The LipidWeb blank

Hydroxyeicosatetraenoic Acids and Related
Mono-Oxygenated Fatty Acids



The oxygenated metabolites or oxylipins derived from arachidonic and related fatty acids are produced through a series of complex interrelated biosynthetic pathways often termed the 'eicosanoid cascade'. The prostanoids (prostaglandins, thromboxanes and prostacyclins) have distinctive ring structures in the centre of the molecule and are discussed on their own web page. Here, the linear hydroxyeicosatetraenes and related mono-oxygenated metabolites are described, together with octadecanoids produced from linoleate and similar fatty acids from eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. While these are relatively simple in structure, they are precursors for families of more complex molecules, such as the leukotrienes and lipoxins. The two main enzymatic pathways for production of these eicosanoid utilize lipoxygenases (LOXs) and enzymes of the cytochrome P-450 family.


1.   Lipoxygenases and Hydroxyeicosatetraenoic Acids

Lipoxygenases are a family of enzymes that can be characterized as non-heme iron proteins or dioxygenases, which catalyse the abstraction of hydrogen atoms from bis-allylic positions of fatty acids while adding oxygen to generate hydroperoxide products. They occur widely in plants, fungi, some prokaryotes (cyanobacteria and proteobacteria) and animals, but not in the archaea and perhaps insects. The plant lipoxygenases have distinctive substrates and products, and they are described in our web page dealing with plant oxylipins rather than here, although interesting parallels can be drawn with the mechanisms and functions of the animal enzymes.

Animal lipoxygenases that utilize arachidonic acid as substrate are of great biological and medical relevance, because of the functions of the products in signalling or in inducing structural or metabolic changes in the cell. For example, they react with arachidonic acid per se to produce specific hydroperoxides and thence by downstream processing the plethora of eicosanoids, each with distinctive functions, which are described in these pages. However, they can also react directly with phospholipids in membranes to produce hydroperoxides that perturb the membrane structure. Thence, programmed structural changes in the cell can be induced, as in the maturation of red blood cells. In addition, phospholipid hydroperoxides can stimulate the formation of secondary products. Lipoxygenases can attack low-density lipoproteins directly with major implications for the onset of atherosclerosis.

The nomenclature of animal lipoxygenases is based on the specificity of the enzymes with respect to the products of the reaction with arachidonate (not the initial point of hydrogen abstraction); for example, 12-LOX oxygenates arachidonic acid at carbon-12. The stereochemistry of the reaction can be specified when necessary (e.g. 12R-LOX or 12S-LOX), although the more important enzymic hydroperoxides have the S-configuration. Where more than one enzyme has the same specificity, it may be named after the tissue in which it is found, and there are platelet, leukocyte and epidermal types of 12-LOX, for example.

As the research in this area has developed, this simplistic nomenclature has become confusing. It has become evident that some enzymes can oxygenate more than one position and that this can vary with the chain-length of the polyunsaturated substrate and the positions of the double bonds. Enzymes with specificities for four different positions in arachidonic acid occur in animal tissues, i.e. 5-LOX, 8-LOX, 12 LOX, and 15-LOX, although some of these have dual specificities, while many iso-forms exist depending on species. There are now considered to be six main lipoxygenase family members in humans (5-LOX,12-LOX,12/15-LOX (15-LOX type 1), 15-LOX type 2, 12(R)-LOX, and epidermal LOX) and seven in mice. Orthologues of the same gene have different reaction specificities in different species. For example, mice do not express a distinct 15 LOX but rather a leukocyte-derived 12 LOX with some 15-LOX activity, so it can be difficult to extrapolate from animal experiments to human conditions. The positions at which the enzymes interact with arachidonic acid and the main products are illustrated in the figure below.

Reactions of lipoxygenases with arachidonic acid

Each of the lipoxygenase proteins in animal tissues has a single polypeptide chain with a molecular mass of 75-80 kDa. They have a N‑terminal 'β‑barrel' domain, which is believed to function in the acquisition of the substrate, and a larger catalytic domain containing a single atom of non-heme iron, which is bound to conserved histidine residues and to the carboxyl group of a conserved isoleucine at the C-terminus of the protein. For catalysis, the iron component of the enzymes must be oxidized to the active ferric state.

