THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 268, No. 34, Issue of December 5, PP . 25439-25448, 1993 Printed in U. S. A.

Metabolism of Leukotriene B4 by Cultured Human Keratinocytes FORMATION OF GLUTATHIONE CONJUGATES AND DIHYDRO METABOLITES*

(Received for publication, May 17, 1993, and in revised form, August 2, 1993)

Pat Wheelan$, Joseph A. ZirrolliS, Joseph G . MorelliQ, and Robert C. MurphySIl From the $Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine and the §Department of Dermatology, University of Colorado School of Medicine, Denver, Colorado 80206

Six previously unidentified leukotriene (LT) B4 me- tabolites formed during incubation of LTB4 with hu- man keratinocytes in primary culture indicate the im- portance of the 12-hydroxyeicosanoid dehydrogenase pathway in LTB4 metabolism. The ultraviolet absorp- tion spectra obtained for all keratinocyte metabolites revealed the presence of a conjugated diene structural moiety rather than the conjugated triene structure of LTB4. Metabolites were characterized using fast atom bombardment-mass spectrometry, gas chromatogra- phy-mass spectrometry of the pentafluorobenzyl ester, trimethylsilyl ether derivatives and specific degrada- tion reactions. The previously identified 10,l l-dihy- dro-LTB4 and 10,ll-dihydro-12-epi-LTB4 were ob- served as well as 20-OH-10,ll-dihydro-LTB4, consist- ent with the reductase pathway of LTB4 metabolism. This pathway involves initial formation of 12-oxo- LTB4 catalyzed by 12-hydroxyeicosanoid dehydrogen- ase followed by reduction by A’’-reductase. The most lipophilic metabolite of LTB4 was identified as. 10- hydroxy-4,6,12-octadecatrienoic acid which could re- sult from &oxidation reactions of LTB4 following re- duction of the 10,ll-double bond. One of the most abundant metabolites was characterized as 3,7,14-tri- hydroxy-8,10,16-docosatrienoic acid which could re- sult from fatty acid elongation following reduction of the l0,ll-double bond. Additional abundant LTB4 me- tabolites were identified that result from glutathione conjugation of 12-oxo-LTB4. These were characterized using fast atom bombardment-mass spectrometry and by chemical degradation using hypochlorous acid as well as transpeptidases. These metabolites were iden- tified as 5,12-dihydroxy-6-glutathionyl-7,9,14-eicos- atrienoic acid (c-LTB3), 5,12-dihydroxy-6-cysteinyl- glycyl-7,9,14-eicosatrienoic acid (d-LTBs), and 5,12- dihydroxy-6-cysteiny1-7,9,14-eicosatrienoic acid (e- LTBs). We propose that these metabolites result from a 1,s Michael-type addition of glutathione to the 12- oxo-LTB4 intermediate.

Neutrophil infiltration and elevated production of leuko-

* This work was supported in part by National Institutes of Health Grant HL25785. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll TO whom correspondence should be addressed: Dept. of Pediat- rics K929, National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson St., Denver, CO 80206.

triene B4 (LTB,)’ characterize skin inflammatory disease states (1, 2). Stimulated neutrophils produce LTB, (3) and a recent study has suggested a synergistic interaction between keratinocytes and neutrophils results in elevated production of LTB, not seen by either keratinocytes or neutrophils alone (4). LTB,, a 5-lipoxygenase product of arachidonic acid me- tabolism, is a potent chemotactic agent ( 5 ) , and its presence may account for continued neutrophil infiltration. In addition, the presence of LTB, may result in increased keratinocyte proliferation (6,7) and postinflammatory melanocyte pigmen- tation (8) which are characteristic of psoriasis. Keratinocyte metabolism of LTB, may be then a critical factor related to the pathogenosis of skin inflammatory diseases.

Metabolism of LTB4 has been shown to occur by two pathways. Human PMN metabolize LTB, by cytochrome P- 450-dependent w-oxidation to produce 20-OH LTB, and 20- COOH LTB, (9). These products have also been identified as LTB, metabolites in isolated rat hepatocytes as well as prod- ucts resulting from further &oxidation of these metabolites (10, 11). Metabolism of LTBl has also been shown to occur by reduction of the l0,ll-double bond in the conjugated triene system of LTB, resulting in the formation of l0,ll-dihydro metabolites. The 10,11-reductase pathway for LTB, initially involves oxidation of LTB, to 12-oxo-LTB, catalyzed by 12- hydroxyeicosanoid dehydrogenase followed by a two-step re- duction. This pathway has been identified in porcine and rat PMN (12,13) and several murine cells (14). Human cell types that have been shown to metabolize LTB, by the 10,ll- reductase pathway include human lung macrophages (15), monocytes (16), and kidney mesangial cells (17), as well as whole lung (18). In terms of biological activity, both pathways produce metabolites that are generally much less potent than the parent LTB, in the effect on PMN function (19-22).

In the present work, human keratinocytes were shown to metabolize LTB4 by the reductase pathway producing 10,11- dihydro-LTB, metabolites. Additional metabolites in this pathway were identified that result from both chain elonga- tion and chain shortening of dihydro-LTB, and from w- oxidation of 10,ll-dihydro-LTB,. In addition, a novel meta- bolic pathway was discovered that results in glutathione con- jugation of LTB,. This LTB, metabolite is analogous to LTC, and is further metabolized by y-glutamyltranspeptidase and dipeptidase to LTD, and LTE, analogs.

The abbreviations used are: LTB,, leukotriene B,; ECI, electron capture ionization (negative ion mass spectra); El, electron ionization (positive ion mass spectra); CF-FAB, continuous flow fast-atom bom- bardment; CID, collision induced decomposition; PFB/TMS, penta- fluorobenzyl ester/trimethylsilyl ether; HPLC, high performance liq- uid chromatography; RP, reverse-phase; NP, normal phase; GC/MS, gas chromatography/mass spectometry; PMN, polymorphonuclear leukocytes. Nomenclature and abbreviations of leukotrienes and re- lated fatty acids follow proposed guidelines (23).

25439

25440 Keratinocyte Leukotriene Bg Metabolism

EXPERIMENTAL PROCEDURES

Materials-Leukotriene B, (LTB,) and [6,7,14,15-d4]LTB, were purchased from Biomol Research Laboratories (Plymouth, PA). [5,6,8,9,11,12,14,15-3HslLTB4 (195 Ci/mmol) was purchased from DuPont-New England Nuclear. LTC,, LTD,, LTE,, and 20-OH LTB4 were purchased from Cayman Chemical (Ann Arbor, MI). Synthetic lO,lI-dihydro-LTB, and 10,ll-dihydro-12-epi-LTB, were generous gifts of Dr. J. R. Falck (Southwestern Medical Center, University of Texas). All solvents were HPLC grade obtained from Fisher Scientific (Fair Lawn, NJ).

