Metabolomics allows the discrimination of the pathophysiological relevance of hyperinsulinism in obese prepubertal children.

BACKGROUND/OBJECTIVES: Insulin resistance (IR) is the cornerstone of the obesity-associated metabolic derangements observed in obese children. Targeted metabolomics was employed to explore the pathophysiological relevance of hyperinsulinemia in childhood obesity in order to identify biomarkers of IR with potential clinical application. SUBJECTS/METHODS: One hundred prepubertal obese children (50 girls/50 boys, 50% IR and 50% non-IR in each group), underwent an oral glucose tolerance test for usual carbohydrate and lipid metabolism determinations. Fasting serum leptin, total and high molecular weight-adiponectin and high-sensitivity C-reactive protein (CRP) levels were measured and the metabolites showing significant differences between IR and non-IR groups in a previous metabolomics study were quantified. Enrichment of metabolic pathways (quantitative enrichment analysis) and the correlations between lipid and carbohydrate metabolism parameters, adipokines and serum metabolites were investigated, with their discriminatory capacity being evaluated by receiver operating characteristic (ROC) analysis. RESULTS: Twenty-three metabolite sets were enriched in the serum metabolome of IR obese children (P<0.05, false discovery rate (FDR)<5%). The urea cycle, alanine metabolism and glucose-alanine cycle were the most significantly enriched pathways (PFDR<0.00005). The high correlation between metabolites related to fatty acid oxidation and amino acids (mainly branched chain and aromatic amino acids) pointed to the possible contribution of mitochondrial dysfunction in IR. The degree of body mass index-standard deviation score (BMI-SDS) excess did not correlate with any of the metabolomic components studied. In the ROC analysis, the combination of leptin and alanine showed a high IR discrimination value in the whole cohort (area under curve, AUCALL=0.87), as well as in boys (AUCM=0.84) and girls (AUCF=0.91) when considered separately. However, the specific metabolite/adipokine combinations with highest sensitivity were different between the sexes. CONCLUSIONS: Combined sets of metabolic, adipokine and metabolomic parameters can identify pathophysiological relevant IR in a single fasting sample, suggesting a potential application of metabolomic analysis in clinical practice to better identify children at risk without using invasive protocols.

Allergic asthma: an overview of metabolomic strategies leading to the identification of biomarkers in the field.

Allergic asthma is a prominent disease especially during childhood. Indoor allergens, in general, and particularly house dust mites (HDM) are the most prevalent sensitizers associated with allergic asthma. Available data show that 65-130 million people are mite-sensitized world-wide and as many as 50% of these are asthmatic. In fact, sensitization to HDM in the first years of life can produce devastating effects on pulmonary function leading to asthmatic syndromes that can be fatal. To date, there has been considerable research into the pathological pathways and structural changes associated with allergic asthma. However, limitations related to the disease heterogeneity and a lack of knowledge into its pathophysiology have impeded the generation of valuable data needed to appropriately phenotype patients and, subsequently, treat this disease. Here, we report a systematic and integral analysis of the disease, from airway remodelling to the immune response taking place throughout the disease stages. We present an overview of metabolomics, the management of complex multifactorial diseases through the analysis of all possible metabolites in a biological sample, obtaining a global interpretation of biological systems. Special interest is placed on the challenges to obtain biological samples and the methodological aspects to acquire relevant information, focusing on the identification of novel biomarkers associated with specific phenotypes of allergic asthma. We also present an overview of the metabolites cited in the literature, which have been related to inflammation and immune response in asthma and other allergy-related diseases.

Insulin resistance in prepubertal obese children correlates with sex-dependent early onset metabolomic alterations.

BACKGROUND: Insulin resistance (IR) is usually the first metabolic alteration diagnosed in obese children and the key risk factor for development of comorbidities. The factors determining whether or not IR develops as a result of excess body mass index (BMI) are still not completely understood. OBJECTIVES: This study aimed to elucidate the mechanisms underpinning the predisposition toward hyperinsulinemia-related complications in obese children by using a metabolomic strategy that allows a profound interpretation of metabolic profiles potentially affected by IR. METHODS: Serum from 60 prepubertal obese children (30 girls/30 boys, 50% IR and 50% non-IR in each group, but with similar BMIs) were analyzed by using liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry following an untargeted metabolomics approach. Validation was then performed on a group of 100 additional children with the same characteristics. RESULTS: When obese children with and without IR were compared, 47 metabolites out of 818 compounds (P<0.05) obtained after data pre-processing were found to be significantly different. Bile acids exhibit the greatest changes (that is, approximately a 90% increase in IR). The majority of metabolites differing between groups were lysophospholipids (15) and amino acids (17), indicating inflammation and central carbon metabolism as the most altered processes in impaired insulin signaling. Multivariate analysis (OPLS-DA models) showed subtle differences between groups that were magnified when females were analyzed alone. CONCLUSIONS: Inflammation and central carbon metabolism, together with the contribution of the gut microbiota, are the most altered processes in obese children with impaired insulin signaling in a sex-specific fashion despite their prepubertal status.

