Eicosanoids: what they are and how they are studied

Eicosanoids are lipid mediators derived from polyunsaturated fatty acids, mainly arachidonic acid. They include prostaglandins, thromboxanes, leukotrienes and lipoxins, alongside endocannabinoids and other bioactive lipids. They are not stored in the cell: they are produced on the spot from the arachidonic acid that cytosolic phospholipase A2 releases from membrane phospholipids when the cell is stimulated. They act in an autocrine and paracrine way on surface receptors, have half-lives from seconds to minutes, and regulate inflammation, platelet aggregation, vascular tone and many cell-signalling pathways. They are at once pharmacological targets — the cyclooxygenases are the target of NSAIDs — and biomarkers in research on inflammation, pain, oncology and cardiovascular and metabolic disease.

Where an eicosanoid comes from: the three arachidonic acid routes

Once released, arachidonic acid can follow three distinct enzymatic routes. The cyclooxygenase route (COX-1 and COX-2) converts it into the unstable endoperoxide PGH2, from which specific synthases produce prostaglandin E2, D2 and F2α, prostacyclin (PGI2) and thromboxane A2. The lipoxygenase route produces, through 5-LOX and the accessory protein FLAP, leukotriene A4 and from it leukotriene B4 and the cysteinyl leukotrienes; 12- and 15-lipoxygenase instead generate the hydroxyeicosatetraenoic acids (HETEs) and, through the sequential action of several lipoxygenases, the lipoxins. The third route runs through the cytochromes P450, producing epoxyeicosatrienoic acids (EETs) and 20-HETE. Beside these there is a non-enzymatic route: free-radical peroxidation of arachidonic acid generates the isoprostanes.

Why stable metabolites are measured instead of the mediators

Some eicosanoids are too unstable to be measured directly. Thromboxane A2 hydrolyses spontaneously with a half-life of about 30 seconds into the biologically inactive thromboxane B2; prostacyclin, with a half-life of about 2 minutes at physiological pH and temperature, hydrolyses into 6-keto prostaglandin F1α. In both cases the stable product is measured and the activity of the pathway is inferred from it. The practical consequence is that sampling has to be designed so as not to generate in vitro what is being measured: accidental platelet activation during collection produces thromboxane that was not there in the patient.

ELISA or LC-MS/MS?

The two techniques answer different questions. The competitive enzyme immunoassay measures one analyte per plate, needs small volumes and no dedicated instrument, and is the usual choice for many samples against a known target; its limit is antibody cross-reactivity with structurally similar metabolites, which has to be assessed from the manufacturer's data. Liquid chromatography coupled to tandem mass spectrometry measures dozens of oxylipins in a single run, resolves isomers and quantifies by isotope dilution with deuterated standards, but needs more sample, more method development time and a dedicated instrument. In practice the two are used in sequence: immunoassay screening across large series, confirmation and extended profiling by mass spectrometry.

How are eicosanoid standards stored?

They are polyunsaturated lipids and they oxidise: solutions must be protected from light and oxygen and kept under the conditions stated in the batch certificate of analysis, which also reports solvent, concentration and expiry date. Mass spectrometry needs, besides the unlabelled standard for the curve, the matching deuterated analogue added to the sample before extraction, which compensates for extraction losses and ion suppression. Ready-made oxylipin mixtures serve to check retention times and transitions when a multi-analyte method is set up. The same applies to the bioactive lipids covered in the guide to endocannabinoids.

Standards and kits in the catalogue

The references below come from Cayman Chemical, long specialised in lipid chemistry, and are listed in the CABRU catalogue. The list is partial: the full range of standards, tracers, antibodies and ELISA kits for eicosanoids and oxylipins is in the product catalogue and in the biochemicals category.

To check which reference suits the matrix and method in use, it is enough to state the code or the analyte and write to CABRU.