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BACKGROUND: Metabolomics is an emerging and powerful technology that offers a comprehensive view of an organism's physiological status. Although widely applied in human medicine, it is only recently making its introduction in veterinary medicine. As a result, validated metabolomics protocols in feline medicine are lacking at the moment. Since biological interpretation of metabolomics data can be misled by the extraction method used, species and matrix-specific optimized and validated metabolomic protocols are sorely needed. RESULTS: Systematic optimization was performed using fractional factorial experiments for both serum (n = 57) and urine (n = 24), evaluating dilution for both matrices, and aliquot and solvent volume, protein precipitation time and temperature for serum. For the targeted (n = 76) and untargeted (n = 1949) validation of serum respectively, excellent instrumental, intra-assay and inter-day precision were observed (CV ≤ 15% or 30%, respectively). Linearity deemed sufficient both targeted and untargeted (R2 ≥ 0.99 or 0.90, respectively). An appropriate targeted recovery between 70 and 130% was achieved. For the targeted (n = 69) and untargeted (n = 2348) validation of the urinary protocol, excellent instrumental and intra-assay precision were obtained (CV ≤ 15% or 30%, respectively). Subsequently, the discriminative ability of our metabolomics methods was confirmed for feline chronic kidney disease (CKD) by univariate statistics (n = 41 significant metabolites for serum, and n = 55 for urine, p-value<0.05) and validated OPLS-DA models (R2(Y) > 0.95, Q2(Y) > 0.65, p-value<0.001 for both matrices). SIGNIFICANCE: This study is the first to present an optimized and validated wholistic metabolomics methods for feline serum and urine using ultra-high performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry. This robust methodology opens avenues for biomarker panel selection and a deeper understanding of feline CKD pathophysiology and other feline applications.
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Metabolômica , Gatos , Animais , Metabolômica/métodos , Medicina Veterinária/métodos , Cromatografia Líquida de Alta Pressão , Urinálise/métodosRESUMO
BACKGROUND: IgE-mediated cow's milk allergy (IgE-CMA) is one of the first allergies to arise in early childhood and may result from exposure to various milk allergens, of which ß-lactoglobulin (BLG) and casein are the most important. Understanding the underlying mechanisms behind IgE-CMA is imperative for the discovery of novel biomarkers and the design of innovative treatment and prevention strategies. METHODS: We report a longitudinal in vivo murine model, in which two mice strains (BALB/c and C57Bl/6) were sensitized to BLG using either cholera toxin or an oil emulsion (n = 6 per group). After sensitization, mice were challenged orally, their clinical signs monitored, antibody (IgE and IgG1) and cytokine levels (IL-4 and IFN-γ) measured, and fecal samples subjected to metabolomics. The results of the murine models were further extrapolated to fecal microbiome-metabolome data from our population of IgE-CMA (n = 22) and healthy (n = 23) children (Trial: NCT04249973), on which polar metabolomics, lipidomics and 16S rRNA metasequencing were performed. In vitro gastrointestinal digestions and multi-omics corroborated the microbial origin of proposed metabolic changes. RESULTS: During mice sensitization, we observed multiple microbially derived metabolic alterations, most importantly bile acid, energy and tryptophan metabolites, that preceded allergic inflammation. We confirmed microbial dysbiosis, and its associated effect on metabolic alterations in our patient cohort, through in vitro digestions and multi-omics, which was accompanied by metabolic signatures of low-grade inflammation. CONCLUSION: Our results indicate that gut dysbiosis precedes allergic inflammation and nurtures a chronic low-grade inflammation in children on elimination diets, opening important new opportunities for future prevention and treatment strategies.