All of the enzymes appear to include the fatty acid substrate within a tight channel with smaller channels that direct molecular oxygen toward the selected carbon, facilitating the formation of specific hydroperoxy-eicosatetraenes (HPETEs). In other words, the regiospecificity is regulated by the orientation and depth of substrate entry into the active site, while stereospecificity is controlled by switching the position of oxygenation on the reacting pentadiene of the substrate at a single active enzyme site, which is conserved as an alanine residue in S‑lipoxygenases and a glycine residue in the rarer R‑lipoxygenases. There is evidence that two amino acids opposite the catalytic iron ion determine the orientation of the substrate for entry into the enzyme channel. With 5-LOX and 8-LOX, the carboxyl group of arachidonic acid enters the active site, while with 12-LOX and 15-LOX the ω-terminus enters the site and facilitates the activity. It should be noted that the specificities of the enzymes are not always absolute and can differ between species.

Lipoxygenase action is believed to proceed in four steps - hydrogen abstraction (1), radical rearrangement (2), oxygen insertion (3), and peroxy radical reduction (4), all occurring under steric control, as illustrated.

Mechanism of lipoxygenase action

For example, in the action of 5-LOX, the first and rate-limiting step is the abstraction of a hydrogen atom from carbon 7 by ferric hydroxide, involving a proton-coupled electron transfer in which the electron is transferred directly to the iron(III) to produce a substrate radical, while the iron atom is reduced to the ferrous form. The cis-double bond in position 5 migrates to position 6 with a change to the trans-configuration. The structure of this radical is uncertain, as are the details of the next steps in which the di-oxygen moiety (from molecular oxygen) is added, leaving the hydroperoxyl moiety in position 5. With the reduction step, the resulting product is 5S-hydroperoxy-6t,8c,11c,14c-eicosatetraenoic acid (5-HPETE). In the process, the iron atom is re-oxidized to its ferric form.

HPETE have a short half-life and are rapidly metabolized, for example leading to hydroxy-eicosatetraenes (HETE) with the same stereochemistry via reduction by glutathione peroxidases. Alternatively, isomerization reactions can occur to produce leukotrienes and lipoxins. While their primary function is to act as intermediates in the biosynthesis of other eicosanoids, HPETE also have some biological activities of their own.

5-LOX is found only in cells derived from bone marrow (leukocytes, macrophages, etc) and it is of particular interest as the product is the primary precursor for the leukotrienes. In contrast to other lipoxygenases, it requires the presence of a specific activator protein - lipoxygenase-activating protein (FLAP) - on the perinuclear membrane. This facilitates the transfer of arachidonic acid to the active site on 5-LOX and is believed to effect the functional coupling of phospholipase A2 (cPLA2) to 5-LOX at the membrane. It is noteworthy that both cPLA2 and 5-LOX are Ca2+-dependent. The activities of 5-LOX and related enzymes are regulated by the concentration and availability of the substrates and by phosphorylation-dephosphorylation by protein kinases.

Scottish thistle8-, 12- and 15-LOX operate in a similar way to give analogous products. 15-LOX has a broader specificity, and in human leukocytes it is sometimes termed the 12/15-LOX (or 15-LOX-1) as it can also produce some 12-HETE, 8,15-diHETE and eoxin A4. It is able to oxidize linoleate to 13‑hydroperoxyoctadecadienoate (and in part to the 9-isomer), and it differs from the others in that it can utilize arachidonate bound to phospholipids as a substrate, hence the interest in the role of the enzyme in membrane disruption and in disease states. Uniquely, it synthesises both pro- and anti-inflammatory molecules.

12-LOX from human platelets and leukocytes was one of the first lipoxygenases to be characterized, but a rather different enzyme is present in the epidermis. Although lipoxygenase metabolites generally have a hydroperoxide moiety in the S-configuration, lipoxygenases in mammalian skin can produce the R-form. Indeed, 12R-HETE was first characterized as a component of psoriatic lesions. One of the enzymes responsible is a second form of the human 15-lipoxygenase (15-LOX-2), but there is also a 12R-LOX with quite specific functions in keratinocytes and certain other tissues, especially in relation to linoleate metabolism. Enzymes related to the latter are common in aquatic invertebrates. Mouse skin produces a lipoxygenase that is structurally related to 15-LOX-2, but generates 8S-HETE and 8S,15S-diHPETE from arachidonic acid, so it is closer in its properties to human 12-lipoxygenase.

