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Copyright of Berichte aus dem Julius Kühn-Institut is the property of Julius Kuehn Institut and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The distinctive aroma of onions, consisting primarily of sulfur-containing compounds, is one of the reasons for the popularity of the vegetable. The rapid enzymatic and chemical reactions that occur after the destruction of onion bulb tissue render the analysis of the volatile profile challenging. Therefore, sample preparation is a crucial step in the analysis of the onion volatilome, but it often does not receive the necessary attention in the literature. In this work, we focused on two aspects: Firstly, we compared different sample preparation approaches to maximize the volatile yield and to enable the description of the onion volatile profile as it would emerge after a solid-phase microextraction (SPME) Arrow sampling procedure. For headspace (HS)-gas chromatography-mass spectrometry analysis, onion juice with the addition of an ammonium sulfate solution proved to be the best option. Secondly, we optimized the HS sampling and desorption parameters (enrichment time, enrichment temperature, agitator speed, desorption time) for onion volatiles using the efficient design of experiments (DoE) approach. The optimal conditions for the analysis with HS-SPME Arrow were an enrichment time of 75 min at 60°C with an agitator speed of 713 rpm and a desorption time of 120 s., (© 2024 The Author(s). Journal of Separation Science published by Wiley‐VCH GmbH.)

Domesticated strawberry is susceptible to sudden frost episodes, limiting the productivity of this cash crop in regions where they are grown during early spring. In contrast, the ancestral woodland strawberry (Fragaria vesca) has successfully colonized many habitats of the Northern Hemisphere. Thus, this species seems to harbour genetic factors promoting cold tolerance. Screening a germplasm established in the frame of the German Gene Bank for Crop Wild Relatives, we identified, among 70 wild accessions, a pair with contrasting cold tolerance. By following the physiological, biochemical, molecular, and metabolic responses of this contrasting pair, we identified the transcription factor Cold Box Factor 4 and the dehydrin Xero2 as molecular markers associated with superior tolerance to cold stress. Overexpression of green fluorescent protein fusions with Xero2 in tobacco BY-2 cells conferred cold tolerance to these recipient cells. A detailed analysis of the metabolome for the two contrasting genotypes allows the definition of metabolic signatures correlated with cold tolerance versus cold stress. This work provides a proof-of-concept for the value of crop wild relatives as genetic resources to identify genetic factors suitable to increase the stress resilience of crop plants., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Soil salinity is progressively impacting agriculture, including viticulture. Identification of genetic factors rendering grapevine (Vitis vinifera L.) resilience that can be introgressed into commercial varieties is necessary for safeguarding viticulture against the consequences of global climate change. To gain insight into the physiological and metabolic responses enabling salt tolerance, we compared a salt-tolerant accession of Vitis sylvestris from Tunisia, "Tebaba", with "1103 Paulsen" rootstock widely used in the Mediterranean. Salt stress was slowly increased, simulating the situation of an irrigated vineyard. We determined that "Tebaba" does not sequester sodium in the root but can cope with salinity through robust redox homeostasis. This is linked with rechanneling of metabolic pathways toward antioxidants and compatible osmolytes, buffering photosynthesis, such that cell-wall breakdown can be avoided. We propose that salt tolerance of this wild grapevine cannot be attributed to a single genetic factor but emerges from favorable metabolic fluxes that are mutually supportive. We suggest that introgression of "Tebaba" into commercial varieties is preferred over the use of "Tebaba" as a rootstock for improving salt tolerance in grapevine., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Endurance exercise alters whole-body as well as skeletal muscle metabolism and physiology, leading to improvements in performance and health. However, biological mechanisms underlying the body's adaptations to different endurance exercise protocols are not entirely understood. Methods: We applied a multi-platform metabolomics approach to identify urinary metabolites and associated metabolic pathways that distinguish the acute metabolic response to two endurance exercise interventions at distinct intensities. In our randomized crossover study, 16 healthy, young, and physically active men performed 30 min of continuous moderate exercise (CME) and continuous vigorous exercise (CVE). Urine was collected during three post-exercise sampling phases (U01/U02/U03: until 45/105/195 min post-exercise), providing detailed temporal information on the response of the urinary metabolome to CME and CVE. Also, fasting spot urine samples were collected pre-exercise (U00) and on the following day (U04). While untargeted two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) led to the detection of 608 spectral features, 44 metabolites were identified and quantified by targeted nuclear magnetic resonance (NMR) spectroscopy or liquid chromatography-mass spectrometry (LC-MS). Results: 104 urinary metabolites showed at least one significant difference for selected comparisons of sampling time points within or between exercise trials as well as a relevant median fold change >1.5 or <0. 6 ¯ (NMR, LC-MS) or >2.0 or <0.5 (GC×GC-MS), being classified as either exercise-responsive or intensity-dependent . Our findings indicate that CVE induced more profound alterations in the urinary metabolome than CME, especially at U01, returning to baseline within 24 h after U00. Most differences between exercise trials are likely to reflect higher energy requirements during CVE, as demonstrated by greater shifts in metabolites related to glycolysis (e.g., lactate, pyruvate), tricarboxylic acid cycle (e.g., cis -aconitate, malate), purine nucleotide breakdown (e.g., hypoxanthine), and amino acid mobilization (e.g., alanine) or degradation (e.g., 4-hydroxyphenylacetate). Discussion: To conclude, this study provided first evidence of specific urinary metabolites as potential metabolic markers of endurance exercise intensity. Future studies are needed to validate our results and to examine whether acute metabolite changes in urine might also be partly reflective of mechanisms underlying the health- or performance-enhancing effects of endurance exercise, particularly if performed at high intensities., Competing Interests: Author SA was employed by the company TSG ResearchLab gGmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kistner, Mack, Rist, Krüger, Egert, Biniaminov, Engelbert, Seifert, Dörr, Ferrario, Neumann, Altmann and Bub.)

Methane emissions by ruminants contribute to global warming and result in a loss of dietary energy for the animals. One possibility of reducing methane emissions is by dietary strategies. In the present trial, we investigated the long-term effects of Mootral, a feed additive consisting of garlic powder ( Allium sativum ) and bitter orange extracts ( Citrus aurantium ), on fermentation parameters and the microbial community in the rumen simulation technique (RUSITEC) system. The experiment lasted 38 days and was divided into three phases: an equilibration period of 7 days, a baseline period (BL) of 3 days, and experimental period (EP) of 28 days. Twelve fermentation vessels were divided into three groups ( n = 4): control (CON), short-term (ST), and long-term (LT) application. From day 11 to day 27, 1.7 g of Mootral was added to the ST vessels; LT vessels received 1.7 g of Mootral daily for the entire EP. With the onset of Mootral application, methane production was significantly reduced in both groups until day 18. Thereafter, the production rate returned to the initial quantity. Furthermore, the short chain fatty acid fermentation profile was significantly altered by Mootral application; the molar proportion of acetate decreased, while the proportions of propionate and butyrate increased. Metabolomic analysis revealed further changes in metabolite concentrations associated with the Mootral supplementation period. The methyl coenzyme-M reductase gene copy number was reduced in the liquid and solid phase, whereas the treatment did not affect the abundance of bacteria. At the end of the BL, Methanomicrobia was the most abundant archaeal class. Mootral supplementation induced an increase in the relative abundance of Methanomassiliicoccales and a reduction in the relative abundance of Methanomicrobia, however, this effect was transient. Abundances of bacterial families were only marginally altered by the treatment. In conclusion, Mootral has the transient ability to reduce methane production significantly due to a selective effect on archaea numbers and archaeal community composition with little effect on the bacterial community., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Brede, Peukert, Egert, Breves and Brede.)