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Microbiota , Hipersensibilidade a Leite , Humanos , Criança , Pré-Escolar , Bovinos , Feminino , Camundongos , Animais , Disbiose , RNA Ribossômico 16S , Inflamação , Alérgenos , Lactoglobulinas , Imunoglobulina E , MetabolomaRESUMO
Intestinal fibrostenosis in patients with Crohn's disease (CD) is a common and untreatable comorbidity that is notoriously difficult to monitor. We aimed to find metabolites associated with the presence of fibrostenosis in patients with CD using targeted and untargeted metabolomics analyses of serum and primary cell cultures using hyphenated ultra-high performance liquid chromatography high-resolution mass spectrometry. Targeted metabolomics revealed 11 discriminating metabolites in serum, which were enriched within the arginine and proline metabolism pathway. Based on untargeted metabolomics and discriminant analysis, 166 components showed a high predictive value. In addition, human intestinal fibroblasts isolated from stenotic tissue were characterized by differential levels of medium-chain dicarboxylic acids, which are proposed as an energy source through beta-oxidation, when oxidative phosphorylation is insufficient. Another energy providing pathway in such situations is anaerobic glycolysis, a theory supported by increased expression of hexokinase 2 and solute carrier family 16 member 1 in stenotic fibroblasts. Of interest, four (unannotated) metabolic components showed a negative correlation with hexokinase 2 gene expression. Together, this study provides a discriminative metabolic fingerprint in the serum and in intestinal fibroblasts of stenotic and non-stenotic patients with CD suggestive for increased production of building blocks for collagen synthesis and increased glycolysis.
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Doença de Crohn , Humanos , Doença de Crohn/metabolismo , Hexoquinase/metabolismo , Metabolômica/métodos , Constrição Patológica/complicações , MetabolomaRESUMO
BACKGROUND: Prostate cancer patients treated with radiotherapy are susceptible to acute gastrointestinal (GI) toxicity due to substantial overlap of the intestines with the radiation volume. Due to their intimate relationship with GI toxicity, faecal microbiome and metabolome dynamics during radiotherapy were investigated. MATERIAL & METHODS: This prospective study included 50 prostate cancer patients treated with prostate (bed) only radiotherapy (PBRT) (n = 28) or whole pelvis radiotherapy (WPRT) (n = 22) (NCT04638049). Longitudinal sampling was performed prior to radiotherapy, after 10 fractions, near the end of radiotherapy and at follow-up. Patient symptoms were dichotomized into a single toxicity score. Microbiome and metabolome fingerprints were analyzed by 16S rRNA gene sequencing and ultra-high-performance liquid chromatography hybrid high-resolution mass spectrometry, respectively. RESULTS: The individual α-diversity did not significantly change over time. Microbiota composition (ß-diversity) changed significantly over treatment (PERMANOVA p-value = 0.03), but there was no significant difference in stability when comparing PBRT versus WPRT. Levels of various metabolites were significantly altered during radiotherapy. Baseline α-diversity was not associated with any toxicity outcome. Based on the metabolic fingerprint, no natural clustering according to toxicity profile could be achieved. CONCLUSIONS: Radiation dose and treatment volume demonstrated limited effects on microbiome and metabolome fingerprints. In addition, no distinctive signature for toxicity status could be established. There is an ongoing need for toxicity risk stratification tools for diagnostic and therapeutic purposes, but the current evidence implies that the translation of metabolic and microbial biomarkers into routine clinical practice remains challenging.
