©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Metabolism of 6-trans-Isomers of Leukotriene B in Cultured Hepatoma Cells and in Human Polymorphonuclear Leukocytes
IDENTIFICATION OF A Delta^6-REDUCTASE METABOLIC PATHWAY (*)

(Received for publication, March 3, 1995; and in revised form, June 23, 1995)

Pat Wheelan Robert C. Murphy (§)

From the Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The intermediate metabolic events which degrade hydroxy polyunsaturated fatty acids is largely unknown. Such molecules are common products of lipid peroxidation and lipoxygenase catalyzed oxidation of arachidonic acid. Metabolism of two 5,12-dihydroxyeicosatetraenoic acids, 6-trans-LTB(4) (leukotriene B(4)), and 6-trans-12-epi-LTB(4) was studied in HepG2 cells (a human-derived hepatoma cell line). Extensive metabolism was observed with a major metabolite identified as 4-hydroxy-6-dodecenoic acid for both epimers. Incubation of 6-trans-LTB(4) epimers at shorter times revealed the formation of intermediate metabolites, including 6-hydroxy-4,8-tetradecadienoic acid and 8-hydroxy-4,6,10-hexadecatrienoic acid suggesting beta-oxidation as the major pathway leading to the formation of the common terminal metabolite. Two additional metabolites were structurally elucidated as 5-oxo-6,7-dihydro-LTB(4) and 6,7-dihydro-LTB(4) which have not been previously described. Formation of 5-oxo-6,7-dihydro-LTB(4) and 6,7-dihydro-LTB(4) were also observed during metabolism of 6-trans-12-epi-LTB(4) in human polymorphonuclear leukocytes. Of particular interest is the metabolism of these compounds by beta-oxidation from the carboxyl terminus, a process which is not observed with leukotriene B(4) or leukotriene C(4). Identification of these metabolites suggested the operation of the 5-hydroxyeicosanoid dehydrogenase pathway followed by a Delta^6-reductase metabolic pathway which has not been previously described. This pathway of beta-oxidation may limit the activity of various 5,12-diHETEs including nonenzymatic hydrolysis products of LTA(4) and also the recently described B(4)-isoleukotrienes.


INTRODUCTION

Various isomeric 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acids (5,12-diHETEs) (^1)can be formed within mammalian cells both by enzymatic and nonenzymatic processes. Several isomers of this family of arachidonic acid metabolites have potent biological activities and are thought to play significant roles as lipid mediators. The first committed step in leukotriene biosynthesis is the formation of leukotriene A(4) (LTA(4)) by 5-lipoxygenase(1) . Leukotriene A(4) hydrolase enzymatically hydrates this triene epoxide to leukotriene B(4) (LTB(4)) (2) which is thought to play an important role as a chemotactic factor for the human polymorphonuclear leukocyte(3) . In addition, there is a competitive nonenzymatic hydrolysis of LTA(4) leading to the formation of two epimeric dihydroxyeicosatetraenoic acids, 6-trans-LTB(4) and 6-trans-12-epi-LTB(4). These two leukotrienes are substantially less potent compared to the enzymatic product LTB(4), but there is evidence to suggest they retain significant biological activity(4) . Recently, we have identified the formation of a family of free radical-derived 5,12-diHETEs which have substantial biological activity(5) . These compounds, which have been termed B(4)-isoleukotrienes, are not enzymatic products of arachidonic acid metabolism, but rather are a result of reactive oxygen species attack on arachidonic acid while esterified to phospholipids.

Termination of the biological activity of 5,12-diHETE species is thought to be a result of metabolic inactivation. The most widely studied is LTB(4) for which a specific cytochrome P-450(6, 7, 8) catalyzes the formation of 20-hydroxy-LTB(4) and 20-carboxy-LTB(4) in the human polymorphonuclear leukocyte. LTB(4) is also rapidly taken up by the hepatocyte and metabolized by cytochrome P-450 -oxidation and by subsequent beta-oxidation in both mitochondria and peroxisomes(9, 10, 11) . Recently, it was found that for those cells which do not express the high affinity P-450, an alternative pathway of LTB(4) metabolism exists. A 12-hydroxyeicosanoid dehydrogenase/Delta-reductase pathway leads to the conversion of the conjugated triene in LTB(4) to a conjugated diene and numerous subsequent beta-oxidation metabolites and glutathione conjugates result(12, 13) .

Considerably less is known about the metabolism of 5,12-diHETE isomers that are not substrates for the cytochrome P-450. Neither 6-trans-LTB(4) epimer is efficiently metabolized by the specific cytochrome P-450(14) . This observation reveals the importance of the double bond geometry at carbon 6 when comparing the metabolic fate of LTB(4) with these isomeric 5,12-diHETEs. Furthermore, it is interesting to note that the metabolism of prostaglandins proceeds by beta-oxidation from the carboxyl terminus(15) . Such C-1 beta-oxidation has not been observed as a major pathway for LTB(4) metabolism. The only exception was the identification of a beta-oxidation intermediate, 3-hydroxy-LTB(4), formed when LTB(4) was metabolized in rat hepatocytes in the presence of ethanol(16, 17) .

