Evaluation of tauroursodeoxycholic acid in liver cells' cultures by MEKC: Initial hints to comprehend its role in diabetes mellitus of obese individuals.

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.

COVID-19 and Comorbidities: What Has Been Unveiled by Metabolomics?

The COVID-19 pandemic has brought about diverse impacts on the global population. Individuals with comorbidities were more susceptible to the severe symptoms caused by the virus. Within the crisis scenario, metabolomics represents a potential area of science capable of providing relevant information for understanding the metabolic pathways associated with the intricate interaction between the viral disease and previous comorbidities. This work aims to provide a comprehensive description of the scientific production pertaining to metabolomics within the specific context of COVID-19 and comorbidities, while highlighting promising areas for exploration by those interested in the subject. In this review, we highlighted the studies of metabolomics that indicated a variety of metabolites associated with comorbidities and COVID-19. Furthermore, we observed that the understanding of the metabolic processes involved between comorbidities and COVID-19 is limited due to the urgent need to report disease outcomes in individuals with comorbidities. The overlap of two or more comorbidities associated with the severity of COVID-19 hinders the comprehension of the significance of each condition. Most identified studies are observational, with a restricted number of patients, due to challenges in sample collection amidst the emergent situation.

Untargeted Metabolomics Studies of H9c2 Cardiac Cells Submitted to Oxidative Stress, ß-Adrenergic Stimulation and Doxorubicin Treatment: Investigation of Cardiac Biomarkers.

One of the biggest challenges in the search for more effective treatments for diseases is understanding their etiology. Cardiovascular diseases (CVD) are an important example of this, given the high number of deaths annually. Oxidative stress (the imbalance between oxidant and antioxidant species in biological system) is one of the factors responsible for CVD occurrence, demanding extensive investigation. Excess of reactive oxygen species (ROS) are primarily responsible for this condition, and clinical and scientific literature have reported a significant increase in ROS when therapeutic drugs, such as doxorubicin and isoproterenol, are administered. In this context, the aim of this study is the investigation of potential biomarkers that might be associated with oxidative stress in cardiomyocytes. For this purpose, H9c2 cardiomyocytes were submitted to oxidative stress conditions by treatment with doxorubicin (DOX), isoproterenol (ISO) and hydrogen peroxide (PER). Metabolomics analyses of the cell extract and the supernatant obtained from the culture medium were then evaluated by CE-ESI(+)-TOF-MS. Following signal processing, statistical analyses, and molecular features annotations, the results indicate changes in the aspartate, serine, pantothenic acid, glycerophosphocholine and glutathione metabolism in the cell extract.

Comprehending Cardiac Dysfunction by Oxidative Stress: Untargeted Metabolomics of In Vitro Samples.

Cardiovascular diseases (CVDs) are noncommunicable diseases known for their complex etiology and high mortality rate. Oxidative stress (OS), a condition in which the release of free radical exceeds endogenous antioxidant capacity, is pivotal in CVC, such as myocardial infarction, ischemia/reperfusion, and heart failure. Due to the lack of information about the implications of OS on cardiovascular conditions, several methodologies have been applied to investigate the causes and consequences, and to find new ways of diagnosis and treatment as well. In the present study, cardiac dysfunction was evaluated by analyzing cells' alterations with untargeted metabolomics, after simulation of an oxidative stress condition using hydrogen peroxide (H2O2) in H9c2 myocytes. Optimizations of H2O2 concentration, cell exposure, and cell recovery times were performed through MTT assays. Intracellular metabolites were analyzed right after the oxidative stress (oxidative stress group) and after 48 h of cell recovery (recovery group) by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in positive and negative ESI ionization mode. Significant alterations were found in pathways such as "alanine, aspartate and glutamate metabolism", "glycolysis", and "glutathione metabolism", mostly with increased metabolites (upregulated). Furthermore, our results indicated that the LC-MS method is effective for studying metabolism in cardiomyocytes and generated excellent fit (R2Y > 0.987) and predictability (Q2 > 0.84) values.

The New Omics Era into Systems Approaches: What Is the Importance of Separation Techniques?

