Eicosanoids and related compounds: structures, nomenclature and biosynthetic pathways

Hartmut Kühn and Sabine Borngräber

Institute of Biochemistry, University Clinics (Charité), Humboldt University, Hessische Str. 3-4, 10115 Berlin, Germany

Introduction

Eicosanoids and oxylipins comprise a family of structurally related lipid mediators that exhibit in teresting biological activities in animals and in the plant kingdom, respectively. Eicosanoids are synthesized from arachidonic acid (AA) that is released upon cell stimulation from membrane phospholipids. However, AA is not the only substrate for eicosanoid synthesis. Even in mammals, where it is one of the major polyenoic fatty acids, other fatty acids with different chain lengths and different degrees of unsaturation may be used as substrate (Fig. I). In higher plants, AA only occurs in small amounts and thus eicosanoids are usually not formed. In stead, the C-18 fatty acids (linoleic acid and α-linolenic acid) which occur in plants in large amounts are converted into oxylipins via several oxidative pathways. In the early days of eicosanoid research only a small number of bioactive lipids were known and there was no need for a systematic classification. How ever, during the last 20 years a large variety of eicosanoids and structurally related lipid mediators have been identified and for most of them the biosynthetic route has been investigated. To manage this structural multiplicity, a systematic classification and a comprehensive nom enclature for eicosanoids and related com pounds is required.

In this introductory chapter the basic rules for the currently used classification and nomenclature of eicosanoids and related compounds are summarized. For more detailed information the reader is referred to several reviews in which the nomenclature of Hcosanoids and oxylipins is explained. Moreover, the recommendations of the Committee on Eicosanoid Nomenclature may be consulted. In this paper suggestions for the nomenclature of enzymes involved in eicosanoid metabolism are also provided. These suggestions have been considered for revision of the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes.

Eicosanoids and related compounds in animals

The arachidonic acid cascade

As indicated above, the major source of eicosanoids and related com pounds in animals is the AA cascade (Fig. 2). Upon cell stimulation, AA, or under certain circumstances other precursor fatty acids, are liberated from the membrane phospholipids via activation of lipid-cleaving enzymes, such as phospholipase A 2 The free fatty acids are subsequently metabolized via three different pathways (Fig. 2). (i) The cyclooxygenase (COX) pathway, forming prostaglandins, thromboxanes or prostacyclins, (ii) the lipoxygenase (LOX) pathway, forming leukotrienes, lipoxins, hepoxilins and hydro(pero)xy fatty acids and (iii) the cytochrome P-450 (cyt P-450) pathway, forming hydroxylated fatty acids and epoxy derivatives. In these metabolic routes the initial reaction is an oxygenation of the fatty acid substrate. During the COX reaction two molecules of dioxygen are introduced, one at C11 and the second at C15 of the AA backbone. In contrast, the LOX reaction involves the introduction of one molecule of dioxygen at different positions of the substrate molecule, which are determined by the positional specificity of the enzymes. During cyt P-450-catalysed oxygenation, atomic oxygen is introduced, leading to fatty acid hydroxylation or to epoxidation of double bonds. Both the COX and LOX reactions are initiated by hydrogen abstractions from doubly allylic methylene groups, forming fatty acid radicals. This radical formation may be regarded as fatty acid activation. In contrast, the cyt-P-450-catalysed oxygenation involves activation of atmospheric dioxygen, destabilizing the O-O bond. After this, one oxygen atom is transferred to the fatty acid substrate, the other one is reduced, forming water.

The cyclooxygenase pathway

More than 50 years ago a com pound was discovered in the seminal fluid and in the prostate which caused contraction of smooth muscle cells. Although the chemical structure of this factor remained unclear for many years, it was named prostaglandin because of its organ source. Since then the chemical structures of a variety of prostanoids have been identified, and we also know that the prostate is not the only, and not even the major, source of prostaglandin (PG) formation. Moreover, most enzymes involved in prostaglandin biosynthesis have been well characterized.

The initial enzyme for prostagland information is prostaglandin endoperoxide synthase which, for simplicity, is called cyclooxygenase. This enzyme, the three-dimensional structure of which has been reported is a haemoprotein and exhibits both cyclooxygenase and peroxidase activity. It introduces two molecules of dioxygen into the fatty acid substrate, forming the cyclic endoperoxide PGG2 which is subsequently reduced to the m ore stable PGH2 (Scheme 1). PGH2 serves as substrate for the formation of the classical prostaglandins PGD2, PGE2 and PGF2α which exhibit interesting bioactivities.

Two isoenzymes of COX have been shown to exist: COX-1 is constitutively expressed in many mammalian cells and tissues and appears to be responsible for the formation of prostaglandins involved in the regulation of physiological events. COX-2 is an inducible form of the enzyme that is low-level expressed in inflammatory cells under basal conditions but is strongly induced in response to inflammatory stimuli. This induction suggested an involvement of the enzyme in the pathogenesis of inflammation and suggested COX-2 as a major target for the development of non-steroidal anti-inflammatory drugs.

