Essential Fatty Acids And The Biosynthesis Of Eicosanoids

Some types of polyunsaturated fatty acids, the so-called essential fatty acids, are converted into oxygenated fatty acids with potent physiological effects. These include effects on muscle contraction, cell adhesion, immune function and vascular tone.

In Section 2.2.5 we described the biosynthesis of the polyunsaturated fatty acids and indicated that mammals lack the A12 and A15-desaturases. They cannot, therefore, synthesize linoleic or a-linolenic acids. It turns out, however, that these poly-unsaturated fatty acids are absolutely necessary for the maintenance of growth, reproduction and good health and must, therefore, be obtained in the diet from plant foods. They are called essential fatty acids and their role in nutrition and health will be described in detail in Section 4.2.3. They are precursors of a variety of oxygenated 20C fatty acids with potent biological activities now generally known collectively as the eicosanoids.

As early as 1930, two American gynaecologists, Kurzrok and Lieb, reported that the human uterus, on contact with fresh human semen, was provoked into either strong contraction or relaxation. Both von Euler in Sweden and Goldblatt in England subsequently discovered marked stimulation of smooth muscle by seminal plasma. Von Euler then showed that lipid extracts of ram vesicular glands contained the activity and this was associated with a fatty acid fraction. The active factor was named prostaglandin and was shown to possess a variety of physiological and pharmacological properties.

In 1947, the Swede, Bergstrom, started to purify these extracts and soon showed that the active principle was associated with a fraction containing unsaturated hydroxy acids. The work then lapsed until 1956, when with the help of an improved test system (smooth muscle stimulation in the rabbit duodenum) Bergstrom isolated two prostaglandins in crystalline form (PGE1 and PGFla). Their structure, as well as that of a number of other prostaglandins, was elucidated by a combination of degradative, mass spectrometric, X-ray crystal-lographic and NMR studies (Fig. 2.38). The nomenclature is based on the fully saturated 20C acid with C8 to C12 closed to form a 5-membered ring; this is called prostanoic acid. Thus PGE1 is designated 9-keto-11a,15a-dihydroxyprost-13-enoic acid. The 13,14 double bond has a trans configuration; all the other double bonds are cis. Figure 2.38 clearly brings out the difference between the 'E' and

Prostanoic Acid Eicosanoids
Fig. 2.38 Structures of prostaglandins E and F and their precursors.

'F series, which have a keto and hydroxyl group at position 9, respectively; 'a' refers to the stereochemistry of the hydroxyl, and the suffix 1, 2 or 3 is related to the precursor fatty acid from which they are derived. That is, the 1, 2 or 3 refer to how many double bonds are contained in the prostaglandin structure. The name prostaglandin (and the related prostanoic acid structure) derives from the fact that early researchers believed that the prostate gland was the site of their synthesis.

2.4.1 The pathways for prostaglandin synthesis are discovered

Although prostaglandins were the first biologically active eicosanoids to be identified, it is now known that the essential fatty acids are converted into a number of different types of eicosanoids. (Eicosa-noid is a term meaning a 20C fatty acid derivative.) The various eicosanoids are important examples of local hormones. That is, they are generated in situ and, because they are rapidly metabolized, only have activity in the immediate vicinity. A summary of the overall pathway for generation of eicosanoids is shown in Fig. 2.39. Essential fatty acids can be attacked by lipoxygenases, which give rise to leu-kotrienes or hydroxy fatty acids and lipoxins.

Alternatively, metabolism by cyclooxygenase gives cyclic endoperoxides from which the classical prostaglandins or thromboxanes and prostacyclin can be synthesized. A third possibility is via cytochrome P450 oxygenation where atomic oxygen is introduced leading to fatty acid hydroxylation or epoxidation of double bonds. Whereas both the lipoxygenase and cyclooxygenase reactions arise from the formation of a fatty acid radical, in P450-oxygenation activation of atmospheric molecular oxygen is involved. After this, one oxygen is transferred to the fatty acid substrate and one is reduced forming water. Because, historically, prostaglandin synthesis was elucidated before that of the other eicosanoids, we shall describe pros-taglandin formation first.