Omics sciences have been facing challenges in different fields, especially in life sciences. One of these challenges involves assessing biology into systems interpretation. With the advance of genomics, molecular biology has been projected into the realm of systems biology. In a different direction, systems approaches are making definitive strides toward scientific understanding and biotechnological applications. Separation techniques provided meaningful progress in the omics era, conducting the classical molecular biology to contemporary systems biology. In this introductory chapter, the relevance of these techniques to the development of different omics sciences, within the systems biology context, will be discussed.

Metabolomics Data Treatment: Basic Directions of the Full Process.

The present chapter describes basic aspects of the main steps for data processing on mass spectrometry-based metabolomics platforms, focusing on the main objectives and important considerations of each step. Initially, an overview of metabolomics and the pivotal techniques applied in the field are presented. Important features of data acquisition and preprocessing such as data compression, noise filtering, and baseline correction are revised focusing on practical aspects. Peak detection, deconvolution, and alignment as well as missing values are also discussed. Special attention is given to chemical and mathematical normalization approaches and the role of the quality control (QC) samples. Methods for uni- and multivariate statistical analysis and data pretreatment that could impact them are reviewed, emphasizing the most widely used multivariate methods, i.e., principal components analysis (PCA), partial least squares-discriminant analysis (PLS-DA), orthogonal partial least square-discriminant analysis (OPLS-DA), and hierarchical cluster analysis (HCA). Criteria for model validation and softwares used in data processing were also approached. The chapter ends with some concerns about the minimal requirements to report metadata in metabolomics.

Metabolite mass spectrometry profiling of cacao genotypes reveals contrasting resistances to Ceratocystis cacaofunesta phytopathogen.

Ceratocystis wilt is a lethal disease of cacao, and the search for resistant genotypes may provide the best way to deal with the disease. Resistance or susceptibility behavior of some cacao genotypes when infected by Ceratocystis cacaofunesta is not yet understood. Herein, we report an LC-MS metabolomic screening analysis based on high-resolution MS to obtain comprehensive metabolic profile associated with multivariate data analysis of PLS-DA, which was effective to classify CCN-51 and TSH-1188 as resistant genotypes to C. cacaofunesta fungus, while CEPEC2002 was classified as a susceptible one. Using reversed-phase LC method, electrospray interface, and high-resolution tandem MS by the quadrupole-TOF analyzer, the typical profiles of metabolites, such as phenylpropanoids, flavonoids, lipids, alkaloids, and amino acids, were obtained. Untargeted metabolite profiles were used to construct discriminant analysis by partial least squares (PLS-DA)-derived loading plots, which placed the cacao genotypes into two major clusters related to susceptible or resistant groups. Linolenic, linoleic, oleic, stearic, arachidonic, and asiatic acids were annotated metabolites of infected, susceptible, and resistant genotypes, while methyl jasmonate, jasmonic acid, hydroxylated jasmonic acid, caffeine, and theobromine were annotated as constituents of the resistant genotypes. Trends of these typical metabolites levels revealed that CCN51 is susceptible, CEPEC2002 is moderately susceptible, and TSH1188 is resistant to C. cacaofunesta. Therefore, profiles of major metabolites as screened by LC-MS offer an efficient tool to reveal the level of resistance of cacao genotypes to C. cacaofunesta present in any farm around the world.

Gas chromatography-mass spectrometry untargeted profiling of non-Hodgkin's lymphoma urinary metabolite markers.

Non-Hodgkin's lymphoma (NHL) is a cancer of the lymphatic system where the lymphoid and hematopoietic tissues are infiltrated by malignant neoplasms of B, T, and natural killer lymphocytes. Effective and less invasive methods for NHL screening are urgently needed. Herein, we report an untargeted gas chromatography-mass spectrometry (GC-MS) method to investigate metabolic changes in non-volatile derivatized compounds from urine samples of NHL patients (N = 15) and compare them to healthy controls (N = 34). Uni- and multivariate data analysis showed 18 endogenous metabolites, including amino acids and their metabolites, sugars, small organic acids, and vitamins, as statistically significant for group differentiation. A receiver operating characteristic curve (ROC) generated from a support vector machine (SVM) algorithm-based model achieved 0.998 of predictive accuracy, displaying the potential and relevance of GC-MS-detected urinary non-volatile compounds for predictive purposes. Furthermore, a specific panel of key metabolites was also evaluated, showing similar results. All in all, our results indicate that this robust GC-MS method is an effective screening tool for NHL diagnosis and it is able to highlight different pathways of the disease. Graphical Abstract.