AA, which contains four double bonds (Fig. 1), is converted via the COX pathway into the prostaglandins of the 2-series (PGD2, PGE2, PGF2α, PGI2, TXA2). If (8Z,11Z,14Z)-eicosa-8,l1,14-trienoic acid or (5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaenoic acid are used as substrate, the prostaglandins of the 1-series (PGE1, PGF1a etc.) and those of the 3-series (PGE3, PGF3α, etc.) are formed, respectively. Polyenoic fatty acids containing only two double bonds, such as linoleic acid or (11Z ,14Z)-eicosa-l1,14-dienoic acid, cannot be converted into prostaglandins.

In Scheme I the biosynthesis and the chemical structures of the classical prostaglandins (PGD2, PGE2, PGF2α) are summarized. It can be seen that these compounds differ from each other with respect to the chemical nature of the substituents at the prostanering and in their stereochemistry. In Table I the scientific names of the biologically most relevant prostanoids and of other eicosanoids and related compounds are summarized.

The unstable endoperoxide PGH2 formed via the COX-reaction can also be converted into prostacyclin (PGI2) and thromboxane (TX) A2 (Scheme 2). In mammals, TXA2 is mainly produced by activated blood platelets and induces vasoconstriction, cell adhesion to the vessel wall and platelet aggregation, PGI2 is formed by vascular endothelial cells and antagonizes the TXA2-induced effects. Thus, the TXA2-PGI2 steady state appears to be important for systemic blood pressure regulation and for the pathogenesis of thrombosis.

In addition to the classical prostaglandins (PGD2, PGE2, PGF2α, TXA2, PGI2), the biological effects of which have been well investigated, several other prostanoids (PGA2, PGB2, PGJ2) have been detected (Fig. 3), but little is know n as to their biological importance.

The bioactive PGs are further metabolized to decomposition products that are subsequently eliminated from the body by excretion in urine. This metabolization increases the structural multiplicity of the prostanoids. It should be emphasized that some of the metabolization products may still be bioactive. The decomposition pathways for various prostanoids are different but there are common principles for prostanoid metabolization: (i) oxidation of the OH-group at C-15, forming the corresponding keto derivatives; (ii) β-oxidation of the carboxyl terminus, forming the di-nor prostaglandins and (iii) ω-oxidation of the methyl terminus, forming dicarboxylic compounds. TXA2 and PGI2 are rather unstable prostanoids that rapidly undergo decomposition and, thus, their direct quantification is rather problematic. However, their stable metabolization products 6-keto-PGF1α (for PGI2) and TXB2 (for TXA2) may be determined as measures for PGI2 and TXA2 synthesis.

The lipoxygenase pathway

Lipoxygenases (LOXs) catalyse the dioxygenation of polyunsaturated fatty acids containing at least one 1,4-pentadienyl system to their corresponding 1-hydroperoxy derivatives (Scheme 3). According to the currently used nomenclature, lipoxygenases are classified with respect to their positional specificity of AA oxygenation. Arachidonic acid 5-LOXs introduce molecular oxygen at C5 of the AA backbone, forming (6E,8Z,11Z,14Z)-(5S)-hydroperoxyeicosa-6,8,ll, l4-tetraenoic acid (5S-HpETE). In contrast, 15-LOXs catalyse the formation of (5Z,8Z,11Z,13E)-(15S)-hydroperoxyeicosa-5,8,11, 13-tetraenoic acid (Scheme 4). In mammalian cells, three major types of LOXs (5-LOX, 12-LOX and 15-LOX) may be classified, but there are also reports of an inducible 8-LOX in mouse skin.

The primary LOX metabolites, the hydroperoxy fatty acids, are further metabolized to an array of secondary products that may be classified in to several groups according to their chemical structures: (i) leukotrienes containing three conjugated double bonds, (ii) mono- and double-oxygenated polyenoic fatty acids that are not formed via a leukotriene intermediate and (iii) lipoxins and hepoxilins.

Leukotrienes

The name leukotriene was introduced for two reasons: (i) they were discovered in leucocytes and (ii) they contain a conjugated triene chromophore. Leukotrienes are abbreviated to LT follow ed by a capital letter (A, B, C, D or E) and a subscript. The capital letter indicates the chemical structure of the substituents and the subscript the number of double bonds which is the same as in the fatty acid substrate used for leukotriene synthesis. AA, which contains four double bonds, is converted in to leukotrienes of the 4-series. In contrast, PGs of the 2-series are formed from this substrate. The reasons for the different degree of unsaturation of PGs and leukotrienes is the fact that during PG formation two double bonds are lost whereas all double bonds are retained during leukotriene synthesis. When (5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaenoic acid is used as substrate, leukotrienes of the 5-series are formed. As for the prostaglandins, the geometry of the double bonds and the configuration of the chiral centres are not indicated by the abbreviations. The Committee on Eicosanoid Nomenclature has published several guidelines for the nom enclature of leukotrienes and related oxygenated fatty acids.