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Neoplasias da Próstata , Masculino , Humanos , Estudos Prospectivos , RNA Ribossômico 16S , Neoplasias da Próstata/radioterapia , Próstata/efeitos da radiação , MetabolomaRESUMO
OBJECTIVES: The objective of this study was to evaluate the presence of traces of thiamazole in the urine of owners of hyperthyroid cats treated with antithyroid drugs. METHODS: Urine was collected from 24 owners of hyperthyroid cats, five human patients treated with thiamazole and five healthy humans without any contact with antithyroid drugs. All owners of hyperthyroid cats were asked to fill out a questionnaire. Urine of hyperthyroid cats was collected by spontaneous micturition. All urine samples were stored at -20°C until analysis by ultra-high-performance liquid chromatography coupled to high-resolution quadrupole Orbitrap mass spectrometry. RESULTS: These owners were assessed to have a lot of contact with their cat. Adherence to antithyroid medication handling guidelines was rather poor. High concentrations of thiamazole were detected in all feline samples (median concentration 2818 ng/ml; range 104-15,127) and in the urine of all human patients treated with thiamazole (median concentration 4153 ng/ml; range 1826-5009). No thiamazole was detected in the urine of owners of hyperthyroid cats (limit of detection 3.88 ng/ml; limit of quantification 11.75 ng/ml). CONCLUSIONS AND RELEVANCE: The results regarding the potential exposure of owners of hyperthyroid cats to antithyroid drugs are reassuring. Nevertheless, prudence is still warranted when administering antithyroid drugs. Whether these results can be extrapolated to the use of transdermal application requires further investigation.
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Doenças do Gato , Hipertireoidismo , Administração Cutânea , Animais , Antitireóideos/uso terapêutico , Doenças do Gato/tratamento farmacológico , Gatos , Humanos , Hipertireoidismo/tratamento farmacológico , Hipertireoidismo/veterinária , Metimazol/uso terapêutico , Inquéritos e QuestionáriosRESUMO
BACKGROUND: The alarming trend of paediatric obesity deserves our greatest awareness to hinder the early onset of metabolic complications impacting growth and functionality. Presently, insight into molecular mechanisms of childhood obesity and associated metabolic comorbidities is limited. This systematic review aimed at scrutinising what has been reported on putative metabolites distinctive for metabolic abnormalities manifesting at young age by searching three literature databases (Web of Science, Pubmed and EMBASE) during the last 6 years (January 2015-January 2021). Global metabolomic profiling of paediatric obesity was performed (multiple biological matrices: blood, urine, saliva and adipose tissue) to enable overarching pathway analysis and network mapping. Among 2792 screened Q1 articles, 40 met the eligibility criteria and were included to build a database on metabolite markers involved in the spectrum of childhood obesity. Differential alterations in multiple pathways linked to lipid, carbohydrate and amino acid metabolisms were observed. High levels of lactate, pyruvate, alanine and acetate marked a pronounced shift towards hypoxic conditions in children with obesity, and, together with distinct alterations in lipid metabolism, pointed towards dysbiosis and immunometabolism occurring early in life. Additionally, aberrant levels of several amino acids, most notably belonging to tryptophan metabolism including the kynurenine pathway and its relation to histidine, phenylalanine and purine metabolism were displayed. Moreover, branched-chain amino acids were linked to lipid, carbohydrate, amino acid and microbial metabolism, inferring a key role in obesity-associated insulin resistance. CONCLUSIONS: This systematic review revealed that the main metabolites at the crossroad of dysregulated metabolic pathways underlying childhood obesity could be tracked down to one central disturbance, i.e. impending insulin resistance for which reference values and standardised measures still are lacking. In essence, glycolytic metabolism was evinced as driving energy source, coupled to impaired Krebs cycle flux and ß-oxidation. Applying metabolomics enabled to retrieve distinct metabolite alterations in childhood obesity(-related insulin resistance) and associated pathways at early age and thus could provide a timely indication of risk by elucidating early-stage biomarkers as hallmarks of future metabolically unhealthy phenotypes.
