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Genetic factors, diet, lifestyle, and other factors lead to various complications in the body, such as obesity and other chronic diseases. The inflammatory state caused by excessive accumulation of body fat affects the pathways related to the control of glycemic homeostasis, leading to a high demand for insulin, to subsequent failure of stressed ß cells, and development of type 2 diabetes mellitus (T2DM). The study of new endocrine signalers, such as bile acids (BAs), becomes necessary as it allows the development of alternatives for T2DM treatment. In this work, a methodology was developed to quantify tauroursodeoxycholic BA (TUDCA) in liver cells of the HepG2 strain treated in hyperlipidic medium. This BA helps to improve insulin clearance by increasing the expression of the insulin-degrading enzyme, restoring sensitivity to this hormone, and making it viable for treating T2DM. Herein, a targeted metabolomic method for TUDCA determination in extracellular medium of hepatocyte matrices by micellar electrokinetic chromatography-UV was optimized, validated, and applied. The optimized background electrolyte was composed of 40 mmol/L sodium cholate and 30 mmol/L sodium tetraborate at pH 9.0. The following figures of merit were evaluated: linearity, limit of quantification, limit of detection, accuracy, and precision. Data obtained with the validated electrophoretic method showed a self-stimulation of TUDCA production in media supplemented only with BA. On the other hand, TUDCA concentration was reduced in the hyperlipidic medium. This suggests that, in these media, the effect of TUDCA is reduced, such as self-stimulated production and consequent regulation of glycemic homeostasis. Therefore, the results reinforce the need for investigating TUDCA as a potential T2DM biomarker as well as its use to treat several comorbidities, such as obesity and diabetes mellitus.

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Extracellular chemical cues constitute much of the language of life among marine organisms, from microbes to mammals. Changes in this chemical pool serve as invisible signals of overall ecosystem health and disruption to this finely tuned equilibrium. In coral reefs, the scope and magnitude of the chemicals involved in maintaining reef equilibria are largely unknown. Processes involving small, polar molecules, which form the majority components of labile dissolved organic carbon, are often poorly captured using traditional techniques. We employed chemical derivatization with mass spectrometry-based targeted exometabolomics to quantify polar dissolved phase metabolites on five coral reefs in the U.S. Virgin Islands. We quantified 45 polar exometabolites, demonstrated their spatial variability, and contextualized these findings in terms of geographic and benthic cover differences. By comparing our results to previously published coral reef exometabolomes, we show the novel quantification of 23 metabolites, including central carbon metabolism compounds (e.g., glutamate) and novel metabolites such as homoserine betaine. We highlight the immense potential of chemical derivatization-based exometabolomics for quantifying labile chemical cues on coral reefs and measuring molecular level responses to environmental stressors. Overall, improving our understanding of the composition and dynamics of reef exometabolites is vital for effective ecosystem monitoring and management strategies.

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As sessile organisms, plants develop the ability to respond and survive in changing environments. Such adaptive responses maximize phenotypic and metabolic fitness, allowing plants to adjust their growth and development. In this study, we analyzed the metabolic plasticity of Arabidopsis thaliana in response to nitrate deprivation by untargeted metabolomic analysis and using wild-type (WT) genotypes and the loss-of-function nia1/nia2 double mutant. Secondary metabolites were identified using seedlings grown on a hydroponic system supplemented with optimal or limiting concentrations of N (4 or 0.2 mM, respectively) and harvested at 15 and 30 days of age. Then, spectral libraries generated from shoots and roots in both ionization modes (ESI +/-) were compared. Totals of 3407 and 4521 spectral signals (m/z_rt) were obtained in the ESI+ and ESI- modes, respectively. Of these, approximately 50 and 65% were identified as differentially synthetized/accumulated. This led to the presumptive identification of 735 KEGG codes (metabolites) belonging to 79 metabolic pathways. The metabolic responses in the shoots and roots of WT genotypes at 4 mM of N favor the synthesis/accumulation of metabolites strongly related to growth. In contrast, for the nia1/nia2 double mutant (similar as the WT genotype at 0.2 mM N), metabolites identified as differentially synthetized/accumulated help cope with stress, regulating oxidative stress and preventing programmed cell death, meaning that metabolic responses under N starvation compromise growth to prioritize a defensive response.

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Roselle (Hibiscus sabdariffa) is rich in phenolic compounds with antiobesogenic and antidiabetic effects. In this study, the effects of aqueous extracts of two varieties of Hibiscus sabdariffa, Alma blanca (white-yellow color) and Cuarenteña (purple color), were evaluated for the prevention of obesity and insulin resistance in rats fed a high-fat and high-fructose diet (HFFD), identifying targeted molecules through global metabolomics. After sixteen weeks, both roselle aqueous extracts prevented body weight gain, and white roselle extract ameliorated insulin resistance and decreased serum free fatty acid levels. Moreover, white roselle extract decreased 18:0 and 20:4 lysophosphatidylethanolamines and purple roselle extract increased 16:0 and 20:4 lysophosphatidylinositol compared to HFFD-fed rats. These results demonstrate that roselle's beneficial health effects are variety-dependent. Interestingly, the white roselle extract showed a greater beneficial effect, probably due to its high contents of organic and phenolic acids, though its consumption is not as popular as that of the red/purple varieties.

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BACKGROUND: While mammographic density is one of the strongest risk factors for breast cancer, little is known about its determinants, especially in young women. We applied targeted metabolomics to identify circulating metabolites specifically associated with mammographic density in premenopausal women. Then, we aimed to identify potential correlates of these biomarkers to guide future research on potential modifiable determinants of mammographic density. METHODS: A total of 132 metabolites (acylcarnitines, amino acids, biogenic amines, glycerophospholipids, sphingolipids, hexose) were measured by tandem liquid chromatography/mass spectrometry in plasma samples from 573 premenopausal participants in the Mexican Teachers' Cohort. Associations between metabolites and percent mammographic density were assessed using linear regression models, adjusting for breast cancer risk factors and accounting for multiple tests. Mean concentrations of metabolites associated with percent mammographic density were estimated across levels of several lifestyle and metabolic factors. RESULTS: Sphingomyelin (SM) C16:1 and phosphatidylcholine (PC) ae C30:2 were inversely associated with percent mammographic density after correction for multiple tests. Linear trends with percent mammographic density were observed for SM C16:1 only in women with body mass index (BMI) below the median (27.4) and for PC ae C30:2 in women with a BMI over the median. SM C16:1 and PC ae C30:2 concentrations were positively associated with cholesterol (total and HDL) and inversely associated with number of metabolic syndrome components. CONCLUSIONS: We identified new biomarkers associated with mammographic density in young women. The association of these biomarkers with mammographic density and metabolic parameters may provide new perspectives to support future preventive actions for breast cancer.

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Altered cell metabolism is a hallmark of cancer and critical for its development. Particularly, activation of one-carbon metabolism in tumor cells can sustain oncogenesis while contributing to epigenetic changes and metabolic adaptation during tumor progression. We assessed whether increased one-carbon metabolism activity is a metabolic feature of invasive ductal carcinoma (IDC). Differences in the metabolic profile between biopsies from IDC (n = 47) and its adjacent tissue (n = 43) and between biopsies from different breast cancer subtypes were assessed by gas spectrometry in targeted (Biocrates Life Science ® ) and untargeted approaches, respectively. The metabolomics data were statistically treated using MetaboAnalyst 4.0, SIMCA P+ (version 12.01), Statistica 10 software and t test with p < 0.05. The Cancer Genome Atlas breast cancer dataset was also assessed to validate the metabolomic profile of IDC. Our targeted metabolomics analysis showed distinct metabolomics profiles between IDC and adjacent tissue, where IDC displayed a comparative enrichment of metabolites involved in one-carbon metabolism (serine, glycine, threonine, and methionine) and a predicted increase in the activity of pathways that receive and donate carbon units (i.e., folate, methionine, and homocysteine). In addition, the targeted and untargeted metabolomics analyses showed similar metabolomics profiles between breast cancer subtypes. The gene set enrichment analysis identified different transcription-related functions between IDC and non-tumor tissues that involved one-carbon metabolism. Our data suggest that one-carbon metabolism may be a central pathway in IDC and even in general breast tumors, representing a potential target for its treatment and prevention.