The 5,12-diHETEs are hydroxy, unsaturated fatty acids that could be substrates for beta-oxidation from the carboxyl terminus. Studies of the metabolism of structurally related monohydroxyeicosatetraenoic acids, 12-HETE and 5-HETE, revealed beta-oxidation from the C-1 terminus to be a major pathway of metabolism. Metabolism of 12-HETE by beta-oxidation in mouse peritoneal macrophages led to the formation of both 8-hydroxy-hexadecatrienoic acid and 4-hydroxy-dodecenoic acid as major metabolites(18) . Recently, a microsomal 5-hydroxyeicosanoid dehydrogenase activity was reported to exist within human polymorphonuclear leukocytes that reversibly converted 5(S)-HETE to 5-oxo-ETE and also converts 6-trans-12-epi-LTB(4) to a 5-oxo metabolite(19) . This 5-oxo metabolite was further converted to dihydro products identified as 5-oxo-6,11-dihydro-LTB(4) and 6,11-dihydro-LTB(4)(19) .

In the present work, metabolism of 6-trans-12-epi-LTB(4) and 6-trans-LTB(4) was examined in a human-derived hepatoma cell line, HepG2 cells. These cells metabolize LTB(4) by a unique beta-oxidation process from C-1 with the formation of 10-hydroxy-octadecatetraenoic acid(20) . However, the metabolism of the 6-trans epimers of LTB(4) was completely different from that previously described for LTB(4). The results reported here reveal that beta-oxidation for these 5,12-diHETEs is dependent on the geometry of the first double bond (Delta^6) in the conjugated triene. Such metabolic processes could play an important role in limiting the activity of free radical-derived B(4)-isoleukotrienes generated in vivo.


EXPERIMENTAL PROCEDURES

Materials

6-trans-12-epi-Leukotriene B(4) (6-trans-12-epi-LTB(4), 5(S),12(S)-dihydroxy-6E,8E,10E,14Z-eicosatetraenoic acid), 6-trans-leukotriene B(4) (6-trans-LTB(4), 5(S),12(R)-dihydroxy-6E,8E,10E,14Z-eicosatetraenoic acid), and leukotriene A(4) (LTA(4)) methyl ester were purchased from Cayman Chemical (Ann Arbor, MI). [14,15-^3H(2)]LTA(4) methyl ester (42.0 Ci/mmol) and [5,6,8,9,11,12,14,15-^3H(8)] LTB(4) (195 Ci/mmol) were purchased from DuPont NEN (Boston, MA). [14,15-^3H(2)]6-trans-LTB(4) and [14,15-^3H(2)]6-trans-12-epi-LTB(4) were synthesized from [14,15-^3H(2)]LTA(4) methyl ester as follows. LTA(4) methyl ester (10 µg) was combined with [14,15-^3H(2)]LTA(4) methyl ester (10 µCi) and the solvent removed under a stream of nitrogen. Dilute hydrochloric acid (1%, 200 µl) was added followed by the addition of ethanol (50 µl). After 30 min at room temperature, water (2 ml) was added and the solution extracted three times with ethyl acetate (2 ml). The combined ethyl acetate layers, containing greater than 90% of the radioactivity, were evaporated under nitrogen. The methyl ester was hydrolyzed using the procedure of Carrier et al.(21) . Briefly, acetone (1.6 ml) and 0.25 N sodium hydroxide (0.4 ml) were added and the reaction kept at room temperature for 3 h. Acetone was evaporated under nitrogen and water (2 ml) was added and the solution acidified to pH < 3 by the addition of hydrochloric acid. The solution was extracted three times with ethyl acetate (2 ml) and the combined ethyl acetate layers evaporated under nitrogen to near dryness. The sample was reconstituted in the initial reverse phase HPLC solvent and immediately analyzed using the RP HPLC conditions described below. The two radioactive products displaying a UV triene chromophore ((max) = 269 nm) at retention times of 32.8 and 33.5 min corresponding to 6-trans-LTB(4) and 6-trans-12-epi-LTB(4), respectively, were separately collected. All solvents were HPLC grade purchased from Fisher Scientific (Fair Lawn, NJ).

HepG2 Culture and Incubation

HepG2 cells, a line derived from a human hepatocellular carcinoma, were obtained from American Type Culture Collection (Rockville, MD). Cells were cultured at 37 °C in 5% CO(2) atmosphere in 75-cm^2 tissue culture flasks with 15 ml of minimum essential medium (Eagle's) containing Earle's balanced salt solution, non-essential amino acids, and sodium pyruvate (BioWhittaker, Walkersville, MD) with added fetal bovine serum (10%). Cells were subcultured every 4-5 days. The substrates, 6-trans-LTB(4) or 6-trans-12-epi-LTB(4) containing radiolabeled substrate (0.5 µCi), were added to cells 4 days after subculture when cells were nearing confluency. Tissue culture medium was removed and the substrate, lyophilized to dryness and reconstituted in 15 ml of medium (0.9 µM) containing 0.1% bovine serum albumin and without fetal bovine serum, was added. Following incubation at 37 °C in 5% CO(2) for 24 h, medium was removed, 35 ml of cold ethanol was added to the medium, and the sample stored at -20 °C for at least 3 h to precipitate protein. Samples were centrifuged and the supernatants decanted and evaporated to near dryness. Samples were reconstituted in 1 ml of initial reverse phase HPLC solvent and immediately analyzed. For time course studies, cells were grown in 25-cm^2 tissue culture flasks using 5 ml of medium. Substrates were added in 5 ml of medium (0.9 µM) containing radiolabeled substrate (0.2 µCi) and the cells incubated for 3, 5, 7, 9, 15, and 24 h. Following incubation at these time points, the medium was removed and analyzed as above.

