RESUMEN
The human gastrointestinal tract is inhabited by the largest microbial community within the human body consisting of trillions of microbes called gut microbiota. The normal flora is the site of many physiological functions such as enhancing the host immunity, participating in the nutrient absorption and protecting the body against pathogenic microorganisms. Numerous investigations showed a bidirectional interplay between gut microbiota and many organs within the human body such as the intestines, the lungs, the brain, and the skin. Large body of evidence demonstrated, more than a decade ago, that the gut microbial alteration is a key factor in the pathogenesis of many local and systemic disorders. In this regard, a deep understanding of the mechanisms involved in the gut microbial symbiosis/dysbiosis is crucial for the clinical and health field. We review the most recent studies on the involvement of gut microbiota in the pathogenesis of many diseases. We also elaborate the different strategies used to manipulate the gut microbiota in the prevention and treatment of disorders. The future of medicine is strongly related to the quality of our microbiota. Targeting microbiota dysbiosis will be a huge challenge.
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Microbioma Gastrointestinal , Microbiota , Probióticos , Disbiosis/terapia , Tracto Gastrointestinal , Humanos , Prebióticos , Probióticos/uso terapéuticoRESUMEN
Estimating the postmortem interval (PMI) has remained the subject of investigations in forensic medicine for many years. Every kind of death results in changes in metabolites in body tissues and fluids due to lack of oxygen, altered circulation, enzymatic reactions, cellular degradation, and cessation of anabolic production of metabolites. Metabolic changes may provide markers determining the time since death, which is challenging in current analytical and observation-based methods. The study includes metabolomics analysis of blood with the use of an animal model to determine the biochemical changes following death. LC-MS is used to fingerprint postmortem porcine blood. Metabolites, significantly changing in blood after death, are selected and identified using univariate statistics. Fifty-one significant metabolites are found to help estimate the time since death in the early postmortem stage. Hypoxanthine, lactic acid, histidine, and lysophosphatidic acids are found as the most promising markers in estimating an early postmortem stage. Selected lysophosphatidylcholines are also found as significantly increased in blood with postmortal time, but their practical utility as PMI indicators can be limited due to a relatively low increasing rate. The findings demonstrate the great potential of LC-MS-based metabolomics in determining the PMI due to sudden death and provide an experimental basis for applying this attitude in investigating various mechanisms of death. As we assume, our study is also one of the first in which the porcine animal model is used to establish PMI metabolomics biomarkers.
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Risk factors for type 2 diabetes mellitus (T2DM) consist of a combination of an unhealthy, imbalanced diet and genetic factors that may interact with each other. Single nucleotide polymorphism (SNP) in the prospero homeobox 1 (PROX1) gene is a strong genetic susceptibility factor for this metabolic disorder and impaired ß-cell function. As the role of this gene in T2DM development remains unclear, novel approaches are needed to advance the understanding of the mechanisms of T2DM development. Therefore, in this study, for the first time, postprandial changes in plasma metabolites were analysed by GC-MS in nondiabetic men with different PROX1 genotypes up to 5 years prior to prediabetes appearance. Eighteen contestants (12 with high risk (HR) and 6 with low risk (LR) genotype) participated in high-carbohydrate (HC) and normo-carbohydrate (NC) meal-challenge tests. Our study concluded that both meal-challenge tests provoked changes in 15 plasma metabolites (amino acids, carbohydrates, fatty acids and others) in HR, but not LR genotype carriers. Postprandial changes in the levels of some of the detected metabolites may be a source of potential specific early disturbances possibly associated with the future development of T2DM. Thus, accurate determination of these metabolites can be important for the early diagnosis of this metabolic disease.
