F-2 Isoprostanes

[Colette]

Theisoprostanes are prostaglandin-like compounds formed in vivo from the free radical-catalyzed peroxidationof essential fatty acids (primarily arachidonic acid) without the direct action of cyclooxygenase (COX) enzymes. The compounds were discovered in 1990 by L. Jackson Roberts and Jason D. Morrow in the Division of Clinical Pharmacology at Vanderbilt University.[1][2] [3][4]These nonclassical eicosanoids possess potent biological activity as inflammatory mediators that augment the perception of pain.[5] These compounds are accurate markers of lipid peroxidation in both animal and human models of oxidative stress.

Elevated levels of isoprostanes are suspected of contributing to increased risk of heart attack in patients taking Coxibs[citation needed]. Isoprostanes and their metabolites have also been shown to be elevated in the urine of cigarette smokers, and have been suggested as biomarkers of oxidative stress in smokers.[6]

Polyunsaturated fatty acids other than arachidonic acid are also vulnerable to reactive oxygen species and produce isoprostanes. For example, in addition to the four classes of F2-isoprostanes that can arise from arachidonic acid, peroxidation of eicosapentaenoic acid (EPA) is predicted to lead to the generation of six classes of F3 isoprostanes, α-linolenic and γ-linolenic acids to two classes of E1- and F1-isoprostanes, and docosahexaenoic acid to eight classes of D4-isoprostanes and eight classes of E4-isoprostanes. Each of the classes comprise up to eight racemic isomers, leading to an astounding number of isoprostane molecules.[7]

The isoprostanes (IsoPs) are a unique series of prostaglandin-like compounds formed in vivo via the non-enzymatic, free radical-catalyzed peroxidation of arachidonic acid. These compounds were first discovered in the Division of Clinical Pharmacology at Vanderbilt by Drs. Jack Roberts and Jason Morrow in 1990. Over the course of the past 20 years, we and others have carried out studies defining the basic chemistry and biochemistry involved in the formation and metabolism of IsoPs. Today, one particular class of IsoPs, F2-IsoPs (shown below), is known as the "gold standard" biomarker of endogenous lipid peroxidation resulting from oxidative stress.

Because these molecules are chemically stable and have been identified in every biological matrix analyzed, measurement of F2-IsoPs has revolutionized the ability of investigators to quantify oxidant injury in vivo. In a multi-investigator study sponsored by the National Institute of Environmental Health Sciences (NIEHS), termed the Biomarkers of Oxidative Stress (BOSS) Study, it was found that quantification of plasma or urinary F2-IsoPs by mass spectrometry was the most accurate method to assess endogenous oxidative stress.

A number of mass spectrometric methods have been developed to quantify F2-IsoPs. Our laboratory uses gas chromatography/negative ion chemical ionization mass spectrometry (GC/NICI-MS) employing stable isotope dilution with [2H4]-15-F2t-IsoP as the internal standard. 15-F2t-IsoP, one abundant F2-IsoP regioisomer, and other co-eluting F2-IsoPs are quantified using this method. This approach is that originally developed by Morrow and Roberts and has been widely used in a variety of research and clinical studies. Click here to view the assay methodology as published in Nature Protocols. (Please note that the nomenclature of F2-IsoPs varies in the literature. 15-F2t-IsoP is also referred to as 8-iso-prostaglandin F2α or 8-iso-PGF2α as well as iPF2α-III.)

Importantly, normal levels of F2-IsoPs in healthy humans have been defined. Normal levels in plasma are 35 +/- 6 pg/mL while normal levels in urine are 1.6 +/- 0.6 ng/mg Creatinine. Defining these levels allows for assessment of various disease states on endogenous oxidant tone and allows for the determination of the extent to which therapeutic interventions affect levels of oxidative stress. Levels of F2-IsoPs are increased in human body fluids and tissues in many diseases and disorders including atherosclerosis and associated risk factors including smoking and obesity, ischemia/reperfusion injury, certain types of cancers, neurodegeneration, and asthma, among others. Further, antioxidant supplementation as well as lifestyle changes (ie. cessation of cigarette smoking or weight loss) have been shown to decrease levels of F2-IsoPs.