Bio-synthesized sardine oil concentrate alters the composition of hepatic lipids in rats: A lipidomic approach.

Both preventive and curative therapies have created a considerable demand for n-3 PUFAs (polyunsaturated fatty acids) from fish oil, such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, for human use. Bio-synthesized sardine oil (bioSO) concentrate containing an acylglycerols mixture with 50% n-3 PUFAs was obtained by Candida cylindracea lipase hydrolysis and subsequently used for in vivo tests in animals. Wistar rats received, by gavage, a dose of 0.2 g/kg/day of bioSO or unmodified sardine oil (unSO) or saline solution (control) for three consecutive days and the liver tissue was evaluated by a selective and sensitive lipidomic approach based on ultra-performance liquid chromatography-quadruple time-of-flight mass spectrometry (UPLC-QTOF-MSE) and gas chromatography (GC). In addition, antioxidant parameters, response of oxidative stress marker and estimated fatty acid desaturase indexes were determined. The use of bioSO led to an increase in Cer d18:1/16:0, PE-Cer d14:2/18:0 and highly unsaturated phosphatylcholines (PC 38:4, PC 40:6 and PC 42:8) in the hepatic tissue membranes. There was also an increase in DHA incorporation in animals that received bioSO in comparison with the control animals. No differences in superoxide dismutase and catalase activity levels were observed between the groups, and malondialdehyde levels and delta 5-desaturase activity were higher in animals supplemented with bioSO. These results indicate that bioSO increase the hepatic incorporation of DHA, especially those esterified as PCs, and are probably absorbed and transported more effectively than the unSO. Enzymatically hydrolyzed compounds containing antioxidants may be a viable alternative for obtaining n-3 PUFA-enriched functional lipids.

Targeted metabolomics: Liquid chromatography coupled to mass spectrometry method development and validation for the identification and quantitation of modified nucleosides as putative cancer biomarkers.

A notable change in the body fluids nucleosides of cancer patients has been actively highlighted in searches for new biomarkers to early cancer detection. For this reason, improvements of bioanalytical methods for these compounds focused on a noninvasive sampling trend are of great importance. Therefore, this work aimed firstly to develop efficient methods for nucleoside analysis in urine and serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS), applying different strategies to quantify nine nucleosides, and further identify other untargeted nucleosides. Sample preparation was based on protein precipitation and affinity-solid phase extraction (SPE), whereas quantification was performed using a triple quadrupole (QqQ) mass analyzer operating in the selected reaction monitoring (SRM) mode. Surrogates matrices were proposed as an alternative to standard addition calibration. Specifically, to quantitate creatinine, a simple LC-MS/MS method was validated and used for normalization of urinary metabolites quantitation. To identify the other nucleosides, LC methods using different MS scans modes were evaluated on a quadrupole-time of flight (Q-TOF) or a hybrid triple quadrupole linear ion trap (Q-trap). Validation was performed for nucleosides quantification using the synthetic matrices of urine and serum, and selectivity, linearity, accuracy, reproducibility, matrix effect, LOD's and LOQ's were accessed, providing trustworthy results for bioanalysis purposes. Both LC-Q-Trap/MS and LC-Q-TOF/MS methods showed proper sensitivity for structural characterization on assays with urine and serum samples from healthy volunteers and could also be used in the identification of untargeted nucleosides. The investigated approaches delivered in-depth results and seem promising for future applications on urine and serum samples analyses aiming to validate nucleosides as cancer biomarkers.

Assessment of nucleosides as putative tumor biomarkers in prostate cancer screening by CE-UV.

