CHEMICAL BIOLOGY

Isoprostanes

Ginger L. Milne and Jason D. Morrow,, Division of Clinical Pharmacology, Vanderbilt University Medical Center,, Nashville,, Tennessee

doi: 10.1002/9780470048672.wecb268

The isoprostanes are a unique series of prostaglandin-like compounds formed in vivo via a nonenzymatic mechanism involving the free radical-initiated peroxidation of arachidonic acid. This article summarizes our current knowledge of these compounds. Herein, a historical account of their discovery and the mechanism of their formation are described. Methods by which these compounds can be analyzed and quantified are also discussed, and the use of these molecules as biomarkers of in vivo oxidant stress is summarized. In addition to being accurate indices of lipid peroxidation, some isoprostanes possess potent biological activity. This activity will be discussed in detail. Finally, in more recent years, isoprostane-like compounds have been shown to be formed from polyunsaturated fatty acids, including eicosapentaenoic acid and docosahexaenoic acid. These findings will be summarized as well.

Free radicals, largely derived from molecular oxygen, have been implicated in a variety of human conditions and diseases, including atherosclerosis and associated risk factors, cancer, neurodegenerative diseases, and aging. Damage to tissue biomolecules by free radicals is postulated to contribute significantlly to the pathophysiology of oxidative stress. Measuring oxidative stress in humans requires accurate quantification of either free radicals or damaged biomolecules. The targets of free radical-mediated oxidant injury include lipids, proteins, and DNA. Several methods exist to quantify free radicals and their oxidation products, although many of these techniques suffer from lack of sensitivity and specificity, especially when used to assess oxidant stress status in vivo. In a recent multi-investigator study, termed the Biomarkers of Oxidative Stress Study (BOSS), sponsored by the National Institutes of Health, it was found that the most accurate method to assess in vivo oxidant stress status is the quantification of plasma or urinary isoprostanes (IsoPs) (1). IsoPs, a series of prostaglandin (PG)-like compounds produced by the free radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase, were first discovered by our laboratory in 1990 (2). Since that time, we and others have shown that levels of IsoPs are increased in several human diseases. Furthermore, several of these compounds possess potent biological activity and thus may be mediators of oxidant injury. In recent years, additional related compounds, derived from various polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been discovered to be formed as products of the IsoP pathway. It is the purpose herein to summarize our current knowledge regarding the IsoPs, including their discovery, the chemical biology of their formation, the utility of these compounds as markers of in vivo oxidant stress, and their bioactivity.

Historical Aspects

In the 1960s and 1970s, it was shown that PG-like compounds could be formed by the autoxidation of purified polyunsaturated fatty acids. Seminal studies by Pryor, Porter, and others (see Further Reading) led to a proposed mechanism by which these compounds are generated via bicycloendoperoxide intermediates. However, this work was never carried beyond in vitro studies. Furthermore, it was not determined whether PG-like compounds could be formed in biological fluids containing unsaturated fatty acids.

In the 1980s, we showed that PGD₂ derived from the cyclooxygenase is primarily metabolized in vivo in humans to form 9α, 11β-PGF₂ by the enzyme 11-ketoreductase. In aqueous solutions, however, PGD₂ is an unstable compound that undergoes isomerization of the lower side chain and these isomers can likewise be reduced by 11-ketoreductase to yield isomers of 9α, 11β-PGF₂. In studies undertaken to further characterize these compounds using gas chromatography (GC)/mass spectrometry (MS), we found that when plasma samples from normal volunteers were processed and analyzed immediately, a series of peaks was detected possessing characteristics of F-ring PGs. Interestingly, however, when plasma samples that had been stored at —20 °C for several months were reanalyzed, identical chromatographic peaks were detected but levels of putative PGF₂-like compounds were up to 100-fold higher. Subsequent experiments led to the finding that these PGF₂-like compounds were generated in both freshly processed and stored plasma, not by a COX-derived mechanism, but nonenzymatically by autoxidation of arachidonic acid. Because these compounds contain F-type prostane rings, they are referred to as F₂-IsoPs.

Formation of the F₂-Isoprostanes

A mechanism to explain the formation of the F₂-IsoPs from arachidonic acid is outlined in Fig. 1 and is based on that proposed by Pryor for the generation of bicycloendoperoxide intermediates. After abstraction of a bisallylic hydrogen atom and the addition of a molecule of oxygen to arachidonic acid to form a peroxyl radical, the peroxyl radical undergoes 5-exo cyclization and a second molecule of oxygen adds to the backbone of the compound to form PGG2-like compounds. These unstable bicycloendoperoxide intermediates are then reduced to the F₂-IsoPs. Based on this mechanism of formation, four F₂-IsoP regioisomers aare generated. Compounds are denoted as 5-, 12-, 8-, or 15-series regioisomers depending on the carbon atom to which the side-chain hydroxyl is attached 3. An alternative nomenclature system for the IsoPs has been proposed by Rokach et al. in which the abbreviation iP is used for isoprostane and the regioisomers are denoted as III-VI based on the number of carbons between the omega carbon and the first double bond 4.

