Lipidomic profiling of the Brazilian yellow scorpion venom: new insights into inflammatory responses following Tityus serrulatus envenomation.

Due to the high prevalence and clinical relevance, scorpionism is a critical public health issue in several Brazilian regions. Tityus serrulatus, commonly known as the Brazilian yellow scorpion, is the most venomous genus found in Brazilian fauna and associated with severe clinical manifestations such as localized pain, hypertension, sweating, tachycardia and complex hyperinflammatory responses. In general, T. serrulatus venom contains a complex mixture of active compounds, including proteins, peptides, and amino acids. Although knowledge of the protein fractions of scorpion venom is available, venom lipid components are not yet comprehensively known. The aim of the present study was to determine and characterize the lipid constituents/profile of the T. serratus venom utilizing liquid chromatography coupled with high-resolution mass spectrometry. Lipid species (164 in total) belonging to 3 different lipid categories, glycerophospholipids, sphingolipids, and glycerolipids, were identified. A further search on MetaCore/MetaDrug platform, which is based upon a manually curated database of molecular interactions, molecular pathways, gene-disease associations, chemical metabolism, and toxicity information, exhibited several metabolic pathways for 24 of previously identified lipid species, including activation of nuclear factor kappa B and oxidative stress pathways. Further several bioactive compounds, such as plasmalogens, lyso-platelet-activating factors, and sphingomyelins, associated with systemic responses triggered by T. serrulatus envenomation were detected. Finally, lipidomic data presented provide advanced and valuable information to better comprehend the mechanisms underlying the complex pathophysiology induced by T. serrulatus envenomation.

A lipidomics approach reveals new insights into Crotalus durissus terrificus and Bothrops moojeni snake venoms.

Snakebite envenomation causes > 81,000 deaths and incapacities in another 400,000 people worldwide every year. Snake venoms are complex natural secretions comprised of hundreds of different molecules with a wide range of biological functions that after injection cause local and systemic manifestations. Although several studies have investigated snake venoms, the majority have focused on the protein portion (toxins), without significant attention paid to the lipid fraction. Therefore, an untargeted lipidomic approach based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) was applied to investigate the lipid constituents of venoms of the snake species Crotalus durissus terrificus and Bothrops moojeni. Phosphatidylcholines (PC), Lyso-PCs, phosphatidylethanolamines (PE), Lyso-PE, phosphatidylserine (PS), phosphatidylinositol (PI), ceramides (Cer), and sphingomyelin (SM) species were detected in the analyzed snake venoms. The identified lipids included bioactive compounds such as platelet-activating factor (PAF) precursor, PAF-like molecules, plasmalogens, ceramides, and sphingomyelins with long fatty acid chain lengths, which may be associated with the systemic responses triggered by C. d. terrificus and B. moojeni envenomation. These responses include platelet aggregation, activation of intercellular adhesion molecule 1 (ICAM1), apoptosis, as well as the production of pro-inflammatory lipid mediators, cytokines, and reactive species. The newly proposed lipidomics strategy provided valuable information regarding the lipid profiles of viperid venoms, which could lead to increased understanding of the complex pathology promoted by snakebite envenomation.

Electrospun protein fibers loaded with yerba mate extract for bioactive release in food packaging.

BACKGROUND: Yerba mate extract was encapsulated in electrospun zein fibers. Solutions were prepared with 30% (w/v) zein, and yerba mate extract was added at concentrations of 1%, 3%, and 5% (w/w). The rheology and electrical conductivity of the polymer solutions were evaluated. The extract and the fibers were characterized through an analysis of total and individual phenolic compounds, antioxidant activity, and Fourier-transform infrared (FTIR) spectra. Morphology, size distribution, and thermal stability were also evaluated. The release kinetics of zein fibers loaded with different concentrations of yerba mate were evaluated in a hydrophilic food-simulant medium (10% ethanol). RESULTS: Yerba mate extract had a total phenolic compound content of 1287.76 ± 11.55 mg of gallic acid 100 g-1 yerba mate extract. The major individual phenolic compounds obtained were chlorogenic acid and rutin, quantified by high-performance liquid chromatography and mess spectrometry (HPLC-MS). Zein fibers loaded with 5% extract exhibited higher antioxidant activity with 83.0% inhibition. The fibers with different concentrations of yerba mate displayed homogeneous morphology. Yerba mate extract encapsulated in zein fibers had greater thermal stability than the free extract. Zein fibers comprising 5% yerba mate extract, when in contact with a hydrophilic food simulant medium, showed a release of approximately 49% of extract within 50 h. CONCLUSION: Zein fibers containing yerba mate extract may be used as antioxidant releasers for food packaging. © 2020 Society of Chemical Industry.

