Eicosanoids and Other Oxylipins

An Introduction

The chemistry, biochemistry, pharmacology and molecular biology of eicosanoids and related lipids, including the octadecanoids, 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 the relevant metabolites is oxylipin, defined as a family of oxygenated natural products that are formed from unsaturated fatty acids by pathways with at least one step of mono- or dioxygen-dependent oxidation and so can contain hydroperoxyl, hydroxyl, epoxy, oxo and/or endoperoxide substituents. Simplistically, they are usually described as being synthesised in situ when needed in response to biological stimuli while having a short half-life and acting locally via interactions with receptors or other intracellular effectors. Professors Bengt Samuelsson, Sune Bergström and John Vane were honoured by the award of the Nobel Prize for Medicine in 1982 for their discoveries in this field.

Scottish thistleThe term eicosanoid is used to embrace those lipid mediators derived from C20 polyunsaturated fatty acids, including prostaglandins, thromboxanes, leukotrienes, hydroxyeicosatetraenoic acids and related oxygenated derivatives. Note that the preferred IUPAC name is ‘icosanoid’, but this is largely ignored in the scientific literature. The main precursor fatty acid is 5c,8c,11c,14c-eicosatetraenoic (arachidonic or 20:4(n‑6)), although 8c,11c,14c-eicosatrienoic (dihomo-γ-linolenic or 20:3(n‑6)) and 5c,8c,11c,14c,17c-eicosapentaenoic (20:5(n‑3) or EPA) acids are also relevant (see our web page on 'polyunsaturated fatty acids'). While these have been of special significance from a historical perspective, it is now impossible to discuss such lipids without considering the docosanoids, i.e., 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 other polyunsaturated fatty acids of the n‑3 family (E‑series resolvins are eicosanoids). Nor can octadecanoids (C18) derived from linoleate be ignored. Related C20 and C22 products formed by non-enzymic means (autoxidation) include 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. While unesterified oxylipins are of primary importance, it is becoming evident that some may operate in esterified forms, including as oxidized phospholipids, endocannabinoids and cholesterol esters (all with separate web pages on this site).

It is noteworthy that the precursors for all these metabolites belong to either the omega-6 (n-6) or the omega-3 (n-3) families of polyunsaturated fatty acids. As oxylipins are so numerous and operate in so many different ways, they must provide a substantial component of the reason for the essentiality of these fatty acids for the survival and well-being of animals.

Other oxygenated fatty acids occur in nature that may not be lipid mediators, and these are discussed in a further web page here..., while the Fatty Acid esters of Hydroxy Fatty Acids (FAHFA), discovered relatively recently, should perhaps be classified as oxylipins now from a functional perspective. Radical nitrogen species can react with unsaturated fatty acids to produce nitro fatty acids, which are anti-inflammatory agents, and those aldehydes produced by cleavage of fatty acid hydroperoxides must be discussed in this context also.

A further collective term - the epilipidome - is increasingly being applied that encompasses these and other lipid mediators, i.e., a subset of the natural lipidome formed by lipid modifications via enzymatic and non-enzymatic reactions (e.g., oxidation, nitration, sulfation, halogenation) and required to regulate complex biological actions.

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. In this document, I introduce some basic concepts and discuss the primary rate-limiting enzyme for eicosanoid production in animal tissues, i.e., phospholipase A2. The mechanism for catabolism of oxylipins is common to most eicosanoid classes and for convenience is discussed below. Further web pages in this series 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, signalling or physiology, where the balance between different oxylipins and their pro- and anti-inflammatory effects is crucial. As many as 500 oxylipins have been detected in human plasma (if not fully characterized), and each of these may have its own role in metabolism. Those requiring a deeper insight should consult the publications cited below and the other documents and references in this section of the website.