Cancer is responsible for millions of deaths worldwide, but most base diseases may be cured if detected early. Screening tests may be used to identify early-stage malignant neoplasms. However, the major screening tool for prostate cancer, the prostate-specific antigen test, has unsuitable sensitivity. Since cancer cells may affect the pattern of consumption and excretion of nucleosides, such biomolecules are putative biomarkers that can be used for diagnosis and treatment evaluation. Using a previously validated method for the analysis of nucleosides in blood serum by capillary electrophoresis with UV-vis spectroscopy detection, we investigated 60 samples from healthy individuals and 42 samples from prostate cancer patients. The concentrations of nucleosides in both groups were compared and a multivariate partial least squares-discriminant analysis classification model was optimized for prediction of prostate cancer. The validation of the model with an independent sample set resulted in the correct classification of 82.4% of the samples, with sensitivity of 90.5% and specificity of 76.7%. A significant downregulation of 5-methyluridine and inosine was observed, which can be indicative of the carcinogenic process. Therefore, such analytes are potential candidates for prostate cancer screening. Graphical Abstract Separation of the studied nucleosides and the internal standard 8-Bromoguanosine by CE-UV (a); classification of the external validation samples (30 from healthy volunteers and 21 from prostate cancer patients) by the developed Partial Least Square - Discriminant Analysis (PLS-DA) model with accuracy of 82.4% (b); Receiver Operating Characteristics (ROC) curve (c); and Variable Importance in the Projection (VIP) values for the studied nucleosides (d). A significant down-regulation of 5- methyluridine (5mU) and inosine (I) was observed, which can be indicative of the presence of prostate tumors.

Strategies Involving Mass Spectrometry Combined with Capillary Electrophoresis in Metabolomics.

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.

CE-UV for the characterization of passion fruit juices provenance by amino acids profile with the aid of chemometric tools.

A separation method was developed in order to quantify free amino acids in passion fruit juices using CE-UV. A selective derivatization reaction with FMOC followed by MEKC analysis was chosen due to the highly interconnected mobilities of the analytes, enabling the separation of 22 amino acids by lipophilicity differences, as will be further discussed. To achieve such results, the method was optimized concerning BGE composition (concentrations, pH, and addition of organic modifier) and running conditions (temperature and applied voltage). The optimized running conditions were: a BGE composed by 60 mmol/L borate buffer at pH 10.1, 30 mmol/L SDS and 5 % methanol; running for 40 min at 23°C and 25 kV. The method was validated and applied on eight brands plus one fresh natural juice, detecting 12 amino acids. Quantification of six analytes combined with principal component analysis was capable to characterize different types of juices and showed potential to detect adulteration on industrial juices. Glutamic acid was found to be the most concentrated amino acid in all juices, exceeding 1 g/L in all samples and was also crucial for the correct classification of a natural juice, which presented a concentration of 22 g/L.

Higher detectability method for the analysis of nucleosides, putative tumor biomarkers, in blood serum samples by CE-UV with reversed EOF.

The development and validation of methodologies for the analysis of biological samples is of outcome importance in order to obtain trustworthy results. This work reports a novel CE-UV method for the assessment of nucleosides, putative tumor biomarkers, in blood serum. The separation of seven nucleosides within c.a. 20 min has been achieved with: BGE 30 mmol/L borate at pH 9.90, 50 mmol/L CTAB, and 10% methanol; V = -10 kV; T = 20°C; and capillary dimensions of 56 cm × 50 µm. The sample plug was concentrated by a modified large volume sample stacking strategy that provided better detectability. Validation showed that the method is suitable for bioanalytical purposes and initial applications in serum samples from healthy subjects are also presented. Finally, statistical methods were applied to verify the effect of characteristics such as age, smoking habits, and alcohol consumption on nucleoside concentrations in blood serum. Univariate statistical analysis tests emphasized the need for age matching, which was confirmed by PCA-DA and PLS-DA. Cancer history in the nearby family may also interfere in nucleoside levels in blood serum, since adenosine concentrations were statistically higher for volunteers who declared having diseased relatives.

Unveiling differences between patients with acute coronary syndrome with and without ST elevation through fingerprinting with CE-MS and HILIC-MS targeted analysis.

