C3 to leukotriene Da. Liver and kidney homogenates did not catabolize leukotriene Cs appreciably due to inhi- bition of y-glutamyl transpeptidase in these ...
BIOUH~ICAL CHEMISTRY Vol. 256, No. 18, h u e of September 25, pp. 9573-9578,1981 Printed in U.S.A.
THE JOURNAL OF
Metabolism of Leukotriene C3in the GuineaPig IDENTIFICATION OF METABOLITES FORMED BY LUNG, LIVER, AND KIDNEY* (Received for publication, March 4,1981)
Sven Hammarstrom From theDepartment of Chemistry, Karolinska Znstitutet, S-10401 Stockholm, Sweden reverse phase HPLC' (8) was 1.5 mCi and thespecific activity was 50 [6,6,8,9,11,12-3He]leukotriene C3 was converted to polar metabolites which were excreted in feces and urine Ci/mmol based on radioactivity and UV absorbance measurements. The labeled material co-chromatographed on HPLC with unlabeled for 4-6 daysaftersubcutaneousadministrationto leukotriene C3 and after treatment with y-glutamyl transpeptidase guinea pigs. Lung homogenates converted leukotriene with leukotriene DS. To prepare tritium-labeled leukotriene D3, 10 C3to leukotriene Da. Liver and kidney homogenatesdid pCi of [3&]leukotriene C3 was treated with 0.2 mg/ml of partiaUy not catabolize leukotriene Csappreciably due to inhi- purified porcine kidney y-glutamyl transpeptidase (Sigma) in 0.05 ml bition of y-glutamyl transpeptidasein these tissuesby of 0.1 M T&.HC1/0.01 M MgCh, pH 8.5, a t 37 "C for 30 min. Methanol endogenousglutathione.Liverandkidneyhomogecontaining 0.5% acetic acid (0.05 ml) was added and themixture was nates metabolized ['He]leukotiene D3 to the cysteine injected onto a CI8 Nucleosil (5-pm particles, Machery-Nagel Co., analog, leukotriene E3 (Bernstrom, K., andHammar- Diiran, Germany; 250 X 4.6 m m ) HPLC column using methanol/ In ad- water, 7 3 (v/v), plus 0.1%acetic acid adjusted to pH5.4 with NH4OH strom, S . (1981) J. BioL Chem. 266, 9679-9682). dition leukotriene Ca and products of greater polarity as solvent. The yield of [3&]leukotriene D3 (elution volume, 22 m l ) 8 pCi. Other materials were the same as described before (8). were formed. The conversion of leukotrieneDs to leu- was Animal Experiments-One to 20 pCi of [3&]leukotriene C3, diskotiene C3was catalyzed by y-glutamyl transpeptidase solved in 0.9% NaC1,were injected subcutaneously in the back of male which performed a reverse reaction due to the high guinea pigs weighing430-550 g. Urine and feces were collected sepatissue concentrations of glutathione. The results sug- rately (urine via a piece of plastic tubing to vials containing toluene). gest that the degradation of leukotriene C3 to leuko- After the collecting period, the specimens were stored frozen at -20 triene Ds is important for the further metabolism of OC. Feces specimens were suspended in 5 volumes of 80% ethanol and this leukotrienein liver and kidney. refluxed for 1 h. The extraction procedure was repeated twice and aliquots of the combined filtered extracts (or urine specimens) were removed for radioactivity determination after addition of Instagel (Packard). Counting efficiencies were determined after addition of Slow reacting substance of anaphylaxis (SRS-A) is a smooth 3H20 (feces) or by external standardization (urine) using a Packard muscle contracting factor presumed to be a mediator of al- Model 3385 liquid scintillation counter. Incubations-Lung, liver, and kidneys were rapidly excised, lergic symptoms, e.g. in asthma(reviewedin Ref. l).Although SRS-A was first described in 1938 (2)its structure