Identification of markers for dietary intake and health status by GC-MS based metabolite profiling approaches

Markers are compounds that can be used as indicators of an exposure, a metabolic state, or any other effect. Metabolomics and metabolite profiling approaches for marker discovery will increasingly gain significance. In the context of food, diet, and health, these approaches allow among others the identification of dietary intake markers, which can complement and verify traditional dietary assessment methods in epidemiologic studies. Consequently, the investigation of associations between diet and health status in general, and also in particular diet-related diseases will be improved. Compared to classical biomarker studies, metabolomics enables a more comprehensive investigation of clinical markers for diagnosis, prognosis and monitoring of diseases, such as type 2 diabetes mellitus. Especially, early diagnosis in pre-disease states, where symptoms are not yet evident, are of particular interest. The aim of this thesis was to evaluate the application of GC-MS based metabolite profiling approaches for the identification of markers for dietary intake and health status. In this respect, volatile organic compounds and sugar compounds were analyzed to discover marker candidates in urine and plasma samples from a cross-sectional study with 300 participants, as well as from a human intervention study with diabetic, prediabetic and healthy participants. In the past, the search for markers of dietary intake mostly focused on non-volatile metabolites. To explore the potential of the volatilome, urine samples of a cross-sectional study were analyzed aiming to exemplary identify markers of coffee consumption using an untargeted HS-SPME-GC×GC-MS method. Six marker candidates were identified from a profile of 138 volatile organic compounds with the most robust represented by 3,4-dimethyl-2,5-furandione. Moreover, the correlation with the general dietary intake data highlighted the volatilome as a particularly interesting source for the detection of new dietary markers. The chromatographic separation of sugar compounds is often insufficient due to the high structural similarities. Therefore, in most studies common and well-known sugar compounds are analyzed in human body fluids. Within the scope of this thesis, a semitargeted GC-MS sugar profiling method was developed, which revealed a more complex sugar profile, both in urine and plasma, than described so far or expected. Rare sugar compounds such as psicose and trehalose were detected. However, the knowledge about their origin and presence in urine or plasma is limited to date. Moreover, the maltose concentration in urine was shown to be dependent on sex and menopause status (pre- and post-menopausal) a relationship with the vaginal microbiota is suggested here. In addition, the association of the urinary sugar profile with dietary intake data enabled the identification and confirmation of several new and also known marker candidates as for example, for consumption of avocado, dairy products and alcohol. The plasma sugar profiles of healthy, prediabetic and diabetic volunteers after an oral glucose tolerance test could be clearly distinguished, independent of glucose. Remarkably, a variety of sugar compounds showed marked postprandial differences dependent on health status. For example, trehalose showed a profile similar to the insulin-dependent profile of glucose. However, the origin and underlying biological mechanism for those sugar compounds remain to be elucidated. During the application of the one-dimensional GC-MS sugar profiling method to urine and plasma samples, it became evident that even more sugar compounds might be present, although in low concentrations, but were not detected due to limitations of the analytical method. Therefore, the one-dimensional method was transferred into a two-dimensional GC×GC-MS method. Improved sensitivity and separation finally enabled the detection of 84 instead of 55 sugar compounds in urine. The two-dimensional method was applied in an intervention study with apples, and revealed marker candidates for apple consumption for future validation. Overall, the results illustrate the benefit of a comprehensive analysis of sugar compounds in urine and plasma, including minor and rare sugar derivatives. The GC-MS based metabolite profiling approaches addressing the volatilome and the sugar profile, respectively, were demonstrated to be promising approaches for the identification of markers for dietary intake and health status. Future work should address the identification of unknown compounds, the adaptation of the GC×GC-MS sugar profiling method for quantitative purposes, and especially the validation of the identified marker candidates with respect to their suitability to more accurately assess dietary intake or diabetic state. High priority should also be given to the biochemical mechanisms and the origin of the compounds as well as their physiological or pathophysiological function in human metabolism.Marker sind Substanzen, die als Indikatoren für eine Exposition, einen