Although the initial abstraction of any bisallylic hydrogen atoms of arachidonic acid is equally likely, the different IsoP regioisomers are not formed in equal amounts. When arachidonic acid is oxidized, the 5- and 15-series regoisomers are formed in significantly higher amounts than the 8- and 12-series regoisomers. An explanation for this difference has been elucidated by Yin et al., who demonstrated that the arachidonyl hydroperoxides which give rise to the 8- and 12-series regioisomers readily undergo further oxidation to yield a newly discovered class of compounds that contain both bicycloendoperoxde and cyclic peroxide moieties termed dioxolane-IsoPs (Fig. 2) (5). Overall, 5- and 15-series reioisomers cannot undergo this further oxidation but instead accumulate at higher concentrations in tissues and fluids. Analytical methods allowing the separation and quantification of each of the four classes of IsoP regioisomers, as well as their individual stereoisomers, have recently been developed in our laboratory.

An important structural distinction between IsoPs and cyclooxygenase-derived PGs is that the former contain side chains that are predominantly oriented cis to the prostane ring, whereas the latter possess exclusively trans side chains. A second important difference between IsoPs and PGs is that IsoPs are formed in situ esterified to phospholipids and are subsequently released by a phospholipase(s), whereas PGs are generated only from free arachidonic acid.

Figure 1. Mechanism of formation of the F₂-isoprostanes.

Figure 2. Formation of dioxolane isoprostanes.

Quantification of F₂-Isoprostanes

Several methods have been developed to quantify the F₂-IsoPs. Our laboratory uses a gas chromatographic/negative ion chemical ionization-mass spectrometric (GC/NICI-MS) approach employing stable isotope dilution (6). For quantification purposes, we measure the F₂-IsoP, 15-F_2t-IsoP, and other F₂-IsoPs that coelute with this compound. Several internal standards are available from commercial sources to quantify the IsoPs. In our assays, we typically use either [²H₄]-15-F_2t-IsoP ([²H₄]-8-iso- PGF_2a) or [²H₄]-PGF_2a as internal standards. The advantages of mass spectrometry over other approaches include its high sensitivity and specificity, which yields quantitative results in the low picogram range. Its drawbacks are that it is labor intensive and requires considerable expenditures on equipment.

Several alternative mass spectrometric assays have been developed by different investigators, including Rokach et al. (4, 7, 8). Like our assay, these methods quantify F₂-IsoPs using stable isotope dilution GC/NICI-MS and require solid-phase extraction using a C18 column, thin layer chromatography (TLC) purification, and chemical derivatization. These assays, however, measure F₂-IsoP isomers other than 15-F₂-IsoP, but they are comparable with ours in terms of utility. In addition to these GC/MS assays, several liquid chromatographic (LC) MS methods for F₂-IsoPs have been developed. One advantage of LC/MS methods is that the sample preparation for analysis is simpler than that for GC/MS because no derivatization of the molecule is required. The method reported earlier this year by Taylor et al. is the first of these LC/MS methods to be validated for quantitation of IsoPs in biological fluids (9). In this assay, a gradient reverse-phase LC tandem mass spectrometric approach is used to separate several 15-series IsoP stereoisomers for quantitation with [²H₄]-PGF_2a as the internal standard. The outcomes of this assay correlated significantly with GC/MS results, and the coefficients of variation in the measurements were lower for their LC/MS assay than for their GC/MS assay. The authors thus suggest that this LC/MS method potentially offers greater precision than existing methods while allowing for the quantitation of more compounds with simpler sample preparation.

Alternative methods have also been developed to quantify IsoPs using immunological approaches (6, 10). Antibodies have been generated against 15-F_2t-IsoP, and at least three immunoassay kits are commercially available. Although mass spectrometric methods of IsoP quantification are considered the best methods for analysis, immunoassays have expanded research in this area due to their low cost and relative ease of use. Only limited information is currently available regarding the precision and accuracy of immunoassays. In addition, little data exist comparing IsoP levels determined by immunoassay with MS. though Wang et al. offers one example of an MS-validated immunoassay (11) out laboratory’s experiments have not validated the commercical available kits. Analogous to immunological methods to quantify cyclooxygenase-derived PGs, immunoassays for IsoPs suffer from a lack of specificity. Furthermore, the sensitivity and/or specificity of these kits vary substantially between manufacturers.

F₂-Isoprostanes as an Index of Oxidant Stress In Vivo

The true utility of the F₂-IsoPs is in the quantification of lipid peroxidation and thus oxidant stress status in vivo. F₂-IsoPs are stable, robust molecules and are detectable in all human tissues and biological fluids analyzed, including plasma, urine, bronchoalveolar lavage fluid, cerebrospinal fluid, and bile (6). The quantification of F₂-IsoPs in urine and plasma, however, is most convenient and least invasive. And, based on available data, quantification of these compounds in either plasma or urine is representative of their endogenous production and thus gives a highly precise and accurate index of in vivo oxidant stress. Although measurement of F₂-IsoPs in plasma is indicative of their endogenous formation at a specific point in time, analysis of these compounds in urine is an index of systemic or “whole-body” oxidant stress integrated over time. It is important to note that the measurement of free F₂-IsoPs in urine can be confounded by the potential contribution of local IsoP production in the kidney. In light of this issue, we have identified the primary urinary metabolite of 15-F_2t-IsoP to be 2,3-dinor-57 5,6-dihydro-15-F_2t-IsoP, and we have developed a highly sensitive and accurate mass spectrometric assay to quantify this molecule. However, the extent to which 15-F_2t-IsoP is converted to the urinary metabolite remains unclear. Nevertheless, the quantification of 2,3-dinor-5,6-dihydro-15-F_2t-IsoP may represent a truly noninvasive, time-integrated measurement of systemic oxidation status that can be applied to living subjects.

