ISOPROSTANES


CHEMISTRY AND BIOLOGY


1.   Structures and Occurrence

Isoprostanes are prostaglandin-like compounds produced primarily from esterified arachidonic acid in tissues by non-enzymatic reactions catalysed by free radicals in vivo. Thus, they do not require cyclooxygenases (COX-1 and COX-2) for their formation. Such autoxidation reactions lack specificity and lead to the formation of many different structural and stereo-isomers. Although isoprostanes have a short half-life, some of them have potent biological activities, especially in the lungs and kidney, and they may even function in normal physiology. They are believed to be useful markers for oxidative stress, and importantly they can be assayed by non-invasive means. Related compounds are formed from eicosapentaenoic and docosahexaenoic acids in animals and from α-linolenic acid in plants. Isoprostanes were first produced in the test tube as long ago as 1967, but it was more than twenty years later before it was realized that they were also formed in appreciable amounts in vivo and had important biological properties.

In contrast to the conventional prostanoids, which are produced mainly in the form of the free acids, the isoprostanes are synthesised in an ester-bound state in position sn-2 of phospholipids. However, for practical convenience they are illustrated mainly as the free acids here.

Isoprostanes resemble normal prostanoids in some ways, and the most abundant form is analogous to prostaglandin F, while analogues of PGD2 and PGE2 are also found. However, they differ in many aspects of their stereochemistry. For example, the side-chains are mainly cis to the cyclopentane ring, although trans isomers (as in normal prostanoids) also exist. Four regioisomers of the F-, D- and E-series isoprostanes are possible, and each of these are produced in eight distinct diastereomeric forms, i.e. 64 distinct isomers can exist of each series. To distinguish them from the normal prostaglandins, it is recommended that they are each given the abbreviation 'IsoP', with a prefix determined mainly by the location of the hydroxyl group in the side-chain (5, 8, 12 or 15) that defines the structure further. In addition, the structures can be distinguished more precisely according to the cis- or trans-configuration of the side-chain relative to the ring (normally cis as this is more stable thermodynamically), whether the ring hydroxyls are above (ent) or below (α) the ring (normally the latter), and by the absolute configuration of the hydroxyl group in the side chain (S for the α-series, and R for the ent-series). The basic structures of the four main F2-IsoPs are illustrated below.

Formulae of F2 isoprostanes

Isoprostanes have been found in very many animal tissues and most biological fluids, including plasma and urine, although the basal levels vary appreciably among species and among individuals, depending on the degree of oxidative stress. As an example, the level of F2-isoprostanes in the plasma of healthy humans is typically in the range of 20 to 30pg per ml, and this is roughly ten times greater than that of COX-derived PGF. In urine, the concentration of PGF is forty times higher than that in plasma and most of this is derived from the isoprostane pathway as two enantiomers are present in equal amounts. The 5- and 15-series isoprostanes are most abundant because the precursors that lead to the 8- and 12-series can undergo further oxidation. In plasma, F2-isoprostanes are transported in esterified form mainly in the high-density lipoproteins (HDL), with some preference for the HDL3 fraction.

F-ring IsoPs (F2-dihomo-IsoPs) are also generated from adrenic acid (22:4(n-6)), which is highly enriched in the white matter of brain and is associated with myelin.

A neuroprostaneIsoprostane-like compounds, i.e. F3, A3 and J3 isoprostanes, the last two with cyclopentenone rings, are formed from the oxidation of eicosapentaenoic acid (20:5(n-3)) in the heart muscle of mice in vivo. In addition, brain tissue contains relatively high proportions of docosahexaenoic acid (22:6(n-3)), and this gives rise to isoprostane-like compounds that have been characterized and termed neuroprostanes. There is a special interest in F4 neuroprostanes, which appear to be promising biomarkers for a variety of neurodegenerative disorders, including Alzheimer's disease. Indeed, related compounds can be formed from any lipid with a 1,4,7-octatriene unit, although the additional double bonds in eicosapentaenoic and docosahexaenoic acids mean that there is a much wider range of products, even if the general mechanisms are the same. However, omega-3 isoprostanes are not easy to quantify in urine.

Isothromboxanes have only been detected in significant amounts in animals subjected to severe oxidative stress, for example after oxidative injury caused by carbon tetrachloride administration, and the mechanism for their formation is still a matter of conjecture.

An isolevuglandinIsolevuglandins (isoLGs) are a family of reactive γ-ketoaldehydes, analogous to the levuglandins (see our web pages on prostanoids)), which are generated from isoprostanes by opening of the cyclopentane ring. They are distinguished from the levuglandins on the basis of their variable geometry. Isolevuglandins are highly reactive and were overlooked in biological samples for many years until discovered as protein-adducts by an immunological approach. The 4-oxoaldehyde unit is highly reactive towards primary amines. Isolevuglandins form Schiff bases and pyrroles rapidly with the ε-amino groups of lysyl residues in proteins, and these can undergo further oxidation with production of lactam and hydroxylactam end products. Such adducts disturb the functions of the proteins, and also inhibit their catabolism. Similar reactions occur with phosphatidylethanolamine in cell membranes. Related neuroketones are produced from docosahexaenoic acid in brain.

Formula of an isofuranIsofurans are oxidation products of arachidonate that contain substituted tetrahydrofuran rings (isomers with tetrahydropyran rings are also known). Two mechanisms have been described for formation of these compounds, involving either cleavage of a cyclic peroxide intermediate or hydrolysis of an epoxide, and these lead to the formation of eight regioisomers in total. Production of these is favoured relative to isoprostanes under conditions of high oxygen tension, since the final step involves an attack of molecular oxygen rather than an intramolecular rearrangement.

Isoprostanes derived from linolenic acid, and termed phytoprostanes, have been found in plants, but these are discussed in our web-page on plant oxylipins.


2.   Biosynthesis

Synthesis of isoprostanes in animal tissues in vivo is brought about by a series of free radical-catalysed reactions, most of which do not involve enzymes, and any fatty acid with three or more double bonds can be a substrate. The main route via an endoperoxide intermediate is illustrated below with the synthesis of 15-F2-IsoP isomers as the example.

Synthesis of isoprostanes

In order to commence isoprostane formation, there is a requirement for reactive oxygen species, such as peroxyl radicals, singlet oxygen and so forth, which can abstract a hydrogen atom from bis-allylic methylene groups of polyunsaturated fatty acids under aerobic conditions in vivo in animals (and plants). These are produced in increasing amounts under conditions of oxidative stress. As this radical generation is not enzymatic, all methylene groups between two cis double bonds can potentially be involved in the reaction, although not necessarily to the same degree.

After hydrogen abstraction, the pentadienyl radical formed combines with an oxygen molecule to generate a racemic peroxy radical that has a propensity to rearrange to form equivalent amounts of α,α- and β,β-bicyclic endoperoxy radicals, which are configured almost exclusively cis with respect to the cyclopentane ring. In the next step, the bicyclic endoperoxy radical reacts on either face of the side-chain with a further oxygen molecule to produce racemic hydroperoxy bicyclic endoperoxy radicals. The radical chain reaction is terminated by abstraction of hydrogen from an appropriate donor molecule such as a polyunsaturated fatty acid or glutathione. The product is an IsoPG, i.e. an analogue of PGG2, which can be reduced to the stable F2-isoP. Analogous reactions occur to produce isoprostanes with the hydroxyl group in positions 5, 8 or 12.

Isoprostanes of the IsoPF series are produced in limited amounts only in vitro, but are major metabolites in vivo, through the reduction of IsoPGs via natural endogenous reductants such as glutathione, hematin, lipoic acid, polyunsaturated fatty acids or glutathione peroxidase. Thromboxane-like compounds are also formed in vivo, and the catalyst in this instance is probably complexed iron, but PGI analogues are not produced. When the biosynthesis of isoprostanes proceeds via this endoperoxide route, all 64 possible stereoisomers can be produced.

As the G and H-ring endoperoxide structures are highly labile compounds with a half-life of only a few minutes, they can isomerize rapidly to give a variety of products, including analogues of PGE2 and PGD2. These are formed competitively with F2-IsoPs, and when cellular reducing agents, such as glutathione (GSH) or α-tocopherol, are depleted formation of E2/D2-IsoPs is favoured. The latter can spontaneously dehydrate to form isoprostanes containing cyclopentenone rings, i.e. with a double bond and carbonyl group on the prostane ring and related to PGA2 and PGJ2. These are highly reactive electrophiles and readily form Michael adducts with thiols such as are found on cysteine residues in proteins and glutathione, and they are are rapidly metabolized in vivo to water-soluble glutathione conjugates.

Formation of 15A2-isoprostane

A number of other biosynthetic pathways have been identified. For example, a second mechanism for isoprostane synthesis illustrated below involves a free-radical-catalysed dioxetane mechanism. It has been demonstrated in vitro and is presumed to operate in vivo also, although this is a matter of controversy.