metabolischen Zustand oder einen Effekt herangezogen werden. Metabolomics und Metabolite profiling-Ansätze gewinnen in der Markerforschung zunehmend an Bedeutung. Metabolomics ermöglicht die Identifizierung von Markern für den Lebensmittelverzehr, die in Zukunft unter anderem in epidemiologischen Studien zur Ergänzung und Überprüfung traditioneller Ernährungserhebungsmethoden Verwendung finden werden. In der Konsequenz können Zusammenhänge zwischen Ernährung und Gesundheit im Allgemeinen, sowie ernährungsabhängigen Erkrankungen im Speziellen, besser beschrieben werden. Außerdem können mittels Metabolomics auch Marker identifiziert werden, die im klinischen Rahmen eine Diagnose, Prognose oder Überwachung von Behandlungsmaßnahmen für eine Erkrankung ermöglichen, wie z.B. bei Typ 2 Diabetes mellitus. Von besonderem Interesse sind dabei Marker, die eine frühzeitige Diagnose, das heißt vor der Manifestation von Symptomen, ermöglichen. Ziel der vorliegenden Arbeit war es, die Verwendung von GC-MS basierten Metabolite profiling-Ansätzen zur Identifizierung von Markern für den Lebensmittelverzehr und den Gesundheitsstatus zu prüfen. Einen besonderen Schwerpunkt bildeten dabei volatile organische Verbindungen und Zuckerverbindungen, die in Urin- und Plasmaproben einer Querschnittsstudie mit 300 Probanden sowie einer humanen Interventionsstudie mit Diabetikern, Prädiabetikern und Gesunden analysiert wurden. Bei der bisherigen Suche nach Markern für den Lebensmittelverzehr lag das Augenmerk vor allem auf nicht-volatilen Metaboliten. Um das Potential des Volatiloms zu eruieren, wurden Urinproben einer Querschnittsstudie mithilfe einer ungerichteten HS-SPME-GC×GC-MS Methode analysiert und darin beispielhaft nach Markern für den Kaffeekonsum gesucht. Aus dem Urinprofil mit 138 volatilen Verbindungen wurden sechs plausible Kandidaten identifiziert, von denen sich 3,4-Dimethyl-2,5-furandion als der robusteste Marker erwies. Mittels einer Korrelationsanalyse anhand von Verzehrsdaten weiterer Lebensmittel wurde darüber hinaus gezeigt, dass das Volatilom eine vielversprechende Quelle neuer Marker für den Lebensmittelverzehr ist. Zucker lassen sich aufgrund ihrer strukturellen Ähnlichkeit häufig nur unzureichend chromatographisch trennen, daher werden in humanen Matrices bisher mehrheitlich nur wenige bekannte Zuckerverbindungen erfasst. Im Rahmen dieser Arbeit wurde eine semi-gerichtete GC-MS Zuckerprofiling-Methode entwickelt, mit der gezeigt werden konnte, dass das humane Zuckerprofil im Urin und im Plasma erheblich komplexer ist, als bisher beschrieben und angenommen. Verschiedene Zuckerverbindungen, wie beispielsweise Psicose oder Trehalose, über deren Herkunft und Vorhandensein im Urin oder in Plasma fast nichts bekannt ist, wurden nachgewiesen. Im Urin zeigten sich darüber hinaus Unterschiede in der Maltosekonzentration in Abhängigkeit vom Geschlecht sowie dem prä- und postmenopausalen Status, die vermutlich im Zusammenhang mit der vaginalen Mikrobiota stehen. Die Assoziation der Zuckerprofile mit dem Lebensmittelverzehr ermöglichte zudem die Identifizierung neuer und Bestätigung bekannter Marker, beispielsweise für den Verzehr von Avocado und Milchprodukten, sowie für Alkoholkonsum. Im Plasma von Gesunden, Prädiabetikern und Diabetikern wurden nach einem oralen Glucosetoleranztest deutliche Unterschiede im Zuckerprofil festgestellt. Interessanterweise zeigten eine Reihe neuer Zuckerverbindungen markante postprandiale Unterschiede abhängig vom Gesundheitszustand. Beispielsweise zeigte Trehalose ein ähnliches Profil wie die insulinabhängige Glucose. Jedoch ist weder über den Mechanismus noch zur Herkunft dieser Zucker etwas bekannt. Bereits die bisherigen Ergebnisse des Zuckerprofilings in Urin und Plasma zeigten, dass zusätzliche Zuckerverbindungen, wenn auch in sehr geringer Konzentration, vorhanden sind. Daher wurde die eindimensionale Methode zu einer zweidimensionalen GC×GC-MS-Methode mit verbesserter Sensitivität und Trennung weiterentwickelt, was nun die Erfassung von 84 statt 55 Zuckerverbindungen in Urin ermöglicht. Erste Auswertungen der Messdaten einer Interventionsstudie mit Äpfeln zeigten, dass diese Methode die Identifizierung von potentiellen Markern für den Verzehr von Äpfeln ermöglicht. Die Ergebnisse verdeutlichen, welches Potential in der umfassenden Analyse von Zuckern, einschließlich seltener Verbindungen, steckt. GC-MS basierte Metabolite profiling-Ansätze, wie hier für das Volatilom und das Zuckerprofil gezeigt, sind geeignete Methoden für die Identifizierung von Markern des Lebensmittelverzehrs und des Gesundheitsstatus. Die Identifizierung bisher unbekannter Verbindungen, die Weiterentwicklung der Zuckeranalytik zu einer quantitativen Methode und insbesondere die Validierung der identifizierten Marker bezüglich ihrer Eignung, den Lebensmittelverzehr bzw. den diabetischen Status akkurater zu erfassen, sind zukünftige Ziele. Besonders herausfordernd ist es dabei, die mechanistischen Zusammenhänge aufzuklären, insbesondere im Hinblick auf Herkunft, Vorhandensein und Funktion der detektierten Zuckerverbindungen im menschlichen Metabolismus

