New perspectives on 'Breathomics': metabolomic profiling of non-volatile organic compounds in exhaled breath using DI-FT-ICR-MS.

Breath analysis offers tremendous potential for diagnostic approaches, since it allows for easy and non-invasive sample collection. "Breathomics" as one major research field comprehensively analyses the metabolomic profile of exhaled breath providing insights into various (patho)physiological processes. Recent research, however, primarily focuses on volatile compounds. This is the first study that evaluates the non-volatile organic compounds (nVOCs) in breath following an untargeted metabolomic approach. Herein, we developed an innovative method utilizing a filter-based device for metabolite extraction. Breath samples of 101 healthy volunteers (female n = 50) were analysed using DI-FT-ICR-MS and biostatistically evaluated. The characterisation of the non-volatile core breathome identified more than 1100 metabolites including various amino acids, organic and fatty acids and conjugates thereof, carbohydrates as well as diverse hydrophilic and lipophilic nVOCs. The data shows gender-specific differences in metabolic patterns with 570 significant metabolites. Male and female metabolomic profiles of breath were distinguished by a random forest approach with an out-of-bag error of 0.0099. Additionally, the study examines how oral contraceptives and various lifestyle factors, like alcohol consumption, affect the non-volatile breathome. In conclusion, the successful application of a filter-based device combined with metabolomics-analyses delineate a non-volatile breathprint laying the foundation for discovering clinical biomarkers in exhaled breath.

Reduced iron and cobalt levels in response to curcumin supplementation are not responsible for the prolonged larval development and do not affect the oxidative stress tolerance and polyamine status of D. melanogaster.

Recent reports indicated that the phytochemical curcumin possesses iron-chelating activity. Here, by employing the fruit fly Drosophila melanogaster, we conducted feeding studies supplementing curcumin or, as a control, the iron chelator bathophenanthroline (BPA). First, the absorption and further metabolization of dietary curcuminoids were proved by metabolomics analyses. Next, we found that 0.2% dietary curcumin, similar to BPA, lowered the iron but also the cobalt content, and to a lesser extent affected the manganese and zinc status. Supplementation during larval stages was required and sufficient for both compounds to elicit these alterations in adult animals. However, curcumin-induced retarded larval development was not attributable to the changed trace metal status. In addition, a reduction in the iron content of up to 70% by curcumin or BPA supplementation did not reduce heme-dependent catalase activity and tolerance toward H2 O2 in D. melanogaster. Moreover, polyamines were not influenced by curcumin treatment and decreased iron levels. This was confirmed for selected organs from 0.2% curcumin-treated mice, except for the spleen. Here, elevated spermidine level and concomitant upregulation of genes involved in polyamine production were associated with a putatively anemia-derived increased spleen mass. Our data underline that the metal-chelating property of curcumin needs to be considered in feeding studies.

Evaluating the Effect of Data Merging and Postacquisition Normalization on Statistical Analysis of Untargeted High-Resolution Mass Spectrometry Based Urinary Metabolomics Data.

Urine is one of the most widely used biofluids in metabolomic studies because it can be collected noninvasively and is available in large quantities. However, it shows large heterogeneity in sample concentration and consequently requires normalization to reduce unwanted variation and extract meaningful biological information. Biological samples like urine are commonly measured with electrospray ionization (ESI) coupled to a mass spectrometer, producing data sets for positive and negative modes. Combining these gives a more complete picture of the total metabolites present in a sample. However, the effect of this data merging on subsequent data analysis, especially in combination with normalization, has not yet been analyzed. To address this issue, we conducted a neutral comparison study to evaluate the performance of eight postacquisition normalization methods under different data merging procedures using 1029 urine samples from the Food Chain plus (FoCus) cohort. Samples were measured with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS). Normalization methods were evaluated by five criteria capturing the ability to remove sample concentration variation and preserve relevant biological information. Merging data after normalization was generally favorable for quality control (QC) sample similarity, sample classification, and feature selection for most of the tested normalization methods. Merging data after normalization and the usage of probabilistic quotient normalization (PQN) in a similar setting are generally recommended. Relying on a single analyte to capture sample concentration differences, like with postacquisition creatinine normalization, seems to be a less preferable approach, especially when data merging is applied.