1.   The Elements

Arachidonic acidOf the relevant 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 such as phosphatidylinositol, it is a factor in the integrity of cellular membranes. The four cis-double bonds mean that the molecule is highly flexible, and this helps to confer an appropriate degree of fluidity to membranes. Even when arachidonic acid is esterified, lipids can sometimes act biologically, and for example, diacylglycerols enriched in arachidonic acid and derived from phosphatidylinositol are cellular messengers. Anandamide or N‑arachidonoylethanolamine is an endogenous cannabinoid or 'endocannabinoid', which produces neurobehavioral responses like those induced by the phytocannabinoids from cannabis and has 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 to anandamide. Indeed, there are suggestions that arachidonic acid per se may have some distinctive function in animal tissues; for example, the cellular level of unesterified arachidonic acid may be a mechanism by which apoptosis is regulated. It is reported to be a safe protective agent against blood flukes of the genus Schistosoma by inducing the tegument-associated neutral sphingomyelinase of the parasites to disrupt the membrane.

The oxygenated metabolites derived from arachidonic and related fatty acids are produced through a series of complex interrelated biosynthetic pathways that is sometimes termed the 'arachidonate or eicosanoid cascade', and the structures of some of these eicosanoids are illustrated below.

Structures of some eicosanoids

The prostanoids (prostaglandins, thromboxanes and prostacyclins) have 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. Most eicosanoids are produced enzymatically with great stereochemical precision, and this is necessary for their functions. They are highly potent in the nanomolar range in vitro in the innumerable activities that have been defined, mainly 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.

There are selective mechanisms for incorporation of arachidonate into membrane phospholipids that include the formation of coA esters and remodelling by the Lands cycle before this can be released for oxylipin production. Biosynthesis of eicosanoids then involves the action of multiple enzymes, several of which can be rate limiting. In the first step, arachidonic acid is released from membrane phospholipids in tissues 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, via cyclooxygenases (COXs), lipoxygenases (LOXs) and epoxygenases of the cytochrome P-450 family (CYPs); the last are also used for sterol and bile acid oxidation. 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 characteristic synthases. The figure below summarizes in simplistic terms the various pathways for formation of eicosanoids from phospholipid precursors.

Biosynthetic pathways for eicosanoid production

There are several lipoxygenases that act upon different positions of arachidonic acid, mainly 5, 8, 12 and 15, to produce various hydroperoxyeicosatetraenoic acids (HPETEs) and thence the 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 (HETEs) and epoxides (EETs) as the primary products (with dihydroxy acids or DHET as secondary metabolites). While many of the requisite enzymes, precursors and products are only present in certain types of cells, the proximity of some cell types can facilitate the transfer of eicosanoids between them for further metabolism, and some of the leukotrienes are produced by trans-cellular mechanisms.

Most cell types can produce eicosanoids from phospholipid-derived precursors in this way, although much research has been concerned with those cells that are part of the innate immune system. 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, and this can be released by adipose triacylglycerol lipase as a substrate for production of eicosanoids when the cells are stimulated appropriately. Indeed, there are now suggestions that lipid droplets in all cell types are crucial for the response mechanisms to cellular stress and act as hubs to integrate metabolic and inflammatory processes. Via their lipolytic machinery, they regulate the availability of fatty acids for production of oxylipins from polyunsaturated fatty acids and thence for the induction of signalling pathways.

Eicosanoids are generated mainly from unesterified fatty acids, not the CoA esters, and they act in this form, but it is increasingly recognized that they may occur and that some may indeed be synthesised while esterified to other lipids. The latter may simply be inert storage molecules that are available for when a rapid response is required, but some may even act in ester form, especially with oxygenated forms of endocannabinoids. Ultimately, many of the eicosanoids produced in this way are directed to the cellular membranes where they can interact with receptors, which include cell-surface G-protein-linked receptors (GPCRs) and peroxisome proliferator-activated receptors (PPARs).

While the eicosanoids were the first to be identified and studied intensively and I have used them for illustrative purposes in this web page, there is now a body of evidence that docosanoids (protectins, resolvins and maresins, or collectively 'specialized pro-resolving mediators' (SPMs)) derived from the n‑3 family of fatty acids such as EPA and DHA may be equally relevant, often opposing the actions of the eicosanoids. Similarly, octadecanoids derived from linoleate have vital signalling properties. All of these are oxylipins are produced by related means, often using the same enzymes as in eicosanoid production, and they are essential elements of this story.

Biosynthesis of specialized pro-resolving mediators

Formula of jasmonic acidMany parallels can be drawn between the eicosanoids in animals and the structures and functions of plant oxylipins, which are derived primarily from C18 fatty acids like α-linolenic acid (18:3(n-3)), the most abundant polyunsaturated fatty acid in photosynthetic tissues. Jasmonic acid contains a cyclopentanone ring analogous to that in some prostaglandins, and the jasmonates have signalling properties in relation to plant development and defence against pathogens and abiotic stresses of various kinds. Plants do not have the COX enzymes, but they do have lipoxygenases, which in fact were first characterized from plants, and a variety of other oxidases/oxygenases.

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. Fungal species such as Aspergillus, Candida and Cryptococcus spp. that are pathogenic to animals and parasitic protozoa such as Trypanosoma cruzi can produce the same oxylipins as are present in the host, including both eicosanoids and docosanoids, as well as some unique to the organisms by utilizing enzymes that are functionally comparable but evolutionarily distinct from those in animals.

2.   Autoxidation

As well as the enzymatic reactions, lipids can be oxidized non-enzymatically by reactive oxygen and nitrogen species (ROS and RNS, respectively) in all animal and plant tissues in an uncontrolled manner by mechanisms that start with an attack by free radicals on the fatty acid components, followed by chain propagation and ultimately termination steps as discussed more fully here... Such reactions occur on intact lipids in tissues, and the result is usually a complex mixture of positional and stereo isomers of hydroperoxides that can rapidly rearrange or react to form other products that may act metabolically in both the lipid-bound and free states, and they are a major cause of food spoilage. Those autoxidation products generated from ROS are discussed in a number of our web pages, and they include the oxidized phospholipids, isoprostanes and secondary oxygenated metabolites, often with reactive electrophilic carbonyl groups, such as short-chain aldehydes. Indeed any unsaturated lipid, including sterols, can be a source of oxidized metabolites. While myeloperoxidase can catalyse addition of hypochlorous acid (HOCl) to double bonds in unsaturated fatty acyl chains via radical intermediates to form chlorohydrins of significance to disease states, a more important reaction is with the vinyl ether bond in plasmalogens discussed in that web page. Analogous reactions with RNS produce nitro fatty acids.

Pathways leading to reactive oxygen/nitrogen species and to free radicals

Tissues have developed complex defensive measures involving antioxidants and antioxidant enzymes to counter the potentially deleterious effects of lipid radicals and other autoxidation products, and these are discussed in our web pages on tocopherols (vitamin E) and coenzyme Q.

3.   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 by hydrolysis of the ester bond at the sn-2 position of membrane phospholipids, which are usually enriched in this acid; the other products are lysophospholipids. Depending on the tissue and physiological conditions, phosphatidylcholine and phosphatidylethanolamine can be substrates for arachidonic acid release in the same way. This reaction sets in motion a cascade of cellular processes that utilize cyclooxygenases, lipoxygenases and cytochrome P450 oxidases, the key enzymes in the biosynthesis of oxylipins of all kinds.

Generation of arachidonic acid and eicosanoids from phosphatidylinositol

Many enzymes of the phospholipase A2 type have been characterized in a PLA2 superfamily with 16 sub-families (classified on the chronology of their discovery), and three main types have been identified that are relevant here: cytosolic calcium-dependent PLA2 (cPLA2), cytosolic calcium-independent PLA2 (iPLA2, Group VIA) and secreted PLA2 (sPLA2, Group V). They are all water-soluble enzymes with structures that enable association with membranes, and each has a characteristic catalytic site, a Ser-Asp dyad where the substrate binds, and an interfacial surface that facilitates contact with cellular membranes (or supramolecular structures such as micelles, vesicles and liposomes in an aqueous environment). In general, different isoforms of these and other phospholipases of this type have characteristic locations in tissues, cell types or organelles, where they each have particular substrate specificities and presumably regulatory mechanisms. In humans, cPLA2 has a preference for phospholipids containing arachidonic acid, whereas iPLA2 favours EPA, and sPLA2 prefers DHA as substrate, all in position sn-2. The nature of the alkyl ester moiety in position sn-1 is sometimes relevant in that cPLA2 selects the vinyl ether phospholipid (plasmalogen) over the diester form, while iPLA2 has comparable activity with both, and sPLA2 prefers diester phospholipids.