Human Keratinocyte Isolation and Incubation-Human keratino- cytes were isolated from neonatal foreskins as previously described (24). Keratinocytes were grown in 75-cmZ tissue culture flasks at 37 'C, 5% CO2 in keratinocyte serum-free medium (Life Technologies, Inc.) containing bovine pituitary extract (25 mg/500 ml). Cells were grown until fully confluent a t which time cultures contained 12-15 X lo6 cells/flask. LTB, (60 pg) and [3Hs]LTB4 (1.6 X IO6 disintegra- tions/min) were lyophilized to dryness and reconstituted in 50 ml of culture medium (3.58 pM LTB,). Culture medium was removed from four flasks of confluent cells and replaced with 12.5 ml of LTB4- containing medium/flask. Following incubation at 37 "C for 24 h, the supernatants were removed and stored at -70 "C. For some experi- ments, the added LTB, was a mixture of do-LTB4 and [6,7,14,15-d,] LTB,. The d0/d4 ratio was determined after constituting the LTB4 for incubation by analysis of the PFB/TMS derivatives by ECI GC/

cubated for 24 h in 12.5 ml of medium containing l0,ll-dihydro- MS. In other experiments, confluent keratinocyte cultures were in-

LTB, (20-25 pg) or lO,ll-dihydro-12-epi-LTB4 (20-25 pg). As a control experiment, confluent keratinocytes were boiled for 10 min then cooled to 37 "C. LTB, (3.58 p ~ ) containing [3Hs]LTB4 was added and the cells incubated at 37 "C for 24 h. Cell supernatant was removed and stored at -70 'C.

Metabolite Purification and HPLC-Protein was precipitated from thawed supernatants by the addition of four volumes of ethanol and storage at -20 "C for 3 h. Following centrifugation, the supernatants were decanted and combined from four flasks of keratinocyte cultures for LTB, incubations or combined from two flasks each for dihydro LTB, incubations. The supernatants were taken to near dryness by rotary evaporation and the residue taken up in 1.5-2.0 ml of the initial RP-HPLC solvent. RP-HPLC analysis was performed using an Ultremex column (4.6 X 250 mm, 5-atomic mass units C-18 Phenomenex, Rancho Palos Verdes, CA). The mobile phase consisted of methanol/water, 0.05% acetic acid (pH adjusted to 5.75 with ammonium hydroxide) at an initial composition of 30% methanol and at a flow rate of 1 ml/min. A linear gradient to 70% methanol from 0-35 min was used followed by a second linear gradient to 100% methanol over 10 min. Radioactive metabolites from 200-400 pl of sample were detected using an on-line radioactive detector (Flow One/Beta, Radiomatic, Tampa, FL). The remainder of the sample was chromatographed and UV absorbance monitored (HP-1040A photodiode array detector, Hewlett-Packard, Palo Alto, CA). Frac- tions (0.5 ml) were collected, and 50-pl aliquots of each fraction were counted on a scintillation counter. Normal-phase HPLC separation of lO,ll-dihydro-LTB, and l0,ll-dihydro-12-epi-LTB, was per- formed using an Ultremex column (4.6 X 250 mm, 5 atomic mass units of silica; Phenomenex, Rancho Palos Verdes, CA) and an isocratic solvent system at 3 ml/min consisting of hexane/isopropa- nol/acetic acid (96:4:0.1).

Continuous Flow-Fast Atom BombardmentlMass Spectrometry (CF-FAB/MSJ"egative ion FAB spectra were obtained on a Fin- nigan TSQ 70 (San Jose, CA) triple quadrupole mass spectrometer using continuous flow conditions. HPLC fractions containing radio- activity were concentrated in uacuo to 60-100 ng/pl (calculated from specific radioactivity). Aliquots (1 p1) were introduced into the mass spectrometer through a 1 meter X 50-atomic mass units fused silica transfer capillary with methanol/water/glycerol (40:lOl) as the sol- vent at a flow rate of 5 pl/min. The FAB gun was operated at 1 mA with xenon accelerated to 6 kV. Spectra were acquired from 200-700 atomic mass units at 1 scan/s. For collision-induced dissociation and tandem mass spectrometry analyses, argon was used as the collision gas at a pressure which resulted in a 7040% reduction in the abundance of the precursor ion. The collision offset energy (EM) was 30 eV. Product ion spectra were acquired from 50 atomic mass units to the precursor ion at 1 scan/2 s. Gas Chromatography/Mass Spectrometry-Lyophilized samples

were derivatized for GC/MS analysis by the addition of a 10% solution (v/v) of N,N-diisopropylethylamine (Aldrich) in acetonitrile (50 pl)

followed by the addition of a 10% solution (v/v) of pentafluorobenzyl bromide (Aldrich) in acetonitrile (50 pl). The samples were kept at room temperature for 30 min and evaporated under NP. The dried samples were further derivatized with the addition of acetonitrile (50 pl) and bis(trimethylsily1)trifluoroacetamide (Supelco, Inc.) (50 pl) and kept at 60 "C for 5 min followed by evaporation under Nz. ECI was employed to gain molecular weight information as well as infor- mation on the number of hydroxy substituents of each metabolite from production of the abundant carboxylate anion and ions derived from losses of trimethylsilyl alcohol (25, 26). For this sensitive mode of operation, the derivatized samples were redissolved in acetonitrile at 2-10 ng/pl. Electron ionization (EI) GC/MS analysis was employed to provide detailed structural information regarding hydroxy substit- uent position from abundant fragmentations adjacent to trimethyl- silyl ether positions (27). For E1 GC/MS analysis, samples were redissolved in acetonitrile at 25-50 ng/pl. A Finnigan SSQ 70 (San Jose, CA) was employed in both modes of sample ionization; electron capture (ECI) mass spectra (negative ions) were obtained in the chemical ionization mode with methane as the moderating gas and E1 mass spectra (positive ions) were obtained using an electron energy of 70 eV. The GC capillary column was a 10 m X 0.25-mm DB-1 (J&W Folsom, CA) column with 0.25-atomic mass units film thick- ness. For less volatile metabolite derivatives, a 5 m X 0.25-mm DB-1 column was used. The injector temperature was maintained at 275 "C and the transfer line at 300 "C. PFB/TMS derivatized samples (1 p1) were injected into the gas chromatograph using an initial column temperature of 100 "C followed by a 15 "C/min ramp to 310 "C. Equivalent carbon values (EC values) were determined by comparison to standard PFB derivatives of straight chain fatty acids.

Catalytic Reduction-HPLC fractions were stripped of solvent then dissolved in methanol (400 p l ) containing 5% Rh/A1203 catalyst (0.2- 0.4 mg). Hydrogen gas was bubbled through the mixture for 2 min at room temperature. The methanol solution was removed from the catalyst after centrifugation and the catalyst washed with additional methanol. The combined methanol extracts were dried under nitrogen and the sample derivatized for GC/MS analysis.

Oxidative Ozonolysis-Samples (400-500 ng) were evaporated in 1.2 X- 100-mm capillary tubes. The dried samples were exposed to a stream of ozone in oxygen for 1 min followed by exposure to vapor from a heated solution (60 "C) of formic acid (200 pl) and hydrogen peroxide (100 pl) for 30 min. Samples were analyzed by ECI GC/MS following preparation of the PFB/TMS derivatives.

