- Email: [email protected]
(30] Leukotriene metabolism by isolated rat hepatocytes
[30]
LEUKOTRIENE METABOLISM
277
the purified enzyme exhibits peroxidase activity. This is further confirmed by incubating 5-HPETE with the purified enzyme under anerobic conditions where a significant amount of 5-HETE formation is observed in the reaction mixture. The pH of the reaction has a profound influence on the product profile. At pH values below 6.5, the ratio between HPETEs and DiH(P)ETEs (judged by ratio of absorption at 235 and 268 nm) is ~2.5, whereas at pH 7.3 the DiH(P)ETE formation is significantly reduced. At pH 9.0, the lipoxygenase activity is only about 1% of the activity at pH 6.3, but the formation of 8-HPETE exceeds that of 5-HPETE, while the other HPETEs drop to undetectable levels. In addition to the six HPETEs identified during L~-catalyzed oxidation of arachidonic acid, all possible diH(P)ETEs are detected, with each exhibiting a distinct absorption spectrum. The diH(P)ETEs include those that are directly generated by dual lipoxygenase activity and those that result from the nonenzymatic hydrolysis of LTA4s. Furthermore, when 5(OOH), 15-(OH)-, or 5(OH),15(OOH)-arachidonic acid are incubated with the Ll form, all possible isomers of lipoxin A and lipoxin B are isolated from the reaction. We have demonstrated further that the potato lipoxygenase can act at all possible positions of eicosapentaenoic acid and docosahexaenoic acid to give rise to their respective hydroperoxy compounds. T M Given its ease of preparation and the wide range of compounds it can produce, potato arachidonate 5-1ipoxygenase is useful in the preparation of many experimentally interesting metabolites of arachidonic acid and other PUFAs. t5 j. Whelan, P. Reddanna, G. Prasad, M. K. Rap, and C. C. Reddy, Ann. N . Y. Acad. Sci. 524, 391 (1988). 16 j. Whelan, P. Reddanna, G. Prasad, and C. C. Reddy, in "Proceedings of the Short Course on Polyunsaturated Fatty Acids and Eicosanoids" (W. E. M. Lands, ed.), p. 468. AOCS Press, Champaign, IL, 1987.
[30] L e u k o t r i e n e M e t a b o l i s m b y Isolated R a t H e p a t o c y t e s By M I C H A E L
A . SHIRLEY a n d DANNY O . STENE
Leukotrienes are potent biologically active molecules, yet the roles played by these substances as mediators of inflammation remain unclear. One approach to the study of eicosanoid involvement in physiological and pathological processes has been to assess the production of eicosanoids in association with particular normal or disease states. A crucial part of such METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
278
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[30]
an approach for leukotrienes must focus on their metabolic fate in vivo. Metabolites which may accumulate in urine or bile might serve as indicators of leukotriene synthesis at remote sites such as the lung or skin, Recently, as a model of in vivo metabolism, isolated rat hepatocytes have been shown to metabolize leukotrienes to polar metabolites. ~This chapter will report on the methods used for the production of leukotriene metabolites by isolated rat hepatocytes. Isolation and Purification of Hepatocytes
Animals Male Sprague-Dawley rats fed ad libitum on lab chow and tap water, and ranging from 200 to 650 g, are used. Neither the size of the animal, nor the time of day at which cells are isolated, appears to affect the results of leukotriene metabolism studies. However, smaller animals tend to give better cell yields and viabilities than larger animals.
Buffers The buffers used are a perfusion buffer (buffer A) containing 116 mM NaC1, 6 mM KCI, 0.74 mM KH2PO4, 0.6 mM MgSO4, 12 mM NaHCO3, 15/~M glucose, pH 7.1-7.4, equilibrated with 95% 02, 5% CO2 (O2/CO2); a wash buffer (buffer B) containing 126 mM NaCI, 5.2 mM KCI, 3 mM NazPO4, 0.9 mM MgSO4, 0.12 mM CaC12, 10/zM glucose, pH 7.1-7.4, equilibrated with O2/CO2; and an incubation buffer (buffer C) prepared by adding Tris [2-amino-2-(hydroxymethyl)-l,3-propanediol] and CaCI2 to buffer B, to the final concentrations of 10 mM Tris, and I mM CaC12, pH 7.4. 2
Reagents for Metabolite Purification Leukotriene E4 [LTE4, (5S)-hydroxy-(6R)-S-cysteinyl-(7E,9E, 11Z, 14Z)-eicosatetraenoic acid] was received as a gift from Dr. J. Rokach (Merck-Frosst, Canada Inc.), or is purchased from BioMol (Philadelphia, PA). Leukotriene B4 [LTB4, (5S, 12R)-dihydroxy-(6Z,8E, 10E, 14Z)-eicosatetraenoic acid] is purchased from Cayman Chemical Co. (Ann Arbor, MI). [3H]LTE4 [(5S)-hydroxy-(6R)-S-cysteinyl-(7E,9E, 11Z, 14Z)-[14,153H2]eicosatetraenoic acid, 39 Ci/mmol] and [3H]LTB4 [(5S,12R)-dihydroxy-(6Z,8E, 10E, 14Z)-[5,6,8,9,11,12,14,15-3H8 ]eicosatetraenoic acid, i D. O. Stene and R. C. Murphy, J. Biol. Chem. 263, 2773 (1988). 2 M. J. Garrity, E. P. Brass, and E. P. Robertson, Biochim. Biophys. Acta 796, 136 (1984).