Structural elucidation of derivatives of polyfunctional metabolites after methyl chloroformate derivatization by high-resolution mass spectrometry gas chromatography. Application to microbiota metabolites.

Metabolomics has become an essential discipline in the study of microbiome, emerging gas chromatography coupled to mass spectrometry as the most mature, robust, and reproducible analytical technique. Silylation is the most widely used chemical derivatization strategy, although it has some limitations. In this regard, alkylation by alkyl chloroformate offers some advantages, such as a rapid reaction, milder conditions, better reproducibility, and the generation of more stable derivatives. However, commercial spectral libraries do not include many of the alkyl derivatives, mainly for polyfunctional metabolites, which can form multiple derivatives. That introduces a huge bias in untargeted metabolomics leading to common errors such as duplicates, unknowns, misidentifications, wrong assignations, and incomplete results from which non-reliable findings and conclusions will be retrieved. For this reason, the purpose of this study is to overcome these shortcomings and to expand the knowledge of metabolites in general and especially those closely related to the gut microbiota through the thorough study of the reactivity of the different functional groups in real matrix derivatized by methyl chloroformate, a common representative alkylation reagent. To this end, a systematic workflow has been developed based on exhaustive structural elucidation, along with computational simulation, and taking advantage of the high sensitivity and high-resolution gas chromatography-mass spectrometry. Several empirical rules have been established according to chemically different entities (free fatty acids, amino acids, polyols, sugars, amines, and polyfunctional groups, etc.) to predict the number of derivatives formed from a single metabolite, as well as their elution order and structure. In this work, some methyl chloroformate derivatives not previously reported as well as the mechanisms to explain them are given. Extremely important is the interconversion of E- and Z- geometric isomers of unsaturated dicarboxylic acids (case of fumaric-maleic and case of citraconic-mesaconic acids), or the formation of cycled derivatives for amino acids, as well as common metabolites, as in the case of serine and cysteine, and many others.

Early Leishmania infectivity depends on miR-372/373/520d family-mediated reprogramming of polyamines metabolism in THP-1-derived macrophages.

Leishmania amazonensis is a protozoan that primarily causes cutaneous leishmaniasis in humans. The parasite relies on the amino acid arginine to survive within macrophages and establish infection, since it is a precursor for producing polyamines. On the other hand, arginine can be metabolized via nitric oxide synthase 2 (NOS2) to produce the microbicidal molecule nitric oxide (NO), although this mechanism does not apply to human macrophages since they lack NOS2 activity. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at posttranscriptional levels. Our previous work showed that mmu-miR-294 targets Nos2 favoring Leishmania survival in murine macrophages. Here, we demonstrate that human macrophages upregulate the hsa-miR-372, hsa-miR-373, and hsa-miR-520d, which present the same seed sequence as the murine mmu-miR-294. Inhibition of the miR-372 impaired Leishmania survival in THP-1 macrophages and the effect was further enhanced with combinatorial inhibition of the miR-372/373/520d family, pointing to a cooperative mechanism. However, this reduction in survival is not caused by miRNA-targeting of NOS2, since the seed-binding motif found in mice is not conserved in the human 3'UTR. Instead, we showed the miR-372/373/520d family targeting the macrophage's main arginine transporter SLC7A2/CAT2 during infection. Arginine-related metabolism was markedly altered in response to infection and miRNA inhibition, as measured by Mass Spectrometry-based metabolomics. We found that Leishmania infection upregulates polyamines production in macrophages, as opposed to simultaneous inhibition of miR-372/373/520d, which decreased putrescine and spermine levels compared to the negative control. Overall, our study demonstrates miRNA-dependent modulation of polyamines production, establishing permissive conditions for intracellular parasite survival. Although the effector mechanisms causing host cell immunometabolic adaptations involve various parasite and host-derived signals, our findings suggest that the miR-372/373/520d family may represent a potential target for the development of new therapeutic strategies against cutaneous leishmaniasis.