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Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/etiología , Dieta , Susceptibilidad a Enfermedades , Proteínas de Homeodominio/genética , Estado Prediabético/epidemiología , Estado Prediabético/etiología , Proteínas Supresoras de Tumor/genética , Alelos , Biomarcadores , Diabetes Mellitus Tipo 2/diagnóstico , Cromatografía de Gases y Espectrometría de Masas , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Metaboloma , Metabolómica/métodos , Polonia/epidemiología , Polimorfismo de Nucleótido Simple , Estado Prediabético/diagnósticoRESUMEN
OBJECTIVES: Down syndrome is the most common human chromosomal aberration. It is commonly known that it is a genetic- based disease, but still, pathomechanisms which lead to observed disorders have not been explained. The objective of this study was to determine the metabolic fingerprinting of the amniotic fluid women carrying foetuses with Down syndrome (DS). MATERIAL AND METHODS: The study and control groups consisted of women who underwent routine amniocentesis between the 15th and 18th week of gestation. After analysis of the karyotyping results, 13 women with foetal DS were chosen. For the control group, 13 healthy patients with uncomplicated pregnancies who delivered healthy newborns at term was selected. Amniotic fluid was analyzed using liquid chromatography combined with high resolution mass spectrometry. RESULTS: In the amniotic fluid of women with foetal DS compared to patients with healthy foetuses, we reported significant differences in the level of four metabolites: methylhistidine, hexanoylcarnitine, diacetylspermine and p-cresol sulfate which may be connected with improper development of nervous system and muscles. We detected bacterial metabolite, which support the latest thesis about non-sterile intrauterine environment. CONCLUSIONS: Based on our findings, we hypothesise that differences in the level of four metabolites in the amniotic fluid may play role in the pathogenesis of DS. Defining their potential as biochemical pathogenic factors of DS requires further investigation of the biological pathways involving in the foetal development.
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Líquido Amniótico , Síndrome de Down , Amniocentesis , Líquido Amniótico/metabolismo , Aberraciones Cromosómicas , Femenino , Humanos , Recién Nacido , Embarazo , Atención PrenatalRESUMEN
The gerbil is a well-known model for studying cerebral ischemia. The CA1 of the hippocampus is vulnerable to 5 min of ischemia, while the CA2-4 and dentate gyrus (DG) are resistant to it. Short-lasting ischemia, a model of transient ischemic attacks in men, results in CA1 neuron death within 2-4 days of reperfusion. Untargeted metabolomics, using LC-QTOF-MS, was used to enrich the knowledge about intrinsic vulnerability and resistance of hippocampal regions and their early post-ischemic response (IR). In total, 30 significant metabolites were detected. In controls, taurine was significantly lower and guanosine monophosphate was higher in CA1, as compared to that in CA2-4,DG. LysoPG and LysoPE were more abundant in CA1, while LysoPI 18:0 was detected only in CA2-4,DG. After IR, a substantial decrease in the citric acid level in CA1, an accumulation of pipecolic acid in both regions, and opposite changes in the amount of PE and LysoPE were observed. The following metabolic pathways were identified as being differentially active in control CA1 vs. CA2-4,DG: metabolism of taurine and hypotaurine, glycerophospholipid, and purine. These results may indicate that a regulation of cell volume, altered structure of cell membranes, and energy metabolism differentiate hippocampal regions. Early post-ischemia, spatial differences in the metabolism of aminoacyl-tRNA biosynthesis, and amino acids and their metabolites with a predominance of those which upkeep their well-being in CA2-4,DG are shown. Presented results are consistent with genetic, morphological, and functional data, which may be useful in further study on endogenous mechanisms of neuroprotection and search for new targets for therapeutic interventions.
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Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Metabolómica , Daño por Reperfusión/metabolismo , Animales , Análisis Discriminante , Gerbillinae , Análisis de los Mínimos Cuadrados , Masculino , Redes y Vías Metabólicas , Metaboloma , Especificidad de ÓrganosRESUMEN
Ulcerative colitis (UC) and Crohn's disease (CD) represent inflammatory bowel diseases (IBD) with multifactorial pathogenesis, involving genetic, environmental and microbial factors. Interactions between gut microbiota and immune system result in changes in metabolic pathways. Metabolomics is a comprehensive and quantitative (or semi-quantitative) analysis of metabolites synthetized in human's biological system. It has been shown that metabolic profiling might be used to identify disease biomarkers. Recent findings confirmed alterations in the number of metabolites in patients with IBD. However, most of the studies included adult individuals with ongoing treatment which might have affected the metabolite profiling. Therefore, the aim of our study was to collect the knowledge about metabolomics in paediatric patients with CD or UC based on the currently published literature.