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Obesidade Infantil/metabolismo , Aminoácidos/metabolismo , Metabolismo dos Carboidratos , Humanos , Metabolismo dos Lipídeos , Redes e Vias MetabólicasRESUMO
SCOPE: Immunoglobulin E-mediated food allergies (IgE-FA) are characterized by an ever-increasing prevalence, currently reaching up to 10.4% of children in the European Union. Metabolomics has the potential to provide a deeper understanding of the pathogenic mechanisms behind IgE-FA. METHODS AND RESULTS: In this work, literature is systematically searched using Web of Science, PubMed, Scopus, and Embase, from January 2010 until May 2021, including human and animal metabolomic studies on multiple biofluids (urine, blood, feces). In total, 15 studies on IgE-FA are retained and a dataset of 277 potential biomarkers is compiled for in-depth pathway mapping. Decreased indoleamine 2,3-dioxygenase-1 (IDO- 1) activity is hypothesized due to altered plasma levels of tryptophan and its metabolites in IgE-FA children. In feces of children prior to IgE-FA, aberrant metabolization of sphingolipids and histidine is noted. Decreased fecal levels of (branched) short chain fatty acids ((B)SCFAs) compel a shift towards aerobic glycolysis and suggest dysbiosis, associated with an immune system shift towards T-helper 2 (Th2) responses. During animal anaphylaxis, a similar switch towards glycolysis is observed, combined with increased ketogenic pathways. Additionally, altered histidine, purine, pyrimidine, and lipid pathways are observed. CONCLUSION: To conclude, this work confirms the unprecedented opportunities of metabolomics and supports the in-depth pathophysiological qualification in the quest towards improved diagnostic and prognostic biomarkers for IgE-FA.
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Anafilaxia , Hipersensibilidade Alimentar , Animais , Disbiose , Humanos , Imunoglobulina E , Metabolômica/métodosRESUMO
Hirschsprung's disease (HD) is a congenital structural abnormality of the colon seen in approximately 1 to 5000 live births. Despite surgical correction shortly after presentation, up to 60% of patients will express long-term gastrointestinal complaints, including potentially life-threatening Hirschsprung-associated enterocolitis (HAEC). In this study fecal samples from postoperative HD patients (n = 38) and their healthy siblings (n = 21) were analysed using high-resolution liquid chromatography-mass spectrometry aiming to further unravel the nature of the chronic gastrointestinal disturbances. Furthermore, within the patient group, we compared the faecal metabolome between patients with and without a history of HAEC as well as those diagnosed with short or long aganglionic segment. Targeted analysis identified several individual metabolites characteristic for all HD patients as well as those with a history of HAEC and long segment HD. Moreover, multivariate models based on untargeted data established statistically significant (p < 0.05) differences in comprehensive faecal metabolome in the patients' cohort as a whole and in patients with a history of HAEC. Pathway analysis revealed the most impact on amino sugar, lysine, sialic acid, hyaluronan and heparan sulphate metabolism in HD, as well as impaired tyrosine metabolism in HAEC group. Those changes imply disruption of intestinal mucosal barrier due to glycosaminoglycan breakdown and dysbiosis as major metabolic changes in patients' group and should be further explored for potential diagnostic or treatment targets.
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Doença de Hirschsprung/metabolismo , Metaboloma , Estudos de Casos e Controles , Criança , Pré-Escolar , Enterocolite/etiologia , Enterocolite/metabolismo , Fezes/química , Feminino , Gânglios/anormalidades , Doença de Hirschsprung/complicações , Doença de Hirschsprung/cirurgia , Humanos , Mucosa Intestinal/metabolismo , Intestino Grosso/anormalidades , Intestino Grosso/inervação , Masculino , Redes e Vias Metabólicas , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/metabolismo , Período Pós-OperatórioRESUMO
Of the many metabolites involved in any clinical condition, only a narrow range of biomarkers is currently being used in the clinical setting. A key to personalized medicine would be to extend this range. Metabolic fingerprinting provides a more comprehensive insight, but many methods used for metabolomics analysis are too complex and time-consuming to be diagnostically useful. Here, a rapid evaporative ionization mass spectrometry (REIMS) system for direct ex vivo real-time analysis of biofluids with minor sample pretreatment is detailed. The REIMS can be linked to various laser wavelength systems (such as optical parametric oscillator or CO2 laser) and with automation for high-throughput analysis. Laser-induced sample evaporation occurs within seconds through radiative heating with the plume guided to the MS instrument. The presented procedure includes (i) laser setup with automation, (ii) analysis of biofluids (blood/urine/stool/saliva/sputum/breast milk) and (iii) data analysis. We provide the optimal settings for biofluid analysis and quality control, enabling sensitive, precise and robust analysis. Using the automated setup, 96 samples can be analyzed in ~35-40 min per ionization mode, with no intervention required. Metabolic fingerprints are made up of 2,000-4,000 features, for which relative quantification can be achieved at high repeatability when total ion current normalization is applied. With saliva and feces as example matrices, >70% of features had a coefficient of variance ≤30%. However, to achieve acceptable long-term reproducibility, additional normalizations by, e.g., LOESS are recommended, especially for positive ionization.