Benzoate X receptor zinc-finger gene switches for drug-inducible regulation of transcription.

Targeted zinc-finger (ZF) DNA-binding domains in conjunction with nuclear receptor ligand-binding domains (LBDs) produce chemically inducible gene switches that have applications in gene therapy and proteomic and genomic research. The benzoate X receptor-ß (BXRß) LBD was used to construct homodimer and single-chain ZF transcription factors (ZF(TF)s). These ZF(TF)s specifically regulated the transcription of target genes in response to two ligands, ethyl-4-hydroxybenzoate and propyl-4-hydroxybenzoate, in a dose-dependent manner. The ZF(TF)s also regulated the expression of endogenous intercellular adhesion molecule-1 in response to either ligand. The advantage of BXRß-based ZF(TF)s is that the ligands are inexpensive and easily synthetically modified, making the system a base for creation of orthogonal ligand-receptor pairs and expanding the gene-switch toolbox.

Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1.

How well antibodies can protect against disease due to HIV-1 infection remains a pivotal but unresolved issue with important implications for vaccine design and the use of prophylactic antibody to prevent infection after accidental exposure to the virus and to interrupt transmission of virus from mother to child. Strong doubts about the possible utility of antibodies in vivo have been raised because of the relative resistance of primary viruses to antibody neutralization in vitro. Primary viruses are likely to be close to the viruses transmitted during natural infection in humans. Vaccine studies have been of little value in assessing antibody efficacy in vivo because none of the strategies described to date have elicited significant neutralizing antibody responses to primary viruses. Passive immunization studies are similarly hindered by the paucity of reagents able to neutralize primary viruses effectively and a single study has suggested some benefit. Here we describe experiments to explore the ability of passive antibody to protect against primary virus challenge in hu-PBL-SCID mice. In this model, severe combined immunodeficient (SCID) mice are populated with human peripheral blood mononuclear cells (PBMCs) and infected with HIV-1. We find that the potent neutralizing human monoclonal antibody IgG1b12 at high dose is able to completely protect even when given several hours after viral challenge. The results are encouraging for antibody-based postexposure prophylaxis and support the notion that antibody induction could contribute to an effective vaccine.

Bidirectional correlation of NMR and capillary electrophoresis fingerprints: a new approach to investigating Schistosoma mansoni infection in a mouse model.

We demonstrate the statistical integration of nuclear magnetic resonance (NMR) spectroscopy and capillary electrophoresis (CE) data in order to describe a pathological state caused by Schistosoma mansoni infection in a mouse model based on urinary metabolite profiles. Urine samples from mice 53 days post infection with S. mansoni and matched controls were analyzed via NMR spectroscopy and CE. The two sets of metabolic profiles were first processed and analyzed independently and were subsequently integrated using statistical correlation methods in order to facilitate cross assignment of metabolites. Using this approach, metabolites such as 3-ureidopropionate, p-cresol glucuronide, phenylacetylglycine, indoxyl sulfate, isocitrate, and trimethylamine were identified as differentiating between infected and control animals. These correlation analyses facilitated structural elucidation using the identification power of one technique to enhance and validate the other, but also highlighted the enhanced ability to detect functional correlations between metabolites, thereby providing potential for achieving deeper mechanistic insight into the biological process.

Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes.

Antibodies that catalyze the aldol reaction, a basic carbon-carbon bond-forming reaction, have been generated. The mechanism for antibody catalysis of this reaction mimics that used by natural class I aldolase enzymes. Immunization with a reactive compound covalently trapped a Lys residue in the binding pocket of the antibody by formation of a stable vinylogous amide. The reaction mechanism for the formation of the covalent antibody-hapten complex was recruited to catalyze the aldol reaction. The antibodies use the epsilon-amino group of Lys to form an enamine with ketone substrates and use this enamine as a nascent carbon nucleophile to attack the second substrate, an aldehyde, to form a new carbon-carbon bond. The antibodies control the diastereofacial selectivity of the reaction in both Cram-Felkin and anti-Cram-Felkin directions.

Immune versus natural selection: antibody aldolases with enzymic rates but broader scope.

