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(The FASEB Journal. 2004;18:1791-1800.)
© 2004 FASEB

Isoprostanes: markers and mediators of oxidative stress

PAOLO MONTUSCHI, PETER J. BARNES* and L. JACKSON ROBERTS, II{dagger},1

Department of Pharmacology, School of Medicine, Catholic University of the Sacred Heart, Rome, Italy;
* Department of Thoracic Medicine, Imperial College, Faculty of Medicine at the National Heart and Lung Institute, London, UK; and
{dagger} Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, Tennessee, USA

1 Correspondence: Department of Pharmacology, 522 RRB, 23rd & Pierce Ave., Vanderbilt University, Nashville, TN 37232-6602, USA. E-mail: jack.roberts{at}vanderbilt.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 OXIDATIVE STRESS AND LIPID...
 THE ISOPROSTANE PATHWAYS
 MEASURING OXIDATIVE STRESS:...
 ISOPROSTANES AS MARKERS OF...
 BIOLOGICAL EFFECTS
 MECHANISMS OF ACTION
 THERAPEUTIC IMPLICATIONS
 CONCLUSIONS
 REFERENCES
 
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.—Montuschi, P., Barnes, P. J., Roberts, L J., II. Isoprostanes: markers and mediators of oxidative stress.


Key Words: lipid peroxidation • F2-isoprostanes • neuroprostanes • F-ring


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
OXIDATIVE STRESS AND LIPID...
 THE ISOPROSTANE PATHWAYS
 MEASURING OXIDATIVE STRESS:...
 ISOPROSTANES AS MARKERS OF...
 BIOLOGICAL EFFECTS
 MECHANISMS OF ACTION
 THERAPEUTIC IMPLICATIONS
 CONCLUSIONS
 REFERENCES
 
OXIDATIVE STRESS is characterized by an imbalance between increased exposure to free radicals, principally derived from oxygen, and antioxidant defenses, comprised of both small molecular weight antioxidants, such as glutathione, and antioxidant enzymes, such as superoxide dismutase. Free radicals can be generated endogenously from various sources (for example, mitochondria and oxidative burst during phagocyte activation) or derived from exogenous sources such as environmental toxins and cigarette smoke. Free radicals cause direct damage to critical biomolecules including DNA, lipids, and proteins. Oxidative stress is now recognized to be a prominent feature of many acute and chronic diseases including cancer, cardiovascular disease, neurodegenerative disease, lung disease and even the normal aging process. However, definitive evidence for this association has often been lacking due to recognized shortcomings with methods available to assess oxidant stress status in vivo in humans (1) .

Several in vitro markers of oxidative stress are available, but most are of limited value in vivo because they lack sensitivity and/or specificity or require invasive methods (2) . Isoprostanes are prostaglandin (PG) -like substances that are produced in vivo independently of cyclooxygenase (COX) enzymes, primarily by free radical-induced peroxidation of arachidonic acid (3) . The formation of PG-like compounds during auto-oxidation of polyunsaturated fatty acids was first reported in the mid-1970s (4) , but isoprostanes were not discovered to be formed in vivo in humans until 1990 (3) . F2-isoprostanes are a group of 64 compounds isomeric in structure to cyclooxygenase-derived PGF2{alpha}. Other products of the isoprostane pathway are also formed in vivo by rearrangement of labile PGH2-like isoprostane intermediates. These include E2- and D2-isoprostanes (5) , cyclopentenone-A2- and J2-isoprostanes (6) , and highly reactive acyclic-ketoaldehydes (isoketals) (7) (Fig. 1 ). Oxidation of docosahexaenoic acid, an abundant unsaturated fatty acid in the central nervous system, results in the formation of isoprostane-like compounds, termed neuroprostanes (8) .