[30]
LEUKOTRIENEMETABOLISM
279
200 Ci/mmol] are purchased from New England Nuclear (Boston, MA). Supplies for chromatography include octadecylsilyl (ODS) flash chromatography cartridges (Sep-Pak C18) from Waters Associates (Milford, MA), reversed-phase high-performance liquid chromatography (HPLC) cartridge columns from Jones Chromatography (Columbus, OH), and HPLC solvents from Fisher (Fairlawn, N J). Surgery and Cell Isolation The surgical procedure described by Wagle and Ingebretsen, as revised by Brass, is used. 3'4 The perfusion apparatus used in these studies is improvised from standard lab glassware. It allows the perfusion buffer to be warmed and gassed, and maintained at a perfusion pressure of approximately 17 cm water. Unlike the apparatus described by Wagle and Ingebretsen, the buffer is gassed directly, and the apparatus is not enclosed. Following intraperitoneal injection of sodium pentobarbital (60 mg/kg), the anesthetized rat is placed in a dissecting tray and the abdomen opened by a midline scissor incision from just above the bladder to the xiphoid process. Anterolateral incisions are made on each side from the level of the bladder to the level of the kidneys and the resulting tissue flaps pulled aside to expose the peritoneal cavity. After exposing the posterior peritoneum by pulling the intestines to the fight of the animal, ligatures are placed around the portal vein and inferior vena cava as described, 3 and the portal vein is cannulated with an 18-gauge 5.1 cm Quik-Cath (Travenol) attached to the outlet of the perfusion apparatus with intravenous tubing (Travenol, Deerfield, IL). After securing the portal vein cannula, the liver is perfused with buffer A, which is maintained at 37° and equilibrated with O2/CO2 by the perfusion system. The perfusion system outlet is 40 cm above the dissecting tray. As the liver perfusion is started, the inferior vena cava is cut well below the kidney to provide the necessary fluid exit. The ligamentum teres hepatis and diaphragm are then cut, and the anterior thoracic wall removed by making parallel cuts of the rib cage along the midclavicular line. A loose ligature is placed around the inferior vena cavajust superior to the diaphragm, as is another just inferior to the right atrium. A small incision is made in the vessel between the two ligatures and an 18-gauge catheter (with the stylet removed and cut to about 1 cm) is inserted and secured. The ligature around the inferior vena cava in the abdomen is then tied. The liver is dissected from the animal by first freeing the lower margin of the abdominal vena cava just below the ligature. The perfusion line is 3 S. R. Wagle and W. R. lngebretsen, Jr., this series, Vol. 35, p. 579. 4 E. Brass, personal communication, 1986.
280
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[30]
then moved to the thoracic catheter, and the portal vein and liver are dissected free from the surrounding mesentery. The perfusion line is then returned to the portal vein, and the liver dissected free from the posterior abdominal wall. The tubing connecting the liver to the perfusion apparatus is removed and the liver is attached to the outlet of the perfusion apparatus by way of the portal vein catheter. The perfusion buffer is adjusted to 1 mM CaCIz by addition of 2 ml 100 mM CaCI2, and 3 units/ml collagenase (Type II, Cooper Biomedical, Freehold, N J) is added. The liver is perfused with this collagenase buffer for 10 min. via the portal vein, 5 min via the inferior vena cava, and then 5 min with collagenase-free/Ca2÷-free buffer via the portal vein. The liver is then disconnected from the perfusion system and placed into a 250-ml silanized glass beaker containing about 30 ml of buffer A warmed to 37°. [All glassware used during the purification of hepatocytes is silanized by rinsing with neat dimethyldichlorosilane (Pierce, Rockford, IL) followed by rinsing with dry methanol.] Hepatocytes are teased from the liver into the buffer using forceps. The resulting cell suspension is filtered through nylon mesh (70 mesh, Nytex nylon) into a 200-ml Erlenmeyer flask and placed into a water bath for 10 min at 37° with gentle shaking and continuous O2/CO2 ventilation. Cells are poured from the flask through the nylon mesh into 50-ml plastic centrifuge tubes chilled on ice, the tubes are filled with buffer B (4°), and then are centrifuged at 65 g for 3 min. After removing the supernatant, the pellet is very gently resuspended in chilled buffer B followed by recentrifugation. This is repeated, and the washed hepatocytes are gently resuspended in chilled buffer C. The cells are counted, and the viability evaluated by the trypan blue exclusion test. 5 Cells used for metabolism studies are always >85% viable. Generation and Purification of Leukotriene Metabolites Incubation Conditions
Incubations with LTE4 are conducted with 3 x 10 6 cells/ml in a gently shaking water bath at 37 ° with O2/CO2 ventilation. For studies of substrate concentration effects, [3H]LTE4, at concentrations of 0.05, 0.5, 10, 25, and 50/zM, is incubated with 2 or 5 ml of cells in a 25-ml plastic tissue culture flask for 30 min. For studies of the time course of LTE4 metabolism, 15 ml of cells in a 50-ml flask are incubated with 5/xM [3H]LTE4 for up to 60 min. Incubations with LTB4 (3/zM) are conducted with 5 × 10 6 cells/ml for 45 min as above. 5 p. O. Seglen, Methods Cell Biol. 13, 29 (1976).