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Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Redes y Vías Metabólicas , Metaboloma , Niño , HumanosRESUMEN
BACKGROUND: The transcription factor 7-like 2 (TCF7L2) gene confers one of the strongest genetic predispositions to type 2 diabetes, but diabetes development can be modified by diet. OBJECTIVE: The aim of our study was to evaluate postprandial metabolic alterations in healthy men with a high genetic risk of diabetes, after two meals with varying macronutrient content. METHODS: The study was conducted in 21 homozygous nondiabetic men carrying the high-risk (HR, n = 8, age: 31.2 ± 6.3 y, body mass index (BMI, kg/m2) 28.5 ± 8.1) or low-risk (LR, n = 13, age: 35.2 ± 10.3 y, BMI: 28.1 ± 6.4) genotypes at the rs7901695 locus. During two meal challenge test visits subjects received standardized isocaloric (450 kcal) liquid meals: high-carbohydrate (HC, carbohydrates: 89% of energy) and normo-carbohydrate (NC, carbohydrates: 45% of energy). Fasting (0 min) and postprandial (30, 60, 120, 180 min) plasma samples were analyzed for metabolite profiles through untargeted metabolomics. Metabolic fingerprinting was performed on an ultra-high-performance liquid chromatography (UHPLC) system connected to an iFunnel quadrupole-time-of-flight (Q-TOF) mass spectrometer. RESULTS: In HR-genotype men, after the intake of an HC-meal, we noted a significantly lower area under the curves (AUCs) of postprandial plasma concentrations of most of the phospholipids (-37% to -53%, variable importance in the projection (VIP) = 1.2-1.5), lysophospholipids (-29% to -86%, VIP = 1.1-2.6), sphingolipids (-32% to -47%, VIP = 1.1-1.3), as well as arachidonic (-36%, VIP = 1.4) and oleic (-63%, VIP = 1.3) acids, their metabolites: keto- and hydoxy-fatty acids (-38% to -78%, VIP = 1.3-2.5), leukotrienes (-65% to -83%, VIP = 1.4-2.2), uric acid (-59%, VIP = 1.5), and pyroglutamic acid (-65%, VIP = 1.8). The AUCs of postprandial sphingosine concentrations were higher (125-832%, VIP = 1.9-3.2) after the NC-meal, AUCs of acylcarnitines were lower (-21% to -61%, VIP = 1.1-2.4), and AUCs of fatty acid amides were higher (51-508%, VIP = 1.7-3.1) after the intake of both meals. CONCLUSIONS: In nondiabetic men carrying the TCF7L2 HR genotype, subtle but detectable modifications in intermediate lipid metabolism are induced by an HC-meal. This trial was registered at www.clinicaltrials.gov as NCT03792685.
