Hydroxyeicosatetraenoic Acids and Related Compounds

1.   Hydroxyeicosatetraenoic Acids

The function of lipoxygenases in generating 5-, 8-, 11- and 15-hydroperoxyeicosatetraenoic acids (‘HPETE’) is discussed in the Introduction to this series of web pages. These are rapidly reduced in tissues to the corresponding hydroxyeicosatetraenoic acids (‘HETE’), i.e. 5S-hydroxy-6t,8c,11c,14c-, 8S-hydroxy-5c,9t,11c,14c-, 12S-hydroxy-5c,8c,10t,14c- and 15S-hydroxy-5c,8c,11c,13t-eicosatetraenoic acids, two of which are illustrated below as examples. Of these, the 5-hydroxy isomer is of particular importance as the precursor of the leukotrienes and lipoxins, which are discussed elsewhere in these pages, and of 5-oxo-eicosatetraenoic acid (see below). The hydroxyl group has the S-configuration usually, but R-enantiomers are also formed in some circumstances and this is very important for the biological activities.

Structural formulae of representative HETE

In addition, arachidonic acid (not esterified forms) can be oxidized by various cytochrome P450 mixed-function oxidases to form different 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 reaction is NADPH-dependent, requiring transfer of electrons from NADPH to the P450 heme iron (lipoxygenases use nonheme iron), and it is catalysed by a membrane-bound enzyme, NADPH-cytochrome P450 reductase. 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. For example, 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. 12(R)-HETE as opposed to the 12(S)-isomer is the main product of the reaction, and this was at one time thought to be a distinguishing feature, but some other lipoxygenases are now known to produce the former enantiomer.

Secondly, there are ω/ω-1 hydroxylases that introduce a hydroxyl group into positions 20 or 19 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-form CYP4F3B is the main enzyme 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.

Production of omega- and omega-1-HETE

Fungi and yeasts are able to produce 3R- and/or 3S-HETE and 3,18-di-HETE, when supplied with exogenous arachidonic acid. In this instance, the 3-hydroxyl group is introduced by partial β-oxidation by mitochondrial enzymes, a process that is inhibited by aspirin and can also occur in mammalian tissues. 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.

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. In aortic endothelial cells, 20-HETE is incorporated into several phospholipids, while 15-HETE is again found in phosphatidylinositol and 12-HETE occurs predominantly in phosphatidylcholine in microsomal membranes. In neutrophils, 5-HETE is incorporated mainly into phosphatidylcholine and triacylglycerols. In addition, appreciable amounts of 15-HETE can be formed by a direct action of 15-LOX upon arachidonate esterified to phosphatidylethanolamine.

 

2.   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, all 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% 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. Free epoxyeicosatrienes are released when required following activation of phospholipase A by neuronal, hormonal or chemical stimuli. 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.

While they may simply constitute an inert storage pool as part of the mechanism of regulation, there is also a possibility that esterified epoxy-eicosanoids may have a biological function within membranes. In addition, phospholipids containing EET are substrates for the production of lipid mediators such as 2-epoxyeicosatrienoylglycerols (analogous to 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.

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, nonenzymic lipid peroxidation has been observed in erythrocytes in vitro, and some EETs may arise by this route.

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.

Linoleic acid (9,12-18:2), the biosynthetic precursor of arachidonic acid, is also 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 diols.

 

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 an oxidative process involving a 5-hydroxyeicosanoid 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 nonenzymic route. Its signalling functions are mediated via a specific receptor, leading to increased intracellular calcium concentrations and inhibition of cAMP production.

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 importance 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 oxidation in positions 19 or 20.

 

4.   Cytochrome P450 Metabolites of Eicosapentaenoic and Docosahexaenoic Acids

It has taken longer to recognize the importance of the metabolites of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids 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-epoxydocosapenatenoate. 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. 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.

Reaction of cytochrome P450 isoforms with EPA and DHA

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. It has been suggested that they may be responsible for some of the beneficial effects associated with dietary n-3 fatty acid intake.

 

5.   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 nonreceptor 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. Animal models can have very different isoforms of enzymes, which can make it 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, but a few of the more important are described briefly below. To my knowledge there has been no substantial published review that correlates the properties of these primary products of lipoxygenases and cytochrome P450 oxidases.

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. 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 that may have clinical utility.

Arachidonate 8(S)-lipoxygenase and its product 8S-hydroxy-5c,9t,11c,14c-8-eicosatetraenoic acid (8S-HETE) has only been found in the skin of mice and rats. It is a potent activator of the peroxisome proliferators-activated receptor PPARα, it is an antitumourogenic agent towards skin cancer, and it promotes wound healing in the cornea. A human orthologue of this enzyme is found in a few tissues but produces 15S-HETE.

12S-Hydroxy-5c,8c,10t,14c-eicosatetraenoic acid 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. 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. In contrast, 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.

15S-Hydroxy-5c,8c,11c,13t-eicosatetraenoic acid 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 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. It appears to contribute to the development of Hodgkin lymphoma, colorectal and many other cancers, but it also activates PPARγ, a nuclear transcription factor involved in epithelial differentiation, which may explain an antiproliferative 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. Eicosapentaenoic and docosahexaenoic acids are potent inhibitors of the biosynthesis of this compound, suggesting that this may be a partial explanation for the physiological role of (ω-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 tumour growth, but 8- and 11-HETE have antitumour activities.

The 3-hydoxy-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.

As 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 antihypertensive, fibrinolytic, and antithrombotic 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 profibrinolytic 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 neurohormones and neuropeptides. There 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 of dormancy of primary cancer tumours, 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 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.

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 anticancer agents.

Esterified HETE. Many of these lipoxygenase and oxidase products are found naturally in membrane phospholipids where they may perturb the membrane structure and effect secondary oxygenations, which could induce changes in cells. For example, 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, especially in relation to immune regulation, signalling and blood coagulation.

 

Recommended Reading

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Updated January 24, 2014