Metabolism of leukotriene B4 in isolated rat hepatocytes. Identification of a novel 18-carboxy-19,20-dinor leukotriene B4 metabolite.

Isolated rat heptocytes were found to metabolize leukotriene B4 (LTB4) to a number of products which could be separated by reverse phase high performance liquid chromatography (HPLC). After incubation of LTB4 with hepatocytes for 15 min, the known omega-oxidized metabolites, 20-hydroxy- and 20-carboxy-LTB4, were identified by HPLC retention time and gas chromatography-mass spectrometry. An early fraction corresponding to 15% of the initial LTB4 was structurally characterized as a novel metabolite, 18-carboxy-19,20-dinor-LTB4, by ultraviolet spectroscopy and gas chromatography-mass spectrometry of the derivatized and derivatized, reduced metabolite. The short HPLC retention time of this metabolite was consistent with its reduced lipophilicity. An additional minor metabolite was tentatively identified as 3-hydroxy-LTB4. These two novel metabolites provide evidence for beta-oxidation as an important route of hepatic biotransformation of LTB4 and 20-hydroxy-LTB4.

Radiolabeled leukotriene (LT) B., was incubated with isolated rat hepatocytes in order to assess the metabolism of this chemotactic leukotriene by the liver. At least eight radioactive metabolites were observed, three of which were previously identified as 20-hydroxy-, 20-carboxy-, and 18-carboxy-19,20-dinor-LTBI.
A less lipophilic major metabolite (designated Hw) was purified by two reverse phase high performance liquid chromatography separations and was found to exhibit maximal UV absorbance at 269 nm with shoulders at 260 and 280 indicating the presence of a conjugated triene chromophore. Negative ion electron capture gas chromatography/mass spectrometry analysis of the pentafluorobenzyl ester, trimethylsilyl ether derivative of HIV, and positive ion electron ionization mass spectra of the methyl ester trimethylsilyl derivative were consistent with a structure of this metabolite being 16-carboxy-14,15-dihydro-17,18, 19,20- tetraenoic acid, LTB,)' is a metabolite of arachidonic acid from the 5-lipoxygenase pathway which possesses potent biological activities on white blood cells including being chemotactic and chemokinetic for neutrophils (1) as well as an inducer of helper and suppressor T-cells (2,3). LTB, is formed from arachidonic acid by the action of the enzyme arachidonate 5-lipoxygenase (EC 1.13.11.34) which initially forms the reactive allylic epoxide, leukotriene Ad. Leukotriene Aq hydrolase, a soluble enzyme that stereospecifically adds water to LTA, to form LTB, (4,5), is found in several cells including the neutrophil (6), macrophage (7), and red blood cell (8). A great deal of interest in the 5-lipoxygenase products has centered around the specific role that these molecules play in several inflammatory disorders and understanding of their metabolic fate (9). LTB, is metabolized in various cells, including neutrophils which convert LTB, into 20-hydroxy-LTB, (10) by a specific cytochrome P-450 isozyme (11). This initial metabolite can be further oxidized to 20-carboxy-LTB, via the 20-aldehyde intermediate presumably mediated by the same enzyme (12). During this process of w-oxidation, substantial reduction in biological activity occurs (13,14). Other cells are also known to metabolize LTB, including the hepatocyte which can w-oxidize LTB, (15). In these cells, there is evidence that alcohol dehydrogenase and aldehyde dehydrogenase can oxidize 20-hydroxy-LTB, to 20-carboxy-LTB, (16). Hepatocytes were also shown to carry out P-oxidation of 20-carboxy-LTB4 with the identification of 1%carboxy-LTB4 (15).
Metabolism of radiolabeled LTB, in the intact animal has been studied, and experiments revealed extensive metabolism of LTB, with excretion of radioactive metabolites into urine (17). The only metabolite identified was 20-hydroxy-LTB4 and a substantial amount of radioactivity from tritium-labeled LTB, converted into volatile counts, presumably water. These results suggest that substantial metabolism of LTB, occurs in uiuo at carbon positions which are typically labeled with tritium atoms. Thus, more extensive metabolism probably occurs in uiuo than that which is suggested from identified metabolites.

RESULTS
Metabolite Isolation and Purification-Incubation of LTB, with rat hepatocytes led to the production of metabolites including several which have not been previously structurally characterized.
