Towards Chiralomics: Targeted and Untargeted Gas Chromatography-Mass Spectrometry based Enantioselective Metabolome Analysis

Chirality is not a rare phenomenon in metabolomes. Nevertheless, common metabolomics approaches still detect enantiomers as a single signal. However, they are individual metabolites due to different stereospecific enzymes catalyzing their metabolism. The here presented methods consider the configuration of chiral metabolites in the interpretation of metabolomics data to investigate the following biomedical hypotheses. D-Amino acids (D-AAs), albeit much lower abundant than their antipodes, are potential diagnostic markers for diseases affecting the liver, the gut and the gut flora. The other assumption of this thesis was that the comprehensive resolution of stereoisomers from metabolomes, providing more detailed metabolic fingerprints than conventional approaches, would facilitate the differentiation of groups. In order to investigate the former hypothesis two different GC-MS techniques were used for the implementation, optimization and validation of quantitative AAE analysis, primarily in urine and serum. The GC-qMS method using MeOH/Methylchloroformate (MCF) derivatization and a gamma-CD derivative for enantiomer separation was chosen, among several tested derivative/chiral selector combinations, in my diploma thesis as the most effective GC-qMS method for quantitative amino acid enantiomer (AAE) analysis with respect to the number of baseline separated proteinogenic AAE pairs and peak intensities. Sample preparation was optimized to allow the accurate quantification of exclusively free AAEs. In addition injection parameter and IS contents were optimized to decrease LLOQs and the method was validated by comparison to a non-chiral GC-qMS method for AA quantification. Finally, the method was subjected to biomedical applications that underlined the potential of D-AAs as diagnostic markers due to changed D-AA levels found in mouse serum and mouse liver tissues as a consequence of pathological changes of the liver. The method separated ten pairs of AAEs, but it failed to separate Phe enantiomers, D-Ile/L-Leu, L-Thr/L-Asp, and L-Ser/D-Met. For L-Thr, L-Asp and D-Met a specific m/z enabled their accurate quantification in SIM mode but the quantification of the other coeluting enantiomers was impeded. Moreover, not each D-AA that was baseline separated from its antipode could be quantified in all samples of interest due to insufficient LLOQs. Thus, the potential of comprehensive GC×GC-TOFMS was tested for quantitative AAE analysis in urine and serum. The same derivative/chiral selector combination was used and a ZB-AAA column provided best AAE resolution as the second dimension selector from two different selectors tested. Upon optimization of the temperature program, baseline separation was accomplished for all detected AAEs except for Phe enantiomers. The method was validated by comparison to the chiral GC-qMS method and pros and cons of both approaches were discussed. The application of chiral GC×GC-TOFMS revealed increased D-AA serum levels in patients with liver cirrhosis when compared to respective serum levels of healthy individuals. Finally, performing a chiral fingerprinting approach, it was demonstrated that comprehensive enantiomer resolution assisted with the differentiation of different NAFLD stages in the analysis of urine samples from respective mouse models. There was observed a clear group separation of the three investigated groups after PCA of differentiating features and D-Val, D-2-Hydroxyglutaric acid and D-allo-Ile levels significantly differentiated NASH from hepatic steatosis. There were identified further enantiomers of hydroxydiacids and AA derivatives that significantly differentiated the diseased groups from controls and besides D-Val six other separated AAEs differentiated the experimental groups. This chiral GC-qMS based fingerprinting approach introduced a new field of investigation that I named ‘chiralomics’ as it expanded metabolomics to the chiral dimension

Lactonization of the Oncometabolite D-2-Hydroxyglutarate Produces a Novel Endogenous Metabolite

In recent years, onco-metabolites like D-2-hydroxyglutarate, which is produced in isocitrate dehydrogenase-mutated tumors, have gained increasing interest. Here, we report a metabolite in human specimens that is closely related to 2-hydroxyglutarate: the intramolecular ester of 2-hydroxyglutarate, 2-hydroxyglutarate-γ-lactone. Using 13C5-L-glutamine tracer analysis, we showed that 2-hydroxyglutarate is the endogenous precursor of 2-hydroxyglutarate-lactone and that there is a high exchange between these two metabolites. Lactone formation does not depend on mutated isocitrate dehydrogenase, but its formation is most probably linked to transport processes across the cell membrane and favored at low environmental pH. Furthermore, human macrophages showed not only striking differences in uptake of 2-hydroxyglutarate and its lactone but also in the enantiospecific hydrolysis of the latter. Consequently, 2-hydroxyglutarate-lactone may play a critical role in the modulation of the tumor microenvironment