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Espectrometria de Massas/métodos , Metabolômica/métodos , Líquidos Corporais/química , Humanos , Lasers de Gás , Lasers de Estado SólidoRESUMO
The probiotic Bacillus subtilis strain 29784 (Bs29784) has been shown to improve performance in broilers. In this study, we used a metabolomic and 16S rRNA gene sequencing approach to evaluate effects of Bs29874 in the broiler intestine. Nicotinic acid and hypoxanthine were key metabolites that were produced by the strain in vitro and were also found in vivo to be increased in small intestinal content of broilers fed Bs29784 as dietary additive. Both metabolites have well-described anti-inflammatory effects in the intestine. Furthermore, Bs29784 supplementation to the feed significantly altered the ileal microbiome of 13-day-old broilers, thereby increasing the abundance of genus Bacillus, while decreasing genera and OTUs belonging to the Lactobacillaceae and Enterobacteriacae families. Moreover, Bs29784 did not change the cecal microbial community structure, but specifically enriched members of the family Clostridiales VadinBB60, as well as the butyrate-producing families Ruminococcaceae and Lachnospiraceae. The abundance of various OTUs and genera belonging to these families was significantly associated with nicotinic acid levels in the cecum, suggesting a possible cross-feeding between B. subtilis strain 29784 and these beneficial microbes. Taken together, the data indicate that Bs29784 exerts its described probiotic effects through a combined action of its metabolites on both the host and its microbiome.
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Gram-negative bacteria have a well-known impact on the disease state of neonatal calves and their mortality. This study was the first to implement untargeted metabolomics on calves' fecal samples to unravel the effect of Gram-negative bacterial endotoxin lipopolysaccharide (LPS). In this context, calves were challenged with LPS and administered with fish oil, nanocurcumin, or dexamethasone to evaluate treatment effects. Ultra-high-performance liquid-chromatography high-resolution mass spectrometry (UHPLC-HRMS) was employed to map fecal metabolic fingerprints from the various groups before and after LPS challenge. Based on the generated fingerprints, including 9650 unique feature ions, significant separation according to LPS group was achieved through orthogonal partial least squares discriminant analysis (Q2 of 0.57 and p-value of 0.022), which allowed the selection of 37 metabolites as bacterial endotoxin markers. Tentative identification of these markers suggested that the majority belonged to the subclass of the carboxylic acid derivatives-amino acids, peptides, and analogs-and fatty amides, with these subclasses playing a role in the metabolism of steroids, histidine, glutamate, and folate. Biological interpretations supported the revealed markers' potential to aid in disease diagnosis, whereas beneficial effects were observed following dexamethasone, fish oil, and nanocurcumin treatment.