was eluci- weighed, and minced with a pair of scissors in 4 volumes of cold 20 dated only recently (3, 4). The parent compound is a novel n m KHzP04/72 n m K*HP0&3 mM nicotinamide/3.6 m~ MgC12, pH 7.4 (11).The suspensions were homogenized (Potter-Elvehjem type) derivative of arachidonic acid containing a glutathione sub- and centrifuged a t 950 X g for 15 min. In some experiments, the stituent. It has been designated leukotriene C4 (5).Leuko- supernatant was further centrifuged a t 8,500 X g for 15 min and the triene D4, the corresponding cysteinylglycine-containingde- new supematant was centrifuged a t 104,OOO X g for 60 min. Each rivative of arachidonic acid, has also been identified (6,7). In incubation mixture contained 5 ml of tissue supernatant (950 X g addition, leukotrienes having different numbers of double unless otherwise stated) prewarmed to 37 "C, and 1pCi of 3H-labeled bonds (leukotrienes C3,D3 (8),CS, and Ds (9,lO))have been leukotriene (Cs or D3) was dissolved in 0.02 ml of 80% ethanol. After 30 min at 37 "C (gentle shaking), 4 volumes of ethanol were added. characterized. Tritium-labeled leukotriene C3 of high spe- The mixtures were filtered, concentrated, and subjected to chromacific activity was recently prepared biosynthetically from tography on 10-ml columns of Amberlite XAD-8. These were eluted [5,6,8,9,11,12-3H~]5,8,11-eicosatrienoic acid (8).The present with 20 ml of water and 20 ml of 80% ethanol. report deals with the excretion of this compound after subHPLC-Unless otherwise stated, the column (500X 10 m m ) concutaneous administration and itscatabolism by cell-freeprep- tained CIS Polygosil(5pm; Machery-Nagel) andthe solvent was methanol/water, 75:25 (v/v), plus 0.1% acetic acid, adjusted to pH5.4 arations of lung, liver, and kidney of the guinea pig. with NHIOH. The flow rate was 4.5 ml/min. An LDC Spectromonitor 11, UV detector, set at 280 nm was connected to the column effluent. MATERIALS AND METHODS The pump and injector were Waters Models 6000 A and U6K, ~5,6,8,9,11,12-3H~Leukoh.ienes C3 a n d W-These compounds respectively. Chromatographic fractions were collected on a time were prepared as described before (8). [5,6,8,9,11,12-3&]5,8,11-Ei~~~basis and radioactivity was determined in aliquots of the fractions atrienoic acid (0.1 Ci; 62 Ci/mmol) was incubated with 7. logmasto- after addition of Instagel. cytoma cells in the presence of lo-' M cysteine and 20 p~ A23187 for 20 min at 37 "C. The yield of tritium-labeled leukotriene C3 after RESULTS chromatographic purification on Amberlite XAD-8, silicic acid, and In Vivo Experiments-Radioactivity from [3H6]leukotriene
This work was supported by grants from the Swedish Medical Research Council (Project 03X-5914), the Swedish Cancer Society (Project 1503-02X.3, and Magnus Bergvab Stiftelse. 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.
C3 was quantitatively excreted in feces and urine during 4-5day periods after subcutaneous administration to guinea pigs. Usually about 60% of the radioactivity appeared in feces and 40% appeared in urine (Fig. 1). Unlabeled leukotriene C3 was added to urine or ethanol
' The abbreviation used is HPLC, high performance liquid chromatography.
9573
9574
Metabolism of Leukotrienes
Feces
X
0
0
-
:
c
.-
50-
Urine
"
x
W
-
25
lbo i 0
75
T imc , hours
lis 0
FIG. 1. Excretion of tritium in feces and urine after subcutaneous administration of [5,6,8,9,11,12-*~]1eukotriene Cs(20 pCi, 50 Ci/mmol) to a male guinea pig weighing 430 g.
