Eicosanoids and related metabolites, sometimes referred to as oxylipins, are a group of structurally diverse metabolites that derive from the oxidation of PUFAs, including arachidonic acid, linoleic acid, and linolenic acid, dihomo linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid. They are locally acting bioactive signaling lipids that regulate a diverse set of homeostatic and inflammatory processes. Given the important regulatory functions in numerous physiological and pathophysiological states, the accurate measurement of eicosanoids and other oxylipins is of great clinical interest and lipidomics is now widely used to screen effectively for potential disease biomarkers.
Researchers at UC San Diego have developed a rapid and sensitive method for the analysis of esterified oxylipins using alkaline hydrolysis to release them followed by ultra-performance liquid chromatography coupled with mass spectrometric analysis. Detailed evaluation of the data revealed that several oxylipins are susceptible to alkaline-induced degradation. Nevertheless, of the 136 metabolites we examined, 56 were reproducibly recovered after alkaline hydrolysis. We classified those metabolites that were resistant to alkaline-induced degradation and applied this methodology to quantify metabolite levels in a macrophage cell model and in plasma of healthy subjects. After alkaline hydrolysis of lipids, 34 metabolites could be detected and quantified in resting and activated macrophages, and 38 metabolites were recovered from human plasma at levels that were substantially greater than in free form. By carefully selecting internal standards and taking the observed experimental limitations into account, we established a robust method that can be reliably employed for the measurement of esterified oxylipins in biological samples.
The new invention provides a rapid and sensitive method for the analysis of esterified oxylipins using alkaline hydrolysis to release them followed by ultra-performance liquid chromatography coupled to mass spectrometric analysis. The new method can be used to quantitate esterified eicosanoids and fatty acids in biological samples and provide a new way for differentiating between non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.
The invention demonstrates that a reduced alkaline-induced degradation will yield a much better recovery of metabolites both in terms of sensitivity (e.g., the metabolites that we were able to detect before at 60% we measure now at 100%) and the overall number of metabolites that we are now able to measure. In addition, the new method has much better reproducibility and precision. This is important for application to human material as it improves the statistics of the data making them more reliable
This invention demonstrates that this method can be used to accurately measure the sum total, free and esterified oxylipins, in human plasma and in biological samples.
This technology is patent pending and available for licensing and/or research sponsorship.
Lipidomics, oxidized lipids, oxylipins, eicosanoids, metabolites, alkaline hydrolysis, biomarkers