The study aimed to investigate possible systematic effects in the basic underlying variability of individual metabolomic data. In this context, the extent of gender- and genotype-dependent differences reflected in the metabolic composition of three tissues in fattening pigs was determined. The 40 pigs belonged to the genotypes PIx(LWxGL) and PIxGL with gilts and boars, respectively. Blood and tissue samples from M. longissimus dorsi and liver were directly taken at the slaughtering plant and directed to GC × GC qMS metabolite analysis. Differences were observed for various metabolite classes like amino acids, fatty acids, sugars, or organic acids. Gender-specific differences were much more pronounced than genotype-related differences, which could be due to the close genetic relation of the fattening pigs. However, the metabolic dimorphism between gilts and boars was found to be genotype-dependent, and vice versa metabolic differences between genotypes were found to be gender-dependent. Most interestingly, integration into metabolic pathways revealed different patterns for carbon (C) and nitrogen (N) usage in boars and gilts. We suppose a stronger N-recycling and increased energy metabolism in boars, whereas, in gilts, more N is presumably excreted and remaining carbon skeletons channeled into lipogenesis. Associations of metabolites to meat quality factors confirmed the applicability of metabolomics approaches for a better understanding about the impact of drivers (e.g., gender, age, breed) on physiological processes influencing meat quality. Due to the huge complexity of the drivers-traits-network, the derivation of independent biomarkers for meat quality prediction will hardly be possible.

The macronutrient potassium (K) has vital physiological functions in plants and its availability can strongly impact quality of crops like tomato. The impact of K nutrition on conventional tomato fruit quality parameters has been described several times, but detailed investigations on the effect of K supply on the fruit metabolite profile are still rare. To fill this gap, we investigated the influence of K fertilization on the metabolite profile of tomato fruits. For this purpose, an outdoor pot experiment with three different cocktail tomato cultivars was performed. A fertilization regimen with five K levels was applied, ranging from deficiency to sufficient supply. Fruit samples were analyzed by untargeted GC×GC-MS to cover the primary metabolite profile as well as some secondary metabolites. As verified using ICP-OES, fruit K content was highly proportional to the supplied amount of K. At the metabolite profile level, the most prominent and cultivar-independent effect of increased K fertilization was the rise of tricarboxylic acid (TCA) cycle intermediates. Further effects were more cultivar-specific, for example an increase of the mobile nitrogen pool (e.g. amines like putrescine and amides like asparagine), changes in the profile of minor sugars (especially disaccharides) as well as higher levels of some secondary metabolites. Pronounced response patterns were mainly observed in the cultivars Primavera and Yellow Submarine that were recently characterized as higher yielding, demanding a stronger consideration of cultivar differences in future studies., (Copyright © 2020. Published by Elsevier Masson SAS.)

Scope: Diabetes is thought to primarily represent a disturbance of carbohydrate metabolism; however, population studies employing metabolomics have mainly identified plasma amino acids and lipids, or their products, as biomarkers. In this pilot study, the aim is to analyze a wide spectrum of sugar compounds in the fasting state and during an oral glucose tolerance test (OGTT) in healthy, prediabetic, and type 2 diabetic volunteers., Methods and Results: The three volunteer groups underwent a standard OGTT. Plasma samples obtained in the fasting state, 30 and 90 min after the OGTT, are subjected to a semitargeted GC-MS (gas chromatography-mass spectrometry) sugar profiling. Overall, 40 sugars are detected in plasma, of which some are yet unknown to change during an OGTT. Several sugars (e.g., trehalose) reveal significant differences between the volunteer groups both in fasting plasma and in distinct time courses after the OGTT. This suggests an endogenous production from orally absorbed glucose and/or an insulin-dependent production/removal from plasma., Conclusion: It is demonstrated that more sugars than expected can be found in human plasma. Since some of these show characteristic differences depending on health status, it may be worthwhile to assess their usability as biomarkers for diagnosing early-stage insulin resistance and type 2 diabetes., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Banana is one of the most widely consumed fruits in the world. However, information regarding its health effects is scarce. Biomarkers of banana intake would allow a more accurate assessment of its consumption in nutrition studies., Objectives: Using an untargeted metabolomics approach, we aimed to identify the banana-derived metabolites present in urine after consumption, including new candidate biomarkers of banana intake., Methods: A randomized controlled study with a crossover design was performed on 12 healthy subjects (6 men, 6 women, mean ± SD age: 30.0 ± 4.9 y; mean ± SD BMI: 22.5 ± 2.3 kg/m2). Subjects underwent 2 dietary interventions: 1) 250 mL control drink (Fresubin 2 kcal fiber, neutral flavor; Fresenius Kabi), and 2) 240 g banana + 150 mL control drink. Twenty-four-hour urine samples were collected and analyzed with ultra-performance liquid chromatography coupled to a quadrupole time-of-flight MS and 2-dimensional GC-MS. The discovered biomarkers were confirmed in a cross-sectional study [KarMeN (Karlsruhe Metabolomics and Nutrition study)] in which 78 subjects (mean BMI: 22.8; mean age: 47 y) were selected reflecting high intake (126-378 g/d), low intake (47.3-94.5 g/d), and nonconsumption of banana. The confirmed biomarkers were examined singly or in combinations, for established criteria of validation for biomarkers of food intake., Results: We identified 33 potentially bioactive banana metabolites, of which 5 metabolites, methoxyeugenol glucuronide (MEUG-GLUC), dopamine sulfate (DOP-S), salsolinol sulfate, xanthurenic acid, and 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-β-carboline sulfate, were confirmed as candidate intake biomarkers. We demonstrated that the combination of MEUG-GLUC and DOP-S performed best in predicting banana intake in high (AUCtest = 0.92) and low (AUCtest = 0.87) consumers. The new biomarkers met key criteria establishing their current applicability in nutrition and health research for assessing the occurrence of banana intake., Conclusions: Our metabolomics study in healthy men and women revealed new putative bioactive metabolites of banana and a combined biomarker of intake. These findings will help to better decipher the health effects of banana in future focused studies. This study was registered at clinicaltrials.gov as NCT03581955 and with the Ethical Committee for the Protection of Human Subjects Sud-Est 6 as CPP AU 1251, IDRCB 2016-A0013-48; the KarMeN study was registered with the German Clinical Trials Register (DRKS00004890). Details about the study can be obtained from https://www.drks.de., (Copyright © American Society for Nutrition 2019.)

Scope: The human volatilome has gained high interest for the discovery of potential biomarkers of diseases. However, knowledge about the diet as a crucial factor affecting the volatilome is scarce. Therefore, the search for disease biomarkers, as well as the potential use of volatiles as dietary markers is so far limited. The aim of this study is to investigate the association of the diet with the urinary volatilome with the special task to find potential markers of coffee consumption in 24 h urine samples from the Karlsruhe Metabolomics and Nutrition (KarMeN) study., Methods and Results: Acidified urine samples are analyzed using an approach combining headspace solid phase microextraction (HS-SPME) sampling with untargeted GC×GC-MS. Overall, 138 reliably occurring volatiles are detected. To account for the unequally concentrated urine samples, results of six different commonly used normalization methods are compared. Statistical analysis evidences six potential markers of coffee consumption, the most promising being 3,4-dimethyl-2,5-furandione. A correlation analysis between the 24 h dietary recall data and the urinary volatilome reveals further promising associations., Conclusion: The human urinary volatilome is highly affected by the diet, enabling access to a high level of information about potential diet-related biomarkers. Therefore, it is a very promising source for further investigations on dietary markers., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The life sciences are currently being transformed by an unprecedented wave of developments in molecular analysis, which include important advances in instrumental analysis as well as biocomputing. In light of the central role played by metabolism in nutrition, metabolomics is rapidly being established as a key analytical tool in human nutritional studies. Consequently, an increasing number of nutritionists integrate metabolomics into their study designs. Within this dynamic landscape, the potential of nutritional metabolomics (nutrimetabolomics) to be translated into a science, which can impact on health policies, still needs to be realized. A key element to reach this goal is the ability of the research community to join, to collectively make the best use of the potential offered by nutritional metabolomics. This article, therefore, provides a methodological description of nutritional metabolomics that reflects on the state-of-the-art techniques used in the laboratories of the Food Biomarker Alliance (funded by the European Joint Programming Initiative "A Healthy Diet for a Healthy Life" (JPI HDHL)) as well as points of reflections to harmonize this field. It is not intended to be exhaustive but rather to present a pragmatic guidance on metabolomic methodologies, providing readers with useful "tips and tricks" along the analytical workflow., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Although sugars and sugar derivatives are an important class of metabolites involved in many physiologic processes, there is limited knowledge on their occurrence and pattern in biofluids., Objective: Our aim was to obtain a comprehensive