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Cytochrome P450s (CYPs) are functionally diverse monooxygenases responsible for oxidation of endogenous and xenobiotic compounds. The function of nonmammalian CYPs are largely unknown and tools for characterization limited. CYPs critical for xenobiotic metabolism are prone to catalytic cycle uncoupling resulting in reactive oxygen species (ROS) generation that is highly dependent on the specific CYP isoform and substrate interaction. This study describes the rapid assessment of the activity and coupling efficiency of CYPs using capillary electrophoresis with UV detection. The coupling efficiency of five zebrafish (Danio rerio) CYP1 isoforms with a series of fluorogenic substrate probes was determined by the rate of NADP+ formation and compared with fluorescent product turnover rates. In most cases, NADP+ formation significantly overestimated CYP1 catalytic activity for substrate O-dealkylation suggesting uncoupling. ROS production was confirmed by elevated hydrogen peroxide generation in poorly coupled reactions. Reactions with β-estradiol confirmed that CYP1A, 1C1, and 1C2 have greater catalytic activity and coupling efficiency; CYP1B1 and 1D1 had coupling efficiencies under 4%. This work highlights the wide disparity in uncoupling induced by unproductive substrate binding among different CYP isoforms. [ABSTRACT FROM AUTHOR]

Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal enzyme, β-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-β- d-glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration ( IC) of 47.5 ± 0.1 and 4.6 ± 1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The design of boronic acid sensors for photometric detection of carbohydrates has relied on exploiting differences in the thermodynamic stability of complex formation for molecular recognition. Herein, we introduce a direct method for analysis of sugar alcohols using 3-nitrophenylboronic acid (NPBA) as an electrokinetic probe in capillary electrophoresis (CE). Dynamic complexation of neutral polyols by NPBA during electromigration allows for their simultaneous resolution and UV detection based on formation of an anionic ternary boronate ester complex in phosphate buffer. Unlike conventional boronic acid sensors, thermodynamic and electrokinetic processes in CE allow for improved selectivity for the resolution of sugar alcohol stereoisomers having different vicinal polyol chain lengths even in cases when binding affinity is similar due to differences in their complex mobility. Three complementary approaches were investigated to compare the thermodynamics of polyol chelation with NPBA, namely direct binding assays by CE, UV absorbance spectroscopy and an indirect p K depression method. Overall, CE offers a convenient platform for characterization of reversible arylboronic acid interactions in free solution while allowing for direct analysis of complex mixtures of neutral/UV-transparent polyols without complicated sample handling. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The advent of electrospray-ionization mass spectrometry (ESI-MS) has given rise to expanded newborn screening programs for the early detection of inborn errors of metabolism (IEM). Despite the benefit of high-throughput screening for disease prognosis, conventional ESI-MS methods are limited by inadequate specificity, complicated sample handling, and low positive predictive outcome that can contribute to a high rate of false-positives. Herein, we report a robust strategy for neonatal screening based on capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) that offers a convenient platform for the direct analysis of amino acids, acylcarnitines, and their stereoisomers from dried blood spot (DBS) extracts without chemical derivalization. On-line sample preconcentration with desalting by CE-ESI-MS allowed for improved concentration sensitivity when detecting poorly responsive metabolites in complex biological samples without ionization suppression or isomeric/isobaric interferences. Method validation demonstrated that accurate yet precise quantification can be achieved for 20 different amino acid and acylcarnitine biomarkers associated with IEMs when using a single non-deuterated internal standard. CE-ESI-MS represents a promising second-tier method in newborn screening programs that is compatible with ESI-MS/MS technology in cases when improved specificity and sensitivity is warranted for diagnosis confirmation and subsequent monitoring. [ABSTRACT FROM AUTHOR]