Peptidase Reactions-Metabolites were dried under NZ and redis- solved in 1 ml of Hanks' balanced salt solution (Life Technologies, Inc.). y-Glutamyltranspeptidase (Sigma, 0.25 units in 100 pl of Hanks' balanced salt solution) and leucine aminopeptidase (Sigma, 1.25 units in 100 p1 of Hanks' balanced salt solution) were added and the solutions kept at room temperature overnight. Methanol (400 pl) was added, and the samples were filtered and analyzed by RP-HPLC using the solvent system described above.

Hypochlorow Acid Reactions-Hypochlorous acid was freshly dis- tilled from 5.25% sodium hypochlorite (Clorox") after acidification to pH 7.5 with dilute sulfuric acid (28). Molarity of the hypochlorous acid solution was determined by UV absorption at 235 nm using an extinction coefficient of 100 M" cm" (29). Metabolites (0.5-0.8 pg) were dried, and diluted hypochlorous acid solution (150 pl, 1.4 X lo-' M) was added to give a 2-3-fold molar excess of acid. Reactions were kept at 60 "C for 2 min and immediately analyzed by RP-HPLC using the RP mobile phase at an initial composition of 50% methanol and at a flow rate of 1 ml/min followed by a linear gradient to 100% methanol in 30 min. Products were collected, reduced, and derivatized for ECI GC/MS and El GC/MS analysis.

RESULTS

After a 24-h incubation of LTBl and [3Hs]LTB4 with human keratinocytes, 73 L- 15% (n = 4) of the added radioactivity was recovered in the extracellular supernatant. LTB4 was not altered by 24 h of incubation in medium alone (30). In addition, LTB, incubation with boiled cells did not result in the formation of any of the metabolites found from live cells. Incubation with boiled cells did produce a 30% conversion of LTB4 to 6-trans-LTB4, most likely due to metal catalyzed isomerization of the 6-cis double bond (data not shown). Following protein precipitation, centrifugation, and sample lyophilization, 52 f 9% ( n = 4) of nonvolatile radioactivity

Keratinocyte Leukotriene B, Metabolism 25441

LTB4 h

0.35 $1 c 4

LT84

h B. HK2 km=230nm

4

210 230 250 270 290 $ 1 . * . I-_,,-.

210 230 250 270 290

C. HK9 km=232nm

a

210 230 250 270 290 Wavelength (nm)

FIG. 2. Ultraviolet absorbance spectra of selected metabo- lites. Spectra were recorded during elution of the metabolite from the HPLC column. A , metabolite HK1; B, metabolite HK2; C, me- tabolite HK9.

Time (rnin)

FIG. 1. Reverse-phase HPLC separation of LTBI metabo- lites produced by intact human keratinocytes. Human keratin- ocytes (12-15 X lo6 cells) were incubated in tissue culture medium containing 3.58 p~ LTB, for 24 h at 37 "C. Supernatants from the incubations were treated with ethanol to precipitate proteins, taken to dryness, and injected onto the RP-HPLC column. The column was eluted with a linear gradient from 30 to 70% methanol in 35 min then to 100% methanol to 45 min. A , radioactivity eluting from the column was detected with a liquid scintillation radioactivity detector. HPLC effluent was monitored for ultraviolet absorption at 270 nm ( B ) and 232 nm (C). The abbreviations used for the metabolites proceed from the most lipophilic metabolite ( H K l ) to the least lipophilic metabolite (HK12) .

was recovered and analyzed by RP-HPLC. Fig. L4 shows a typical radiochromatogram, displayed from 25-50 min, ob- tained during RP-HPLC analysis. This HPLC elution range contained most of the major radioactive compounds except for a band of radioactivity which eluted within the first 7 min at the solvent front (14 f 2% of the total radioactivity analyzed by RP-HPLC (n = 6)). The amount of radioactivity corresponding to nonmetabolized LTB, ranged from 0 to 8% of the total radioactivity by HPLC analysis. Specific wave- lengths of UV absorption monitored during the RP-HPLC analysis are shown in Fig. 1B (270 nm) and 1C (232 nm). No major radioactive peak other than recovered LTB, retained the chromophore indicative of a conjugated triene.

10-HOTrE (lO-hydroxy-4,6,12-octadecatrienoic acid) (HK1)"Metabolite HK1 was more lipophilic than LTB, and had the UV spectrum shown in Fig. 2.4 with a Amax at 232 nm suggesting a conjugated diene chromophore. Such a chromo- phore could only result following reduction of one of the double bonds of the conjugated triene structure in LTB,. Negative ion CF-FAB/MS analysis of this radioactive fraction revealed an ion at m/z 293 which was 42 atomic mass units lower than the observed carboxylate anion for LTB, Such a mass shift would be consistent with a monohydroxy C-18 carboxylic acid with three double bonds. This assignment was confirmed by ECI GC/MS analysis of the PFB/TMS deriva- tive (Fig. 3A) (EC value = 19.65) which showed the carbox- ylate anion (A-) at m/z 365 (M- - PFB) with additional ions at m/z 293 (A- - CHz = Si(CH&), and m/z 275 (A- - TMSOH). Analysis of the PFB/TMS derivative of the re- duced metabolite had a carboxylate ion at m/z 371, confirming the presence of three double bonds. Due to the unexpected structure of this metabolite and the possibility that this

compound may have been a contaminant coeluting with the radioactive fraction and unrelated to LTB4 metabolism, ker- atinocytes were incubated with a mixture of do-LTB4 and d4- LTB,. The initial do-LTB4/d4-LTB4 ratio (5:2) was deter- mined by ECI GC/MS analysis of the derivatized starting LTB, mixture (Fig. 3A) using m/z 479 (LTB4 carboxylate anion) and m/z 483 (d4-LTB4 carboxylate anion). The PFB/ TMS derivative of HK1 from this incubation (Fig. 3A) clearly contained the isotope label indicative of an LTB,-derived metabolite. E1 GC/MS analysis revealed an odd electron ion at m/z 456 (M+' - TMSOH) and an abundant ion at m/z 259 (loss of the neutral radical CsF&HzO' from m/z 456) (Fig. 3B). This analysis also showed an ion which was consistent with a C-10 hydroxy position at m/z 435 (TMSO+=CH(CH& (CH)4(CHz)zC0zPFB) following a-fragmentation at the TMS ether position. Location of the hydroxy group was further confirmed by E1 GC/MS analysis of the PFB/TMS derivative of the reduced metabolite (Fig. 3C) which showed both a-trimethylsilyl ether fragment ions at m/z 215 (CH3(CHz)7CH='OTMS) and at m/z 439 (TMSO'=CH(CHZ)SCO~PFB). Oxidative ozonolysis of this metabolite followed by derivatization of the reaction products and analysis by negative ion mass spectrometry resulted in an abundant ion at m/z 413 (PFBOzCCHz CH(OTMS)(CH,)zCOz-). The GC retention time and mass spectrum were identical to the product obtained from oxida- tive ozonolysis of synthetic 10,11-dihydro-LTB4 (see discus- sion below) and was consistent with monohydroxy hexane- dioic acid. This established the position of the conjugated diene at C-4 and C-6 as shown in Fig. 3B with an additional unsaturation at C-12. The above data were consistent with the assignment of metabolite HK1 as 10-HOTrE.