Human Polymorphonuclear Leukocyte Incubation with 6-trans-12-epi-LTB(4)

Human polymorphonuclear leukocytes were isolated using a Percoll gradient as described previously(22) . 6-trans-12-epi-LTB(4) and [14,15-^3H(2)]6-trans-LTB(4) (0.5 µCi), evaporated under nitrogen and reconstituted in Hank's balanced salt solution (10 ml, Life Technologies, Inc., Grand Island, NY) to give a substrate concentration of 2.0 µM, was added to pelleted cells (2 10^8 cells) and the cells gently resuspended. Incubation was continued for 50 min at 37 °C at which time cells were pelleted and the supernatant removed and combined with 4 volumes of cold ethanol. Supernatants were treated as described above followed by reverse phase HPLC analysis.

Reverse Phase HPLC (RP HPLC) Analysis of Metabolites

RP HPLC analysis was performed using an Ultremex column (4.6 250 mm, 5-µm C-18; Phenomenex, Rancho Palos Verdes, CA) with a mobile phase of methanol:water, 0.05% acetic acid (pH adjusted to 5.8 with ammonium hydroxide) at an initial composition of 30% methanol. A flow rate of 1 ml/min was used and a linear gradient to 70% methanol in 35 min was immediately started followed by a second linear gradient to 100% methanol at 45 min. RP HPLC analysis was monitored by a photodiode array detector or by an on-line radioactivity detector.

Mass Spectrometric Analysis of Metabolites

Collected RP HPLC fractions containing radioactive metabolites were analyzed by gas chromatography/mass spectrometry (GC/MS) following derivatization to the pentafluorobenzyl ester/trimethylsilyl ether compounds. Samples were dried under nitrogen and derivatized by the addition of a 10% solution (v/v) of N,N-diisopropylethylamine (Aldrich) in acetonitrile (50 µl) followed by the addition of a 10% solution (v/v) of pentafluorobenzyl bromide (Aldrich) in acetonitrile (50 µl). Samples were kept at room temperature for 20 min and then taken to dryness under a stream of nitrogen. Samples were further derivatized by the addition of acetonitrile (50 µl) and either bis(trimethylsilyl)trifluoroacetamide (50 µl) (Sigma) or bis(trimethyl [^2H(9)] silyl)acetamide (50 µl) (MSD Isotopes, Montreal, Canada) and heated at 60 °C for 5 min. Samples were evaporated under nitrogen and acetonitrile added to give a metabolite concentration of 1 ng/µl for analysis by negative ion GC/MS and a concentration of 10-20 ng/µl for analysis by positive ion GC/MS. GC/MS analyses were performed on a Finnigan SSQ 70 (San Jose, CA) using methane as the moderating gas for negative ion GC/MS analyses and an electron energy of 70 eV for positive ion GC/MS analyses. A 10 m 0.25-mm DB-1 or a 5 m 0.25-mm DB-1 (0.25 µm film thickness) capillary GC column was used (J & W, Folsom, CA) with an injector temperature of 275 °C and a transfer line temperature of 300 °C. The initial column temperature was 150 °C followed by a linear ramp to 300 °C at 15 °C/min. Equivalent carbon values (EC value) were obtained by comparison to retention times of standard straight chain fatty acids derivatized as the PFB esters.

Catalytic hydrogenation of metabolites was performed as described earlier (23) prior to PFB/TMS derivatization. Oxidative ozonolysis of 200-400 ng of sample was performed without solvent as described earlier (13) prior to PFB/TMS derivatization as described above.


RESULTS

Hepatoma G2 Metabolism of 6-trans-LTB(4)and 6-trans-12-epi-LTB

Following a 24-h incubation of HepG2 cells with 6-trans-LTB(4) (0.9 µM) containing [14,15-^3H(2)]6-trans-LTB(4) (0.5 µCi), 94% of the added radioactivity was recovered in the extracellular supernatant. With 6-trans-12-epi-LTB(4) (0.9 µM) containing [14,15-^3H(2)]6-trans-12-epi-LTB(4) (0.5 µCi) as the substrate, 93% of the added radioactivity was recovered in the extracellular supernatant. RP HPLC analysis of the supernatant fractions resulted in recovery of 56 ± 1% (n = 2) of the radioactivity as a single metabolite observed at a retention time of 20.6 min for incubations using 6-trans-LTB(4) as the substate. For incubations using 6-trans-12-epi-LTB(4) as the substrate, 67 ± 7% (n = 2) of the recovered radioactivity was contained in the same RP HPLC peak (Fig. 1A). No UV triene chromophore indicative of the starting substrates ((max) = 269 nm) was observed at the expected retention times of 32.8 min for 6-trans-LTB(4) or at 33.5 min for 6-trans-12-epi-LTB(4). With shorter incubation times, intermediate radioactive compounds were detected as shown in Fig. 1B for the RP HPLC analysis of the extracellular supernatant from HepG2 cells incubated for 5 h with 6-trans-LTB(4) (0.9 µM) containing [14,15-^3H(2)]6-trans-LTB(4) (0.2 µCi). No metabolites contained a UV chromophore indicative of a conjugated triene moiety. Radioactive metabolites 3, 4, and 5 (Fig. 1B) displayed a UV absorption with (max) at 230 nm indicative of a conjugated diene chromophore. Metabolites 1 and 2 showed no UV absorption above 205 nm. RP HPLC analysis of a 5-h incubation of HepG2 cells with 6-trans-12-epi-LTB(4) (0.9 µM) containing [14,15-^3H(2)]6-trans-12-epi-LTB(4) (0.2 µCi) showed a similar profile of radioactive products.


