The LipidWeb blank

Eicosanoids and Related Oxylipins

An Introduction

The chemistry, biochemistry, pharmacology, and molecular biology of eicosanoids and related lipids, including the docosanoids and plant oxylipins, are vast, complex and occasionally contradictory subjects that continue to develop at an extraordinarily rapid rate. The only generic term that adequately covers all of the relevant metabolites is oxylipin, defined as a family of oxygenated natural products that are formed from unsaturated fatty acids by pathways involving at least one step of dioxygen-dependent oxidation. Simplistically, they are usually described as being synthesised in situ when needed in response to stimuli while having a short half-life and acting locally via interactions with specific receptors or intracellular effectors.

The term eicosanoid is used to embrace those biologically active lipid mediators derived from C20 fatty acids, including prostaglandins, thromboxanes, leukotrienes and related oxygenated derivatives. Note that the preferred IUPAC name is ‘icosanoid’, but this is largely ignored in the scientific literature. The key precursor fatty acids are 8c,11c,14c-eicosatrienoic (dihomo-γ-linolenic or 20:3(n-6)), 5c,8c,11c,14c-eicosatetraenoic (arachidonic or 20:4(n-6)) and 5c,8c,11c,14c,17c-eicosapentaenoic (20:5(n-3) or EPA) acids (see our web page on 'polyunsaturated fatty acids'). While these have been of special importance from a historical perspective, it is now impossible to discuss such lipids and their biological activities properly without also considering the docosanoids (protectins, resolvins and maresins or 'specialized pro-resolving mediators') derived from 4c,7c,10c,13c,16c,19c-docosahexaenoic acid (22:6(n-3) or DHA), and related C20 and C22 products formed by non-enzymic means the (isoprostanes). Similarly, plant products such as the jasmonates and other oxylipins derived from 9c,12c,15c-octadecatrienoic (α-linolenic or 18:3(n‑3)) acid have some comparable structural features and functions. It is noteworthy that the precursors for all of these belong to either the omega-6 or the omega-3 families of polyunsaturated fatty acids.

These pages are intended only as a broad overview of the topic that can be understood by scientists with some knowledge of lipids in general but not of oxylipins in particular. In this document, I introduce some basic concepts and discusses the primary rate-limiting enzyme for eicosanoid production in animal tissues, i.e. phospholipase A2. Further web pages explore the chemistry, biochemistry and function of the various types of oxylipin, although I am conscious that my approach may not adequately describe the true complexity of the interactions that occur in terms of metabolism and signalling or of physiological functions, especially the balance between pro- and anti-inflammatory effects. Those requiring a deeper insight should consult the publications cited below and at the end of the further web documents in this series.

1.   The Elements

Arachidonic acidOf these fatty acids, arachidonic acid has been by far the most studied, and it is special in many ways. It is an essential fatty acid in that it cannot be synthesised de novo in animals, and linoleic acid from the diet is required as the primary precursor. As a major component of phospholipids, and especially of phosphatidylinositol, it is important for the integrity of cellular membranes. The four cis-double bonds mean that the molecule is highly flexible, and this helps to confer the correct degree of fluidity in membranes. When the arachidonic acid is esterified, the lipids often have distinctive biological activities. For example, diacylglycerols enriched in arachidonic acid and derived from phosphatidylinositol are important cellular messengers. Anandamide or N-arachidonoylethanolamine is an endogenous cannabinoid or 'endocannabinoid', which produces neurobehavioral effects similar to those induced by cannabis and may have important signalling roles in the central nervous system, especially in the perception of pain and in the control of appetite. 2-Arachidonoyl-glycerol has similar properties. Indeed, there are suggestions that arachidonic acid per se may have some biological importance in animal tissues; for example, the cellular level of unesterified arachidonic acid may be a mechanism by which apoptosis is regulated.