Hydroxy fatty acids produced in this way can be further oxidized to their keto analogues (c.f. 5-oxo-eicosatetraenoic acid below) or to dihydroxy derivatives that include the leukotrienes discussed in a separate web page. In addition, 5-, 12- and 15-HETEs can be esterified to phospholipids in tissues, often with some specificity. For example, 15-HETE is selectively esterified to phosphatidylinositol in lung and kidney epithelial cells and in aortic endothelial cells, while 12-HETE occurs predominantly in phosphatidylcholine in microsomal membranes. In neutrophils, 5-HETE is incorporated mainly into phosphatidylethanolamine plasmalogens and phosphatidylcholine. Similarly, appreciable amounts of 15-HETE can be formed by a direct action of 15-LOX upon arachidonate esterified to phosphatidylethanolamine. There are suggestions that cholesteryl arachidonate in the lipoprotein LDL is a good substrate for 12/15-lipoxygenase and other oxidizing agents and that the products are a causative factor in atherosclerosis; some transfer of the HETE component to phospholipids is believed to occur. Non-enzymatic oxidation (autoxidation) can also occur by the initial steps described in our web page on isoprostanes.


Fungal enzymes: Fungi and yeasts are able to produce 3R- and/or 3S-HETE and 3,18-di-HETE, when supplied with exogenous arachidonic acid. The biochemical mechanism is unclear, and there are reports that implicate lipoxygenases, cyclooxygenases and mitochondrial β-oxidation. With some pathogenic fungi, the 3-hydroxyeicosanoids produced in infected cells can be acted upon by the host COX-2 enzyme to form a family of 3-hydroxy-prostaglandins, which are at least as active biologically as the normal compounds. Also, Candida albicans and some related species appear to produce prostanoids, including PGE2, by a cyclooxygenase pathway.


2.   Cytochrome P450 Oxidases and Hydroxyeicosatetraenoic Acids

Arachidonic acid per se (not esterified forms) can be oxidized by several cytochrome P450 mixed-function oxidases to produce various HETE isomers (the name was coined to describe the first such enzyme to be characterized and was based on an unusual absorbance peak at 450 nm from its carbon monoxide-bound form). These enzymes are membrane-bound hemoproteins that catalyse the activation of molecular oxygen and the transfer of a single atomic oxygen to a substrate carbon atom, i.e. they are monooxygenases. The result is the introduction of either a hydroxyl or an epoxyl group into the molecule. The catalytic turnover of the reaction is NADPH-dependent, requiring transfer of electrons from NADPH to the P450 heme iron (lipoxygenases use non-heme iron), for which a membrane-bound enzyme, NADPH-cytochrome P450 reductase, is essential.

Cytochrome P450 oxidases are found in all mammalian cell types and indeed appear to be ubiquitous in higher organisms, although the number and distribution of particular forms of the enzymes are specific both to cell type and the species. In addition to their role in generating HETE isomers, enzymes of this kind have a more general function in the eicosanoid cascade in the metabolism of prostanoids, and they are involved in cholesterol and steroid metabolism. The contribution of cytochrome P450 oxidases to HETE production relative to that of the lipoxygenases has still to be determined.

Three types of reaction have been observed in animal cells, leading to the formation of three distinct families of eicosanoids. For example, one series of reactions occurs at bis-allylic centres and is lipoxygenase-like in the nature of the ultimate HETE products, although hydroperoxy intermediates are not involved. Thus, microsomal cytochrome P450 oxidases can react with arachidonic acid to produce six regioisomeric cis,trans-conjugated dienols, i.e. with the hydroxyl group in positions 5, 8, 9, 11, 12 or 15. The mechanism is believed to involve bis-allylic oxidations at either carbon-7, 10 or 13, followed by acid-catalysed rearrangement to the cis,trans dienol. Two examples of the products are illustrated. 12(R)-HETE as opposed to the 12(S)-isomer is the main product of the reaction, and this was at one time though to be a distinguishing feature, but some other lipoxygenases are now know to produce the former enantiomer.