10
20
I iver
extracts of feces containing excreted radioactivity from [3H] leukotriene C3. The mixtures were purified by chromatography on Amberlite XAD-8, Sephadex G-15, and silicic acid. The final analyses by HPLC showed that theexcreted material did not contain leukotriene C3;the radioactive metabolites were eluted before leukotriene C3, close to thesolvent front. Catabolism of Leukotriene C3 by Lung, Liver, a n d Kidney Homogenates-Identical amounts of [3H6]leukotriene C3 (1 pCi)were incubated at 37 "C for 30 min with 950 X g supernatant fractions from lung, liver, and kidney homogenates; 75-81% of the radioactivity was adsorbed on XAD-8. After elution with 80%ethanol, this material was analyzed by reverse phase HPLC (Fig. 2). In experiments with lung homogenates, three radioactive components were seen. The first peak co-chromatographed with unlabeled leukotriene C3, added as an internal reference. The remaining two peaks (CI and CII)had longer elution times. Little or no products more polar than leukotriene C3had been formed and no radioactivity was eluted with the methanol, used routinely at the end of each analysis. After incubations with liver and kidney homogenates, most or all of the radioactivity co-chromatographed with leukotriene C3. After incubations with kidney homogenates, small amounts of two products with the same chromatographic properties as the lung metabolites were formed. The major lung metabolite (CI) hada relative elution time compared to leukotriene CSof1.5. This is similar to the relative elution time of leukotriene D3 compared to leukotriene C3 (1.6 Ref. 8). Fig. 3 shows HPLC recordings of CI mixed with unlabeled leukotriene D3 (upper) and the same mixture after partial conversion to leukotriene E3 by a dipeptidase from porcine kidney (Lowe? the preparation of enzyme and the characterization of leukotriene E3 are described in an accompanying paper (12)). The results indicate that CI and leukotriene DS are identical. CII co-chromatographed with leukotriene E3 on reverse phase HPLC (see also below under "Catabolism of Leukotriene D3 by Lung, Liver, and Kidney Homogenates). Subfractionation of lung homogenates by differential centrifugation showed that the leukotriene C3-degradingactivities were enriched in a fraction sedimenting between 8,500 and 104,Ooo x g. Effect of GLutathwne on the Conversion of Leukotriene C to Leukotriene D byy-GLutamyL Transpeptidase-Fig. 4 illustrates the effect of glutathione on the conversion of leukotriene C4 to leukotriene D4 by partially purified y-glutamyl transpeptidase from swine kidney (Sigma). Significant inhibition of the reaction was observed at concentrations of 0.5
Elution time, rnin
LTC3
kidney
1
1
1
70 Elutlontime.
mln
FIG.2. Metabolism of leukotriene Csby guinea pig tissue. The diagrams show HPLC recordings of products obtained after incubations of [3Hs]leukotriene C3 (LTC,, 1 pCi, 50 Ci/mmol) with supernatant fractions from guinea pig lung, liver, and kidney homogenates (950 X g,5 ml, 37 "C, 5 min). Radioactivity was determined by liquid scintillation counting of aliquota of chromatographic fractions. glutathione l l ~ and higher. The formation of leukotriene Ed, due to dipeptidase activity in this enzyme preparation, also decreased in the presence of glutathione. Catabolism of Leukotriene D3 by Lung, Liver, a n d Kidney Homogenates-Identical amounts of [3Hs]leukotriene D3 (1 pCi) were incubated with 950 X g supernatant fractions from lung, liver, and kidney homogenates. Experimental conditions were the same as those used above for the incubations with leukotriene CB. The amounts of radioactivity adsorbed to Amberlite XAD-8 showed greater variation than after incubations with leukotriene C3. This was due to more extensive l i be shown below. Fig. conversion to polar metabolites as w ~
9575
Metabolism of Leukotrienes A.
3-
i;
'0 x c
2x
-I
lo
io
io
30
Elutiontirn*.min
LTD3 x
so
I
io
60
Iiver
"
sc FIG. 3. Co-chromatography of CI (Fig,2 ) and leukotriene Ds. The HPLCrecordings were obtained with CI from lung and unlabeled leukotriene D3 (LTDa, upper) and the same mixture after partial conversion by kidney cysteinylglycinase ( l o w e r ; cf. Ref. 12). Radioactivity (X-X) was determined on aliquots of HPLC fractions, and light absorbance a t 280 nm (-) was continuously monitored at the column effluent.
1 .fa 0
~
0
10
.
20
30
J
40
Elution time, rnin
j
50
60
J
70
~
kidney
Elution time. rnin
0
3
1
,
1 2
LTEk r
k
8
[GSH] rnM
FIG. 4. Effects of glutathione on the conversion of leukotriene C, to leukotriene D, by y-glutamyl transpeptidase (partially purified). Leukotriene C, (1 m o l , L T G ) and enzyme (20 pg) in 0.1 ml of 0.1 M Tris-HCl/lO m~ MgCldO-8 m~ glutathione, pH 8.5, were incubated at 37 "C for 30 min. Reactions were stopped with
FIG. 5. Metabolism of leukotriene Dsby guinea pig tissues. The diagrams show HPLC recordings of products obtained after incubations of [3Hs]leukotriene D3 (LTD3, 1 pCi, 50 Ci/mmol) with supernatant fractions (950 X g, 5 ml) from guinea pig lung, liver, and kidney homogenates (conditions as in Fig. 2).
min had been formed. Twenty-five % of the radioactivity from the XAD-8 chromatography appeared in the water eluate. Approximately half (54%) was due toleukotriene Ds, and 20% was due to polar products, eluted close to the solvent front. methanol/0.5% acetic acid (0.1 m l ) and product compone composiMore extensive conversion of leukotriene D3 was observed tions were determined by HPLC (conditions as for the preparation of after incubations with liver homogenates. The same metabo[3Hs]leukotriene Ds; see text). LTD,, leukotiene D,; LTEI, leukolites as were formed by lung (DI and DII) were formed in triene E,. greater amounts (Fig. 5). There was also increased radioactiv5 shows the results of HPLC analyses of 80%ethanol eluates ity eluting close to thesolvent front. Fifty-five % of the tritium from XAD-8 columns. Most of the radioactivity from experi- was eluted with water from XAD-8. It consisted of polar ments with lung homogenates co-chromatographed with un- products (elution time, 5-16 &, 42%), leukotriene Da (28%), labeled leukotriene D3. Two products, DI and DII, hadelution and DII (19%). Kidney homogenates also transformed leukotriene D3 effitimes similar to those of leukotrienes C3 and E , respectively. In addition, more polar products, eluting between 7 and 17 ciently to other products. The major one was DII (Fig. 5).
Metabolism of Leukotrienes
n
I-
u
U
m
2 est
Elution time (mlnl
n 4
FIG. 6. Co-chromatography of DI from lung (Fig. 6 ) and unlabeled leukotriene CS (LTCS).The diagrams show HPLC recordings before ( A ) and after treatment with y-glutamyl transpeptidase ( B ) .
50I
FIG. 8. Conversion of [S&]leukotriene Ds to [S&]leukotriene CSby 7-glutamyl transpeptidase in the presence of glutathione. The diagram shows an HPLC of products from an incubation (37 "C, 30 min) of [3Hs]leuktrieneD3 (LTD,) with y-glutamyl transpeptidase (0.2 mg/ml) and glutathione (8 m).Unlabeled leuktriene CS (LTC3) was added as internal reference after the incubation.
6-
I
10
2'0 $0 Elution time, min
io
50
FIG. 7. HPLC of DII from kidney (Fig. 5) and unlabeled leukotriene Es.LTE3, leukotriene Es.