Arachidonic acid has only rarely been encountered in higher plants, but it is a constituent of some algae, fungi and moulds. During fungal infections of plants, it is known to elicit the production of plant defence compounds (phytoalexins), probably after conversion to oxygenated metabolites.

The oxygenated metabolites derived from arachidonic and related fatty acids are produced through a series of complex interrelated biosynthetic pathways sometimes termed the 'arachidonate or eicosanoid cascade'. They are so numerous and have such a range of biological activities that they must provide a substantial component of the reason for the essentiality of the latter to the survival and well-being of animals. The structures of some examples of the important eicosanoid classes are illustrated below. The prostanoids (prostaglandins, thromboxanes and prostacyclins) have distinctive ring structures in the centre of the molecule. While the hydroxyeicosatetraenes are apparently simpler in structure, they are precursors for families of more complex molecules, such as the leukotrienes and lipoxins.

Structures of some eicosanoids

The 'natural' eicosanoids are produced enzymatically with great stereochemical precision, and this is essential for their biological functions. They are highly potent in the nanomolar range in vitro in the innumerable activities that have been defined, especially in relation to inflammatory responses, pain and fever. Most organs and cell types produce them, but with a high degree of tissue specificity, and some are even synthesised cooperatively between cells.

Biosynthesis of eicosanoids involves the action of multiple enzymes, several of which can be rate limiting. The figure below summarizes in simplistic terms the various pathways for the formation of eicosanoids. The first step in their biosynthesis is the production of free arachidonic acid in tissues from membrane phospholipids upon stimulation of the enzyme phospholipase A2 by various physiological and pathological factors, including hormones and cytokines. There are then three main enzymatic pathways for eicosanoid formation, involving cyclooxygenases (COXs), lipoxygenases (LOXs) and oxygenases of the cytochrome P-450 family. The COX pathway (two isoforms denoted COX-1 and COX-2) produces the prostaglandins PGG2 and PGH2, which are subsequently converted into further prostaglandins, prostacyclin and thromboxanes (TXs) by distinct synthases.

Biosynthetic pathways for eicosanoid production

There are several lipoxygenases that act upon different positions on arachidonic acid, mainly 5, 12 and 15, although an 8-lipoxygenase is also relevant, to produce various hydroperoxyeicosatetraenoic acids (HPETEs) and thence into hydroxyeicosatetraenoic acids (HETEs) and further products. For example, leukotriene LTA4 is produced from 5-HETE and is in turn a precursor for leukotriene LTB4, cysteinyl-leukotrienes (CysLTs) and lipoxins (LXs). The cytochrome P-450 epoxygenase pathway produces hydroxyeicosatetraenoic acids and epoxides as the primary products. It should be noted that many of the requisite enzymes, precursors and products are specific to particular types of cells.

Most cell types can produce eicosanoids from phospholipid precursors in this way. In addition, triacylglycerols in cytoplasmic lipid droplets of human mast cells, which are potent mediators of immune reactions and influence many inflammatory diseases, have a high content of arachidonic acid, which can be released by adipose triacylglycerol lipase as a substrate for production of specific eicosanoids, when the cells are stimulated appropriately.

Eicosanoids are generated mainly from unesterified fatty acids, not the CoA esters, and they function in this form, but it is increasingly recognized that they may occur and some may indeed be synthesised while esterified to other lipids. These may simply be an inert storage form available when a rapid response is required, but some also be active biologically as esters. This is especially true for the endocannabinoids. Ultimately, many of the eicosanoids produced in this way return to the membranes where they can interact with specific receptors.

While the eicosanoids were the first to be identified and studied intensively and I have used them as the main examples in this web page for illustrative purposes, it is now recognized that docosanoids (protectins, resolvins and maresins or 'specialized pro-resolving mediators') derived from the n-3 family of fatty acids such as DHA may be just as important in their biological functions often opposing the actions of the eicosanoids, while octadecanoids derived from linoleate also have essential biological properties. These are produced in a related manner, often using the same enzymes as in eicosanoid production, so they are an important element of this story as of course are the plant oxylipins.