Figure 1: Reverse phase HPLC analysis with radioactivity monitoring of HepG2 metabolites released from the cells following: (A) 24 h incubation with 6-trans-12-epi-LTB(4) and (B) 5 h incubation with 6-trans-LTB(4).



The time course for the formation of the major metabolite of 6-trans-12-epi-LTB(4) (Metabolite 1) is shown in Fig. 2A and the time courses for the formation of the remaining intermediate metabolites are shown in Fig. 2B. At all time points, metabolite 1 was the major radioactive metabolite. Metabolites 2, 3, and 4 reached a maximum concentration of approximately 5% of the total radioactivity between 3 and 9 h of incubation and declined to less than 1% by 24 h. Metabolite 5 reached a maximum concentration of approximately 15% of the total radioactivity between 3 and 9 h and thereafter declined and was not detected at 24 h. Similar time courses were obtained when HepG2 cells were incubated with 6-trans-LTB(4). Radioactive metabolites were analyzed by GC/MS analysis in both the negative ion mode and positive ion mode following derivatization to the pentafluorobenzyl ester/trimethylsilyl ether (PFB/TMS) compounds.


Figure 2: Formation of HepG2 extracellular metabolites during incubation with 6-trans-12-epi-LTB(4) from 3 to 24 h expressed as a percent of the total recovered radioactivity for each metabolite identified in Fig. 1. A, time course for formation of the major metabolite, metabolite 1. B, time course for the formation of the remaining metabolites, metabolites 2, 3, 4, and 5.



4-Hydroxy-6-dodecenoic Acid (Metabolite 1)

Analysis of the PFB/TMS derivative of metabolite 1 by negative ion GC/MS revealed an abundant [M-PFB] ion at m/z 285 (data not shown) and a less abundant ion at m/z 195 (loss of TMSOH). The EC value for this derivative was 13.8 and combined with the negative ion GC/MS data was consistent with a 12-carbon structure containing one OTMS substituent and one double bond. Positive ion GC/MS analysis of this derivative resulted in an observed ion at m/z 355 (base peak) consistent with a C-4 OTMS substituent and a double bond at C-6 (Fig. 3A). Such a favorable fragmentation for OTMS substituted unsaturated PFB esters is always observed for derivatized hydroxy fatty acids containing unsaturation two carbons removed from the OTMS carbon and results in loss of a neutral stabilized allylic radical(23) . The above data is consistent with assignment of the metabolite 1 structure as 4-hydroxy-6-dodecenoic acid.


Figure 3: Positive ion electron ionization (70 eV) mass spectra of the PFB/TMS derivatives of: A, 4-hydroxy-6-dodecenoic acid (metabolite 1); B, 6-hydroxy-4,8-tetradecadienoic acid (metabolite 2); and C, 8-hydroxy-4,6,10-hexadecatrienoic acid (metabolite 3) with UV spectrum shown in inset.



6-Hydroxy-4,8-tetradecadienoic Acid (Metabolite 2)

Negative ion GC/MS analysis of the PFB/TMS derivative of metabolite 2 showed an abundant [M-PFB] ion at m/z 311 (data not shown) with a less abundant ion observed at m/z 221 (loss of TMSOH). The gas chromatographic retention data (EC value = 15.9) for this derivative was consistent with the negative ion mass spectral data for a PFB-derivatized 14-carbon fatty acid containing one OTMS substituent and two double bonds. Positive ion GC/MS analysis (Fig. 3B) resulted in a base peak ion observed at m/z 381 which was the expected alpha-fragmentation ion for a derivative containing a C-6 OTMS substituent with one double bond at C-8 as described for metabolite 1. The second double bond, positioned between C-1 and C-6, is suggested to be at the C-4 position. A C-3 position for this double bond, a position which would place the unsaturation two carbon atoms removed from the OTMS substituted carbon, would be expected to result in facile fragmentation of the C-5,C-6 bond in positive ion GC/MS analysis resulting in an abundant ion observed at m/z 213 with loss of a neutral allylic stabilized radical, bulletCH(2)CH = CHCH(2)CO(2)PFB. The double bond could be at the C-2 position, however, the likely mechanism leading to the formation of this metabolite by beta-oxidation is more consistent with the C-4 position (see below). The structure of metabolite 2 was thereby determined as 6-hydroxy-4,8-tetradecadienoic acid.

8-Hydroxy-4,6,10-hexadecatrienoic Acid (Metabolite 3)

An abundant [M-PFB] ion at m/z 337 and a less abundant ion at m/z 247 (loss of TMSOH) observed during negative ion GC/MS analysis of the PFB/TMS derivative of metabolite 3 (data not shown) combined with an EC value of 17.9 suggested that metabolite 3 was a 16-carbon fatty acid containing one hydroxy substituent and three double bonds. Positive ion GC/MS analysis revealed an abundant ion at m/z 407 (Fig. 3C) which suggested a double bond position at C-10 and an OTMS substituent at C-8. The positions of the remaining two double bonds were determined as the C-4 and C-6 positions by observation of a conjugated diene moiety by UV analysis with (max) at 230 nm (insetFig. 3C) which precluded a 2,4 position for the diene in conjugation with the carboxylic acid which would result in an expected (max) at significantly higher wavelength(24) . Also, lack of an abundant fragment ion observed at m/z 213 resulting from C-7,C-8 fragmentation precluded a C-3,C-5 position. The structure of metabolite 3 was determined based on the above data as 8-hydroxy-4,6,10-hexadecatrienoic acid.

5-oxo-12-Hydroxy-8,10,14-eicosatrienoic Acid (5-oxo-6,7-Dihydro-LTB(4)) (Metabolite 4)

Negative ion GC/MS analysis of the PFB/TMS derivative of metabolite 4 revealed an abundant [M-PFB] ion at m/z 407 (Fig. 4A) with a less abundant ion observed at m/z 317 (loss of TMSOH). Analysis of this metabolite derivatized as the PFB ester with [^2H(9)]TMS derivatization of hydroxy groups resulted in an abundant observed ion at m/z 416 with a less abundant ion at m/z 317 (loss of [^2H(9)]TMSOH), suggesting the presence of a single hydroxy substituent. Additionally, analysis of hydrogenated metabolite 4 followed by PFB/TMS derivatization revealed an abundant ion at m/z 413 with a less abundant ion observed at m/z 323 (loss of TMSOH) consistent with the presence of three double bonds in metabolite 4. An EC value of 23.0 combined with the above data suggested this metabolite was a 20-carbon fatty acid containing three double bonds, one hydroxy substituent, and one oxo substituent. Positive ion GC/MS analysis of the PFB/TMS derivative revealed an abundant ion at m/z 477 (base peak) with an additional ion at m/z 167 (Fig. 4B). Both of these ions shifted by 9 daltons to m/z 486 and 176 when the PFB/[^2H(9)]TMS derivative was analyzed by positive ion GC/MS. The abundant ion at m/z 477 located the single hydroxy substituent at C-12 with a double bond at C-14 (see above). The oxo group was located at the C-5 position resulting from oxidation of the original 5-hydroxy moiety. The conjugated diene, (max) at 230 nm (Fig. 4A, inset), was determined as the C-8,C-10 position based on the lack of an observed fragmentation of the C-11,C-12 bond, which would be expected for a 7,9 position, and the lack of a UV chromophore consistent with a C-6,C-8 position in conjugation with the 5-oxo substituent. The observed ion at m/z 167 (Fig. 4B) containing the OTMS substituent must arise by internal fragmentation. A possible mechanism leading to the formation of this ion is given in Fig. 5. Rearrangement of the molecular ion, ionized at the OTMS substituent, by 1,6-hydride transfer of the C-12 hydrogen to the C-8 position results in formation of a distonic ion. This ion may rearrange through a six-membered ring intermediate involving transfer of the C-8 hydrogen to the 5-oxo substituent resulting in loss of a neutral enolized ketone fragment (CH(2)=C(OH)(CH(2))(3)CO(2)PFB). The resulting distonic ion undergoes rapid rearrangement with loss of a neutral allylic-stabilized radical (CH(3)(CH(2))(3)CH=CHCH(2)) and formation of the resonance stabilized ion observed at m/z 167. The above data is consistent with assignment of metabolite 4 structure as 5-oxo-12-hydroxy-8,10,14-eicosatrienoic acid (or 5-oxo-6,7-dihydro-LTB(4)).


Figure 4: Mass spectral analysis of the PFB/TMS derivative of 5-oxo-6,7-dihydro LTB(4) (metabolite 4). A, negative ion chemical ionization mass spectrum with UV spectrum shown in the inset. B, positive ion electron ionization (70 eV) mass spectrum.




Figure 5: Proposed mechanism leading to formation of the ion observed at m/z 167 in the electron ionization mass spectrum of the PFB/TMS derivative of 5-oxo-6,7-dihydro-LTB(4).



5,12-Dihydroxy-8,10,14-eicosatrienoic Acid (6,7-Dihydro-TB(4)) (Metabolite 5)