The cytosolic Ca2+-dependent phospholipase A2 (the isoform 'cPLA2α' or 'Group IVA cPLA2') has a marked preference for phospholipids containing arachidonic acid in the sn-2 position, and there is clear evidence that it is necessary for 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 the 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. It is believed that the specificity of cPLA2α is due to an interaction between certain of its aromatic residues with the double bonds of arachidonic acid, although also with EPA but perhaps surprisingly not towards DHA (which does not have a Δ5-double bond).

For this purpose, 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. As well as control via transcriptional regulation, the enzyme is responsive to various stimuli, such as hormones, cytokines and neurotransmitters. The lipids ceramide-1-phosphate and phosphatidylinositol 4,5‑bisphosphate bind to the enzyme, the latter in a 1:1 stoichiometry, and this is required for activation and translocation of the enzyme to the site of action. Ceramide-1-phosphate increases the inflammatory response and can inhibit wound healing by stimulating release of arachidonic acid directly via cPLA2α with translocation of this enzyme to the Golgi apparatus. Lactosylceramide stimulates the enzyme but sphingomyelin is inhibitory.

Scottish thistlecPLA2α 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. In this manner, cPLA2 is responsible for the massive production of pro-inflammatory eicosanoids that accompanies inflammation induced by systemic flagellin or the lethal anthrax toxin, so prostaglandin synthesis via cyclooxygenase-1 can threaten life when produced systemically rather than acutely. On the other hand, this enzyme is responsible for the release of 15‑hydroxyeicosatetraenoic acid (15-HETE) from a storage form in phospholipids of macrophages when required for the synthesis of the pro-resolving lipid mediators, the lipoxins, i.e., it can switch the formation of eicosanoids of a pro-inflammatory type to those that inhibit inflammation. In healthy mitochondria, the main phospholipase responsible for Ca2+-activated release of arachidonic acid is phospholipase A2ζ (cPLA2ζ)

sPLA2 occurs as a diverse family of enzymes with varying tissue and cellular distributions that hydrolyses phospholipids to release lysophospholipids and free fatty acids when there is an increase in intracellular calcium concentration. It is an inducible enzyme that enhances cPLA2 to control the magnitude and duration of elevated free fatty acid levels including that of arachidonic acid. It acts primary on the sn‑2 position of negatively charged phospholipids, but some isoforms may hydrolyse other positions. In contrast to cPLA2α, Ca2+ is required both for binding of the substrate phospholipids and for activation of most isoforms of the enzyme. A peroxisomal Ca2+‑independent phospholipase has only recently been identified, but it may be of special relevance to eicosanoid production in that it generates arachidonoyl species, such as 2-arachidonoyl-lysophosphatidylcholine. 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 subsequent formation of pro-resolving lipid mediators (SPMs) from EPA and DHA. In effect, it is an immunosuppressive sPLA2 that tips the micro-environmental lipid balance toward an anti-inflammatory state.

iPLA2 has no preference for arachidonic acid, and it appears to have only a minor role in eicosanoid production. Rather, it is used mainly in phospholipid re-modelling or general catabolism, where it ensures the availability of the required substrates. However, there is increasing evidence that the Ca2+‑independent iPLA2β mobilizes DHA, especially in the brain, for the biosynthesis of SPMs.

It should not be forgotten that the other products of the phospholipase A2 action, lysophospholipids, have their own physiological properties, while the reverse reaction in which lysophosphatidylinositol is re-acylated can occur to regenerate phosphatidylinositol. As in the Lands cycle for remodelling of phospholipids, a membrane-bound O-acyltransferase (MBOAT7) acting upon lysophosphatidylinositol with a marked preference for arachidonoyl-CoA has been characterized from neutrophils, and this may be a means by which free arachidonic acid, eicosanoid and phosphoinositide levels are regulated. All members of the phospholipase A2 family are of course required for many other purposes as part of the normal turnover of membrane lipids, and for digestion of dietary phospholipids, host defence against bacterial infections and the production of lysophospholipids as signalling mediators.