[30]
LEUKOTRIENE METABOLISM
281
Metabolite Purification Hepatocyte incubations are stopped by the addition of 4 volumes chilled ethanol. After storage overnight at - 7 0 ° , precipitated proteins are pelleted by centrifugation at room temperature. For the purification of LTE4 metabolites, the ethanolic supernatant is diluted by addition of l0 volumes of reversed-phase HPLC solvent A (see below), and partially purified by solid-phase extraction on an ODS Sep-Pak. The Sep-Pak cartridges are washed with solvent A and eluted with methanol. The methanol eluent is evaporated under vacuum and redissolved in 80% solvent A : 20% methanol for analysis by reversed-phase HPLC. The radioactivity content of the sample before and after evaporation is determined to quantitate the production of volatile counts. The recovery of tritium-labeled metabolites from the Sep-Paks averages 85% throughout these experiments. The ethanolic supernatant containing LTB4 metabolites is taken to dryness under vacuum and the residue dissolved in water (acidified to pH 3.5 with acetic acid). This sample is taken for direct analysis by reversedphase HPLC. LTE4 metabolites are subjected to reversed-phase HPLC using a linear gradient from 80% solvent A to 100% solvent B. Solvent A is water with 16.6 mM ammonium acetate, pH 4.5, and 132 /~M tetrasodium ethylenediaminetetraacetic acid (EDTA); solvent B is 90% methanol, 10% solvent A, 0.1% acetic acid, and 132/zM EDTA. For LTB4 metabolites, the same reversed-phase HPLC system is used except that ammonium formate is the buffer, the gradient is started at 90% solvent A, and EDTA is not included. The reversed-phase HPLC effluent is monitored by a photodiode array detector (HP-1040A, Hewlett-Packard, Palo Alto, CA), and/or a radioactivity detector (FLO-ONE Beta, model IC radioactive flow detector, Radiomatic Instruments, Tampa, FL). The UV wavelengths monitored are at 270 nm (for LTB4 metabolites), 280 nm, or a broad-band UV signal which sums the absorbance from 260 to 340 nm (for LTE4 metabolites). One assumption made for these experiments is that, during purification and reversed-phase HPLC of leukotriene metabolites, there are no differences in the recoveries of the various metabolites. Total recovery of leukotriene metabolites from reversed-phase HPLC ranges from 50 to 80%. The recoveries of LTE4 metabolites are best when the columns are washed prior to use with water/methanol (90 : 10, v/v) and 132 mM EDTA as described. 6 6 F. S. Anderson, J. Y. Westcott, J. A. Zirrolli, and R. C. Murphy, Anal. Chem. 55, 1837 (1983).
282
BIOSYNTHESIS,
ENZYMOLOGY,
AND
CHEMICAL
SYNTHESIS
[30]
=,..-, 0
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•- . ~
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.
[30]
LEUKOTRIENEMETABOLISM
283
LTE4 Metabolism LTE4 has been shown to be metabolized by isolated rat hepatocytes to six polar metabolites (Fig. 1). 1 The initial N-acetylation of LTE4 is followed by to-oxidation and/3-oxidation from the to terminus. All of these metabolites retain the typical leukotriene UV chromophore with absorption maxima at 280 nm, except for Metabolite E which is a conjugated tetraene with a UV absorption maximum at 309 nm. The metabolism of LTE4 is dependent on the concentration of LTE4 exposed to the isolated hepatocytes (Figs. 2 and 3). When 50/xM LTE4 is used, the only metabolite observed is N-acetyl-LTE4. However, as the concentration of LTE4 decreases, the conversion of LTE4 to to/fl-oxidized metabolites increases. When 25/xM LTE4 is used, to/fl-oxidized metabolites appear, but N-acetyl-LTE4 is still the dominant metabolite. At low substrate concentrations (less than 5/~M), only to/fl-oxidized metabolites are present after 30-rain incubation. Loss of the tritium label is up to 45% of the total indicating metabolism of LTE4 beyond the 16-carboxydihydro
[ 50wM
LTE4
D
go • 5~M
B
I0
15
20
25
3O
25~M
35
15
I0
211
25
30
35
60
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-m
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10
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2O
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TIME(min)
3o
.
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.
.
.
,
15
.
.
.
.
,
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.
.
.
.
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.
.
.
.
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,
30
.
,
,~.._1
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TIME(min)
FIG. 2. LTE4 concentration effects on metabolism. Reversed-phase HPLC radiochromatograms of [3H]LTE4 metabolites from isolated rat hepatocytes (3 × 106 cells/ml, 30 min) at the LTE4 concentrations indicated. The identities of the metabolites are determined by comparison with biologically derived and synthetic standards (see Fig. 1). Reversed-phase HPLC conditions are as noted in the text.
284
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS lOO
0 ~ 0 ~ _ . _ _ _ _ _
[30l
0
5¢~ 50
•1"" ~ ~ ~ O . . . . . .
