An arrhythmogenic metabolite in atrial fibrillation.

BACKGROUND: Long-chain acyl-carnitines (ACs) are potential arrhythmogenic metabolites. Their role in atrial fibrillation (AF) remains incompletely understood. Using a systems medicine approach, we assessed the contribution of C18:1AC to AF by analysing its in vitro effects on cardiac electrophysiology and metabolism, and translated our findings into the human setting. METHODS AND RESULTS: Human iPSC-derived engineered heart tissue was exposed to C18:1AC. A biphasic effect on contractile force was observed: short exposure enhanced contractile force, but elicited spontaneous contractions and impaired Ca2+ handling. Continuous exposure provoked an impairment of contractile force. In human atrial mitochondria from AF individuals, C18:1AC inhibited respiration. In a population-based cohort as well as a cohort of patients, high C18:1AC serum concentrations were associated with the incidence and prevalence of AF. CONCLUSION: Our data provide evidence for an arrhythmogenic potential of the metabolite C18:1AC. The metabolite interferes with mitochondrial metabolism, thereby contributing to contractile dysfunction and shows predictive potential as novel circulating biomarker for risk of AF.

Identification of potential human urinary biomarkers for tomato juice intake by mass spectrometry-based metabolomics.

PURPOSE: Dietary biomarkers allow the accurate and objective determination of the dietary intake of humans and can thus be valuable for investigating the relation between consumption of foods and biochemical as well as physiological responses. The objective of this study was the identification of potential urinary biomarkers for consumption of tomato juice. METHODS: In the course of a dietary intervention study, the human urine metabolome of a study cohort was compared between a tomato-free diet and after intake of tomato juice by application of an LC-HRMS-based metabolomics approach. The data acquisition was achieved using an orbitrap mass spectrometer, followed by multistage data processing and univariate as well as multivariate statistical analysis to identify discriminating features. RESULTS: Statistical analysis revealed several unique features detectable after tomato juice intake. The most discriminating markers were putatively identified as hydroxylated and sulfonated metabolites of esculeogenin B, aglycone of the steroidal glycoalkaloid esculeoside B recently found in tomato juice. Furthermore, the ß-carboline alkaloids tangutorid E and F and glucuronidated derivatives thereof were identified in urine. CONCLUSIONS: Steroidal glycoalkaloids in tomato juice are cleaved after ingestion, and hydroxylated and sulfonated metabolites of their aglycones might serve as urinary biomarkers for tomato juice intake. Similarly, ß-carboline alkaloids and glucuronidated derivatives were identified as potential urinary biomarkers. Both the aglycones of the steroidal alkaloids and the ß-carboline alkaloids might exhibit biological activities worth investigating.

Artificial vs natural Stachybotrys infestation-Comparison of mycotoxin production on various building materials.

The genus Stachybotrys belongs to filamentous fungi found in indoor environment, mostly on cellulose-rich substrates after water-damage. The major purpose of this study was to investigate the influence of different building materials in case of mold infestation on the mycotoxin production of Stachybotrys species. Fifteen Stachybotrys mycotoxins including satratoxins, phenylspirodrimanes, and recently discovered stachybotrychromenes were in the focus of the investigations. Artificial and natural infestations were compared to determine whether environmental factors, for example, time of growth, temperature, humidity, and material additives have an influence on the observed mycotoxin profiles. It turned out that mycotoxin profiles from Stachybotrys spp. on building materials can be influenced by cellulose, paints, and paste of the materials. The total toxin levels of artificially and naturally contaminated gypsum board samples ranged up to 30 µg/cm2 , whereas wallpaper samples showed total toxin levels in the range of 20-66 µg/cm2 . A naturally infested sample disclosed the conversion of the dialdehyde components to the corresponding lactone isomers under the influence of light.

A Fungal N-Dimethylallyltryptophan Metabolite from Fusarium fujikuroi.

The range of secondary metabolites (SMs) produced by the rice pathogen Fusarium fujikuroi is quite broad. Several polyketides, nonribosomal peptides and terpenes have been identified. However, no products of dimethylallyltryptophan synthases (DMATSs) have been elucidated, although two putative DMATS genes are present in the F. fujikuroi genome. In this study, the in vivo product derived from one of the DMATSs (DMATS1, FFUJ_09179) was identified with the help of the software MZmine 2. Detailed structure elucidation showed that this metabolite is a reversely N-prenylated tryptophan with a rare form of prenylation. Further identified products probably resulted from side reactions of DMATS1. The genes adjacent to DMATS1 were analyzed; this showed no influence on the biosynthesis of the product.

