Introduction

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Arachidonic acid serves as a substrate for two different enzymatic cascades, the PGH synthase cascade and the 5-lipoxygenase cascade, to yield a diverse family of lipid mediators, namely the prostaglandins (Smith, 1992) and leukotrienes (Henderson, 1994), respectively. These molecules exert potent biological activities relevant to intracellular communication through specific G protein-linked receptors (Yokomizo et al., 1997; Narumiya, 1995). The concentrations of these lipid mediators within tissues and the resultant biological response elicited by these eicosanoids are regulated in large measure by the rate at which these compounds are metabolized to inactive compounds. Both leukotrienes and prostaglandins are metabolized by a variety of enzymatic processes, including peroxisomal -oxidation and microsomal cytochrome P450-dependent -oxidation. Other specific enzymatic pathways include the 15-hydroxy prostaglandin dehydrogenase pathway, which inactivates PGE₂ and PGF₂, and the 12-hydroxyeicosanoid dehydrogenase pathway, which metabolizes LTB₄ (Hansen, 1976; Wainwright and Powell, 1991; Yokomizo et al., 1993), but these pathways are not expressed in the rat hepatocyte.

Previously, the metabolism of LTB₄ in the hepatocyte was found to be mediated by a cytochrome P450-dependent -oxidation to 20-hydroxy-LTB₄ (Murphy and Wheelan, 1997; Kikuta et al., 1994) followed by alcohol dehydrogenase (Baumert et al., 1989; Shirley and Murphy, 1990) and an aldehyde dehydrogenase-dependent (Sumimoto and Minakami, 1990) conversion of the -hydroxy metabolite to 20-carboxy-LTB₄. Once a carboxylic acid moiety is formed at the terminal carbon atom of LTB₄, it is converted to a CoA thioester where it can undergo subsequent -oxidation to 18-carboxy-LTB₄ (Shirley and Murphy, 1990). Relatively small quantities of ethanol present during hepatocyte incubations have been found to reduce substantially the rate of LTB₄ metabolism (Baumert et al., 1989; Shirley et al., 1992) with subsequent accumulation of 20-hydroxy-LTB₄ and formation of a C-1 -oxidation product, 3-hydroxy-LTB₄ (Shirley et al., 1992; Shirley and Murphy, 1992). Both of these LTB₄ metabolites retain significant biological activity as chemotactic agents (Shirley et al., 1992). This alteration in LTB₄ metabolism at low concentrations of ethanol could play an important role in the development of liver disease in human subjects because of the development of a concentration gradient of chemotactic agents within the hepatocyte (Shirley et al., 1992).

Prostaglandins (PGE₂ and PGF₂) as well as thromboxane B₂ are metabolized by peroxisomal CoA-dependent -oxidation (Diczfalusy, 1994). An intermediate step of -oxidation involves oxidation of a 3-hydroxy-acyl-CoA ester that requires NAD⁺ as cofactor. In addition, a common metabolic pathway for these prostanoids, before chain shortening, involves reduction of the double bond at carbon-5 in a series of complex enzymatic steps that ultimately require intracellular reducing equivalents (Smeland et al., 1992). In spite of these requirements of both oxidizing and reducing equivalents for prostaglandin metabolism, little is known about whether ethanol or acetaldehyde affects prostaglandin metabolism through either direct or indirect biochemical interactions.

There have been no investigations of whether acetaldehyde metabolism through aldehyde dehydrogenase is linked to an alteration in the metabolic processing of LTB₄. Furthermore, little is known about the effect of ethanol on prostaglandin metabolism in the hepatocyte. Previous interest in the interaction between ethanol and eicosanoids has focused largely on the effect of ethanol in altering the biosynthesis of these cyclooxygenase products (Pennington, 1985; Nebert, 1994). The effect of ethanol in altering -oxidation of PGE₂ in the isolated rat hepatocyte has not been previously reported.

	Materials and Methods

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Materials. The following drugs and chemicals were kindly provided by or obtained from the sources indicated: PGE₂, LTB₄, 20-carboxy LTB₄ and 20-hydroxy-LTB₄ (Cayman Chemical Co., Ann Arbor, MI), synthetic 3-hydroxy-LTB₄ (Prof. J.R. Falck, University of Texas, Dallas, TX, tritium-labeled PGE₂ ([5,6,8,11,12,14,15-3H(N)]PGE₂, 171 Ci/mmol) (DuPont-New England Nuclear Co., Boston, MA), all solvents used for HPLC and sample dilution (Fisher Scientific, Fair Lawn, NJ), scintillation cocktail used for radioactive detection (Packard Instrument Co., Meriden, CT) and HBSS (Gibco BRL Life Technologies, Gaithersburg, MD).

Rat hepatocyte preparation. Rat hepatocytes were prepared for use at the University of Colorado Hepatobiliary Center according to the collagenase perfusion procedure described by Li et al., (1991). Cells (4 × 10⁶/ml) were suspended in modified Waymouth's MB 752/1 cell medium and stored at 4°C. Within 1 hr of preparation, cells were pelleted at 112 × g to resuspend them in the incubation buffer.

Metabolism of LTB₄ and PGE₂: cell incubation. Stock solutions of LTB₄ (20 µM + 0.2% BSA) and PGE₂ (20 µM PGE₂ with 0.4 µCi/mL ³H-PGE₂) were prepared in HBSS and refrigerated before obtaining cells. Solutions of ethanol in HBSS (0 mM, 50 mM, 100 mM, 200 mM and 400 mM) and acetaldehyde in HBSS (0.2 mM, 0.6 mM, 2.0 mM, 60 mM and 200 mM) were prepared ahead of time in a cold room (7.2°C) and then sealed and stored on ice. Isolated rat hepatocytes (2 × 10⁶ cells) were gently resuspended in each of eleven 15-ml polypropylene centrifuge tubes containing 0.5 ml of the different ethanol/acetaldehyde stock solutions as well as a control tube containing 0.5 ml of HBSS alone. To each of the 11 tubes in sequence, we added 0.5 ml of LTB₄ stock solution for final concentrations of 10 µM LTB₄ along with 2 × 10⁶ rat hepatocytes/ml. The final concentrations of ethanol/acetaldehyde in each experiment were half the concentrations of the corresponding stock solutions. The same sequence of events was followed using PGE₂ as substrate in 11 additional 1-ml incubations for final concentrations of 10 µM PGE₂ along with 2 × 10⁶ rat hepatocytes/ml. After incubation of the capped tubes for 25 min in a shaking water bath (37°C), we added 4 ml of cold 100% ethanol to each tube to terminate cell activity, precipitate cellular proteins and extract eicosanoids. The ethanolic solutions were stored for 2 hr at 20°C, after which the reaction contents were centrifuged at 224 × g, and the supernatant was separated from the residual pellet and retained for analysis. These incubations for both PGE₂ and LTB₄ were repeated in four separate experiments (four different rat livers).

Metabolite separation: reverse-phase HPLC. Reverse-phase HPLC with gradient elution coupled with mass spectrometry was used to separate and identify standard compounds and metabolites of eicosanoids. The extracts from the incubation samples were dried by rotary evaporation and brought up to 1.0 ml with a mixture of solvents matching initial HPLC conditions [75% A and 25% B; A = 8.3 mM acetic acid buffered at pH 5.0 with NH₄OH, B = acetonitrile/methanol (65:35 v/v)]. These 1-ml suspensions were filtered (0.2-µm syringe filters Nalge; Rochester, NY) and 500-µL aliquots were set up for HPLC analysis using a Gilson Auto-injector 231-401 (Gilson Medical Electronics, Middletown, WI). Reverse-phase HPLC was carried out using a 4.6 × 250 mm Ultremex C-18 column (Phenomenex, Rancho Palos Verdes, CA) at a flow rate of 1.0 ml/min and a linear gradient from 25% B to 75% B in 25 min. The HPLC column was connected to a radioactivity monitor (Flow One/Beta Radiomatic, Tampa, FL) for quantification of radioactive PGE₂ metabolites. Components were identified as metabolites of LTB₄ by the characteristic UV absorbance pattern centered at a wavelength of 270 nm using an HPLC photodiode array detector.

Statistical analysis. The statistical analysis of the combined data from four separate experiments was performed using JMP software (SAS Institute, Cary, NC). The S.E.M. of four replicate experiments was calculated for each concentration of ethanol and acetaldehyde employed. The statistical results are expressed as error bars on the subsequent figures and are individually described in the figure legends. The significance of the difference between data-points was established with application of an analysis of variance using Fisher's protected measure of least significant difference with a multiple comparison procedure.

	Results

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LTB₄ metabolism and ethanol. As previously reported (Shirley et al., 1992), the incubation of LTB₄ (10 µM) with rat hepatocytes resulted in rapid -oxidation that yielded 20-hydroxy-LTB₄, which was subsequently oxidized to the major metabolites 20-carboxy-LTB₄ and 18-carboxy-LTB₄ (fig. 1A). The identity of the LTB₄ metabolites was confirmed by HPLC retention time and mass spectrometric data. Dramatic changes in the abundance of these metabolites were observed when increasing concentrations of ethanol were added to the hepatocyte incubation (fig. 2). In the presence of 25 mM ethanol (fig. 1B), the major metabolite was observed to be 20-hydroxy-LTB₄, and the quantities of 20-carboxy-LTB₄ and 18-carboxy-LTB₄ significantly decreased over initial levels of hepatocytes not exposed to ethanol. Furthermore, there was a doubling of the residual amount of LTB₄ remaining in the incubation supernatant. A small increase of 3-OH-LTB₄ was observed with 25 mM ethanol present in the incubation media. These major effects on LTB₄ metabolism evident at 25 mM ethanol did not appear to be significantly altered when the concentration of ethanol was increased up to 100 mM (fig. 2). The abundance of 3-hydroxy-LTB₄ observed at 25 mM LTB₄ only slightly increased when higher concentrations of ethanol were present in the incubation media. The effect of ethanol on -oxidation of 20-hydroxy-LTB₄ was assessed by relating the quantity of 18-COOH-LTB₄ to total -oxidized products (18-COOH-LTB₄ and 20-COOH-LTB₄) formed at various concentrations of ethanol (table 1). There was no apparent inhibition of the metabolism of 20-COOH-LTB₄ to 18-COOH-LTB₄ even with high ethanol concentrations (100-200 mM) present in the incubation media.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Separation and identification of metabolites of LTB4 (10 µM) in isolated rat hepatocytes (2 × 106 cells/ml) using reverse-phase HPLC with effluent monitored at 270 nm. HPLC peaks were structurally characterized by mass spectrometry in separate experiments. A) Representative control incubation with no ethanol or acetaldehyde added. Metabolite 20-hydroxy-LTB4 (20-OH-LTB4) is a product of -oxidation that subsequently undergoes -oxidation leading to the formation of the major metabolites 20-carboxy-LTB4 (20-COOH-LTB4) and 18-carboxy-LTB4 (18-COOH-LTB4). Several small metabolites appear in UV trace that were not considered in this work, along with an impurity i at 24 min that did not have the characteristic triene absorbance centered at 270 nm. B) Formation of LTB4 metabolites produced during incubation with rat hepatocytes and addition of 25 mM ethanol. Note the increase in 20-hydroxy-LTB4 with decreases in 20- and 18-carboxy-LTB4. C) Formation of LTB4 metabolites produced during incubation with rat hepatocytes and addition of 100 mM acetaldehyde.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Quantity of major metabolites formed from LTB4 during incubation with rat hepatocytes in relation to increasing ethanol concentrations in incubation medium. Data-points on the log scale correspond to ethanol concentrations of 0, 25, 50, 100 and 200 mM. Error bars represent the S.E.M. of four separate experiments. The increase in 20-hydroxy-LTB4 observed above 25 mM ethanol and the decreases in 20-carboxy-LTB4 and 18-carboxy-LTB4 are all significantly different from control incubations (*P < .05; **P < .01).