Population divergence in heat and drought responses of a coastal plant: from metabolic phenotypes to plant morphology and growth.

Studying intraspecific variation in multistress responses is central for predicting and managing the population dynamics of wild plant species under rapid global change. Yet, it remains a challenging goal in this field to integrate knowledge on the complex biochemical underpinnings for the targeted 'non-model' species. Here, we studied divergence in combined drought and heat responses among Northern and Southern European populations of the dune plant Cakile maritima, by combining comprehensive plant phenotyping with metabolic profiling via FT-ICR-MS and UPLC-TQ-MS/MS. We observed pronounced constitutive divergence in growth phenology, leaf functional traits, and defence chemistry (glucosinolates and alkaloids) among population origins. Most importantly, the magnitude of growth reduction under drought was partly weaker in southern plants and associated with divergence in plastic growth responses (leaf abscission) and the modulation of primary and specialized metabolites with known central functions not only in plant abiotic but also in biotic stress responses. Our study indicates that divergent selection has shaped the constitutive and drought-/heat-induced expression of numerous morphological and biochemical functional traits to mediate higher abiotic stress resistance in southern Cakile populations, and highlights that metabolomics can be a powerful tool to explore the underlying mechanisms of local adaptation in 'non-model' species.

A Prospective Analysis of the Metyrapone Short Test Using Targeted and Untargeted Metabolomics.

INTRODUCTION: The present study aimed to prove the metyrapone short test in a day clinic to be suitable for examining the integrity of the hypothalamic-pituitary-adrenal (HPA) axis in patients with suspected secondary and tertiary adrenal insufficiency and to identify novel effector molecules in acute stress response. METHODS: 44 patients were prospectively enrolled. Based on stimulated 11-deoxycortisol levels, patients were divided into a physiological (11-deoxycortisol ≥70 µg/L) and a pathological (11-deoxycortisol <70 µg/L) response group. Clinical follow-up examination was performed for validation. Ultraperformance liquid chromatography tandem mass spectrometry and a Fourier-transform-ion-cyclotron-resonance-mass-spectrometry were used for targeted and untargeted steroid metabolomics. RESULTS: At baseline, lower levels of cortisone (42 vs. 50 nmol/L, p = 0.048) and 17-OH-progesterone (0.6 vs. 1.2 nmol/L, p = 0.041) were noted in the pathological response group. After metyrapone administration, the pathological response group exhibited significantly lower 11-deoxycortisol (39.0 vs. 94.2 µg/L, p < 0.001) and ACTH (49 vs. 113 pg/mL, p < 0.001) concentrations as well as altered upstream metabolites. Untargeted metabolomics identified a total of 76 metabolites to be significantly up- or downregulated by metyrapone. A significant increase of the bile acid glycochenodeoxycholic acid (GCDC, p < 0.01) was detected in both groups with an even stronger increase in the physiological response group. After a mean follow-up of 17.2 months, an 11-deoxycortisol cut-off of 70 µg/L showed a high diagnostic performance (sensitivity 100%, specificity 96%). CONCLUSION: The metyrapone short test is safe and feasible in a day clinic setting. The alterations of the bile acid GCDC indicate that the liver might be involved in the acute stress response of the HPA axis.

Microbiome and Metabolome Insights into the Role of the Gastrointestinal-Brain Axis in Parkinson's and Alzheimer's Disease: Unveiling Potential Therapeutic Targets.

Neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's disease (AD), the prevalence of which is rapidly rising due to an aging world population and westernization of lifestyles, are expected to put a strong socioeconomic burden on health systems worldwide. Clinical trials of therapies against PD and AD have only shown limited success so far. Therefore, research has extended its scope to a systems medicine point of view, with a particular focus on the gastrointestinal-brain axis as a potential main actor in disease development and progression. Microbiome and metabolome studies have already revealed important insights into disease mechanisms. Both the microbiome and metabolome can be easily manipulated by dietary and lifestyle interventions, and might thus offer novel, readily available therapeutic options to prevent the onset as well as the progression of PD and AD. This review summarizes our current knowledge on the interplay between microbiota, metabolites, and neurodegeneration along the gastrointestinal-brain axis. We further illustrate state-of-the art methods of microbiome and metabolome research as well as metabolic modeling that facilitate the identification of disease pathomechanisms. We conclude with therapeutic options to modulate microbiome composition to prevent or delay neurodegeneration and illustrate potential future research directions to fight PD and AD.

Microbiota independent effects of oligosaccharides on Caco-2 cells -A semi-targeted metabolomics approach using DI-FT-ICR-MS coupled with pathway enrichment analysis.

Milk oligosaccharides (MOS) and galactooligosaccharides (GOS) are associated with many benefits, including anti-microbial effects and immune-modulating properties. However, the cellular mechanisms of these are largely unknown. In this study, the effects of enriched GOS and MOS mixtures from caprine and bovine milk consisting mainly 6'-galactosyllactose, 3'-sialyllactose, and 6'-sialyllactose on Caco-2 cells were investigated, and the treatment-specific metabolomes were described. In the control, the cells were treated with a sugar mix consisting of one-third each of glucose, galactose and lactose. A local metabolomics workflow with pathway enrichment was established, which specifically addresses DI-FT-ICR-MS analyses and includes adaptations in terms of measurement technology and sample matrices. By including quality parameters, especially the isotope pattern, we increased the precision of annotation. The independence from online tools, the fast adaptability to changes in databases, and the specific adjustment to the measurement technology and biomaterial used, proved to be a great advantage. For the first time it was possible to find 71 active pathways in a Caco-2 cell experiment. These pathways were assigned to 12 main categories, with amino acid metabolism and carbohydrate metabolism being the most dominant categories in terms of the number of metabolites and metabolic pathways. Treatment of Caco-2 cells with high GOS and glucose contents resulted in significant effects on several metabolic pathways, whereas the MOS containing treatments resulted only for individual metabolites in significant changes. An effect based on bovine or caprine origin alone could not be observed. Thus, it was shown that MOS and GOS containing treatments can exert microbiome-independent effects on the metabolome of Caco-2 cells.

Cohort profile: the Food Chain Plus (FoCus) cohort.

The Food Chain Plus (FoCus) cohort was launched in 2011 for population-based research related to metabolic inflammation. To characterize this novel pathology in a comprehensive manner, data collection included multiple omics layers such as phenomics, microbiomics, metabolomics, genomics, and metagenomics as well as nutrition profiling, taste perception phenotyping and social network analysis. The cohort was set-up to represent a Northern German population of the Kiel region. Two-step recruitment included the randomised enrolment of participants via residents' registration offices and via the Obesity Outpatient Centre of the University Medical Center Schleswig-Holstein (UKSH). Hence, both a population- and metabolic inflammation- based cohort was created. In total, 1795 individuals were analysed at baseline. Baseline data collection took place between 2011 and 2014, including 63% females and 37% males with an age range of 18-83 years. The median age of all participants was 52.0 years [IQR: 42.5; 63.0 years] and the median baseline BMI in the study population was 27.7 kg/m2 [IQR: 23.7; 35.9 kg/m2]. In the baseline cohort, 14.1% of participants had type 2 diabetes mellitus, which was more prevalent in the subjects of the metabolic inflammation group (MIG; 31.8%). Follow-up for the assessment of disease progression, as well as the onset of new diseases with changes in subject's phenotype, diet or lifestyle factors is planned every 5 years. The first follow-up period was finished in 2020 and included 820 subjects.

Differential Effects of Obesity, Hyperlipidaemia, Dietary Intake and Physical Inactivity on Type I versus Type IV Allergies.