Normal levels of F₂-IsoPs in healthy humans have been defined (6, 12, 13). Defining these levels is particularly important in that it allows for an assessment of the effects of diseases on endogenous oxidant tone and allows for the determination of the extent to which various therapeutic interventions affect levels of oxidant stress. Elevations of IsoPs in human body fluids and tissues have been found in a diverse array of human disorders, some of which include atherosclerosis, hypercholesterolemia, diabetes, obesity, cigarette smokng, neurodegenerative diseases, and rheumatoid arthritis. Furthermore treatments for some of these conditions, including antioxidant supplementaton, c treaments, cessation of smoking, and even weight loss, have been shown to decrease production of F₂-IsoPs. Thus, the clinical utility of F₂-IsoPs has been great and continues to grow. For manuscripts and comprehensive reviews on IsoP detection in human disease, please refer to the Further Reading Section.

Biological Activities of the F₂-Isoprostanes

In addition to being robust markers of in vivo oxidant stress, F₂-IsoPs can exert potent biological activity and potentially mediate some adverse effects of oxidant injury. As mentioned, IsoPs are initially formed in vivo esterified to glycerophospholipids. Molecular modeling of IsoP-containing phospholipids reveals them to be remarkably distorted molecules. Thus, the formation of these abnormal phospholipids would be expected to exert profound effects on membrane fluidity and integrity, well-known sequelae of oxidant injury. All studies exploring their bioactivity, however, have been performed using unesterified IsoPs. Recent studies by Stafforini et al. have shown that F₂-IsoP hydrolysis from phospholipids is regulated, at least in part, by the platelet-activating factor (PAF) acetylhydrolases I and II (14).

One particular F₂-IsoP that is produced abundantly in vivo and has been extensively tested for biological activity is 15-F_2t-IsoP (8-iso-PGF_2a), which differs from cyclooxygenase-derived PGF_2a only in the inversion of the upper side-chain stereochemistry. This IsoP has been found to be a potent vasoconstrictor in a variety of vascular beds, including the kidney, lung, heart, and brain. In addition, 15-F_2t-IsoP induces endothelin release and proliferation of vascular smooth muscle cells. There is also additional evidence that this molecule can increase resistance to aspirin inhibition of platelet aggregation in platelets as well as inhibit platelet aggregation in human whole blood. These vasoactive effects of 15-F_2t-IsoP have been shown to result from interaction with the thromboxane receptor, a G-protein-coupled transmembrane eicosanoid receptor, based on the finding that these effects can be abrogated by thromboxane receptor antagonists.

The testing of other F₂-IsoPs for biological activity has been limited. It has been shown, however, that 15-F_2c-IsoP (12-iso-PGF_2a) activates the PGF_2a receptor and induces hypertrophy in cardiac smooth muscle cells.

Formation of Isoprostanes with Alternative Ring Structures

Since the initial discovery of the F₂-IsoPs, our laboratory has shown that the IsoP pathway provides a mechanism for the generation of various classes of IsoPs from arachidonic acid, which differ in regard to the functional groups on the prostane ring. In addition to undergoing reduction to yield F₂-IsoPs, the arachidonyl endoperoxide intermediate can undergo isomerization to yield E- and D-ring IsoPs (Fig. 3), which are isomeric to PGE₂ and PGD₂, respectively 15 E₂/D₂-IsoPs are formed competitively with F₂-IsoPs, and recent studies have demonstrated that the depletion of cellular reducing agents, such as glutathione (GSH) or α-tocopherol, favors the formation of E₂/D₂-IsoPs over that of reduced F₂-IsoPs. Importantly, depletion of GSH and α-tocopherol occurs in various human tissues under conditions of oxidant injury, including the brains of patients with Alzheimer’s disease (AD). Thus, the ratios of F-ring to E/D-ring IsoPs in postmortem brain tissues from patients with AD were examined, and not only were levels of both E₂/D₂-and F₂-IsoPs significantly elevated, but also E₂/D₂-IsoPs were the favored products of the IsoP pathway in affected brain regions. This increased ratio of E₂/D₂-IsoPs to F₂-IsoPs provides information not only about lipid peroxidation in a given organ but also about the reducing environment in that tissue.

E₂/D₂-IsoPs, however, are not terminal products of the IsoP pathway. These compounds readily dehydrate in vivo to yield A₂/J₂-IsoPs (Fig. 3), which are also known as cyclopentenone IsoPs because they contain an α, β-unsaturated cyclopentenone ring structure (16, 17). A₂/J₂-IsoPs are highly reactive electrophiles that readily form Michael adducts with cellular thiols, including those found on cysteine residues in proteins and glutathione. These cyclopentenone IsoPs are rapidly metabolized in vivo by glutathione transferase enzymes to water-soluble modified glutathione conjugates. The major urinary cyclopentenone IsoP metabolite in rats, a 15-A_2t-IsoP mercapturic acid sulfoxide conjugate, was recently identified in our laboratory 18.