Each year the Eicosanoid Core Laboratory works with investigators around the world to quantify F2-IsoPs. If you are interested in measuring F2-IsoPs in your research, please contact Dr. Ginger Milne with any questions regarding study design, sample collection/storage, or pricing. We look forward to working with you!

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Some years ago it was discovered that prostaglandin F2-like compounds are formed in vivo by nonenzymatic free radical-catalyzed peroxidation of arachidonic acid. Because these compounds are a series of isomers that contain the prostane ring of prostaglandins, they were termed F2-isoprostanes. Intermediates in the isoprostane pathway are prostaglandin H2-like compounds that become reduced to form F2-isoprostanes but also undergo rearrangement in vivo to form E2-, D2-, A2-, J2-isoprostanes, isothromboxanes, and highly reactive gamma-ketoaldehydes, termed isoketals. Analogous compounds have also been shown to be formed from free radical mediated oxidation of docosoahexaenoic acid. Because docosahexaenoic acid is highly enriched in neurons, these compounds have been termed neuroprostanes and neuroketals. An important aspect of the discovery of isoprostanes is that measurement of F2-isoprostanes has emerged as one of the most reliable approaches to assess oxidative stress status in vivo, providing an important tool to explore the role of oxidative stress in the pathogenesis of human disease. Measurement of F4-neuroprostanes has also proved of value in exploring the role of oxidative stress in neurodegenerative diseases. Products of the isoprostane pathway have been found to exert potent biological actions and therefore may participate as physiological mediators of disease.

We recently reported the discovery of a series of bioactive prostaglandin F2-like compounds (F2-isoprostanes) that are produced in vivo by free radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme. Inasmuch as phospholipids readily undergo peroxidation, we examined the possibility that F2-isoprostanes may be formed in situ on phospholipids. Initial support for this hypothesis was obtained by the finding that levels of free F2-isoprostanes measured after hydrolysis of lipids extracted from livers of rats treated with CCl4 to induce lipid peroxidation were more than 100-fold higher than levels in untreated animals. Further, increased levels of lipid-associated F2-isoprostanes in livers of CCl4-treated rats preceded the appearance of free compounds in the circulation, suggesting that the free compounds arose from hydrolysis of peroxidized lipids. This concept was supported by demonstrating that free F2-isoprostanes were released after incubation of lipid extracts with bee venom phospholipase A2 in vitro. When these lipid extracts were analyzed by HPLC, fractions that yielded large quantities of free F2-isoprostanes after hydrolysis eluted at a much more polar retention volume than nonoxidized phosphatidylcholine. Analysis of these polar lipids by fast atom bombardment mass spectrometry established that they were F2-isoprostane-containing species of phosphatidylcholine. Thus, unlike cyclooxygenase-derived prostanoids, F2-isoprostanes are initially formed in situ on phospholipids, from which they are subsequently released preformed, presumably by phospholipases. Molecular modeling of F2-isoprostane-containing phospholipids reveals them to be remarkably distorted molecules. Thus, the formation of these phospholipid species in lipid bilayers may contribute in an important way to alterations in fluidity and integrity of cellular membranes, well-known sequelae of oxidant injury.

Using muscle bath techniques, we examined the inhibitory activities of several E- and F-ring isoprostanes in canine and porcine airway smooth muscle. 8-Isoprostaglandin E1 and 8-isoprostaglandin E2 (8-iso PGE2) reversed cholinergic tone in a concentration-dependent manner, whereas the F-ring isoprostanes were ineffective. Desensitization with 8-iso-PGE2 and PGE2 implicated isoprostane activity at the PGE2 receptor (EP). We found that the inhibitory E-ring isoprostane responses were significantly augmented by rolipram (a type IV phosphodiesterase inhibitor), while 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) had no effect, suggesting a role for cAMP in isoprostane-mediated relaxations. 8-Iso-PGE2 did not reverse KCl tone, suggesting that voltage-dependent Ca2+ influx and myosin light chain kinase are not suppressed by isoprostanes. Patch-clamp studies showed marked suppression of K+ currents by 8-iso-PGE2. We conclude that E-ring isoprostanes exert PGE2 receptor-directed, cAMP-dependent relaxations in canine and porcine airway smooth muscle. This activity is not dependent on K+ channel activation or the direct inhibition of voltage-operated Ca2+ influx or myosin light chain kinase.

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