Synthesis of isoprostanes

In this instance, the primary substrates are 1'- and 8'-hydroperoxy radicals rather than the 4'- and 5'-hydroperoxy radicals required for the endoperoxide mechanism. It is noteworthy that this second pathway is much more stereo-selective than the first and yields two regioisomers, each of which consists of two racemic diastereomers with either cis or trans substitution at the cyclopentane ring. The relative contributions of the two pathways to isoprostane synthesis have still to be determined, but the latter is probably a minor route.

As the isoprostanes in animal tissues are formed from arachidonic acid predominantly in position 2 of phospholipids in membranes, they must be released by the action of phospholipase A2 and related enzymes before they can exert their main physiological effects. In the free acid form, they can circulate in the plasma and interact with membrane receptors. However, it is possible that they may also have some biological functions while still linked to phospholipids.

Isoprostanes appear to be de-activated or catabolized by similar enzymic mechanisms to those for the prostanoids. Circulating F2-isoprostanes are filtered in the kidney and appear in the urine. They can also undergo metabolism in the liver to produce metabolites such as 2,3-dinor-15-F2t- and 2,3-dinor-5,6-dihydro-15-F2t-isoprostanes, which are also excreted in the urine.


3.   Isoprostanes and Oxidative Stress

There is an intriguing hypothesis that during evolution in primitive cells, isoprostane formation resulted from the increasing aerobic conditions and these molecules were selected as a means of signalling more specific imbalances in the redox state of the cells. Only later did truly enzymatic pathways evolve to produce eicosanoids as signalling molecules, but isoprostane formation has been retained as a back-up system.

Isoprostanes are believed to be valuable indicators of oxidative stress in animal tissues, which has been defined as "a disturbance in the prooxidant–antioxidant balance in favour of the former", i.e. there is an excessive production of lipid peroxidation products, which may be involved in the development or exacerbation of cancer, and cardiovascular and neurological diseases, for example. They have been detected in all biological fluids and tissues analysed to date. In particular, there is growing acceptance that measurement of the relatively stable F2-isoprostanes and their metabolites in urine is a reliable non-invasive approach to the determination of the degree of oxidative stress in patients. There is no need to store samples at −70șC, nor to be concerned about further artefactual formation of isoprostanes on storage.

Scottish thistleNormal levels of isoprostanes in healthy humans have been defined, so that the effects of disease states and subsequent therapeutic intervention can be determined. Measurement of isoprostanes has been termed by some to be the ‘gold standard’ by which oxidative damage and stress can be assessed, a conclusion borne out by a recent multi-investigator study, termed the Biomarkers of Oxidative Stress (BOSS) Study, sponsored by the National Institute of Health in the USA. Thus, increased levels of urinary isoprostanes have been measured in many conditions that have been associated with excessive generation of free radicals, including poisoning with paraquat and carbon tetrachloride, smoking, alcoholism and cirrhosis of the liver. They have been implicated in the pathophysiology of many human disease states, including brain degeneration, kidney diseases, ischemia-reperfusion injury, atherosclerosis and diabetes. For example, there is good clinical evidence that the concentration of F2-isoPs in urine is an independent and cumulative marker of coronary heart disease, reflecting high concentrations of this metabolite in atherosclerotic plaques, where like the thromboxanes it may activate the TP receptor. It is also reported to be a reliable indicator of oxidative stress in type 2 diabetes.

Lipid peroxidation is believed to be a factor in many disease states associated with the brain, where IsoPA2 and IsoPJ2 are usually considered to be the preferred products of the isoprostane pathway; they have potent effects on neuronal apoptosis and exacerbate neurodegeneration caused by other insults at concentrations as low as 100 nM. One reason for this is that the distinctive functional group of the cyclopentenone isoprostanes can react with the cysteine residue of glutathione and with cysteine in cellular proteins with harmful consequences. In other tissues, IsoPA2 and IsoPJ2 may have anti-inflammatory effects. Arachidonate-derived isoprostanes and isofurans and neuroprostanes derived from docosahexaenoic acid have been shown to increase in concentration in diseased regions of brains from patients who have died from advanced Alzheimer's and Parkinson's diseases. The levels of these compounds increase also in cerebrospinal fluid of patients with the early stages of Alzheimer's and Huntington's diseases, findings that are of diagnostic value and may assist in the evaluation of experimental therapies. As the levels of F2 isoprostanes derived from adrenic acid are markedly increased under conditions of oxidant stress, they may be a selective marker of white matter injury in vivo. Similarly, D2 and E2-isoprostanes have been detected in elevated concentrations in brain tissues affected by trauma.

It has become apparent that isoprostane levels in plasma and tissues are highest during fetal and early neonatal life in comparison to adults, and that they may have important roles in development and in the transition to postnatal life. The placenta is believed to be an important source of isoprostanes.