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Hydrophilic divinylbenzene (DVB) (Bakerbond) has surfaced as a promising sorbent for active sampling of analytes from aqueous matrices over a very broad polarity range. Given this, hydrophilic DVB may likewise offer potential for passive sampling, if sorbent/water partitioning coefficients (Ksw) were to be available. In this work, static exposure batch experiments were performed to quantitatively study the equilibrium sorption of 131 environmentally relevant organic contaminants (P values ranging from -1.30 to 9.85) on hydrophilic DVB. The superior affinity of hydrophilic DVB, as compared to Oasis HLB, for compounds with a broad polarity range was confirmed by functional Fourier-transform infrared spectroscopy and Raman characterization, demonstrating the presence of carboxyl moieties. Concentration effects were studied by increasing compound concentrations in mixture experiments and resulted in the steroidal endocrine disrupting compounds in higher Ksw, while lower Ksw were obtained for the (alkyl)phenols, personal care products, pesticides, pharmaceuticals, and phthalates. Nevertheless, Ksw remained constant in the said design for equilibrium water concentrations at environmentally relevant seawater levels. An independent analysis of thermodynamic parameters (change in enthalpy, entropy, and Gibbs free energy) revealed the nature of the main partitioning processes. While polar (log P < 4) compounds were mainly served by physisorption, nonpolar (log P > 4) compounds also exhibited binding by multiple hydrogen bonding. In conclusion, this research facilitates the future application of hydrophilic DVB for active as well as passive sampling in the analysis of organic contaminants for monitoring purposes and for toxicity testing.
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Praguicidas , Poluentes Químicos da Água , Interações Hidrofóbicas e Hidrofílicas , Compostos de VinilaRESUMO
A generic extraction and UHPLC-Q-Orbitrap™-HRMS method was developed for four insect species (mealworm, grasshopper, house cricket and black soldier fly) analyzing a large spectrum of organic chemical contaminants, including pesticides (nâ¯=â¯25), (veterinary) drugs (nâ¯=â¯29), and mycotoxins (nâ¯=â¯23). To prove the method as 'fit-for-purpose', a successful validation was performed, both qualitatively, by determining the screening detection limit (SDL), selectivity and specificity, as well as semi-quantitatively, by assessing the within-day precision (relative standard deviation (RSD)) and recovery. For both the mealworm, grasshopper, house cricket and black soldier fly, 64, 61, 59 and 62 compounds were detected at the respective SDL levels (1-100⯵gâ¯kg-1), predominantly below existing maximum residue limits for other edible matrices. Mean recoveries ranged between 70% and 120% and RSD-values were in line with European regulations (CD 2002/657/EC; SANCO). Finally, the potential of the screening methodology was demonstrated on real insect samples, revealing minor to no contamination.
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Cromatografia Líquida de Alta Pressão/métodos , Insetos/química , Espectrometria de Massas/métodos , Micotoxinas/análise , Praguicidas/análise , Drogas Veterinárias/análise , Animais , Inocuidade dos Alimentos , Insetos/metabolismo , Limite de Detecção , Micotoxinas/isolamento & purificação , Praguicidas/isolamento & purificação , Extração em Fase Sólida , Drogas Veterinárias/isolamento & purificaçãoRESUMO
In recent years, metabolomics has surfaced as an innovative research strategy in human metabolism, whereby selection of the biological matrix and its inherent metabolome is of crucial importance. However, focusing on a single matrix may imply that relevant molecules of complementary physiological pathways, covered by other matrices, are missed. To address this problem, this study presents a unique multi-matrix platform for polar metabolic fingerprinting of feces, plasma and urine, applying ultra-high performance liquid-chromatography coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry, that is able to achieve a significantly higher coverage of the system's metabolome and reveal more significant results and interesting correlations in comparison with single-matrix analyses. All three fingerprinting approaches were proven 'fit-for-purpose' through extensive validation in which a number of endogenous metabolites were measured in representative quality control samples. For targeted and untargeted validation of all three matrices, excellent linearity (coefficients of determination R2â¯≥â¯0.99 or 0.90 respectively), recovery and precision (coefficients of varianceâ¯≤â¯15% or 30% respectively) were observed. The potential of the platform was demonstrated by subjecting fecal, urine and plasma samples (collected within one day) from ten healthy volunteers to metabolic fingerprinting, yielding respectively 9 672, 9 647, and 6122 components. Orthogonal partial least-squares discriminant analysis provided similar results for feces and plasma to discriminate according to gender (p-value, R2(X), R2(Y) and Q2(Y)), suggesting feces as an excellent alternative biofluid to plasma. Moreover, combining the different matrices improved the model's predictivity, indicating the superiority of multi-matrix platforms for research purposes in biomarker detection or pathway elucidation and in the selection of the most optimal matrix for future clinical purposes.