Combined Untargeted and Targeted Fingerprinting by Comprehensive Two-Dimensional Gas Chromatography to Track Compositional Changes on Hazelnut Primary Metabolome during Roasting

This study focuses on the detectable metabolome of high-quality raw hazelnuts (Cory- lus avellana L.) and on its changes after dry-roasting. Informative fingerprinting was obtained by comprehensive two-dimensional gas chromatography with fast-scanning quadrupole mass spectrom- etry (GC×GC-qMS) combined with dedicated data processing. In particular, combined untargeted and targeted (UT) fingerprinting, based on pattern recognition by template matching, is applied to chromatograms from raw and roasted samples of Tonda Gentile Trilobata and Anakliuri hazelnuts harvested in Italy and Georgia. Lab-scale roasting was designed to develop a desirable organoleptic profile matching industrial standards. Results, based on 430 peak features, reveal that phenotype expression is markedly correlated to cultivar and pedoclimatic conditions. Discriminant components between cultivars are amino acids (valine, alanine, glycine, and proline); organic acids (citric, aspartic, malic, gluconic, threonic, and 4-aminobutanoic acids); and sugars and polyols (maltose, xylulose, xylitol, turanose, mannitol, scyllo-inositol, and pinitol). Of these, alanine, glycine, and proline have a high informational role as precursors of 2-acetyl- and 2-propionylpyrroline, two key-aroma com- pounds of roasted hazelnuts. Roasting has a decisive impact on metabolite patterns—it caused a marked decrease (−90%) of alanine, proline, leucine and valine, and aspartic and pyroglutamic acid and a −50% reduction of saccharose and galactose

Combined Untargeted and Targeted Fingerprinting by Comprehensive Two-Dimensional Gas Chromatography to Track Compositional Changes on Hazelnut Primary Metabolome during Roasting

This study focuses on the detectable metabolome of high-quality raw hazelnuts (Cory- lus avellana L.) and on its changes after dry-roasting. Informative fingerprinting was obtained by comprehensive two-dimensional gas chromatography with fast-scanning quadrupole mass spectrom- etry (GC×GC-qMS) combined with dedicated data processing. In particular, combined untargeted and targeted (UT) fingerprinting, based on pattern recognition by template matching, is applied to chromatograms from raw and roasted samples of Tonda Gentile Trilobata and Anakliuri hazelnuts harvested in Italy and Georgia. Lab-scale roasting was designed to develop a desirable organoleptic profile matching industrial standards. Results, based on 430 peak features, reveal that phenotype expression is markedly correlated to cultivar and pedoclimatic conditions. Discriminant components between cultivars are amino acids (valine, alanine, glycine, and proline); organic acids (citric, aspartic, malic, gluconic, threonic, and 4-aminobutanoic acids); and sugars and polyols (maltose, xylulose, xylitol, turanose, mannitol, scyllo-inositol, and pinitol). Of these, alanine, glycine, and proline have a high informational role as precursors of 2-acetyl- and 2-propionylpyrroline, two key-aroma com- pounds of roasted hazelnuts. Roasting has a decisive impact on metabolite patterns—it caused a marked decrease (−90%) of alanine, proline, leucine and valine, and aspartic and pyroglutamic acid and a −50% reduction of saccharose and galactose

Image6_Acute effects of moderate vs. vigorous endurance exercise on urinary metabolites in healthy, young, physically active men—A multi-platform metabolomics approach.JPEG

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 2.0 or 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.</p

Image7_Acute effects of moderate vs. vigorous endurance exercise on urinary metabolites in healthy, young, physically active men—A multi-platform metabolomics approach.JPEG

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 2.0 or 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.</p

Table6_Acute effects of moderate vs. vigorous endurance exercise on urinary metabolites in healthy, young, physically active men—A multi-platform metabolomics approach.DOCX

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 2.0 or 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.</p

Image2_Acute effects of moderate vs. vigorous endurance exercise on urinary metabolites in healthy, young, physically active men—A multi-platform metabolomics approach.JPEG

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 2.0 or 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.</p

Image5_Acute effects of moderate vs. vigorous endurance exercise on urinary metabolites in healthy, young, physically active men—A multi-platform metabolomics approach.JPEG

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 2.0 or 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.</p