Philip Britz-McKibbin

Comprehensive plasma thiol redox status determination for metabolomics.

Thiol homeostasis plays an important role in human health and aging by regulation of cellular responses to oxidative stress. Due to major constraints that hamper reliable plasma thiol/disulfide redox status assessment in clinical research, we introduce an improved strategy for comprehensive thiol speciation using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) that overcomes sensitivity, selectivity and dynamic range constraints of conventional techniques. This method integrates both specific and nonspecific approaches toward sensitivity enhancement for artifact-free quantification of labile plasma thiols without complicated sample handling. A multivariate model was developed to predict increases in ionization efficiency for reduced thiols when conjugated to various maleimide analogs based on their intrinsic physicochemical properties. Optimization of maleimide labeling in conjunction with online sample preconcentration allowed for simultaneous analysis of nanomolar levels of reduced thiols and free oxidized thiols as their intact symmetric or mixed disulfides. Identification of low-abundance thiols and various other polar metabolites detected in plasma was supported by prediction of their relative migration times using CE as a qualitative tool complementary to ESI-MS. Plasma thiol redox status determination together with untargeted metabolite profiling offers a systemic approach for elucidation of the causal role of dysregulated thiol metabolism in the etiology of human diseases.

Serum and adipose tissue amino acid homeostasis in the metabolically healthy obese.

A subgroup of obese individuals, referred to as metabolically healthy obese (MHO), have preserved insulin sensitivity and a normal lipid profile despite being obese. The molecular basis for this improved cardiometabolic profile remains unclear. Our objective was to integrate metabolite and gene expression profiling to elucidate the molecular distinctions between MHO and metabolically unhealthy obese (MUO) phenotypes. A subset of individuals were selected from the Diabetes Risk Assessment study and classified into three groups using anthropometric and clinical measurements: lean healthy (LH), MHO, and MUO. Serum metabolites were profiled using gas chromatography coupled to mass spectrometry. Multivariate data analysis uncovered metabolites that differed between groups, and these were subsequently validated by capillary electrophoresis coupled to mass spectrometry. Subcutaneous adipose tissue (SAT) gene expression profiling using microarrays was performed in parallel. Amino acids were the most relevant class of metabolites distinguishing MHO from MUO individuals. Serum levels of glutamic acid, valine, and isoleucine were positively associated (i.e., LH < MHO < MUO) with homeostasis model assessment-insulin resistance (HOMA-IR) and glycated hemoglobin (HbA1c) values, while leucine was only correlated with HOMA-IR. The glutamine-to-glutamic acid ratio and glycine were inversely correlated (i.e., LH > MHO > MUO) with HbA1c values. Concomitantly, SAT gene expression profiling revealed that genes related to branched-chain amino acid catabolism and the tricarboxylic acid cycle were less down-regulated in MHO individuals compared to MUO individuals. Together, this integrated analysis revealed that MHO individuals have an intermediate amino acid homeostasis compared to LH and MUO individuals.

High quality drug screening by capillary electrophoresis: A review

High quality assays are needed in drug discovery to reduce the high attrition rate of lead compounds during primary screening. Capillary electrophoresis (CE) represents a versatile micro-separation technique for resolution of enzyme-catalyzed reactions, including substrate(s), product(s), cofactor(s) and their stereoisomers, which is needed for reliable characterization of biomolecular interactions in free solution. This review article provides a critical overview of new advances in CE for drug screening over the past five years involving biologically relevant enzymes of therapeutic interest, including transferases, hydrolases, oxidoreductases, and isomerases. The basic principles and major configurations in CE, as well as data processing methods needed for rigorous characterization of enzyme inhibition are described. New developments in functional screening of small molecules that modulate the activity of disease-related enzymes are also discussed. Although inhibition is a widely measured response in most enzyme assays, other important outcomes of ligand interactions on protein structure/function that impact the therapeutic potential of a drug will also be highlighted, such as enzyme stabilization, activation and/or catalytic uncoupling. CE offers a selective platform for drug screening that reduces false-positives while also enabling the analysis of low amounts of complex sample mixtures with minimal sample handling.