View this table: [in this window] [in a new window]   TABLE 1 The extent of -oxidation of 20-carboxy-LTB4 during incubation of LTB4 with isolated rat hepatocytes. Values represent the ratio of picomoles of 18-carboxy-LTB4 to total picomoles of -carboxy-LTB4 metabolites (18-carboxy-LTB4 × 100/18-carboxy-LTB4 + 20-carboxy-LTB4) Error represents S.E.M. of four trials.

LTB₄ metabolism and acetaldehyde. Addition of acetaldehyde to the incubation medium of isolated rat hepatocytes did not affect the apparent rate of metabolism or disposition of LTB₄ into different metabolites when concentrations up to 1 mM were present in the incubation media (fig. 3). At higher concentrations of acetaldehyde (30 mM), there was a decrease in abundance of 18-carboxy-LTB₄ with a corresponding increase in the concentration of precursors 20-carboxy-LTB₄ and LTB₄. At 100 mM acetaldehyde, there were decreases in all metabolites and increases in unmetabolized LTB₄ (fig. 1C). The extent of -oxidation was altered significantly when acetaldehyde in the incubation media was greater than 1 mM, which suggests that acetaldehyde affects some steps of 20-carboxy-LTB₄ -oxidation (table 1).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Quantity of major metabolites of LTB4 in isolated rat hepatocytes observed at increasing acetaldehyde concentrations in the incubation medium. Data-points on the log scale correspond to acetaldehyde concentrations of 0, 0.1, 0.3, 1, 30 and 100 mM. Error bars represent the S.E.M. of four separate experiments. The increase in relative abundance of LTB4 and the decrease in 18-carboxy-LTB4 between 1 and 100 mM acetaldehyde were statistically significant (*P < .05; **P < .01).

PGE₂ metabolism and ethanol. Incubation of PGE₂ with isolated rat hepatocytes for 25 min resulted in the rapid metabolism of this prostaglandin as revealed by the HPLC separation of radioactive components (fig. 4A). Five major radioactive peaks could be separated, and they were identified via mass spectrometric techniques as unchanged PGE₂, dinor-PGE₁, dinor-PGE₂, tetranor-PGE₁ and an HPLC peak (retention time 8.5 min) corresponding to two coeluting taurine conjugates, tauro-dinor-PGE₁ and tauro-dinor-PGE₂, as previously described (Hankin et al., 1997). The absolute magnitude of the taurine conjugates of the dinor-PGE metabolites was found to vary 4-fold between experimental days, from 7% to 30% of the total metabolites. However, the absolute quantity of total metabolites was remarkably similar in day-to-day hepatocyte preparations. No dose-dependent effect of ethanol or acetaldehyde was observed on the extent of taurine conjugation (data not shown). The quantity of dinor-PGE₁ and dinor-PGE₂ as taurine conjugates was calculated by proportioning the total amount of coeluting taurine conjugates by the ratio of dinor-PGE₁ to dinor-PGE₂ observed in these experiment. Independent mass spectrometric analysis of the taurine conjugate HPLC peak in four separate samples revealed the molecular ion ratios of tauro-dinor-PGE₁ to tauro-dinor-PGE₂ to be constant relative to the ratios of the HPLC radioactive peak for dinor-PGE₁/dinor-PGE₂ found in these samples. Thus the effect of ethanol on formation of the -oxidized products dinor-PGE₁, dinor-PGE₂ and tetranor-PGE₂ could be assessed independently of the conjugation reaction.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Distribution of radioactive metabolites of PGE2 with [5,6,8,11,12,14,15-3H(N)]PGE2 (171 Ci/mmol) incubated with isolated rat hepatocytes and varying concentrations of ethanol and acetaldehyde. Metabolites separated by HPLC were detected with an online radioactivity monitor. A) Control metabolite profile with 10 µM PGE2 and 2 × 106 cells/ml. B) Metabolite profile after incubation of cells in medium containing 25 mM ethanol. C) Metabolite profile after incubation of cells in medium containing 100 mM acetaldehyde.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Abundance of major metabolites of PGE2 in isolated rat hepatocytes in relation to increasing ethanol concentrations in incubation medium. Data-points on the log scale correspond to ethanol concentrations of 0, 25, 50, 100 and 200 mM. Error bars represent the S.E.M. from four separate experiments. The increase in abundance of dinor-PGE1 at 25 mM ethanol and the corresponding decrease in tetranor-PGE1 at 50 mM ethanol concentrations were significantly different from control incubations (*P < .05; **P < .01).