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Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Carbohidratos de la Dieta/administración & dosificación , Metabolismo de los Lípidos/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Adulto , Estudios Cruzados , Predisposición Genética a la Enfermedad , Genotipo , Homocigoto , Humanos , Lípidos/sangre , Masculino , Metabolómica , Polimorfismo de Nucleótido Simple , Periodo Posprandial/genética , Periodo Posprandial/fisiología , Factores de Riesgo , Método Simple CiegoRESUMEN
The prospero homeobox 1 (PROX1) gene may show pleiotropic effects on metabolism. We evaluated postprandial metabolic alterations dependently on the rs340874 genotypes, and 28 non-diabetic men were divided into two groups: high-risk (HR)-genotype (CC-genotype carriers, n = 12, 35.3 ± 9.5 years old) and low-risk (LR)-genotype (allele T carriers, n = 16, 36.3 ± 7.0 years old). Subjects participated in two meal-challenge-tests with high-carbohydrate (HC, carbohydrates 89%) and normo-carbohydrate (NC, carbohydrates 45%) meal intake. Fasting and 30, 60, 120, and 180 min after meal intake plasma samples were fingerprinted by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In HR-genotype men, the area under the curve (AUC) of acetylcarnitine levels was higher after the HC-meal [+92%, variable importance in the projection (VIP) = 2.88] and the NC-meal (+55%, VIP = 2.00) intake. After the NC-meal, the HR-risk genotype carriers presented lower AUCs of oxidized fatty acids (-81-66%, VIP = 1.43-3.16) and higher linoleic acid (+80%, VIP = 2.29), while after the HC-meal, they presented lower AUCs of ornithine (-45%, VIP = 1.83), sphingosine (-48%, VIP = 2.78), linoleamide (-45%, VIP = 1.51), and several lysophospholipids (-40-56%, VIP = 1.72-2.16). Moreover, lower AUC (-59%, VIP = 2.43) of taurocholate after the HC-meal and higher (+70%, VIP = 1.42) glycodeoxycholate levels after the NC-meal were observed. Our results revealed differences in postprandial metabolites from inflammatory and oxidative stress pathways, bile acids signaling, and lipid metabolism in PROX1 HR-genotype men. Further investigations of diet-genes interactions by which PROX1 may promote T2DM development are needed.
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Ácidos y Sales Biliares/sangre , Diabetes Mellitus Tipo 2/genética , Carbohidratos de la Dieta/metabolismo , Proteínas de Homeodominio/genética , Inflamación/sangre , Metabolismo de los Lípidos/genética , Polimorfismo de Nucleótido Simple , Proteínas Supresoras de Tumor/genética , Acetilcarnitina/sangre , Adulto , Alelos , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/etiología , Dieta , Interacción Gen-Ambiente , Genotipo , Humanos , Ácidos Linoleicos/sangre , Masculino , Comidas , Metaboloma , Ornitina/sangre , Estrés Oxidativo , Periodo Posprandial , Transducción de Señal , Esfingosina/sangreRESUMEN
BACKGROUND: Metabolic profiling might be used to identify disease biomarkers. The aim of our study was to determine the usefulness of untargeted metabolomics analysis to detect differences in serum metabolites between newly diagnosed and untreated pediatric patients with Crohn's disease (CD) or ulcerative colitis (UC) in comparison with a control group (Ctr). Moreover, we investigated the potential of profiling metabolomics and inflammatory markers to improve the noninvasive diagnosis of CD and UC in children. METHODS: Metabolic fingerprinting of serum samples was estimated with liquid chromatography coupled with mass spectrometry in children with CD (n = 9; median age, 14 years), UC (n = 10; median age, 13.5 years), and controls (n = 10; median age, 12.5 years). RESULTS: The majority of chemically annotated metabolites belonged to phospholipids and were downregulated in CD and UC compared with the Ctr. Only 1 metabolite, lactosylceramide 18:1/16:0 (LacCer 18:1/16:0), significantly discriminated CD from UC patients. Interestingly, combining LacCer 18:1/16:0 with other inflammatory markers resulted in a significant increase in the area under the curve with the highest specificity and sensitivity. CONCLUSIONS: Using serum untargeted metabolomics, we have shown that LacCer 18:1/16:0 is a very unique metabolite for CD patients.
