The Journal of Nutrition Vol. 128 No. 2 February 1998, pp. 434S-438S

Eicosanoids and Isoeicosanoids: Indices of Cellular Function and Oxidant Stress^1,2

Muredach P. Reilly, John A. Lawson, and Garret A. FitzGerald³

The Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, PA 19104

	ABSTRACT

Abstract Introduction References

Arachidonic acid (AA) is an unsaturated fatty acid constituent of the phospholipid domain of cell membranes. It is subject to release via mobilization of phospholipases, particularly a cytoplasmic phospholipase A₂. Thereafter, it may be metabolized by at least two cyclooxygenase (COX) isoforms to prostaglandins and related compounds, via lipoxygenases to leukotrienes and via p450-catalyzed metabolism to epoxyeicosatrienoic acids. Collectively, these bioactive lipids are termed eicosanoids. All of these lipids express potent bioactivity in vitro. Clinical studies have already demonstrated the importance of COX and lipoxygenase (LOX) products in human disease. The generation of models of COX, LOX and prostaglandin receptor gene inactivation is likely to broaden our insight into the importance of these compounds in vivo. Crystallization of the biosynthetic enzymes is likely to facilitate the development of highly specific inhibitors, as is the case already for COX-2. AA possesses intrinsic biological properties. It is also subject to free radical attack, generating isomeric eicosanoid species, the isoeicosanoids. These compounds may also express biological activity in vitro, although their importance in vivo is unclear. Development of specific assays for these compounds in urine suggests their utility as noninvasive indices of oxidant stress in vivo.

	INTRODUCTION

Abstract Introduction References

In 1934, von Euler identified a lipid-soluble substance from semen that would stimulate uterine smooth muscle contractions and named it prostaglandin (von Euler 1934). Bergström and Sjovall (1960) isolated prostaglandin E from sheep prostate glands and showed that it was a 20-carbon fatty acid with blood pressure-lowering activity. Bergström et al (1964) demonstrated the enzymatic conversion of arachidonic acid to prostaglandin E₂. Because arachidonic acid is synthesized from linoleic acid in humans, these discoveries established prostaglandins as a product of the metabolism of essential fatty acids.

	EICOSANOIDS: FORMATION AND BIOLOGICAL ACTIVITY

It is now known that arachidonic acid (AA)⁴ is subject to metabolism by a wide array of bioactive lipid mediators. Two isoforms of the prostaglandin (PG) G/H synthase, colloquially known as cyclooxygenases (COX), catalyze the formation of PG and related compounds. Vane (1971) first demonstrated that COX was the target for aspirin inhibition of PG formation. Inhibition of COX-1 in platelets, with consequent suppression of formation of thromboxane (Tx) A₂, underlies the efficacy of aspirin in the treatment of platelet-dependent vascular occlusion (Patrono 1994).

Although the expression of COX-1 may be regulated, it is usually expressed constitutively. Similarly, although COX-2 expression may be constitutive, particularly in the cells of the reproductive tract and in the nervous system (Yamagata et al. 1993), its expression is usually tightly regulated, particularly by cytokines, growth factors and tumor-promoting agents (Fu et al. 1990). These observations have implicated COX-2 in PG generation in inflammation and, perhaps, cancer (Tsujii et al. 1997). COX-1, by contrast, is expressed constitutively in normal gastric epithelium. Inhibition of COX-1 is thought to underlie the gastrointestinal side effects of commonly available nonsteroidal anti-inflammatory drugs (NSAID), all of which are quite nonselective between the two COX isoforms (Smith et al. 1996). Thus, the development of highly selective COX-2 inhibitors (Seibert et al. 1994) may promise compounds that are better tolerated and more efficacious than conventional NSAIDs. A potential caveat is that an evoked inflammatory response is impaired in mice deficient in COX-1, but not COX-2. The latter mice were also able to mount an inflammatory response to incidental infection (Langenbach 1995; Morham 1995). Both COX isoforms have now been crystallized (Browner 1996, Loll et al. 1995).

Although G protein-coupled receptors for all of the PG have been cloned (Narumiya 1994), only the human pharmacology of specific antagonists of the TxA₂ receptor (TP) has been characterized to date. It is likely that the recent generation of mice deficient in each of these receptors will further elucidate their in vivo biology. Similarly, there have been suggestions that eicosanoids might activate nuclear receptors (Forman et al. 1997). However, issues of specificity and concentration-response relationships with compounds actually formed in vivo must be resolved.