0
10-1
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O'.*
101
10e
Substrate Concentration (pM) FIG. 3. LTE4 concentration effects on metabolism. The effect of substrate concentration on the extent of LTE4 metabolism by isolated rat hepatocytes is plotted. Cells are incubated with from 0.05 to 50 p.M LTE4 for 30 rain as described in the text. Data points represent, for the to/B-oxidized metabolites (©), the sum of the reversed-phase HPLC UV peak heights of the metabolites divided by the sum of the peak heights for all metabolites and LTE4. For LTE4 (CD)and N-acetyl-LTE4 (O), the data points represent the reversed-phase HPLC peak heights divided by the sum of the peak heights for all metabolites and LTE4.
metabolite. When the relative amount of to/fl-oxidized metabolites is plotted versus the LTE4 concentration, it is apparent that at substrate concentrations about 10/zM, the rate of LTE4 metabolism by isolated rat hepatocytes is nonlinear. When LTE4 is incubated with hepatocytes at 5/zM, the extent of LTE4 metabolism increases as a function of time. In order to determine the extent to which metabolism continues past the formation of Metabolite D, the broad-band monitoring capability of the photodiode array detector is used to generate a signal derived from monitoring 260 to 340 nm. Although the extinction coefficient of the conjugated tetraene chromophore of Metabolite E is not known, it is estimated to be approximately 80,000 at 309 nm based values reported in the literature. 7 The extinction coefficient for LTE4 has been determined to be 40,000 at 280 nm. 8 The relative changes in the broad band signals are assumed to be linear for all metabolites, and the peak heights for Metabolite E are divided by 2 as a correction for the higher extinction coefficient of this metabolite. Using this approach, the relative amounts of Metabolites C, D, and E change as a function of time (Fig. 4; LTE4, and Metabolites A and B are not shown). Clearly, Metabolite E is a major LTE4 metabolite at 60 rain. It is also apparent that the relative amount of volatile tritium increases over time, and this increase parallels the increases in Metabolites D and E. 7 G. A. J. Pitt and R. A. Morton, Prog. Chem. Fats Lipids 4, 227 (1957). 8 R. A. Lewis, J. M. Drazen, K. F. Austen, D. A. Clark, and E. J. Corey, Biochem. Biophys. Res. Commun. 96, 271 (1980).
[30]
LEUKOTRIENEMETABOLISM
60 Met. E
~]Met. C 15
• ........
I0
285
zo
~_...
"'""""•_ __... •
,, ,""
20 ]tl
,,""
0
,o. Met. O
._~ 6o
10
20
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50
60
70
va.N
e ~
30-
"~40
•
ILl
/
y" 1O
o/
,"
.."
/ 20
30
40
50
60
70
e0
O
10
20
30
40
50
60
70
Time in Minutes FIG. 4. Metabolism of LTEa as a function of time. The amount of Metabolite E increases relative to other metabolites as a function of time during metabolism of LTE4 by isolated rat hepatocytes (5 p~M LTE4, 3 × 106 ceUs/ml). A broad-band UV signal from 260 to 340 nm is used to measure metabolites by reversed-phase HPLC. The relative amount of volatile tritium increases in parallel with the increases in Metabolites D and E. The data are from two experiments, the open points from one experiment, and the closed points from another (see the text for an explanation of how the data is calculated).
LTB4 Metabolism Rat hepatocyte metabolism of LTB4 results in three major metabolites as assessed by reversed-phase HPLC (Fig. 5). As with LTE4 metabolism, LTB4 is also to- and/3-oxidized from the to terminus. The formation of 20-COOH-LTB4 and 18-COOH-LTB4 has been reported previously. 9 Preliminary mass spectral data indicate that 16-COOH-14,15dihydrotetranor-LTB4 is also formed. Volatile products account for approximately 30% of the starting radiolabel. One or more highly polar metabolites, representing 11% of the nonvolatile radiolabel, elute with the 9 T. W. Harper, M. J. Garrity, and R. C. Murphy, J. Biol. C h e m . 261, 5414 (1986).
t]O
286
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
5000
[31]
8-coo.
4000.
, s-coo.
2000
2o-coo.
1000.
0
l0
20
90 40 50 TIME (MINUTES)
50
70
FIo. 5. LTB4 metabolism by isolated rat hepatocytes. The reversed-phase HPLC radiochromatogram of [3H]LTB4 metabolites formed by hepatocytes (5 ml cells, 5 x 106 cells/ml) incubated with LTB4 (3/zM) for 45 min. The identity of 20-COOH-LTB4 is determined by coelution with a synthetic standard. 18-COOH-LTB4 and 16-COOH-LTB4 are identified by electron impact mass spectrometry. Reversed-phase HPLC conditions are as outlined in the text.
solvent front of the gradient reversed-phase HPLC system. Further studies on the metabolism of LTB4 by isolated rat hepatocytes are in progress. The relevance of the isolated rat hepatocyte model of LTE4 metabolism to the fate of leukotrienes in vivo has been supported by the identification of 20-COOH-LTE4 (Metabolite B) and 16-COOH-dihydro-LTE4 (Metabolite D) in rats injected with LTE4. ~o This hepatocyte system serves as a biosynthetic source of these metabolites. to p. Perrin, J. Zirrolli, D. O. Stene, J. P. Lellouche, J. P. Beaucourt, and R. C. Murphy, Prostaglandins 37, 53 (1989).