The absolute amount of each metabolite depended upon the incubation conditions employed, concentration of LTB4, and incubation time. Furthermore, each preparation of hepatocytes was somewhat variable in the relative amounts of each metabolite observed, likely due to the exact cell viability for each hepatocyte preparation. In a large incubation of LTB, (119 nmol of LTB4, 5 x lo6 cells/ml, 10 ml) carried out for 30 min, the majority of the radioactivity was recovered in the ethanolic supernatant of the hepatocyte suspension. As seen in Table I, 8% of the initial radioactivity from LTB, remained cell-associated in the pellet. Some radioactivity in the cell pellet could be extracted by exhaustive sonication in methanol/water, but this pellet-associated ra- showed the same reverse-phase HPLC metabolic profile as that of the supernatant (see below). Evaporation of the supernatant resulted in a loss. of 33% of the initial radioactivity as volatile material, most likely water, while 59% was nonvolatile at reduced pressure (0.1 Torr for over 1 h). This residue was dissolved in acidic water and applied to a reversephase solid-phase extraction system with approximately 56% of the starting material (95% of the added radioactivity) being retained and then eluted with methanol. Reverse-phase HPLC separation (System 1) of this partially purified extract revealed four major and three minor radioactive components (Fig. 1). The reverse-phase conditions were altered from that previously reported (15) in order to retain the more polar metabolites longer on the HPLC column. The radioactivity which was not retained by the reverse-phase SepPak was designated Hx and corresponded to 3% of the initial LTB, starting material in this experiment.
The radioactive peaks observed (Fig. 1) also were found to absorb ultraviolet light at 270 nm, consistent with the retention of the conjugated triene chromophore in each metabolite structure (Fig. 1). As seen in Fig. 1, five metabolites were designated Hrr through Hvi, respectively, in order of decreasing lipophilicity, based upon their relative reverse-phase HPLC retention times. The relative amount of each metabolite obtained from several experiments is shown in Table II. In other experiments, specific incubation conditions were optimized for production of one or more metabolites in order to increase the quantity available for structural studies. A second HPLC purification step (System 2) was employed to further separate these metabolites from other biochemical impurities.
This reverse-phase HPLC separation employed an acetonitrilelwater gradient system, and separation of interfering substances was followed by UV absorbance. In addition, analysis of the metabolites following this second reverse-phase HPLC separation showed a substantially reduced number of extraneous GC/MS components during subsequent mass spectrometric analyses. When incubations were carried out for longer times or with a lower concentration of LTB.,, a larger production of the component labeled Hx was observed. This component was not retained by the reverse-phase HPLC system and therefore it is not known if one or more components are present in this peak.

ZO-OH-LTB4 and
ZO-COOH-LTB4--Although no 20-hydroxy-LTB1 remained in the supernatant following the 30min incubation seen in Fig. 1 System 1 was used for the separation as outlined in the text. Prostaglandin B, (100 ng) was added to the ethanolic supernatant prior to evaporation as an internal standard. Spectra were obtained using an on-line UV detector, from experiments using 12 pM LTB,, 5 X lo7 cells/l0 ml, for 30 min. A, metabolite Hi"; B, metabolite Hive; C, metabolite Hv; and D, metabolite Hvl. tion with synthetic standards, the identity of 20-hydroxy-LTB, was established.
Correspondingly, the identity of 20-COOH-LTB, (metabolite Hii, Fig. 1) was established by the UV absorption spectrum and co-elution with synthetic standards. In addition, the electron ionization mass spectrum of the dimethyl ester, bis(trimethylsily1) ether derivative (Me, TMS) and the negative ion electron capture mass spectrum of the bis(pentafluorobenzy1) ester, bis(trimethylsily1) ether (PFB, TMS) derivative were identical for both the isolated hepatocyte metabolite (Hii) and synthetic 20-carboxy-LTB4 (data not shown). The   radioactive metabolite Hiii had a UV absorbance spectrum similar to that observed for LTB, and 20-carboxy-LTB4.

18-COOH-dinor-LTB4-
Furthermore, the electron ionization mass spectrum of the Me, TMS derivative was identical with that which has been previously published (15)  HIV---The radioactive metabolite eluting with a HPLC retention time slightly less than that of the previously characterized 18-COOH-LTB4 had a UV absorbance maximum at 269 nm with shoulders at 260 and 280 nm, consistent with the presence of a conjugated triene moiety (Fig. 24). GC/MS analysis of the Me, TMS derivative using electron ionization Hiv. Since P-oxidation typically results in the loss of two carbon units as methylene units, this would lead to a loss of 28 mass units from a homologous series of poxidation metabolites. However, as shown for the P-oxidation of LTB, (19), the chain-shortening process of P-oxidation results in loss of only 26 mass units when an isolated double bond is present two carbons from the corresponding CoA ester moiety. Based upon HPLC retention time and mass spectrometric analyses, the structure of metabolite Hiv was consistent with a further p-oxidized metabolite of LTB, which has one double bond saturated during the P-oxidation process. Considering this metabolite retained a triene chromophore, the double bond reduced would correspond to the double bond in the 14,15-position. Assuming that the double bonds in the conjugated triene remain in their original conformation, we conclude that compound Hiv is 18,19,.

Met&o&e
Hv-It is noteworthy that metabolite Hv was the most variable metabolite in terms of yield. In some experiments, this metabolite became a major metabolite, although, as shown in Fig. 1, in other experiments it was rather minor. Hv retained the conjugated triene structure as evidenced by the UV absorption spectrum with maximal absorbance at 270 nm and shoulders at 261 and 281 nm (Fig. 2C). Attempts to analyze this molecule by gas chromatography/mass spectrometry as described for the previous metabolites, either through formation of the methyl ester, trimethylsilyl ether derivative, or pentafluorobenzyl ester, trimethylsilyl ether derivative, failed to reveal any gas chromatographic peaks which might be related to a LTB, metabolite.
In contrast, negative ion  (Fig. 6B). Since these ions were observed at even mass, this suggested an anion (M-H-) containing an odd number of nitrogen atoms.
One of the possibilities for metabolite Hv was that of a taurine conjugate.
Therefore, hepatocytes were prelabeled with ['*C]taurine prior to incubation with [3H]LTBl for 30 min. After isolation of LTB, metabolites, as described above, HPLC analysis revealed the co-elution of 14C, using an online scintillation detector, with metabolite Hv at the correct retention time as indicated by the tandem UV monitor (Fig.   FIG. 6. A, continuous flow negative ion fast atom bombardment mass-spectrum of metabolite Hv (tauro-18-COOH-LTB,) obtained using a Finnigan TSQ-70B. 65 ng were injected in 1~1 of mobile phase methanol/water/glycerol (4O:lO:l). Xenon was used as the particle source at 7 kV accelerating potential. The mass indicated depicts that which was obtained using high resolution analysis (see "Results"). B, mass spectrum of metabolite Hv following methylation with ethereal diazomethane for 5 min, using identical MS conditions. Methvl ester (33 ng) was injected as outlined above. 7). As expected, there was a doublet at the retention time in the tritium channel resulting from separation of [3H]Hv from the [YJ]HV (label from taurine) detected at 4% efficiency in the tritium channel. This separation was due to a tritium isotope effect on HPLC retention times. anion has also been observed.' Other collisioninduced dissociation ions from Hv-Me correspond to positions which would be expected to be particularly labile either by being allylic to double bonds or cy to a hydroxyl moiety. The carboxy-19,20-dinor-LTB4. The failure to observe this molecule during GC/MS analysis would be consistent with the inability to form a volatile derivative of taurine conjugates (20).
Metatwlites HvI and H,va-In all experiments, metabolites Hvi and H,vB were obtained as minor metabolites (Fig. 1). Nevertheless, it was possible to obtain UV spectra (Fig. 2 Leukotriene Bq is metabolized in the isolated rat hepatocyte into several metabolites by w-and P-oxidation (Fig. 9). In addition, taurine conjugation of one metabolite has been observed. The w-oxidation of LTB, has been studied in some detail in the human polymorphonuclear leukocyte where a unique cytochrome P-450Lrs has been reported to catalyze both w-oxidation of LTB4 as well as the formation of 20-CHO-LTBI and 20-COOH-LTB,. w-Hydroxylation of LTB, has also been studied in the hepatic microsomal preparations which lead to the formation of 20-hydroxy-as well as . These studies suggest that a unique isozyme or P-450 may exist which is different from the cytochrome P-450s which catalyze similar o-oxidations of prostaglandins and lauric acid. The conversion of 20-OH-LTB, to 20-COOH-LTB, in rat hepatocytes has been shown to be due to enzymatic activity in the cytosol which could be inhibited with 4-methylpyrazole, an inhibitor of alcohol dehydrogenase (16). These studies have implicated alcohol dehydrogenase and aldehyde dehydrogenase in the conversion of 20-OH-LTB, to 20-COOH-LTB4. Cytosolic enzymes in hepatocytes may therefore play an important role in the metabolic formation of 20-COOH-LTB4.