Urinary isoprostane analysis has also been used to assess the efficacy of antioxidants in vivo and to establish the value of antioxidant administration in clinical trials. For example, measurement of plasma F2-IsoP levels has been employed to study the efficacy of vitamin E as an antioxidant in demonstrating that doses of 1600 IU/day or greater of α-tocopherol are required. Surprisingly, supplements of vitamin C do not alter IsoP levels in humans.

In addition, in their esterified form in membranes, isoprostanes are long-lasting markers of oxidative damage and they enable the site of endogenous lipid peroxidation to be identified. Indeed, it is possible that in this form they have effects on the fluidity of membranes and may be responsible for some membrane dysfunction.


4.   Other Biological Activities

While isoprostanes have been observed to have innumerable physiological functions in vitro, the extent of their importance in vivo is uncertain and controversial. Often it is not clear whether the isoprostanes are simply markers for oxidative events or are mediators. The biological activity of 15-F2t-IsoP, the first isoprostane to be available commercially, has been most studied, and following intravenous administration it has been shown to be a vasoconstrictor in most species and vascular beds, including kidney, blood vessels, lymphatic vessels, the bronchi, the gastrointestinal tract and the uterus. In addition, it stimulates the induction of mitosis in certain vascular smooth muscle cells, and there is evidence that it inhibits the pro-aggregatory effects of thromboxanes via an interaction with the receptors for the latter.

In the lung, many different tissues or cell types respond to isoprostanes in various ways. The effects can be excitatory or inhibitory, depending on the nature and concentration of the specific type of isoprostane, as well as the nature of the cell and animal species. As in other tissues, oxidative stress is an important factor and isoprostanes are believed to be involved in various disease states of the lung. Indeed, it has been suggested that they are not merely markers for oxidative stress, but may be a novel class of inflammatory mediators, perhaps acting in the regulation of vascular smooth muscle tone, especially during fetal development. Similarly, isoprostanes have been implicated in oxidative damage to the liver, where they are markers or more controversially mediators of the effects.

The concentrations of protein-adducts of isolevuglandins have been shown to increase greatly in plasma from patients with advanced atherosclerosis. Such lipid-protein conjugates from both epithelial cells and plasma proteins, including apoprotein A1, may accumulate over a considerable time so could serve as a cumulative index for oxidative injury. Because of their irreversibly reaction with proteins, the isolevuglandins together with the levuglandins are highly neurotoxic.

Isoprostanes derived from eicosapentaenoic acid are also known to have important biological activities. For example, supplementation of the diet with this fatty acid was found to reduce the levels of pro-inflammatory arachidonate-derived F2-isoprostanes by a substantial amount in experimental animals. This effect may have a bearing on the reputed cardio-protective effects of eicosapentaenoic acid. Isoprostanes derived from docosahexaenoic acid may promote apoptosis of cancer cells, while isoprostane analogues of the cyclopentenone neuroprostanes are potent anti-inflammatory mediators.

It should not be forgotten that isoprostanes are formed first as a component of phospholipids rather than in the free form, and that they may function in this state. For example, phosphatidylcholine esterified with isoprostane analogues of PGJ2 and phosphatidylethanolamine adducted to levuglandins with appreciable biological activity have been found in tissues. Molecular models suggested that these adopt highly twisted conformations and may be disruptive in membranes, or it may enable them to act as ligands to specific receptors. Each of the different oxidized phospholipids may affect different signalling pathways.

formula of 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine

In particular, 1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine, derived from the arachidonoyl analogue, has been shown to modulate the expression of a large number of genes in human aortic endothelial cells in vitro, and it is also a potent activator of the peroxisome-proliferator-activated receptor (PPARα). These activities are observed at concentrations of less than 1 μg/mL, ten-fold lower than those observed in vascular cell walls. It has both pro- and anti-inflammatory effects via specific receptors in endothelial cells and macrophages, and there is a suggestion that it may have therapeutic properties for treatment of inflammatory diseases of the lung.


5.   Analysis of Isoprostanes

Gas chromatography allied to mass spectrometry (negative-ion chemical-ionization (GC/NICI-MS) and stable isotope dilution), after appropriate extraction and derivatization, is undoubtedly the most accurate and specific method for identifying and quantifying individual isoprostanes in biological fluids, although HPLC linked to tandem mass spectrometry is increasingly being used. However, these techniques are time consuming and costly, and radioimmunoassay procedures are often favoured in clinical applications. While these lack the specificity of chromatographic methods, minimal sample preparation is required and large numbers of samples can be assayed quickly at relatively low cost.


Suggested Reading


W.W. Christie

James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.

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© AOCS Credits/disclaimer Updated: January 22nd, 2014 Author