tamyl Transpeptidase in the Presenceof Gl~tathione-[~H~] Leukotriene D3 (0.2 pci) in 0.05 ml of 0.1 M Tris-HCl/lO m~ MgC12/8 m~ glutathione/0.2 mg/ml of y-glutamyl transpeptidase (Sigma) was incubated for 30 min at 37 "C. A control experiment without glutathione was performed in the same way. After addition of 0.05 ml of 0.5% acetic acid in methanol and 1.5 nmol of unlabeled leukotriene C3, the mixtures were analyzed by HPLC. Fig. 8 shows a chromatogram from an incubation performed in the presence of glutathione. The major radioactive component co-chromatographed with the unlabeled leukotriene C3. The other radioactive component was unchanged leukotriene D3. No formation of radioactive leukotriene C3 was observed in the absence of glutathione. DISCUSSION
Leukotriene C3 is a glutathione-containing derivative of eicosatrienoic acid (n-9). It has been previously demonstrated Polar products, a small amount of DI, and a product eluting that this compound is converted by y-glutamyltranspeptidase after DII were formed too. Forty-six % of the radioactivity from porcine kidney to a corresponding cysteinylglycine-conwas eluted with water from XAD-8.Its composition was: polar taining derivative, leukotriene D3 (8).Moreover, a dipeptidase metabolites, 41% leukotriene D3, 9 % DII, 26%; and product from the Same organ was recently shown to convert leukoeluting after DII, 15%. triene D3by enzymatic removal of glycine to a cysteine DI from lung, liver, or kidney incubations was mixed with derivative of eicosatrienoic acid, leukotriene E3 (12). Similar unlabeled leukotriene C3 and reanalyzed by HPLC. The ra- enzymes are involved in the catabolism of glutathione (redioactive and unlabeled compounds from these analyses were viewed in Ref. 13). The present results showed that labeled collected, treated with y-glutamyl transpeptidase (0.2 mg/ml; products were excreted both in feces (-60%) and in urine 37 "C, 30 min) and analyzed again by HPLC. Fig. 6 shows the (-40%) following administration to guinea pigsof tritiumresults obtained with DI from lung. The results were similar labeled leukotriene C3 ofhigh specific radioactivity. Chromatif DI from liver or kidney incubations wasused instead, ographic analyses of excreted metabolites revealed that leusuggesting that DIand leukotriene CSare identical. kotriene C3 had been completely transformed to more polar DII from lung, liver, and kidney incubations was mixedwith products. unlabeled leukotriene E3. This cysteine derivative of 5-hyIncubations with tissue homogenates demonstrated that droxy-7,9,11-eicosatnenoicacid was prepared from leuko- leukotriene C3was rapidly converted to a less polar metabolite triene D3by treatment with a dipeptidase from porcine kidney by lung. The product was identified as the cysteinylglycine as described in an accompanying report (12). The results of analog of leukotriene C3, leukotriene D3 (Fig. 9). This product an HPLC analysis of DII from kidney are shown in Fig. 7. was not metabolized further to a large extent by lung prepaSimilar results were obtained with DII from lung and liver rations. Some conversion to leukotrienes C3 and E3 was obincubations. Under these conditions, 11-trans-leukotriene D3 served and w l ibe discussed below. (8)was eluted just before leukotriene E3.The results indicate Leukotriene C3 was hardly metabolized at all by liver and kidney homogenates. This was surprising because both tissues that DIIand leukotriene E3 are identical. Conversion of Leukotriene 4 to Leukotriene C3 by y-Glu- from several animal species contain y-glutamyl transpeptidase
9577
Metabolism of Leukotrienes &COOH
H
S"CH2
&cooH
1
S"CH2 CHCONHCH2COOH I
CH CONHCHZ COOH
I
I
NH2
NHCO(CH212 CHCOOH
I
NH2
Leukot r iene 03
Leukotriene C3
CHCOOH
I
CHCONHCH2 COOH
I
NH2 Leukot riene D3
CI FIG. 9. Conversion of leukotriene C3 to leukotriene Da by guinea pig lung.