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  		Figure 1. Isoprostane (IsoP) pathway of free radical-induced oxidation of arachidonic acid. Tx: thromboxane. IsoK: isoketals

The discovery of isoprostanes has important implications for medicine (5) : First, it has now been established that measurement of F2-isoprostanes is the most reliable approach to assess oxidative stress status in vivo, providing an important tool to explore the role of oxidative stress in the pathogenesis of human disease. In addition, products of the isoprostane pathway have been found to exert potent biological actions and therefore may be pathophysiologic mediators of disease. This review is timely since the number of publications related to isoprostanes is increasing at an exponential rate (9) . We summarize the biological importance of lipid peroxidation and oxidative stress and how they are currently measured. We focus on the advantages of measuring F2-isoprostanes as biomarkers of lipid peroxidation, the clinical utility of measuring F2-isoprostanes for monitoring disease and response to therapy, their potential role as mediators of oxidative stress, and the therapeutic implications of this knowledge.


   OXIDATIVE STRESS AND LIPID PEROXIDATION
TOP
 ABSTRACT
 INTRODUCTION
 OXIDATIVE STRESS AND LIPID...
THE ISOPROSTANE PATHWAYS
 MEASURING OXIDATIVE STRESS:...
 ISOPROSTANES AS MARKERS OF...
 BIOLOGICAL EFFECTS
 MECHANISMS OF ACTION
 THERAPEUTIC IMPLICATIONS
 CONCLUSIONS
 REFERENCES
 
Lipids are a major target of free radical attack, which induces lipid peroxidation. Lipid peroxidation is a self-propagating phenomenon terminated by antioxidants (see below; R•=free radical species, L=lipid, A=antioxidant).

  1. Initiation: R• + LH -> RH + L•
  2. Propagation: L• + O2 ->
  3. LOO• LOO• + LH -> LOOH + L•
  4. Termination: L• + AH -> LH + A•
  5. A• + LOO• -> LOO-A

Free radical-induced peroxidation of membrane lipids can be very damaging because it leads to alterations in the biophysical properties of the membrane, such as the degree of fluidity, and can lead to inactivation of membrane-bound receptors or enzymes, which in turn may impair normal cellular function. Moreover, generation of highly reactive secondary aldehyde products of lipid peroxidation—e.g., isoketals from the isoprostane pathway and 4-hydroxynonenal (10) —may contribute to and amplify cellular damage due to their ability to covalenty modify critical biomolecules. Therefore, measurement of products of lipid peroxidation has been commonly used to assess oxidative stress/injury.


   THE ISOPROSTANE PATHWAYS
TOP
 ABSTRACT
 INTRODUCTION
 OXIDATIVE STRESS AND LIPID...
 THE ISOPROSTANE PATHWAYS
MEASURING OXIDATIVE STRESS:...
 ISOPROSTANES AS MARKERS OF...
 BIOLOGICAL EFFECTS
 MECHANISMS OF ACTION
 THERAPEUTIC IMPLICATIONS
 CONCLUSIONS
 REFERENCES
 
After arachidonic acid peroxidation, three arachidonyl radicals are produced and undergo endocyclization to form four PGH2-like bicyclic endoperoxide intermediate regioisomers (Fig. 1) . These are reduced to four F-ring regioisomers, each consisting of eight racemic diastereoisomers. The Eicosanoid Nomenclature Committee has approved a nomenclature system for isoprostanes in which the different regioisomer classes are designated by the carbon number of the side chain where the hydroxyl is located, with the carboxyl carbon designated as C-1 (Fig. 1) (11) . In this review, we will use this nomenclature system. F2-Isoprostanes are initially formed esterified on phospholipids and then released in free form by phospholipase(s) (12) . Isoprostane endoperoxides may undergo rearrangement in vivo to form an E- or D-ring and thromboxane-ring compounds (5) . E2- and D2-isoprostanes undergo dehydration in vivo to form reactive cyclopentenone A2- and J2-isoprostanes (6) . Highly reactive {gamma}-ketoaldehydes (isoketals or isolevuglandins) may be formed as products of isoprostane endoperoxide rearrangement (7) (Fig. 1) . The metabolic fate of isoprostanes is mostly unknown except for 15-F2t-isoprostane (8-iso-PGF2{alpha}). 2,3-Dinor-5,6-dihydro-15-F2t-isoprostane is the major urinary metabolite of 15-F2t-isoprostane in humans (13) . Docosahexaenoic acid (C22:6{omega}3) is the most abundant unsaturated fatty acid in the central nervous system. Oxidation of this 22-carbon fatty acid may result in the formation of isoprostane-like compounds, termed neuroprostanes, which may be unique markers of oxidative neuronal injury (8) . As with isoprostanes, neuroprostane bicyclic endoperoxide intermediates are reduced to F-rings (F4-neuroprostanes) or undergo rearrangement to E- and D-ring compounds (E4/D4-neuroprostanes) (14) . The latter can undergo dehydration, resulting in the formation of cyclopentenone neuroprostanes, termed A4/J4-neuroprostanes (15) . Endoperoxides may undergo rearrangement to form highly reactive isoketal-like compounds termed neuroketals (16) . Limited to 15-F2t-isoprostane, an enzymatic synthesis via COX-1 and/or COX-2 has been reported in vitro (17 18 19) and in vivo (20) . However, nonselective COX inhibition does not affect urinary excretion of 15-F2t-isoprostane in healthy subjects (21) or in patients with pathophysiologic conditions associated with platelet activation and increased F2-isoprostane production (22 , 23) . Moreover, COX activation contributed undetectably to 15-F2t-isoprostane formation in healthy subjects who were given endotoxin to induce inflammation (24) . Current evidence indicates that the COX pathway is a trivial contributor to overall 15-F2t-isoprostane generation in vivo (5 , 25) . 5-F2t-isoprostane exhibits no capacity for enzymatic synthesis (26) .


   MEASURING OXIDATIVE STRESS: ADVANTAGES OF THE ISOPROSTANES
TOP
 ABSTRACT
 INTRODUCTION
 OXIDATIVE STRESS AND LIPID...
 THE ISOPROSTANE PATHWAYS
 MEASURING OXIDATIVE STRESS:...
ISOPROSTANES AS MARKERS OF...
 BIOLOGICAL EFFECTS
 MECHANISMS OF ACTION
 THERAPEUTIC IMPLICATIONS
 CONCLUSIONS
 REFERENCES
 
Despite the importance of measuring lipid peroxidation to explore the potential role of oxidative stress in the pathogenesis of human diseases, no previously existing assay of lipid peroxidation was considered "ideal." Assays that had been developed had several shortcomings related to 1) the specificity of the assay itself for the product of lipid peroxidation being measured, 2) the product being measured was not a specific product of lipid peroxidation, 3) the lack of sufficient sensitivity to detect levels of the product being measured in normal subjects, thus allowing the definition of a normal range, 4) levels of the product being measured being influenced by external factors, such as the lipid content of the diet, or 5) the assay being too invasive for human investigation (27) . The most widely used test for oxidative stress is measurement of malondialdehyde (MDA), a product of lipid peroxidation, by a thiobarbituric acid-reacting substances (TBARS) assay (10) . However, the use of this assay to assess oxidative stress status is problematic because MDA is not a specific product of lipid peroxidation and the TBARS assay is not specific for MDA (27) . Another method of assessing lipid peroxidation in vivo is measurement of exhaled volatile alkanes, such as ethane and pentane (28) . However, the accuracy of exhaled pentane as a marker of endogenous lipid peroxidation has been questioned (29) : these hydrocarbon gases are minor end-products of peroxidation and their concentrations are influenced by the breakdown rate of peroxides (27) . Various methods have been used to measure lipid hydroperoxides, but marked inconsistencies have been found with levels detected, for example, in human plasma, raising questions regarding accuracy of assay methodology (30) . Lipid hydroperoxydes cannot not be detected in the circulation even under conditions of severe oxidative stress using a highly accurate and sensitive gas chromatography/mass spectrometry (GC/MS) assay (2) , rendering this approach for assessing oxidative stress status in humans of little or no value.