10,ll-dihydro-LTB, and 10,ll-dihydro-12-epi-LTB, fHK2)"Negative ion CF-FAB/MS analysis of the radioactive fraction corresponding to metabolite HK2 (Fig. 1C) revealed an ion at m/z 337 suggesting reduction of one of the double bonds in LTB,. This metabolite was slightly more lipophilic than LTB,, and the UV maximum at 230 nm (Fig. 2B) indicated that one of the double bonds of the conjugated triene structure had been reduced. Tandem mass spectromet- ric analysis of the product ions following collision induced decomposition (CID) of m/z 337 revealed ions at m/z 319 (loss of H20) and m/z 115 (O=CH(CHZ),COz-) (data not shown). This product ion spectrum was identical to that

25442

A. ECI

Keratinocyte Leukotriene B4 Metabolism

A. ECZ

3. E l 1

mlz

COz PFB

L MW = 546

259

C. EI

7 456

m/Z

loo” l3

aJ 181 02 r 1 8 l PFB

2’5

- I - 21s Mw = 5J2

9 1c nl 439 .t I s rr” u

I

600

FIG. 3. Mass spectral analysis of the PFB/TMS derivative of metabolite HK1. A, electron capture ionization mass spectrum (negative ions) with A.1 showing the initial (I,-LTB,/c&-LTB4 carbox- ylate ion region and A.2 showing the carboxylate ions of metabolite HK1 obtained following incubation with deuterium labeled LTB4. B, electron ionization mass spectrum (positive ions). C, electron ioniza- tion mass spectrum (positive ions) of reduced, derivatized HK1.

obtained from CID of the carboxylate anions of synthetic 10,11-dihydro-LTB4 and from synthetic lO,ll-dihydro-12- epi-LTB,. ECI GC/MS analysis of the PFB/TMS derivative (EC value = 22.7) confirmed the presence of two hydroxy groups with the carboxylate anion (A-) at m/z 481 (M- - PFB) and additional ions at m/z 409 (A- - (CH2=Si(CH&)), m/z 391 (A- - TMSOH), m/z 319 (A- - TMSOTMS), and m/z 301 (A- - Z(TMS0H)) (Fig. 4A). The loss of TMSOTMS has been previously described in the mass spectrum of poly- trimethylsilyl ether derivatives (31). ECI GC/MS of the PFB/ TMS-derivatized, reduced metabolite confirmed the presence of three double bonds from the observed carboxylate anion at m/z 487, six mass units higher than the starting metabolite (data not shown). In addition, analysis of the PFB/TMS derivative of HK2 obtained following incubation with LTB, containing d4-LTB4 clearly established it as an LTB4-derived metabolite with the carboxylate anions observed at m/z 481 and m/z 485 (Fig. 4A). E1 GC/MS analysis of the PFB/TMS derivative produced the mass spectrum shown in Fig. 4B. The odd electron molecular ion was observed at m/z 662 with

[A--90] 3.9 1

” 100

B. EI 1 81

I

. . 300 400 500

m / Z

c- I

n ,1O.ll-dihydro-l2-epi-LTBq 10.ll-dihydro-LT.Bq

Time (min)

FIG. 4. Analysis of metabolite HK2. A, electron capture ioni- zation mass spectrum (negative ions) of the PFB/TMS derivative with A.l showing the carboxylate ion region following incubation containing deuterium labeled LTB,. €3, electron ionization mass spec- trum (positive ions). C, normal-phase HPLC separation of metabolite HK2 isolated using reverse-phase HPLC. This apparent single me- tabolite was further separated on a column of silicic acid using hexane/isopropanol/acetic acid in an isocratic system (see “Experi- mental Procedures”). The HPLC elution was monitored at 232 nm.

additional odd electron ions observed at m/z 572 (loss of TMSOH from m/z 662) and at m/z 482 (loss of TMSOH from m/z 572). Loss of 15 atomic mass units (‘CH,) from the molecular ion resulted in an observed ion at m/z 647. Frag- mentation at positions immediately adjacent to trimethylsilyl ether groups (a-fragmentation) were observed at m/z 213 (CH3)(CH2)4-(CH)2CH2CH=‘OTMS), at m/z 551 (TMSO+= CH(CH2)2-(CH),CH(OTMS)(CH2)3COzPFB), and at m/z 395

CHz(CH)z(CH2),CH3). Additional ions were observed at m/z 461 (loss of TMSOH from m/z 551), m/z 371 (loss of P(TMS0I-I) from m/z 551), and at m/z 305 (loss of TMSOH from m/z 395). This mass spectrum was identical to the E1 GC/MS spectrum of the PFB/TMS derivatives of synthetic 10,ll-dihydro-LTB4 and of synthetic lO,ll-dihydro-12-epi- LTB,. The presence of double bonds at positions 6,8, and 14 was confirmed by oxidative ozonolysis and ECI GC/MS analy- sis of the PFB/TMS derivatives of the reaction products. The ion at m/z 413 was identified and was consistent with the PFB/TMS derivative of a C-6 monohydroxy dicarboxylic acid product. The retention time of this derivatized ozonolysis

(TMSO’=CH(CH)4(CH2)2CH(OTMS)

Keratinocyte Leukotriene B4 Metabolism 25443

product was identical to the derivatized fragment obtained from oxidative ozonolysis of synthetic 10,ll-dihydro-LTB4. No other products were observed in the oxidative ozonolysis of either HK2 or synthetic 10,ll-dihydro-LTB4. The ion at m/z 413 is consistent with both 2-OH-hexanedioic acid, which would arise from the C-1 to C-6 fragment in l0,ll-dihydro- LTB,, and with 3-OH-hexanedioic acid, which would originate from the C-9 to c-14 fragment in 10,11-dihydro-LTB4. These two isomeric hydroxydioates were apparently not separated by the GC conditions. Oxidative ozonolysis of authentic LTB4 followed by GC/MS analysis revealed two ozonolysis prod- ucts, one with the same retention time and abundant ion at m/z 413 (2-OH-hexanedioic acid) and a second product ion at m/z 385 (C-11 to C-14 fragment, 2-OH-butanedioic acid).

Normal phase HPLC analysis of metabolite HK2 (Fig. 4C) revealed this metabolite to be a mixture of two compounds. The earlier eluting metabolite coeluted with synthetic 10,ll- dihydro-LTB, and the later eluting metabolite coeluted with synthetic 10,11-dihydro-12-epi-LTB4.

The above mass spectral, HPLC, and UV data were con- sistent with the assignment of metabolite HK2 as a mixture of lO,ll-dihydro-LTB, and 10,ll-dihydro-12-epi-LTB4.

3,7,14-triHDoTrE (3,7,14-Trihydroxy-8,10,16-docosatri- emic acid) (HK3)"The UV spectrum for HK3, a metabolite that was slightly less lipophilic than LTB4, also showed a AmSx at 230 nm and was identical to the UV spectrum of HK2 shown in Fig. 2B. ECI GC/MS analysis of the PFB/TMS derivative (EC value = 25.6) suggested the presence of three hydroxy groups with the carboxylate anion (A-) observed at m/z 597 (Fig. 5A) and additional ions at m/z 525 (A- - CH2=Si(CH&), m/z 507 (A- - TMSOH), m/z 435 (A- - TMSOTMS), m/z 417 (A- - 2(TMSOH)), m/z 345 (A- - TMSOH - TMSOTMS), and at m/z 327 (A- - 3(TMSOH)). The mass spectrum of the derivatized metabolite from kera- tinocyte incubation containing deuterium-labeled LTB4 also showed the expected ratio of d0/d4 carboxylate ions (Fig. 5A). Additionally, the presence of three double bonds was estab- lished by ECI GC/MS analysis of the derivatized, reduced metabolite which produced ions 6 atomic mass units higher with (A-) observed at m/z 603. Reduction of one double bond (+2 atomic mass units) and addition of one hydroxy group (+88 atomic mass units, OTMS) to LTB, would result in a carboxylate anion at m/z 569. Observation of the carboxylate anion for HK3 at 28 atomic mass units higher suggested the addition of two methylene groups. E1 GC/MS analysis of the PFB/TMS derivatized metabolite (Fig. 5B) suggested the additional hydroxy group and two methylene groups had added by chain elongation at the carboxyl terminus. Frag- mentation at positions adjacent to trimethylsilyl ether sub- stituents suggested an hydroxy substituent a t (2-14 by the observed ions at m/z 213 (TMSO'=CHCH2CHCH(CH2),CH,) and a t m/z 577 (TMSO'=CH(CH,),(CH),CH (OTMS)(CHZ)~CH(OTMS)CH~CO,PFB - TMSOH) and fur- ther loss of TMSOH from m/z 577 resulting in the ion observed at m/z 487 and loss of 2(TMSOH) resulting in the ion observed at m/z 397. An hydroxy substituent at C-7 was suggested by the observed ion at m/z 305

- TMSOH). This structure assignment was further confirmed by electron ionization mass spectral analysis of the deriva- tized, reduced metabolite (Fig. 5C). The hydroxy substituent position at C-14 resulted in the production of a-trimethylsilyl ether fragment ions at m/z 215 (TMSO+=CH(CH2),CH3) and m/z 671 (TMSO+=CH(CH2),CH(OTMS)(CH2)&H (OTMS)CH&O,PFB) with additional loss of TMSOH from this ion to give the observed ion at m/z 581. The hydroxy

(TMSO'=CH(CH),(CHz)zCH(OTMS)CH2(CH)2(CHZ)4CH3

A. ECI IA-l

89 I#-2701 321 iA"1801 507

(A"901

100 2M) 300 400 SO0 600 700 mlz

B. EI

100 400 600

C. EI m / Z

OTUS 671 215 401

485 341

I 413 Mw J 784

" 100 200 300 4 0 500 600 700 mlt

FIG. 5. Mass spectral analysis of the PFB/TMS derivative of metabolite HK3. A, electron capture ionization mass spectrum (negative ions) of the PFB/TMS derivative with A.1 showing the carboxylate ion region following incubation containing deuterium- labeled LTB,. B, electron ionization mass spectrum (positive ions). C, electron ionization mass spectrum (positive ions) of reduced deriv- atized HK3.

substituent at position C-7 resulted in a-trimethylsilyl ether fragment ions at m/z 485 (TMSO+=CH(CH2)3- CH(OTMS)CH2C02PFB) and at m/z 401 (TMSO' =CH(CHz)&H(OTMS)(CH2)7CH3). Additional fragment ions related to fragmentation at C-7 were observed at m/z 413 (loss of CHZ=Si(CH& from m/z 485), m/z 323 (loss of TMSOTMS from m/z 4851, and m/z 311 (loss of TMSOH from m/z 401). The hydroxy substituent at C-3 resulted in a minor a-trimethylsilyl ether fragment ion at m/z 341 (TMSO+=CHCH2C02PFB). The above data were consistent with the structure assignment of metabolite HK3 as the chain elongated 3,7,14-trihydroxy-8,10,16-docosatrienoic acid. 20-OH-l0,ll-dihydro-LTB4 (5,12,20-trihydroxy-6,8,14-ei-

cosatrienoic acid) (HK10)"Negative ion CF-FAB/MS analy- sis of the RP-HPLC fraction containing metabolite HK10, a metabolite that was slightly more lipophilic than synthetic 20-OH LTB,, yielded an abundant ion at m/z 353. At 18 atomic mass units higher than the carboxylate anion for LTB,, this suggested the presence of one additiona1 hydroxy group, consistent with the observed HPLC retention time, and reduction of one double bond from the starting LTB4.

25444 Keratinocyte Leukotriene B4 Metabolism

The UV spectrum for this metabolite had a Amax at 230 nm, identical to the spectrum for 10,ll-dihydro-LTB, (Fig. 2B). Negative ion GC/MS analysis of the PFB/TMS derivative of this metabolite (Fig. 6A) (EC value = 25.5) confirmed the presence of three hydroxy groups with the observed carbox- ylate anion (A-) at m/z 569 and additional ions at m/z 479 (A- - TMSOH), m/z 407 (A- - TMSOTMS), m/z 389 (A- - 2(TMSOH)), m/z 317 (A- - TMSOH - TMSOTMS), and m/z 299 (A- - 3(TMSOH)). Following incubation with deu- terium-labeled LTB,, the carboxylate anion of the derivatized metabolite also showed the addition of four deuterium atoms at mlz 573 (Fig. 6A). The positive ion mass spectrum of this PFB/TMS derivatized metabolite (Fig. 6B) revealed odd elec- tron ions at m/z 660 (M+ ' - TMSOH) and at m/z 570 (M+ ' - 2(TMSOH)). In addition, a-trimethylsilyl ether fragment ions suggesting a C-12 hydroxy substituent were observed at m/z 551 (TMSO+=CH(CHz)2(CH)4CH(OTMS)(CH2)3C02- PFB) and at m/z 301 (TMSO'=CHCH,(CH),(CH,),- CH,OTMS). An additional fragment ion related to m/z 551 was observed at mlz 461 (loss of TMSOH from m/z 551). Fragment ions suggesting a C-5 hydroxy substituent position

A. ECZ /A-1

569

I [A'-180]

' I I I 479 389 407 [A'.90]

100 200 300 400 500 600 m/Z

B. EZ 73

100 u)O 300 400 500 600 700 -1.

c. Ez 181

'"1 I

were observed at mlz 369 (TMSO+=CH(CHZ),COzPFB) and at mlz 393 (TMSO+=CH(CH),(CHz)zCH(OTMS)CHz(CH)z- (CHZ)&H~OTMS - TMSOH). The positive ion mass spec- trum of the derivatized, reduced metabolite (Fig. 6C) was identical to that of derivatized, reduced synthetic 20-OH LTB, with the most abundant a-trimethylsilyl ether fragment ions observed at m/z 303 (TMSO+=CH(CHz)7CH20TMS) and at m/z 369 (TMSO+=CH(CH2)&O2PFB). Additional fragment ions were observed at mlz 555 (TMSO+=CH (CHz)6CH(OTMS)(CHz)3C0zPFB), m/z 465 (loss of TMSOH from m/z 555), m/z 489 (TMSO'=CH(CH,),CH(OTMS) (CHZ)~CHZOTMS), m/z 399 (loss of TMSOH from m/z 489), and at mlz 375 (loss of 2(TMSOH) from mlz 555). A low abundant but significant ion observed at m/z 103 (TMSO+=CHz) located the third hydroxy substituent at C- 20. From the ultraviolet absorption data and the composite mass spectral data, the structure of metabolite HKlO was established as 20-OH-10,ll-dihydro-LTB, (5,12,20-trihy- droxy-6,8,14-eicosatrienoic acid).