The chemical reactivity of cyclopentenone IsoPs suggested that these compounds might be biologically active. The recent synthesis of two cyclopentenone IsoPs regioisomers, 15-A₂-IsoP and 15-J₂-IsoP, has allowed us to examine their bioactivity. Studies employing primary cortical neuronal cultures demonstrated that both 15-A₂-IsoP and 15-J₂-IsoP potently induce neuronal apoptosis and exacerbate neurodegeneration caused by other insults at concentrations as low as 100 nM (19). These compounds elicit neuronal apoptosis via a discreet signaling pathway that overlaps extensively with cell death signaling cascades triggered by other oxidative insults. Thus, A₂/J₂-IsoPs, or the downstream signaling pathways activated by these compounds, might represent novel neuroprotective therapeutic targets.

Cyclopentenone IsoPs also exert biological effects in non-neural tissue. Recent studies employing macrophages reveal that 15-A₂-IsoPs potently suppress lipopolysacharide (LPS)-induced inflammatory signaling via inhibition of the NF-KB pathway (20). 15-A₂-IsoPs abrogate inducible nitric oxide synthase and cyclooxygenase-2 expression in response to LPS, as well as the elaboration of several pro-inflammatory cytokines. Similar anti-inflammatory effects were observed with 15-J₂-IsoPs. However, 15-J₂-IsoPs also activate the peroxisome proliferators activated receptor-gamma (PPARy) with an EC₅₀ of approximately 3 μM. This receptor modulates a wide variety of biological processes, including inflammatory signaling and fatty acid metabolism. 15-J₂-IsoPs also induce macrophage apoptosis at low micromolar concentrations in a PPAR_γ-independent manner. Thus, there is a diversity of actions among cyclopentenone IsoP isomers. It seems that these compounds could act as negative-feedback regulators of the inflammatory response, because oxidative stress and lipid peroxidation often occur under conditions of chronic inflammation.

Figure 3. Formation of E₂/D₂-isoprostanes and A₂/J₂-isoprostanes.

Formation of Isoprostanes from Other PUFAs

Arachidonic acid is not the only polyunsaturated fatty acid that can be oxidized to generate IsoPs. The basic requirement for cyclization to occur is the presence of at least three double bonds. F-ring IsoPs have been shown to be generated from the peroxidation of linolenic acid [C18:3, ω-3, F₁-IsoPs] (21), EPA [C20:5, ω-3, F₃-IsoPs] (22), and DHA [C22:6, ω-3, F₄-IsoPs or F₄-neuroprostanes (NPs)] (23). Similar to the distribution of F₂-IsoP regioisomers, certain F₃-IsoP and F₄-NP regioisomers are more abundant than others (24). The 5-series and 18-series F₃-IsoPs are the most abundant because the 8-, 12-, and 15-series compounds can further oxidize (unpublished data). Analogously, the 4-series and 20-series F4-NPs are formed in the largest amounts. In addition to F-ring compounds, E- and D-ring as well as A- and J-ring, or cyclopentenone, IsoPs are generated from the oxidation of EPA (unpublished data) and DHA.

In recent years, emerging evidence has implicated increased dietary intake of fish oil, which contains large amounts of EPA and DHA, as being beneficial in the prevention and treatment of several diseases, including atherosclerotic cardiovascular disease and sudden death, neurodegeneration, and various inflammatory disorders. Furthermore, recent data have suggested that the anti-inflammatory effects and other biologically relevant properties of ω-3 fatty acids are due, in part, to the generation of various bioactive oxidation products (25, 26). We thus hypothesized that EPA- and DHA-derived IsoPs could contribute to the beneficial biological effects of fish oil supplementation. Indeed, one report states that the EPA-derived IsoP, 15-F_3t-IsoP, possesses activity that is different from 15-F_2t-IsoP in that it does not affect human platelet shape change or aggregation. The lack of activity of 15-F_3t-IsoP is consistent with observations regarding EPA-derived PGs in that these latter compounds exert either a weaker agonist or no effects in comparison with arachidonic acid-derived PGs.

Interestingly, our laboratory has recently shown that the levels of these compounds generated from the oxidation of EPA significantly exceeded those of F₂-IsoPs generated from arachidonic acid, perhaps because EPA contains more double bonds and is therefore more easily oxidizable. Additionally, in vivo in mice, levels of F₃-IsoPs in tissues such as heart were virtually undetectable at baseline but supplementation of animals with EPA markedly increased quantities up to 27.4 ± 5.6-ng/g heart. But, of particular note, we found that EPA supplementation markedly reduced levels of arachidonate-derived F₂-IsoPs by up to 64% (p < 0.05). Furthermore, in a recent small clinical trial, termed the KANWU study, of 162 healthy men and women, plasma F₂-IsoP levels significantly decreased (by as much as 29%) after 3 months of supplementation with 3.6-g/day fish oil (27). Together these observations are significant because F₂-IsoPs are generally considered to be proinflammatory molecules associated with the pathophysiological sequelae of oxidant stress (28-31). It is thus intriguing to propose that part of the mechanism by which EPA prevents certain diseases is its ability to decrease F₂-IsoP generation. In addition, it suggests that supplementation with fish oil may be of benefit to populations associated with increased levels of F₂-IsoPs.

