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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. S25-S30, (2002)
© 2002 American Thoracic Society

Article

The Isoprostanes

Their Role as an Index of Oxidant Stress Status in Human Pulmonary Disease

Jason D. Morrow and L. Jackson Roberts

Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee

Correspondence and requests for reprints should be addressed to Jason D. Morrow, M.D., 526 RRB, Vanderbilt University, 23rd and Pierce Avenues, Nashville, TN 37232–6602. E-mail: jason.morrow{at}mcmail.vanderbilt.edu

ABSTRACT

The isoprostanes are a unique series of prostaglandin-like compounds formed in vivo from the free radical–initiated peroxidation of arachidonic acid independent of the cyclooxygenase enzyme. This article summarizes selected aspects regarding our current knowledge of these compounds and what are considered avenues for future research. Novel aspects related to the biochemistry of isoprostane formation are discussed first, followed by a summary of methods by which these compounds are analyzed. A considerable portion of this article deals with the utility of measuring isoprostanes as markers of oxidant injury in vitro and in vivo, particularly in pulmonary diseases. Studies performed over the past decade have shown that these compounds are extremely accurate measures of lipid peroxidation in animals and humans and have illuminated the role of oxidant injury in a number of human diseases, including those related to the lung.

Key Words: lipid peroxidation • eicosanoids • isoprostanes • lipid

The development of specific, reliable, and noninvasive methods for measuring oxidative stress in humans is of fundamental importance for establishing the role of free radicals in human diseases (1). Lipid peroxidation is a central feature of oxidant stress and can be assessed by a number of methods that include the quantification of either primary or secondary peroxidation end products. Primary end products include conjugated dienes and lipid hydroperoxides. Secondary end products that can be quantified include thiobarbituric reactive substances, gaseous alkanes, and a group of prostaglandin (PG)F2-like products termed "F2-isoprostanes" (F2-IsoPs) (1, 2). Quantification of these various compounds has proven highly useful for the study of free radical–mediated lipid peroxidation in in vitro model systems. On the other hand, the F2-IsoPs appear to be a significantly more accurate marker of oxidative stress in vivo in humans and animals than other compounds (3, 4). The purpose of this article is to acquaint the reader with the IsoPs from a biochemical perspective and to provide information regarding the utility of quantifying these molecules as indicators of oxidant stress, in particular, in association with human pulmonary disease.

MECHANISM OF FORMATION OF THE ISOPROSTANES

IsoPs are PG-like compounds formed from the peroxidation of arachidonic acid (2–5). Unlike PGs, however, they do not require the cyclooxygenase enzyme for their formation. Figure 1 outlines the mechanism by which IsoPs are generated. After abstraction of a bis-allylic hydrogen atom and the addition of a molecule of oxygen to arachidonic acid to form a peroxyl radical, endocyclization occurs, and an additional molecule of oxygen is added to form PGG2-like compounds. These unstable bicycloendoperoxide intermediates are then converted to parent IsoPs. Based on this mechanism of formation, four IsoP regioisomers are generated (2, 5). Compounds are denoted as either 5-, 12-, 8-, or 15-series regioisomers depending on the carbon atom to which the side chain hydroxyl is attached (6). IsoPs that contain the F-type prostane ring are isomeric to PGF2{alpha} and are thus referred to as F2-IsoPs. An alternative nomenclature system for the IsoPs has been proposed by Rokach and colleagues (7). In this nomenclature, the abbreviation iP is used for isoprostane. Regioisomers are denoted as III–VI based on isomer structure.



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  		Figure 1. Mechanism of formation of the F2-IsoPs. Four regioisomers are formed each consisting of eight racemic diastereomers. Stereochemistry is not indicated.

 
An important structural distinction between IsoPs and cyclooxygenase-derived PGs is that the former contains side chains that are predominantly oriented cis to the prostane ring, whereas the latter possess exclusively trans side chains (2, 5). A second important difference between IsoPs and PGs is that IsoPs are formed completely in situ in phospholipids and are subsequently released by a phospholipase(s) (3, 4). In contrast, PGs are generated only from free arachidonic acid.

Although outside the scope of this discussion, it should be recognized that in addition to the F2-IsoPs, other classes of IsoPs can be formed from the peroxidation of arachidonate, including those with D-type and E-type prostane rings and A-type and J-type prostane rings. F2-IsoPs, however, have been the most studied class of IsoPs and, because of their stability, afford the most accurate measure of oxidant stress (4).