Glutathione Conjugation (HK9, HK5, and HK4)"The UV spectra of LTB, metabolites HK9 (Fig. 2C), HK5, and HK4 were identical and showed a Amax at 232 nm. These spectra differed from the UV spectra of the dihydro metabolites (Fig. 2, A and B) with a broad absorption envelope extending up to 270 nm with significant absorption at 250 nm. No data were obtained from either positive or negative ion GC/MS analysis of the PFB/TMS derivatives suggesting an LTB, modification that was not readily amenable to gas chromato- graphic analysis. Following catalytic reduction, the positive ion mass spectra of the PFB/TMS derivatives were identical to the spectrum of derivatized, reduced LTB4, suggesting an LTB, modification that was also susceptible to catalytic re- duction. The negative ion FAB mass spectra of metabolites HK9, HK5, and HK4 yielded ions at m/z 642, m/z 513, and m/z 456, respectively, and also had ions at 4 atomic mass units higher for the corresponding metabolites obtained fol- lowing LTB, incubation with deuterium-labeled LTB4. These ions are 18 atomic mass units higher than the ions observed for synthetic LTC, (mlz 624), LTD, (mlz 495), and for LTE, (m/z 438).

Suspecting that HK9 may be a glutathione conjugate anal- ogous to LTC, which could be further metabolized by tran- speptidases to HK5 and KH4 (LTD, and LTE, analogs) metabolites HK9 and HK5 were treated with y-glutamyl transpeptidase and leucine aminopeptidase (32). RP-HPLC analysis of the reaction products from the fraction containing HK9 showed approximately 40% of this fraction was unaf- fected by treatment with the transpeptidases. This was likely

,,"I

due to contamination from metabolite HK8. The remaining 181 60% of this fraction coeluted with metabolite HK4. RP-HPLC

CO&B analysis of the transpeptidase reaction with HK5 showed 100% conversion to a compound which coeluted with metab- olite HK4.

CF-FAB mass spectral analysis of metabolite HK9 resulted in a product ion at mlz 272 following CID of m/z 642. The same ion transition was observed in the product ion spectrum of LTC, carboxylate anion and is indicative of the glutathione

in abundant product ions observed at m/z 177 and at mlz 143. These ion transitions were also observed in the product ion

3 69

375 465 489 5s5 I ! I 3 9 9 I / I moiety (33). CID of m/z 513 from metabolite HK5 resulted

FIG. 6. Mass spectral analysis of the PFB/TMS derivative spectrum of the carboxylate anion of LTD4 related to frag- of metabolite HK10. A, electron capture ionization mass spectrum mentation of the dipeptide moiety. Thus, the product ion

carboxylate ion region following incubation containing deuterium- (negative ions) of the PFB/TMS derivative with A.1 showing the spectra of LTC, and LTD, and the analogous LTB, metabo-

labeled LTB,. B , electron ionization mass spectrum (positive ions). lites, HK9 and HK57 were dominated by product ions C, electron ionization mass spectrum (positive ions) of the reduced only to the Peptide moieties. However, the product ion spec- PFB/TMS derivative. trum of LTE, carboxylate anion is dominated by ions retain-

Keratinocyte Leukotriene B4 Metabolism 25445

Metabolite HK4 CF-FABIMSIMS

100

3 A-= 456

T 229

$, , .

" 100 200 300 400

mlz FIG. 7. Tandem mass spectrometry (MS/MS) of underiva-

tized metabolite HK4. Product ion spectrum following CID of the carboxylate anion, m/z 456, obtained by CF-FAB ionization. See text for descriptions of fragment ions.

ing the carbon skeleton (34). The same fragmentation pattern was observed in the tandem mass spectral analysis of metab- olite HK4 (Fig. 7). CID of the carboxylate anion (m/z 456) of metabolite HK4 resulted in formation of the product ion at m/z 369, likely due to a @-elimination mechanism involving loss of ethylene amine and COz as proposed for the product ion formation from LTE,. Loss of HzO from this ion would produce the ion observed at m/z 351, a decomposition product ion also observed in the tandem mass spectrum of LTE,. Additional loss of HzO produced an ion observed at m/z 333 and loss of the neutral O=CH(CH2)&OZH produced the ion at m/z 235. Both of these decompositions were also observed in the product ion spectrum of LTE,. Another product ion common to LTE, and HK4 was observed at m/z 115 (O=CH(CHZ)&O2-). In contrast to LTE,, where the most abundant product ion (m/z 333) does not involve carbon- carbon skeleton fragmentation, the most abundant fragment ion observed during CID of HK4 (m/z 229) involved fragmen- tation of the C-l1,C-12 bond. This was likely due to a favor- able charge remote fragmentation (35,36) of m/z 369 in which the C-12 hydroxy hydrogen is transferred to the C-9 position, resulting in a terminal alkene containing the carboxylate moiety with loss of the neutral nine carbon aldehyde fragment. Loss of HzO from this ion produced the ion observed at m/z 211.

The product ions at m/z 235 and m/z 229 formed by CID of the carboxylate anion of HK4 suggested a C-6 position for the glutathione conjugation of LTB, with a conjugated diene at C-7,C-9. This was verified by analysis of the products obtained from the reaction of HK9 with hypochlorous acid (HOCl). The reaction of HOCl with LTC, results in the formation of diastereomeric suifoxides and 5,12-dihydroxy eicosatetraenoic acids (37). The analogous reaction of HOCl with HK9 would be expected to result in the formation of trihydroxy eicosatrienoic acids as well as in the formation of sulfoxides. Determination of the position of the third hydroxy group, which would add to the end of the conjugated diene system with loss of glutathione following HOCl treatment of HK9, would then indicate both the positions of double bonds and of the glutathione moiety. Metabolite HK9 was purified by RP-HPLC using the RP mobile phase at an initial com- position of 50% MeOH followed by a linear gradient to 100% MeOH in 30 min (retention time = 11.7 min). RP-HPLC analysis of the products formed during the reaction of HOC1 and HK9 showed two minor products more lipophilic than HK9 at retention times 16.8 and 17.4 min (Fig. 8). UV analysis of these products revealed absorption spectra identical to that

1 UVar6.7mi'n

l-4 1- = 244 nm

I W 0 10 20

Time (rnin} FIG. 8. Reverse-phase HPLC analysis with UV absorbance

monitored at 232 nm following reaction of metabolite HK9 (7 pM) with HOCl (14 MM). Products at retention times 6.3 and 6.7 min (S1 and S,) had the UV spectrum shown in the inset with X,, = 244 nm, consistent with sulfoxide formation. GC/MS analysis of the reduced compounds was also consistent with this assignment (see text). Products at retention times 16.8 and 17.4 min (Tl and Tz) were identified as trihydroxy products by mass spectral analysis (see Fig. 9).

