In vitro reactivity and metabolism of the glucose degradation product 3,4-dideoxyglucosone-3-ene under physiological conditions and its browning potential upon sterilization

3,4-dideoxyglucosone-3-ene (3,4-DGE) is a highly reactive α,β-unsaturated dicarbonyl compound formed due to dehydration of its precursor 3-deoxyglucosone (3-DG) during heat-treatment or storage of glucose-containing foods and medicinal preparations. Since 3-DG is present also in vivo, the first aim of this thesis was to elucidate if its thermal degradation to 3,4-DGE might also occur under physiological and febrile conditions, respectively. In fact, upon in vitro incubation and thanks to analysis via UHPLC DAD the formation of 3,4-DGE could be observed, which was even enhanced at 39 °C. Since 3-DG levels are known to be increased in serum under diabetic conditions, 3,4-DGE might be formed systemically. The following aim was to analyze in vitro the interaction of 3,4-DGE with certain components of the human blood circulation. This GDP is also found in infusion fluids which are administered intravenously and might therefore interact directly with blood and vessels. Therefore, 3,4-DGE was incubated with physiological concentrations of selected targets, and free 3,4-DGE was quantified after defined time points. First, human serum albumin (HSA) was applied as a target, which reacted quickly with 3,4-DGE: Only 8.5 % of the initial concentration were still present after seven hours. This resulted in the loss of about 47 % accessible free thiol groups in HSA, as assessed via Ellman’s assay. Since the protein’s ketoprofen-binding ability and esterase-like activity were not influenced, it was deduced that arginine 410 of the Sudlow site II was not critically modified. Glutathione was selected as a second target compound and a prompt formation of two stereoisomeric adducts was observed; until now, the formation of only one conjugate had been reported. 3,4-DGE reacted also with immunoglobulin G (IgG), but not with immobilized collagen type IV. On the one hand, these results suggest that the highly reactive 3,4-DGE might be efficiently trapped by certain components present in human blood, possibly preventing further reactions, and aiding its clearance. Simultaneously, the loss of free thiol groups in HSA and glutathione suggests that their original activity against both oxidative and electrophilic stress in the human body is adversely affected by 3,4-DGE. It is to be further investigated if the resulting modification of HSA and IgG might reduce their functions, in the latter case even immune response. The next aim was to elucidate the metabolism of free 3,4-DGE and its glutathione conjugates. First, a literature- and in-silico-based prediction of its biotransformation was performed. These results were later used to aid interpretation of the successive in vitro experiments. Before their execution, 3,4-DGE was purified via semi-preparative HPLC. Finally, 3,4-DGE and its glutathione conjugates were subjected to in vitro setups comprising phase I and II metabolism. Chromatographic separation of the sample compounds via microLC coupled with high-resolution mass spectrometry revealed the formation of a possible follow-up metabolite of the glutathione conjugates in the setup containing liver microsomes: The observed hydrolysis product might have been formed via γ-glutamyltranspeptidase-assisted enzymatic catalysis. Moreover, it can be regarded as a possible precursor of mercapturic acids, typical metabolites of glutathione conjugates. If found in blood or - especially in the case of metabolites - urine, the observed adducts of 3,4-DGE with HSA, IgG and glutathione as well as possible metabolites might serve as markers for either exposure to 3,4-DGE or even its in vivo formation from 3-DG, signalizing dicarbonyl stress. Glucose-based infusion fluids and peritoneal dialysis fluids develop a brown color during heat-treatment and storage. The scope of the second project of this thesis was to determine the contribution of 3,4-DGE to browning of these fluids and thus its reactivity under sterilization conditions in absence of proteins, compared to glucose. First, fluids containing either 3,4-DGE or glucose were heat-sterilized. Analysis via UHPLC-DAD revealed mainly five compounds in the heat-treated 3,4-DGE solutions which absorb visible light and are expected to be conducive to browning of the fluids. Similar UV-Vis spectra hinted at structural similarities between three of the observed molecules. Due to their absorption behavior, the compounds in the glucose fluids were negligible and ignored in the following experiments. To gain further insight into the target analytes found in the 3,4-DGE-based solutions, detection was switched to mass spectrometry, enabling the assignment of three compounds to molecular masses and possible sum formulas. It was suggested that the underlying mechanism of formation is the oligomerization of up to four molecules of either 3,4-DGE or 5-HMF; combinations of both substances cannot be excluded. Subsequently, the heat-treated fluids containing either 3,4-DGE or glucose were infused directly into a high-resolution mass spectrometer. The resulting data permitted an overview over the entirety of sterilization products to which sum formulas were assigned. In summary, the findings presented in this thesis expand the knowledge regarding the high reactivity and toxicological potential of the glucose degradation product 3,4-dideoxyglucosone-3-ene under physiological conditions; additionally, they also suggest its possible contribution to browning of glucose-containing fluids. The observed high reactivity towards components of the human blood may be the reason why free 3,4-DGE has not yet been detected in this bodily fluid. Attention towards this compound and the risks it poses to the human organism will hopefully be enhanced by these results; a welcome progress may be the further reduction of 3,4-DGE content in glucose-based medicinal fluids, improving biocompatibility, reducing side effects, and thus benefitting the health condition of patients dependent on these treatments.