[3 1] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n of H u m a n L u n g L e u k o t r i e n e A4 H y d r o l a s e
By N O B U Y A
O H I S H I , TAKASHI I Z U M I , YOUSUKE SEYAMA, TAKAO S H I M I Z U
and
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) catalyzes enzymatic hydration of LTA4 [5(S)-trans-5,6-oxido-7,9-trans-ll,14-cis-eicosatetraenoic acid] to LTB4 [5(S), 12 (R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid], a potent chemotactic substance. This reaction on the epoxide differs from that of epoxide hydrolases (EC 3.3.2.3). The latter METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
LEUKOTRIENE METABOLISM
277
the purified enzyme exhibits peroxidase activity. This is further confirmed by incubating 5-HPETE with the purified enzyme under anerobic conditions where a significant amount of 5-HETE formation is observed in the reaction mixture. The pH of the reaction has a profound influence on the product profile. At pH values below 6.5, the ratio between HPETEs and DiH(P)ETEs (judged by ratio of absorption at 235 and 268 nm) is ~2.5, whereas at pH 7.3 the DiH(P)ETE formation is significantly reduced. At pH 9.0, the lipoxygenase activity is only about 1% of the activity at pH 6.3, but the formation of 8-HPETE exceeds that of 5-HPETE, while the other HPETEs drop to undetectable levels. In addition to the six HPETEs identified during L~-catalyzed oxidation of arachidonic acid, all possible diH(P)ETEs are detected, with each exhibiting a distinct absorption spectrum. The diH(P)ETEs include those that are directly generated by dual lipoxygenase activity and those that result from the nonenzymatic hydrolysis of LTA4s. Furthermore, when 5(OOH), 15-(OH)-, or 5(OH),15(OOH)-arachidonic acid are incubated with the Ll form, all possible isomers of lipoxin A and lipoxin B are isolated from the reaction. We have demonstrated further that the potato lipoxygenase can act at all possible positions of eicosapentaenoic acid and docosahexaenoic acid to give rise to their respective hydroperoxy compounds. T M Given its ease of preparation and the wide range of compounds it can produce, potato arachidonate 5-1ipoxygenase is useful in the preparation of many experimentally interesting metabolites of arachidonic acid and other PUFAs. t5 j. Whelan, P. Reddanna, G. Prasad, M. K. Rap, and C. C. Reddy, Ann. N . Y. Acad. Sci. 524, 391 (1988). 16 j. Whelan, P. Reddanna, G. Prasad, and C. C. Reddy, in "Proceedings of the Short Course on Polyunsaturated Fatty Acids and Eicosanoids" (W. E. M. Lands, ed.), p. 468. AOCS Press, Champaign, IL, 1987.
[30] L e u k o t r i e n e M e t a b o l i s m b y Isolated R a t H e p a t o c y t e s By M I C H A E L
A . SHIRLEY a n d DANNY O . STENE
Leukotrienes are potent biologically active molecules, yet the roles played by these substances as mediators of inflammation remain unclear. One approach to the study of eicosanoid involvement in physiological and pathological processes has been to assess the production of eicosanoids in association with particular normal or disease states. A crucial part of such METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
278
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[30]
an approach for leukotrienes must focus on their metabolic fate in vivo. Metabolites which may accumulate in urine or bile might serve as indicators of leukotriene synthesis at remote sites such as the lung or skin, Recently, as a model of in vivo metabolism, isolated rat hepatocytes have been shown to metabolize leukotrienes to polar metabolites. ~This chapter will report on the methods used for the production of leukotriene metabolites by isolated rat hepatocytes. Isolation and Purification of Hepatocytes
Animals Male Sprague-Dawley rats fed ad libitum on lab chow and tap water, and ranging from 200 to 650 g, are used. Neither the size of the animal, nor the time of day at which cells are isolated, appears to affect the results of leukotriene metabolism studies. However, smaller animals tend to give better cell yields and viabilities than larger animals.
Buffers The buffers used are a perfusion buffer (buffer A) containing 116 mM NaC1, 6 mM KCI, 0.74 mM KH2PO4, 0.6 mM MgSO4, 12 mM NaHCO3, 15/~M glucose, pH 7.1-7.4, equilibrated with 95% 02, 5% CO2 (O2/CO2); a wash buffer (buffer B) containing 126 mM NaCI, 5.2 mM KCI, 3 mM NazPO4, 0.9 mM MgSO4, 0.12 mM CaC12, 10/zM glucose, pH 7.1-7.4, equilibrated with O2/CO2; and an incubation buffer (buffer C) prepared by adding Tris [2-amino-2-(hydroxymethyl)-l,3-propanediol] and CaCI2 to buffer B, to the final concentrations of 10 mM Tris, and I mM CaC12, pH 7.4. 2
Reagents for Metabolite Purification Leukotriene E4 [LTE4, (5S)-hydroxy-(6R)-S-cysteinyl-(7E,9E, 11Z, 14Z)-eicosatetraenoic acid] was received as a gift from Dr. J. Rokach (Merck-Frosst, Canada Inc.), or is purchased from BioMol (Philadelphia, PA). Leukotriene B4 [LTB4, (5S, 12R)-dihydroxy-(6Z,8E, 10E, 14Z)-eicosatetraenoic acid] is purchased from Cayman Chemical Co. (Ann Arbor, MI). [3H]LTE4 [(5S)-hydroxy-(6R)-S-cysteinyl-(7E,9E, 11Z, 14Z)-[14,153H2]eicosatetraenoic acid, 39 Ci/mmol] and [3H]LTB4 [(5S,12R)-dihydroxy-(6Z,8E, 10E, 14Z)-[5,6,8,9,11,12,14,15-3H8 ]eicosatetraenoic acid, i D. O. Stene and R. C. Murphy, J. Biol. Chem. 263, 2773 (1988). 2 M. J. Garrity, E. P. Brass, and E. P. Robertson, Biochim. Biophys. Acta 796, 136 (1984).
[30]
LEUKOTRIENEMETABOLISM
279
200 Ci/mmol] are purchased from New England Nuclear (Boston, MA). Supplies for chromatography include octadecylsilyl (ODS) flash chromatography cartridges (Sep-Pak C18) from Waters Associates (Milford, MA), reversed-phase high-performance liquid chromatography (HPLC) cartridge columns from Jones Chromatography (Columbus, OH), and HPLC solvents from Fisher (Fairlawn, N J). Surgery and Cell Isolation The surgical procedure described by Wagle and Ingebretsen, as revised by Brass, is used. 3'4 The perfusion apparatus used in these studies is improvised from standard lab glassware. It allows the perfusion buffer to be warmed and gassed, and maintained at a perfusion pressure of approximately 17 cm water. Unlike the apparatus described by Wagle and Ingebretsen, the buffer is gassed directly, and the apparatus is not enclosed. Following intraperitoneal injection of sodium pentobarbital (60 mg/kg), the anesthetized rat is placed in a dissecting tray and the abdomen opened by a midline scissor incision from just above the bladder to the xiphoid process. Anterolateral incisions are made on each side from the level of the bladder to the level of the kidneys and the resulting tissue flaps pulled aside to expose the peritoneal cavity. After exposing the posterior peritoneum by pulling the intestines to the fight of the animal, ligatures are placed around the portal vein and inferior vena cava as described, 3 and the portal vein is cannulated with an 18-gauge 5.1 cm Quik-Cath (Travenol) attached to the outlet of the perfusion apparatus with intravenous tubing (Travenol, Deerfield, IL). After securing the portal vein cannula, the liver is perfused with buffer A, which is maintained at 37° and equilibrated with O2/CO2 by the perfusion system. The perfusion system outlet is 40 cm above the dissecting tray. As the liver perfusion is started, the inferior vena cava is cut well below the kidney to provide the necessary fluid exit. The ligamentum teres hepatis and diaphragm are then cut, and the anterior thoracic wall removed by making parallel cuts of the rib cage along the midclavicular line. A loose ligature is placed around the inferior vena cavajust superior to the diaphragm, as is another just inferior to the right atrium. A small incision is made in the vessel between the two ligatures and an 18-gauge catheter (with the stylet removed and cut to about 1 cm) is inserted and secured. The ligature around the inferior vena cava in the abdomen is then tied. The liver is dissected from the animal by first freeing the lower margin of the abdominal vena cava just below the ligature. The perfusion line is 3 S. R. Wagle and W. R. lngebretsen, Jr., this series, Vol. 35, p. 579. 4 E. Brass, personal communication, 1986.
280
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[30]
then moved to the thoracic catheter, and the portal vein and liver are dissected free from the surrounding mesentery. The perfusion line is then returned to the portal vein, and the liver dissected free from the posterior abdominal wall. The tubing connecting the liver to the perfusion apparatus is removed and the liver is attached to the outlet of the perfusion apparatus by way of the portal vein catheter. The perfusion buffer is adjusted to 1 mM CaCIz by addition of 2 ml 100 mM CaCI2, and 3 units/ml collagenase (Type II, Cooper Biomedical, Freehold, N J) is added. The liver is perfused with this collagenase buffer for 10 min. via the portal vein, 5 min via the inferior vena cava, and then 5 min with collagenase-free/Ca2÷-free buffer via the portal vein. The liver is then disconnected from the perfusion system and placed into a 250-ml silanized glass beaker containing about 30 ml of buffer A warmed to 37°. [All glassware used during the purification of hepatocytes is silanized by rinsing with neat dimethyldichlorosilane (Pierce, Rockford, IL) followed by rinsing with dry methanol.] Hepatocytes are teased from the liver into the buffer using forceps. The resulting cell suspension is filtered through nylon mesh (70 mesh, Nytex nylon) into a 200-ml Erlenmeyer flask and placed into a water bath for 10 min at 37° with gentle shaking and continuous O2/CO2 ventilation. Cells are poured from the flask through the nylon mesh into 50-ml plastic centrifuge tubes chilled on ice, the tubes are filled with buffer B (4°), and then are centrifuged at 65 g for 3 min. After removing the supernatant, the pellet is very gently resuspended in chilled buffer B followed by recentrifugation. This is repeated, and the washed hepatocytes are gently resuspended in chilled buffer C. The cells are counted, and the viability evaluated by the trypan blue exclusion test. 5 Cells used for metabolism studies are always >85% viable. Generation and Purification of Leukotriene Metabolites Incubation Conditions
Incubations with LTE4 are conducted with 3 x 10 6 cells/ml in a gently shaking water bath at 37 ° with O2/CO2 ventilation. For studies of substrate concentration effects, [3H]LTE4, at concentrations of 0.05, 0.5, 10, 25, and 50/zM, is incubated with 2 or 5 ml of cells in a 25-ml plastic tissue culture flask for 30 min. For studies of the time course of LTE4 metabolism, 15 ml of cells in a 50-ml flask are incubated with 5/xM [3H]LTE4 for up to 60 min. Incubations with LTB4 (3/zM) are conducted with 5 × 10 6 cells/ml for 45 min as above. 5 p. O. Seglen, Methods Cell Biol. 13, 29 (1976).
[30]
LEUKOTRIENE METABOLISM
281
Metabolite Purification Hepatocyte incubations are stopped by the addition of 4 volumes chilled ethanol. After storage overnight at - 7 0 ° , precipitated proteins are pelleted by centrifugation at room temperature. For the purification of LTE4 metabolites, the ethanolic supernatant is diluted by addition of l0 volumes of reversed-phase HPLC solvent A (see below), and partially purified by solid-phase extraction on an ODS Sep-Pak. The Sep-Pak cartridges are washed with solvent A and eluted with methanol. The methanol eluent is evaporated under vacuum and redissolved in 80% solvent A : 20% methanol for analysis by reversed-phase HPLC. The radioactivity content of the sample before and after evaporation is determined to quantitate the production of volatile counts. The recovery of tritium-labeled metabolites from the Sep-Paks averages 85% throughout these experiments. The ethanolic supernatant containing LTB4 metabolites is taken to dryness under vacuum and the residue dissolved in water (acidified to pH 3.5 with acetic acid). This sample is taken for direct analysis by reversedphase HPLC. LTE4 metabolites are subjected to reversed-phase HPLC using a linear gradient from 80% solvent A to 100% solvent B. Solvent A is water with 16.6 mM ammonium acetate, pH 4.5, and 132 /~M tetrasodium ethylenediaminetetraacetic acid (EDTA); solvent B is 90% methanol, 10% solvent A, 0.1% acetic acid, and 132/zM EDTA. For LTB4 metabolites, the same reversed-phase HPLC system is used except that ammonium formate is the buffer, the gradient is started at 90% solvent A, and EDTA is not included. The reversed-phase HPLC effluent is monitored by a photodiode array detector (HP-1040A, Hewlett-Packard, Palo Alto, CA), and/or a radioactivity detector (FLO-ONE Beta, model IC radioactive flow detector, Radiomatic Instruments, Tampa, FL). The UV wavelengths monitored are at 270 nm (for LTB4 metabolites), 280 nm, or a broad-band UV signal which sums the absorbance from 260 to 340 nm (for LTE4 metabolites). One assumption made for these experiments is that, during purification and reversed-phase HPLC of leukotriene metabolites, there are no differences in the recoveries of the various metabolites. Total recovery of leukotriene metabolites from reversed-phase HPLC ranges from 50 to 80%. The recoveries of LTE4 metabolites are best when the columns are washed prior to use with water/methanol (90 : 10, v/v) and 132 mM EDTA as described. 6 6 F. S. Anderson, J. Y. Westcott, J. A. Zirrolli, and R. C. Murphy, Anal. Chem. 55, 1837 (1983).
282
BIOSYNTHESIS,
ENZYMOLOGY,
AND
CHEMICAL
SYNTHESIS
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LEUKOTRIENEMETABOLISM
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LTE4 Metabolism LTE4 has been shown to be metabolized by isolated rat hepatocytes to six polar metabolites (Fig. 1). 1 The initial N-acetylation of LTE4 is followed by to-oxidation and/3-oxidation from the to terminus. All of these metabolites retain the typical leukotriene UV chromophore with absorption maxima at 280 nm, except for Metabolite E which is a conjugated tetraene with a UV absorption maximum at 309 nm. The metabolism of LTE4 is dependent on the concentration of LTE4 exposed to the isolated hepatocytes (Figs. 2 and 3). When 50/xM LTE4 is used, the only metabolite observed is N-acetyl-LTE4. However, as the concentration of LTE4 decreases, the conversion of LTE4 to to/fl-oxidized metabolites increases. When 25/xM LTE4 is used, to/fl-oxidized metabolites appear, but N-acetyl-LTE4 is still the dominant metabolite. At low substrate concentrations (less than 5/~M), only to/fl-oxidized metabolites are present after 30-rain incubation. Loss of the tritium label is up to 45% of the total indicating metabolism of LTE4 beyond the 16-carboxydihydro
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FIG. 2. LTE4 concentration effects on metabolism. Reversed-phase HPLC radiochromatograms of [3H]LTE4 metabolites from isolated rat hepatocytes (3 × 106 cells/ml, 30 min) at the LTE4 concentrations indicated. The identities of the metabolites are determined by comparison with biologically derived and synthetic standards (see Fig. 1). Reversed-phase HPLC conditions are as noted in the text.
