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Management of phenylketonuria (PKU) requires lifelong restriction of phenylalanine (Phe) intake using specialized medical foods to prevent neurocognitive impairment in affected patients. However, dietary adherence is challenging to maintain while ensuring adequate nutrition, which can lead to sub-optimal clinical outcomes. Metabolomics offers a systematic approach to identify new biomarkers of disease progression in PKU when using urine as a surrogate for blood specimens that is more accurate than self-reported diet records. Herein, the plasma and urine metabolome of a cohort of classic PKU patients (median age = 11 years; n = 22) mainly prescribed (78%) a Phe-restricted diet were characterized using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Overall, there was good mutual agreement between plasma Phe and tyrosine (Tyr) concentrations measured from PKU patients when using an amino acid analyzer based on UPLC-UV as compared to MSI-CE-MS with a mean bias of 12% (n = 82). Longitudinal measurements of recently diagnosed PKU infants (n = 3) revealed good long-term regulation of blood Phe with dietary management, and only occasional episodes exceeding the recommended therapeutic range (>360 μM) unlike older PKU patients. Plasma metabolomic studies demonstrated that non-adherent PKU patients had lower circulating concentrations of Tyr, arginine, 2-aminobutyric acid, and propionylcarnitine (q < 0.05, FDR) that were inversely correlated to Phe (r ≈ -0.600 to -0.830). Nontargeted metabolite profiling also revealed urinary biomarkers associated with poor dietary adherence among PKU patients, including elevated concentrations of catabolites indicative of Phe intoxication (e.g., phenylpyruvic acid, phenylacetylglutamine, hydroxyphenylacetic acid). Additionally, PKU patients with poor blood Phe control had lower excretion of urinary compounds derived from co-metabolism of Tyr due to microbiota activity (e.g., cresol sulfate, phenylsulfate), as well as several metabolites associated with inadequate nutrient intake, including low carnitine and B vitamin status (e.g., folic acid, vitamin B12). Interestingly, an unknown urinary metabolite was strongly correlated with Phe excretion in PKU patients (r = 0.861), which was subsequently identified as imidazole lactic acid when using high resolution MS/MS. Overall, urine profiling offers a non-invasive approach for better treatment monitoring of individual PKU patients, which can also guide the design of novel therapies that improve adherence to Phe-restricted diets without acquired nutritional deficiencies.

A large body of evidence has linked unhealthy eating patterns with an alarming increase in obesity and chronic disease worldwide. However, existing methods of assessing dietary intake in nutritional epidemiology rely on food frequency questionnaires or dietary records that are prone to bias and selective reporting. Herein, metabolic phenotyping was performed on 42 healthy participants from the Diet and Gene Intervention (DIGEST) pilot study, a parallel two-arm randomized clinical trial that provided complete diets to all participants. Matching single-spot urine and fasting plasma specimens were collected at baseline, and then following two weeks of either a Prudent or Western diet with a weight-maintaining menu plan designed by a dietician. Targeted and nontargeted metabolite profiling was conducted using three complementary analytical platforms, where 80 plasma metabolites and 84 creatinine-normalized urinary metabolites were reliably measured (CV < 30%) in the majority of participants (>75%) after implementing a rigorous data workflow for metabolite authentication with stringent quality control. We classified a panel of metabolites with distinctive trajectories following two weeks of food provisions when using complementary univariate and multivariate statistical models. Unknown metabolites associated with contrasting dietary patterns were identified with high-resolution MS/MS, as well as co-elution after spiking with authentic standards if available. Overall, 3-methylhistidine and proline betaine concentrations increased in both plasma and urine samples after participants were assigned a Prudent diet ( q < 0.05) with a corresponding decrease in the Western diet group. Similarly, creatinine-normalized urinary imidazole propionate, hydroxypipecolic acid, dihydroxybenzoic acid, and enterolactone glucuronide, as well as plasma ketoleucine and ketovaline increased with a Prudent diet ( p < 0.05) after adjustments for age, sex, and BMI. In contrast, plasma myristic acid, linoelaidic acid, linoleic acid, α-linoleic acid, pentadecanoic acid, alanine, proline, carnitine, and deoxycarnitine, as well as urinary acesulfame K increased among participants following a Western diet. Most metabolites were also correlated ( r > ± 0.30, p < 0.05) to changes in the average intake of specific nutrients from self-reported diet records reflecting good adherence to assigned food provisions. Our study revealed robust biomarkers sensitive to short-term changes in habitual diet, which is needed for accurate monitoring of healthy eating patterns in free-living populations, and evidence-based public health policies for chronic disease prevention.

Mass spectrometry (MS)-based metabolomic initiatives that use conventional separation techniques are limited by low sample throughput and complicated data processing that contribute to false discoveries. Herein, we introduce a new strategy for unambiguous identification and accurate quantification of biomarkers for inborn errors of metabolism (IEM) from dried blood spots (DBS) with quality assurance. A multiplexed separation platform based on multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) was developed to provide comparable sample throughput to flow injection analysis-tandem MS (FIA-MS/MS) but with greater selectivity as required for confirmatory testing and discovery-based metabolite profiling of volume-restricted biospecimens. Mass spectral information is encoded temporally within a separation by serial injection of three or more sample pairs, each having a unique dilution pattern, alongside a quality control (QC) that serves as a reference in every run to facilitate between-sample comparisons and/or batch correction due to system drift. Optimization of whole blood extraction conditions on DBS filter paper cut-outs was first achieved to maximize recovery of a wide range of polar metabolites from DBS extracts. An interlaboratory comparison study was also conducted using a proficiency test and retrospective neonatal DBS that demonstrated good agreement between MSI-CE-MS and validated FIA-MS/MS methods within an accredited facility. Our work demonstrated accurate identification of various IEM based on reliable measurement of a panel of primary or secondary biomarkers above an upper cutoff concentration limit for presumptive screen-positive cases without stable isotope-labeled reagents. Additionally, nontargeted metabolite profiling by MSI-CE-MS with temporal signal pattern recognition revealed new biomarkers for early detection of galactosemia, such as N-galactated amino acids, that are a novel class of pathognomonic marker due to galactose stress in affected neonates.

A major constraint in large-scale mass spectrometry (MS)-based metabolomic initiatives is the low sample throughput associated with chromatographic or electrophoretic separations. Herein, we introduce multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) as a multiplexed separation platform for metabolomics that increases sample throughput up to one order of magnitude while improving overall data fidelity. We demonstrate that serial injection of seven or more discrete sample segments can be performed within a single capillary while maintaining isomeric resolution without ion suppression when using a high mass resolution time-of-flight-MS. Customized injection sequences can be devised to encode information temporally within a separation based on signal pattern recognition, which enables unambiguous identification and accurate quantification (mean bias <10%) of polar metabolites in human plasma with good reproducibility (CV ≈ 10%, n = 70). False discoveries are avoided when using a rigorous dilution trend filter to reject spurious signals and background peaks that comprise the majority (≈65%) of total detectable features. MSI-CE-MS offers an unprecedented approach to enhance sample throughput analogous to direct infusion-MS (≈3 min/sample) while delivering far greater selectivity, quantitative performance, and data quality since the same ion from different samples migrates into the ion source within a short time interval (≈2-6 min). These distinct analytical and bioinformatic merits are achieved without column switching, isotopic labeling, hardware modifications, or costly infrastructure investments.

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 preconcentration 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 (E(GSSG/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-dinitrobenzene (CDNB) and N-acetyl-L-cysteine (NAC) were found to significantly alter E(GSSG/2GSH) and/or glutathione oxidation kinetics (e.g., k(GSSG)) 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.

Characterization of unknown low-abundance metabolites in biological samples is one the most significant challenges in metabolomic research. In this report, an integrative strategy based on capillary electrophoresis-electrospray ionization-ion trap mass spectrometry (CE-ESI-ITMS) with computer simulations is examined as a multiplexed approach for studying the selective nutrient uptake behavior of E. coli within a complex broth medium. On-line sample preconcentration with desalting by CE-ESI-ITMS was performed directly without off-line sample pretreatment in order to improve detector sensitivity over 50-fold for cationic metabolites with nanomolar detection limits. The migration behavior of charged metabolites were also modeled in CE as a qualitative tool to support MS characterization based on two fundamental analyte physicochemical properties, namely, absolute mobility (muo) and acid dissociation constant (pKa). Computer simulations using Simul 5.0 were used to better understand the dynamics of analyte electromigration, as well as aiding de novo identification of unknown nutrients. There was excellent agreement between computer-simulated and experimental electropherograms for several classes of cationic metabolites as reflected by their relative migration times with an average error of <2.0%. Our studies revealed differential uptake of specific amino acids and nucleoside nutrients associated with distinct stages of bacterial growth. Herein, we demonstrate that CE can serve as an effective preconcentrator, desalter, and separator prior to ESI-MS, while providing additional qualitative information for unambiguous identification among isobaric and isomeric metabolites. The proposed strategy is particularly relevant for characterizing unknown yet biologically relevant metabolites that are not readily synthesized or commercially available.

Key points: Maternal obesity has been associated with shifts in intestinal microbiota, which may contribute to impaired barrier functionImpaired barrier function may expose the placenta and fetus to pro‐inflammatory mediatorsWe investigated the impacts of diet‐induced obesity in mice on maternal and fetal intestinal structure and placental vascularizationDiet‐induced obesity decreased maternal intestinal short chain fatty acids and their receptors, impaired gut barrier integrity and was associated with fetal intestinal inflammation.Placenta from obese mothers showed blood vessel immaturity, hypoxia, increased transcript levels of inflammation, autophagy and altered levels of endoplasmic reticulum stress markers.These data suggest that maternal intestinal changes probably contribute to adverse placental adaptations and also impart an increased risk of obesity in the offspring via alterations in fetal gut development. Shifts in maternal intestinal microbiota have been implicated in metabolic adaptations to pregnancy. In the present study, we generated cohorts of female C57BL/6J mice fed a control (17% kcal fat, n = 10–14) or a high‐fat diet (HFD 60% kcal from fat, n = 10–14; ad libitum) aiming to investigate the impact on the maternal gut microbiota, intestinal inflammation and gut barrier integrity, placental inflammation and fetal intestinal development at embryonic day 18.5. HFD was associated with decreased relative abundances of short‐chain fatty acid (SCFA) producing genera during pregnancy. These diet‐induced shifts paralleled decreased maternal intestinal mRNA levels of SCFA receptor Gpr41, modestly decreased cecal butyrate, and altered mRNA levels of inflammatory cytokines and immune cell markers in the maternal intestine. Maternal HFD resulted in impaired gut barrier integrity, with corresponding increases in circulating maternal levels of lipopolysaccharide (LPS) and tumour necrosis factor. Placentas from HFD dams demonstrated blood vessel immaturity and hypoxia; decreased free carnitine, acylcarnitine derivatives and trimethylamine‐N‐oxide; and altered mRNA levels of inflammation, autophagy, and ER stress markers. HFD exposed fetuses had increased activation of nuclear factor‐kappa B and inhibition of the unfolded protein response in the developing intestine. Taken together, these data suggest that HFD intake prior to and during pregnancy shifts the composition of the maternal gut microbiota and impairs gut barrier integrity, resulting in increased maternal circulating LPS, which may ultimate contribute to changes in placental vascularization and fetal gut development. Key points: Maternal obesity has been associated with shifts in intestinal microbiota, which may contribute to impaired barrier functionImpaired barrier function may expose the placenta and fetus to pro‐inflammatory mediatorsWe investigated the impacts of diet‐induced obesity in mice on maternal and fetal intestinal structure and placental vascularizationDiet‐induced obesity decreased maternal intestinal short chain fatty acids and their receptors, impaired gut barrier integrity and was associated with fetal intestinal inflammation.Placenta from obese mothers showed blood vessel immaturity, hypoxia, increased transcript levels of inflammation, autophagy and altered levels of endoplasmic reticulum stress markers.These data suggest that maternal intestinal changes probably contribute to adverse placental adaptations and also impart an increased risk of obesity in the offspring via alterations in fetal gut development. [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]