Untargeted metabolomics of strawberry (Fragaria x ananassa 'Camarosa') fruit from plants grown under osmotic stress conditions.

BACKGROUND: Plants activate defense mechanisms to cope with adverse environmental conditions, leading to the accumulation and / or depletion of general and specialized metabolites. In this study, a multiplatform untargeted metabolomics strategy was employed to evaluate metabolic changes in strawberry fruit of cv. Camarosa grown under osmotic stress conditions. Liquid chromatography-mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) data from strawberries grown under two water-deficit conditions, irrigated at 95% crop evapotranspiration (ETc) and 85% ETc, and one excess salt condition with a 80 mmol L-1 NaCl solution, were analyzed to determine treatment effects on fruit metabolism. RESULTS: Multivariate principal component analysis, orthogonal projections to latent structures - discriminant analysis (OPLS-DA), and univariate statistical analyses were applied to the data set. While multivariate analyses showed group separation by treatment, T-tests and fold change revealed 12 metabolites differentially accumulated in strawberries from different treatments - among them phenolic compounds, glycerophospholipids, phytosterols, carbohydrates, and an aromatic amino acid. CONCLUSION: Untargeted metabolomic analysis allowed for the annotation of compounds differentially accumulated in strawberry fruit from plants grown under osmotic stress and non-stressed plants. The metabolic disturbance in plants under stress involved metabolites associated with the inhibition of reactive oxygen species and cell-wall and membrane lipid biosynthesis, which might serve as osmotic stress biomarkers. © 2019 Society of Chemical Industry.

Recent advances in the application of capillary electromigration methods for food analysis and Foodomics.

This review work presents and discusses the main applications of capillary electromigration methods in food analysis and Foodomics. Papers that were published during the period February 2013-February 2015 are included following the previous review by Garcia-Cañas et al. (Electrophoresis, 2014, 35, 147-169). Analysis by CE of a large variety of food-related molecules with different chemical properties, including amino acids, hazardous amines, peptides, proteins, phenols, polyphenols, lipids, carbohydrates, DNAs, vitamins, toxins, contaminants, pesticides, residues, food additives, as well as small organic and inorganic compounds. This work includes recent results on food quality and safety, nutritional value, storage, bioactivity, as well as applications of CE for monitoring food processing. The use, among other CE developments, of microchips, CE-MS, and chiral CE in food analysis and Foodomics is also discussed.

Potential of prodendronic polyamines with modulated segmental charge density as novel coating for fast and efficient analysis of peptides and basic proteins by CE and CE-MS.

In this work, the suitability of a new polymer family has been investigated as capillary coatings for the analysis of peptides and basic proteins by CE. This polymer family has been designed to minimize or completely prevent protein-capillary wall interactions and to modify the EOF. These coating materials are linear polymeric chains bearing as side cationizable moiety a dentronic triamine derived from N,N,N',N'-tetraethyldiethylenetriamine (TEDETA), which is linked to the backbone through a spacer (unit labeled as TEDETAMA). Four different polymers have been prepared and evaluated: a homopolymer which comprised only of those cationizable repetitive units of TEDETAMA, and three copolymers that randomly incorporate TEDETAMA together with neutral hydrosoluble units of N-(2-hydroxypropyl) methacrylamide (HPMA) at different molar percentages (25:75, 50:50 and 75:25). It has been demonstrated that the composition of the copolymers influences the EOF and therefore the separation of the investigated biopolymers. Among the novel polymers studied, poly-(TEDETAMA-co-HPMA) 50:50 copolymer was successfully applied as coating material of the inner capillary surface in CE-UV and CE-MS, providing EOF reversing together with fast and efficient baseline separation of peptides and basic proteins. Finally, the feasibility of the polymer-coated capillary was shown through the analysis of lysozyme in a cheese sample.

The role of direct high-resolution mass spectrometry in foodomics.

Foodomics has been defined as a global discipline in which advanced analytical techniques and bioinformatics are combined to address different questions in food science and nutrition. There is a growing number of works on the development and application of non-targeted omics methods in foodomics, which reflects that this emerging discipline is already considered by the scientific community to be a valuable approach to assess food safety, quality, and traceability as well as for the study of the links between food and health. As a result, there is a clear need for more rapid, high-throughput MS approaches for developing and applying non-targeted studies. Nowadays, direct MS analysis is one of the main choices to achieve high throughput, generating a set of information from the largest possible number of samples in a fast and straightforward way. The use of high- and ultrahigh-resolution MS greatly improves the analytical performance and offers a good combination of selectivity and sensitivity. By using a range of methods for direct sample introduction/desorption/ionization, high-throughput and non-target analysis of a variety of samples can be obtained in a few seconds by HRMS analysis. In this review, a general overview is presented of the main characteristics of direct HRMS-based approaches and their principal applications in foodomics.

Integration of pressurized liquid extraction and in-line solid-phase extraction to simultaneously extract and concentrate phenolic compounds from lemon peel (Citrus limon L.).

This work aimed to develop an integrated method to extract and fractionate phenolic compounds from lemon (Citrus limon L.) peel by in-line coupling pressurized liquid extraction and solid-phase extraction (PLE-SPE). The effect of the adsorbent used in the SPE (Sepra™ C18-E, Sepra™ NH2, and PoraPak Rxn), the combination of organic extraction-elution solvents (water-ethanol and water-ethyl lactate), extraction temperature (40-80 °C), and extraction water pH (4.0, 6.0, and 7.0) were the investigated variables. The highest yield and separation degree were observed using Sepra™ C18-E and the water-ethanol combination as the extraction solvent-eluent. Higher temperatures led to higher yields but negatively affected the retention of less polar compounds, hesperidin, and narirutin during the extraction step. The lower pH improved the yield of most evaluated compounds; however, it did not improve the adsorbent retention at high temperatures. Thus, the developed PLE-SPE method resulted in higher extraction yields from lemon peel, especially total less polar compounds (20.2100 ± 0,0050 mg/g) and hesperidin (12.8120 ± 0.0006 mg/g) and allowed the separation of polar compounds and less polar compounds in distinct extract fractions. Besides, PLE-SPE resulted in higher yields compared to other extraction methods. The integrated approach allowed obtaining extract fractions with different chemical composition through an environmentally friendly procedure. The research outcomes may be helpful for natural products chemistry, and industrial processes.

Targeted analysis of eicosanoids derived from cytochrome P450 pathway by high-resolution multiple-reaction monitoring mass spectrometry.

Cytochrome P450 (CYP450) pathway is one of the critical enzymatic via eicosanoid biosynthesis. Nevertheless, their metabolites are far less explored. This pathway plays a crucial role in converting arachidonic acid to hydroxyeicosatetraenoic (HETEs), epoxyeicosatrienoic (EETs), dihydroxyeicosatetraenoic acids (DiHETEs), and dihydroxyeicosatrienoic acids (DiHETrEs), which mediate several physiological and pathological functions. However, CYP450-derived eicosanoids are structurally complex, making those analyses a challenge in lipidomics studies. Herein, a high-resolution multiple-reaction monitoring (MRMHR ) method has been proposed as a powerful tool for the simultaneous analysis of CYP450-eicosanoids on different biological samples. The developed liquid chromatography (LC)-MRMHR method was partially validated according to the Food and Drug Administration (FDA) criteria, demonstrating adequate specificity, linearity, precision, and accuracy. Besides, several biological samples were analyzed to guarantee the feasibility of the method. The proposed strategy may improve the understanding of CYP450-derived eicosanoids in biological systems, which could be fundamental to reveal new aspects of those in physiologic and pathologic conditions.

Susceptibility and resistance of Sporothrix brasiliensis to branded and compounded itraconazole formulations.

Itraconazole is the first drug of choice for the treatment of sporotrichosis and it is available at different concentrations for veterinary patients. However, therapeutic failure has been reported, limiting clinical treatment. This study evaluated the in vitro efficacy of brand-name and compounded itraconazole formulations against Sporothrix brasiliensis and estimated the itraconazole content in each tested formulation. Oral capsules were acquired from two brand-name products for human (H-IND) and veterinary (V-IND) uses, and three from compounding pharmacies in Pelotas, RS, for human (H-COMP1/H-COMP2) and veterinary (V-COMP) uses. Capsule purity was analyzed by liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS). Antifungal activity was determined against 29 Sporothrix brasiliensis by the M38-A2 guideline of CLSI. H-IND/H-COMP1/H-COMP2 had high efficacy against S. brasiliensis (approximately 70% of total isolated susceptible), V-COMP showed moderate efficacy (51.7%), and V-IND was the least effective formulation (37.9%). Thirty-four percent of the total isolates were resistant to all formulations. Furthermore, itraconazole content did not match the concentration indicated by the manufacturers, ranging from 387.70 to 7.81 µg/mg (H-COMP2 > V-COMP > H-IND > H-COMP1 > V-IND). Therefore, it is possible that the formulations showed different in vitro efficacy due to the difference in their itraconazole contents. Given the emergence of antifungal resistance for all formulations, the choice product to be used must follow susceptibility testing. Stringent quality control measures are recommended for product manufactures to assure drug content uniformity.

