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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Detection of Novel Cytotoxic Imidazole Alkaloids in Tomato Products by LC-MS/MS.

Imidazole alkaloids represent a rather small group of alkaloids and are assumed not to be of significance to the human food chain so far. In this study, novel imidazole alkaloids occurring in tomato products were synthesized and structurally characterized by nuclear-magnetic-resonance spectroscopy and high-resolution mass spectrometry. These alkaloids are amides of either histidinol or histamine and short-chain fatty acids and could be quantitated in all of the 28 analyzed tomato products using a newly developed and validated LC-MS/MS-based method. Levels ranged from approximately 5 µg/kg to almost 3 mg/kg in the analyzed tomato products, and N-caprylhistidinol and its putative isomer were shown to occur in the highest amounts. These imidazole alkaloids are thus regularly ingested in considerable amounts as part of the human diet. In the course of evaluating their effects on the viability of HT-29 cells, all compounds were shown to significantly reduce cell viability starting at concentrations of 100 µM.

Large-Scale Screening of Foods for Glucose-Derived ß-Carboline Alkaloids by Stable Isotope Dilution LC-MS/MS.

The occurrence of glucose-derived ß-carboline alkaloids tangutorid E (Tan E) and tangutorid F (Tan F) as well as their dehydroxy-derivatives (DH-Tan E/F) was investigated in a broad variety of foodstuffs by LC-MS/MS-based stable isotope dilution analysis (SIDA). For that purpose, the target compounds and their 13C6-stable isotope-labeled analogues were synthesized from l-tryptophan and (13C6-)d-glucose and used to develop a rapid LC-MS/MS-SIDA method. After validation for several food matrices, the method was applied to the analysis of these ß-carbolines in 80 food items. Quantitative amounts were detected in 46.3, 50.0, and 42.5% of the samples regarding Tan E, Tan F, and DH-Tan E/F, respectively, with corresponding ranges of 0.01-6.75, 0.01-5.07, and 0.01-0.75 mg/kg; the highest amounts were found in processed tomato products. A combination of the obtained occurrence data in foods with average food consumption data led to the calculation of rough estimates for the chronic daily intake of those alkaloids, yielding values of 0.44, 0.36, and 0.13 µg/kg body weight/day for Tan E, Tan F, and DH-Tan E/F, respectively. Evidently, the consumption of processed tomato-based products accounts for the majority of the total daily intake of the investigated ß-carbolines; the potential bioactivities of Tan E, Tan F, and DH-Tan E/F have yet to be investigated.

Exploring Secondary Metabolite Profiles of Stachybotrys spp. by LC-MS/MS.

The genus Stachybotrys produces a broad diversity of secondary metabolites, including macrocyclic trichothecenes, atranones, and phenylspirodrimanes. Although the class of the phenylspirodrimanes is the major one and consists of a multitude of metabolites bearing various structural modifications, few investigations have been carried out. Thus, the presented study deals with the quantitative determination of several secondary metabolites produced by distinct Stachybotrys species for comparison of their metabolite profiles. For that purpose, 15 of the primarily produced secondary metabolites were isolated from fungal cultures and structurally characterized in order to be used as analytical standards for the development of an LC-MS/MS multimethod. The developed method was applied to the analysis of micro-scale extracts from 5 different Stachybotrys strains, which were cultured on different media. In that process, spontaneous dialdehyde/lactone isomerization was observed for some of the isolated secondary metabolites, and novel stachybotrychromenes were quantitatively investigated for the first time. The metabolite profiles of Stachybotrys species are considerably influenced by time of growth and substrate availability, as well as the individual biosynthetic potential of the respective species. Regarding the reported adverse effects associated with Stachybotrys growth in building environments, combinatory effects of the investigated secondary metabolites should be addressed and the role of the phenylspirodrimanes re-evaluated in future research.

Interaction of the mycotoxin metabolite dihydrocitrinone with serum albumin.

Citrinin (CIT) is a nephrotoxic mycotoxin produced by Penicillium, Monascus, and Aspergillus species. CIT appears as a contaminant in cereals, cereal-based products, fruits, nuts, and spices. During the biotransformation of CIT, its major urinary metabolite dihydrocitrinone (DHC) is formed. Albumin interacts with several compounds (including mycotoxins) affecting their tissue distribution and elimination. CIT-albumin interaction is known; however, the complex formation of DHC with albumin has not been reported previously. In this study, we aimed to investigate the interaction of DHC with albumin, employing fluorescence spectroscopy, circular dichroism, and molecular modeling studies. Furthermore, species differences and thermodynamics of the interaction as well as the effects of albumin on the acute in vitro toxicity of DHC and CIT were also tested. Our main observations/conclusions are as follows: (1) Fluorescence signal of DHC is strongly enhanced by albumin. (2) Formation of DHC-albumin complexes is supported by both fluorescence spectroscopic and circular dichroism studies. (3) DHC forms similarly stable complexes with human albumin (K~105 L/mol) as CIT. (4) DHC-albumin interaction did not show significant species differences (tested with human, bovine, porcine, and rat albumins). (5) Based on modeling studies and investigations with site markers, DHC occupies the Heme binding site (subdomain IB) on human albumin. (6) The presence of albumin significantly decreased the acute in vitro cytotoxic effects of both DHC and CIT on MDCK cell line.

Stachybotrychromenes A-C: novel cytotoxic meroterpenoids from Stachybotrys sp.

In the course of gaining new insights into the secondary metabolite profile of various Stachybotrys strains, in particular concerning triprenyl phenol-like compounds, so far, unknown metabolites with analogous structural features were discovered. Three novel meroterpenoids containing a chromene ring moiety, namely stachybotrychromenes A-C, were isolated from solid culture of the filamentous fungus Stachybotrys chartarum DSMZ 12880 (chemotype S). Their structures were elucidated by means of comprehensive spectroscopic analysis (1D and 2D NMR, ESI-HRMS, and CD) as well as by comparison with spectroscopic data of structural analogues described in literature. Stachybotrychromenes A and B exhibited moderate cytotoxic effects on HepG2 cells after 24 h with corresponding IC50 values of 73.7 and 28.2 µM, respectively. Stachybotrychromene C showed no significant cytotoxic activity up to 100 µM. Moreover, it is noteworthy that stachybotrychromenes A-C are produced not only by S. chartarum chemotype S but also S. chartarum chemotype A and Stachybotrys chlorohalonata.

Large-scale total synthesis of 13C3-labeled citrinin and its metabolite dihydrocitrinone.

The analysis of the nephrotoxic mycotoxin citrinin in food, feed, and physiological samples is still challenging. Nowadays, liquid chromatography coupled with mass spectrometry is the method of choice for achieving low limits of detection. But matrix effects can present impairments for this method. Stable isotope dilution analysis can prevent some of these problems. Therefore, a stable isotopically labeled standard of citrinin for use in stable isotope dilution analysis was synthesized on large scale. The improved diastereoselective total synthetic strategy offered the possibility to introduce three 13C-labels in two steps by ortho-toluate anion chemistry. This led to a mass difference of 3 Da, sufficient for preventing spectral overlap. Additionally, a stable isotopically labeled form of dihydrocitrinone, the main urinary metabolite of citrinin, was synthesized with the same mass difference. This was achieved by a sequence of cyclisation, oxidation, deprotection, and carboxylation reactions starting from a protected intermediate of the labeled citrinin synthesis. Thus, this method also offers a complete way to synthesize dihydrocitrinone from citrinin on large scale.