Abstract
Oxidised lipids, covering enzymatic and auto-oxidation-synthesised mediators, are important signalling metabolites in inflammation while also providing a readout for oxidative stress, both of which are prominent physiological processes in a plethora of diseases. Excretion of these metabolites via urine is enhanced through the phase-II conjugation with glucuronic acid, resulting in increased hydrophilicity of these lipid mediators. Here, we developed a bovine liver-β-glucuronidase hydrolysing sample preparation method, using liquid chromatography coupled to tandem mass spectrometry to analyse the total urinary oxidised lipid profile including the prostaglandins, isoprostanes, dihydroxy-fatty acids, hydroxy-fatty acids and the nitro-fatty acids. Our method detected more than 70 oxidised lipids biosynthesised from two non-enzymatic and three enzymatic pathways in urine samples. The total oxidised lipid profiling method was developed and validated for human urine and was demonstrated for urine samples from patients with rheumatoid arthritis. Pro-inflammatory mediators PGF2α and PGF3α and oxidative stress markers iPF2α- IV, 11-HETE and 14-HDoHE were positively associated with improvement of disease activity score. Furthermore, the anti-inflammatory nitro-fatty acids were negatively associated with baseline disease activity. In conclusion, the developed methodology expands the current metabolic profiling of oxidised lipids in urine, and its application will enhance our understanding of the role these bioactive metabolites play in health and disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9742-2) contains supplementary material, which is available to authorized users.
Highlights
Oxidised lipids are important signalling mediators in health and disease, capable of providing quantitative readouts relating to inflammatory and oxidative stress status
For determining the optimal enzymatic deconjugation procedure for urinary oxidised lipids, three GUSs derived from H. pomatia, E. coli and bovine liver were investigated
No increase in metabolite levels were achieved through increasing the hydrolysis time beyond 2 h or from increasing the temperature
Summary
Oxidised lipids are important signalling mediators in health and disease, capable of providing quantitative readouts relating to inflammatory and oxidative stress status. The auto-oxidation pathway of oxidised lipids is interlinked with reactive oxygen species (ROS) or reactive nitrogen species (RNS), leading to the peroxidation of fatty acids in membrane-bound phospholipids and producing the isoprostanes (IsoPs) [1, 2] or nitrofatty acids (NO2-FAs) [3]. The lipid peroxidation readout from IsoPs are considered the golden standard for measuring oxidative stress in biological systems [4, 5]. NO2FAs potentiate diverse anti-inflammatory signalling actions regarded as beneficial within health and disease [3]. These peroxidised lipids impair membrane and organelle integrity and are subsequently excreted from the cell into systemic circulation via cellular repair mechanisms [1]
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