After the structures of PGE and PGF had been defined, the subsequent rapid exploitation of this field, including the unravelling of the biosynthetic pathways, was done almost entirely by two research teams led by van Dorp in Holland and by Bergstrom and Samuelsson in Sweden. Both realized that the most likely precursor of PGE2 and PGF2a was arachidonic acid. This was then demonstrated by both groups simultaneously (with the same preparation of tritiated arachidonic acid) by incubation with whole homogenates of sheep

Phospholipid

Precursor fatty acid (Arachidonate)

Stimulus

Lipoxygenases

Leukotrienes + hydroxy-fatty acids, lipoxins hepoxilins

Cyclooxygenase

Prostacyclin synthetase

Prostacyclin (PGI2)

cyt P450 epoxygenase cyt P450 epoxygenase

Cyclooxygenase

Prostacyclin synthetase

Hydroxy fatty acids, fatty acid epoxides

Thromboxane synthetase

Fig. 2.39 Overall pathway for conversion of essential fatty acids into eicosanoids.

Hydroxy fatty acids, fatty acid epoxides

Prostaglandins (PGD, PGE, PGF mainly)

Thromboxane synthetase

Thromboxanes (TXs)

Fig. 2.39 Overall pathway for conversion of essential fatty acids into eicosanoids.

vesicular glands. An important point that was noted, was that the unesterified fatty acid was the substrate and not an activated form (see later for discussion about release of free fatty acids in tissues). The ability of the arachidonic acid chain to fold allows the appropriate groups to come into juxtaposition for the ring closure to occur (Fig. 2.38 and Fig. 2.40). The reactions take place in the microsomal fraction, but a soluble heat-stable factor is required. This cofactor can be replaced by reduced glutathione. The reaction also requires molecular oxygen. Labelling with 1802 demonstrated that all three oxygen atoms in the final prostaglandin (Fig. 2.40) are derived from the gas.

This key initial reaction in prostaglandin formation is catalysed by the enzyme prostaglandin endoperoxide synthase. For simplicity this is usually known as cyclooxygenase (COX; Figs 2.39 and 2.40). There are two major isoforms and crystal structures for both have been obtained. The cyclooxygenases are haemoproteins and they

Cyclooxygenase Pathway Structures

Arachidonic acid

Fig. 2.40 Mechanism of biosynthesis of the cyclic endoperoxide, PGH2.

oh exhibit both cyclooxygenase and peroxidase activities. Cyclooxygenase-1 is constitutively expressed in many mammalian cells and tissues, and appears to be responsible for the formation of pros-taglandins involved in the general regulation of physiological events. On the other hand, cycloox-ygenase-2 is present at low basal levels in inflammatory cells. It is strongly induced by inflammatory stimuli such as cytokines, endotoxins, tumour promoters and some lipids. Both isoforms have similar Vmax and Km values for arachidonate, undergo suicide inactivation and their reactions are initiated by hydroperoxide. However, COX-2 needs much lower levels of hydroperoxide and has somewhat different substrate selectivity than COX-1. Both enzymes will, nevertheless, utilize a broad spectrum of polyunsaturated fatty acid substrates such as linoleate, y-linolenate, a-linolenate and arachidonate. Thus, they can give rise to a variety of endoperoxides and, therefore, to a host of different prostaglandins.

Arachidonic acid

} cyclooxygenase activity pgg2

peroxidase activity pgh2

Fig. 2.40 Mechanism of biosynthesis of the cyclic endoperoxide, PGH2.