Lipoxygenases generate leukotrienes and related compounds. These lipids express biological properties of likely relevance to inflammatory responses in vivo. This is consistent with the phenotype expressed by mice deficient in the 5-lipoxygenase enzyme (Chen et al. 1994). Specific antagonists of sulfidopeptide leukotrienes have found efficacy in human asthma, as have specific inhibitors of the 5-lipoxygenase (O'Byrne 1994). Leukotrienes and related compounds have also been implicated in neuronal function, atherogenesis, cellular proliferation and the regulation of vascular tone in vitro. Again, the recent availability of mice deficient in specific lipoxygenases may elucidate the in vivo relevance of these observations.

Much less is known of the biological importance of the epoxygenase catalyzed formation of epoxyeicosatrienoic acids (EET) and related products, probably by p450 isozymes with high affinity for arachidonic acid as a substrate (McGiff 1991). These compounds are potent regulators of epithelial ion transport and vascular tone in vitro (Oyekan et al. 1994). However, specific receptors for EET have yet to be cloned, and animals deficient in the AA-specific p450 isoforms (Wu et al. 1996) have yet to be generated. Data suggestive of their importance in hypertension have been obtained with nonspecific inhibitors (Makita et al. 1994). However, given the absence of a specific means of pharmacological inhibition of their synthesis or action, their role in pathophysiology is speculative at present.

In addition to these observations, cells may interact to generate novel transcellular products of AA (Marcus 1990). Similarly, AA itself may directly modify cellular function. Thus, arachidonoylation of cellular proteins such as G proteins (Hallak et al. 1994) or miniglucagon (Sauvadet et al. 1997) may modify their effects. It may directly regulate ion channels (Damron et al. 1993) or influence gene expression (Barry et al. 1997). Differential allosteric regulation of the two COX isoforms by AA can cause dramatic differences in isoform selectivity for inhibitors, as a function of AA concentration (Swinney et al. 1997).

A recent area of interest has been the potential importance of oxidized lipids in the modification of cellular function (Lehr et al. 1997). The focus of this review will be on candidate members of this species, a family of free radical-catalyzed products of arachidonic acid, the isoeicosanoids.

	THE ISOEICOSANOIDS

Isoeicosanoids, isomers of enzymatically derived eicosanoids, are free radical-catalyzed products of arachidonic acid (Nugteren and Christ-Hazelhof 1980, O'Connor et al. 1984). The existence of the F₂ isoprostanes, isomers of PGF₂, in human plasma and urine was first described by Morrow et al. (1990). More recently, the free radical-dependent formation of E₂ and D₂ isoprostanes, isothromboxanes and isoleukotrienes has been reported.

Four classes of F₂ isoprostanes (IPF) have been described (Fig. 1; Waugh and Murphy 1996). The relative abundance of formation of specific isomers in vivo is unknown, but to date, the most extensively studied is a member of the IPF-IV class, 8-epi PGF₂. This potent vasoconstrictor and mitogen is present in atherosclerotic plaque, but not in healthy vascular tissue (Pratico et al. 1997). These effects of 8-epi PGF₂ are blocked by thromboxane receptor antagonists. Unlike thromboxane analogs, 8-epi PGF₂ induces platelet shape change without irreversible aggregation; it also acts with threshold concentrations of conventional agonists to induce irreversible aggregation (Pratico et al. 1996). However, high concentrations of 8-epi PGF₂ are necessary to activate the cloned splice variants of the human thromboxane receptor; its importance as an autacoid in vivo is unclear.

View larger version (14K): [in this window] [in a new window]   Fig 1. The F2-isoprostane family of isomers can be divided into four classes; 8-epi PGF2 belongs to class IV and IPF2-I to class I. Although 8-epi PGF2 is a minor by-product of the cyclooxygenase (COX) pathways in vitro, there is no evidence that IPF2-I can be generated in this manner.

