The Lipid Library

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 (phytoprostanes) in plants. Isoprostanes were first produced in the test tube as long ago as 1967 (Nugteren, Vonkeman, and van Dorp), but it was more than twenty years later before it was realized that they were also formed 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.

Isoprostanes resemble normal prostanoids in many ways, and the most abundant form is analogous to prostaglandin F_2α, 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.

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_2α. In urine, the concentration of PGF_2α 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.

Isoprostane-like compounds (F₃, A₃ and J₃ and 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, and 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. This effect may have a bearing on the reputed cardio-protective effects of eicosapentaenoic acid. 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. These can have ring structures analogous to those of PGA and PGJ. 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 wider range of products, even if the general mechanisms are the same. However, omega-3 isoprostanes are not easy to quantify in urine.

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.

In order to commence isoprostane formation, the presence of reactive oxygen species is required, which can abstract a hydrogen atom from bis-allylic methylene groups of polyunsaturated fatty acids under aerobic conditions in vivo in animals (and plants). 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. As the G and H-ring endoperoxide structures are highly labile compounds with a half-life of only a few minutes, they isomerize rapidly to give a variety of products, including analogues of PGE and PGD.

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.

A second mechanism 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.

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 has still to be determined, but the latter is probably a minor route.

Isolevuglandins (isoLGs) are a family of reactive γ-ketoaldehydes, analogous to the levuglandins (see our web pages on prostanoids), generated via bicyclic endoperoxyl intermediates by free radical oxidation of arachidonate-containing lipids by  mechanisms believed to be related to isoprostane formation. 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. They form Schiff bases and pyrroles rapidly with the ε-amino groups of lysyl residues in proteins, before they are oxidized to lactam and hydroxylactam end products.

Isofurans are oxidation products of arachidonate that contain substituted tetrahydrofuran rings. 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. Related compounds formed from docosahexaenoic acid have been characterized from the brain cortex of a mouse model of Alzheimer disease.

Isoprostanes that are structurally related to PGE₂ and PGD₂ 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.

As the isoprostanes in animal tissues are formed from arachidonic acid predominantly in position 2 of phospholipids, they must be released by the action of phospholipase A₂ 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.

3.   Biological Activity

Isoprostanes are believed to be valuable indicators of oxidative stress in animal tissues, i.e. when 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. There is growing acceptance that measurement of the relatively stable F₂-isoprostanes, and 8-IsoPF_2α in particular, in urine is a reliable non-invasive approach to the determination of the degree of oxidative stress in patients. 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, cirrhosis of the liver, brain degeneration, ischemia–reperfusion injury, atherosclerosis and diabetes. 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. 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.

While isoprostanes have been observed to have innumerable physiological functions in vitro, the extent of their importance in vivo is uncertain and controversial. The biological activity of 15-F_2t-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 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. Similarly, isoprostanes have been implicated in oxidative damage to the liver, where they are markers or more controversially mediators of the effects.

Lipid peroxidation is believed to be a factor in many disease states associated with the brain. IsoPA₂ and IsoPJ₂ are usually considered to be the preferred products of the isoprostane pathway in brain; 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.

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, 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.

The concentrations of protein-adducts of isolevuglandins have been shown to increase greatly in plasma from patients with advanced atherosclerosis. Such lipid-protein conjugates 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.

4.   Analysis of Isoprostanes

Gas and liquid chromatography allied to mass spectrometry, after appropriate extraction and derivatization, are the most accurate methods for identifying and quantifying individual isoprostanes in biological fluids. However, such methods 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

• Basu, S. Isoprostanes. In: Bioactive Lipids. pp. 265-285 (edited by A. Nicolaou and G. Kokotos, Oily Press, Bridgwater) (2004).
• Cracowski, J.L. Isoprostanes: an emerging role in vascular physiology and disease? Chem. Phys. Lipids, 128, 75-83 (2004).
• Fam, S.S. and Morrow, J.D. The isoprostanes: unique products of arachidonic acid oxidation - A review. Curr. Med. Chem., 10, 1723-1740 (2003).
• Fessel, J.P. and Roberts, L.J. Isofurans: novel products of lipid peroxidation that define the occurrence of oxidant injury in settings of elevated oxygen tension. Antioxid. Redox Signal., 7, 202-209 (2005).
• Janssen, L.J. The pulmonary biology of isoprostanes. Chem. Phys. Lipids, 128, 101-116 (2004).
• Milne, G.L., Musiek, E.S. and Morrow, J.D. The cyclopentenone (A2/J2) isoprostanes - unique, highly reactive products of arachidonate peroxidation. Antioxid. Redox Signal., 7, 210-220 (2005).
• Montine, K.S., Quinn, J.F., Zhang, J., Fessel, J.P., Roberts, L.J., Morrow, J.D. and Montine, T.J. Isoprostanes and related products of lipid peroxidation in neurodegenerative diseases. Chem. Phys. Lipids, 128, 117-124 (2004).
• Musiek, E.S., Yin, H.Y., Milne, G.L. and Morrow, J.D. Recent advances in the biochemistry and clinical relevance of the isoprostane pathway. Lipids, 40, 987-994 (2005).
• Pratico, D., Rokach, J., Lawson, J. and FitzGerald, G.A. F₂-isoprostanes as indices of lipid peroxidation in inflammatory diseases. Chem. Phys. Lipids, 128, 165-171 (2004).
• Roberts, L.J. and Fessel, J.P. The biochemistry of the isoprostane, neuroprostane, and isofuran pathways of lipid peroxidation. Chem. Phys. Lipids, 128, 173-186 (2004).
• Salomon, R.G. Levuglandins and isolevuglandins: stealthy toxins of oxidative injury. Antioxid. Redox Signal., 7, 185-201 (2005).
• Taber, D.F., Morrow, J.D. and Roberts, L.J. A nomenclature system for the isoprostanes. Prostaglandins, 53, 63-67 (1997).
• Yin, H., Brooks, J.D., Gao, L., Porter, N.A. and Morrow, J.D. Identification of novel autoxidation products of the ω-3 fatty acid eicosapentaenoic acid in vitro and in vivo. J. Biol. Chem., 282, 29890-29901 (2007).
• Yin, H.Y. and Porter, N.A. New insights regarding the autoxidation of polyunsaturated fatty acids. Antioxid. Redox Signal., 7, 170-184 (2005).

Updated: 5/5/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland