Click-reactive Lipid Analysis – Efficient and optimized click-reaction for the detection and determination of Lipids

The study of metabolic overload diseases has become an important focus of biomedical research, driven by the need to understand the consequences of over-caloric westernized diet. Since metabolic overload usually leads to obesity, studies of fatty acid metabolism are a central aspect of current metabolism research.

While the need for detailed quantitative information about fatty acid metabolism from all available model systems, such as purified proteins, cultivated cell lines, primary cells, isolated organs and whole organisms is steadily increasing, we face a concomitant decrease of the accessibility and acceptance of a major technology used to obtain these data in the last 60 years, namely the use of radiolabeled fatty acids for monitoring fatty acid metabolism because of the related costs and safety concerns. Another drawback is the limited sensitivity of experiments using radioactive fatty acids. The relevant isotopes, 3H and 14C, have moderate or low specific activities and require long exposure times in order to obtain a meaningful result. Click-chemistry allows the sensitive and specific detection of compounds containing azido groups or terminal alkynes. Both can be integrated into fatty acids without major disturbance of the structure of the hydrophobic hydrocarbon chains. Click-labeled precursors have already been proposed to replace radioactive molecules in metabolic labeling experiments, including amino acids, nucleotides or lipids. However, the use of click-labeled fatty acids to monitor cellular lipid metabolism has been hampered by the lack of existing protocols that allow sensitive detection of click-labeled lipids. The present invention solves this problem by use of a fluorogenic click-reaction combined with an optimized protocol for the detection reaction, TLC separation, and fluorescence detection. By the use of this invention, it is possible to generate an image of the original metabolism as such.

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