Browsing by Subject "GC-MS"

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Identification of markers for dietary intake and health status by GC-MS based metabolite profiling approaches
(2020) Mack, Carina I.; Kulling, Sabine E.
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.
Weinblattmetabolite als Resistenzmarker für eine Plasmopara viticola Widerstandsfähigkeit
(2024) Grünwald, Maike; Vögele, Ralf
Downy mildew of grapevine is one of the most important diseases of the European grapevine Vitis vinifera LINNÉ supsp. vinifera. It is caused by the obligate biotrophic oomycete Plasmopara viticola Berl. & De Toni. American grapevines are largely resistant to downy mildew and may contribute in to the protection of susceptible vines against P. viticola. Therefore, this work deals with the metabolite profiling of resistance markers (RM) from volatile secondary metabolites of susceptible and resistant grapevines. 10 genotypes with different resistance to P. viticola were analysed. 3 different Vitis species (V. vinifera, V. riparia, V. labrusca) and some hybrid vines were analysed. The constitutive markers of 3 developmental stages (BBCH6, BBCH8, BBCH9) were determined. In addition, induced markers were analysed. Furthermore, the relationship between leaf position and the occurrence of RM was investigated. The used metabolomic methods were also applied to identify markers for leaf position. For metabolite profiling the grape leaves were analysed using GC-MS and were evaluated using non-targeted and targeted analytical methods. The comparison of the metabolite profiles showed that the developmental stage has the strongest influence on the metabolite profile and the influence of the leaf position is so small that it can be neglected. A total of 41 constitutive RMs were identified. The metabolites identified came from the substance classes of green leaf volatiles (GLV), norisoprenoids, benzoate derivatives, monoterpenoids, a furan and a sesquiterpene. It was elaborated, that GLV, norisoprenoids, benzoate derivatives and 2-ethylfuran were almost exclusively present in higher concentrations in resistant genotypes. Monoterpenoids and the sesquiterpene calacorene were mainly detected in higher concentrations in the susceptible genotypes of V. vinifera. Furthermore, it could be shown, that monoterpenoids were detected in significantly increased concentrations at the developmental stage BBCH6 in the susceptible V. vinifera. calacorene was never detected in BBCH6. It was only found in significantly increased concentrations in the later developmental stages of V. vinifera. GLVs are RM in the resistant genotypes such as V. labrusca and occur here exclusively at the later developmental stages, mainly at BBCH9. For the constitutive RMs from the classes of norisoprenoids, benzoate derivatives and furan, no dependence of the developmental stages and their occurrence was observed. 3 other metabolites were identified as RMs that showed a strong correlation with the developmental stage. They appeared initially in BBCH6 in significantly increased concentrations in susceptible V. vinifera, but in BBCH9 they showed significantly increased concentrations in the resistant V. labrusca genotypes. These RMs with changing correlation were geranyl acetone, terpineol, and (Z)-3-hexenal. The correlation between occurrence of the constitutive RMs and the developmental stages has not been described before. It is a new finding of this work that monoterpenoids occur as RMs in grapevines mainly at BBCH6 and GLV as well as sesquiterpenes were found as RMs mainly at BBCH9. Norisoprenoids, benzoate derivatives and furan occurred as RMs at all developmental stages tested. Furthermore, terpenoids occurred species-specifically more frequently in V. vinifera as constitutive RMs, whereas the furan 2-ethylfuran never appeared as a RM in V. vinifera. Benzoate derivatives and GLVs were most frequently detected as RMs in V. labrusca genotypes. Norisoprenoids appeared most frequently, but not exclusively, as RMs in V. riparia. The correlation between the occurrence of norisoprenoids as RMs and species specificity to V. riparia is a new finding of this work. Nevertheless, it should be briefly mentioned, that this species specificity refers exclusively to the classification as a constitutive RM. Monoterpenes are also part of the metabolite profile of V. labrusca as well as norisoprenoids are important flavour compounds in quality wines. 24 induced RMs were determined. Most of the induced RMs occurred in the fungus-resistant cultivar Regent and only six induced RMs in the resistant V. labrusca hybrid Blue Isabella and in V. riparia. For all RMs identified, it was searched for reports on bioactivity in publications. For 2-ethylfuran, an isomer mix of (Z)- and (E)-ocimene as well as cyclocitral, an inhibitory effect on P. viticola has already been published. Still, 20 compounds have been associated here for the first time with a resistance response to P. viticola infection. These are theaspirane, (E)-damascone, (E)-damascenone, dihydroedulan I, megastigmatrienone, sulcatone, carvomenthenal A, nonanol , (Z)-3-hexenyl acetate, p-cymenene, p-cymene, limonene, alloocimene, myrcene, citronellol, hotrienol, (Z)-rose oxide, geranium oxide and calacorene.