Negative ion GC/MS analysis of the PFB/TMS derivative of metabolite 5 resulted in an abundant [M-PFB] ion at m/z 481 (data not shown) with less abundant ions observed at m/z 391 (loss of TMSOH), m/z 319 (loss of CH(2) = Si(CH(3))(2) and TMSOH) and at m/z 301 (loss of 2TMSOH) (EC value = 23.2). At two mass units higher than the fragment ions observed for derivatized LTB(4), this suggested metabolite 5 was a dihydro analog of LTB(4). Analysis of the PFB/TMS derivative of hydrogenated metabolite 5 revealed an abundant [M-PFB] ion at m/z 487 with less abundant ions observed at m/z 397, 325, and 307 confirming the presence of three double bonds in this metabolite. UV analysis showed the presence of a conjugated diene moiety ((max) = 230 nm, inset, Fig. 6A) which suggested one of the double bonds of the conjugated triene moiety had been reduced. Positive ion GC/MS analysis of the PFB/TMS derivative revealed fragment ions at m/z 551, m/z 461 (loss of TMSOH from m/z 551), and at m/z 371 (loss of 2TMSOH from m/z 551) which located one of the OTMS substituents at C-12 with a double bond at C-14 (Fig. 6A). These fragment ions were observed at m/z 569, 470, and 371 when the PFB/[^2H(9)]TMS derivative was analyzed (Fig. 6B). Location of the second OTMS substituent was confirmed as at the C-5 position by the observed fragment ion at m/z 369 which shifted to m/z 378 when the PFB/[^2H(9)]TMS derivative was analyzed. The position of the conjugated diene may be C-6,C-8, C-7,C-9, or C-8,C-10; however, the positive ion mass spectrum for PFB/TMS derivatized 10,11-dihydro-LTB(4) (positions C-6 and C-8 for the conjugated diene) has been previously published and differs markedly from that of the dihydro derivative reported here(13) . The positive ion spectrum of the methyl ester/TMS derivative of a dihydro metabolite reported as 6,11-dihydro-LTB(4) (positions C-7 and C-9 for the conjugated diene) has also been reported (19, 25) and the observed fragmentations agree closely with those reported here when the differences between a PFB ester and a methyl ester are considered (a difference which does not significantly effect mass spectral fragmentation(23) ). The dihydro metabolite is assigned the 6,7-dihydro-LTB(4) structure (positions C-8 and C-10 for the conjugated diene), however, based on the following data. The observed ion at m/z 279 (m/z 288 for the PFB/[^2H(9)] TMS derivative) most likely resulted from fragmentation of the C-6,C-7 bond with charge retention on the C-7 to C-20 fragment. A possible mechanism for the formation of this ion is given in Fig. 7. The mechanism involves the intermediate formation of an ion that is observed at low abundance at m/z 395. The ion at m/z 395 leading to the formation of m/z 279 would contain two OTMS substituents and analysis of the PFB/[^2H(9)]TMS derivative would be expected to result in an observed ion for this fragmentation at m/z 413. This ion was observed, however, the ratio of m/z 404:413, both of which are low abundant ions, was approximately 2:1 in the PFB/[^2H(9)]TMS derivatized metabolite, suggesting that m/z 395 results from two different fragmentations: one containing two OTMS groups and possibly further fragmenting to the ion observed at m/z 279 and a second m/z 395 ion containing only one OTMS group. This second ion may be due to rearrangement as was observed earlier in the positive ion GC/MS spectra of PFB/TMS derivatized LTB(4) and 10,11-dihydro-LTB(4)(23) . If the double bonds were located at the C-7,C-9 positions, the ion at m/z 279 would correspond to an apparent vinylic fragmentation which is unlikely for this structure (see discussion on apparent vinylic fragmentation in (23) ).


Figure 6: Electron ionization (70 eV) mass spectra of: (A) the PFB/TMS derivative of 6,7-dihydro-LTB(4) (metabolite 5) with UV spectrum shown in the inset and (B) the PFB/[^2H(9)] TMS derivative of 6,7-dihydro-LTB(4).




Figure 7: Proposed mechanism leading to formation of the ions observed at m/z 395 and 279 in the electron ionization mass spectrum of the PFB/TMS derivative of 6,7-dihydro-LTB(4).



The observed ion at m/z 435 also contained one OTMS group as evidenced by a shift of 9 atomic mass units to m/z 444 in the analysis of the PFB/[^2H(9)]TMS derivative. This ion may be due to loss of acetylene (26 atomic mass units) from the base peak at m/z 461. This would be a favorable process with loss of a small neutral molecule possibly involving loss of carbons 11 and 12 with OTMS transfer to C-10. The corresponding ion in the methyl ester/TMS derivative, m/z 269, may likewise be due to loss of 26 atomic mass units from m/z 295 rather than an unlikely vinylic fragmentation of the C-10,C-11 bond with the conjugated diene at C-7 and C-9 as was previously suggested (19, 25) . A C-7,C-9 position for the conjugated diene moiety would also be expected to result in an abundant fragment ion due to fragmentation of the C-11,C-12 bond with loss of a neutral allylic stabilized radical (C-1 though C-11) and formation of an observed ion at m/z 213 (TMSO=CHCH(2)CH = CHC(5)C). Lack of an observed ion at m/z 213 then implies in the spectra reported here as well as in the spectra of the previously identified 6,11-dihydro-LTB(4) metabolite derivatized as the methyl ester/TMS compound (19, 25) that location of the conjugated diene is best supported by positive ion GC/MS data as a 6,7-dihydro-LTB(4) structure.

In order to unambiguously assign double bond location, metabolite 5 was subjected to oxidative ozonolysis and negative ion GC/MS analysis of the products following PFB/TMS derivatization. With double bonds located at C-8 and C-10, the expected dicarboxylic acids were a 4-carbon monohydroxy fragment corresponding to C-11 through C-14 and an 8-carbon monohydroxy fragment corresponding to C-1 through C-8. Following PFB/TMS derivatization, these fragments were observed at m/z 385 and 441 with EC values of 13.4 and 17.9, respectively (Fig. 8). The spectrum of the derivatized 4-carbon fragment is identical to that obtained from oxidative ozonolysis followed by PFB/TMS derivatization of LTB(4) with an observed base peak at m/z 385 (loss of one PFB group) and minor ions observed at m/z 341 (loss of 44 atomic mass units) and at m/z 295 (loss of TMSOH from m/z 385) (Fig. 8A). The spectrum of the PFB/TMS derivatized 8-carbon fragment is also dominated by an observed ion resulting from loss of one PFB group at m/z 441 (Fig. 8B). Expected fragments resulting from oxidative ozonolysis of a dihydro metabolite containing double bonds at positions C-7 and C-9, a 5-carbon monohydroxy diacid and a 7-carbon monohydroxy diacid resulting in expected PFB/TMS derivatized fragments observed at m/z 399 and 427, respectively, were not observed. The above data for metabolite 5 is then consistent with the structure 5,12-dihydroxy-8,10,14-eicosatrienoic acid (6,7-dihydro-LTB(4)).


