The Lipid Library

HYDROXYEICOSATETRAENOIC ACIDS AND RELATED COMPOUNDS

Chemistry and Biology

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,11c14c-, 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 (see below) and this is very important for the biological activities.

In addition, arachidonic acid (not esterified forms) can be oxidized by various cytochrome P450 mixed-function oxidases to form various HETE isomers. 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, 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 distribution of particular forms of the enzymes is specific both to cell type and the species. In addition to their role in generating HETE isomers, these enzymes 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 though to be a distinguishing feature, but some lipoxygenases are now know to produce the former enantiomer also.

Secondly, there are ω/ω-1 hydroxylases that introduce a hydroxyl group into positions 20 and 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.

Fungi and yeasts are able to produce 3R- and/or 3S-HETE, when supplied with exogenous arachidonic acid. In this instance, they are formed 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.

Appreciable amounts of 15-HETE can be formed by a direct action upon arachidonate esterified to phosphatidylethanolamine, and it is possible that  as part of an intact lipid this exert biological effects, which are distinct from that of the free eicosanoid.

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.

Various isozyme of the cytochrome P450 epoxygenase exist, and they produce all four EET regioisomers. The enzymes are located in the endoplasmic reticulum of the cell, 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.

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 the different isozymes produce 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. There is also a possibility that esterified epoxy-eicosanoids may have a biological function within membranes.

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.

EETs are further metabolized to the corresponding dihydroxyeicosatrienoic acids (DHET) by epoxide hydrolases, of which isozyme 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.

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

5-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), especially of eosinophils and neutrophils, and of platelets. 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.

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 oxidation in positions 19 or 20.

4.   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. 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. 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 has biological functions in its own right although these can be difficult to disentangle from those of its metabolites. 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.

Arachidonate 8(S)-lipoxygenase and its product 8S-hydroxy-5c,9t,11c14c-8-eicosatetraenoic acid ((S)-HETE) has only been found in the skin of mice and rats. It is a potent activator of the peroxisome proliferators-activated receptor PPARa, it is an anti-tumorogenic agent towards skin cancer, and it promotes wound healing in the cornea.

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 in 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. The enantiomeric compound 12R-HETE is believed to be involved in the pathophysiology of psoriasis and similar skin diseases, but it may also function in 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, and one of these 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, but its precise role in each of these processes is still unclear.

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.

The various epoxyeicosatrienoic acids have major functions as autocrine and paracrine effectors in the cardiovascular and renal systems. Because of the anti-hypertensive, fibrinolytic, and anti-thrombotic properties of EETs, their presence in red blood cells may have important implications for the control of circulation and the physical properties of the circulating blood. 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.

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.

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.

Recommended Reading

• Brash, A.R. Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate. J. Biol. Chem., 274, 23679-23682 (1999).
• Capdevila, J.H., Falck, J.R. and Harris, R.C. Cytochrome P450 and arachidonic acid bioactivation: molecular and functional properties of the arachidonate monooxygenase. J. Lipid Res., 41, 163-181 (2000).
• Coon, M.J. Omega oxygenases: nonheme-iron enzymes and P450 cytochromes. Biochem. Biophys. Res. Commun., 338, 378-385 (2005).
• Fürstenberger, G., Marks, F. and Krieg, P. Arachidonate 8(S)-lipoxygenase. Prostaglandins Other Lipid Mediators, 68-69, 235-243 (2002).
• Kock, J.L.F., Sebolai, O.M., Pohl, C.H., van Wyk, P.W.J. and Lodolo, E.J. Oxylipin studies expose aspirin as antifungal. FEMS Yeast Res., 7, 1207-1217 (2007).
• Kuhn, H., Walther, M. and Kuban, R.J. Mammalian arachidonate 15-lipoxygenases. Structure, function, and biological implications. Prostaglandins Other Lipid Mediators, 68-69, 263-290 (2002).
• Powell, W.S. and Rokach, J. Biochemistry, biology and chemistry of the 5-lipoxygenase product 5-oxo-ETE. Prog. Lipid Res., 44, 154-183 (2005).
• Spector, A.A. and Norris, A.W. Action of epoxyeicosatrienoic acids on cellular function. Am. J. Physiol. Cell Physiol., 292, C996-C1012 (2007).
• Yamamoto, S., Suzuki, H. and Ueda, N. Arachidonate 12-lipoxygenases. Prog. Lipid Res., 36, 23-41 (1997).

Updated: 3/12/2007 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland