The Metabolism of Leukotriene B₄ in Lewis Lung Carcinoma Porcine Kidney Cells

JOSEPH A. HANKIN and ROBERT C. MURPHY

Department of Pediatrics, Division of Basic Sciences, National Jewish Medical and Research Center, Denver, Colorado

	INTRODUCTION

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Leukotriene B₄ [5(S),12(R)-dihydroxy-6(Z),8(E),10(E),14(Z)- eicosatetraenoic acid, (LTB₄)] is a product of the 5-lipoxygenase pathway of arachidonic acid metabolism operational in several cell types, including the human polymorphonuclear leukocyte (1, 2). Because of its purported biological function to mediate neutrophil infiltration, there is considerable interest in an understanding of the biosynthesis of this eicosanoid during inflammatory reactions and, in particular, during inflammatory reactions in the lung. Several studies have shown that LTB₄ can be biosynthesized in the challenged human lung and measured in bronchoalveolar lavage fluid (3).

LTB₄, like other eicosanoids, is rapidly metabolized in vivo by multiple pathways into biologically inactive products. Certain cells, including the human polymorphonuclear leukocyte, are known to express a specific cytochrome P-450 (CYP4F3) that carries out -oxidation of LTB₄ to form 20-hydroxy-LTB₄ (6). -Oxidation is known to be a predominant pathway of LTB₄ metabolism in the hepatocyte after the initial -oxidation of the methyl terminus and alcohol dehydrogenase-mediated formation of an -carboxyl moiety (7, 8). Several other enzymatic pathways have been discovered that transform LTB₄ into inactive products, including the 12-hydroxyeicosanoid dehydrogenase (9) and 10,11-reductase pathways (10), which ultimately convert the conjugated triene into a conjugated diene moiety. Most recently, the formation of a glucuronide of LTB₄ has been described (13).

Emergence of electrospray tandem mass spectrometry has facilitated the studies of eicosanoid metabolism including the metabolism of LTB₄ (14, 15). This sensitive technique permits direct analysis of metabolites as they are separated and eluted from a high-performance liquid chromatography (HPLC) column. With the implementation of microbore HPLC columns, concentration of metabolites can be maintained at high levels, ensuring maximal sensitivity of this mass spectrometric technique. While electrospray ionization generates abundant negative ions such as the carboxylate anion, tandem mass spectrometry provides structural information necessary to characterize the metabolites.

Interest in the production of LTB₄ within pulmonary tissue has increased, but the means by which one can assess in vivo production of LTB₄ in the lung has been problematic because the lung is a difficult organ to sample. One strategy that has been used for the assessment of prostaglandin production in vivo is the measurement of specific prostaglandin metabolites excreted into the urine (16). Rates of endogenous LTC₄ production have been estimated by measuring urinary LTE₄ excretion (17). Therefore, the identification of stable metabolites of LTB₄ as markers of LTB₄ production and a more complete understanding of the ultimate metabolic fate of LTB₄ has become of interest. The metabolism of eicosanoids within the intact organism could involve multiple tissues, for example, an eicosanoid could be metabolized directly in the tissue responsible for its biosynthesis and subsequently enter the circulation and be transported to a secondary site such as the liver or kidney. Some studies, which have suggested that LTB₄ rapidly exits the lung once it enters the pulmonary circulation (18), bring into focus other tissue sites of LTB₄ metabolism before excretion into urine. The liver can rapidly metabolize LTB₄ in part because of an active transport system that extracts LTB₄ from the circulating blood (19). The kidney is another organ of particular interest when one is concerned about excretion of metabolites, and several studies have reported on the potential metabolism of LTB₄ by renal homogenate preparations (9, 20, 21) and kidney-derived cells such as mesangial cells (22). We report here the use of electrospray tandem mass spectrometric techniques to study the metabolism of LTB₄ in cultured porcine renal tubule epithelial cells that produce numerous metabolites, several of which have not been previously structurally characterized.

	METHODS

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Materials

Leukotriene B₄ (LTB₄) and deuterium-labeled LTB₄ ([6,7,14,15-²H]LTB₄, 98 atom% ²H₄) were purchased from Cayman Chemical (Ann Arbor, MI). Tritium-labeled LTB₄ ([5,6,8,9,11,12,14,15-³H(N)]LTB₄, 171 Ci/ mmol) was purchased from DuPont-New England Nuclear (Boston, MA). Hanks' balanced salt solution (HBSS) was purchased from GIBCO-BRL Life Technologies (Gaithersburg, MD). All solvents used for HPLC and sample dilution were HPLC grade and purchased from Fisher Scientific (Fair Lawn, NJ). Water used in solutions was both deionized and distilled before use.

Cell Culture

Lewis lung carcinoma pig kidney cells (LLCPK-1), a cell line of renal tubule epithelial cells, were obtained from the American Type Culture Collection (Rockville, MD) and grown in RPMI medium supplemented with 6% calf bovine serum, 20 mM HEPES, streptomycin (100 µg/ml), and penicillin (100 µg/ml). Cells were passed twice weekly using 0.05% trypsin and 0.01% EDTA and were plated onto 75-cm² culture plates and allowed to proliferate to confluency (72-96 h). The cells were maintained at 37° C in a humidified atmosphere of 95% air and 5% CO₂.

Metabolism of LTB₄

The eicosanoids for these experiments were supplied by the manufacturers as concentrated solutions in ethanol, which was removed by a stream of dry nitrogen before the addition of 10 ml of HBSS to a final concentration of 5 µM LTB₄, 5 µM d₄-LTB₄, and 1 µCi of [³H]LTB₄ as the incubating medium. For each experiment, the growth medium in a 75-cm² culture plate confluent with LLCPK-1 cells was decanted and replaced with the solutions of LTB₄ substrates. The culture plate was placed in a heated (37° C) shaking water bath for 9-12 h. Aliquots (100 µl) of the reaction mixture were removed from the incubation, diluted to 500 µl with HPLC solvents at initial gradient composition, and analyzed by HPLC with on-line UV and radioactivity counting at 1, 3, 7, 9, and 12 h (see RP-HPLC section) to follow the disappearance of LTB₄ and the appearance of metabolites. The incubation was terminated by the addition of 30 ml of ethanol when most LTB₄ had disappeared. After 1 h at 4° C, the reaction contents with ethanol were centrifuged at 224 × g and the supernatant separated from the residual pellet and retained for analysis. The ethanolic extract was dried by rotary evaporation, reconstituted in 100% water, and purified by solid-phase extraction as previously described (14).

RP-HPLC with On-line UV, Radioactivity Detection, and Mass Spectrometry

Reversed-phase HPLC (RP-HPLC) was used to separate the metabolites by gradient elution, with mobile phase A containing 8.3 mM acetic acid buffered at pH 5.0 with NH₄OH and mobile phase B composed of CH₃CN-methanol (65:35, vol/vol), and performed with a 2.0 × 150 mm Ultremex C₁₈ RP-HPLC column (Phenomenex, Rancho Palos Verdes, CA). Metabolites were eluted from the column at 200 µl/ min with a linear gradient from 30% B to 60% B in 50 min to 100% B at 60 min. The eluent was split postcolumn for on-line analysis, with a flow of 40 µl/min going to the electrospray ion source of the mass spectrometer (see the next section). The majority of the flow was directed through a scanning UV monitor (Linear Instruments, Reno, NV) and then into a radioactivity monitor (Flow One/Beta Radiomatic, Tampa, FL). This on-line analysis permitted a precise alignment of UV, mass spectral, and radioactivity content of chromatographically separated metabolites. Fractions were also collected with the same column and HPLC gradient for more in depth mass spectral analysis.

Electrospray Mass Spectrometry (Negative Ions)

Collected fractions as well as the split eluent from the on-line analysis were analyzed by electrospray ionization mass spectrometry, using a Sciex API-III⁺ triple quadrupole mass spectrometer (PE-Sciex, Thornhill, ON, Canada). The mass range m/z 160 to 700 was scanned at a rate of 0.33 scans/s. Spray voltage was 2900 V, the orifice voltage was maintained at 50 V, and a collisional offset potential of 20 eV was maintained for collision-induced dissociation (CID) experiments along with a collision gas thickness (argon) of 240 × 10¹³ molecules/ cm². For flow injection of isolated fractions, an eluent solution of 50:50 A/B was used at a flow rate of 10 µl/min through a 0.5 m × 50 µm fused silica capillary tube.

	RESULTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

When Lewis lung carcinoma porcine renal tubule epithelial cells were incubated with LTB₄, there was a time-dependent increase in the formation of metabolites readily observed after a 3-h incubation. Three of the most lipophilic metabolites (A, B, and C) were readily formed even in this short incubation period and increased through 7 h of incubation. At this time, nearly all of the initial substrate was gone. Between 9 and 12 h a decrease in metabolites A, B, and C was observed with a subsequent increase in metabolites that eluted earlier in the HPLC separation. Detailed analysis of the metabolites formed from LTB₄ after 9 h of incubation revealed more than 12 separable metabolic products. Incubations were carried out with two different isotopic variants of LTB₄, namely tritium-labeled LTB₄ as well as the stable isotopic variant of LTB₄ containing deuterium atoms at carbons 6, 7, 14, and 15. The use of these different isotopic variants permitted a rapid evaluation of the separation of metabolites using the radiolabeled tracer and radioactive monitoring of the HPLC trace, whereas the deuterium-labeled variant permitted an unambiguous identification of metabolites eluting from the HPLC column when analyzed by electrospray mass spectrometry. Metabolites appeared as mass doublets in their mass spectra since an equal molar quantity of both deuterium-labeled and non-deuterium-labeled LTB₄ was added at the beginning of the experiment. On the basis of the extraction of radioactivity from the cell incubations, it was observed that greater than 95% of the total radioactivity appeared in the supernatant that was decanted from the cell incubation system.

Reversed-phase HPLC separation of the LTB₄ metabolites is shown in Figure 1 as the UV absorption monitoring of the HPLC effluent (270 and 235 nm) and sequential radioactive monitoring to detect the elution of radioactive metabolites. The majority of the metabolites displayed an ultraviolet absorption spectrum that maximized at 235 nm (Figure 1, middle panel ), indicative of a conjugated diene structure. An example of a UV spectrum of a typical metabolite is shown in the inset in Figure 1 (middle panel ). There were only a few metabolites that displayed the conjugated triene chromophore with a maximum absorbance at 270 nm typical of the starting material, which contains a conjugated triene. Table 1 summarizes the relative abundance of the metabolites formed during this 9-h incubation period as well as the reversed-phase HPLC retention time, the major carboxylate anion obtained by electrospray mass spectrometry (see below), and the results of the structural characterization of these metabolites.

View larger version (30K): [in this window] [in a new window]   Figure 1.   Distribution of metabolites from a 9-h incubation of LTB4 in Lewis lung carcinoma porcine tubule epithelial cells (LLCPK-1). Metabolites were separated by RP-HPLC with gradient elution, on-line radioactive counting, and UV monitoring at the diagnostic wavelengths for conjugated dienes (235 nm) and conjugated trienes (270 nm). Structures of major metabolites (alphabetically labeled) are described in text.

Metabolite A

The most abundant metabolite formed during the 9-h incubation was found to be metabolite A. The ultraviolet absorption spectra revealed the presence of a conjugated diene and its electrospray mass spectrometry indicated a molecular anion at m/z 337, suggesting reduction of one double bond in the conjugated triene chromophore of LTB₄. Tandem mass spectrometric analysis showed a distribution of product ions identical to that previously described for 10,11-dihydro-LTB₄ (23).