Electrochemical simulation of metabolic reactions of the secondary fungal metabolites alternariol and alternariol methyl ether.

Mycotoxins are secondary plant metabolites that have been found to cause severe diseases in humans and livestock. Exposure can take place on a daily basis since mycotoxins can be found not only in food, animal food, and dietary supplements but also in materials used in buildings. For this work, the Alternaria toxins alternariol (AOH) and alternariol methyl ether (AME) are chosen as representatives for this relevant compound class and are investigated regarding their oxidative phase I metabolism using a combination of electrochemical (EC) oxidation and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). This previously established method has been proven to be a valuable tool for the electrochemical simulation of certain phase I metabolic reactions. A comparison of the electrochemically generated products with those formed during microsomal incubation demonstrates the potential of the method for the successful prediction of the main phase I metabolic reactions of mycotoxins. It can thus find use as a supportive method in the elucidation of the metabolic pathways of various mycotoxins.

Evaluation of Food Intake Biomarkers for Red Bell Peppers in Human Urine Based on HPLC-MS/MS Analysis.

SCOPE: The validation of dietary biomarkers is essential for the use in objective and quantitative assessment of the human dietary intake. In this study, the urinary excretion of previously identified potential biomarkers after intake of red bell peppers is analyzed. METHODS AND RESULTS: The urine samples obtained after a two-phase dietary intervention study in which 14 volunteers participated are quantitatively analyzed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) after an extensive validation. In the first phase, the volunteers abstain completely from bell peppers and paprika products (control group) and in the second phase, the volunteers consume a defined amount of fresh red bell peppers (case group). After analysis, all potential biomarkers show high dispersions of their concentration, indicating interindividual differences. The glucuronidated apocarotenoid (compound 1), which probably resulted from the main carotenoids of red Capsicum fruits, shows a rapid urinary excretion. The other glucuronidated metabolites (compounds 2-8), described as potential derivatives of capsianosides from Capsicum, show a slightly delayed but longer urinary excretion. CONCLUSIONS: A correlation between an intake of red bell pepper and the urinary excretion of recently described potential biomarkers is observed. Due to large interindividual differences, it is reasonable to assume that at least the qualitative detection of the consumption of bell peppers and possibly all Capsicum fruits is feasible.

Human Intervention Study: Alkylresorcinol Metabolites as Potential Biomarkers for Grain Intake and the Occurrence of Alkylresorcinols in Commonly Consumed Foods in the German Retail Sector.

Grains are one of the primary nutrients and are associated with many health benefits. To reflect the intake of grain-based products, two promising potential biomarkers are alkylresorcinol (AR) metabolites 3,5-dihydroxybenzoic acid (3,5-DHBA) and 3-(3,5-dihydroxyphenyl)-propanoic acid (3,5-DHPPA). The aim of this study was to validate the occurrence of AR in food samples and investigate the suitability of their metabolites as potential biomarkers in human intervention studies. In the first step, the AR content in different grain products from the German retail sector was analyzed by GC-MS. ARs were found in higher concentrations in whole grain products and in moderate contents in refined grains and quinoa. Based on these results, human intervention studies were performed in the next step, and the AR metabolites 3,5-DHBA and 3,5-DHPPA were analyzed by LC-MS/MS in urine samples. The intake of only whole grain products leads to an increasing level of both potential biomarkers, while a refined grain diet shows decreasing levels of the AR metabolites. The excretion of 3,5-DHBA after a whole grain-rich diet differs significantly (p = 0.043) from no grain intake.

Micro-scale screening of genetically modified Fusarium fujikuroi strain extends the apicidin family.

Apicidins are a class of naturally occurring cyclic tetrapeptides produced by few strains within the Fusarium genus. These secondary metabolites have gained significant attention due to their antiprotozoal activity through HDAC inhibition, thereby highlighting their potential for the treatment of malaria. Predominantly, apicidins have been isolated from Fusarium semitectum, offering a deep insight into the biosynthetic pathway responsible for their formation. A similar biosynthetic gene cluster has also been identified in the rice pathogenic fungus F. fujikuroi, leading the discovery of three additional apicidins through genetic manipulation. Routine mass spectrometric screening of these compound-producing strains revealed another metabolite structurally related to previously studied apicidins. By optimizing culture conditions and developing an effective isolation method, we obtained a highly pure substance, whose chemical structure was fully elucidated using NMR and HRMS fragmentation. Further studies were conducted to determine cytotoxicity, antimalarial activity, and HDAC inhibitory activity of this new secondary metabolite alongside the previously known apicidins. This work not only expands the apicidin class with a new member but also provides extensive insights and comparative analysis of apicidin-like substances produced by F. fujikuroi.