F2-Isoprostanes are considered the best available biomarkers of oxidative stress status and lipid peroxidation in vivo (2) ; other products of the isoprostane pathway such as D2- and E2-isoprostanes are less suitable as they are less stable (2) . Although isoprostanes are not a major product of lipid peroxidation, current methodology is able to readily detect their steady-state levels in vivo (5) . F2-Isoprostanes are initially formed in situ esterified in phospholipids, then released in free form by phospholipase action (12) . F2-Isoprostanes are detectable in their esterified form in all normal biological tissues and in free form in all normal biological fluids, indicating "physiological" levels of oxidative stress (2 , 5 , 25) . Approaches to assess endogenous production of F2-isoprostanes in humans include measurement of free unmetabolized F2-isoprostanes in biological fluids such as plasma and urine, measurement of esterified F2-isoprostanes in biopsy specimens and plasma lipoproteins, and measurement of the major urinary metabolite of the F2-isoprostane 15-F2t-isoprostane, 2,3-dinor-5,6-dihydro-15-F2t-isoprostane, which may provide an accurate assessment of total endogenous F2-isoprostane production (2) . GC/MS is the reference analytical method for isoprostane measurements in biological fluids and tissues (2) , but it is time-consuming and expensive. For these reasons, immunoassays for the measurement of isoprostanes have been developed and are commercially available. A radioimmunoassay has been quantitatively validated by GC/MS analysis of 15-F2t-isoprostane and cross-reactivity with other F2-isoprostane isomers is very low (21) . However, the immunoassays have not been tested for cross-reactivity with F2-isoprostane metabolites, which might be formed in vivo (25) . While some immunoassays have been shown to correlate fairly well with GC/MS measurements (21) , others have not (31) .

Measurement of F2-isoprostanes has several advantages over other quantitative markers of oxidative stress (2) . F2-Isoprostanes are 1) are chemically stable, 2) specific products of peroxidation, 3) are formed in vivo, 4) are present in detectable amounts in all normal tissues and biological fluids, thus allowing definition of a normal range; 5) levels increase substantially in animal models of oxidant injury, 6) are unaffected by lipid content in the diet (32 33 34) , and 7) might provide a sensitive biochemical basis in dose-finding studies with antioxidants (2) . Isoprostanes are frequently measured in urine, because it is noninvasive, isoprostanes are not formed artifactually by auto-oxidation in urine, and they are very stable in urine (35) . There is no significant daily variability of urinary isoprostane concentrations in healthy subjects (21 , 32 , 36) ; day-to-day variability in healthy and disease states is relatively limited (22 , 26 , 37 , 38) , although one study reported significant variability (39) . F2-Isoprostanes have been detected in exhaled breath condensate (EBC), a completely noninvasive method to collect secretions from the airways suitable for repeated measures of inflammation and oxidative stress in patients with lung disease, including young children and adults with severe disease (40 , 41) . Measurement of F2-isoprostanes esterified in plasma lipoproteins may provide a useful approach to assess oxidation of LDL in vivo (18 , 42) , a central feature of atherosclerosis. Measurement of F2-isoprostanes in tissues and/or biological fluids provides a valuable new approach to the quantification of oxidative stress as well as a biochemical basis for assessing therapeutic intervention. In fact, a key issue in performing an intervention trial with an antioxidant is to establish whether the treatment decreases oxidative stress in study subjects and at what dose(s) (27) . Measurement of the level of lipid peroxidation as reflected by F2-isoprostane concentrations in biological fluids may help to identify those patients most likely to benefit from antioxidant treatment.

One shortcoming associated with measurement of F2-isoprostanes has been recognized. Because of the mechanism by which F2-isoprostanes are generated, their formation is impaired at elevated oxygen tensions, so that measurement of F2-isoprostanes is an insensitive marker of lipid peroxidation in settings of elevated oxygen tension, such as hyperoxia-induced lung injury. However, the recent discovery of isofurans, products of lipid peroxidation with a substituted tetrahydrofuran ring whose formation is favored as oxygen tension increases, overcomes this limitation with measurement of F2-isoprostanes (43) . Future studies may indicate that combined measurement of both F2-isoprostanes and isofurans affords the best assessment of oxidative stress under all circumstances than either measurement alone.


   ISOPROSTANES AS MARKERS OF OXIDATIVE STRESS IN HUMAN DISEASES
TOP
 ABSTRACT
 INTRODUCTION
 OXIDATIVE STRESS AND LIPID...
 THE ISOPROSTANE PATHWAYS
 MEASURING OXIDATIVE STRESS:...
 ISOPROSTANES AS MARKERS OF...
BIOLOGICAL EFFECTS
 MECHANISMS OF ACTION
 THERAPEUTIC IMPLICATIONS
 CONCLUSIONS
 REFERENCES
 
The measurement of isoprostanes in biological fluids and/or tissue specimens has important clinical implications. Measurement of F2-isoprostanes has implicated a role of free radicals and oxidant injury in a wide variety of human diseases, including cardiovascular, pulmonary, neurological, renal, and liver diseases (Table 1 ) (2 , 5 , 25) . Although the association between increased oxidative stress and disease does not necessarily imply a causative link, the fact that the increase in F2-isoprostane levels is an early event in asthma (44 , 45) , hepatic cirrhosis (46) , scleroderma (47) , and Alzheimer’s disease (48 , 49) suggests a causative role for oxidative stress at least in these diseases. Chronic healthy smokers have higher free and esterified F2-isoprostane plasma concentrations and urinary excretion of F2-isoprostane metabolites compared than healthy nonsmokers, indicating pro-oxidant effects of smoking in vivo (22 , 50) . This conclusion is further supported by the fact that isoprostane levels return to baseline values 2 wk after smoking cessation (50) . Since LDL oxidation may lead to atherosclerosis, these findings may provide a causative link between smoking and the development of atherosclerosis (5 , 50) . Measurement of isoprostanes may have prognostic value in those diseases in which a role for oxidative stress has been implicated.


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  		Table 1. Disorders and pathophysiological conditions in which measurement of F2-soprostanes has implicated a role for oxidative stress in humansa

The issue of whether lipid peroxidation is part of the pathogenesis of Alzheimer’s disease or a consequence of neurodegeneration is still uncertain. However, current evidence suggests that oxidative stress is an early event that may play an important role in the pathogenesis of Alzheimer’s disease (48 , 49 , 51) . F2-Isoprostane concentrations in cerebrospinal fluid are elevated early in the course of dementia (52) , and correlate with disease severity (53 , 54) and progression (Fig. 2 ). F2-Isoprostanes are elevated in urine in young patients with Down’s syndrome, which is associated with precocious Alzheimer’s disease-like pathology and dementia (55) . Pericardial F2-isoprostane concentrations increase with the functional severity of heart failure and are associated with ventricular dilatation, suggesting a possible role for in vivo oxidative stress on ventricular remodeling and the progression to heart failure (56) .



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  		Figure 2. Concentrations of F2-isoprostanes in postmortem ventricular fluid obtained from 23 patients with Alzheimer’s disease and 11 age-matched controls (54) (left panel). Horizontal lines are means. F2-Isoprostane levels were significantly higher in patients with Alzheimer’s disease than controls (P<0.01). Mean F2-isoprostane concentrations (±SE) in ventricular fluid were plotted against cortical atrophy in the same patients and control subjects (54) (right panel). Cortical atrophy was graded as absent (degree 0, n=15), mild (degree 1, n=8), moderate (degree 2, n=8), or severe (degree 3, n=4) in all patients with Alzheimer’s disease and controls. Spearman’s ranked correlation gave P < 0.01. Analysis restricted to Alzheimer’s disease patients only was statistically significant (n=23, P<0.05).

In patients with lung diseases, 15-F2t-isoprostane concentrations in EBC reflect the degree of airway inflammation (44 , 57 , 58) , with the highest levels reported in patients with acute lung injury/adult respiratory distress syndrome (59) (Fig. 3 ). Likewise, urinary isoprostane concentrations correlate with disease severity in scleroderma (60) .



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  		Figure 3. 8-Isoprostane (15-F2t-isoprostane) concentrations in exhaled breath condensate in healthy nonsmoker (normal, n=10) (57) , healthy current smokers (HS, n=12) (57) , patients with mild (n=12), moderate (n=15), and severe (n=17) asthma (44) , patients with chronic obstructive pulmonary disease (COPD) who were ex-smokers (n=25) or current smokers (n=15) (57) , and patients with cystic fibrosis (n=19) (58) . 8-Isoprostane concentrations were measured by an enzyme immunoassay (58) . Values are expressed as means ± SE.