Figure 8: Negative ion chemical ionization GC/MS analysis of the PFB/TMS derivatives of ozonolysis products of 6,7-dihydro-LTB(4). A, ion profile at m/z 385 with the inset showing negative ion mass spectrum of fragment containing C-11 through C-14 observed at scan 153 with EC value = 13.4, and B, ion profile at m/z 441 with the inset showing negative ion mass spectrum of fragment containing C-1 through C-8 observed at scan 297 with EC value = 17.9.



It was previously observed that oxo compounds elute during RP HPLC analysis after the corresponding hydroxy compound when acetonitrile is present in the organic phase(19, 26) , but in the present system using only methanol in the organic phase, the 5-oxo compound eluted slightly before the corresponding hydroxy compound. The reverse order of elution depending on the presence or absence of acetonitrile was also observed using synthetic 12-oxo-LTB(4) which eluted from RP HPLC slightly before LTB(4) when only methanol was used in the organic phase but eluted after LTB(4) when the organic phase consisted of 1:1 methanol:acetonitrile.

Incubation of Human Polymorphonuclear Leukocytes with 6-trans-12-epi-LTB(4)

RP HPLC analysis of extracellular supernatants following incubation of human polymorphonuclear leukocytes with 6-trans-12-epi-LTB(4) containing [14,15-^3H(2)]6-trans-12-epi-LTB(4) is shown in Fig. 9. The profile of radioactive metabolites is similar to that reported earlier(19, 27) . Radioactive products containing a triene chromophore were observed at 10.8 and 19.2 min. These products are likely the -oxidation metabolites, 20-carboxy-6-trans-12-epi-LTB(4) and 20-hydroxy-6-trans-12-epi-LTB(4), and were not further characterized. The radioactive metabolite observed at 35.7 min contained the UV chromophore indicative of a conjugated diene. Mass spectrometric analysis as described above revealed that this metabolite was identical to the 6,7-dihydro-LTB(4) metabolite (metabolite 5) isolated from HepG2 incubations. In addition, UV analysis monitoring at 230 nm revealed a second metabolite containing a UV chromophore indicative of a conjugated diene which eluted just after 6-trans-12-epi-LTB(4) and slightly before 6,7-dihydro-LTB(4) (inset, Fig. 9). Mass spectrometric analysis of this metabolite revealed that it was identical to metabolite 4 isolated from HepG2 cells and was assigned the structure 5-oxo-6,7-dihydro-LTB(4).


Figure 9: Reverse phase HPLC analysis with radioactivity monitoring of extracellular metabolites produced by human polymorphonuclear leukocytes following 50 min incubation with 6-trans-12-epi-LTB^4. Inset shows the UV monitor trace at 230 nm revealing the conjugated diene-containing metabolites, 5-oxo-6,7-dihydro-LTB(4)(4) and 6,7-dihydro-LTB(4)(5) , with end absorption of 6-trans-12-epi-LTB(4) ((max) = 269 nm). Resolution at the UV spectrophotometer is better than observed at the radioactivity monitor due to on-line mixing of the HPLC effluent with scintillation mixture prior to radioactivity detection.




DISCUSSION

The metabolism of 6-trans-LTB(4) and 6-trans-12-epi-LTB(4) by HepG2 cells was quite similar both qualitatively and quantitatively. Based on the chemical structures of the identified metabolites, the sequence of metabolic events is proposed in Fig. 10. Initial metabolism likely involved oxidation of the 5-hydroxy group to form 5-oxo-LTB(4) which has been previously described(19) . This ketone could be reduced to 5-oxo-6,7-dihydro-LTB(4) by an enzymatic activity which converts an alpha,beta-unsaturated keto moiety to the corresponding saturated ketone. We have termed this activity Delta^6-reductase. This same enzymatic pathway was found in human polymorphonuclear leukocytes when metabolism of 6-trans-12-epi-LTB(4) was examined and exact double bond location in the observed metabolites determined by oxidative ozonolysis. It is likely that the initially described reductase pathways suggested to lead to the formation of 6,11-dihydro-LTB(4)(19, 27) actually resulted in the formation of 6,7-dihydro-LTB(4).


Figure 10: Proposed metabolism of 6-trans-LTB(4) and 6-trans-12-epi-LTB(4) in HepG2 cells involving 5-hydroxydehydrogenase/Delta^6-reductase and beta-oxidation.



The reduction of alpha,beta-unsaturated ketones, which are metabolites of various eicosanoids, is a major metabolic process. The metabolism of LTB(4) by 12-hydroxyeicosanoid dehydrogenase leads to the formation of an alpha,beta-unsaturated ketone, 12-oxo-LTB(4). Subsequent metabolism involving Delta-reductase results in the formation of 10,11-dihydro metabolites which have been identified in several human cell types(13, 28, 29, 30) . A major route of metabolism of many prostaglandins involves initial oxidation of the 15-hydroxy group with formation of 15-oxo prostanoids which are alpha,beta-unsaturated ketones. This process is catalyzed by a widely distributed enzyme, 15-hydroxyprostaglandin dehydrogenase ( (31) and references therein). Further metabolism by 15-keto-Delta-reductase results in formation of 13,14-dihydro-15-oxo metabolites(32, 33) .