Formula of protectin D1

2.   Phospholipase A2

Most of the arachidonic acid (and other polyunsaturated fatty acids) in animal tissues is in esterified form, mainly in phospholipids and in phosphatidylinositol and the polyphosphoinositides in particular. Before this arachidonate can be used for eicosanoid synthesis, it must be released by the action of the enzyme phospholipase A2. In addition to phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine can be substrates for arachidonic acid release, depending on the tissue and physiological conditions.

Generation of arachidonic acid and eicosanoids from phosphatidylinositol

A large number of enzymes with phospholipase A2 activity have been characterized, and three main types have been identified that are relevant here: cytosolic calcium-dependent PLA2 (cPLA2), cytosolic calcium-independent PLA2 (iPLA2) and secreted PLA2 (sPLA2). iPLA2 shows no specificity for arachidonic acid in particular, and it appears to have only a minor role in eicosanoid production. Rather, it is involved in phospholipid re-modelling or general catabolism, where it ensures the availability of the required substrates. On the other hand, the cytosolic Ca2+-dependent phospholipase A2 (specifically the isoform 'cPLA2α' or 'Group IVA cPLA2') does have a marked specificity for phospholipids containing arachidonic acid in the sn-2 position, and there is clear evidence that the enzyme plays a key role in the release of this fatty acid for generation of prostanoids and related metabolites. Indeed, it is believed to be rate limiting for eicosanoid production in many tissues. It makes use of a catalytic Ser-Asp dyad to hydrolyse fatty acids, and it contains a so-called ‘C2’ domain that facilitates a calcium-dependent translocation of the enzyme from the cytosol to the membrane surface, where the phospholipid substrate and the key enzymes of eicosanoid biosynthesis are located. In fact, there are six isoenzymes with molecular masses in the range of 60 to 100kDa, which are regulated by phosphorylation, although only the cPLA2α form is relevant here. These enzymes are of course required for other purposes, for example digestion of dietary phospholipids and host defence against bacterial infections.

To function in this way, cPLA2α must translocate from the cytosol to the perinuclear and endoplasmic reticulum membranes in response to an increase in cytosolic Ca2+, where it is phosphorylated and activated by mitogen-activated protein kinases. In addition to control via transcriptional regulation, the activity of the enzyme is responsive to various stimuli, such as hormones, cytokines and neurotransmitters. In particular, it has been demonstrated that ceramide-1-phosphate and phosphatidylinositol 4,5-bisphosphate bind to the enzyme. The latter is bound in a 1:1 stoichiometry and is required for activation and translocation of the enzyme to the site of action. Ceramide-1-phosphate stimulates release of arachidonic acid through direct activation of cPLA2α, and it also induces the translocation of cPLA2α to the Golgi apparatus. Lactosylceramide has similar effects, but sphingomyelin is inhibitory.

cPLA2α is usually considered a pro-inflammatory enzyme in that it is the first step that leads to the production of the prostaglandins and leukotrienes, which tend to stimulate inflammatory processes. On the other hand, this enzyme is also responsible for the specific release of 15-hydroxyeicosatetraenoic (15-HETE) acid from a storage form in phospholipids of macrophages when required for the synthesis of the pro-resolving lipid mediators, the lipoxins. It is noteworthy that activation of cPLA2 is responsible for the massive production of pro-inflammatory eicosanoids that accompanies inflammation induced by systemic flagellin or the lethal anthrax toxin. Thus, prostaglandin synthesis via cyclooxygenase-1 can threaten life when produced systemically rather than acutely.

sPLA2 is an inducible enzyme that enhances the effects of cPLA2 to control the magnitude and duration of elevated free fatty acid levels including that of arachidonic acid. A peroxisomal Ca2+-independent phospholipase has only recently been identified, but may be of particular importance for eicosanoid production in that it generates arachidonoyl species, such as 2-arachidonoyl lysophosphatidylcholine, with high specificity.