Fluidized-bed drying of black rice grains: Impact on cooking properties, in vitro starch digestibility, and bioaccessibility of phenolic compounds.

This study evaluated the influence of different fluidized-bed drying temperatures (20, 60, and 100 °C) on the cooking properties, in vitro starch digestibility, and phenolic bioaccessibility of black rice. The results indicated that the formation of fissures in the grains dried at or above 60 °C reduced the physical integrity of the grains after cooking, increasing the starch digestion and the rehydration ratio, and reduced the cooking time, the hardness and adhesiveness. Due to the higher digestibility of grains dried at higher temperatures, an increase in the bioaccessibility of ferulic acid, which was previously associated with the polysaccharides, was observed. Caffeic acid was the only phenolic compound whose levels decreased when the drying temperature increased. At high temperatures and in the gastric phase, cyanidin chalcones were formed due to the deglycosylation of cyanidin-3-O-glucoside. PRACTICAL APPLICATION: The results of this study provide information to the food industry about the effects of different fluidized-bed drying temperatures on the rice structure after cooking and that, consequently, affect the availability of bioactive compounds after digestion and the glycemic index of black rice.

Cake of brown, black and red rice: Influence of transglutaminase on technological properties, in vitro starch digestibility and phenolic compounds.

This study aimed to evaluate the influence of transglutaminase addition on the technological properties and in vitro starch digestibility of gluten-free cakes of brown, black, and red rice, as well as the effect of baking on the content of phenolic compounds. Transglutaminase addition exerted significant effect in the technological properties only in the brown rice cake, resulting in a decrease in crumb firmness and an increase in the specific volume. Red rice cakes treated with transglutaminase presented a lower glucose release rate (k) compared to cakes without the enzyme. Cakes from pigmented rice varieties had lower crumb firmness and k values than brown rice cakes. Baking reduced only the contents of ferulic and p-coumaric acids and significantly increased the extractability of hydroxybenzoic, caffeic, caftaric, and protocatechuic acids. However, the addition of the enzyme resulted in a slight decrease in the total phenolic content of the cakes.

Metabolomics study of early metabolic changes in hepatic HepaRG cells in response to rosemary diterpenes exposure.

Rosemary diterpenes have demonstrated diverse biological activities, such as anti-cancer, antiinflammatory, as well as other beneficial effects against neurological and metabolic disorders. In particular, carnosic acid (CA), carnosol (CS) and rosmanol (RS) diterpenes have shown interesting results on anti-cancer activity. However, little is known about the toxic effects of rosemary diterpenes at the concentrations needed to exert their antiproliferative effect on cancer cells. In our study, CA, CS and RS exhibited a concentration-dependent effect on cell viability of two human colon cancer cell lines (HT-29 and HCT116) after 24 h exposure. HT-29 cell line was more resistant to the inhibitory effect of the three diterpenes than HCT116 cell line. Among the three diterpenes, RS exerted the strongest effect in both cell lines. To investigate the hepatotoxicity of CA, CS and RS, undifferentiated and differentiated HepaRG cells were exposed to increasing concentrations of the diterpenes (from 10 to 100 µM). Differentiated cells were found to be more resistant to the toxic activity of the three diterpenes than undifferentiated HepaRG, probably related to a higher detoxifying function of differentiated HepaRG cells compared with the undifferentiated cells. The metabolic profiles of differentiated HepaRG cells in response to CA, CS and RS were examined to determine biochemical alterations and deepen the study of the effects of rosemary phenolic diterpenes at molecular level. A multiplatform metabolomics study based on liquid- and gas-chromatography hyphenated to high resolution mass spectrometry revealed that rosemary diterpenes exerted different effects when HepaRG cells were treated with the same concentration of each diterpene. RS revealed a greater metabolome alteration followed by CS and CA, in agreement with their observed cytotoxicity. Metabolomics provided valuable information about early events in the metabolic profiles after the treatment with the investigated diterpenes from rosemary.

Background correction in separation techniques hyphenated to high-resolution mass spectrometry - Thorough correction with mass spectrometry scans recorded as profile spectra.

Separation techniques hyphenated with high-resolution mass spectrometry have been a true revolution in analytical separation techniques. Such instruments not only provide unmatched resolution, but they also allow measuring the peaks accurate masses that permit identifying monoisotopic formulae. However, data files can be large, with a major contribution from background noise and background ions. Such unnecessary contribution to the overall signal can hide important features as well as decrease the accuracy of the centroid determination, especially with minor features. Thus, noise and baseline correction can be a valuable pre-processing step. The methodology that is described here, unlike any other approach, is used to correct the original dataset with the MS scans recorded as profiles spectrum. Using urine metabolic studies as examples, we demonstrate that this thorough correction reduces the data complexity by more than 90%. Such correction not only permits an improved visualisation of secondary peaks in the chromatographic domain, but it also facilitates the complete assignment of each MS scan which is invaluable to detect possible comigration/coeluting species.

Anionic metabolite profiling by capillary electrophoresis-mass spectrometry using a noncovalent polymeric coating. Orange juice and wine as case studies.

In several metabolomic studies, it has already been demonstrated that capillary electrophoresis hyphenated to mass spectrometry (CE-MS) can detect an important group of highly polar and ionized metabolites that are overseen by techniques such as NMR, LC-MS and GC-MS, providing complementary information. In this work, we present a strategy for anionic metabolite profiling by CE-MS using a cationic capillary coating. The polymer, abbreviated as PTH, is composed of a poly-(N,N,N',N'-tetraethyldiethylenetriamine, N-(2-hydroxypropyl) methacrylamide, TEDETAMA-co-HPMA (50:50) copolymer. A CE-MS method based on PTH-coating was optimized for the analysis of a group of 16 standard anionic metabolites. Separation was achieved within 12min, with high separation efficiency (up to 92,000 theoretical plates per meter), and good repeatability, namely, relative standard deviation values for migration times and peak areas were below 0.2 and 2.1%, respectively. The optimized method allowed the detection of 87 metabolites in orange juice and 142 metabolites in red wine, demonstrating the good possibilities of this strategy for metabolomic applications.

Algorithm for comprehensive analysis of datasets from hyphenated high resolution mass spectrometric techniques using single ion profiles and cluster analysis.

Various algorithms have been developed to improve the quantity and quality of information that can be extracted from complex datasets obtained using hyphenated mass spectrometric techniques. While different approaches are possible, the key step often consists in arranging the data into a large series of profiles known as extracted ion profiles. Those profiles, similar to mono-dimensional separation profiles, are then processed to detect potential chromatographic peaks. This allows extracting from the dataset a large number of peaks that are characteristics of the compounds that have been separated. However, with mass spectrometry (MS) detection, the response is usually a complex signal whose pattern depends on the analyte, the MS instrument and the ionization method. When converted to ionic profiles, a single separated analyte will have multiple images at different m/z range. In this manuscript we present a hierarchical agglomerative clustering algorithm to group profiles with very similar feature. Each group aims to contain all profiles that are due to the transport and monitoring of a single analyte. Clustering results are then used to generate a 2 dimensional representation, called clusters plot, which allows an in-depth analysis of the MS dataset including the visualization of poorly separated compounds even when their intensity differs by more than two orders of magnitude. The usefulness of this new approach has been validated with data from capillary electrophoresis time of flight mass spectrometry hyphenated via an electrospray ionization. Using a mixture of 17 low molecular endogenous compounds it was verified that ionic profiles belonging to each compounds were correctly clustered even with very low degree of separation (R below 0.03). The approach was also validated using a urine sample. While with the total ion profile 15 peaks could be distinguished, 70 clusters were obtained allowing a much thorough analysis. In this particular example, the total computing took less than 10 min.

Capillary Electrophoresis in Food and Foodomics.

Quality and safety assessment as well as the evaluation of other nutritional and functional properties of foods imply the use of robust, efficient, sensitive, and cost-effective analytical methodologies. Among analytical technologies used in the fields of food analysis and foodomics, capillary electrophoresis (CE) has generated great interest for the analyses of a large number of compounds due to its high separation efficiency, extremely small sample and reagent requirements, and rapid analysis. The introductory section of this chapter provides an overview of the recent applications of capillary electrophoresis (CE) in food analysis and foodomics. Relevant reviews and research articles on these topics are tabulated including papers published in the period 2011-2014. In addition, to illustrate the great capabilities of CE in foodomics the chapter describes the main experimental points to be taken into consideration for a metabolomic study of the antiproliferative effect of carnosic acid (a natural diterpene found in rosemary) against HT-29 human colon cancer cells.