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Biomarcadores/análisis , Colitis Ulcerosa/diagnóstico , Enfermedad de Crohn/diagnóstico , Heces/química , Mediadores de Inflamación/metabolismo , Metaboloma , Adolescente , Estudios de Casos y Controles , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Femenino , Estudios de Seguimiento , Humanos , MasculinoRESUMEN
Different kinds of gastrointestinal tract modulations known as "bariatric surgery" are actually the most effective treatment for obesity and associated co-morbidities, such as type 2 diabetes (T2DM). The potential causes of those effects have yet to be explained. In our study, we focused on molecular changes evoked by laparoscopic sleeve gastrectomy leading to T2DM remission. Two complementary metabolomics techniques, namely, liquid chromatography coupled with mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS), were used to study those effects in a group of 20 obese patients with T2DM selected from a cohort of 372 obese individuals who underwent bariatric surgery and did not receive anti-diabetic treatment afterward. Modified levels of carnitines, lipids, amino acids (including BCAA) and α- and ß-hydroxybutyric acids were detected. Presented alterations suggest a major role of mitochondria activity in T2DM remission process. Moreover, some of the observed metabolites suggest that changes in gut microbiota composition may also correlate with the tempo of diabetes recovery. Additional analyses confirmed a relationship between biochemical and clinical parameters and the aforementioned metabolites, thereby, highlighting a role of mitochondria and microbes. Our data suggests that there is a previously undescribed relationship between mitochondria and gut microbiota, which changes after the bariatric surgery. More investigations are needed to confirm and explore the observed findings.
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Cirugía Bariátrica/métodos , Diabetes Mellitus Tipo 2/cirugía , Gastrectomía/métodos , Metaboloma , Obesidad Mórbida/cirugía , Adulto , Aminoácidos/sangre , Cirugía Bariátrica/instrumentación , Glucemia/metabolismo , Carnitina/sangre , Cromatografía Liquida , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/microbiología , Femenino , Cromatografía de Gases y Espectrometría de Masas , Gastrectomía/instrumentación , Microbioma Gastrointestinal/fisiología , Humanos , Hidroxibutiratos/sangre , Laparoscopía , Lípidos/sangre , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Mitocondrias/metabolismo , Obesidad Mórbida/sangre , Obesidad Mórbida/complicaciones , Obesidad Mórbida/microbiología , Inducción de RemisiónRESUMEN
OBJECTIVE: The objective of the study was to perform maternal plasma metabolic fingerprinting to evaluate differences in plasma metabolites between healthy and Down syndrome (DS) pregnancies and to indicate novel non-invasive markers for DS prenatal diagnostics. METHODS: This was a case-control study of pregnancies between 15th and 18th gestational week. LC-MS-based metabolic fingerprinting of plasma samples was performed. RESULTS: Levels of five metabolites were significantly lower in the plasma of DS pregnancies. The majority of the statistically significant metabolites may be connected with fetal brain and central nervous system development (eg, fatty acid amides). According to the receiver operating characteristic (ROC), the combination of linoleamide and piperine has the highest diagnostic potential: area under the curve (AUC) = 0.878, sensitivity of 100%, and specificity of 73.3%. CONCLUSIONS: The study indicates disturbances in maternal metabolic pathways evoked by fetal DS. Novel potential maternal plasma metabolomic markers for non-invasive prenatal diagnostics of fetal DS are proposed.
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Síndrome de Down , Enfermedades Fetales/metabolismo , Metaboloma , Plasma/metabolismo , Adulto , Estudios de Casos y Controles , Femenino , Humanos , EmbarazoRESUMEN
Bariatric surgery was born in the 1950s at the University of Minnesota. From this time, it continues to evolve and, by the same token, gives new or better possibilities to treat not only obesity but also associated comorbidities. Metabolomics is also a relatively young science discipline, and similarly, it shows great potential for the comprehensive study of the dynamic alterations of the metabolome. It has been widely used in medicine, biology studies, biomarker discovery, and prognostic evaluations. Currently, several dozen metabolomics studies were performed to study the effects of bariatric surgery. LC-MS and NMR are the most frequently used techniques to study main effects of RYGB or SG. Research has yield many interesting results involving not only clinical parameters but also molecular modulations. Detected changes pertain to amino acid, lipids, carbohydrates, or gut microbiota alterations. It proves that including bariatric surgery to metabolic surgery is warranted. However, many molecular modulations after those procedures remain unexplained. Therefore, application of metabolomics to study this field seems to be a proper solution. New findings can suggest new directions of surgery technics modifications, contribute to broadening knowledge about obesity and diseases related to it, and perhaps develop nonsurgical methods of treatment in the future.
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Cirugía Bariátrica , Metaboloma , Metabolómica/métodos , Obesidad/metabolismo , Obesidad/cirugía , Humanos , Resultado del TratamientoRESUMEN
Mechanisms responsible for metabolic gains after bariatric surgery are not entirely clear. The purpose of this study was evaluation of metabolic changes after laparoscopic Roux-en-Y gastric bypass or laparoscopic sleeve gastrectomy in semi-annual follow up. The study participants were selected from obese patients with T2DM who underwent one of the mentioned bariatric procedures. Serum metabolic fingerprinting by use of liquid and gas chromatography with mass spectrometry detection was performed on samples obtained from studied patients before, one, and six months post-surgery. Performed analyses resulted in 49 significant and identified metabolites. Comparison of the two described procedures has allowed to detect metabolites linked with numerous pathways, processes and diseases. Based on the metabolites detected and pathways affected, we propose a "gear mechanism" showing molecular changes evoked by both bariatric procedures. Critical evaluation of clinical data and obtained metabolomics results enables us to conclude that both procedures are very similar in terms of general clinical outcome, but they strongly differ from each other in molecular mechanisms leading to the final effect. For the first time general metabolic effect of bariatric procedures is described. New hypotheses concerning molecular mechanisms induced by bariatric surgeries and new gut microbiota modulations are presented.
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Diabetes Mellitus Tipo 2/metabolismo , Gastrectomía , Derivación Gástrica , Metabolómica/métodos , Obesidad/metabolismo , Adulto , Índice de Masa Corporal , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/cirugía , Femenino , Estudios de Seguimiento , Cromatografía de Gases y Espectrometría de Masas/métodos , Microbioma Gastrointestinal/fisiología , Humanos , Laparoscopía/métodos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Obesidad/sangre , Obesidad/cirugía , Pérdida de Peso/fisiologíaRESUMEN
Cataract is the leading cause of blindness worldwide. Epidemiological studies revealed up to a fivefold increased prevalence of cataracts in diabetic subjects. Metabolomics is nowadays frequently implemented to understand pathophysiological processes responsible for disease occurrence and progression. It has also been used recently to study the metabolic composition of aqueous humor (AH). AH is a transparent fluid which fills the anterior and posterior chambers of the eye. It supplies nutrients and removes metabolic waste from avascular tissues in the eye. The aim of this study was to use metabolomics to compare the AH of diabetic and non-diabetic patients undergoing cataract surgery. Several antioxidants (methyltetrahydrofolic acid, taurine, niacinamide, xanthine, and uric acid) were found decreased (-22 to -61%, p-value 0.05-0.003) in AH of diabetics. Also amino acids (AA) and derivatives were found decreased (-21 to -36%, p-value 0.05-0.01) while glycosylated AA increased (+75-98%, p-value 0.03-0.009) in this group of patients. Metformin was detected in AH of people taking this drug. To our knowledge, this is the first metabolomics study aiming to assess differences in AH composition between diabetic and non-diabetic patients with cataract. An increased oxidative stress and perturbations in amino acid metabolism in AH may be responsible for earlier cataract onset in diabetic patients.
