Genetic perturbations and foreign chemicals can result in a multitude of changes across a wide range of biochemical processes in a biological system. These perturbations may affect the metabolome, the small molecule metabolites in an organism. Recently, liquid-chromatography coupled to mass spectrometry (LC-MS) technology has been used to quantify large proportions of the metabolome, however standardized protocols are not yet available for use with Drosophila melanogaster. Here, I developed an ion-pairing LC-MS protocol for the metabolomic characterization of D. melanogaster and demonstrated its implementation in establishing the metabolomic profile of flies under oxidative stress and in the metabolic profiles of four different Drosophila species. I demonstrated that this new method allows for the detection of otherwise difficult metabolites and that it is repeatable and sensitive with acceptable levels of ionsuppression, matrix effects, limits of detection and quantification. I then used this method to determine and quantify the metabolomic fingerprints of loss of Superoxide dismutase activity and paraquat-induced stress. Comparing and contrasting the effects of these two sources of oxidative stress, I document both similarities and stressor-specific effects.

Universiẗat, Diss., 2006--Frankfurt (Main). / Enth. Sonderabdr. aus Zeitschr. - Beitr. teilw. dt., teilw. eng. - Zsfassung in dt. und engl. Sprache.

Tübingen, Univ., Diss., 2005.

Würzburg, Univ., Diss., 2009. / Zsfassung in engl. Sprache.

Universiẗat, Diss., 2008--Frankfurt (Main). / Engl. Übers. des Hauptsacht.: Evaluation of the bioavailability of Boswellic acids in vitro and in vivo by LC-MS.

Universiẗat, Diss., 2003--Bayreuth.

Insects represent very useful experimental model in various branches of biological research. The investigation is driven by economic importance of many insect species, and also by biological features of insects as model organisms such as short period of reproduction, easy breeding and manipulation and, in particular, the minimal regulatory requirements which are associated to the management of vertebrates. Here we report robust and efficient LC/MS/MS methodology for the determination of the physiologically important lipid molecular species in insects. The target metabolites represent polar glycerophos-phopholipids (GPL) and nonpolar lipids diacylglycerols (DG) and triacylglycerols (TG). Combination of the LC/MS data with the subsequent GC fatty acid analysis enables complete structural elucidation of particular lipid species including their fatty acid compositions. The developed methodology was applied to studies of the chill tolerance of the firebug Pyrhocorris appterus. Fields and laboratory experiments were conducted to separate the triggering effects of low temperature, desiccation and diapause progression on the physiological characteristics related to chill tolerance with emphasis on the restructuring of GPL composition. The same effect on the GPL composition was observed during acclimatization in the field and cold acclimation in laboratory. By contrast, the GPL changes related to desiccation and diapause progression were relatively small (Tomčala et al, 2006). In adults of Drosophila melanogaster it has been found that acclimation at 15, 20 and 25°C during preimaginal development affects thermal tolerance and composition of membrane GPLs. Low temperature acclimation was associated with increase in proportion of ethanolamine at the expense of choline in GPLS. Relatively small, but statistically significant changes in lipid molecular compositon were observed with decreasing acclimation temperature (Overgard et al, 2008). Hormonal treatment studies on insect model Locusta migratoria showed a heterogeneous distribution of individual DGs in haemolymph after the hormone application and revealed that mobilization of the DGs is molecular species-specific with the highest proportion of DG 16:0/18:1 and forming in summary about 20% of the total mobilized DG content. Additional analysis of fat body triacylglycerols revealed that the AKH mobilizes the DGs specifically with the preference of those possessing the unsaturated C18 fatty acids (FAs). The fat body FAs with more than 18 carbons did not participate on the mobilization (Tomcala et al, 2009). The LC/MS methodology was further applied to lipid composition studies of several samples with very diverse biological origin (fish, human blood etc.) and was proved to be universally applicable to the wide scope of biological samples.

abstract: In the United States, two-thirds of adults are considered hypertensive orprehypertensive. In addition, chronic illness, such as hypertension, cardiovascular disease, and type II diabetes, results in $3.5 trillion in annual healthcare cost and is the primary cause of disability and death. As a result, many individuals seek cheaper and simpler alternatives to combat their conditions. In this exploratory analysis, a study assessing nitrate intake and its effects on vascular function in 39 young adult males was investigated for underlying metabolic variations through a liquid chromatography – mass spectrometry-based large-scale targeted metabolomics approach. A two-way repeated measures ANOVA was used, and 18 significant metabolites were discovered across the time, treatment, and time & treatment groups, including prostaglandin E2 (p<0.001), stearic acid (p=0.002), caprylic acid (p=0.016), pentadecanoic acid (p=0.027), and heptadecanoic acid (p=0.005). In addition, log-transformed principal component analysis and orthogonal partial least squares – discriminant analysis models demonstrated distinct separation among the treatment, control, and time variables. Moreover, pathway and enrichment analyses validated the effect of nitrate intake on the metabolite sets and its possible function in fatty acid oxidation. This better understanding of altered metabolic pathways may help explicate the benefits of nitrate on vascular function and reveal any unknown mechanisms of its supplementation. / Dissertation/Thesis / Masters Thesis Nutrition 2020

A highly sensitive and rapid method for the analysis of isradipine in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed. The procedure involves a simple liquid-liquid extraction of isradipine and amlodipine (IS, internal standard) with methyl-t-butyl ether after alkaline treatment and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 372.1 → m/z 312.2 and m/z 408.8 → m/z 237.9, for quantification of isradipine and IS, respectively. The standard calibration curves showed good linearity within the range of 10 to 5000 pg/mL (r2 ≥ 0.9998). The lower limit of quantitation (LLOQ) was 10 pg/mL. The retention times of isradipine (0.81 min) and IS (0.65 min) suggested the potential for high throughput of the proposed method. In addition, no significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence studies of 5 mg of sustained-release isradipine in 24 healthy Korean volunteers.

Widespread sedentary lifestyle and unhealthy eating habits in the last few decades have resulted in a dramatic increase of the number of people affected by obesity, type 2 diabetes, and cardiovascular diseases. The study of these pathological conditions revealed that impaired metabolism often causes these disorders. Lipid metabolism research has contributed significantly to determining mechanisms underlying metabolic disorders. Omega-3 fatty acids are an interesting target for lipidomics studies because they were shown to lower risk of cardiovascular diseases and are hypothesized to regulate lipid metabolism. In this work, I optimized lipid extraction and chemical modification methods for analysis of fatty acids profile of tissue samples and biofluids using comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GCxGC-MS). At first, I evaluated the relative amount of omega-3 fatty acids in red blood cells (Omega-3 index) of people living in Czech Republic in either the capital city (n=476) or the rural region (n=388). For this large-scale project, I extracted phospholipids from red blood cell (RBC) membranes, transesterified them into fatty acid methyl esters (FAMEs), and measured their profile by GCxGC-MS. The mean Omega-3 index was 3.56 mol % and I detected no significant...