   BIOLOGICAL EFFECTS
TOP
 ABSTRACT
 INTRODUCTION
 OXIDATIVE STRESS AND LIPID...
 THE ISOPROSTANE PATHWAYS
 MEASURING OXIDATIVE STRESS:...
 ISOPROSTANES AS MARKERS OF...
 BIOLOGICAL EFFECTS
MECHANISMS OF ACTION
 THERAPEUTIC IMPLICATIONS
 CONCLUSIONS
 REFERENCES
 
Isoprostanes are not only biomarkers of oxidative stress but have numerous biological effects, suggesting they may function as pathophysiologic mediators of oxidant injury. Most of the current knowledge on the biological actions of F2-isoprostanes is limited to 15-F2t-isoprostane, which is a potent vasoconstrictor. In the rat, F2-isoprostanes reduce glomerular filtration rate and renal blood flow by 40–45% in the low nanomolar range (Fig. 4 ) (3 , 61) . The E-ring isoprostane 15-E2t-isoprostane is a potent renal vasoconstrictor (5) . In experimental animals, 15-F2t-isoprostane has constrictor effects in other vascular beds including pulmonary artery (3) , coronary arteries (62) , cerebral arterioles (63) , retinal vessels (64) , and portal vein (65) . F2-Isoprostanes potently induce retinal vascular endothelial cell death in newborn rats and piglets (66) . F2-Isoprostanes cause contraction of human bronchial smooth muscle in vitro (67) and induce airflow obstruction and plasma exudation in guinea pigs in vivo (68) . It has been found that the major urinary metabolite of 15-F2t-isoprostane is a potent constrictor of retinal and brain microvessels comparable to that observed with 15-F2t-isoprostane (69) .



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  		Figure 4. Reduction of renal blood flow during infusion of 8-isoprostane (15-F2t-isoprostane) in the renal artery of rats (3 , 61) . Values are expressed as means ± SE.

Isoprostanes have important in vitro activities that could be relevant to the pathophysiology of atherosclerosis (70) . F2-Isoprostanes promote platelet activation (70) and induce mitogenesis in vascular smooth muscle cells (61) . Moreover, F2-isoprostane formation is increased during LDL oxidation in vitro (42) and they are major contributors to the proadhesive effect induced by minimally oxidatively modified LDL on neutrophils (71) . Isoprostanes stimulate proliferative responses in fibroblasts (72) , cause hypertrophy of rat neonate ventricular myocytes (73) , and can alter endothelial cell biology as indicated by proliferative effects and increased endothelin-1 expression in bovine aortic endothelial cells (74) . At present it is not known whether the concentrations of isoprostanes reached locally in vivo are sufficient to exert biological effects, because no specific inhibitor of their biological actions exists (vide infra). However, their overproduction may contribute to the increased platelet activation in patients with diabetes (75) , bronchoconstriction in patients with asthma (67) , decreased renal blood flow in the hepatorenal syndrome (5 , 76) and rhabdomyolysis-induced renal failure (77 , 78) , fetal/newborn hypoxic-ischemic encephalopathies (63) , and in some complications of cardiac reperfusion injury such as myocardial stunning (70) . Isoketals, highly reactive products of the isoprostane pathway, and isoketal adducted proteins inhibit proteasome activity and, if produced in excess, may have relevance to the pathogenesis of neurodegenerative diseases and other diseases involving oxidative stress associated with impaired proteasome function (79) .