Germination of cereal grains as a way to improve the nutritional value: a review.

Whole grain cereals have been found to be a good source of nutritionally valuable substances, such as antioxidants, minerals, vitamins, and dietary fiber. A wide range of these compounds is affected by germination. While some compounds, such as beta-glucans are degraded, others, like vitamins can be increased by means of malting. Therefore, germination and malting of cereals is a way not only to produce fermentable extract for the brewing and distilling industries, but can also be a way to produce ingredients enriched with health promoting compounds. Malt extracts have also been shown to be good substrates for the growth and application of probiotic bacteria.

In Vitro Metabolism of Phenylspirodrimanes Derived from the Indoor Fungus Stachybotrys.

Fungi belonging to the genus Stachybotrys are frequently detected in water-damaged indoor environments, and a potential correlation between emerging health problems of inhabitants of affected housing and the fungi is controversially discussed. Secondary metabolites (i.e., mycotoxins) produced by Stachybotrys, such as the highly toxic macrocyclic trichothecenes (MCTs), are of potential concern to human health. The present study, however, focused on the potential effects of the more broadly and abundantly formed group of phenylspirodrimanes (PSDs). The phase I and II metabolism of four structurally different PSDs were investigated in vitro using hepatic models in combination with high-performance liquid chromatography high-resolution mass spectrometry (HPLC-HRMS) analysis. In addition to metabolite detection by HRMS, isolation and structure elucidation by nuclear magnetic resonance spectroscopy (NMR) was part of the conducted study as well.

Re-examination of the anion derivatives of isoflavones by radical fragmentation in negative electrospray ionization tandem mass spectrometry: experimental and computational studies.

This paper reports theoretical and experimental studies of gas-phase fragmentation reactions of four naturally occurring isoflavones. The samples were analyzed in negative ion mode by direct infusion in ESI-QqQ, ESI-QqTOF and ESI-Orbitrap systems. The MS/MS and MS(n) spectra are in agreement with the fragmentation proposals and high-resolution analyses have confirmed the formulae for each ion observed. As expected, compounds with methoxyl aromatic substitution have showed a radical elimination of •CH(3) as the main fragmentation pathway. A second radical loss (•H) occurs as previously observed for compounds which exhibit a previous homolytic •CH(3) cleavage (radical anion) and involves radical resonance to stabilize the anion formed. However, in this study we suggest another mechanism for the formation of the main ions, on the basis of the enthalpies for each species. Compounds without methoxy substituent dissociate at the highest energies and exhibit the deprotonated molecule as the most intense ion. Finally, energy-resolved experiments were carried out to give more details about the gas-phase dissociation reaction of the isoflavones and the results are in agreement with the theoretical approaches.

Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium.

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.

Identification of Potential Urinary Biomarkers for Bell Pepper Intake by HPLC-HRMS-Based Metabolomics and Structure Elucidation by NMR.

Dietary biomarkers show great promise for objectively assessing the food intake in humans. In this study, potential urinary biomarkers for red bell pepper intake were identified based on a dietary intervention study and a comprehensive metabolomics approach. Spot urine samples from 14 volunteers were collected in the two phases of the study (control phase: abstaining from any bell pepper/paprika products; case phase: consumption of a defined amount of fresh red bell pepper and abstaining from any further bell pepper/paprika products) and analyzed by high-performance liquid chromatography high-resolution mass spectrometry (HPLC-HRMS). Comparison of the obtained metabolomics data using statistical analysis revealed that the respective urine metabolomes differ significantly, which was attributable to the bell pepper intake. Some of the most discriminating metabolites were selected and isolated from human urine for unequivocal structure elucidation by nuclear magnetic resonance (NMR) spectroscopy. Herein, seven novel glucuronidated metabolites most likely derived from capsanthin and capsianosides were identified, implying their potential application as dietary biomarkers for the entire Capsicum genus.

Characterization of Oligomeric Proanthocyanidin-Enriched Fractions from Aronia melanocarpa (Michx.) Elliott via High-Resolution Mass Spectrometry and Investigations on Their Inhibitory Potential on Human Topoisomerases.

Aronia melanocarpa (MICHX.) ELLIOTT, which belongs to the Rosaceae family, has increasingly come into focus of research due to the high content of polyphenols. In addition to antioxidative properties, further health-promoting effects of these polyphenols are still of interest. Especially, the proanthocyanidins offer thereby huge opportunities due to their high structural heterogeneity. Therefore, the present study focuses on the topoisomerase inhibiting effects of oligomeric proanthocyanidins (PACs), which are potentially depended on their degree of polymerization. The investigated PACs isolated from Aronia berries were characterized by chromatographic techniques and liquid chromatography-high-resolution mass spectrometry. Four PAC enriched fractions were obtained from Aronia pomace containing 47 PACs with a degree of polymerization from three to six. Due to the low yield of hexamers, the potential inhibiting effects against human topoisomerase were investigated for the trimer to pentamer fractions. The relaxation and decatenation assays were performed to examine the inhibiting effect on topoisomerases under cell-free conditions. Moreover, rapid isolation of topoisomerase cleavage complexes in human colon carcinoma HT29 cells was performed to evaluate the effect on topoisomerases in a cell-based system. The fractions demonstrated inhibitory potential on topoisomerases I and II. In sum, an increasing effect strength depending on the degree of polymerization was shown.

Urinary Biomarkers for Orange Juice Consumption.

SCOPE: As orange juice belongs to one of the most consumed juices worldwide, a human study is performed to identify urinary biomarkers for the consumption of orange juice in order to differentiate between low, medium, and high intake. METHODS AND RESULTS: The 32 study participants abstained from citrus fruits, juices and products thereof, except for one portion of orange juice, for eight days. Throughout the study, spot urine samples are collected and quantitatively analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) regarding their content of several potential biomarkers for orange juice intake after enzymatic treatment with ß-glucuronidase. Proline betaine is determined as a long-term biomarker: based on its urinary excretion, orange juice consumption is traceable for at least 72 h after intake. Naringenin and hesperetin are identified as qualitative short-term biomarkers. Synephrine sulfate also showed a fast increase and decrease in a semi-quantitative approach. In the case of phloretin, no correlation between orange juice consumption and the urinary concentration is observed. CONCLUSION: Proline betaine is the most promising biomarker for orange juice consumption and allows to differentiate between low, medium, and high intake. Hesperetin and naringenin (as well as synephrine) are applicable as supporting biomarkers, whereas phloretin does not represent a reliable biomarker for orange juice consumption.

Ion identity molecular networking for mass spectrometry-based metabolomics in the GNPS environment.

Molecular networking connects mass spectra of molecules based on the similarity of their fragmentation patterns. However, during ionization, molecules commonly form multiple ion species with different fragmentation behavior. As a result, the fragmentation spectra of these ion species often remain unconnected in tandem mass spectrometry-based molecular networks, leading to redundant and disconnected sub-networks of the same compound classes. To overcome this bottleneck, we develop Ion Identity Molecular Networking (IIMN) that integrates chromatographic peak shape correlation analysis into molecular networks to connect and collapse different ion species of the same molecule. The new feature relationships improve network connectivity for structurally related molecules, can be used to reveal unknown ion-ligand complexes, enhance annotation within molecular networks, and facilitate the expansion of spectral reference libraries. IIMN is integrated into various open source feature finding tools and the GNPS environment. Moreover, IIMN-based spectral libraries with a broad coverage of ion species are publicly available.

Identification of Novel Iso-Esculeoside B from Tomato Fruits and LC-MS/MS-Based Food Screening for Major Dietary Steroidal Alkaloids Focused on Esculeosides.