QUANTIFICATION OF F2-ISOPS

Several methods exist to quantify the F2-IsoPs. We measure them using a gas chromatographic/negative ion chemical ionization mass spectrometric approach employing stable isotope dilution (3). For quantification purposes, we measure the F2-IsoP, 15-F2t-IsoP, and other F2-IsoPs that coelute with that compound. Other investigators measure different F2-IsoP isomers (7). [2H4]15-F2t-IsoP ([2H4] 8-iso- PGF2{propto}) and [2H4]-PGF2{propto} are available from commercial sources for use as internal standards for these assays. Mass spectrometry is a highly sensitive method to measure IsoPs and yields quantitative results in the low picogram range. Its drawbacks are that it is labor intensive and that it requires substantial capital expenditure.

Alternative methods have been developed to quantify IsoPs using immunologic approaches (8). Antibodies have been generated against 15-F2t-IsoP, and at least three immunoassay kits are commercially available. A potential drawback of these methods is that limited information is currently available regarding their precision and accuracy. In addition, few data exist comparing IsoP levels determined by immunoassay to mass spectrometry. Analogous to immunologic methods to quantify cyclooxygenase-derived PGs, it might be predicted that immunoassays for IsoPs will suffer from a lack of specificity. On the other hand, it is likely that the quantification of IsoPs using immunoassays will expand research in this area, as these techniques are affordable and relatively easy to perform.

ISOPROSTANE FORMATION IN VIVO

We have defined normal levels of F2-IsoPs in human biologic fluids such as plasma and urine (3, 4, 8). It is important to note that quantities of these compounds exceed those of cyclooxygenase-derived PGs by at least an order of magnitude, suggesting that IsoPs are a major pathway of arachidonic acid disposition. Furthermore, it is important to consider the relevance of the finding that levels of F2-IsoPs are sufficient to be detected in every normal biologic fluid that has been assayed including plasma, urine, bronchoalveolar lavage fluid, cerebrospinal fluid, and bile (8). In addition, Montuschi and colleagues (9) have successfully developed methods to quantify IsoPs in exhaled breath condensates and have reported that this method is an accurate index to assess oxidative stress status in various pulmonary disorders (vide infra).

Previously, using other methods to assess lipid peroxidation, there was no definitive evidence indicating that lipid peroxidation occurs in vivo except under situations of oxidant stress. However, the finding of significant levels of F2-IsoPs in normal animal and human biologic fluids and tissues indicates that there is ongoing lipid peroxidation that is incompletely suppressed by antioxidant defenses, even in normal humans and animals. This finding lends support to the hypothesis that the normal aging process is due to enhanced oxidant damage of relevant biologic molecules over time. In this regard, a previous study has suggested that IsoP levels in normal humans increase with age, although a more recent report refutes this (7, 10).

Over the past decade, a number of studies involving the quantification of F2-IsoPs as an index of lipid peroxidation or oxidant stress both in vitro and in vivo have been performed. This work is particularly important regarding in vivo oxidant stress, as it has been previously recognized that one of the greatest needs in the field of free radical research is a reliable noninvasive method to assess lipid peroxidation in vivo in humans (1, 11, 12). In this respect, most methods available to assess oxidant stress in vivo previously have suffered from a lack of sensitivity and/or specificity or are unreliable. However, a substantial body of evidence indicates that measurement of IsoPs in body fluids such as plasma provides a reliable approach to assess lipid peroxidation in vivo and represents a major advance in our ability to assess oxidative stress status in animals and humans (11, 12). Specific examples where IsoPs have been used by us as measures of oxidant stress in association with human disease or animal models of human disease are discussed subsequently.

