Detection Methods for Arachidonic Acid

What is Arachidonic Acid？

Arachidonic acid (AA) is an all-cis 5,8,11,14-eicosatetraenoic acid, which is an unsaturated fatty acid. It is one of the most abundant and widely distributed polyunsaturated essential fatty acids in living organisms. In the arachidonic acid cascade, AA has three main metabolic pathways in living organisms: through cyclooxygenases (COXs), lipoxygenases (LOX) or cytochrome P-450 (CYP-450) to produce prostaglandin (PG), thromboxane (TX), and leukotriene (LT) or hydroxyfatty acid (OHFA). Among them, COXs are divided into 3 forms: COX-1 (constitutive), COX-2 (mitogen-induced form) and COX-3 (variant of COX-1).

• Prostaglandin metabolites (PGs): PGs are important inflammatory mediators mediated by COXs and produced by AA transformation, and are also important lipid mediators known to regulate physiological functions including immunity and inflammation. PGs increase capillary permeability, enhance edema caused by other inflammatory conditions, and enhance pain and swelling caused by histamines and bradykinin-like substances.
• Leukotriene metabolites (LTs): LTs are a class of biologically active compounds that structurally contain three conjugated double bonds and belong to a family of lipid agents. They have a dominant role in hypersensitivity reactions, inflammation and proliferation-related disease processes, and are particularly highlighted in the pathogenesis and progression of allergic asthma disease.
• Thromboxane metabolites (TXs): AA is reduced by peroxidase to produce PGH2, which in turn is reduced by thromboxane synthase to produce TXs-like substances. TXs are produced by platelets and have platelet coagulation and vasoconstrictive effects. Like PGs, TXs include a variety of compounds with different structural functions (thromboxane derivatives), such as TXA2, TXA3, TXB1, TXB2, TXB3, etc.
• Hydroxy fatty acid metabolites (OHFAs): AA can also produce OHFAs metabolites through the CYP450 enzyme pathway: epoxyeicosatrienoic acid (EET), hydroxyeicosatetraenoic acid (20-HETE), eicosatetraenoic acid (ETE), dihydroxyeicosatetraenoic acid (DHET), etc.

Illustrative overview of the AA cascade (Willenberg et al., 2015)

AA is released from membrane phospholipids via phospholipase A2s and has been shown to be involved in pathophysiological events related to cell injury, inflammation and apoptosis. Therefore, accurate qualitative and quantitative analysis is important in the development mechanism, diagnosis and prediction of AA-related diseases.

Arachidonic Acid Test Method

The main analytical methods for the determination of arachidonic acid and its metabolites in biological samples are gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS ), liquid chromatography-electrospray mass spectrometry (LC-ESI/MS), liquid chromatography-nuclear magnetic resonance (LC-NMR) capillary electrophoresis-mass spectrometry (CE-MS ), and enzyme-linked immunosorbent assay (ELISA).

GC-MS analysis generally requires a more complex pretreatment step before analysis, including sample purification and derivatization. This method is not suitable for the analysis of thermally unstable metabolites such as epoxyeicosatetraenoic acids (EETs).

Enzyme-linked immunosorbent assay (ELISA) is the most widely used technique, focusing on endpoint metabolomics (PGE2, LTE4, etc.). However, this method is too complex and lacks good reproducibility.

LC-MS/MS as a major technical tool in metabolomics has become an effective tool for measuring small molecule metabolites (relative molecular mass less than 1500) in biological samples. The development and application of this analytical technique in AA detection has been a popular research area.

LC-MS analytical strategies do not require excessive sample pre-treatment methods. After purification and enrichment by protein precipitation, liquid-liquid extraction or solid-phase extraction, biological samples can be directly used for LC-MS detection and analysis. LC-MS/MS is the main technique used for quantitative analysis of endogenous compounds in metabolomics, with the advantages of short analysis time, full automation, minimal sample preparation (nodulation), and is suitable for endogenous analytes with a wide range of relative molecular masses and polarities. It has been widely used for the qualitative and quantitative analysis of AA metabolites and their structural analogs, steroids and other components.

Reference

1. Willenberg, I., Ostermann, A. I., & Schebb, N. H. (2015). Targeted metabolomics of the arachidonic acid cascade: current state and challenges of LC–MS analysis of oxylipins. Analytical and bioanalytical chemistry, 407(10), 2675-2683.

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