Metabolite B

The next most lipophilic metabolite of significant abundance was designated metabolite B and it as well had a conjugated diene UV absorption chromophore. This metabolite formed a carboxylate anion at m/z 339, suggesting that only two double bonds (as a conjugated diene) remained in this metabolite. Collisional activation of the ion at m/z 339 (Figure 2A) as well as the d₄-metabolite B (m/z 341; Figure 2B) yielded an abundant product ion at m/z 115 that did not contain any of the deuterium atoms. The similarity of this mass spectrum to that of metabolite A suggested that the double bond that was further reduced in this metabolite was the 14,15 double bond. The mass spectrometric data were consistent with the identity of this metabolite as 10,11,14,15-tetrahydro-LTB₄, which had been previously identified as a product of LTB₄ metabolism in porcine leukocyte and a crude cytosolic preparation from porcine kidney (9).

View larger version (20K): [in this window] [in a new window]   Figure 2.   Electrospray ionization, negative ion tandem mass spectra of (A) metabolite B after collisional activation of the precursor ion at m/z 339 and (B) the corresponding d4 isotope-labeled metabolite at m/z 343.

Metabolite C

The second most abundant metabolite was somewhat more lipophilic than the tetrahydro-LTB₄ metabolite B eluting 1.2 min after B. The ultraviolet absorption spectra suggested the presence of a conjugated diene, but the carboxylate anion doublet observed for this metabolite was at m/z 293/297. The collision-induced decomposition mass spectrum of metabolite C is shown in Figure 3, which had a prominent product ion at m/z 137 and abundant ions at m/z 153 and 111. The increase lipophilicity of metabolite C relative to metabolite B and reduced molecular weight were suggestive of a hydroxyeicosatetraenoic (HETE)-type structure. Indeed, the product ions were consistent with a previously identified metabolite, 10- hydroxy-4,6,12-octadecatrienoic acid (10-HOTrE) (23).

View larger version (16K): [in this window] [in a new window]   Figure 3.   Electrospray ionization, negative ion tandem mass spectra of (A) metabolite C after collisional activation of the precursor ion at m/z 293 and (B) the corresponding d4 isotope-labeled metabolite at m/z 297.

Metabolite D

The earliest major metabolite, eluting at 14.6 min, had a UV mass spectrum of a conjugated diene. The electrospray mass spectrum of this metabolite revealed an isotope doublet at m/z 353 and 357. The presence of one additional oxygen atom, consistent with the decreased retention time on reversed-phase HPLC and the collisional activation of this metabolite in the tandem mass spectrometer (Figure 4A), revealed the formation of several product ions derived from the carboxyl terminus, similar in abundance to that observed for metabolite A (m/z 179, 163, 225, and 115). A diagnostically different ion resulting from cleavage between carbons at positions 10 and 11 (observed at m/z 139 in metabolite A) was shifted to m/z 155, suggesting that an addition of oxygen took place again on the methyl terminus of the original LTB₄ structure. The physical chemical data and deuterium isotope labeling pattern as well as the HPLC retention time were consistent with this metabolite being 10,11-dihydro-20-hydroxy-LTB₄, which has not been previously described.

View larger version (18K): [in this window] [in a new window]   Figure 4.   Electrospray ionization, negative ion tandem mass spectra of (A) metabolite D after collisional activation of the precursor ion at m/z 353 and (B) the corresponding d4 isotope-labeled metabolite at m/z 357.