MEASUREMENT OF THE MAJOR URINARY METABOLITE OF THE ISOP 15-F2T-ISOP AS AN INDEX OF SYSTEMIC OXIDANT STATUS

Unmetabolized IsoPs can be quantified either as free compounds in biologic fluids or esterified in tissue lipids (3). Although applicable to animal studies, the measure of these compounds in human trials is potentially limited by invasive procedures necessary to obtain tissues or biologic fluids. In addition, levels of IsoPs in a particular organ or body cavity likely do not represent an index of systemic or "whole-body" oxidation. Whereas unmetabolized urinary IsoPs can be quantified, the interpretation of measuring these compounds as an index of total systemic production of IsoPs is confounded by the potential contribution of local IsoP production in the kidney (8, 11, 12). It is well established in the PG field that measurement of the urinary excretion of metabolites of PGs represents the most reliable approach to assess total endogenous PG production (13). Accordingly, quantification of the urinary excretion of an F2-IsoP metabolite should also afford an accurate measure of endogenous production of IsoPs and allow for urine collected over a number of hours to provided an integrated index of IsoP production.

We previously undertook a study to identify a urinary metabolite of an F2-IsoP that could be measured. One of the F2-IsoPs that we have shown is formed in vivo is 15-F2t-IsoP (8-iso-PGF2{alpha}) (14). Metabolism of PGs has usually has been found to produce a number of metabolites; however, we found that a single metabolite predominated in the profile of compounds produced from metabolism of 15-F2t-IsoP. This metabolite was identified by mass spectrometry as 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15). We synthesized this compound and converted it to an [18O4] derivative for use as an internal standard and developed and refined a stable isotope dilution-negative ion chemical ionization mass spectrometric assay (16, 17). The urinary excretion of this metabolite in normal humans was found to be 0.46 ± 0.18 ng/mg creatinine (mean ± 2 SD). Excretion of the metabolite increases markedly in animal models of oxidant stress. Furthermore, urinary levels of the metabolite increased by a mean of 1.9-fold above levels in humans with polygenic hypercholesterolemia, a condition associated with enhanced IsoP formation (17). These increases were suppressed by a mean of 48% after 8 weeks of treatment with a combination of vitamin E, vitamin C, and ß-carotene. These data support the contention that measurement of the urinary excretion of 2,3-dinor-5,6-dihydro-15-F2t-IsoP will contribute importantly to the assessment of free radical–mediated lipid peroxidation in vivo. This approach may also provide an important tool to assess oxidative stress status in large clinical trials in which the logistics of obtaining blood or tissues are limited. Development of an immunoassay method for the measurement of this metabolite is currently being undertaken. If the accuracy of the immunoassay for this compound can be validated by mass spectrometry, this may result in the wide availability of quantification of this metabolite as a facile, noninvasive means to assess oxidant stress.

QUANTIFICATION OF ISOPROSTANES AS AN INDEX OF OXIDANT STRESS IN VITRO STUDIES

Many studies have been performed by others and us involving the quantification of F2-IsoPs in in vitro systems of lipid peroxidation and F2-IsoP formation has been compared with other markers of lipid peroxidation. This work has demonstrated the utility of measuring these compounds as a reliable index of lipid peroxidation in vitro and has provided a scientific basis to explore their role as markers of oxidant stress in vivo. Some of these in vitro studies are briefly summarized later here.

The formation of F2-IsoP has been compared with malondialdehyde in iron/adenosine diphosphate/ascorbate induced peroxidation of rat liver microsomes (18). Malondialdehyde is one of the most commonly used measures of lipid peroxidation and was quantified in these studies by measuring thiobarbituric acid reacting substances. Both F2-IsoP and malondialdehyde formation increased in parallel in a time-dependent manner and correlated with the loss of arachidonic acid and with increasing oxygen concentrations up to 21%. Although the formation of F2-IsoP correlated with other measures of lipid peroxidation in this in vitro model, we have reported that measurement of F2-IsoPs is far superior to measurements of malondialdehyde as an index of lipid peroxidation in vivo (18).