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, so enzymes that can migrate to the perinuclear and endoplasmic reticulum membranes, where this phospholipase is located, can participate preferentially in arachidonic acid metabolism. For the synthesis of prostanoids, the newly mobilized arachidonic acid must cross the membrane bilayer from the cytosolic to the lumenal side where the cyclooxygenase enzymes are located. Some passive diffusion is possible but transport by fatty acid-binding proteins (FABP) is also likely.

Other relevant enzymes: Adipose tissue lipase may hydrolyse triacylglycerols in cytoplasmic droplets of mast cells to provide unesterified arachidonic acid for eicosanoid biosynthesis, while catabolism (hydrolysis) of endocannabinoids will also release arachidonic acid. In plants and fungi, release of fatty acids by the action of various acylhydrolases on membrane complex lipids is the first step towards the production of oxylipins for signalling purposes.

4.   Catabolism of Eicosanoids

Efficient mechanisms for catabolism and deactivation of eicosanoids are necessary for the regulation of their metabolism. While thromboxanes and some leukotrienes have their own catabolic enzymes, there is one major type of catabolic pathway that is common to most if not all other eicosanoids and thus is a control on their signalling. The first step occurs in the cytoplasm of cells and with prostanoids consists in the oxidation of the 15S‑hydroxyl group by 15‑hydroxyprostaglandin dehydrogenase, one of a family of oxidoreductases that acts on the CH-OH group of a donor molecule with NAD+ or NADP+ as acceptor in a process mediated by solute carrier organic anion transport protein family member 2A1 (SLCO2A1). This enzyme metabolizes E-series prostaglandins, lipoxins, 15‑HETE, 5,15‑diHETE, 8,15‑diHETE and probably many others to the corresponding 15-keto compounds. A further enzyme, dehydrogenase reductase 9 (SDR9C4), recognizes a broad spectrum of lipid mediator oxylipins as substrates and oxidizes hydroxyl groups at carbons C9 and C13 of octadecanoids, C12 and C15 of eicosanoids (but not C5), and C14 of docosanoids. As an example, the process is illustrated for prostaglandin PGE2 with PGE-M as an end-product.

Catabolism of eicosanoids

The second catabolic step with prostanoids consists of reduction of the Δ13 double bond by a Δ13-15-ketoprostaglandin reductase (NADPH/NADH dependent) to give an inert product. This reductase was first identified 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 by peroxisomal enzymes, i.e., via the carboxyl end of the molecule (although some oxidation of the terminal methyl group can occur), leading to the formation of short-chain metabolites, which are excreted in the urine often following glucuronidation. 5-HETE and leukotrienes undergo beta-oxidation from the omega-terminus following an initial omega-hydroxylation, and such variations to the general mechanism are discussed in the web pages dealing with each class of oxylipin.

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 properties. In mammals, 15‑hydroxyprostaglandin dehydrogenase is a key regulatory enzyme in many physiological and pathological processes. It is considered to be a potential pharmacological target for preventing organ damage and enhancing tissue regeneration, and for resisting the complex pathology of aging-associated diseases as it accumulates in aging tissues. As a tumour suppressor, it inhibits proliferation of cancer cells, including colorectal, lung and breast cancers.

There is now evidence that many oxylipins are not processed by this route, but rather carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of main-stream fatty acids, can remove oxylipins from cells during inflammation in vitro and in vivo for beta-oxidation. Indeed, it has been proposed that mitochondrial β-oxidation is a regulatory metabolic checkpoint for oxylipins.

5.   Analysis

As eicosanoids and other oxylipins tend to occur at low levels only in tissues, have such a wide range of structures of varying stereochemistry and short half-lives, analysis has become a rather specialized task for gas chromatography linked to mass spectrometry in the early years but now increasingly of HPLC linked to tandem mass spectrometry with electrospray ionization. Chiral chromatography has a role as the stereochemical forms of oxylipins are often crucial (as is the cis/trans geometry of double bonds).

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