Metabolite E

A group of metabolites that eluted closely together on RP-HPLC were designated E₁ to E₄ since they showed patterns of relatedness to each other (Figure 5). E₁ and E₃ both had significant precursor ions at m/z 353 and both had a UV maximum at 235 nm with an HPLC retention time separation of 1.4 min. Metabolites E₂ and E₄ had precursor ion masses of m/z 355, and both had UV absorbances at _max 235 nm and shared a retention time difference of 1.4 min. Tandem mass spectrometry of E₁ m/z 353 (Figure 6) showed similarities to the previously published mass spectrum of 10,11,14,15-tetrahydro-12-oxo-LTB₄ (m/z 337) with carboxyl-terminus ions having the same mass (m/z 115 and 129) and methyl-terminus ions having 16 daltons greater mass (m/z 221  237, m/z 181  197, m/z 155  171). This suggested a structure for metabolite E₁ similar to that of 10,11,14,15-tetrahydro-12-oxo-LTB₄ with addition of an oxygen atom at a position on the carbon chain that did not readily alter collision-induced decomposition. The structure of metabolite E₁ has been postulated to be a novel metabolite, 20-hydroxy-10,11,14,15-tetrahydro-12-oxo-LTB₄.

View larger version (22K): [in this window] [in a new window]   Figure 5.   Reversed-phase HPLC separation of metabolites E1, E3, and E4 shown with (A) UV absorption chromatogram at 235 nm and alignment of (B) mass chromatograms of ions at m/z 353 (d4 isotope-labeled metabolite at m/z 357) and m/z 355 (d4 isotope-labeled metabolite at m/z 359).

View larger version (20K): [in this window] [in a new window]   Figure 6.   Electrospray ionization, negative ion tandem mass spectra of (A) metabolite E1 after collisional activation of the precursor ion at m/z 353 and (B) the corresponding d4 isotope-labeled metabolite at m/z 357.

Metabolite E₂ had a precursor ion doublet at m/z 355/359 and showed product ions similar to the previously identified 10,11,14,15-tetrahydro-LTB₄ (m/z 339) (Figure 7). Ions retaining the carboxyl terminus were identical (m/z 115, 179, 197) and the methyl terminus-retaining product ions were increased by 16 mass units (m/z 339  355, 141  157). This information was consistent with metabolite E₂ as 20-hydroxy-10,11,14,15-tetrahydro-LTB₄.

View larger version (21K): [in this window] [in a new window]   Figure 7.   Electrospray ionization, negative ion tandem mass spectra of (A) metabolite E2 after collisional activation of the precursor ion at m/z 355 and (B) the corresponding d4 isotope-labeled metabolite at m/z 359.

Metabolites E₃ and E₄ both had tandem mass spectra similar to those of metabolites E₁ and E₂, respectively. The consistent retention time difference between E₁ and E₃ and between E₂ and E₄, along with identical product ion spectra, suggested a similar chemical process responsible for creating distinct species chromatographically, yet retaining identity by mass spectroscopy and UV absorbance. One possibility is that these molecules were diasteromeric pairs, but insufficient material was available for further characterization.

Metabolite F

The metabolite eluting from the HPLC at approximately 38 min also had a conjugated diene ultraviolet spectrum. The on-line electrospray mass spectra revealed an abundant mass doublet at m/z 381 and 385, which was 46 mass units above that of the parent LTB₄. The collision-induced decomposition mass spectrum of m/z 381 (385) yielded product ions similar to that previously observed for the chain-elongated metabolite of LTB₄, which retained a hydroxyl group at the original carbon-1 of LTB₄. Specific ions were consistent with this observation, in particular the ion at m/z 139, suggesting that the methyl terminus up to carbon-12 had not been modified. The most abundant product ion from the 10,11-dihydro-LTB₄ metabolites described above (m/z 115) was shifted to m/z 161, consistent with the addition of the 46 mass units on the carboxyl terminus of this metabolite. On the basis of the HPLC retention time and the mass spectrometric data, the identification of metabolite F was consistent with the previously identified 3,7,14-trihydroxy-dodecatrienoic acid (12).

Metabolite G

One of the early eluting metabolites (22 min), which had a UV spectrum consistent with a conjugated diene, was found by tandem electrospray mass spectrometry to have a molecular anion at m/z 513 that decomposed after collisional activation to yield an abundant ion at m/z 143. This molecular weight and the unique collision-induced decomposition mass spectrum were consistent with 5,12-dihydroxy-6-cysteinyl-glycyl-7,9,14-eicosatrienoic acid (d-LTB₃). Such a metabolite had been previously reported to be formed in human keratinocytes by the addition of glutathione to 12-oxo-LTB₄ and the subsequent cleavage of the -glutamic acid residue from the glutathione moiety (12, 23).