Differences in the degree and severity of Acute Coronary Syndrome, associated to differences in the electrocardiogram, together with blood tests of biomarkers classify patients for diagnosis and treatment. Cases where the electrocardiogram and/or biomarkers are not conclusive still appear, and there is a need for complementary biomarkers for routine determinations. Metabolomics approaches with blind fingerprinting could reveal differences in metabolites, which must be confirmed by means of targeted determinations. CE-MS and HILIC-MS are well suited for the determination of highly polar compounds, like those from to the intermediate metabolism, altered due to acute stress induced by myocardial infarction. Serum from patients with ST-elevated and non-ST elevated myocardial infarction was collected at intensive care and emergency units, and fingerprinted with CE-MS. Data pretreatment and analysis showed up carnitine-related compounds and amino acids differentially present in both groups. Acylcarnitines and amino acids were then quantitatively measured with HILIC-MS-QqQ. The significance of the differences and the sensitivity/specificity of each compound were individually evaluated. The ratio of free carnitine to acylcarnitines, together with the ratios of acetylcarnitine to betaine, to threonine, and to citrulline, showed high significance and area under the curve in the respective receiver operating characteristic curves. This study opens new possibilities for defining new sets of biomarkers for refining the diagnosis of the patients with difficult classification.

Simple and rapid CE-UV method for the assessment of trail pheromone compounds of leaf-cutting ants' venom glands.

Leaf-cutting ants cause large losses in several crops around the world. In the ant species Atta sexdens, each colony comprises up to 5 million individuals. In order to keep a close connection among such a large number of individuals, an efficient chemical communication system is necessary. Among other different substances, these animals use alkylpyrazines to mark their trails and to guide ant workers from the nest to their sources of food. In this study, CE-UV was used to apply a method for qualitative analysis of venom gland components of leaf-cutting ants. Mobility of these compounds proved to be a function of the ionization capability of these bases as well as their volumes. Migration order was thoroughly explained in terms of such parameters. The best analysis conditions were achieved with a BGE composed by 0.8% formic acid plus 20% methanol in water, hydrodynamic injection, and application of external pressure. Such analysis conditions may be easily applied in CE-MS analyses as well. CE-UV analyses proved to be as adequate as GC to analyze such compounds due to system detectability (LD ≅ 0.005 mmol/L), separation efficiency (from 5.07×10(4) to 1.23×10(5) theoretical plates), and resolution (minimum of 2.35). In addition, analysis time was ca. 15 min, which shows another advantage of CE analysis when compared with GC. Although the analytes are found in concentrations as low as 50 ng/venom glands, four putative pyrazine ring moiety substances could be detected in real samples, due to sample stacking and use of a capillary with extended detection cell.

Characterization of protein hydrolysates of cosmetic use by CE-MS.

Protein hydrolysates have been used as active principles in cosmetic products conferring different properties to the final formulations, which are mostly controlled by the peptide size and its amino acid sequence. In this work, capillary electrophoresis coupled to mass spectrometry analyses were carried out in order to investigate such characteristics of protein hydrolysates. Samples of different origins (milk, soy and rice) were obtained from a local company, and were analyzed without a previous preparation step. The background electrolyte (BGE) and sheath liquid compositions were optimized for each sample. The best BGE composition (860 mmol/L formic acid--pH 1.8--in 70:30 v/v water/methanol hydro-organic solvent) was chosen based on the overall peak resolution whereas the best sheath liquid was selected based on increased sensitivity and presented different compositions to each sample (10.9-217 mmol/L formic acid in 75:25-25:75 v/v water/methanol hydro-organic solvent). Most of the putative peptides in the hydrolysate samples under investigation presented molecular masses of 1000 Da or less. De novo sequencing was carried out for some of the analytes, revealing the hydrophobicity/polarity of the peptides. Hence, the technique has proved to be an advantageous tool for the quality control of industrial protein hydrolysates.

CE-MS and related techniques as a valuable tool in tumor biomarkers research.

Cancer has been a disease of great concern because it is the second main cause of death in the world. Cures for most cancer pathologies have not yet been found, and an accurate and early diagnosis is essential for successful treatment. Therefore, research on tumor biomarkers has noticeably increased in recent years. The determination of such biomolecules, together with the routinely used laboratory exams for cancer diagnosis, would constitute a more reliable approach, known as systems biology. The "omics" era has corroborated in such investigations since the development of new technologies has arisen along with it. One of the techniques applied to the investigation of tumor biomarkers is CE, and the increasing applications of CE-MS in this field are also observed. This review covers the published literature on tumor biomarker investigations by CE-MS and related techniques, mostly within the last decade, but not limited to it. For didactic reasons this review is divided into the tumor biomarkers chemical classes, namely, proteins and related molecules, DNA adducts and modified nucleosides.