urinary sugar profile of healthy participants and to demonstrate the wide applicability and usefulness of this sugar profiling approach for nutritional as well as clinical studies., Design: In the cross-sectional KarMeN study, the 24-h urine samples of 301 healthy participants on an unrestricted diet, assessed via a 24-h recall, were analyzed by a newly developed semitargeted gas chromatography-mass spectrometry (GC-MS) profiling method that enables the detection of known and unknown sugar compounds. Statistical analyses were performed with respect to associations of sex and diet with the urinary sugar profile., Results: In total, 40 known and 15 unknown sugar compounds were detected in human urine, ranging from mono- and disaccharides, polyols, and sugar acids to currently unknown sugar-like compounds. A number of rarely analyzed sugars were found in urine samples. Maltose was found in statistically higher concentrations in the urine of women compared with men and was also associated with menopausal status. Further, a number of individual sugar compounds associated with the consumption of specific foods, such as avocado, or food groups, such as alcoholic beverages and dairy products, were identified., Conclusions: We here provide data on the complex nature of the sugar profile in human urine, of which some compounds may have the potential to serve as dietary markers or early disease biomarkers. Thus, comprehensive urinary sugar profiling not only has the potential to increase our knowledge of host sugar metabolism, but can also reveal new dietary markers after consumption of individual food items, and may lead to the identification of early disease biomarkers in the future. The KarMeN study was registered at drks.de as DRKS00004890.

Physiological and functional parameters, such as body composition, or physical fitness are known to differ between men and women and to change with age. The goal of this study was to investigate how sex and age-related physiological conditions are reflected in the metabolome of healthy humans and whether sex and age can be predicted based on the plasma and urine metabolite profiles. In the cross-sectional KarMeN (Karlsruhe Metabolomics and Nutrition) study 301 healthy men and women aged 18-80 years were recruited. Participants were characterized in detail applying standard operating procedures for all measurements including anthropometric, clinical, and functional parameters. Fasting blood and 24 h urine samples were analyzed by targeted and untargeted metabolomics approaches, namely by mass spectrometry coupled to one- or comprehensive two-dimensional gas chromatography or liquid chromatography, and by nuclear magnetic resonance spectroscopy. This yielded in total more than 400 analytes in plasma and over 500 analytes in urine. Predictive modelling was applied on the metabolomics data set using different machine learning algorithms. Based on metabolite profiles from urine and plasma, it was possible to identify metabolite patterns which classify participants according to sex with > 90% accuracy. Plasma metabolites important for the correct classification included creatinine, branched-chain amino acids, and sarcosine. Prediction of age was also possible based on metabolite profiles for men and women, separately. Several metabolites important for this prediction could be identified including choline in plasma and sedoheptulose in urine. For women, classification according to their menopausal status was possible from metabolome data with > 80% accuracy. The metabolite profile of human urine and plasma allows the prediction of sex and age with high accuracy, which means that sex and age are associated with a discriminatory metabolite signature in healthy humans and therefore should always be considered in metabolomics studies.

Scope: L-carnitine has been advertised as a fat-lowering and performance-enhancing supplement, although scientific evidence for its effectiveness is lacking. The uptake of about 1-2 g of L-carnitine per day may result in the formation of metabolites like trimethylamine-N-oxide (TMAO), which in turn may be converted to potential carcinogens or promote the development of cardiovascular diseases., Methods and Results: To assess whether an L-carnitine supplementation changes overall metabolism or causes the formation of previously unknown metabolites, we analyzed plasma samples from Fischer 344 rats originating from a previous study using a multi-platform metabolomics approach comprising LC-MS/MS and GC×GC-MS methods. Despite an intake of up to 352 mg L-carnitine/kg body weight/day for 1 year, plasma concentrations of only 29 out of 359 metabolites were significantly influenced, the induced concentration changes being often comparatively small. Nevertheless, a clear dose-response relationship and a substantial concentration increase were observed for TMAO, i.e. a tenfold higher TMAO level was measured in the high-dose group when compared to the control (2.5 versus 25.0 μM)., Conclusion: Although L-carnitine supplementation did not cause large changes in the plasma metabolome, a higher risk for cardiovascular disease due to chronically elevated TMAO plasma concentrations cannot be excluded., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)