The overall reaction is shown in Fig. 2.40. First, prostaglandin endoperoxide synthase (PES) inserts two molecules of oxygen to yield a 15-hydro-peroxy-9,ll-endoperoxide with a substituted cyclopentane ring (PGG; Fig. 2.40). This is the cyclooxygenase activity of the enzyme. The peroxidase activity then reduces PGG to its 15-hydroxy analogue, PGH.

As mentioned above, the cyclooxygenase activity requires a hydroperoxide activator to remove a hydrogen atom from position 13 on the incoming fatty acid, and therefore allow attack by oxygen. This is an unusual mechanism because, usually, oxygenases work by activating the oxygen substrate. There are other interesting features of the enzyme. For example, the free radical intermediates generated can also inactivate the enzyme. This self-deactivation of the cyclooxygenase occurs in vivo as well as for purified preparations. It may ensure that only a certain amount of endoperoxide is generated even when large quantities of precursor fatty acid are available.

Both the cyclooxygenases have a dual localization in the cell, being present in both the endo-plasmic reticulum and the nuclear envelope. A classical inhibitor of the cyclooxygenases is aspirin and, indeed, much of our knowledge of these enzymes has come from studies with the non-ster-oidal anti-inflammatory drugs (NSAIDs). In addition, gene disruption and overexpression of COX isoforms have confirmed that, as a generalization, the therapeutic anti-inflammatory action of the NSAIDs is due to inhibition of COX-2 while simultaneous inhibition of COX-1 causes most of the unwanted side-effects such as gastric ulceration.

Of the various non-steroidal anti-inflammatory drugs (such as aspirin, ibuprofen, indomethacin and diclofenac) aspirin is the best known. Aspirin competes with arachidonate for binding to the cyclooxygenase active site. Although arachidonate binds about 10000 times better than aspirin, once bound aspirin acetylates a serine residue (serine 530) at the active site to cause irreversible cycloox-ygenase inactivation (Fig. 2.41). Thus, the cell can only restore COX activity by making fresh enzyme. In COX-1 the serine 530 residue, although at the active site, is not needed for catalysis and acetyla-tion by aspirin results in steric hinderance to prevent arachidonate binding. For COX-2, acetylation by aspirin still permits oxygenation of arachido-nate, but the usual product PGH2 is not made.

Not only is aspirin used for pain relief but low-dose therapy is used for a selective inhibition of platelet thromboxane formation, which reduces platelet aggregation and, hence, blood clotting.

Although the other non-steroidal anti-inflammatory drugs inhibit cyclooxygenase activity, most of them cause reversible enzyme inhibition by competing with arachidonate for binding. A well-known example of a reversible NSAID is ibuprofen. The use of NSAIDs is a huge part of the pharmaceutical market and currently accounts for over £3 billion in annual sales - or to put it in another way, approximately 15 x 1012 tablets of aspirin are consumed annually! Because NSAIDs inhibit both cyclooxygenases, there has been active development of COX-2-specific drugs, which should pos

Fig. 2.41 Mechanism for the inactivation of the cyclooxygenase-1 by aspirin.

sess anti-inflammatory properties without the unwanted side-effects of common NSAIDs. The first such drugs have recently become available for patients.

Prostaglandin H is the key intermediate for conversion to various active eicosanoids. The enzymes responsible for its further metabolism are present in catalytic excess to COX and, hence, are not regulatory except in the sense that the balance of their activities determines the pattern of prostaglandins and thromboxanes, which are formed in a given tissue. Thus, although Fig. 2.42 depicts some possible conversions of PGH2 into various biologically active eicosanoids, prostanoid synthesis is cell-

specific. For example, platelets form mainly the thromboxane TxA2; endothelial cells produce PGI2 (prostacyclin) as their major prostanoid; while kidney tubule cells synthesize predominantly PGE2.

2.4.2 Cyclic endoperoxides can be converted into different types of eicosanoids

The various eicosanoids produced from PGH2 (Fig. 2.42) have a remarkable range of biological activities. Moreover, the effect of a given eicosanoid varies from tissue to tissue. In the absence of

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