Internal cytochrome P450 hydroxylation of arachidonic acid

Secondly, there are ω- and (ω-1)-hydroxylases that introduce a hydroxyl group into positions 20 and 19, respectively, of arachidonic acid mainly, although enzymes are present in liver that can react at positions 16, 17 and 18 also. The reaction was first observed with medium-chain saturated fatty acids, such as lauric, where it may play a role in oxidative catabolism. Some isoenzymes are specific for laurate, others for arachidonate, and some will utilize both fatty acids as substrates. In humans, the iso-forms CYP4A and CYP4F are the main enzymes involved in ω‑hydroxylation of polyunsaturated fatty acids, including both arachidonic and eicosapentaenoic acids, while the CYP1A1, CYP2C19, and CYP2E1 forms effect (ω‑1)-hydroxylation. In aortic endothelial cells, 20-HETE has been detected in esterified form in several phospholipid classes.

Production of omega- and omega-1-HETE

Epoxyeicosatrienoic acids: The third series of reactions of P450 arachidonic acid monooxygenases involves the formation of epoxytrienoic acids (‘EET’) from arachidonic acid, i.e. four cis-epoxyeicosatrienoic acids (14,15-, 11,12-, 8,9-, and 5,6-EETs). Apart from the 5,6-isomer, they are relatively stable molecules.

Several iso-enzymes of the cytochrome P450 epoxygenase exist, with CYP2C and CYP2J as the most active, and they can produce all four EET regioisomers, although one isomer usually tends to predominate. For example, epoxygenases that produce 14,15-EET as the main isomer also synthesise a significant amount of 11,12-EET and a little 8,9-EET. The epoxygenase attaches an oxygen atom to one of the carbons of a double bond of arachidonic acid, and as the epoxide forms the double bond is reduced. The enzymes are located in the endoplasmic reticulum of endothelial cells mainly, and they make use of arachidonic acid that is hydrolysed from phospholipids when the Ca2+-dependent phospholipase A2 is activated and translocated from the cytosol to intracellular membranes.

Biosynthesis of epoxy-eicosanoids

The proportions of the various isomers depend on tissue and species, although the 11,12- and 14,15-EET generally tend to predominate. In the rat, 14,15-EET amounts to about 40% of those produced in the heart, while 11,12-EET represents 60% of those produced in the kidney, for example. In addition, each of these regioisomers is a mixture of R,S- and S,R-enantiomers, and each iso-enzyme produces variable proportions, differing even among regioisomers. Eight isomers can be formed, therefore, each with somewhat different biological activities.

The epoxygenases require the fatty acid substrate to be in the unesterified form, but the products can be esterified later. Thus, significant amounts of epoxyeicosatrienes are found esterified to position sn-2 of phospholipids, including phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, perhaps as a storage form that is available when a rapid response is required. For example, free epoxyeicosatrienes are released following activation of phospholipase A2 by neuronal, hormonal or chemical stimuli. There is also a possibility that esterified epoxy-eicosanoids may have a biological function within membranes. The presence of esterified EETs in plasma, suggests that some exchange between tissues is possible, although most are believed to be produced close to the site of action. In many tissues, the esterified epoxy-eicosanoids are so similar in composition to those in the free form, that the conclusion must be that they are entirely products of enzyme action. On the other hand, non-enzymic lipid peroxidation has been observed in erythrocytes in vitro, and some EETs may arise by this route.

In addition, phospholipids containing EET are substrates for the production of lipid mediators such as 2-epoxyeicosatrienoylglycerols (analogous to the endocannabinoid 2-arachidonoylglycerol). Kidney and spleen, for example, synthesise 2-glycerol derivatives containing 11,12-EET or 14,15-EET, which are endocannabinoids and exert biological effects by activating the CB1 and CB2 receptors. Similarly, phospholipids containing EET are probable substrates for synthesis of EET-ethanolamide in the liver and kidney.

EETs are rapidly metabolized in vivo to the corresponding dihydroxyeicosatrienoic acids (DHET) by epoxide hydrolases, of which iso-enzyme forms are known with different cellular locations, i.e. cytosolic or membrane bound. The reaction is illustrated below for the conversion of 14,15-EET to 14,15-DHET.

Action of epoxy-hydrolase

This enzyme metabolizes 8,9-, 11,12- and 14,15-EET efficiently, but 5,6-EET is a poor substrate. In addition, 11,12- and 14,15-EET can undergo partial β-oxidation to form C16 epoxy-fatty acids, or they can be elongated to C22 products. 5,6- and 8,9-EET are substrates for cyclooxygenase. While DHETs were once believed to be merely deactivation products of EETs, they are now known to have some biological effects in their own right.