Plants from the Solanaceae family are known to be sources of several nutritionally relevant steroidal glycoalkaloids (SGAs). With the aim of quantitatively investigating the occurrence of the main SGA from tomatoes, eggplants, and potatoes in various food samples and evaluating their relevance in the human diet, a rapid single-step extraction liquid chromatography-tandem mass spectrometry method was developed. Over the course of isolating several commercially unavailable SGAs from tomato products to use them as reference standards, a previously unknown derivative was detected, structurally characterized, and identified as a novel isomer of esculeoside B-1 and B-2. After validation of the method, 36 food items exclusively derived from Solanaceae plants were analyzed for their SGA contents and a specific occurrence of each alkaloid in tomato, eggplant, or potato products was revealed. This is the first study reporting quantitative data on the occurrence of esculeoside A, B-1, B-2, and iso-esculeoside B in tomato products obtained by using appropriate reference compounds rather than applying a semi-quantitative approach based on α-tomatine as a reference. Some of the analyzed tomato products contained the esculeosides in concentrations of >500 mg/kg, clearly indicating their relevance in the human diet and the need of investigating their potential bioactivities in the future.

Identification of a novel N-caprylhistamine-ß-glucoside from tomato fruits and LC-MS/MS-based food screening for imidazole alkaloids.

The present study aimed at the identification of novel imidazole alkaloids derived from histamine or histidinol and generally investigating the occurrence of suchlike alkaloids in a variety of foodstuffs. Herein, N-caprylhistamine was synthesized and the glucosidic derivative N-caprylhistamine-ß-glucoside was isolated from ripe tomato fruits and structurally characterized. The obtained reference standards were used for the extension of an established LC-MS/MS-based method for the quantitation of several imidazole alkaloids in tomato products. After validation for the two additional analytes and demonstrating the applicability of the method to nine other food matrices, 104 food items were screened for the occurrence of the described imidazole alkaloids. Remarkably, all of the investigated alkaloids were only quantifiable in tomato-based products and the occurrence of N-caprylhistamine and N-caprylhistamine-ß-glucoside was reported for the first time. These imidazole alkaloids could thus be applicable as specific intake biomarkers for tomatoes and their biological activities as well as metabolic fate should be investigated in future research.

Mass Spectrometry-Based Analysis of Urinary Biomarkers for Dietary Tomato Intake.

SCOPE: In this study, the applicability of several ß-carboline, imidazole, and steroidal alkaloids as biomarkers for tomato juice intake is evaluated. METHODS AND RESULTS: Over the course of a 2-week crossover dietary intervention study, 14 volunteers were given low and high doses of tomato juice after 3 days of avoiding tomato-based products. On the day of consumption and the following days, volunteers provided urine samples that were quantitatively analyzed by high-performance liquid chromatography-tandem mass spectrometry. Herein, glucose-derived ß-carboline alkaloids are determined as supporting, yet non-specific dietary biomarkers for tomato juice intake. Several imidazole alkaloids represent further biomarkers, which are shown to specifically indicate consumption of tomato juice for 24 h and partly >24 h. Additionally, steroidal alkaloids derived from esculeogenin B are determined to be specific biomarkers for tomato juice detectable for at least 48 h after consumption. The intake of low and high amounts of tomato juice is significantly distinguishable based on the urinary excretion of all determined biomarkers as well. CONCLUSIONS: The dietary intake of tomato juice is conclusively traceable based on urinary excretion of multiple ß-carboline, imidazole, and steroidal alkaloids, and can be determined for up to 48 h after consumption. Furthermore, different intake doses can clearly be distinguished based on their urinary excretion.

Metabolomics Study on Pathogenic and Non-pathogenic E. coli with Closely Related Genomes with a Focus on Yersiniabactin and Its Known and Novel Derivatives.

The Escherichia coli (E. coli) strains Nissle 1917 (EcN), 83972 and CFT073 are closely related but differ in their phenotypes and pathogenicity. The aim of this study was to compare the metabolome of these strains based on metabolomic data analysis of bacterial samples using liquid chromatography-high resolution mass spectrometry (LCHRMS). The strains were cultivated in minimum essential medium at 37 °C for 6 h. The sterilized culture supernatant was analyzed, followed by data processing to create feature lists, and statistical analysis to identify discriminating features in the metabolomes of the three strains. Metabolites were identified using the exact masses, isotope patterns, and fragmentation spectra. The results showed that the metabolome of EcN differs significantly from the metabolomes of E. coli 83972 and CFT073. Based on the analysis, yersiniabactin (Ybt), its metal complexes, and its known structural derivatives escherichelin and ulbactin B were identified as discriminating features; the latter has not been described for E. coli before. Additionally, novel Ytb derivatives were found and tentatively identified by LC-MS/HRMS. All these metabolites were determined in significantly higher levels in the metabolome of EcN compared to E. coli 83972, which may explain a large part of the observed differences of the metabolomes.