PGE₂ metabolism and acetaldehyde. When the incubation of PGE₂ was carried out in isolated rat hepatocytes in the presence of acetaldehyde (0, 0.1, 0.3, 1, 30 and 100 mM), the relative abundances of dinor-PGE₁, dinor-PGE₂ and tetranor PGE₁ metabolites remained relatively constant from 0 to 0.3 mM acetaldehyde (fig. 6), which are concentrations of acetaldehyde that may be generated in vivo (Weiner, 1997). However, all metabolites were observed to decrease when high concentrations of acetaldehyde (fig. 6) were incubated with PGE₂. The amount of nonmetabolized PGE₂ increased in a dose-dependent manner between 1 and 100 mM acetaldehyde.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Abundance of major metabolites of PGE2 in isolated rat hepatocytes in relation to increasing acetaldehyde concentrations in incubation medium. Data-points on the log scale correspond to acetaldehyde concentrations of 0, 0.1, 0.3, 1, 30 and 100 mM. The increase in abundance of PGE2 at 100 mM acetaldehyde and the decrease in tetranor-PGE1 were significantly different from control incubations (*P < .05; **P < .01).

	Discussion

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Leukotrienes and prostaglandins play important roles in mammalian systems in normal physiology as well as in pathophysiology. For example, they are thought to be major lipid mediators of the inflammatory response (Henderson, 1994; Mayatepek and Hoffman, 1995) and to be responsible for many physiological processes (Smith, 1992; Williams and DuBois, 1996). Because they are substances that serve as chemical messengers, communicating events that lead to cellular activation and corresponding biochemical responses, termination of their activity becomes an important consideration. For eicosanoids in general, metabolism is the major process that limits the biological activity of these substances. Metabolism of both prostaglandins and leukotrienes can involve specific metabolic pathways such as 15-hydroxy prostaglandin dehydrogenase and 12-hydroxy eicosanoid dehydrogenase (Hansen, 1976; Wainwright and Powell, 1991) as well as more general noneicosanoid specific pathways, including serving as substrates for cytochrome P450s and -oxidation. The liver is known to metabolize both prostaglandins and leukotrienes rapidly to a series of metabolites involving both of these more general metabolic cascades that lead to biologically inactive metabolites.