Glutathione metabolism plays a fundamental role in maintaining homeostasis and regulating the redox environment of a cell. Despite the widespread interest in quantifying glutathione metabolites in oxidative stress research, conventional techniques are hampered by complicated sample handling procedures to prevent significant oxidation artifacts generated during sample collection, sample pretreatment, and/or chemical analysis. in this report, a simple and validated method for glutathione analysis from filtered red blood cell (RBC) lysates was developed using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in conjunction with fingerprick microsampling and ultrafiltration. About a 3-fold improvement in precision with nanomolar detection limits was achieved when using online sample pre-concentration with CE-ESI-MS via a modified injection sequence, which permitted accurate determination of the intracellular reduced/oxidized glutathione ratio (GSH/ GSSG), as well as other glutathione species, including protein-bound glutathione mixed disulfide (PSSG), free glutathione mixed disulfides (GSSR) and glutathione thioether conjugates (GSX). In this work, the redox status of filtered hemolysates was determined by the equilibrium half-cell reduction potential for glutathione (EGSSG/2GSH), whereas its intrinsic antioxidant capacity was assessed by the apparent rate of metal-catalyzed oxidation of glutathione. In-vitro incubation studies of intact RBCs with 1-chloro-2,4-dinilrobenzene (CDNB) and N-acetyl-L-cysteine (NAC) were found to significantly alter EGSSG/2GSH and/or glutathione oxidation kinetics (e.g., kGSSG/2GSH) relative to normal controls based on their function as a toxic electrophilic compound and a competitive free radical scavenging/reducing agent, respectively. Differential rates of glutathione oxidation (DIRGO) using CE-ESI-MS offers a novel strategy for global assessment of the impact of intrinsic metabolite constituents (i.e., metabolome) and/or extrinsic perturbants on cellular redox status that is relevant to improved understanding of aging and the pathogenesis of acute or chronic disease states. [ABSTRACT FROM AUTHOR]

• This study demonstrates the utility of specific NEFAs and their sub-classes as viable dietary and clinical biomarkers when reported as their relative proportions. • Consistently stronger dietary correlations were observed when expressed as mol%. • Glucose intolerance was positively associated with odd chain NEFAs as expressed in µM but inversely associated when expressed as mol%. • Monounsaturated NEFAs (µM and mol%) had robust positive associations with pre-pregnancy BMI, second trimester skin-fold thickness, glycated hemoglobin, fasting glucose, and glucose intolerance. • More research is needed to investigate inconsistencies between absolute concentrations and relative proportions when reporting fatty acids. Background: Sparse data exists on the utility of individual serum non-esterified fatty acids (NEFAs) as clinical and dietary biomarkers and how reporting methods could affect these associations. We investigated the associations of 19 serum NEFAs expressed as µM or mol%, with self-reported dietary intake data, and cardiometabolic health indicators in pregnant women. Methods: In this cross-sectional study, 273 pregnant women in their second trimester each completed a semi-quantitative food-frequency questionnaire and provided fasting serum samples. Comprehensive serum NEFA analysis was performed by multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry. We evaluated the associations of NEFAs using two different reporting methods, with diet quality, specific foods intake, and measures of adiposity and glucose homeostasis. Results: Consistently stronger dietary correlations were observed when expressed as mol%. Serum ω -3 NEFAs were associated with diet quality and fish/fish oil daily servings (DHA mol%, r= 0.37; p = 4.8e-10), and odd-chain NEFAs were associated with full-fat dairy intake (15:0 mol%, r = 0.23; p = 9.0e-5). Glucose intolerance was positively associated with odd chain NEFAs as expressed in µM (r = 0.21; p= 0.001) but inversely associated when expressed as mol% (r = −0.31; p= 2.2e-7). In contrast, monounsaturated NEFAs (µM and mol%) had robust positive associations with pre-pregnancy BMI, second trimester skin-fold thickness, glycated hemoglobin, fasting glucose, and glucose intolerance. Conclusions: This study demonstrates the utility of specific NEFAs and their sub-classes as viable dietary and clinical biomarkers when reported as their relative proportions. More research is needed to investigate inconsistencies between absolute concentrations and relative proportions when reporting fatty acids. [ABSTRACT FROM AUTHOR]

Metabolomics offers a hypothesis-generating approach for biomarker discovery in clinical medicine while also providing better understanding of the underlying mechanisms of chronic diseases. Clinical metabolomic studies largely rely on human biofluids (e.g., plasma, urine) as a more convenient specimen type for investigation. However, biofluids are non-organ specific reflecting complex biochemical processes throughout the body, which may complicate biochemical interpretations. For these reasons, tissue metabolomic studies enable deeper insights into aberrant metabolism occurring at the direct site of disease pathogenesis. This review highlights new advances in metabolomics for ex vivo analysis, as well as in situ imaging of tissue specimens, including diverse tissue types from animal models and human participants. Moreover, we discuss key pre-analytical and post-analytical challenges in tissue metabolomics for robust biomarker discovery with a focus on new methodological advances introduced over the past six years, including innovative clinical applications for improved screening, diagnostic testing, and therapeutic interventions for cancer. [ABSTRACT FROM AUTHOR]