Structural and mechanistic studies show that when the selection criteria of the immune system are changed, catalytic antibodies that have the efficiency of natural enzymes evolve, but the catalytic antibodies are much more accepting of a wide range of substrates. The catalytic antibodies were prepared by reactive immunization, a process whereby the selection criteria of the immune system are changed from simple binding to chemical reactivity. This process yielded aldolase catalytic antibodies that approximated the rate acceleration of the natural enzyme used in glycolysis. Unlike the natural enzyme, however, the antibody aldolases catalyzed a variety of aldol reactions and decarboxylations. The crystal structure of one of these antibodies identified the reactive lysine residue that was selected in the immunization process. This lysine is deeply buried in a hydrophobic pocket at the base of the binding site, thereby accounting for its perturbed pKa.

Analytical study proving alprazolam degradation to its main impurity triazolaminoquinoleine through Maillard reaction.

Triazolaminoquinoleine is rapidly formed in formulations of alprazolam tablets in presence of excipients, and its generation is speeded up with increasing temperature and humidity. The present paper deals with detailed quantitative and qualitative studies into the nonactive constituents of the formulation in order to determine the excipient (or the mixture) responsible for the degradation. Our studies have demonstrated that reducing carbohydrate excipients play a fundamental role in the generation of triazolaminoquinoleine, with lactose as the main one responsible, through a Maillard reaction. In order to demonstrate the validity of the proposed degradation mechanism, p-nitrobenzaldehyde has been employed as a model of reaction of the nucleophylic attack of amino-opened structure of alprazolam to an aldehyde to generate the first intermediate involved in Maillard reaction, a Schiff base. This model enables the identification of all the intermediates by mass spectrometry and/or nuclear magnetic resonance techniques, with the outcome of this experiment leading to a full understanding of the generation pathway. Calcium carbonate has been proposed as a possible tablet diluent replacing lactose in the pharmaceutical formulation.

Plasma fingerprinting with GC-MS in acute coronary syndrome.

New biomarkers of cardiovascular disease are needed to augment the information obtained from traditional indicators and to illuminate disease mechanisms. One of the approaches used in metabolomics/metabonomics for that purpose is metabolic fingerprinting aiming to profile large numbers of chemically diverse metabolites in an essentially nonselective way. In this study, gas chromatography-mass spectrometry was employed to evaluate the major metabolic changes in low molecular weight plasma metabolites of patients with acute coronary syndrome (n = 9) and with stable atherosclerosis (n = 10) vs healthy subjects without significant differences in age and sex (n = 10). Reproducible differences between cases and controls were obtained with pattern recognition techniques, and metabolites accounting for higher weight in the classification have been identified through their mass spectra. On this basis, it seems inherently plausible that even a simple metabolite profile might be able to offer improved clinical diagnosis and prognosis, but in addition, specific markers are being identified.

Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches.

Chemically inducible gene switches that regulate expression of endogenous genes have multiple applications for basic gene expression research and gene therapy. Single-chain zinc-finger transcription factors that utilize either estrogen receptor homodimers or retinoid X receptor-alpha/ecdysone receptor heterodimers are shown here to be effective regulators of ICAM-1 and ErbB-2 transcription. Using activator (VP64) and repressor (Krüppel-associated box) domains to impart regulatory directionality, ICAM-1 was activated by 4.8-fold and repressed by 81% with the estrogen receptor-inducible transcription factors. ErbB-2 was activated by up to threefold and repressed by 84% with the retinoid X receptor-alpha/ecdysone receptor-inducible transcription factors. The dynamic range of these proteins was similar to the constitutive system and showed negligible basal regulation when ligand was not present. We have also demonstrated that the regulation imposed by these inducible transcription factors is dose dependent, sustainable for at least 11 days and reversible upon cessation of drug treatment. Importantly, these proteins can be used in conjunction with each other with no detectable overlap of activity enabling concurrent and temporal regulation of multiple genes within the same cell. Thus, these chemically inducible transcription factors are valuable tools for spatiotemporal control of gene expression that should prove valuable for research and gene therapy applications.

Ultra rapid liquid chromatography as second dimension in a comprehensive two-dimensional method for the screening of pharmaceutical samples in stability and stress studies.