284
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS lOO
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Substrate Concentration (pM) FIG. 3. LTE4 concentration effects on metabolism. The effect of substrate concentration on the extent of LTE4 metabolism by isolated rat hepatocytes is plotted. Cells are incubated with from 0.05 to 50 p.M LTE4 for 30 rain as described in the text. Data points represent, for the to/B-oxidized metabolites (©), the sum of the reversed-phase HPLC UV peak heights of the metabolites divided by the sum of the peak heights for all metabolites and LTE4. For LTE4 (CD)and N-acetyl-LTE4 (O), the data points represent the reversed-phase HPLC peak heights divided by the sum of the peak heights for all metabolites and LTE4.
metabolite. When the relative amount of to/fl-oxidized metabolites is plotted versus the LTE4 concentration, it is apparent that at substrate concentrations about 10/zM, the rate of LTE4 metabolism by isolated rat hepatocytes is nonlinear. When LTE4 is incubated with hepatocytes at 5/zM, the extent of LTE4 metabolism increases as a function of time. In order to determine the extent to which metabolism continues past the formation of Metabolite D, the broad-band monitoring capability of the photodiode array detector is used to generate a signal derived from monitoring 260 to 340 nm. Although the extinction coefficient of the conjugated tetraene chromophore of Metabolite E is not known, it is estimated to be approximately 80,000 at 309 nm based values reported in the literature. 7 The extinction coefficient for LTE4 has been determined to be 40,000 at 280 nm. 8 The relative changes in the broad band signals are assumed to be linear for all metabolites, and the peak heights for Metabolite E are divided by 2 as a correction for the higher extinction coefficient of this metabolite. Using this approach, the relative amounts of Metabolites C, D, and E change as a function of time (Fig. 4; LTE4, and Metabolites A and B are not shown). Clearly, Metabolite E is a major LTE4 metabolite at 60 rain. It is also apparent that the relative amount of volatile tritium increases over time, and this increase parallels the increases in Metabolites D and E. 7 G. A. J. Pitt and R. A. Morton, Prog. Chem. Fats Lipids 4, 227 (1957). 8 R. A. Lewis, J. M. Drazen, K. F. Austen, D. A. Clark, and E. J. Corey, Biochem. Biophys. Res. Commun. 96, 271 (1980).
[30]
LEUKOTRIENEMETABOLISM
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Time in Minutes FIG. 4. Metabolism of LTEa as a function of time. The amount of Metabolite E increases relative to other metabolites as a function of time during metabolism of LTE4 by isolated rat hepatocytes (5 p~M LTE4, 3 × 106 ceUs/ml). A broad-band UV signal from 260 to 340 nm is used to measure metabolites by reversed-phase HPLC. The relative amount of volatile tritium increases in parallel with the increases in Metabolites D and E. The data are from two experiments, the open points from one experiment, and the closed points from another (see the text for an explanation of how the data is calculated).
LTB4 Metabolism Rat hepatocyte metabolism of LTB4 results in three major metabolites as assessed by reversed-phase HPLC (Fig. 5). As with LTE4 metabolism, LTB4 is also to- and/3-oxidized from the to terminus. The formation of 20-COOH-LTB4 and 18-COOH-LTB4 has been reported previously. 9 Preliminary mass spectral data indicate that 16-COOH-14,15dihydrotetranor-LTB4 is also formed. Volatile products account for approximately 30% of the starting radiolabel. One or more highly polar metabolites, representing 11% of the nonvolatile radiolabel, elute with the 9 T. W. Harper, M. J. Garrity, and R. C. Murphy, J. Biol. C h e m . 261, 5414 (1986).
t]O
286
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
5000
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FIo. 5. LTB4 metabolism by isolated rat hepatocytes. The reversed-phase HPLC radiochromatogram of [3H]LTB4 metabolites formed by hepatocytes (5 ml cells, 5 x 106 cells/ml) incubated with LTB4 (3/zM) for 45 min. The identity of 20-COOH-LTB4 is determined by coelution with a synthetic standard. 18-COOH-LTB4 and 16-COOH-LTB4 are identified by electron impact mass spectrometry. Reversed-phase HPLC conditions are as outlined in the text.
solvent front of the gradient reversed-phase HPLC system. Further studies on the metabolism of LTB4 by isolated rat hepatocytes are in progress. The relevance of the isolated rat hepatocyte model of LTE4 metabolism to the fate of leukotrienes in vivo has been supported by the identification of 20-COOH-LTE4 (Metabolite B) and 16-COOH-dihydro-LTE4 (Metabolite D) in rats injected with LTE4. ~o This hepatocyte system serves as a biosynthetic source of these metabolites. to p. Perrin, J. Zirrolli, D. O. Stene, J. P. Lellouche, J. P. Beaucourt, and R. C. Murphy, Prostaglandins 37, 53 (1989).
[3 1] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n of H u m a n L u n g L e u k o t r i e n e A4 H y d r o l a s e
By N O B U Y A
O H I S H I , TAKASHI I Z U M I , YOUSUKE SEYAMA, TAKAO S H I M I Z U
and
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) catalyzes enzymatic hydration of LTA4 [5(S)-trans-5,6-oxido-7,9-trans-ll,14-cis-eicosatetraenoic acid] to LTB4 [5(S), 12 (R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid], a potent chemotactic substance. This reaction on the epoxide differs from that of epoxide hydrolases (EC 3.3.2.3). The latter METHODSIN ENZYMOLOGY,VOL. 187
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