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Humor Acuoso/química , Metabolómica/métodos , Espectrometría de Masas en Tándem/métodos , Anciano , Anciano de 80 o más Años , Humor Acuoso/metabolismo , Catarata/complicaciones , Catarata/metabolismo , Cromatografía Liquida/métodos , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Humanos , MasculinoRESUMEN
The major histologic subtypes of non-small cell lung cancer (NSCLC) include adenocarcinoma (ADC), squamous cell lung carcinoma (SCC), and large-cell carcinoma (LCC). Clinical trials of targeted agents and newer chemotherapy agents yielded differences in outcomes according to histologic subgroups providing a rationale for histology-based treatment in NSCLC. Currently, NSCLC subtyping is performed based on histopathological examinations and immunohistochemistry. However available methods leave about 10% of NSCLC cases as not otherwise specified. The purpose of this study was development of an LC-QTOF-MS method for human lung tissue metabolic fingerprinting that could discriminate NSCLC histological subtypes and propose biomarkers candidates that could support proper NSCLC diagnosis. Metabolites were extracted with acetonitrile or methanol/ethanol and different chromatographic conditions were tested. In the final method 10 mg of lung tissue was homogenized with 50% methanol and metabolites were extracted with acetonitrile. Metabolites were separated on C8-RP and HILIC columns. About 3500 and 2000 of metabolic features (in both ion modes) were detected with good repeatability (CV < 20%) by RP and HILIC methods, respectively. Lung tumor and control tissue samples obtained from NSCLC patients were analyzed with developed methodology. Acylcarnitines, fatty acids, phospholipids, and amino acids were found more abundant in tumor as compared to control tissue. Acylcarnitines, lysophospholipids, creatinine, creatine, and alanine were identified as potential targets enabling classification of NSCLC subtypes.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Cromatografía Liquida/métodos , Neoplasias Pulmonares/metabolismo , Pulmón/metabolismo , Espectrometría de Masas/métodos , Metabolómica/métodos , Biomarcadores/análisis , Biomarcadores/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Análisis de los Mínimos Cuadrados , Pulmón/química , Masculino , MetabolomaRESUMEN
Aqueous humor (AH) is a transparent fluid which fills the anterior and posterior chambers of the eye. It supplies nutrients and removes metabolic waste from avascular tissues in the eye. Proper homeostasis of AH is required to maintain adequate intraocular pressure as well as optical and refractive properties of the eye. Application of metabolomics to study human AH may improve knowledge about the molecular mechanisms of eye diseases. Until now, global analysis of metabolites in AH has been mainly performed using NMR. Among the analytical platforms used in metabolomics, LC-MS allows for the highest metabolome coverage. The aim of this study was to develop a method for extraction and analysis of AH metabolites by LC-QTOF-MS. Different protocols for AH preparation were tested. The best results were obtained when one volume of AH was mixed with one volume of methanol : ethanol (1 : 1). In the final method, 2 µL of extracted sample was analyzed by LC-QTOF-MS. The method allowed for reproducible measurement of over 1000 metabolic features. Almost 250 metabolites were identified in AH and assigned to 47 metabolic pathways. This method is suitable to study the potential role of amino acids, lipids, oxidative stress, or microbial metabolites in development of ocular diseases.
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Lipids are important components of biological systems, and their role can be currently investigated by the application of untargeted, holistic approaches such as metabolomics and lipidomics. Acquired data are analyzed to find significant signals responsible for the differentiation between the investigated conditions. Subsequently, identification has to be performed to bring biological meaning to the obtained results. Lipid identification seems to be relatively easy due to the known characteristic fragments; however, the large number of structural isomers and the formation of different adducts makes it challenging and at risk of misidentification. The inspection of data, acquired for plasma samples by a standard metabolic fingerprinting method, revealed multisignal formations for phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins by the formation of ions such as [M + H](+), [M + Na](+), and [M + K](+) in positive ionization mode and [M - H](-), [M + HCOO](-), and [M + Cl](-) in negative mode. Moreover, sodium formate cluster formation was found for [M + H·HCOONa](+) and [H-H·HCOONa](-). The MS/MS spectrum obtained for each of the multi-ions revealed significant differences in the fragmentation, which were confirmed by the analysis of the samples in two independent research centers. After the inspection of an acquired spectra, a list of characteristic and diagnostic fragments was proposed that allowed for easy, quick, and robust lipid identification that provides information about the headgroup, formed adduct, and fatty acyl composition. This ensures successful identification, which is of great importance for the contextualization of data and results validation.