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Fezes/química , Metabolômica , Cromatografia Líquida de Alta Pressão , Análise Discriminante , Voluntários Saudáveis , Humanos , Espectrometria de MassasRESUMO
The consumption of red meat has been linked to an increased colorectal cancer (CRC) risk. One of the major hypotheses states that heme iron (present in red meat) stimulates the formation of genotoxic N-nitroso compounds (NOCs) and lipid peroxidation products (LPOs). By means of DNA adductomics, chemically induced DNA adduct formation can be mapped in relation to e.g. dietary exposures. In this study, this state-of-the-art methodology was used to investigate alkylation and (lipid per)oxidation induced DNA adduct formation in in vitro red vs. white meat digests. In doing so, 90 alkylation and (lipid per)oxidation induced DNA adduct types could be (tentatively) identified. Overall, 12 NOC- and/or LPO-related DNA adduct types, i.e. dimethyl-T (or ethyl-T), hydroxymethyl-T, tetramethyl-T, methylguanine (MeG), guanidinohydantoin, hydroxybutyl-C, hydroxymethylhydantoin, malondialdehyde-x3-C, O6-carboxymethylguanine, hydroxyethyl-T, carboxyethyl-T and 3,N4-etheno-C were singled out as potential heme-rich meat digestion markers. The retrieval of these DNA adduct markers is in support of the heme, NOC and LPO hypotheses, suggesting that DNA adduct formation may indeed contribute to red meat related CRC risk.
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Colo/metabolismo , Neoplasias Colorretais/genética , Dano ao DNA , Carne Vermelha/efeitos adversos , Adulto , Idoso , Animais , Bovinos , Galinhas , Cromatografia Líquida de Alta Pressão , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Adutos de DNA/química , Adutos de DNA/genética , Digestão , Feminino , Heme/metabolismo , Humanos , Peroxidação de Lipídeos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Compostos Nitrosos/metabolismo , Compostos Nitrosos/toxicidade , Oxirredução , Adulto JovemRESUMO
As lipids are assigned a plethora of biological functions, it is evident that dysregulated lipid metabolism signifies a key element in many pathological conditions. With this rationale, this study presents a validated lipidomics platform to map the fecal lipidome, which integrates unique information about host-gut microbiome interactions, gastrointestinal functionality, and dietary patterns. This particular method accomplished coverage across all eight lipid categories: fatty acyls, glycerolipids, phosphoglycerolipids, polyketides, prenols, saccharolipids, sphingolipids, and sterols. Generic extraction of freeze-dried feces was achieved by solid-liquid extraction using methanol and methyl tert-butyl ether. Extracted components were separated by liquid chromatography, whereby the selected ethylene-bridged hybrid phenyl ultra-high-performance liquid chromatography stationary phase allowed fast separation of both individual lipid species and categories. Detection was achieved by high-resolution full-scan Q-Exactive Orbitrap mass spectrometry and covered a broad m/z scan range (67-2300 Da). Method validation was performed in a targeted fashion to evaluate the analytical performance across all lipid categories, revealing excellent linearity (R2 ≥ 0.9921), acceptable repeatability (coefficients of variance ≤15.6%), and stable recovery (coefficients of variance ≤11.9%). Method suitability for untargeted fingerprinting was verified, demonstrating adequate linearity (R2 ≥ 0.90) for 75.3% and acceptable repeatability (coefficients of variance ≤30%) for 84.5% of about 9000 endogenous fecal compounds. Eventually, the potential of fecal lipidomics was exemplified within a clinical context of type 2 diabetes, thereby revealing significant perturbations [orthogonal partial least-squares discriminant analysis Q2(Y) of 0.728] in the fecal lipidome between participants with normal blood glucose levels (n = 26) and those with type 2 diabetes (n = 17).