The chain shortened metabolites identified during metabolism of both 6-trans-LTB(4) and 6-trans-12-epi-LTB(4) by HepG2 cells suggested that initial metabolism by the 5-hydroxyeicosanoid dehydrogenase/Delta^6-reductase pathway was followed by beta-oxidation as outlined in Fig. 10. It was previously demonstrated that metabolism of 6-trans-12-epi-LTB(4) by human polymorphonuclear leukocytes occurred with formation of dihydro-LTB(4) with loss of the proton label at the C-5 carbon atom suggesting an intermediate formation of 5-oxo-dihydro-LTB(4)(27) . Further beta-oxidation could occur from either dihydro intermediate; however, if beta-oxidation occurred primarily from the 6,7-dihydro-LTB(4) intermediate, identification of an 18-carbon dihydroxy intermediate might be expected. As no chain shortened 18-carbon metabolites were observed, beta-oxidation is suggested to occur primarily from 5-oxo-6,7-dihydro-LTB(4). The probable CoA ester-dependent initial steps in peroxisomal beta-oxidation of 5-oxo-6,7-dihydro-LTB(4) involving acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and beta-ketothiolase would lead to formation of an 18-carbon intermediate containing a keto group 3 carbon atoms removed from the CoA ester moiety. This could be rapidly metabolized by a second beta-ketothiolase step to produce the first chain shortened intermediate metabolite that is released to the medium, 8-hydroxy-4,6,10-hexadecatrienoic acid. Such a pathway would also suggest that the formation of 6,7-dihydro-LTB(4) from 5-oxo-6,7-dihydro-LTB(4) must be reversible. The reversibility of a similar step has been suggested in the metabolism of LTB(4) by human keratinocytes where 12-hydroxyeicosanoid dehydrogenase/Delta-reductase activity resulted in the formation of C-12 epimeric isomers of 10,11-dihydro-LTB(4)(13) . Metabolism of 6,7-dihydro-LTB(4) to 5-oxo-6,7-dihydro-LTB(4) would involve 5-hydroxyeicosanoid dehydrogenase activity. It cannot be determined from the results reported here if this is the same 5-hydroxyeicosanoid dehydrogenase activity that is responsible for metabolism of 6-trans-LTB(4) to 5-oxo-6-trans-LTB(4).

Conversion of 8-hydroxy-4(Z),6(E),10(Z)-hexadecatrienoic acid to 6-hydroxy-4,8-tetradecadienoic acid and 4-hydroxy-6-dodecenoic acid has been reported in metabolism of 12-HETE and was suggested to result from peroxisomal beta-oxidation(18) . The monohydroxy hexadecatrienoic acid identified in the present study may differ in the stereochemistry of the conjugated diene with a 4(E),6(E) stereochemistry unchanged from the trans,trans stereochemistry of the C-8 and C-10 double bonds of 6-trans-LTB(4) and 6-trans-12-epi-LTB(4), but normal pathways of beta-oxidation from this metabolite would also result in formation of the chain shortened metabolites. Subcellular localization of this metabolic activity has not been determined for HepG2 metabolism.

Little is known about the metabolic processes that are involved in degradation of 5,12-diHETEs or other hydroxy polyunsaturated fatty acids that may be formed by lipid peroxidation. An exception is the metabolism of LTB(4) for which there are specific enzymes responsible for metabolic inactivation. Detailed studies of the individual steps in beta-oxidation of polyunsaturated fatty acids is known to be complex as exemplified by the discovery of 2,4-dienoyl-CoA reductase (34) as well as specific enzymes that conjugate double bonds up to 5-carbon atoms removed from the CoA ester moiety(35) . The presence of a hydroxyl group in the fatty acid backbone further alters the normal steps of beta-oxidation. The conjugated diene alcohol structural motif is common to a wide variety of products that have undergone either lipoxygenase-catalyzed oxygenation, autoxidation, or free radical catalyzed mechanisms of lipid peroxidation to form conjugated diene alcohols(36) . The metabolic reaction observed for the 5,12-diHETEs studied here suggests a common reaction pathway for such molecules, that being oxidation of the conjugated diene alcohol to the conjugated dienone followed by reduction to a nonconjugated ketone. beta-Oxidation can then proceed through this functionalized carbon atom.


FOOTNOTES

*
This work was supported by a grant from the National Institutes of Health (HL25785). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pediatrics K929, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1273; Fax: 303-398-1694.

(^1)
The abbreviations used are: 5-HETE, 5-hydroxy-6,8,11,14-eicosatetraenoic acid; LTB(4), leukotriene B(4), 5(S),12(R)-6(Z),8(E),10(E),14(Z)-eicosatetraenoic acid; 6-trans-LTB(4), 5(S),12(R)-6(E),8(E),10(E),14(Z)eicosatetraenoic acid; 6-trans-12-epi-LTB(4), 5(S),12(S)-6(E),8(E),10(E),14(Z)-eicosatetraenoic acid; 5-oxo-ETE, 5-oxo-6,8,11,14-eicosatetraenoic acid; 12-HETE, 12-hydroxy-5,8,10,14-eicosatetraenoic acid; GC/MS, gas chromatography/mass spectrometry; PFB/TMS, pentafluorobenzyl ester/trimethylsilyl ether; HPLC, high performance liquid chromatography; RP, reverse phase.


ACKNOWLEDGEMENTS

We thank Dr. Christina Leslie and Joanna Garritano for their help in maintaining the HepG2 cell cultures.


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