Conclusions

The discovery of the IsoPs as products of nonenzymatic lipid peroxidation has been a major breakthrough in the field of free radical research. The quantification of these molecules has opened up new areas of investigation regarding the role of free radicals in human physiology and pathophysiology, and it seems to be the most useful tool currently available to explore the role of lipid peroxidation in the pathogenesis of human disease. Our understanding of the IsoP pathway continues to expand, providing new insights into the nature of lipid peroxidation in vivo, and revealing new molecules that exert potent biological actions and might serve as unique indices of disease. Basic research into the biochemistry and pharmacology of the IsoPs, coupled with clinical studies employing these molecules as biomarkers, should continue to provide important insights into the role of oxidant stress in human disease.

References

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Further Reading

Free Radicals and Disease

Ames BN. Dietary carcinogens and anticarcinogens. Oxygen radicals and degenerative diseases. Science 1983; 221:1256.

Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: an overview. Meth. Enzymol. 1990; 186:1.

Mechanisms of Lipid Peroxidation

Porter NA, Funk MO. Letter: peroxy radical cyclization as a model for prostaglandin biosynthesis. J. Org. Chem. 1975; 40:3614.

Pryor WA, Stanley JP, Blair E. Autoxidation of polyunsaturated fatty acids: II. A suggested mechanism for the formation of TBA-reactive materials from prostaglandin-like endoperoxides. Lipids 1976; 11:370.

Pryor WA, Stanley JP, Blair E, Cullen GB. Autoxidation of polyunsaturated fatty acids. Part I. Effect of ozone on the autoxidation of neat methyl linoleate and methyl linolenate. Arch. Environ. Health 1976; 31:201.

F₂-Isoprostanes: Formation and Metabolism

Lawson JA, Rokach J, Fitzgerald GA. Isoprostanes: formation, analysis and use as indices of lipid peroxidation in vivo. J. Biol. Chem. 1999; 274:24441.

Liu T, Stern A, Roberts LJ, Morrow JD. The isoprostanes: novel prostaglandin-like products of the free radical-catalyzed peroxidation of arachidonic acid. J. Biomed. Sci. 1999; 6:226.

Morrow JD, Awad JA, Boss HJ, Blair IA, Roberts LJ Jr. Non-cyclooxygenase-derived prostanoids (F₂-isoprostanes) are formed in situ on phospholipids. Proc. Natl. Acad. Sci. U.S.A. 1992; 89:10721.

Morrow JD, Chen Y, Brame CJ, Yang J, Sanchez SC, Xu J, Zackert WE, Awad JA, Roberts LJ. The isoprostanes: unique prostaglandin like products of free radical-catalyzed lipid peroxidation. Drug Metab. Rev. 1999; 31:117.

Morrow JD, Minton TA, Badr KF, Roberts LJ. Evidence that the F₂ isoprostane, 8-epi-prostaglandin F₂ alpha, is formed in vivo. Biochim. Biophys. Acta 1994; 1210:244.

Morrow JD, Zackert WE, Yang JP, Kurhts EH, Callewaert D, Dworski R, Kanai K, Taber D, Moore K, Oates JA, Roberts LJ. Quantification of the major urinary metabolite of 15-F_2t-isoprostane (8-iso-PGF2 alpha) by a stable isotope dilution mass spectrometric assay. Anal. Biochem. 1999; 269:326.

Roberts L Jr, Morrow JD. Products of the isoprostane pathway: unique bioactive compounds and markers of lipid peroxidation. Cell Mol. Life Sci. 2002; 59:808.

Rokach J, Khanapure SP, Hwang SW, Adiyaman M, Lawson JA, Fitzgerald GA. The isoprostanes: a perspective. Prostaglandins 1997; 54:823.

Isoprostanes as Biomarkers of Oxidative Stress

Basu S, Whiteman M, Mattey DL, Halliwell B. Raised levels of F₂-isoprostanes and prostaglandin F₂ alpha in different rheumatic diseases. Ann. Rheum. Dis. 2001; 60:627.

Kadiiska MB, Gladen BC, Baird DD, Graham LB, Parker CE, Ames BN, Basu S, Fitzgerald GA, Lawson JA, Marnett LJ, Morrow JD, Murray DM, Plastaras J, Roberts LJ Jr, Rokach J, Shigenaga MK, Sun J, Walter PB, Tomer KB, Barrett JC, Mason RP. Biomarkers of oxidative stress study III. Effects of the nonsteroidal anti-inflammatory agents indomethacin and meclofenamic acid on measurements of oxidative products of lipids in CCl4 poisoning. Free Radic. Biol. Med. 2005; 38:711.

Roberts LJ, Morrow JD. Measurement of F₂-isoprostanes as an index of oxidative stress in vivo. Free Radic. Biol. Med. 2000; 28:505.

Neurodegeneration

Bohnstedt KC, Karlberg B, Wahlund LO, Jonhagen ME, Basun H, Schmidt S. Determination of isoprostanes in urine samples from Alzheimer patients using porous graphitic carbon liquid chromatography-tandem mass spectrometry. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2003; 796:11.

Montine TJ, Beal MF, Robertson D, Cudkowicz ME, Biaggioni I, O’Donnell H, Zackert WE, Roberts LJ, Morrow JD. Cerebrospinal fluid F₂-isoprostanes are elevated in Huntington’s disease. Neurology 1999; 52:1104.

Montine TJ, Markesbery WR, Morrow JD, Roberts LJ Jr. Cerebrospinal fluid F₂-isoprostane levels are increased in Alzheimer’s disease. Ann. Neurol. 1998; 44:410.

Montine TJ, Montine KS, Reich EE, Terry ES, Porter NA, Morrow JD. Antioxidants significantly affect the formation of different classes of isoprostanes and neuroprostanes in rat cerebral synaptosomes. Biochem. Pharmacol. 2003; 65:611.

Montine TJ, Neely MD, Quinn JF, Beal MF, Markesbery WR, Roberts LJ, Morrow JD. Lipid peroxidation in aging brain and Alzheimer’s disease. Free Radic. Biol. Med. 2002; 33:620.

Montine TJ, Quinn JF, Montine KS, Kaye JA, Breitner JC. Quantitative in vivo biomarkers of oxidative damage and their application to the diagnosis and management of Alzheimer’s disease. J. Alzheimers Dis. 2005; 8:359.

Montine KS, Quinn JF, Zhang J, Fessel JP, Roberts LJ Jr, Morrow JD, Montine TJ. Isoprostanes and related products of lipid peroxidation in 48 neurodegenerative diseases. Chem. Phys. Lipids 2004; 128:117.

Reich EE, Markesbery WR, Roberts LJ Jr, Swift LL, Morrow JD, Montine TJ. Brain regional quantification of F-ring and D-/E-ringsoprostanes and neuroprostanes in Alzheimer’s disease. Am. J. Pathol. 2001;158:293.

Atherosclerosis

Block G, Dietrich M, Norkus EP, Morrow JD, Hudes M, Caan B, Packer L. Factors associated with oxidative stress in human populations. Am. J. Epidemiol. 2002; 156:274.

Bruno RS, Leonard SW, Atkinson J, Montine TJ, Ramakrishnan R, Bray TM, Traber MG. Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation. Free Radic. Biol. Med. 2006; 40:689.

Davi G, Alessandrini P, Mezzetti A, Minotti G, Bucciarelli T, Costantinone F, Cipollione F, Bon GB, Ciabattoni G, Patrono C. In vivo formation of of 8-Epi-prostaglandin F₂ alpha is increased in hypercholesterolemia. Arterioscler. Thromb. Vasc. Biol. 1997; 17:3230.

Davi G, Guagnano MT, Ciabattoni G, Basili S, Falco A, Marinopiccoli M, Nutini M, Sensi S, Patrono C. Platelet activation in obese women: role of inflammation and oxidant stress. JAMA 2002; 288:2008.

Dietrich M, Block G, Hudes M, Morrow JD, Norkus EP, Traber MG, Cross CE, Packer L. Antioxidant supplementation decreases lipid peroxidation biomarker F₂-isoprostanes in plasma of smokers. Cancer Epidemiol. Biomarkers Prev. 2002; 11:7.

Gniwotta C, Morrow JD, Roberts LJ Jr, Kuhn H. Prostaglandin F₂-like compounds, F₂-isoprostanes, are present in increased amounts in human atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 1997; 17:3236.

Gross M, Steffes M, Jacobs DR Jr, Yu X, Lewis L, Lewis CE, Loria CM. Plasma F₂-isoprostanes and coronary artery calcification: the CARDIA Study. Clin. Chem. 2005; 51:125.

Helmersson J, Larsson A, Vessby B, Basu S. Active smoking and a history of smoking are associated with enhanced prostaglandin F(2alpha), interleukin-6 and F₂-isoprostane formation in elderly men. Atherosclerosis 2005; 181:201.

Kaikkonen J, Porkkala-Sarataho E, Morrow JD, Roberts LJ Jr, Nyyssonen K, Salonen R, Tuomainen TP, Ristonmaa U, Poulsen HE, Salonen JT. Supplementation with vitamin E but not with vitamin C lowers lipid peroxidation in vivo in mildly hypercholesterolemic men. Free Radic. Res. 2001; 35:967.

Keaney JF Jr, Larson MG, Vasan RS, Wilson PW, Lipinska I, Corey D, Massaro JM, Sutherland P, Vita JA, Benjamin EJ. Obesity and systemic oxidative stress: clinical correlates of oxidative stress in the Framingham Study. Arterioscler. Thromb. Vasc. Biol. 2003; 23:434.

Morrow JD. Quantification of isoprostanes as indices of oxidant stress and the risk of atherosclerosis in humans. Arterioscler. Thromb. Vasc. Biol. 2005; 25:279.

Patrono C, Falco A, Davi G. Isoprostane formation and inhibition in atherothrombosis. Curr. Opin. Pharmacol. 2005; 5:198.

Patrono C, Fitzgerald GA. Isoprostanes: potential markers of oxidant stress in atherothrombotic disease. Arterioscler. Thromb. Vasc. Biol. 1997; 17:2309.