EI "'U TMSO OTMS 181

3 69 C02 r PFB

100 200 300 400 500 600 I d 2

FIG. 9. Electron ionization mMS spectrum (positive ions) of the PFB/TMS derivative of the reduced trihydroxy products isolated in the reaction of HK9 with HOCI.

in Fig. 2B suggesting HOC1-induced loss of the glutathione moiety. ECI GC/MS analysis of these products following catalytic reduction and PFB/TMS derivatization resulted in an observed carboxylate ion at m/z 575, consistent with a C- 20 carboxylic acid containing three trimethylsilyl ether groups (EC value = 25.0). Electron ionization of the PFB/TMS- derivatized, reduced products (Fig. 9) established the position of the three trimethylsilyl ether substituents at C-12 from m/z 215 (TMSO+=CH(CHz),CH3), at C-10 by m/z 527 (TMSO+=CH(CHz),CH(0TMS)(CH~)&O2PFB) and at C-5 by m/z 369 (TMSO+=CH(CH2)&O2PFB). Major products of the reaction of HOCl with HK9 were observed at RP-HPLC retention times of 6.3 and 6.7 min (Fig. 8). The UV spectra of these products were identical and showed a Amax at 244 nm (inset, Fig. 8), a UV maximum shift consistent with sulfoxide formation (37). Catalytic reduction of these products, which would result in loss of the sulfoxide moiety as well as the saturation of double bonds, followed by PFB/TMS derivati- zation resulted in an observed carboxylate ion at m/z 487 when analyzed by negative ion mass spectrometry. This ion was consistent with PFB/TMS-derivatized 5,12-dihydroxy eicosanoic acid and further suggested the assignment of these products as sulfoxides.

The above data were consistent with the assignment of HK9 as 5,12-dihydroxy-6-glutathionyl-7,9,14-eicosatrienoic acid designated c-LTB3 due to analogy to the combined struc- tures of LTC, and LTB, (Structure I). Data for metabolite

25446 Keratinocyte Leukotriene B4 Metabolism OH OH OH

STRUCTURE I

HK5 were consistent with the structure assignment of 5,12- dihydroxy-6-cysteinylglycyl-7,9,14-eicosatrienoic acid desig- nated d-LTB3. Metabolite HK4 was assigned the structure of 5,12-dihydroxy-6-cysteinyl-7,9,14-eicosatrienoic acid desig- nated e-LTB3.

Insufficient material was available for remaining metabo- lites indicated in Fig. 1 to be identified. In addition, the stereochemistry at chiral centers and double bond geometry for identified metabolites has not been determined with the exception of the stereochemistry at C-12 for the HK2 diaster- eomers which was shown to have both the 12(R) and 12(S) configuration by NP-HPLC.

Keratinocyte Incubation with 10,ll -Dihydro-LTB, and 10,ll-Dihydro-12-epi-LTB,-Incubation of keratinocytes with either 10,ll-dihydro-LTB4 or 10,11-dihydro-12-epi- LTB, resulted in the metabolites shown in Fig. 10, A and B , following HPLC separation. Both incubations produced me- tabolites HK1, HK2, HK3, and HKlO that were identical to metabolites produced during incubation with LTB,. It ap- peared likely from these experiments that metabolite HKlO (20-OH-10,ll-dihydro-LTB,) may be a mixture of epimers containing both the 12(R) and 12(S) configuration. Struc- tures of these metabolites were verified by derivatization and mass spectral analysis as outlined above. Metabolite HK2 from each incubation was further analyzed by NP-HPLC (Fig. 10, A and B ) . Although each incubation was made with

A. 1.0 - H K 3

e -

2 w a u - -2

6 a IO 12 H K l HK2

Time (min)

-3 T

L L L "

25 30 35 40 45 50

g HKlO -3 T

25 30 35 40 45 50

Time (rnin)

25 30 35 40 45 50

Time (min) FIG. 10. Reverse-phase HPLC analysis of supernatants

from keratinocyte cultures (12-16 X 10' cells) incubated with I0,ll-dihydro-LTB, (4.8 PM) ( A ) and l0,ll-dihydro-12-epi- LTB, (4.8 PM) (B) . A.1 and B.1 show normal-phase HPLC analysis of metabolite HK2.

pure 10,ll-dihydro-LTB, or l0,ll-dihydro-12-epi-LTB,, as verified by NP-HPLC of the starting compounds, metabolite HK2 from both incubations was a mixture containing both 10,ll-dihydro-LTB, and l0,ll-dihydro-12-epi-LTB4. Impor- tantly, the glutathione conjugate metabolite produced with LTB, incubation, c-LTB3 (HK9), and metabolites d-LTB3 (HK5) and e-LTB3 (HK4) were not produced when keratin- ocytes were incubated with either 10,ll-dihydro-LTB, or l0,ll-dihydro-12-epi-LTB,.

DISCUSSION

Metabolism of LTB4 by human keratinocytes results in the formation of several novel products, none of which retains the conjugated triene structure of LTB,. These LTB, products may all arise from a common reactive intermediate, 12-oxo- LTB4 (Fig. 11) formed by 12-hydroxyeicosanoid dehydrogen- ase (38). Subsequent metabolism of 12-oxo-LTB, by human keratinocytes proceeds by two distinct pathways. Glutathione conjugation of 12-oxo-LTB4 followed by reduction of the 12- keto moiety results in the formation of the LTC, analog, c- LTB,. This metabolic pathway has not been previously iden- tified. Alternatively, reduction by 10,ll-reductase to form 10,ll-dihydro-12-oxo-LTB4 commits further metabolism to the formation of 10,ll-reduced products resulting from W -

oxidation, carboxyl terminus ,&oxidation, and chain elonga- tion. This was evidenced by incubations with 10,ll-dihydro- LTB, or l0,ll-dihydro-12-epi-LTB, during which all dihydro products were formed but no products arising from the first metabolic pathway, glutathione conjugation, were observed.