	DIFFERENTIAL ISOPROSTANE FORMATION

Rather than estimating the formation of "total" F₂ isoprostanes by using a nonisoprostane internal standard, we have developed specific assays for discrete members of the IPF classes (Pratico et al. 1995). Initially, we focused on the formation of 8-epi PGF₂ by human platelets and monocytes in an attempt to characterize the mechanisms of isoprostane generation. To our surprise, we found that 8-epi PGF₂, but not other F₂ isoprostanes, could be formed as a minor product of the COX-1 enzyme in platelets and the COX-2 isoform monocytes (Pratico et al. 1995, Pratico and FitzGerald 1996). These were potentially important observations, because activation of platelets or monocytes may be a feature of many syndromes putatively associated with oxidant stress. However, administration of an aspirin regimen, designed to inhibit platelet COX-1, failed to suppress the elevated urinary 8-epi PGF₂ levels that we observed in chronic smokers (Reilly et al. 1996a), a syndrome of COX-1 activation. The same regimen significantly suppressed excretion of the 11-dehydro metabolite of thromboxane B₂, which largely derives from platelets. Thus, the capacity of platelets to form 8-epi PGF₂ in a COX-1-dependent manner did not appear to contribute in a measurable way to the urinary index of overall 8-epi PGF₂ biosynthesis. It is unknown to what extent, if any, the COX-2-dependent pathway might contribute to the formation of 8-epi PGF₂ in settings of inflammation and cellular proliferation, in which COX-2 induction might be expected.

Despite the apparent lack of relevance of the enzymatic formation of 8-epi PGF₂ to the use of its urinary excretion as an index of oxidant stress, it seemed prudent to develop methods to measure another F₂-isoprostane that was not susceptible to enzymatic formation. IPF₂-I is a member of a distinct class of F₂-isoprostanes (Waugh and Murphy 1996). It is not formed by COX and its excretion in volunteers is not suppressed by aspirin (Pratico et al 1998). Urinary levels of IPF₂-I and 8-epi PGF₂ are closely correlated (r = 0.57, P < 0.0001) in patients with hypercholesterolemia (Fig. 2). This is a setting of moderate COX-1 activation. These observations are consistent with the hypothesis that excretion of both compounds in urine reflects formation by a common mechanismfree radical-catalyzed generation of prostaglandin isomers. Thus it would appear that enzymatic formation of 8-epi PGF₂ by COX is a trivial contributor to overall 8-epi PGF₂ biosynthesis in vivo and should not detract from its usefulness as an index of oxidant stress.

View larger version (22K): [in this window] [in a new window]   Fig 2. The relationship of urinary 8-epi PGF2 to IPF2-I levels in normocholestrolemic controls and patients with homozygous familial hyper-cholesterolemia (HFH). Correlation (r = 0.57, P < 0.0001; n = 109).

	ISOPROSTANES AND OXIDANT STRESS

Oxidative stress is thought to play an important pathophysiologic role in a variety of human diseases, including atherosclerosis, cancer and neurodegenerative disorders. However, difficulty in assessing radical generation in vivo has proven to be the major limitation to our understanding of this mechanism of human diseases. Traditional in vitro assays, directed against malondialdehyde or lipid hydroperoxides, are thought fallible when applied to clinical investigation, because of such factors as ex vivo generation of products and both the instability and nonspecificity of the analytes involved. Furthermore, it is unclear how ex vivo estimates of free radical generation, such as lipoprotein oxidizability or the formation of adducts detected by spin trapping, relate to oxidant stress in vivo.

The measurement of F₂ isoprostanes may represent an important development in the assessment of free radical generation and oxidant stress in vivo. They are remarkably stable compounds. Coordinate elevation of plasma and urinary isoprostanes in syndromes of extrarenal oxidant stress (Morrow et al. 1995, Pratico and FitzGerald 1996) implies that little is likely to be gained by measurement of metabolites, rather than the parent products, in urine. However, estimates of 8-epi PGF₂ in plasma, where there is an abundance of lipid, may be confounded by its autooxidation ex vivo. Additionally, COX-1-dependent formation by platelets activated ex vivo might also undermine plasma-based measurements as indices of actual formation of 8-epi PGF₂ in vivo. Thus measurement of a metabolite of 8-epi PGF₂ (Roberts et al. 1996) might circumvent this problem if it is not formed in the cells of circulating blood. We have utilized gas chromatography/mass spectrometry to validate immunoassays of the parent compound. More recently, we have adopted an integrated approach that uses the coordinate measurement of urinary 8-epi PGF₂ and IPF₂-I in the evaluation of oxidant stress in specific clinical settings.

