RESOLVINS AND PROTECTINS

Chemistry and Biology

Structural formula of docosahexaenoic acid The omega-3 essential fatty acids are currently the focus of considerable interest among nutritionists, because of a large number of potential beneficial effects for consumers. The mechanisms by which such effects are exerted is still a matter for controversy, but is seems likely that oxygenated metabolites derived from eicosapentaenoic acid (20:5(n-3) or EPA) and docosahexaenoic acid (22:6(n-3) or DHA), the resolvins and (neuro)protectins, must play a significant part as they have potent anti-inflammatory and immunoregulatory actions at concentrations in the nanomolar and picomolar range. The term 'resolvins' or 'resolution-phase interaction products' was coined by Professor Charles N. Serhan and colleagues because these compounds were first encountered in resolving inflammatory exudates. Compounds derived from EPA are designated as resolvins of the E series, while those formed from the precursor DHA are denoted as either resolvins or protectins (formerly 'neuroprotectins') of the D series.

1. 18R Resolvins of the E Series

Biosynthesis of the 18R resolvins of the E series (name derived from EPA) has elements in common with the synthesis of the epi-lipoxins and leukotrienes.

Biosynthesis of the 18R-resolvins

In vascular endothelial cells derived from blood vessels, the cyclooxygenase enzyme COX2 that has been acetylated by aspirin introduces an 18R hydroperoxy-group into the EPA molecule (c.f. the role of aspirin in the biosynthesis of the epi-lipoxins). Alternatively, this can be accomplished by a cytochrome P450 enzyme. The product is reduced to the corresponding hydroxy compound before a 5S-hydroperoxy group is introduced into the molecule by the action of 5-lipoxygenase as in the biosynthesis of the leukotrienes. A further reduction step produces 15S,18R-dihydroxy-EPE or resolvin E2. Alternatively, the 5S-hydroperoxy,18R-hydroxy-EPE intermediate is converted to a 5,6-epoxy fatty acid in polymorphonuclear neutrophils in humans and eventually to 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid or resolvin E1 by process similar to the formation of leukotrienes in leukocytes.

The highly specific stereochemistry of resolvin E1 is required for activation of a ligand-specific receptor and thence for its biological activity down to picomolar concentrations. Resolvin E1 is eventually de-activated in tissues by oxidation to 18-oxo-RvE1, and it is catabolized following omega-hydroxylation.

2. 17R- and 17S-Resolvins of the D Series

DHA is converted to 17R-resolvins by a similar aspirin-triggered COX2 (or cytochrome P450) mechanism to the previous. (In the absence of aspirin, COX2 in human microvascular endothelial cells converts DHA to 13S-hydroxy-DHA ). Thence, enzymatic epoxidation generates either 7S,(8)-epoxy or a 4S,(5)-epoxy intermediate, which are acted upon by 5-lipoxygenase to yield the resolvins. The former produces the aspirin-triggered resolvins D1 and D2 as illustrated (D nomenclature derived from DHA), while the latter produces the aspirin-triggered resolvins D3 and D4. All contain a 17R hydroxyl group. Again, there are parallels in the biosynthesis of resolvins (and protectins below)) with that of epi-lipoxins (aspirin-triggered lipoxins) from arachidonate.

Formulae of aspirin-triggered resolvins D1 and d2

In an alternative reaction in the absence of aspirin, 15-lipoxygenase generates 17S-hydroxy-DHA as the initial product. This is converted to 7S-hydroperoxy,17S-hydroxy-DHA by the action of a lipoxygenase, and thence via an epoxy intermediate to epimeric resolvin (RvD1 or 7S,8R,17S-trihydroxy-docosa-4Z,9E,11E,13Z,15E,19Z-hexaenoic acid) and resolvin D2 (RvD2 or 7S,16,17S-trihydroxy-docosa-4Z,8E,10Z,12E,14E,19Z-hexaenoic acid), i.e. all contain a 17S hydroxyl group.

Once more, an alternative lipoxygenase-generated intermediate from 17S-hydroxy-DHA, i.e. 4S-hydroperoxy,17S-hydroxy-DHA, is transformed via an epoxide to epimeric resolvins D3 and D4.

Formulae of resolvins D3 and D4

Interestingly, both 22:5(n-3) and 22:5(n-6) are also good substrates for 15-lipoxygenase, and the latter gives 17S-hydroxy-22:5(n-6) and 10,17S-dihydroxy-22:5(n-6) as the main products. Both of these (n-6) docosanoids are potent anti-inflammatory agents when administered either intravenously or orally.

3. Protectins (Neuroprotectins)

In studies of resolvin formation in brain tissue in response to aspirin treatment, it was shown that new docosatrienes termed initially ‘neuroprotectins’ were produced. As it is now recognized that the formation and actions of these docosanoids are not restricted to neuronal tissue, it has been suggested that the simpler term ‘protectins’ is preferable. The biosynthetic pathway to neuroprotectin or protectin in brain tissue is illustrated below. The lipoxygenase product 17S-hydroperoxy-DHA is converted first to a 16(17)-epoxide and then to the 10,17-dihydroxy docosatriene (10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid), denoted as 10R,17S-DT or PD1 (or NPD1). As with the leukotrienes, there are three double bonds in conjugation, hence the term ‘triene’, though there are six double bonds in total. Again, it appears that this highly stereospecific structure is essential for biological activity.

Protectin biosynthesis

4. Biological Activity

Acute inflammation in response to infection or tissue damage is usually characterized by heat, redness, swelling and pain at a simple observational level, and by edema, accumulation of leukocytes, and then by accumulation of monocytes and macrophages at a cellular level. Prostaglandins and leukotrienes play a role in the early stages. As tissues return to health, resolution of the inflammation involves removal of the leukocytes, ideally without leaving remnants of the host defences or of the invading microorgansms or other inflammatory initiators.

The resolvins and neuroprotectins are distinctive and highly stereospecific lipids, which are endogenous local mediators with strong anti-inflammatory effects in addition to some immunoregulatory activities at picomolar to nanomolar concentrations. They are part of the molecular mechanisms that contribute to removal of inflammatory cells and restoration of tissue integrity once the need for the inflammatory response is over, i.e. they actively assist in the resolution of inflammation, once thought to be a passive process. It is evident that the presence of aspirin uniquely facilitates the resolution of inflammation. Thus, at local sites of inflammation, aspirin treatment enhances the conversion of the omega-3 fatty acids EPA and DHA to 18R-oxygenated products, i.e. resolvins of the E and D series, which carry potent anti-inflammatory signals. So far two receptors have been identified that mediate the activities of RvE₁.

During inflammation, polymorphonuclear neutrophils are produced which have generally beneficial effects in countering disease, but in the longer term or if malfunctioning they may eventually cause trauma and tissue damage through infiltration into tissues. The resolvins, like the lipoxins, appear to have an important role in regulating and indeed inhibiting these harmful effects. In so doing they oppose the effects of some of the pro-inflammatory prostanoids. For example, nanomolar concentrations of resolvin E1 dramatically reduced dermal inflammation, peritonitis, dendritic cell migration and interleukin production. The 17S-resolvins appear to have very similar functions to those of the 18R-series, while the (neuro)protectins appear to operate in the same way in brain tissue. Thus, (N)PD1 has anti-inflammatory effects and protects retinal epithelial cells from apoptosis induced by oxidative stress. In addition, it has protective effects in animal models of stroke and of Alzheimer's disease. Amongst its activities in non-neuronal tissues, it promotes apoptosis of T cells and it has beneficial effects towards asthma. It is evident that such compounds and their metabolism have considerable potential for therapeutic intervention in acute inflammation or chronic inflammatory disease. They may also mitigate the affects of sepsis.

It is now well established that administration of lipoxins, resolvins and protectins in vivo in animal models can aid the process of recovery from inflammation. >From a nutritional or health standpoint, it has been suggested that dietary supplements of the precursor omega-3 fatty acids, taken together with aspirin, may reduce the clinical symptoms of many disease states, including inflammatory disorders, (such as arthritis), cardiovascular disease, asthma and cancer.

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