The chemical structure and the biosynthetic pathway for the major leukotrienes is shown in Scheme 5. AA, which is liberated from the membrane phospholipids, is oxygenated by the 5-LOX, forming 5S-HpETE. In leucocytes, this com pound is either reduced to the corresponding (5S)-alcohol or converted via the LTA4 synthase activity of the 5-LOX in to LTA4. This reaction involves a hydrogen abstraction from C10 and a rearrangement of two double bonds, forming a conjugated triene system. The unstable LTA4 is subsequently hydrolysed to LTB4 by a LTA4 hydrolase. Alternatively, the epoxide reacts with glutathione, leading to the synthesis of LTC4. The LTA4 hydrolase reaction involves a rearrangement of the complete conjugated triene system and it is of particular interest that the C6-C7 double bond of the LTB4 is of cis geometry. In addition, LTA4 may also undergo non-enzymic hydrolysis, forming inter alia 6-trans-12-epi-LTB4. Although structurally related, the non-enzymic hydrolysis products are not as potent pro-inflammatory mediators as the authentic LTB4.

As indicated above, LTA4 may also be converted into the cysteinyl leukotrienes LTC4, LTD4and LTE4. LTC4 is formed via nucleophilic attack of the epoxide ring by reduced glutathione. LTC4 is then transformed into LTD4 and LTE4 by sequential cleavage of the glutamyl and glycyl residues (Scheme 5).

The naturally occurring leukotrienes are synthesized via the intermediate formation of 5S-HpETE. In principle, 15-HpETE may also constitute a substrate for leukotriene formation. In fact, when 15-HpETE was incubated with a purified 15-LOX a 14,15-LTA4 derivative was detected. It remains to be investigated whether cysteinyl leukotrienes can be form ed from this unstable epoxy intermediate and whether these putative metabolites may be of any biological significance.

Lipoxins and hepoxilins

Lipoxins constitute an additional family of bioactive LOX products which are characterized by a conjugated tetraene structure and three asymmetric carbons to which hydroxy groups are attached. Lipoxins are abbreviated to LX followed by a capital letter (A or B) that indicates the position of the OH-groups, and by a subscript which gives the number of the double bonds. The biosynthesis of lipoxins involves a concerted action of 5- and 15-LOXs or of 5- and 12-LOXs as well as an enzymic hydrolysis of an epoxide intermediate (Scheme 6). However, in vitro experiments with purified rabbit 15-LOX suggested that lipoxin B4 can be formed via consecutive double oxygenations of 15-HETE methyl ester. In biological material, LXA4 and LXB4 are the major lipoxins but, depending on the reaction conditions, their all-E isomers have also been detected. Lipoxins display a profile of bioactions that are unique among eicosanoids, which are not discussed here because of space limitations.

Hepoxilins are epoxyhydroxy compounds that are formed from 12- HpETE, Starting from AA, their biosynthesis involves a 12-LO X reaction and a subsequent isomerization of 12S-HpETE. Two m ajor isomers, the hepoxilins A3 and B3, have been identified (Scheme 7). For along time it has been assumed that the formation of the hepoxilins from 12-HpETE is a non-enzymic reaction that may be catalysed by haemoproteins or metalions, but recently an enzyme responsible for the specific conversion of 12S-HpETE in to hepoxilin A3 has been identified [30a]. Both hepoxilin isomers are bioactive in various assay systems. As an epoxy compound, hepoxilin A3 can be converted into glutathione adducts via a LTC4 synthase-like reaction and the glutathione conjugate appears to be involved in the modulation of neurotransmission.

Other oxygenated fatty acids

When not transformed into epoxy leukotrienes, hydroperoxy fatty acids form ed via the LOX reaction are rapidly reduced to the corresponding alcohols. Alternatively, the hydroperoxy group may undergo homolytic cleavage of the O–O bond, leading to the formation of a complex array of secondary lipid peroxidation products which includes fatty acid dimers, keto dienoic fatty acids, epoxy hydroxy compounds, short-chain aldehydes and alkanes, Hydroperoxy, hydroxy and keto fatty acids are usually abbreviated in scientific publications and these abbreviations may lead to confusion among scientists not working in the eicosanoid field. To avoid such problems, guidelines for the abbreviation of these compounds have been published. In Table 2 some of these guidelines are summarized. According to these the primary product of the 15-LOX reaction with linoleic acid, (9Z,11E)-(13S)-hydroperoxyoctadeca-9,11-dienoic acid, should be abbreviated as 13S-HpODE. The 12-LOX product formed from (5Z,8Z,11Z,14Z,17Z)eicosa-5,8,ll,14,17-pentaenoic acid, the (5Z,8Z,10E,14Z,17Z)-(12S)-hydroperoxyeicosa-5,8,10,14, 17-pentaenoic acid, should be abbreviated as 12S-HpEPE and the corresponding 12-keto derivative [(5Z,8Z,10E,14Z, 17Z)-12-oxoeicosa-5,8,10,14,17-pentaenoic acid] as 12-OxoEPE.