Other Metabolites

Several minor metabolites were found with a molecular anion at m/z 291 as well as a glucuronide of these conjugated metabolites that yielded a characteristic loss of 176 daltons after collisional activation (m/z 467, 469 with d₄ doublets at m/z 471, 473). These glucuronide metabolites eluted in the range of 24- 26 min. The nonglucuronidated metabolites likely eluted from the HPLC after substantially longer retention times, approximately 58 min. Insufficient material was available for further structural characterization of these unique metabolites; however, they were conjugated dienes as determined by their UV absorption maximum at 235 nm.

	DISCUSSION

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Considerable interest has been generated concerning LTB₄ biosynthesis in vivo and in particular in the lung during an inflammatory response. This interest has focused research efforts toward understanding the metabolic fate of LTB₄ and identifying ultimate excretion products in the urine that would reflect in vivo biosynthesis. One clinical study found urinary excretion of intact LTB₄, but this was a result of direct inflammation of the glomerulus (24). Attempts to detect and identify LTB₄ metabolites in urine have been unsuccessful to date (24), suggesting extensive metabolism of this eicosanoid mediator in vivo.

The metabolism of LTB₄ in these cultured renal tubule epithelial cells revealed a robust 12-hydroxyeicosanoid dehydrogenase and 10,11-reductase pathway of LTB₄ metabolism. This pathway of LTB₄ metabolism has now been observed in many cells including keratinocytes (13), macrophages (25), lung parenchyma cells (26), and a previous identification in kidney mesangial cells (22). Of particular interest was the large number of tetrahydro metabolites formed by the further reduction of the 14,15 double bond. The metabolic pathway leading to the formation of these metabolites of LTB₄ in the kidney cells is shown in Figure 8.

View larger version (14K): [in this window] [in a new window]   Figure 8.   Pathways of the metabolism of LTB4 in LLCPK-1 cells. Identification of the HPLC peaks in Figure 1 are indicated under each structure.

It is interesting to note that LTB₄ metabolism in these kidney-derived cells suggested that the primary pathway of LTB₄ metabolism was that of reduction rather than oxidation. There was some evidence for metabolites derived from cytochrome P-450 -oxidation as a result of further processing of reduced metabolites of LTB₄. Yet, -oxidation was not the major pathway of LTB₄ metabolism in these cells either as the intact molecule or as products of the 12-hydroxyeicosanoid dehydrogenase pathway. Operation of the 12-hydroxyeicosanoid dehydrogenase/10,11-reductase pathway of metabolism is in stark contrast to the oxidative pathways responsible for the metabolites observed in hepatocytes where P-450-dependent -oxidation and alcohol dehydrogenase lead to an abundant production of oxidized metabolites including products formed by further -oxidation from the -terminus.

In summary, Lewis lung carcinoma porcine kidney cells efficiently metabolized LTB₄ by the 12-hydroxyeicosanoid dehydrogenase pathway. Some of the most abundant metabolites were the result of further reduction of the 14,15 double bond, leading to a series of tetrahydro metabolites of LTB₄. The abundance of these reduced metabolites of LTB₄ formed by these kidney cells as well as the fact that these metabolites were formed by numerous other cells suggest reduced metabolites might be products excreted into urine that might reflect in vivo production of LTB₄.

	Footnotes

Correspondence and requests for reprints should be addressed to Robert C. Murphy, Ph.D., Department of Pediatrics, Division of Basic Sciences, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: murphyr{at}njc.org.

Acknowledgments: The authors acknowledge the generous assistance of Robert J. Anderson and Carla J. Ray (Denver VA Hospital) for providing the LLCPK-1 cells.

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