Metabolomics Reveals Metabolically Healthy and Unhealthy Obese Individuals Differ in their Response to a Caloric Challenge

Objective To determine if metabolically healthy obese (MHO) individuals have a different metabolic response to a standardized diet compared to lean healthy (LH) and metabolically unhealthy obese (MUO) individuals. Methods Thirty adults (35–70 yrs) were classified as LH, MHO, and MUO according to anthropometric and clinical measurements. Participants consumed a standardized high calorie meal (~1330 kcal). Blood glucose and insulin were measured at fasting, and 15, 30, 60, 90 and 120 min postprandially. Additional blood samples were collected for the targeted analysis of amino acids (AAs) and derivatives, and fatty acids (FAs). Results The postprandial response (i.e., area under the curve, AUC) for serum glucose and insulin were similar between MHO and LH individuals, and significantly lower than MUO individuals (p < 0.05). Minor differences were found in postprandial responses for AAs between MHO and MUO individuals, while three polyunsaturated FAs (linoleic acid, γ-linolenic acid, arachidonic acid) showed smaller changes in serum after the meal in MHO individuals compared to MUO. Fasting levels for various AAs (notably branched-chain AA) and FAs (e.g., saturated myristic and palmitic acids) were found to correlate with glucose and insulin AUC. Conclusion MHO individuals show preserved insulin sensitivity and a greater ability to adapt to a caloric challenge compared to MUO individuals.

Control of hydroxyproline catabolism in Sinorhizobium meliloti

Hydroxyproline (Hyp) in decaying organic matter is a rich source of carbon and nitrogen for microorganisms. A bacterial pathway for Hyp catabolism is known; however, genes and function relationships are not established. In the pathway, trans‐4‐hydroxy‐l‐proline (4‐l‐Hyp) is epimerized to cis‐4‐hydroxy‐d‐proline (4‐d‐Hyp), and then, in three enzymatic reactions, the d‐isomer is converted via Δ‐pyrroline‐4‐hydroxy‐2‐carboxylate (HPC) and α‐ketoglutarate semialdehyde (KGSA) to α‐ketoglutarate (KG). Here a transcriptional analysis of cells growing on 4‐l‐Hyp, and the regulation and functions of genes from a Hyp catabolism locus of the legume endosymbiont Sinorhizobium meliloti are reported. Fourteen hydroxyproline catabolism genes (hyp), in five transcripts hypR, hypD, hypH, hypST and hypMNPQO(RE)XYZ, were negatively regulated by hypR. hypRE was shown to encode 4‐hydroxyproline 2‐epimerase and a hypRE mutant grew with 4‐d‐Hyp but not 4‐l‐Hyp. hypO, hypD and hypH are predicted to encode 4‐d‐Hyp oxidase, HPC deaminase and α‐KGSA dehydrogenase respectively. The functions for hypS, hypT, hypX, hypY and hypZ remain to be determined. The data suggest 4‐Hyp is converted to the tricarboxylic acid cycle intermediate α‐ketoglutarate via the pathway established biochemically for Pseudomonas. This report describes the first molecular characterization of a Hyp catabolism locus.

Capillary Electrophoresis–Electrospray Ionization-Mass Spectrometry (CE–ESI-MS)-Based Metabolomics

Metabolomics is a rapidly emerging field of functional genomics research whose aim is the comprehensive analysis of low molecular weight metabolites in a biological sample. Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) represents a promising hyphenated microseparation platform in metabolomics, since a majority of primary metabolites are intrinsically polar. CE-ESI-MS offers a convenient format for the separation of complex mixtures of cationic, anionic, and/or zwitterionic metabolites, as well as their isobaric/isomeric ions without complicated sample handling. Moreover, online sample preconcentration with desalting is readily integrated during separation prior to ionization, where the migration behavior and ionization response of metabolites can be predicted based on their fundamental physicochemical properties. Herein, we describe recent developments in CE-ESI-MS with emphasis on practical protocols necessary for realizing reliable analyses as applied to targeted metabolite profiling and untargeted metabolomic studies in various biological samples.

Metabolomic studies of radiation-induced apoptosis of human leukocytes by capillary electrophoresis-mass spectrometry and flow cytometry: Adaptive cellular responses to ionizing radiation

There is growing interest in the development of new methods for elucidating the biological effects of low‐dose exposure to ionizing radiation (IR) on human health. Herein, we introduce a strategy for assessment of the impact of radiation‐induced oxidative stress on the intra‐cellular metabolism of human leukocytes. Untargeted metabolomic analyses were performed by CE‐ESI‐MS on irradiated leukocytes exposed to increasing doses of ‐radiation emitted from a Taylor source, which were subsequently incubated for 44 h to allow for cellular recovery. Flow cytometry with dual fluorescence staining revealed a major shift from early‐ to late‐stage apoptosis associated with cell membrane permeability changes as radiation dosage was increased relative to the control, but with a significant attenuation measured at intermediate dose levels. CE‐ESI‐MS analysis of filtered white blood cell lysates was also performed to quantify changes associated with 22 intra‐cellular metabolites, which were consistently measured in leukocytes across all radiation levels. Preliminary experiments demonstrated that there was an overall depletion in metabolites with extended exposure to IR; however, there was a non‐linear upregulation of specific metabolites at the 4 Gy level relative to pre‐irradiated levels, notably for arginine, glutamine, creatine, proline and reduced glutathione. Our studies demonstrate that leukocytes require a minimum threshold level of IR to induce a cytoprotective response in metabolism associated with antioxidant defense, energy homeostasis and cell signaling, which is relevant to improved understanding of the mechanisms of oxidative stress in radiobiology and cancer therapy.

Single-step analysis of low abundance phosphoamino acids via on-line sample preconcentration with chemical derivatization by capillary electrophoresis

New strategies for rapid, sensitive and high-throughput analysis of low abundance metabolites in biological samples are required for future metabolomic research. In this report, a direct method for sub-micromolar analyses of phosphoamino acids was developed using on-line sample preconcentration with 9-fluorenylmethyloxycarbonyl chloride (FMOC) derivatization by capillary electrophoresis (CE) and UV detection. Analyte focusing by dynamic pH junction and FMOC labeling efficiency were influenced by several experimental factors including buffer pH, ionic strength, sample injection length and FMOC concentration. About a 200-fold enhancement in concentration sensitivity was achieved under optimal conditions relative to conventional off-line derivatization, as reflected by a detection limit (S/N approximately 3) of 0.1 microM. In-capillary sample preconcentration with chemical labeling by CE offers a unique single-step analytical platform for high-throughput screening of low abundance metabolites without intrinsic chromophores.

Interlaboratory study to evaluate the robustness of capillary electrophoresis-mass spectrometry for peptide mapping

A collaborative study on the robustness and portability of a capillary electrophoresis-mass spectrometry method for peptide mapping was performed by an international team, consisting of 13 independent laboratories from academia and industry. All participants used the same batch of samples, reagents and coated capillaries to run their assays, whereas they utilized the capillary electrophoresis-mass spectrometry equipment available in their laboratories. The equipment used varied in model, type and instrument manufacturer. Furthermore, different types of sheath-flow capillary electrophoresis-mass spectrometry interfaces were used. Migration time, peak height and peak area of ten representative target peptides of trypsin-digested bovine serum albumin were determined by every laboratory on two consecutive days. The data were critically evaluated to identify outliers and final values for means, repeatability (precision within a laboratory) and reproducibility (precision between laboratories) were established. For relative migration time the repeatability was between 0.05 and 0.18% RSD and the reproducibility between 0.14 and 1.3% RSD. For relative peak area repeatability and reproducibility values obtained were 3-12 and 9-29% RSD, respectively. These results demonstrate that capillary electrophoresis-mass spectrometry is robust enough to allow a method transfer across multiple laboratories and should promote a more widespread use of peptide mapping and other capillary electrophoresis-mass spectrometry applications in biopharmaceutical analysis and related fields.

Multiplexed separations for biomarker discovery in metabolomics: Elucidating adaptive responses to exercise training

High efficiency separations are needed to enhance selectivity, mass spectral quality, and quantitative performance in metabolomic studies. However, low sample throughput and complicated data preprocessing remain major bottlenecks to biomarker discovery. We introduce an accelerated data workflow to identify plasma metabolite signatures of exercise responsiveness when using multisegment injection‐capillary electrophoresis‐mass spectrometry (MSI‐CE‐MS). This multiplexed separation platform takes advantage of customizable serial injections to enhance sample throughput and data fidelity based on temporally resolved ion signals derived from seven different sample segments analyzed within a single run. MSI‐CE‐MS was applied to explore the adaptive metabolic responses of a cohort of overweight/obese women (BMI > 25, n = 9) performing a 6‐wk high‐intensity interval training intervention using a repeated measures/cross‐over study design. Venous blood samples were collected from each subject at three time intervals (baseline, postexercise, recovery) in their naïve and trained states while completing standardized cycling trials at the same absolute workload. Complementary statistical methods were used to classify dynamic changes in plasma metabolism associated with strenuous exercise and training status. Positive adaptations to exercise were associated with training‐induced upregulation in plasma l‐carnitine at rest due to improved muscle oxidative capacity, and greater antioxidant capacity as reflected by lower circulating glutathionyl‐l‐cysteine mixed disulfide. Attenuation in plasma hypoxanthine and higher O‐acetyl‐l‐carnitine levels postexercise also indicated lower energetic stress for trained women.