serum 1H-nuclear magnetic resonance (NMR) spectroscopy. Materials and Methods: The hospital records of all patients with a diagnosis of a liver abscess between June 2015 and December 2016 were retrieved from an electronic hospital database. Eighty-six patients with Klebsiella pneumoniae ( K. pneumoniae ) liver abscesses who underwent percutaneous drainage were identified. Twenty patients with confirmed DRKPLAs were studied. Moreover, we identified 20 consecutive patients with drainage-sensitive Klebsiella pneumoniae liver abscesses (DSKPLAs) as controls. Serum samples from the two groups were analyzed with 1H NMR spectroscopy. Partial least squares discriminant analysis (PLS-DA) was used to perform 1H NMR metabolic profiling. Metabolites were identified using the Human Metabolome Database, and pathway analysis was performed with MetaboAnalyst 3.0. Results: The PLS-DA test was able to discriminate between the two groups. Five key metabolites that contributed to their discrimination were identified. Glucose, lactate, and 3-hydroxybutyrate were found to be upregulated in DRKPLAs, whereas glutamine and alanine were downregulated compared with the DSKPLAs. Pathway analysis indicated that amino acid metabolisms were significantly different between the DRKPLAs and the DSKPLAs. The D-glutamine and D-glutamate metabolisms exhibited the greatest influences. Conclusions: The five key metabolites identified in our study may be potential targets for guiding novel therapeutics of DRKPLAs and are worthy of additional investigation.

Background: Parkinson's disease (PD) is the result of progressive degeneration of the nigrostriatal dopaminergic pathway and depletion of neurotransmitter dopamine in the striatum., Methods: We included 17 patients with PD along with 7 patients of progressive supranuclear palsy (PSP), 6 patients of multiple system atrophy (MSA) and 22 age and sex-matched healthy controls. We analyzed metabolite profiles in the serum of these patients and controls using 1 H NMR spectroscopy., Results: Isoleucine, valine, alanine, glutamine and histidine in PD, PSP and MSA were significantly (P < 0.001) higher than controls, whereas, glutamate and glucose were significantly increased in PD (P < 0.001), PSP and MSA (P < 0.05) vs., Control: Citrate was increased in PD, PSP and MSA (P < 0.05) vs., Control: While, acetone, lactate and formate were higher at P < 0.001, threonine is increased at P < 0.05. The 3D scattered score plot of OPLS-DA model revealed clear differentiation among the groups, R2 = 0.92 and Q2 = 0.78., Conclusion: Significant differences in various metabolite levels were found between control and disease groups. Common amino acids that are significantly higher in all groups include branched chain amino acids, which could increase neuronal excitability., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: To determine the frequency of disorders leading to methylmalonic acidurias., Methods: This cross-sectional study was conducted from January 2013 to April 2016 at the Aga Khan University Hospital, Karachi, and comprised patients diagnosed with methylmalonic acidurias based on urine organic acid analysis. Clinical history and biochemical data was collected from the biochemical genetics laboratory requisition forms. Organic acid chromatograms of all the subjects were critically reviewed by a biochemical pathologist and a metabolic physician. For assessing the clinical outcome, medical charts of the patients were reviewed. SPSS 19 was used for data analysis., Results: Of the 1,778 patients 50(2.81%) were detected with methylmalonic acidurias. After excluding patients with non-significant peaks of methylmalonic acidemia, 41(2.31%) were included in the final analysis. Of these, 20(48.7%) were females, while the overall median age was 11.5 months (interquartile range: 6-41.5). On stratification by type of disorders leading to methylmalonic acidurias, 9(22%) had methylmalonic acidemia, 12(29%) had Cobalamin-related remethylation disorders, nonspecific methylmalonic acidurias in 16(39%), while 2(5%) each had succinyl coenzyme A synthetase and Vitamin B12 deficiency. respectively., Conclusions: Screening tests, including urine organic acid, provided valuable clues to the aetiology of methylmalonic acidurias.