The article focuses on the U.S. Plant, Algae and Microbial Metabolomics Research Coordination Network (PAMM-NET) that has been developed by a Research Coordination Network (RCN) grant from the U.S. National Science Foundation (NSF). It mentions that the PAMM-NET's will promote effective communication, collaboration and indentification of challenges in metabolomics. It also discussed the the U.S. Chapter of the Metabolomics Society and coordinated community efforts suported by the PAMM-NET.

Background: The rate of vitamin D deficiency (VDD) in critically ill children worldwide has been estimated at 50%. These children are at risk of multiple organ dysfunction, chronic morbidity, and decreased health related quality of life (HRQL). Pediatric and adult ICU clinical trials suggest that VDD is associated with worse clinical outcomes, although data from supplementation trials are limited and inconclusive. Our group's phase II multicenter dose evaluation pilot study established the efficacy and safety of an enteral weight-based cholecalciferol loading dose to rapidly restore vitamin D levels in critically ill children. Methods: Our aim is to evaluate the impact of this dosing regimen on clinical outcomes. VITdALIZE-KIDS is a pragmatic, phase III, multicenter, double-blind RCT aiming to randomize 766 critically ill children from Canadian PICUs. Participants are randomized using a 1:1 scheme to receive a single dose at enrollment of enteral cholecalciferol (10,000 IU/kg, max 400,000 IU) or placebo. Eligibility criteria include critically ill children aged newborn (> 37 weeks corrected gestational age) to < 18 years who have blood total 25-hydroxyvitamin D < 50 nmol/L. The primary objective is to determine if rapid normalization of vitamin D status improves HRQL at 28 days following enrollment. The secondary objective is to evaluate the impact of rapid normalization of vitamin D status on multiple organ dysfunction. The study includes additional tertiary outcomes including functional status, HRQL and mortality at hospital discharge and 90 days, PICU and hospital length of stay, and adverse events related to vitamin D toxicity. Additionally, we are performing comprehensive vitamin D speciation and non-targeted metabolite profiling as part of a sub-study for the first 100 participants from whom an enrollment and at least one post-intervention blood and urine sample were obtained. Discussion: The VITdALIZE-KIDS trial is the first phase III, multicenter trial to evaluate whether rapid normalization of vitamin D status could represent a simple, inexpensive, and safe means of improving outcomes following pediatric critical illness. Recruitment was initiated in June 2019 and is expected to continue to March 2026. Trial registration: Clinicaltrials.gov, NCT03742505. Study first submitted on November 12, 2018 https://clinicaltrials.gov/study/NCT03742505 [ABSTRACT FROM AUTHOR]

Rates of pediatric Crohn's disease (CD) and ulcerative colitis (UC) are increasing globally. Differentiation of these inflammatory bowel disease (IBD) subtypes however can be challenging when relying on invasive endoscopic approaches. We sought to identify urinary metabolic signatures of pediatric IBD at diagnosis, and during induction treatment. Nontargeted metabolite profiling of urine samples from CD (n = 18) and UC (n = 8) in a pediatric retrospective cohort study was performed using multisegment injection-capillary electrophoresis-mass spectrometry. Over 122 urinary metabolites were reliably measured from pediatric IBD patients, and unknown metabolites were identified by tandem mass spectrometry. Dynamic changes in sum-normalized urinary metabolites were also monitored following exclusive enteral nutrition (EEN) or corticosteroid therapy (CS) in repeat urine samples collected over 8 weeks. Higher urinary excretion of indoxyl sulfate, hydroxyindoxyl sulfate, phenylacetylglutamine, and sialic acid were measured in CD as compared to UC patients, but lower threonine, serine, kynurenine, and hypoxanthine (p < 0.05). Excellent discrimination of CD from UC was achieved based on the urinary serine:indoxylsulfate ratio (AUC = 0.972; p = 3.21 × 10−5). Urinary octanoyl glucuronide, pantothenic acid, and pyridoxic acid were also identified as specific dietary biomarkers of EEN in pediatric IBD patients who achieved clinical remission. This work may complement or replace existing strategies in the diagnosis and early management of children with IBD. [ABSTRACT FROM AUTHOR]