3.    5-Oxo-Eicosatetraenoic Acid

Structural formula of 5-oxo-eicosatetraenoic acid5-Oxo-6t,8c,11c,14c-eicosatetraenoic acid (5-Oxo-ETE) is a metabolite of 5S-hydroxy-6t,8c,11c,14c-eicosatetraenoic acid (5-HETE), produced by oxidation by 5-hydroxy-eicosanoid dehydrogenase, an enzyme found in the microsomal membranes of white blood cells (leukocytes), platelets and especially of eosinophils and neutrophils. The enzyme requires the presence of a 5S-hydroxyl group and a trans-6 double bond in the eicosanoid, and NADP+ is a cofactor. Synthesis of the metabolite is stimulated during periods of oxidative stress. In addition, some 5-oxo-ETE may be formed directly from 5‑hydroperoxyeicosatetraenoic acid, possibly by a non-enzymic route. In neutrophils, a high proportion is rapidly incorporated into the triacylglycerol fraction.

It appears that 5-hydroxyeicosanoid dehydrogenase can also catalyse the reverse reaction, i.e. the reduction of 5-oxo-ETE, and this seems to be of particular important in platelets. The biological activity of 5-oxo-ETE is of course changed by this reverse reaction, and alternative deactivation can occur by reduction of the double bond in position 6, or by further oxidation either by lipoxygenases or by cytochrome P450 enzymes, the latter in positions 19 or 20.

In fact, all the HETE isomers can be converted to oxo-metabolites by specific hydroxy-eicosanoid dehydrogenases, and the 11-, 12- and 15-isomers possess appreciable biological activity. For example, 15(S)-HETE and 11(R)-HETE are substrates for 15-hydroxyprostaglandin dehydrogenase, the enzyme involved in the first step of prostaglandin catabolism, to yield 15-oxo-ETE and 11-oxo-ETE, respectively, which mediate anti-proliferative properties in endothelial cells. Similarly, 14-hydroxy-docosahexaenoic acid is an good substrate for the enzyme to yield the 14-oxo analogue. 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.


4.   Mono-oxygenated Metabolites of EPA and DHA

Lipoxygenases interact with the other essential polyunsaturated fatty acids of the omega-3 and omega-6 families, especially the former, to give comparable series of metabolites. For example, 5-lipoxygenase generates 4- and 7-hydroxy metabolites from docosahexaenoic acid (22:6(n‑3) or DHA), while 12-lipoxygenase generates 11- and 14-hydroxy metabolites, and 15-lipoxygenase produces a 17-hydroxy metabolite. Further reactions produce the protectins, resolvins and maresins or 'specialized pro-resolving mediators', which have special importance in the resolution of inflammation and have their own web page

The products of the lipoxygenases with arachidonate were soon documented, but it has taken longer to recognize the importance of the metabolites of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, especially the epoxides, produced by the activities of various cytochrome P450 enzymes. Indeed, it is now evident that these n-3 polyunsaturated fatty acids, rather than arachidonic acid, are the preferred substrates for some of the isoforms, specifically the CYP1A, CYP2C, CYP2J and CYP2E subfamily members, which then exhibit very different regio- and stereo-specificities. For example, human CYP1A1 acts mainly as a subterminal hydroxylase with arachidonate producing four different isomers, but with EPA it generates mainly 17(R),18(S)-epoxy-eicosatetraenoate with almost absolute regio- and stereo-selectivity. Similarly, with DHA it epoxidizes the n-3 double bond and produces 19,20-epoxydocosapentaenoate. A number of other isoforms of the cytochrome P450 enzymes produce epoxides by reaction with an n‑3 double bond in the same manner, some much more rapidly than with arachidonate as substrate. One exception is CYP2C9, which oxidizes EPA to 14,15-epoxy-ETE mainly and DHA to 10,11-epoxy-DPE.

Action of cytochrome P450 oxidases on EPA and DHA />

The CYP4A/CYP4F subfamilies are the main enzymes that produce 20-hydroxy-eicosatetraenoic acid from arachidonate in mammals, and they hydroxylate the terminal methyl group in EPA and DHA also at the same rate.

By competing with arachidonate, EPA and DHA may modify the action of the various HETE metabolites, but the oxygenated EPA and DHA compounds have biological properties of their own. For example, significant amounts of DHA epoxides, especially 7,8-epoxydocosapentaenoic acid, are present in the central nervous system of rats, where they ameliorate the effects of inflammatory pain. 17,18-Epoxyeicosatetraenoic acid generated in the gut is an anti-allergic molecule. It has been suggested that such EPA and DHA metabolites may be responsible for some of the beneficial effects associated with dietary n-3 fatty acid intake.


5.   Octadecanoids

The action of lipoxygenases upon linoleic acid in plant tissues is discussed in the web page on plant oxylipins, but this fatty acid is acted upon by lipoxygenases in animal tissues in a similar way to produce 9- and 13-hydroperoxy- and thence hydroxy-octadecadienoic acids (octadecanoids or 'HODEs') of defined stereochemistry. In particular, 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) is generated by the action of 15-lipoxygenase (12/15-LOX) on linoleic acid. Oxo-octadecadienoic acids, epoxy-octadecenoic acid and epoxy-keto-octadecenoic acids are also formed in further reactions.

Reaction of 15-LOX with linoleic acid

12R-LOX reacts readily with linoleate (9,12-18:2) to produce 9R-HPODE. As linoleic acid is a major unsaturated fatty acid in animal tissues, appreciable amounts of these hydroxy and hydroperoxy metabolites can accumulate and influence inflammatory diseases. Indeed, linoleate metabolites are by far the most abundant oxygenated fatty acids in both free and esterified form in human plasma. A further interesting observation is that one of the unique ceramides of skin, O-linoleoyl-ω-hydroxyacyl-sphingosine, is a substrate for 12R-LOX with 9R-hydroperoxy-linoleoyl-ω-hydroxyceramide as the product. This in turn can be converted to hepoxilin-like compounds, i.e. with an epoxyl group, by an enzyme epidermal lipoxygenase 3 (eLOX-3). In addition, 11-HETE and 15-HETE can be produced via the cyclooxygenase pathway. Autoxidation also occurs with linoleate to produce the same types of compound but with more variable stereochemistry.

Enzymes of the cytochrome P450 family make a further contribution and linoleic acid is especially a substrate for CYP epoxygenases yielding the linoleic epoxides 9,10- and 12,13-epoxyoctadecenoic acids, which are further metabolized by epoxide hydrolases to form the diols 9,10- and 12,13-dihydroxyoctadecenoic acids, respectively, which are sometimes termed leukotoxins. Trihydroxy compounds may be formed similarly following sequential action of lipoxygenases and epoxygenases. They are detoxified by conversion to the glucuronides. While similar reactions occur with both α- and γ-linoleate in vitro, the biological significance of these metabolites in vivo is not known.


6.   Biological Activity

A large number of hydroxyeicosatetraenoic acids and related compounds have now been discovered and most of these have some form of biological activity, primarily in signalling. In particular, they modulate ion transport, vascular tone, renal and pulmonary functions, and growth and inflammatory responses through both receptor and non-receptor mechanisms. They are released by the action of growth factors and cytokines, and they attain physiological concentrations much higher than those of prostanoids. This is a field that is developing rapidly and it is evident that the picture is complex and very far from complete. A given eicosanoid of this type can have differing functions in different cell types, and its activity may be opposed or modified by another eicosanoid; the balance between them in a cell may be critical. As animal models can have very different isoforms of enzymes, it is often difficult to translate experiments with other species to human conditions. It is not possible to give a comprehensive picture of these manifold biological activities here, and only a few of the more important are described briefly below.

5S-Hydroxy-6t,8c,11c,14c-eicosatetraenoic acid (5(S)-HETE) is important as the precursor of the leukotrienes and lipoxins, but it has some biological functions in its own right, although these can be difficult to disentangle from those of its metabolites, which are more active. For example, like its metabolite 5-oxo-HETE, 5(S)-HETE activates neutrophils and monocytes. It is also known to stimulate proliferation of cancer cells in a similar manner to certain leukotrienes, and increased amounts are formed in brain tumours, for example. 5-LOX inhibitors have preventive effects.

5-Oxo-6t,8c,11c,14c-eicosatetraenoic acid is a chemo-attractant for eosinophils and neutrophils and has many functions in such cells, including actin polymerization, calcium mobilization, integrin expression and degranulation. Its signalling functions are mediated via a specific receptor ('OXE'), leading to increased intracellular calcium concentrations and inhibition of cAMP production. Thereby, it stimulates the proliferation of prostate tumor cells. In addition, it is believed to be an important mediator in asthma and other allergic diseases, and efforts are underway to find inhibitors of the specific receptor that may have clinical utility.