Pratico D, Tangirala RK, Rader DJ, Rokach J, Fitzgerald GA. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice. Nat. Med. 1998; 4:1189.

Diabetes

Davi G, Chiarelli F, Santilli F, Pomilio M, Vigneri S, Falco A, Basili S, Ciabattoni G, Patrono C. Enhanced lipid peroxidation and platelet activation in the early phase of type 1 diabetes mellitus: role of interleukin-6 and disease duration. Circulation 2003; 107:3199.

Davi G, Ciabattoni G, Consoli A, Mezzetti A, Falco A, Santarone S, Pennese E, Vitacolonna E, Bucciarelli T, Costantini F, Capani F, Patrono C. In vivo formation of 8-iso-prostaglandin F₂ alpha and platelet activation in diabetes mellitus: effects of improved metabolic control and vitamin E supplementation. Circulation 1999; 99:224.

Biological Activities of Isoprostanes

Balapure AK, Rexroad CE Jr, Kawada K, Watt DS, Fitz TA. Structural requirements for prostaglandin analog interaction with the ovine corpus luteum prostaglandin F₂ alpha receptor. Implications for development of a photoaffinity probe. Biochem. Pharmacol. 1989; 38:2375.

Banerjee M, Kang KH, Morrow JD, Roberts LJ, Newman JH. Effects of a novel prostaglandin, 8-epi-PGF₂ alpha, in rabbit lung in situ. Am. J. Physiol. 1992; 263:H660.

Cranshaw JH, Evans TW, Mitchell JA. Characterization of the effects of isoprostanes on platelet aggregation in human whole blood. Br. J. Pharmacol. 2001; 132:1699.

Csiszar A, Stef G, Pacher P, Ungvari Z. Oxidative stress-induced isoprostane formation may contribute to aspirin resistance in platelets. Prostaglandins Leukot. Essent. Fatty Acids 2002; 66:557.

Fukunaga M, Takahashi K, Badr KF. Vascular smooth muscle actions and receptor interactions of 8-iso-prostaglandin E₂, an E₂-isoprostane. Biochem. Biophys. Res. Commun. 1993; 195:507.

Fukunaga M, Yura T, Badr KF. Stimulatory effect of 8-Epi-PGF₂ alpha, an F₂-isoprostane, on endothelin-1 release. J. Cardiovasc. Pharmacol. 1995; 26(Suppl 3):S51.

Fukunaga M, Yura T, Grygorczyk R, Badr KF. Evidence for the distinct nature of F₂-isoprostane receptors from those of thromboxane A₂. Am. J. Physiol. 1997; 272:F477.

Hoffman SW, Moore S, Ellis EF. Isoprostanes: free radical-generated prostaglandins with constrictor effects on cerebral arterioles. Stroke 1997; 28:844.

Kang KH, Morrow JD, Roberts LJ Jr, Newman JH, Banerjee M. Airway and vascular effects of 8-epi-prostaglandin F₂ alpha in isolated perfused rat lung. J. Appl. Physiol. 1993; 74:460.

Kunapuli P, Lawson JA, Rokach J, Fitzgerald GA. Functional characterization of the ocular prostaglandin F₂ alpha (PGF₂ alpha) receptor. Activation by the isoprostane, 12-iso-PGF₂ alpha. J. Biol. Chem. 1997; 272:27147.

Kunapuli P, Lawson JA, Rokach JA, Meinkoth JL, FitzGerald GA. Prostaglandin F₁ alpha (PGF₂ alpha) and the isoprostane, 8, 12-iso-isoprostane F₂ alpha-III, induce cardiomyocyte hypertrophy. Differential activation of downstream signaling pathways. J. Biol. Chem. 1998; 273:22442.

Mobert J, Becker BF, Zahler S, Gerlach E. Hemodynamic effects of isoprostanes (8-iso-prostaglandin F₂ alpha and E₂) in isolated guinea pig hearts. J. Cardiovasc. Pharmacol. 1997; 29:789.

Pratico D, Smyth EM, Violi F, Fitzgerald GA. Local amplification of platelet function by 8-Epi prostaglandin F₂ alpha is not mediated by thromboxane receptor isoforms. J. Biol. Chem. 1996; 271:14916.

Takahashi K, Nammour TM, Fukunaga M, Ebert J, Morrow JD, Roberts LJ Jr, Hoover RL, Badr KF. Glomerular actions of a free radical-generated novel prostaglandin, 8-epi-prostaglandin F₂ alpha, in the rat. Evidence for interaction with thromboxane A₂ receptors. J. Clin. Invest. 1992; 90:136.

Yura T, Fukunaga M, Khan R, Nassar GN, Badr KF, Montero A. Free-radical-generated F₂-isoprostane stimulates cell proliferation and endothelin-1 expression on endothelial cells. Kidney Int. 1999; 56:471.

D- and E-ring Eicosanoids: Formation and Metabolism

Liston TE, Oates JA, Roberts LJ Jr. Prostaglandin D₂ is metabolized in humans to 9-alpha,11-beta-prostaglandin F₂, a novel biologically active prostaglandin. Adv. Prostaglandin Thromboxane Leukot. Res. 1985; 15:365.