BACKGROUND: Alongside metabolic diseases (esp. obesity), allergic disorders are becoming increasingly prevalent. Since both obesity and allergies are highly impacted by environmental determinants, with this study we assessed the potential link between metabolic implications and two distinct types of allergies. METHODS: Using cross-sectional data from the German FoCus cohort, n = 385 allergy cases, either hay fever (=type I allergy, n = 183) or contact allergy (=type IV allergy, n = 202) were compared to age- and sex-matched healthy control subjects (1:1 ratio, in total n = 770) regarding their metabolic phenotype, diet, physical activity, sleep, gut microbial composition, and serum metabolite profile using suitable BMI-adjusted models. RESULTS: Obesity and metabolic alterations were found significantly more prevalent in subjects with allergies. In fact, this relation was more pronounced in contact allergy than hay fever. Subsequent BMI-adjusted analysis reveals particular importance of co-occurring hyperlipidaemia for both allergy types. For contact allergy, we revealed a strong association to the dietary intake of poly-unsaturated fatty acids, particularly α-linolenic acid, as well as the enrichment of the corresponding metabolic pathway. For hay fever, there were no major associations to the diet but to a lower physical activity level, shorter duration of sleep, and an altered gut microbial composition. Finally, genetic predisposition for hyperlipidaemia was associated to both contact allergy and hay fever. CONCLUSIONS: Reflected by higher allergy prevalence, our findings indicate an impaired immune response in obesity and hyperlipidaemia, which is differentially regulated in type I and type IV allergies by an unfavourable lifestyle constellation and subsequent microbial and metabolic dysfunctions.

Vitamin C and Omega-3 Fatty Acid Intake Is Associated with Human Periodontitis-A Nested Case-Control Study.

Vitamins and omega-3 fatty acids (Ω3FA) modulate periodontitis-associated inflammatory processes. The aim of the current investigation was to evaluate associations of oral nutrient intake and corresponding serum metabolites with clinical severity of human periodontitis. Within the Food Chain Plus cohort, 373 periodontitis patients­245 without (POL) and 128 with tooth loss (PWL)­were matched to 373 controls based on sex, smoking habit, age and body mass index in a nested case-control design. The amount of oral intake of vitamins and Ω3FAs was assessed from nutritional data using a Food Frequency Questionnaire. Oral intake and circulatory bioavailability of vitamins and Ω3FA serum metabolomics were compared, using ultra-high-resolution mass spectrometry. Periodontitis patients exhibited a significantly higher oral intake of vitamin C and Ω3FA Docosapentaenoic acid (p < 0.05) compared to controls. Nutritional intake of vitamin C was higher in PWL, while the intake of Docosapentaenoic acid was increased in POL (p < 0.05) compared to controls. In accordance, serum levels of Docosapentaenoic acid were also increased in POL (p < 0.01) compared to controls. Vitamin C and the Ω3FA Docosapentaenoic acid might play a role in the pathophysiology of human periodontitis. Further studies on individualized nutritional intake and periodontitis progression and therapy are necessary.

A dietary carbohydrate - gut Parasutterella - human fatty acid biosynthesis metabolic axis in obesity and type 2 diabetes.

Recent rodent microbiome experiments suggest that besides Akkermansia, Parasutterella sp. are important in type 2 diabetes and obesity development. In the present translational human study, we aimed to characterize Parasutterella in our European cross-sectional FoCus cohort (n = 1,544) followed by validation of the major results in an independent Canadian cohort (n = 438). In addition, we examined Parasutterella abundance in response to a weight loss intervention (n = 55). Parasutterella was positively associated with BMI and type 2 diabetes independently of the reduced microbiome α/ß diversity and low-grade inflammation commonly found in obesity. Nutritional analysis revealed a positive association with the dietary intake of carbohydrates but not with fat or protein consumption. Out of 126 serum metabolites differentially detectable by untargeted HPLC-based MS-metabolomics, L-cysteine showed the strongest reduction in subjects with high Parasutterella abundance. This is of interest, since Parasutterella is a known high L-cysteine consumer and L-cysteine is known to improve blood glucose levels in rodents. Furthermore, metabolic network enrichment analysis identified an association of high Parasutterella abundance with the activation of the human fatty acid biosynthesis pathway suggesting a mechanism for body weight gain. This is supported by a significant reduction of the Parasutterella abundance during our weight loss intervention. Together, these data indicate a role for Parasutterella in human type 2 diabetes and obesity, whereby the link to L-cysteine might be relevant in type 2 diabetes development and the link to the fatty acid biosynthesis pathway for body weight gain in response to a carbohydrate-rich diet in obesity development.

Precision Nutrition in Chronic Inflammation.

The molecular foundation of chronic inflammatory diseases (CIDs) can differ markedly between individuals. As our understanding of the biochemical mechanisms underlying individual disease manifestations and progressions expands, new strategies to adjust treatments to the patient's characteristics will continue to profoundly transform clinical practice. Nutrition has long been recognized as an important determinant of inflammatory disease phenotypes and treatment response. Yet empirical work demonstrating the therapeutic effectiveness of patient-tailored nutrition remains scarce. This is mainly due to the challenges presented by long-term effects of nutrition, variations in inter-individual gastrointestinal microbiota, the multiplicity of human metabolic pathways potentially affected by food ingredients, nutrition behavior, and the complexity of food composition. Historically, these challenges have been addressed in both human studies and experimental model laboratory studies primarily by using individual nutrition data collection in tandem with large-scale biomolecular data acquisition (e.g. genomics, metabolomics, etc.). This review highlights recent findings in the field of precision nutrition and their potential implications for the development of personalized treatment strategies for CIDs. It emphasizes the importance of computational approaches to integrate nutritional information into multi-omics data analysis and to predict which molecular mechanisms may explain how nutrients intersect with disease pathways. We conclude that recent findings point towards the unexhausted potential of nutrition as part of personalized medicine in chronic inflammation.

A fungal pathogen induces systemic susceptibility and systemic shifts in wheat metabolome and microbiome composition.

Yield losses caused by fungal pathogens represent a major threat to global food production. One of the most devastating fungal wheat pathogens is Zymoseptoria tritici. Despite the importance of this fungus, the underlying mechanisms of plant-pathogen interactions are poorly understood. Here we present a conceptual framework based on coinfection assays, comparative metabolomics, and microbiome profiling to study the interaction of Z. tritici in susceptible and resistant wheat. We demonstrate that Z. tritici suppresses the production of immune-related metabolites in a susceptible cultivar. Remarkably, this fungus-induced immune suppression spreads within the leaf and even to other leaves, a phenomenon that we term "systemic induced susceptibility". Using a comparative metabolomics approach, we identify defense-related biosynthetic pathways that are suppressed and induced in susceptible and resistant cultivars, respectively. We show that these fungus-induced changes correlate with changes in the wheat leaf microbiome. Our findings suggest that immune suppression by this hemibiotrophic pathogen impacts specialized plant metabolism, alters its associated microbial communities, and renders wheat vulnerable to further infections.

Inflammation Associated Pancreatic Tumorigenesis: Upregulation of Succinate Dehydrogenase (Subunit B) Reduces Cell Growth of Pancreatic Ductal Epithelial Cells.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most fatal malignancies and its development is highly associated with inflammatory processes such as chronic pancreatitis (CP). Since the succinate dehydrogenase subunit B (SDHB) is regarded as tumor suppressor that is lost during cancer development, this study investigated the impact of M1-macrophages as part of the inflammatory microenvironment on the expression as well as function of SDHB in benign and premalignant pancreatic ductal epithelial cells (PDECs). Immunohistochemical analyses on pancreatic tissue sections from CP patients and control individuals revealed a stronger SDHB expression in ducts of CP tissues being associated with a greater abundance of macrophages compared to ducts in control tissues. Accordingly, indirect co-culture with M1-macrophages led to clearly elevated SDHB expression and SDH activity in benign H6c7-pBp and premalignant H6c7-kras PDECs. While siRNA-mediated SDHB knockdown in these cells did not affect glucose and lactate uptake after co-culture, SDHB knockdown significantly promoted PDEC growth which was associated with increased proliferation and decreased effector caspase activity particularly in co-cultured PDECs. Overall, these data indicate that SDHB expression and SDH activity are increased in PDECs when exposed to pro-inflammatory macrophages as a counterregulatory mechanism to prevent excessive PDEC growth triggered by the inflammatory environment.