We and others have performed studies examining the formation of F2-IsoP in low-density lipoproteins (LDLs) exposed to various oxidizing conditions in vitro. Much of the interest in examining this stems from the hypothesis that oxidation of LDL in vivo converts it to an atherogenic form, which is taken up by macrophages in the vessel wall. Subsequent activation of these cells likely plays an important role in the development and progression of atherosclerotic lesions in humans (19). Thus, we have performed studies examining the formation of F2-IsoP in LDLs that is oxidized to determine whether measurement of F2-IsoP esterified to lipoproteins may provide an approach to assess lipoprotein oxidation in vivo (20). These studies are also of interest because one F2-IsoP, 15-F2t-IsoP (8-iso-PGF2{alpha}), is a vasoconstrictor and induces mitogenesis in vascular smooth muscle cells (12), and these effects may be of relevance to the pathophysiology associated with atherosclerosis. In these studies, either plasma lipids or purified LDLs from humans were peroxidized with Cu2+ or the water-soluble oxidizing agent 2,2-azo-bis(2-amidinopropane) (20). The formation of F2-IsoPs was compared with other markers of lipid peroxidation, including formation of cholesterol ester hydroperoxides, phospholipid hydroperoxides, a loss of antioxidants, and changes in the electrophoretic mobility of LDL. In plasma oxidized with 2,2-azo-bis(2-amidinopropane), increases in the formation of F2-IsoP paralleled increases in lipid hydroperoxide formation and occurred only after depletion of the antioxidants ascorbate and ubiquinol-10. In purified LDL that was oxidized, formation of F2-IsoPs again correlated with increases in lipid hydroperoxides and increases in the electrophoretic mobility of LDL. Furthermore, increased F2-IsoP formation occurred only after depletion of the antioxidants {alpha}-tocopherol and ubiquinol-10. Similar findings have been reported by others (21, 22).

There has been significant interest in the role that the macrophage 12-/15-lipoxygenase enzyme might play in the oxidation of lipoproteins in the vascular wall and the relationship to atherosclerosis (23). In support of a role for this enzyme in the oxidation of LDL in vivo, it has been shown that 15-F2t-IsoP formation in LDL incubated with stimulated macrophages isolated from mice genetically engineered with a targeted disruption of the 12/15-lipoxygenase gene was significantly less than when LDL was incubated with macrophages isolated from control animals (24). Subsequent studies have also shown that IsoP production in vivo is decreased in mice lacking the 12-/15-lipoxygenase (25).

Taken together, these in vitro studies suggest that quantification of F2-IsoP esterified in plasma and tissue lipids can provide a useful approach to assessing oxidation of lipids in vivo.

FORMATION OF ISOPROSTANES IN ANIMAL MODELS OF OXIDANT STRESS

It has been previously shown that the formation of IsoPs increases dramatically in animal models of oxidant stress. When rats were administered carbon tetrachloride (CC14), they undergo free radical–induced injury with the major site of toxicity being the liver. Esterified levels of IsoPs in liver tissue increased by 200-fold within 1 hour of treatment and subsequently decline over 24 hours (26). Plasma-free and lipid-esterified IsoP concentrations increased after liver levels up to 50-fold greater than levels in untreated animals. Formation of IsoPs was proportional to the CCl4 dose given. Administration of the antioxidant lazaroid U78517 to CCl4-treated animals significantly blunted the enhanced formation of IsoPs in this model (12).

Diquat is a dipyridyl herbicide that undergoes redox cycling in vivo generating large amounts of the superoxide anion. This compound causes hepatic and renal injury in rats, and this effect is markedly augmented in animals deficient in selenium (Se), a trace element that is required for the enzymatic activities of glutathione peroxidase and other antioxidant proteins. Previous studies have suggested that lipid peroxidation might be involved in the tissue damage associated with this agent. To study whether F2-IsoPs were generated in increased amounts in association with diquat administration to Se-deficient rats, levels of compounds were quantified in plasma and tissues from Se-deficient rats after diquat administration. Se-deficient rats administered diquat showed 10- to 200-fold increases in plasma F2-IsoPs, and the sources of the IsoPs were determined to be the kidney and liver (27). Further studies disclosed that the extent of tissue injury and IsoP formation directly correlated with the degree of Se depletion (28). Taken together, these studies suggest that quantification of F2-IsoPs in animal models of oxidant injury represents an accurate method to assess lipid peroxidation in vivo.

QUANTIFICATION OF F2-ISOPS IN ASSOCIATION WITH HUMAN PULMONARY DISEASE

A number of studies have been reported examining the utility of quantifying IsoPs as an index of oxidant stress in association with human disease, particularly in pulmonary disorders. IsoP levels have been shown to be increased in humans with chronic obstructive pulmonary disease (29), cystic fibrosis (30), pulmonary hypertension (31), and acute respiratory distress disease (32), among others. Table 1 summarizes lung disorders in humans in which IsoP formation has been measured and is increased, supporting the contention that oxidant stress may play a role in disease pathogenesis.


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  		TABLE 1. Human pulmonary disorders associated with increased isoprostane formation

 
The interest in the quantification of IsoPs as an index of oxidative injury in human pulmonary disease stems, in part, from studies that have explored the biologic activities of these compounds in the pulmonary circulation and the bronchial tree. The physiologic effects of IsoPs in the lung have been recently discussed in an outstanding review by Janssen (33), who noted that these compounds are likely not only markers of oxidant stress but play a role in pulmonary pathophysiology, as they evoke important biologic responses on most cell types in the lung. Furthermore, they exhibit differences with respect to the various species and tissues studied. The two IsoPs examined in greatest detail in the lung are 15-F2t-IsoP and 15-E2t-IsoP (33). In general, these compounds are potent constrictors of pulmonary vascular smooth muscle and airway smooth muscle. Effects are mediated by interactions with the thromboxane receptor, a G-protein–coupled eicosanoid receptor, and potentially other eicosanoid receptors (12, 33). Subsequently, they induce various intracellular second-messenger systems, including phospholipase C/inositol trisphosphate and mitogen-activated protein kinase that result in constriction (34–36). Furthermore, IsoPs activate various inflammatory cells such as neutrophils leading to enhanced adhesion to endothelial cells. The increase in adhesion, however, occurs independently of increased expression of various adhesion molecules (37).

We have extensively studied isoprostane formation in two important pulmonary pathophysiologic conditions—chronic heavy cigarette smoking and allergen-induced asthma—and have shown that levels of these compounds are significantly increased, implying that oxidant stress may play a role in the pathophysiology of these diseases. The salient features of this work are summarized later here and illustrate the utility of quantifying IsoPs in human pulmonary diseases believed associated with oxidant stress. Subsequently, a discussion of the more important work of other investigators regarding IsoP formation in pathophysiologic conditions relevant to the lung is undertaken.

ISOPROSTANE FORMATION IN CHRONIC SMOKERS

A link between cigarette smoking and a risk of pulmonary and cardiovascular disease is well established. However, the underlying mechanism(s) for this effect is not fully understood. The gaseous phase of cigarette smoke contains a number of oxidants, and exposure of the lung to the gaseous phase of cigarette smoke in vitro induces oxidation of tissue and circulating lipids (38). Thus, we explored the hypothesis that smoking induces an oxidative stress and specifically determined whether circulating plasma lipids in individuals who smoke contain higher levels of F2-IsoPs, indicative of a greater degree of oxidative modification. Ten individuals who smoked heavily (more than 30 cigarettes per day) and 10 age- and sex-matched nonsmoking normal volunteers were studied (39). Plasma concentrations of free and esterified F2-IsoPs were significantly elevated in the smokers compared with the nonsmokers (p = 0.02 and p = 0.03, respectively). Confirmation that these differences in levels of F2-IsoPs between smokers and nonsmokers were due to cigarette smoking was obtained by measuring levels of F2IsoPs after 2 weeks of abstinence from smoking in 8 of the 10 smokers who successfully abstained. In all subjects, levels of F2IsoPs both free in the circulation and esterified to plasma lipoproteins were significantly lower after 2 weeks of abstinence from smoking (p = 0.03 and p = 0.02, respectively). The occurrence of enhanced formation of IsoPs in smokers has subsequently been confirmed in studies by others (40, 41). Collectively, these findings suggest strongly that smoking causes an oxidative stress, and the observation that smokers have elevated levels of F2-IsoPs esterified in plasma lipids also supports the hypothesis that the link between smoking and risk of pulmonary and cardiovascular disease may be attributed to enhanced oxidation of tissue and or circulating lipids.