Liston TE, Roberts, LJ Jr. Metabolic fate of radiolabeled prostaglandin D₂ in a normal human male volunteer. J. Biol. Chem. 1985; 260:13172.

Liston TE, Roberts LJ Jr. Transformation of prostaglandin D₂ to 9-alpha,

11-beta-(15S)-trihydroxyprosta-(5Z,13E)-dien-1-oic acid (9-alpha, 11-beta-54 prostaglandin F₂): a unique biologically active prostaglandin produced enzymatically in vivo in humans. Proc. Natl. Acad. Sci. U.S.A. 1985; 82:6030.

Morrow JD, Minton TA, Mukundan CR, Campbell MD, Zackert WE, Daniel VC, Badr KF, Blair IA, Roberts, LJ Jr. Free radical-induced generation of isoprostanes in vivo. Evidence for the formation of D-ring and E-ring isoprostanes. J. Biol. Chem. 1994; 269:4317.

Wendelborn DF, Seibert K, Roberts LJ Jr. Isomeric prostaglandin F₂ compounds arising from prostaglandin D₂: a family of eicosanoids produced in vivo in humans. Proc. Natl. Acad. Sci. U.S.A. 1988; 85:304.

Cyclopentenone (A-and J-ring) Eicosanoids: Formation and Metabolism

Hubatsch I, Mannervik B, Gao L, Roberts LJ, Chen Y, Morrow JD. The cyclopentenone product of lipid peroxidation, 15-A_2t-isoprostaglandin (8-isoprostaglandin A₂), is efficiently conjugated with glutathione by human and rat glutathione transferase A4-4. Chem. Res. Toxicol. 2002; 15:1114.

Milne GL, Gao L, Porta A, Zanoni G, Vidari G, Morrow JD. Identification of the major urinary metabolite of the highly reactive cy- clopentenone isoprostane 15-A_2t-isoprostane in vivo. J. Biol. Chem. 2005; 280:25178.

Milne GL, Musiek ES, Morrow JD. The cyclopentenone (A2/J2) isoprostanes—unique, highly reactive products of arachidonate peroxidation. Antioxid. Redox Signal 2005; 7:210.

Milne GL, Zanoni G, Porta A, Sasi S, Vidari G, Musiek ES, Freeman ML, Morrow, JD. The cyclopentenone product of lipid peroxidation, 15-A2t-isoprostane, is efficiently metabolized by HepG2 cells via conjugation with glutathione. Chem. Res. Toxicol. 2004; 17:17.

Zanoni G, Porta A, Castronovo F, Vidari G. First total synthesis of J2 isoprostane. J. Org. Chem. 2003; 68:6005.

Zanoni G, Porta A, Vidari G. First total synthesis of A2 isoprostane. J. Org. Chem. 2002; 67:4346.

Omega-3 Fatty Acids

Hong S, Gronert K, Devchand PR, Moussignac RL, Serhan CN. Novel docosatrienes and 17S-resolvins generated from docosahexaenoic acid in murine brain, human blood, and glial cells. Autacoids in anti-inflammation. J. Biol. Chem. 2003; 278:14677.

Kris-Etherton PM, Harris WS, Appel LJ. Fish consumption, fish oil, omega-3 fatty acids, and cardiovascular disease. Arterioscler. Thromb. Vasc. Biol. 2003; 23:e20.

Kris-Etherton PM, Harris WS, Appel LJ. Omega-3 fatty acids and cardiovascular disease: new recommendations from the American Heart Association. Arterioscler. Thromb. Vasc. Biol. 2003; 23:151.

Kulkarni PS, Srinivasan BD. Prostaglandins E₃ and D₃ lower intraocular pressure. Invest. Ophthalmol. Vis. Sci. 1985; 26:1178.

Ruxton, C. Health benefits of omega-3 fatty acids. Nurs. Stand. 2004; 18:38.

Ruxton CH, Reed SC, Simpson MJ, Millington KJ. The health benefits of omega-3 polyunsaturated fatty acids: a review of the evidence. J. Hum. Nutr. Diet 2004; 17:449.

Serhan CN, Clish CB, Brannon J, Colgan SP, Chiang N, Gronert K. Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase2-nonsteroidal antiinflammatory drugs and transcellular processing. J. Exp. Med. 2000; 192:1197.

Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, Mirick G, Moussignac RL. Resolvins: afamily of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J. Exp. Med. 2002; 196:1025.

Serhan CN, Levy B. Novel pathways and endogenous mediators in anti-inflammation and resolution. Chem. Immunol. Allergy 2003; 83:115.

Sethi S. Inhibition of leukocyte-endothelial interactions by oxidized omega-3 fatty acids: a novel mechanism for the anti-inflammatory effects of omega-3 fatty acids in fish oil. Redox Rep. 2002; 7:369.

Sethi S, Eastman AY, Eaton JW. Inhibition of phagocyte-endothelium interactions by oxidized fatty acids: a natural anti-inflammatory mechanism? J. Lab. Clin. Med. 1996; 128:27.

See Also

Prostaglandins

Chromatography of Lipids

GC-MS of Lipids

Redox Regulation and Signaling: Reactive Oxygen Species (ROS)

Chemistry of Oxidative DNA Damage