Variability of Pyrrolizidine Alkaloid Occurrence in Species of the Grass Subfamily Pooideae (Poaceae).

Pyrrolizidine alkaloids (PAs) are a class of secondary metabolites found in various unrelated angiosperm lineages including cool-season grasses (Poaceae, subfamily Pooideae). Thesinine conjugates, saturated forms of PA that are regarded as non-toxic, have been described to occur in the two grass species Lolium perenne and Festuca arundinacea (Poaceae, subfamily Pooideae). In a wider screen, we tested various species of the Pooideae lineage, grown under controlled conditions, for their ability to produce thesinine conjugates or related structures. Using an LC-MS based targeted metabolomics approach we were able to show that PA biosynthesis in grasses is limited to a group of very closely related Pooideae species that produce a limited diversity of PA structures. High variability in PA levels was observed even between individuals of the same species. These individual accumulation patterns are discussed with respect to a possible function and evolution of this type of alkaloid.

An extract from the Atlantic brown algae Saccorhiza polyschides counteracts diet-induced obesity in mice via a gut related multi-factorial mechanisms.

In this study we addressed the questions whether an Atlantic brown algae extract (BAE) affects diet induced obesity in mice and which would be the primary targets and underlying key mechanisms. Male C57 BL/6 mice were fed a hypercaloric diet, referred to as high fat diet (HFD), supplemented with a freeze-dried aqueous BAE from Saccorhiza polyschides (5 %) for 8 months. Compared to the control group, dietary BAE supplementation significantly attenuated increase in body weight and fat mass. We observed apparent metabolic improvement including normalization of blood glucose, reduced plasma leptin, reduced fecal bile salt hydrolase activity with lower microbial production of toxic bile acid metabolites in the gut and increased systemic bile acid circulation in BAE-fed mice counteracting adverse effects of long term HFD feeding. Survival of mice receiving dietary BAE supplementation appeared slightly enhanced; however, median and maximal life spans as well as hepatic mTOR activation were not significantly different between BAE and control mice. We suggest that the beneficial metabolic effects of our BAE are at least partly mediated by alterations in gut microbiota associated with fermentation of indigestible polysaccharides that are major components of brown algae such as alginates and fucoidans. We moreover propose a multi-factorial mechanism that involves profound alterations in bile acid homeostasis, changes in intestinal and systemic glucose metabolism likely including increased intestinal gluconeogenesis, increased activity of the intestinally derived hormone GLP-1 contributing to promote systemic insulin sensitivity, and inhibition of α-amylase activity, which expectably limits dietary carbohydrate digestion and glucose release.

Validation of a two-step quality control approach for a large-scale human urine metabolomic study conducted in seven experimental batches with LC/QTOF-MS.

After his study of food science at the Rheinische Friedrich-Wilhelms University of Bonn, Tobias J Demetrowitsch obtained his doctoral degree in the research field of metabolomics at the Christian-Albrechts-University of Kiel. The present paper is part of his doctoral thesis and describes an extended strategy to evaluate and verify complex or large-scale experiments and data sets. Large-scale studies result in high sample numbers, requiring the analysis of samples in different batches. So far, the verification of such LC-MS-based metabolomics studies is difficult. Common approaches have not provided a reliable validation procedure to date. This article shows a novel verification process for a large-scale human urine study (analyzed by a LC/QToF-MS system) using a two-step validation procedure. The first step comprises a targeted approach that aims to examine and exclude statistical outliers. The second step consists of a principle component analysis, with the aim of a tight cluster of all quality controls and a second for all volunteer samples. The applied study design provides a reliable two-step validation procedure for large-scale studies and additionally contains an inhouse verification procedure.