(13). Furthermore, large uptakes of radioactivity were observed in liver and kidney after intravenous administration of [3H6]leukotriene C3 to mice, as determined by whole body autoradiography? Liver and kidney contain high concentrations of glutathione (values of 6-8 and 1-4 m~ have been reported for the rat (14)),suggesting that endogenous glutathione competed with leukotriene C3 for the enzyme. Experimental evidence for this proposal was obtained by determining the effects of glutathione on the conversion of leukotriene C4 to leukotriene D d by partially purified y-glutamyl transpeptidase. The results (Fig. 4) showed pronounced inhibition by 0.5 m~ and higher concentrations of glutathione. Although leukotriene C3was not altered by liverand kidney homogenates, [3&]leukotriene D3 was efficientlymetabolized by these organs. It thus appears that the reaction catalyzed by y-glutamyl transpeptidase is a critical step in the catabolism of leukotriene C3 by the guinea pig. The major product formed from leukotriene D3 by kidney in particular but also by liver had identical chromatographic properties as its cysteine analog, leukotriene E3, suggesting that it was formed by enzymatic removal of glycine (Fig. 10): The characterization of this compound and of corresponding products from leukotrienes D., and D5is described in anaccompanying report (12). Liver and lung (and to a small degree also kidney) homogenates converted leukotriene D3 back to leukotriene C3 (Fig. 11). This reaction was presumably catalyzed by y-glutamyl transpeptidase using glutathione as glutamyl donor. Experimental evidence for this proposal was obtained by incubating [3H6]leukotriene D3 with the porcine kidney enzyme. In the presence of 8 m~ glutathione a significant conversion to leukotriene CS was obtained (Fig. 8) whereas no leukotriene C3 was formed in the absence of glutathione. y-Glutamyl transpeptidase catalyzes a reversible reaction when glutathione is the substrate(15) and kinetic evidence for the formation of a glutamyl-enzyme intermediate has been reported (15). The present results suggest that this intermediate reacted with leukotriene 4 to form leukotriene C3. The small formation of leukotriene C3 from leukotriene D3 by kidney prep-
'L. E. Appelgren and s.Hammarstrom, J. Biol. Chem., in press.
NHp Leukot riene Eg
CJI, DII FIG. 10. Conversion of leukotriene D3 to leukotriene E3 by
guinea pig liver and kidney.
NH2 Leukotriene
D3
I
Leukotriene C3
NHCO (CH 1 CHCOOH 2 2 I *HZ
DI FIG. 11. Conversion of leukotriene D3 to leukotriene C3by
guinea pig lung and liver. triene D3 to leukotriene Ea. In summary, the datapresented in this reportindicate that transformation of leukotriene C3 to leukotriene D3 is important for the further catabolism of this compound in liver and kidney. Moreover, glutathione influences not only leukotriene C biosynthesis but also its metabolic degradation. Thus, a lowering of glutathione concentrations would be expected to deerease the biosynthesis and enhance the catabolism of leukotriene C compounds. Acknowledgments-We technical assistance.
wish to thank Saga Elwe for excellent REFERENCES
1. Austen, K. F. (1977) Harvey Lect. 73,93-161 2. Feldberg, W . , and Kellaway, C. H. (1938) J. Physwl. (Lond.)94, 187-226
9578
Metabolism of Leukotrienes
3. Murphy, R. C., Hammarstrom, S., and Samuelsson, B. (1979) Proc. Natl. Acad,Sei. U.S. A . 76, 4275-4279 4. Hammarstrom, S., Murphy, R. C., Samuelsson, B., Clark, D. A., Mioskowski, C., and Corey, E.J. (1979)Bwchem. Bwphys. Res. Commun. 91,1266-1272 5. Samuelsson,B., and Hammarstrom,S . (1980)Prostaglandins 19, 645-648 6. Orning, L., Hammarstrom, S., and Samuelsson, B. (1980)Proc. Natl. Acad.Sei. U.S. A . 77,2014-2017 7. m g , L., andHammarstrom, S. (1980)J. BioZ. Chem. 265, 8023-8026 8. Hammarstrom, S. (1981)J. BioZ. Chem. 266,2275-2279
9. Hammarstrom, S. (1980)J. Biol. Chem. 256,7093-7094 10. Hammarstrom, S. (1981)Biochim. Biophys. Acta 663,575-577 11. Bucher, N. L. R., and McGmahan, K. (1956)J. Biol. Chem. 222, 1-15 12. Bernstrom, K., and Hammarstrom, 8.(1981)J. Biol. Chem. 266, 9579-9582 13. Meister, A., and Tate, S. S. (1976)Annu. Rev. Bwchem. 46,559604 14. Davidson, B. E., and Hird, F. J. R. (1964)Biochem. J. (Lond.) 93,232-236 15. Tate, S. S., and Meister, A. (1974)J. Biol. Chem. 249, 7593-7602