	F2 ISOPROSTANES IN SPECIFIC CLINICAL SYNDROMES

There is a dose-dependent increase in urinary 8-epi PGF₂ excretion in apparently healthy chronic cigarette smokers, which was not suppressed by aspirin. Both cessation of smoking, with supplementation of nicotine patches and short-term therapy with vitamin C (2000 mg/d), an endogenous antioxidant, attenuated the elevation of urinary 8-epi PGF₂ excretion. Deficiency of vitamin C (Heitzer 1996) may render smokers particularly susceptible to the antioxidant effects of exogenous vitamin supplementation.

Oxidation of LDL is thought to play a critical role in atherogenesis. This hypothesis is based largely on indirect evidence. We have recently immunolocalized 8-epi PGF₂ to monocyte/macrophages and vascular smooth muscle cells in human atherosclerotic plaque and demonstrated increased levels of this compound in atherosclerotic vessels, compared with normal arterial segments (Pratico et al. 1997). Furthermore, we have demonstrated along with others that F₂ isoprostanes are formed in LDL when it is oxidized in vitro. Therefore we designed specific studies to assess the biosynthesis of both 8-epi PGF₂ and IPF₂-I in patients with homozygous familial hypercholesterolemia (HFH) and also in more moderate hypercholesterolemia. Urinary excretion of these isoprostanes was increased in both groups of hypercholesterolemic patients compared with their respective controls. Furthermore, the concentration of 8-epi PGF₂ esterified in LDL was elevated and correlated with urinary excretion of this compound in a subset of these patients (Reilly et al. 1996b). Given the potentially distinct mechanisms that might result in free radical generation in smokers, one might anticipate an even greater increment in dyslipidemic individuals who smoke. Interestingly, the precise role of dietary lipids in isoprostane biosynthesis in normal individuals or in the setting of increased biosynthesis remains to be addressed definitively.

Oxidant stress has been implicated in vascular reperfusion after a period of ischemia. Examples include the regional myocardial stunning seen in animal models of coronary occlusion/reperfusion and in some patients after thrombolytic therapy, as well as in the global myocardial dysfunction seen after coronary artery bypass surgery. We have shown increased urinary 8-epi PGF₂ in a number of syndromes of myocardial reperfusion (Delanty et al. 1997). Levels were significantly elevated, coincident with doppler-documented reperfusion, in a canine model of coronary thrombolysis. Similarly, excretion was enhanced coincident with cross-clamp release compared with preoperative and postoperative values in subjects undergoing elective coronary artery bypass surgery. Recently, we have extended these studies to include patients undergoing reperfusion for myocardial infarction. Urinary excretion of 8-epi PGF₂ and IPF₂-I was markedly increased coincident with angiographically documented reperfusion in patients treated with thrombolytic agents and percutaneous transluminal coronary angioplasty (PTCA) for myocardial infarction. There was a minor increment in isoprostane excretion after diagnostic coronary arteriography and elective PTCA.

The metabolic disposition of isoprostanes in vivo remains to be investigated. However, our experience with indices of eicosanoid biosynthesis suggests that urinary excretion may reflect either renal and extrarenal sources (or both) of isoprostane formation, dependent on the experimental setting or the disease under study.

	CONCLUSIONS

Both enzymatic and nonenzymatic products of AA, as well as the substrate itself, have been implicated as mediators in human biology. Generally, the importance of these bioactive lipids is expressed under pathophysiologic circumstances, in which they tend to subserve a homeostatic function. Inhibition of their biosynthesis may have beneficial effects. An example of this is the anti-inflammatory efficacy of NSAID. Alternatively, the same intervention may have harmful consequences. An example is the deterioration of renal blood flow in patients with renal compromise who are administered NSAID.

Urinary excretion of these bioactive lipids may also be utilized to reflect cellular activation in vivo. Given that the products of enzymatic metabolism of AA express relative cellular specificity, excretion of distinct compounds or their metabolites may reflect activation of particular repertoires of cells. Examples include the use of Tx metabolites to reflect platelet activation and prostaglandin D (PGD) metabolites to reflect mast cell activation. Again, the relative contribution to what is excreted in urine is a function of the precise clinical condition under study. Finally, interest is developing in the use of isoprostane excretion as an approach to the study of lipid peroxidation in vivo.

	FOOTNOTES

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