The intermediate, 12-oxo-LTB,, has been identified during LTB4 metabolism by subcellular fractions of porcine PMN, and this enzymatic oxidation was found to be NAD+ depend- ent (38). Further metabolism by A''-reductase produces a l0,ll-dihydro-12-oxo-LTB4 intermediate with subsequent re- duction by 12-keto reductase to produce the 12-hydroxy- l0,ll-dihydro metabolite. The 12-keto reductase appeared to be stereospecific during LTB, metabolism by the subcellular fractions of porcine PMN resulting in formation of only 10,ll- dihydro-LTB4 and not in the formation of 10,11-dihydro-12- epi-LTB,. This also appeared to be true in an earlier study involving intact porcine PMN where the 10,ll-dihydro-LTB4 metabolite was formed initially and the 12-epi isomer was only detected following longer incubation times (39). How- ever, both dihydro epimers were found during LTB, metabo- lism by human monocytes (16), mesangial cells (17), and by human lung (18). In the present study, both epimers of 10,ll- dihydro-LTB4 were detected following a 24-h incubation of LTBl with keratinocytes. Additional time course studies are necessary to determine if both isomers are formed directly from l0,ll-dihydro-12-oxo-LTB4 (path a , Fig. 11) or if only one isomer is initially formed and direct epimerization at the 12-hydroxy position results in the production of the epimeric metabolite (pa th b, Fig. 11). Incubation of human keratino- cytes with 10,ll-dihydro-LTB4 or with 10,11-dihydro-12-epi- LTB, also resulted in epimerization at the C-12 hydroxy position. This would suggest that if both isomers are formed directly from 10,ll-dihydro-12-oxo-LTB,, the reaction is re- versible. However, this is also consistent with reversible direct epimerization at the 12-hydroxy position.

RP-HPLC analysis of the lO,ll-dihydro-LTB4 produced by keratinocytes following 24 h of incubation with LTB4 revealed 10,1l-dihydro-12-epi-LTB4 present in greater amounts than 10,11-dihydro-LTB4. This may indicate a slower rate of me- tabolism for the 12(R) isomer as compared to the 12(s) isomer. Additional dihydro metabolites may be formed from either of the 10,ll-dihydro isomers or may be formed inde-

Keratinocyte Leukotriene Bq Metabolism

LTB4 * 25447

I I2-hydroxycicosanoid dchydrogenarc

0 OH

FIG. 11. Proposed metabolic path- ways for LTB, by human keratino- cytes.

IZ-OXO-LTB~

n OH

pendently from the 10,11-dihydr0-12-0~0 intermediate. Pre- liminary time course studies suggest that initial formation of the 12-hydroxy-l0,11-dihydro metabolite occurs prior to for- mation of the other dihydro metabolites. Also, the lack of any detectable 12-oxo intermediate would suggest that reduction of the 12-keto group occurs prior to formation of additional metabolites. Whether these metabolites are formed from only one epimer of the 10,ll-dihydro species remains to be deter- mined.

Formation of 20-OH-10,11-dihydro-LTB4 involves w-oxi- dation of either or both 10,ll-dihydro epimers. This metabo- lism may be mediated by a specific cytochrome P-450 as previously reported in the metabolism of LTB, by human PMN (40). Metabolism of dihydro-LTB, to more hydrophilic secondary metabolites was also observed in rat mesangial cells, fibroblast tumor cells, mouse bone marrow-derived mac- rophages and T-lymphocytes, although the structures of these secondary metabolites were not determined (14). In previous studies involving LTB, metabolism by human keratinocytes, two radioactive metabolites were observed with RP-HPLC retention times close to but not identical to the retention times of 20-OH-LTB4 and 20-COOH-LTB4 but the structures of these metabolites were not determined (30, 41).

The formation of 10-HOTrE likely involves one cycle of @- oxidation at the carboxyl terminus of 10,ll-dihydro-LTB, resulting in the formation of a P-hydroxy (the original C-5 hydroxy position) chain-shortened intermediate. Dehydration at the ,&hydroxy position followed by saturation of the double bond would result in the formation of 10-HOTrE. One possi- ble double bond reduction pathway could involve 2,4-dienoyl- CoA reductase (11) and subsequent double bond migration to form a conjugated diene. LTB, metabolism by carboxyl ter- minus @-oxidation has only previously been reported when ethanol was present in the incubation with rat hepatocytes (42). Metabolism by carboxyl terminus @-oxidation of the monohydroxy eicosatetraenoic acid 12(S)-HETE has been reported in vascular smooth muscle cells with production of

I z O ~ ~ l - l O , l l d i h y d r o - L T ~ ~ e J,7,14-hiHDoTrE (HKJ) (HKIO)

8-hydroxy-4,6,10-hexadecatrienoic acid (43). The formation of 3,7,14-triHDoTrE is the first reported

LTB4-derived metabolite resulting from apparent chain elon- gation mechanisms. The formation of both a chain-elongated LTB, metabolite and a chain-shortened metabolite (10- HOTrE) by keratinocytes may indicate involvement of dis- tinct subcellular fractions in the metabolism of l0,ll-dihydro- LTB,. Both metabolites are most likely formed through the intermediate formation of 10,ll-dihydro-LTB, CoA thioester with subsequent chain elongation occurring in the endo- plasmic reticulum or chain shortening occurring by peroxi- somal or mitochondrial @-oxidation.

The intermediate 12-oxo-LTB4 is also the most likely in- termediate leading to formation of the novel glutathione conjugate, c-LTB3. A l,&Michael-type addition of glutathione to this activated intermediate would result in glutathione conjugation at the C-6 position and formation of a noncon- jugated keto group at the C-12 position. Subsequent reduction by a 12-keto reductase would lead to the formation of c-LTB3. The stereospecificity of this 12-keto reductase has not been determined. Further metabolism by transpeptidases forms d- LTB, and e-LTB,. A similar Michael addition of glutathione has been reported for other eicosanoids including 15-keto- prostaglandin Fa* (44), prostaglandin AI, and prostaglandin A2 (45), as well as prostaglandin DS (46, 47). Glutathione-S- transferases are known to catalyze such reactions in contrast to glutathione conjugation of LT& which requires a rather specific LTC, synthase (48).

Detailed structural studies of LTB, metabolism have now been carried out in two human cells, the polymorphonuclear leukocyte and the keratinocyte. These two cells metabolize LTB, by completely different pathways. In the neutrophil LTB4 is initially oxidized at the methyl terminus, while in the human keratinocyte metabolism initially involves oxidation of the 12-hydroxy group followed by formation of either glutathione conjugates or 10,ll-dihydro-LTB, metabolites. The tissue distribution of this unique 12-hydroxy-LTB, oxi-

25448 Keratinocyte Leukotriene B4 Metabolism

dation pathway is presently unknown; however, such metab- olites may provide insight into involvement of cells such as the keratinocyte in LTB, metabolism. Glutathione. conjuga- tion is generally a detoxification mechanism whereby biolog- ically active substances are inactivated. However, glutathione conjugation of LTA, to produce LTC, results in the produc- tion of a lipid mediator possessing potent biological activities (49). Production of LTB,-derived analogs of LTC, and LTD4 by the human keratinocytes warrants investigation into po- tential biological properties of these novel compounds due to the observed bioactivity of LTC, and LTD, on keratinocyte function (7) and on neighboring melanocyte function. LTCl and LTD, are potent mitogens for cultured human neonatal melanocytes (50), and LTC, also stimulates melanocyte mi- gration in uitro (51). LTC, also causes chronic growth stimu- lation of human adult melanocytes and loss of contact inhi- bition with formation of structures resembling tumor spher- oids (52). The biological properties of the novel keratinocyte metabolites are currently under investigation.

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