Scottish thistleArachidonate 8(S)-lipoxygenase and its product 8S-hydroxy-5c,9t,11c14c-eicosatetraenoic acid (8S-HETE) has only been found in the skin of mice. It is a potent activator of the peroxisome proliferator-activated receptor PPARα, it is an anti-tumorogenic agent towards skin cancer, and it promotes wound healing in the cornea. In contrast, a human orthologue of this enzyme (15-LOX-2) is found in skin, sebaceous glands and prostate tissue but produces 15S-HETE.

12S-Hydroxy-5c,8c,10t,14c-eicosatetraenoic acid (12S-HETE) is the precursor of the hepoxilins but has important functions of its own. In nervous tissue, it modulates membrane properties and stimulates melatonin synthesis, for example. Together with 15(S)-HETE, it serves as a secondary messenger in synaptic transmission and is involved in learning and memory processes; increased levels are found in Alzheimer's disease. In leukocytes, it promotes chemotaxis and induces the synthesis of heat-shock protein. It can either stimulate or inhibit aggregation in platelets, depending on species and circumstances, and it stimulates lipoxin synthesis. In addition, 12S-HETE can cause constriction of blood vessels and it deactivates prostacyclin synthase. Particular attention has been devoted to the effects of 12S-HETE on inhibiting the adhesion of cancer cells to endothelial cells, an activity that is linked to metastasis in cancer of the prostate and is mediated via cell surface signalling and activation of protein kinase C. It promotes the proliferation of ovarian cancer cells.

The enantiomeric compound 12R-HETE is believed to be involved in the pathophysiology of psoriasis and similar skin diseases, but it is also essential for the development of normal skin. 12R-HETE produced by cytochrome P450 enzymes may have a function in the eye.

15S-Hydroxy-5c,8c,11c,13t-eicosatetraenoic acid (15S-HETE) is a precursor of the lipoxins and is produced by two enzymes in human tissues, one of which is related structurally to the 12-lipoxygenase of leukocytes. Indeed, this 15-lipoxygenase (15-LOX-1) is unusual in that it produces some 12-HETE in addition to the 15-isomer. The second form of the enzyme was first found in the epidermis, although it is now know to exist in other tissues. It is not clear whether free arachidonic acid is the main substrate in vivo, as the enzyme is certainly able to oxidize arachidonate in phospholipids of membranes and lipoproteins. 15S-HETE has been implicated in cell differentiation, inflammation, asthma, carcinogenesis and atherogenesis. In particular, there is an accumulation of 15-HETE in human carotid plaques, and this is believed to play a role in the induction of atherothrombotic events by increasing platelet aggregation and thrombin generation. 15-HETE appears to contribute to the development of Hodgkin lymphoma, colorectal and many other cancers, but that produced by 15-LOX-2 activates PPARγ, a nuclear transcription factor involved in epithelial differentiation, which may explain an anti-proliferative action on prostate cancer cells.

Of the terminal and near-terminal HETE isomers, 20-HETE is pro-inflammatory and has largely detrimental functions, for example in hypertension, in promoting systemic vasoconstriction and in tumour growth. It regulates vascular smooth muscle and endothelial cells by influencing their proliferation, migration, survival, and tube formation. In the kidney, it blocks re-absorption of sodium by inhibiting the Na+-K+-ATPase. EPA and DHA are potent inhibitors of the biosynthesis of this compound, suggesting that this may be a partial explanation for the physiological role of omega-3 fatty acids. Other HETE isomers appear to act in opposition to 20-HETE, and 18- and 19-HETE, for example, induce vasodilatation by inhibiting the effects of 20-HETE. In addition, they together with 16- and 17-HETE induce re-uptake of sodium in the kidney, and 16‑HETE inhibits neutrophil adhesion so may be important in inflammation. 20-HETE may promote tumor growth, but 8- and 11-HETE have anti-tumor activities. However, no cognate receptor or second messenger has yet been identified for ω-HETE.

The 3-hydroxy-eicosanoids produced by pathogenic fungi may play a role in the inflammatory processes associated with infections by such organisms, as they are strong pro-inflammatory lipid mediators. As they are produced during the reproductive phase of yeast and fungal growth, they may also be important for the organism per se.