   MECHANISMS OF ACTION
TOP
 ABSTRACT
 INTRODUCTION
 OXIDATIVE STRESS AND LIPID...
 THE ISOPROSTANE PATHWAYS
 MEASURING OXIDATIVE STRESS:...
 ISOPROSTANES AS MARKERS OF...
 BIOLOGICAL EFFECTS
 MECHANISMS OF ACTION
THERAPEUTIC IMPLICATIONS
 CONCLUSIONS
 REFERENCES
 
Products of the isoprostane pathway exert their biological effects by receptor-mediated interactions—vasoconstriction in the case of F2-isoprostanes and E2-isoprostanes (5) —and through their inherent chemical reactivity (e.g., adduct formation, in the case of cyclopentenone-isoprostanes and isoketals). Cyclopentenone-isoprostanes, which are PGA2- and PGJ2-like compounds, readily react with thiols forming glutathione and protein adducts (6) . Isoketals rapidly adduct to lysine residues on proteins and induce cross-links at rates that exceed other aldehyde products of lipid peroxidation (7) . The nature of the receptor(s) involved in isoprostane actions is not certain. Whereas the vasoconstricting actions of 15-F2t-isoprostane can be abrogated by thromboxane A2 (TP) receptor antagonists, suggesting that it acts as an incidental ligand at TP receptors (5 , 80) , it acts primarily as an antagonist of thromboxane-induced platelet aggregation (81) . In piglet retinal and brain vasculature, 15-F2t-isoprostane has been shown to cause vasoconstriction by inducing thromboxane formation in the endothelium (63 , 64) . However, F2-isoprostanes activate other prostanoid receptors (FP and ET3) (71 , 72 , 82) and possibly a novel isoprostane receptor (5 , 82 , 83) . Molecular cloning of the isoprostane receptor would clarify the mechanism(s) of action of these compounds.


   THERAPEUTIC IMPLICATIONS
TOP
 ABSTRACT
 INTRODUCTION
 OXIDATIVE STRESS AND LIPID...
 THE ISOPROSTANE PATHWAYS
 MEASURING OXIDATIVE STRESS:...
 ISOPROSTANES AS MARKERS OF...
 BIOLOGICAL EFFECTS
 MECHANISMS OF ACTION
 THERAPEUTIC IMPLICATIONS
CONCLUSIONS
 REFERENCES
 
Measurement of F2-isoprostanes provides investigators with a unique tool to assess the role of free radicals in the pathogenesis of human disease with a degree of reliability that was not possible before. As detailed above, the occurrence of oxidative stress has now been established using measurements of F2-isoprostanes in a wide variety of diseases, the number of which continues to increase. An important question is whether oxidative stress plays a fundamental role in the pathogenesis of these diseases. The definitive answer to that question will be forthcoming only by determining whether the lowering the level of oxidative stress by treatment with effective antioxidants affects the manifestations and progression of the disease. There are demonstrations where administration of antioxidants has in fact ameliorated disease processes in animal models of human disease associated with overproduction of isoprostanes, but this has been less clear in humans. For example, animal models of atherosclerosis, such as apolipoprotein E (ApoE) -deficient mice, have demonstrated that antioxidant therapy reduces the progression or induces the regression of atherosclerosis (84) . Increased F2-isoprostane concentrations in urine, plasma, and vascular tissue are associated with atherogenesis in these mice (84) ; vitamin E administration reduces F2-isoprostane production, aortic atherosclerotic areas, and F2-isoprostane levels in the arterial wall without reducing plasma cholesterol levels (84) . These findings suggest that oxidative stress is important in the evolution of atherogenesis in ApoE-deficient mice. By contrast, the results of large prospective, controlled clinical trials assessing the efficacy of vitamin E supplementation in preventing cardiovascular disease are controversial (85) . Vitamin E supplementation was found to be highly efficacious in two trials (CHAOS and SPACE), but six other trials (ATBC, GISSI, PPP, SECURE, HOPE, and VEAPS) failed to show any benefit (86) . The reasons for this discrepancy are unclear, but factors such as selection of patients with varying levels of oxidative stress and dose of vitamin E may influence outcomes. These trials tested widely varying doses of vitamin E ranging from 55 to 800 I.U./day. It is important to point out that the clinical pharmacology of vitamin E has not been defined, so it is not yet known what doses of vitamin E are necessary to suppress oxidative stress in humans. Moreover, none of these studies incorporated measures of oxidative stress, such as measurement of F2-isoprostanes, to determine the level of oxidative stress and the ability of vitamin E to effectively lower the level of oxidative stress in the study subjects. This makes it difficult to interpret the results of these studies. In small single human studies involving subjects with conditions associated with elevated levels of F2-isoprostanes, some have found that vitamin E supplementation reduces the production of F2-isoprostanes, whereas others have not (23 , 75 , 76 , 87 , 88) . In healthy subjects, vitamin E has no effect on urinary F2-isoprostane concentrations at doses of up to 2000 I.U./day for up to 8 wk (89) . Moreover, a high dose of vitamin C (2500 mg/day) had no effect on lowering F2-isoprostane levels in normal subjects (90) but was found to be effective in suppressing isoprostane formation in smokers (50) . This latter finding may be explained by the fact that plasma concentrations of vitamin C, but not other antioxidants, are markedly reduced in smokers (50) . Smaller doses of vitamin C (500 mg/day) have been shown to suppress isoprostane production in smokers, but only in subjects with high body mass index; supplementation with the same dose of vitamin C in combination with additional antioxidants (vitamin E and lipoic acid) was ineffective (91) . What such findings suggest is that oxidative stress is a complex phenomenon that may be influenced by covariates and that appropriate selection of an antioxidant(s) and dose to effectively suppress oxidative stress in specific situations is less than predictable. On the other hand, it suggests that measurements of F2-isoprostanes may provide a uniquely valuable approach to elucidate these complexities and establish effective antioxidant dose regimens that can then be formally tested in individuals with a variety of disease states to determine whether amelioration of oxidative stress mitigates manifestations of disease.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 OXIDATIVE STRESS AND LIPID...
 THE ISOPROSTANE PATHWAYS
 MEASURING OXIDATIVE STRESS:...
 ISOPROSTANES AS MARKERS OF...
 BIOLOGICAL EFFECTS
 MECHANISMS OF ACTION
 THERAPEUTIC IMPLICATIONS
 CONCLUSIONS
REFERENCES
 