There are interesting differences in metabolism between these two families of eicosanoids. Prostaglandins are largely metabolized by peroxisomal -oxidation after formation of the CoA ester at the C-1 carboxylic acid group in the endoplasmic reticulum (Diczfalusy, 1994). Leukotrienes, and in particular LTB₄, are metabolized initially by cytochrome P450 in the endoplasmic reticulum, leading to the -oxidized product 20-hydroxy-LTB₄. This metabolite is then further oxidized to 20-carboxy-LTB₄, a process that involves alcohol dehydrogenase, aldehyde dehydrogenase and possibly cytochrome P450 (Sumimoto and Minakami, 1990). Omega carboxy-LTB₄ is then a substrate for -oxidation after formation of the -CoA thioester, resulting in a series of chain-shortened /-oxidized metabolites, the major dicarboxylic acid metabolite being 18-carboxy-LTB₄. The isolated rat hepatocyte metabolism of PGE₂ and LTB₄ does not involve the more esoteric metabolic pathways of 15-hydroxy prostaglandin dehydrogenase or 12-hydroxy eicosanoid dehydrogenase found in other tissues, but it does provide a model in which to study the effects of ethanol on hepatic eicosanoid metabolism.

There have been several studies investigating an interaction between ethanol and eicosanoids largely from the standpoint of the effect of ethanol on their biosynthetic pathways. In large part, this is because both synthetic pathways are highly regulated. Ethanol has been found to exert both stimulation and inhibitory effects on prostaglandin synthesis (Pennington, 1985; Nebert, 1994). Ethanol has also been found to affect the synthesis of leukotrienes (Shirley et al., 1992; Shirley and Murphy, 1992). Only few studies have specifically investigated an effect of ethanol on the metabolic inactivation of prostaglandins and leukotrienes. Ethanol has been shown to alter 15-hydroxy prostaglandin dehydrogenase activity after both acute and chronic exposure (Pennington et al., 1980). Because this enzyme requires NAD⁺ as cofactor, this effect of ethanol is thought to be mediated by an altered cellular redox potential, with decreased concentrations of NAD⁺ induced by alcohol dehydrogenase metabolism of ethanol. The effects of ethanol and acetaldehyde on prostaglandin metabolism by -oxidation pathways have not been previously investigated. A significant effect of ethanol on leukotriene metabolism has been previously reported (Baumert et al., 1989; Shirley et al., 1992; Shirley and Murphy, 1992). LTB₄ metabolism has been shown to involve alcohol dehydrogenase as the major enzyme in the -oxidation of 20-hydroxy-LTB₄ to 20-carboxy-LTB₄. Inhibition of this metabolic pathway prevents the inactivation of LTB₄, because 20-hydroxy-LTB₄ and 3-hydroxy-LTB₄ (a -oxidation product from the C-1 terminus), as well as intact LTB₄, are chemotactic for the human polymorphonuclear leukocytes. The conversion of 20-hydroxy-LTB₄ to 20-carboxy-LTB₄ probably involves the intermediate formation of 20-oxo-LTB₄, which has been isolated (Soberman et al., 1988). Two separate mechanisms have been postulated to mediate this aldehyde oxidation. The first involved a NADPH-dependent cytochrome P450 pathway, the second a disulfiram-insensitive NAD⁺-dependent aldehyde dehydrogenase pathway. It was suggested that in the human neutrophil, the NAD⁺-dependent aldehyde dehydrogenase pathway was several-fold higher in this activity than the cytochrome P450 pathway (Sumimoto and Minakami, 1990). No investigations have reported whether acetaldehyde interferes with the metabolic conversion of 20-oxo-LTB₄ to 20-carboxy-LTB₄.