Scottish thistleAs the regioisomers and enantiomeric forms have many similar metabolic and functional properties, epoxyeicosatrienoic acids have often been treated as a single class of compounds, although as knowledge has expanded this view is no longer justifiable. 11,12-EET in particular has a number of distinctive activities. The various EETs have major functions as autocrine and paracrine effectors in the cardiovascular and renal systems, which are believed to be largely beneficial. Because of the anti-hypertensive, fibrinolytic, and anti-thrombotic properties of EETs, their presence in red blood cells has important implications for the control of circulation and the physical properties of the circulating blood. Both cis- and trans-EETs are synthesized and stored in erythrocytes, and they are produced and released in response to a low oxygen concentration as during exercise, for example. In the kidney, they modulate ion transport and gene expression, producing vasodilation. In addition, they have anti-inflammatory and pro-fibrinolytic properties. Significant amounts of EETs are incorporated into phospholipids, from which they are rapidly released in the presence of Ca2+ ionophores. It has therefore been suggested that they may be involved in those signal transduction processes mediated by phospholipases. Some of the activities of epoxy-eicosanoids may require cell-surface receptors, though these have yet to be characterized, but others involve intracellular mechanisms, i.e. by direct interaction with ion channels, signalling proteins or transcription factors. In the central nervous system, epoxyeicosanoids may have additional functions, for example in the regulation of the release of neurohormones and neuropeptides. Their concentrations are controlled by soluble epoxide hydrolases.

Until recently, EETs were considered to be relatively benign molecules. However, it has now been demonstrated in mice that they are powerful stimulants for the release from dormancy of primary cancer tumors, for promoting their growth and for triggering metastasis, i.e. the spread of cancer to other organs. Their various metabolites have also been implicated in cancer progression. The dihydroxyeicosatrienoic acids (DHET) produced by epoxide hydrolases may be pro-inflammatory.

17,18-Epoxyeicosatetraenoic acid is the main epoxide regioisomer synthesised from eicosapentaenoic acid. It is a vasodilator and may be responsible for some of the beneficial effects of dietary omega-3 fatty acids. Similarly, 19,20-epoxy-docosapentaenoic acid, derived from DHA, has been shown to have a number of beneficial functions in tissues. However, much less is known of the function of the oxygenated metabolites of EPA and DHA. They appear to act in opposition to HETE isomers and may be especially important in the cardiovascular system and as anti-cancer agents.

Oxidized linoleate metabolites are believed to be atherogenic through the induction of pro-inflammatory cytokines and by formation of foam cells from macrophages by PPAR activation. Their actions on the regulation of inflammation of relevance to the metabolic processes associated with atherogenesis and cancer are attracting special interest. They may also react non-enzymatically with proteins to form potentially toxic adducts. The epoxides derived from linoleic acid have been associated with multiple organ failure and in adult respiratory distress syndrome in burn patients, and their dihydroxy-metabolites are also toxic. On the other hand, there is evidence that a 15-LOX metabolite 13S-HPODE induces apoptosis in colon cancer cells.

Esterified HETE. Many of these lipoxygenase and oxidase products are found naturally in membrane phospholipids where they may serve as a storage form to be released on appropriate stimulation. All mammalian long-chain acyl-CoA synthetase isoforms have the capacity to activate HETE for further esterification, and for example, three 12-hydroxyeicosatetraenoic acid phosphoinositides have been detected in thrombin-activated platelets. On the other hand, such oxidized lipids have the potential to perturb the membrane structure and effect secondary oxygenations that could induce unwanted changes in cells; oxidation of low-density lipoprotein by this means may be important for the initiation of atherosclerosis. Although studies are at a relatively early stage, it is becoming apparent that esterified HETEs may have specific biological functions of their own, especially in relation to immune regulation, signalling and blood coagulation. Those HETE linked to endocannabinoids are a special case discussed on another web page.


7.   Analysis

The bewildering array of eicosanoids and related oxygenated metabolites in animal tissues provides a daunting task for analysts. Selective extraction, concentration and derivatization steps are required, followed by gas chromatography or high-performance liquid chromatography linked to mass spectrometry. Of the many published procedures, the most comprehensive appears to be that of Wang et al. cited below, who describe the analysis of 184 distinct eicosanoids in a single chromatographic run in only five minutes, with the use of deuterated internal standards and tandem mass spectrometry to ensure accurate quantification (I have no personal experience in this area).


Recommended Reading



Lipid listings Credits/disclaimer Updated: May 29th, 2017 Author: William W. Christie LipidWeb icon