ISOPROSTANE FORMATION IN ALLERGEN-INDUCED ASTHMA

Asthma is a chronic inflammatory disease of the airways that was believed to involve oxidant injury to the lung, although firm evidence to support this contention was lacking. Recently, we undertook a series of studies to quantify IsoP formation in a group of 11 patients with mild atopic asthma after an inhaled allergen challenge (42). The urinary excretion of F2-IsoPs increased significantly by a mean of 73% at 2 hours after allergen challenge and remained significantly elevated in all urine collections for the 8-hour period of the study. Urinary IsoPs did not change after inhaled methacholine challenge. In nine of the atopic patients, F2-IsoPs were quantified in bronchoalveolar lavage fluid at baseline and 24 hours after allergen installation. F2-IsoPs were significantly elevated (mean 27%) late in the lavage fluid. Subsequently, we have also shown significant increases in excretion of the major urinary metabolite of 15-F2t-IsoP in atopic patients with asthma after allergen exposure (43). These findings have been confirmed by other investigators (44). In addition, Montuschi and colleagues have also shown that IsoP levels are significantly increased in exhaled breath condensate from asthmatics compared healthy humans (9). This study is particularly important because it suggests that noninvasive methods such as breath analysis can be used to detect oxidative injury locally in pulmonary tissues. Overall, these observations are extremely important in that they provide new evidence for a role of oxidant stress and lipid peroxidation in allergen-induced airway inflammation and thus identify a potential therapeutic target for the treatment of this common pulmonary disorder.

ISOPROSTANE FORMATION IN OTHER PULMONARY DISORDERS

There has been significant interest by a number of other investigators regarding the role of isoprostane formation and oxidant stress in pulmonary disorders. Table 1 summarizes the lung diseases in which IsoP formation has been documented to be increased. Several groups have found that IsoP levels are significantly elevated in persons with cystic fibrosis. In 1999, Collins and colleagues reported increases in plasma levels of 15-F2t-IsoP in humans with cystic fibrosis and that these increases correlated with decreases in plasma antioxidants (45). Subsequent studies by other investigators also showed significant increases in IsoP levels in exhaled breath condensates and urine from patients with cystic fibrosis (30, 46). Interestingly, Ciabottoni and colleagues showed that vitamin E supplementation decreased urinary IsoP excretion, suggesting that this agent may be useful as adjunctive therapy for this disorder (46).

F2-IsoP generation has also been shown to be increased in humans with chronic obstructive pulmonary disease (47, 48). Pratico and colleagues were also able to correlate enhanced oxidant stress with acute exacerbations of chronic disease, suggesting that prevention of acute exacerbations may result in decreased oxidant stress (47).

F2-IsoP formation has also been shown to be a useful biomarker of oxidative stress in various interestitial lung diseases. In one study, Montuschi and colleagues showed that IsoP levels in bronchoalveolar lavage fluid were fivefold greater than controls in persons with cryptogenic fibrosing alveolitis and fibrosing alveolitis associated with systemic sclerosis (49). Furthermore, they were increased, but to a significantly lower extent, in persons with sarcoidosis. These intriguing findings suggest that oxidant stress varies significantly in different forms of interstitial lung disease.

Increased IsoP formation has also been documented in persons with pulmonary hypertension (31). Interestingly, increases in levels of these compounds correlated inversely with pulmonary vasoreactivity as measured by pulmonary vascular resistance changes in response to inhaled nitric oxide challenge. Other pulmonary conditions reported to increase IsoP formation include acute lung injury (32), respiratory failure in infants (50), and exposure to ozone (51). Taken together, these studies provide clear-cut evidence that oxidative stress is increased in a number of pulmonary disorders and that IsoP generation may play a role in the pathophysiology of these diseases. Nonetheless, it remains to be determined the extent to which modulation of oxidant stress with, for example, antioxidants affects the formation of these bioactive compounds. It is hoped that future research will address this issue.

CONCLUSIONS
The discovery of IsoPs as products of nonenzymatic lipid peroxidation has been a major breakthrough regarding the quantification of oxidant stress in vivo. It has opened up new areas of investigation regarding the role of free radicals in human physiology and pathophysiology, importantly those related to human pulmonary disorders. The measurement of these compounds as markers of oxidative stress status appears to be the most useful tool currently available to explore the role of free radicals in the pathogenesis of human disease. Although considerable information has been obtained since the initial discovery of IsoPs, much remains to be understood about the role of these molecules as markers of oxidant stress in pulmonary disorders in vivo. It is anticipated that additional research in this area over the next several years will provide important insights.

FOOTNOTES

Supported by National Institutes of Health Grants DK48831, CA77839, GM15431, and DK26657 from the United States Public Health Service (J.D.M. is the recipient of a Burroughs-Wellcome Fund Clinical Scientist Award in Translational Research).

Received in original form June 14, 2002; accepted in final form October 7, 2002

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