Previous studies have shown that 1%COOH-LTB4 is also a m/z 219 ion lends evidence to support the conjugation of taurine to the w-terminus carboxylate of 18COOH-LTB4 as opposed to the a-terminus carboxylate (CJ. Conjugation at the C-18 carboxyl would be likely, since a CoA ester at this site would be expected considering the observed P-oxidation products. Based upon the HPLC retention time, the UV absorbance, a single carboxylate moiety, the even mass of the molecular anion (M-H-), high resolution mass spectrometry, and the collision-induced dissociation mass spectrometric results, metabolite Hv was assigned the structure tauro-18-major metabolite in this preparation suggesting that @-oxidation proceeds after w-oxidation of the methyl terminus of LTB, (15). However, several other metabolites were not characterized in these studies and structural studies reported here indicate that further P-oxidation indeed takes place in the isolated rat hepatocyte. In particular, P-oxidation of 18-COOH-LTB, resulted in the formation of 16-COOH-LTB3, a metabolite shortened four carbons from native LTB,. Furthermore, this metabolite had the carbon 14-15 double bond saturated during the process of B-oxidation consistent with the previous observations of the @-oxidation of LTE, and formation of 16-COOH-N-acetyl-LTE3 by isolated rat hepatocytes. The formation of this reduced metabolite of LTB, strongly argues for the operation of 2,4-dienoyl-CoA reductase in the processing of the CoA ester of 1%COOH-LTB4 following oxidation by either acyl-CoA dehydrogenase (mitochondrial process) (22) or acyl-CoA oxidase (peroxisomal process) (23). The resultant diene metabolite is identical with the 2,4dienoyl-CoA esters observed in the metabolism of polyunsaturated fatty acids containing the nearest double bond, an even number of carbon atoms from the acyl-CoA ester (24). Following reduction of the 2,4diene unit to the A3-monoene and operation of ci.s-truns-3,2-enoyl-CoA isomerase, @-oxidation can proceed resulting in cleavage of the two carbon fragments and formation of the chain-shortened CoA ester of 16-COOH-LTB3. Taurine (p-aminoethanesulfonic acid) is a ubiquitous molecule present in large amounts in various tissues. It is primarily recognized as being an important amino acid for con- Tauro jugation of certain carboxylic acids, although it has also been implicated to have roles in the central nervous system (25), the heart (26), and other systems (27). Taurine typically forms conjugates with various bile acids (28), and taurine conjugates have been reported for various xenobiotics (20). Recently, the 15,15-dimethyl prostaglandin E2 analog, trimoprostil, has been reported to be metabolized into four different taurine conjugates in rat (29). These conjugates arise from the various P-oxidation intermediates formed from this prostanoid. Considering the common structural features inherent in leukotrienes and prostaglandins, it is not surprising that leukotriene Bq can be conjugated to taurine, although this is the first reported occurrence of a tauro-conjugate in the leukotriene class of arachidonic acid metabolites. Hepatocytes contain a large amount of taurine (mM) (30) and fatty acid intermediates which undergo coenzyme A thioester formation may serve as substrates for the acyl-CoA amino acid Nacyltransferase responsible for taurine conjugation. This soluble enzyme can also use glycine for the amino nitrogen atom acceptor which suggests that the glycine conjugate of 1% carboxy-LTB* may also be present. There were several minor metabolites observed in these isolated rat hepatocyte preparations, but they were not structurally characterized. Previous studies of [3H]LTB4 metabolism in uivo showed a relatively low excretion of nonvolatile radioactivity into the urine (17). In addition, a fair amount of radioactivity has been found in the bile of mice injected with LTB4.3 It is possible that a significant amount of LTB, remains in the body for quite sometime due to enterohepatic circulation, perhaps involving various amino acid conjugates including taurine conjugates.
Several reduced metabolites of LTB, were observed when LTB, was incubated with porcine leukocytes (31). These cells lack w-oxidation capacity for LTB,. Reduced metabolites of 6truns-LTB4 and 12-epi-6-truns-LTB,,, which are poorly w-" T. W. Harper and R. C. Murphy, unpublished observations. oxidized by the human polymorphonuclear leukocyte, have been observed in human polymorphonuclear incubations (32). Such reduced molecules were not observed in these preparations as major metabolites; however, there was one metabolite eluting after LTB, with a retention time similar to that expected for reduced LTB+ Insufficient quantity of this metabohte was available for structural studies. The results of these hepatocyte studies are consistent with the observation that when an intact w-oxidation pathway for LTB, exists within a cell, this is the preferred pathway of metabolism leading to further @-oxidation.
In conclusion, we have found that the isolated rat hepatocyte can convert LTB, into a variety of metabolites which we have structurally characterized by mass spectrometry and ultraviolet spectroscopy as the &oxidation products and taurine conjugate following w-oxidation.
The biological activities of these novel metabolites are currently unknown. The rapid formation of these metabolites and extensive conversion of LTB, suggest that these may be important pathways for inactivation and elimination of LTB, in uiuo.