Results of the investigations reported here confirm the effect of low-dose ethanol (25 mM) in inhibiting the metabolism of LTB₄ in the isolated rat hepatocyte. In parallel incubations, acetaldehyde had surprisingly minor effects on the metabolism of LTB₄ at concentrations of acetaldehyde that are relevant to ethanol metabolism (Weiner, 1997). These results suggest that conversion of 20-hydroxy-LTB₄ to 20-carboxy-LTB₄ via the formation of 20-oxo-LTB₄ does not involve an aldehyde dehydrogenase that utilizes acetaldehyde as a substrate, such as mitochondrial type II NAD⁺-dependent aldehyde dehydrogenase. Even though LTB₄ is known to be metabolized in mitochondrial -oxidation, alternative pathways of oxidation of 20-oxo-LTB₄ are probably involved. Other possibilities include the peroxisomal or microsomal aldehyde dehydrogenases, which are known to metabolize aldehydes other than acetaldehyde. Ethanol was not found to effect significantly the -oxidation of LTB₄ (20-carboxy-LTB₄ to 18-carboxy-LTB₄). However, acetaldehyde at high concentrations (greater than 1 mM) did inhibit the -oxidation of 20-carboxy-LTB₄. Because ethanol did not have a significant effect on this process, it is unlikely that this inhibition of -oxidation is a result of reducing NAD⁺ concentrations within the peroxisomes, which is required for oxidation of the 20-carboxy-18-hydroxy-LTB₄ acyl thioester to the 20-carboxy-18-oxo-LTB₄ CoA thioester before a thiolase cleavage reaction. Acetaldehyde is probably affecting one of the enzymatic steps involved in the multifunctional -oxidation enzyme complex present in peroxisomes.

The effect of ethanol on PGE₂ metabolism was not as striking as the effect of ethanol on LTB₄ metabolism in the isolated rat hepatocyte. There was an alteration in -oxidation apparent at low doses of ethanol (25 mM) with increased formation of the metabolite dinor-PGE₁ and a corresponding decrease in tetranor-PGE₁. Dinor-PGE₁ is typically the major metabolite of PGE₂ in the isolated rat hepatocyte, and it is a product of multiple steps of enzymatic processing that includes several isomerase reactions and eventual involvement of NADPH-dependent 2,4-dienoyl-CoA reductase (Vasiliou et al., 1995), ultimately leading to saturation of the ⁵ double bond and PGE₂ before the initial steps of -oxidation. The involvement of this reductive step suggests the possibility of enhanced synthesis of the dinor-PGE₁ metabolite in the presence of ethanol. However, we believe this is not the case, because the dinor-PGE₁ metabolite was not enhanced but was decreased to some extent with higher ethanol concentrations. A decrease in the relative abundance of tetranor-PGE₁ also accompanied increases in abundance of dinor-PGE₁. The levels of dinor-PGE₂ remained relatively constant, unlike those of dinor-PGE₁ and tetranor-PGE₁. The results from the experiments reported here suggest that the second round of -oxidation is uniquely susceptible to inhibition by ethanolthat is, the metabolism of dinor-PGE₁ to tetranor-PGE₁. A potential site for the effect of ethanol might be oxidation of the 3-hydroxy-CoA thioester to the 3-oxo thio CoA ester of the dinor-PGE₁ metabolites. This step of -oxidation was found to be sensitive to the inhibition of ethanol in the metabolism of LTB₄.

The effect of acetaldehyde on PGE₂ metabolism was similar to that observed for LTB₄ in that no major alteration in metabolite levels was observed at physiologically relevant concentrations of acetaldehyde. However, above 1 mM there was a significant effect on -oxidation with a decrease in relative abundance of tetranor-PGE₂, the product from a second round of -oxidation of PGE₂.

In conclusion, LTB₄ is uniquely susceptible to inhibition of metabolism in the hepatocyte with low doses of ethanol because of the involvement of alcohol dehydrogenase during hepatic catabolism. Ethanol at these low doses decreased -oxidation of PGE₂, but only in the conversion of dinor-PGE₁ to tetranor-PGE₁. Acetaldehyde had surprisingly minor effects on PGE₂ and LTB₄ metabolism at doses relevant to ethanol metabolism. The oxidation of the aldehyde 20-oxo-LTB₄ to 20-carboxy-LTB₄ apparently does not involve an aldehyde dehydrogenase that uses acetaldehyde as a substrate. Acetaldehyde at relatively high concentrations did appear to affect some steps of -oxidation of both PGE₂ and LTB₄. Ethanol has interesting and complex effects on the oxidative metabolism of eicosanoid, and these effects may play an important role in vivo because they occur at concentrations of ethanol that are readily achieved in the human.