A further isoform of phospholipase A2 (sPLA2-IID), present in lymphoid tissue and skin, has a high specificity for the mobilization of omega-3 fatty acids and the pro-resolving lipid mediators derived from docosahexaenoic acid (DHA). In effect, it is an immunosuppressive sPLA2 that tips the micro-environmental lipid balance toward an anti-inflammatory state.

It should not be forgotten that the other products of the reaction, lysophospholipids, have distinct physiological activities, although the reverse reaction in which lysophosphatidylinositol is re-acylated also occurs. For example, a membrane-bound O-acyltransferase (MBOAT7) specific for lysophosphatidylinositol with a marked preference for arachidonoyl-CoA has been characterized from neutrophils, and this may be a means by which free arachidonic acid and eicosanoid levels are regulated.

Enzyme coupling: As arachidonic acid is relatively mobile and can diffuse out of the cell or be re-incorporated into lipids, most eicosanoid production occurs in very close proximity to cPLA2 activity, so enzymes that can migrate to the perinuclear and endoplasmic reticulum membranes where this phospholipase is located can participate preferentially in arachidonic acid metabolism. This is well established for the synthesis of prostanoids, but is also true for the 5-lipoxygenase pathway. Similarly, the close proximity of some cell types can facilitate the transfer of eicosanoids between cells for further metabolism. In addition, cPLA2 can switch the formation of eicosanoids of a pro-inflammatory type to those that inhibit inflammation.

Other relevant enzymes: Adipose tissue lipase may hydrolyse triacylglycerols in cytoplasmic droplets of mast cells to provide unesterified arachidonic acid for eicosanoid biosynthesis.

3.   Catabolism of Eicosanoids

Efficient mechanisms for catabolism and deactivation of eicosanoids are essential for the regulation of their biological activities. While there are specific catabolic enzymes for thromboxanes and some leukotrienes, there is one major catabolic pathway that is common to most if not all other eicosanoids and is important for the control of their signalling activities. The first step consists in the oxidation of the 15(S)-hydroxyl group by a 15‑hydroxyprostaglandin dehydrogenase, which exists in two forms with an NAD+-dependent enzyme displaying the greater activity. This enzyme metabolizes E-series prostaglandins, lipoxins, 15-HETE, 5,15-diHETE, and 8,15-diHETE, and probably many others to the corresponding 15-keto compounds. However, it is now recognized that α,β-unsaturated keto-eicosanoids generated in this way are electrophilic and may interact with nucleophilic centres in proteins and other molecules to modify their activities.

Catabolism of eicosanoids

The second catabolic step consists of reduction of the Δ13 double bond by a Δ13-15-ketoprostaglandin reductase (NADPH/NADH dependent) to give an inactive product. This reductase was first identified as active against leukotriene B4, but is now known to metabolize many prostaglandins and lipoxins. Further catabolism of prostaglandins, HETEs (except for 5-HETE) and lipoxins occurs by the beta-oxidation pathway common to fatty acids in general, i.e. via the carboxyl end of the molecule, leading to the formation of short-chain metabolites, which are excreted in the urine. As an example, the process is illustrated for prostaglandin PGE2. 5-HETE and leukotrienes undergo beta-oxidation from the omega-terminus following an initial omega-hydroxylation. Some of these eicosanoids are also excreted following glucuronidation.

4.   Analysis

As eicosanoids and other oxylipins tend to occur at low levels only in tissues and they have such a wide range of structures of varying stereochemistry, analysis has become a rather specialized task involving the use in the early years of gas chromatography linked to mass spectrometry but now increasingly of HPLC linked to tandem mass spectrometry with electrospray ionization. Chiral chromatography also has an important role.

Recommended Reading

Lipid listings Credits/disclaimer Updated: May 3rd, 2017 Author: William W. Christie LipidWeb icon