Acetylsalicylic acid (ASA), also known as aspirin, appears to be ineffective in inhibiting platelet aggregation in 20–30% of patients. Light transmission aggregometry (LTA) is a gold standard platelet function assay. In this pilot study, we used LTA to personalize ASA therapy ex vivo in atherosclerotic patients. Patients were recruited who were on 81 mg ASA, presenting to ambulatory clinics at St. Michael's Hospital (n = 64), with evidence of atherosclerotic disease defined as clinical symptoms and diagnostic findings indicative of symptomatic peripheral arterial disease (PAD), with an ankle brachial index (ABI) of <0.9 (n = 52) or had diagnostic features of asymptomatic carotid arterial stenosis (CAS), with >50% stenosis of internal carotid artery on duplex ultrasound (n = 12). ASA compliance was assessed via multisegmented injection-capillary electrophoresis-mass spectrometry based on measuring the predominant urinary ASA metabolite, salicyluric acid. LTA with arachidonic acid was used to test for ASA sensitivity. Escalating ASA dosages of 162 mg and 325 mg were investigated ex vivo for ASA dose personalization. Of the 64 atherosclerotic patients recruited, 8 patients (13%) were non-compliant with ASA. Of ASA compliant patients (n = 56), 9 patients (14%) were non-sensitive to their 81 mg ASA dosage. Personalizing ASA therapy in 81 mg ASA non-sensitive patients with escalating dosages of ASA demonstrated that 6 patients became sensitive to a dosage equivalent to 162 mg ASA and 3 patients became sensitive to a dosage equivalent to 325 mg ASA. We were able to personalize ASA dosage ex vivo in all ASA non-sensitive patients with escalating dosages of ASA within 1 h of testing. [ABSTRACT FROM AUTHOR]

Introduction: Irritable bowel syndrome (IBS), the most commonly diagnosed functional gastrointestinal (GI) disorder in developed countries, is characterized by chronic abdominal pain, and altered bowel habits. Objectives: Accurate and timely diagnosis is challenging as it relies on symptoms and an evolving set of exclusion criteria to distinguish it from other related GI disorders reflecting a complex etiology that remains poorly understood. Herein, nontargeted metabolite profiling of repeat urine specimens collected from a cohort of IBS patients (n = 42) was compared to healthy controls (n = 20) to gain insights into the underlying pathophysiology. Methods: An integrated data workflow for characterization of the urine metabolome with stringent quality control was developed to authenticate reliably measured (CV < 30%) and frequently detected (> 75%) metabolites using multisegment injection-capillary electrophoresis-mass spectrometry. Complementary statistical methods were then used to rank differentially excreted urinary metabolites after normalization to osmolality that were subsequently identified by high resolution tandem mass spectrometry and their electrophoretic migration behavior. Results: Our work revealed ten consistently elevated urinary metabolites in repeat samples collected from IBS patients at two different time points (q < 0.05 after age and Benjamini-Hochberg/FDR adjustment), which were associated with greater collagen degradation and intestinal mucosal turn-over processes likely due to low-grade inflammation. IBS-specific metabolites identified in urine included a series of hydroxylysine metabolites (O-glycosylgalactosyl-hydroxylysine, O-galactosyl-hydroxylysine, lysine), mannopyranosy-l-tryptophan, imidazole propionate, glutamine, serine, ornithine, dimethylglycine and dimethylguanosine. A major limitation in this retrospective case-control study was significant co-morbidity of IBS patients with other illnesses, including depression and prescribed medications as compared to healthy controls. Conclusion: This work provides new mechanistic insights into the pathophysiology of IBS while also offering a convenient way to monitor patient disease progression and treatment responses to therapy based on a panel of urinary metabolites that avoids invasive blood sampling, colonoscopy and/or tissue biopsies. [ABSTRACT FROM AUTHOR]