The initial report of the discovery of F2-isoprostanes was primarily a biochemical curiosity demonstrating that prostaglandin-like compounds can be formed in vivo independent of the cyclooxygenases. However, over the last several years the importance of that discovery has begun to emerge. It had been appreciated that methods to assess oxidative stress status in vivo were unreliable, which prevented investigators from being able to unequivocally establish the occurrence of oxidative stress in human disease. It has since become clear that measurement of F2-isoprostanes provides a valuable and reliable approach to assess oxidative stress status in vivo. Measurement of F2-isoprostanes has firmly established the occurrence of oxidative stress in a wide variety of disease states, often for the first time. Moreover, F2-isoprostanes and other products of the isoprostane pathway have been shown to exert potent biological actions both via receptor-dependent and independent mechanisms, and may therefore participate as mediators of oxidant injury. While establishing the occurrence of oxidative stress in a human disease is an important first step, this alone does not determine whether oxidative stress plays a fundamental role in the pathogenesis of that disease. This can only be forthcoming by demonstrating that amelioration of the oxidative stress by treatment with antioxidants mitigates manifestations of the disease. To accomplish that requires an in-depth understanding of the clinical pharmacology of antioxidant agents that currently is lacking. Unfortunately, many large clinical trials of antioxidants have been conducted that may have used an inappropriate antioxidant for that specific disease or an inappropriate dose. Advances in our understanding of the clinical pharmacology of antioxidants, however, should be attainable using measurements of F2-isorprostanes. This would greatly aid our interpretation of the results of these clinical trials and inform future trials regarding appropriate antioxidant treatment regimens to test. The discovery of isoprostanes advances greatly our ability to explore the role of oxidative stress in the pathogenesis of disease, therefore impacting in an important way on clinical medicine.


   ACKNOWLEDGMENTS
 
This work was supported by grant GM42056 and by Catholic University of the Sacred Heart, Academic grant 2002–2003.

Received for publication July 9, 2004. Accepted for publication August 17, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 OXIDATIVE STRESS AND LIPID...
 THE ISOPROSTANE PATHWAYS
 MEASURING OXIDATIVE STRESS:...
 ISOPROSTANES AS MARKERS OF...
 BIOLOGICAL EFFECTS
 MECHANISMS OF ACTION
 THERAPEUTIC IMPLICATIONS
 CONCLUSIONS
 REFERENCES
 

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