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Chili pepper (Capsicum spp.) is one of the most important horticultural crops worldwide, and its unique organoleptic properties and health benefits have been established for centuries. However, there is little knowledge about how metabolites are distributed throughout fruit parts. This work focuses on the use of liquid chromatography coupled with high resolution mass spectrometry (UHPLC-ESI-HRMS) to estimate the global metabolite profiles of the pericarp, placenta, and seeds of Tabasco pepper fruits (Capsicum frutescens L.) at the red mature stage of ripening. Our main results putatively identified 60 differential compounds between these tissues and seeds. Firstly, we found that pericarp has a higher content of glycosides, showing on average a fold change of 5 and a fold change of 14 for terpenoids when compared with other parts of the fruit. While placenta was the richest tissue in capsaicinoid-related compounds, alkaloids, and tocopherols, with a 35, 3, and 7 fold change, respectively. However, the seeds were richer in fatty acids and saponins with fold changes of 86 and 224, respectively. Therefore, our study demonstrates that a non-targeted metabolomic approach may help to improve our understanding of unexplored areas of plant metabolism and also may be the starting point for a detailed analysis in complex plant parts, such as fruits.

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A capillary electrophoresis with diode array and tandem mass spectrometry detection (CE-UV-MS/MS) method has been developed for the targeted assessment of cardiovascular biomarkers candidates, trimethylamine-N-Oxide (TMAO) and l-carnitine, and creatinine in human urine samples. The dual detection was applied due to the high concentration of creatinine (monitored by UV detection at 200 nm) in relation to TMAO and l-carnitine (quantified by selected reaction monitoring (SRM) mass spectrometry), in human urine. All instrumental parameters, sheath liquid (SHL) and background electrolyte (BGE) compositions were optimized with a pool of urine provided by adult healthy volunteers and evaluated by signal-to-noise ratio (SNR) and peak shape of TMAO. The compositions for the optimized BGE was formic acid at concentration of 0.10 mol L-1, and for SHL was 70:30 MeOH:H2O containing 0.05% (v/v) formic acid, delivered at a flow rate of 5 µL min-1. Limits of detection for TMAO, l-carnitine and creatinine were 0.76, 0.54 and 303 µmol L-1, respectively. Limits of quantification were 2.5, 1.8 and 1000 µmol L-1, respectively. Linearity was evaluated by ANOVA and presented R2 from 0.993 to 0.997. Precision and accuracy were evaluated at three concentration levels. Coefficients of variation (CV) from 1 to 21% were obtained for the intra-day precision evaluation and from 2 to 16% for the inter-day precision evaluation. The recovery ranged from 75 to 116%. Quantitation of TMAO and l-carnitine in infarcted patients urine in comparison to healthy individuals indicated a 2.2 fold increase of TMAO and a 7.0 fold increase of l-carnitine. These results showed the potential applicability of the proposed method for the evaluation of TMAO and l-carnitine in urine within a panel of candidate metabolites in targeted metabolomics studies of cardiovascular diseases among other conditions.

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Amino acids play an important role in clinical analysis. Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) has proven to possess several characteristics that make it a powerful and useful tool for the analysis of amino acids in clinical studies. Here we present a method for the separation and quantitative analysis of 27 amino acids in urine based on CE-ESI-MS. The method presents an improved resolution between the isomers Leu, Ile, and aIle, in comparison to other CE-ESI-MS methods in the literature. This method is fast, selective, and simple and has improved sensitivity by applying a pH-mediated stacking strategy, showing that it can be successfully used for amino acid analysis and probably for other small cationic metabolites.