Separation orthogonality has been explored with respect to comprehensive two-dimensional liquid chromatography (2D-LC) for different reversed-phase stationary phases. The outcome of this study points out an SB-CN x BEH-C18 combination, used in the first and the second dimension, respectively, as the most orthogonal chromatographic system for the samples assayed. The present investigation reports the employment of an ultra high pressure liquid chromatography system (UPLC) as the second chromatographic dimension, increasing the sensitivity and the speed, completing the whole chromatographic separation in a reasonable time frame. Finally, an off-line 2D-LC method with diode array detection based on the UPLC has been optimized, allowing the separation and minimizing the run time. SB-CN and BEH-C18 were employed as first and second dimension, respectively, with gradient elution applied in each dimension. Alprazolam degraded tablets were studied as a proof of concept of the utility of this type of setups for impurity profiling of complex samples.

New perspective of diabetes response to an antioxidant treatment through metabolic fingerprinting of urine by capillary electrophoresis.

Benefits of capillary electrophoresis to provide a comprehensive snapshot of multiple metabolites in biological samples have been exploited. Afterwards, multivariate statistical methods can be employed in order to mine additional information from the data. Urine fingerprints of control and diabetic rats have shown the clear effects of an antioxidant treatment on diabetic animals, which were not seen in controls, in a rapid, simple and cost-effective way without identifying a single marker. The procedure involves the measurement of samples with a relatively inexpensive tool such as CE-UV, without any previous treatment other than filtration and the application of chemometric tools [PCA (principal components analysis) and PLS-DA (partial least squares discriminant analysis)]. Data pre-treatment of electrophoretic profiles (alignment, normalization and baseline correction) has shown to be key for further chemometric treatment. Once developed, the methodology can easily be applied for a rapid in vivo screening of extracts with potential in vitro activity. Classification was supported by that produced after PCA and PLS-DA of target variables obtained with selectively designed, time and reagent consuming methods.

Metabolic fingerprinting with capillary electrophoresis.

Increasingly biomedical studies require a top-down approach that can be achieved by comparing patterns, signatures or "fingerprints" of metabolites that change in response to disease, toxin exposure, environmental or genetic alterations. Capillary electrophoresis is a technique well suited for the analysis of biofluids and extracted tissue. The experimental design requires a multidisciplinary team comprising chemists, informaticians, medics, etc. Here we have reviewed the field of CE fingerprinting and organised the manuscript in four main blocks, Sample treatment is a discussion of the latest methods for extraction of compounds, Analytical methods, deals with the different versions of electrophoretic methods and detection instrumentation, Chemometrics and CE fingerprinting, explains algorithms that have been presented for peak alignment, normalization, data analysis and metabolite identification, and the Applications heading focuses in urine, plasma, organic matter and plant extract studies.

Validated flow-injection method for rapid aluminium determination in anti-perspirants.

A flow-injection (FI) method for the rapid determination of aluminium in anti-perspirants has been developed. The method is based on the spectrophotometric detection at 535nm of the complex formed between Al ions and the chromogenic reagent eriochrome cyanine R. Both the batch and FI methods were validated by checking the parameters included in the ISO-3543-1 regulation. Variables involved in the FI method were optimized by using appropriate statistical tools. The method does not exhibit interference from other substances present in anti-perspirants and it shows a high precision with a R.S.D. value (n=6) of 0.9%. Moreover, the accuracy of the method was evaluated by comparison with a back complexometric titration method, which is currently used for routine analysis in pharmaceutical laboratories. The Student's t-test showed that the results obtained by both methods were not significantly different for a significance level of 95%. A response time of 12s and a sample analysis time, by performing triplicate injections, of 60s were achieved. The analytical figures of merit make the method highly appropriate to substitute the time-consuming complexometric method for this kind of analysis.

Capillary electrophoresis as a metabolomic tool in antioxidant therapy studies.

The development of an approach by which two CE methods operating with opposite polarities and orthogonal capillary electrophoretic separation modes (method 1: normal polarity cyclodextrin modified MEKC (CD-MEKC) and method 2: reversed polarity CZE) for the sequential application to urinary samples from a type I diabetes metabolomics investigation is discussed. During method development, problematic MEKC profile drift issues arising from the high glucose content of the diabetic animal urine samples required some electrolyte modifications involving the use of hexafluoroisopropanol (HFIP) to circumvent the drift. Data derived from both methods were subsequently subjected to alignment, normalization and multi-dimensional scaling (MDS) procedures. In such a way, classification of samples derived from control and diabetic animals receiving a placebo from those receiving an antioxidant nutraceutical, was successfully demonstrated. Such a strategy is a cost effective and comprehensive metabolomics tool useful for describing UV absorbing metabolite disease-related changes in nutra/pharma-ceutical studies.