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Miniaturized sample pretreatments for the analysis of phenolic metabolites in plasma, involving protein precipitation, enzymatic deconjugation, extraction procedures, and different derivatization reactions were systematically evaluated. The analyses were conducted by gas chromatography with mass spectrometry for the evaluation of 40 diet-derived phenolic compounds. Enzyme purification was necessary for the phenolic deconjugation before extraction. Trimethylsilanization reagent and two different tetrabutylammonium salts for derivatization reactions were compared. The optimum reaction conditions were 50 µL of trimethylsilanization reagent at 90°C for 30 min, while tetrabutylammonium salts were associated with loss of sensitivity due to rapid activation of the inert gas chromatograph liner. Phenolic acids extractions from plasma were optimized. Optimal microextraction by packed sorbent performance was achieved using an octadecylsilyl packed bed and better recoveries for less polar compounds, such as methoxylated derivatives, were observed. Despite the low recovery for many analytes, repeatability using an automated extraction procedure in the gas chromatograph inlet was 2.5%. Instead, using liquid-liquid microextraction, better recoveries (80-110%) for all analytes were observed at the expense of repeatability (3.8-18.4%). The phenolic compounds in gerbil plasma samples, collected before and 4 h after the administration of a calafate extract, were analyzed with the optimized methodology.

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Nowadays, there is a growing interest in deeply understanding biological mechanisms not only at the molecular level (biological components) but also the effects of an ongoing biological process in the organism as a whole (biological functionality), as established by the concept of systems biology. Within this context, metabolomics is one of the most powerful bioanalytical strategies that allow obtaining a picture of the metabolites of an organism in the course of a biological process, being considered as a phenotyping tool. Briefly, metabolomics approach consists in identifying and determining the set of metabolites (or specific metabolites) in biological samples (tissues, cells, fluids, or organisms) under normal conditions in comparison with altered states promoted by disease, drug treatment, dietary intervention, or environmental modulation. The aim of this chapter is to review the fundamentals and definitions used in the metabolomics field, as well as to emphasize its importance in systems biology and clinical studies.

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This chapter focuses on the important contribution of CE-MS in metabolomics, describing the nature of CE-MS coupling and the technical improvements that have led to the interfaces used in modern instrumentation. Moreover, it will discourse how the variety of electrolyte compositions and additives, which has conferred CE the exceptional selectivity of its multiple separation modes, has been handled to allow interfacing with MS without compromising ionization efficiency and the spectrometer integrity. Finally, the methodologies of CE-MS in current use for metabolomics will be discussed in detail. To verify the scope of CE-MS in clinical metabolomics, a myriad of representative applications has been compiled.

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Background: Supplementation with essential amino acids (EAAs) + arginine is a promising nutritional approach to decrease plasma triglyceride (TG) concentrations, which are an independent risk factor for ischemic heart disease. Objective: The objective of this study was to examine the effects of 8 wk of EAA supplementation on skeletal muscle basal metabolite concentrations and changes in metabolic response to acute EAA intake, with an emphasis on mitochondrial metabolism, in adults with elevated TGs to better understand the mechanisms of lowering plasma TGs. Methods: Older adults with elevated plasma TG concentrations were given 22 g EAAs to ingest acutely before and after an 8-wk EAA supplementation period. Skeletal muscle biopsy samples were collected before and after acute EAA intake, both pre- and postsupplementation (4 biopsy samples), and targeted metabolomic analyses of organic acids and acylcarnitines were conducted on the specimens. Results: Acute EAA intake resulted in increased skeletal muscle acylcarnitine concentrations associated with oxidative catabolism of the supplement components, with the largest increases found in acylcarnitines of branched-chain amino acid oxidative catabolism, including isovaleryl-carnitine (2200%) and 2-methylbutyryl-carnitine (2400%). The chronic EAA supplementation resulted in a 19% decrease in plasma TGs along with accumulation of long-chain acylcarnitines myristoyl- (90%) and stearoyl- (120%) carnitine in skeletal muscle and increases in succinyl-carnitine (250%) and the late-stage tricarboxylic acid cycle intermediates fumarate (44%) and malate (110%). Conclusions: Supplementation with EAAs shows promise as an approach for moderate reduction in plasma TGs. Changes in skeletal muscle metabolites suggest incomplete fatty acid oxidation and increased anaplerosis, which suggests a potential bottleneck in fatty acid metabolism.