Metabolism and excretion of peptide leukotrienes in the anesthetized rat

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BBA 52648

Biochimica et Biophysics Acta 921(1987) 486-493

Elsevier

Metabolism and excretion of peptide leukotrienes in the anesthetized rat

Aidan Foster a, Brian Fi~simmons b, Joshua Rokach b and L. Gordon Letts a

Departments of a Pha~maco~o~ and b Medicinal Chemistry Merck Frosst Canada Inc., Pointe Claire-Doruaf, Quebec (Curtada)

(Received 20 May 1987)

Key words: N-Acetylleukotriene E,; Biliary metabolism; Leukotriene metabolism; (Rat blood)

The metabolism and excretion of the peptide leukotrines C,, DA, E, and N-acetylleukotriene E, have been studied in the anesthetized rat. The intravenous admi~s~ation of 13~]le~o~ene C, (2.6 l lo- ‘* mol/kg) showed a rapid clearance of radi~ctivi~ from the blood and a time-relate biliary excretion, recovering 69 & 1.6% (n = 6) over 60 min. Less than 1% of total radioactivity was recovered in the urine over the same time period. Similarly, the intravenous administration of [3H]leukotriene D,, (2.5 l lo-” mol/kg), f3H]leukotriene E, (2.5 l 10-l’ mol/kg) and N-acetylj3H]leukotriene E, (2.1 l lo-” mol/kg) showed a 62 ~fr 7.5% (n = 4), 52 f 1.5% (n = 4) and 37 f 4.6% (n = 5) biliary recovery of radioactivity, respectively, after 60 min. Examination of bile identified leukotriene Ill, and IV-acetylleukotriene E, as the main products, although substantial radioactivity, which probably represents unidentified polar products, was present at the solvent fronts of the reverse-phase HPLC. Time course studies indicated a relatively rapid conversion of leukotriene C, to leukotriene D4, while leukotriene D4 metabolism appeared to be much slower. Leukotriene E, was a minor product, suggesting that the N-acetylation process is rapid. Incubation of [ ‘H]le~o~ene C, in rat plasma and whole blood in vitro resulted in a slow conversion of leukotriene C, to leukotriene D4 and leukotriene E, only. These data suggest that the majority of the leukotriene metabolism and excretion in vivo in the anesthetized rat occurs predominantly in the hepatic system. We conclude that this model is suitable for the measurement of in vivo production of peptide leukotrienes.

Introduction

Leukotrienes C,, D4 and E, are sequential metabolites of arachidonic acid produced via the Slipoxygenase pathway [l]. The have individually been shown to have potent biological actions and are thought to be involved in a number of patho- physiological disorders in vivo [2]. Aside from their potent actions, little is known about their mechanisms of detoxification and subsequent routes of elimination.

Correspondence: L.G. Letts, Department of Pharmacology, Merck Frosst Canada Inc., P.O. Box 1005, Pointe Claire-

Dorvai, Quebec H9R 4P8, Canada.

Recent work has focused on the identification and biological activity of a novel biliary metabolite of the peptide leukotrienes, N-acetylleukotriene E,. This compound has been identified in

the bile and feces of the rat, using both chemical and i~unolo~c~ meth~ologies [3,4]. We have recently shown synthetic ~-acetylleukot~ene E, to have minimal biological activity when compared to the peptide leukotrienes C.,, D4 and E, [5] which is consistent with N-acetylation being a mechanism of detoxification [6] (Fig 1).

In order to understand more fully the fate of these potent metabolites, we have administered 3H-labeled leukotrienes C,, D4, E, and N-acetylleukotriene E, intravenously to anesthetized rats and monitored their clearance, elimination and

AS-2760/87/$03.50 0 1987 Elsevier Science Publishers B.V. (Biomedic~ Division)

LTC4

1 Y-GLUTAMYL TRANSPEPTIDASE

NH,

1 DIPEPTIDASE

1 ACETYLTRANSFERASE

LTD4

LTEI

N-ACETYL-LTE4

Fig. 1. Metabolism of leukotriene C, in the rat. LT, leukotriene.

metabolism. We also examined the metabolism of [ 3 Hlleukotriene C, in rat plasma and whole blood in vitro to observe initial kinetics prior to vascular clearance in vivo.

Materials and Methods

Surgical procedures Inbred male Sprague-Dawley rats [7] were

anesthetized with sodium pentobarbital(65 mg/kg intraperitoneally). Following a tracheostomy (xylocaine, 100 ~1 2% solution subcutaneously), the animals were ventilated with room air using a Harvard Respirator (Model 680). Airways pressure was monitored using a modified Konzlett

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Apparatus to measure changes in insufflation pressure. Mean arterial pressure was monitored by cannulation of the right carotid artery with PE-50 (Clay Adams, NJ) polypropylene tubing using a Statham pressure transducer (Model 4357). Heart rate was derived from the blood pressure signal using a biotachometer (Beckman, type 650780). Each of the variables were displayed and recorded on a Beckman Chart recorder (Model 510F).

Intravenous drug administration was carried out following cannulation of the right jugular vein (PE-50 polypropylene tubing), while the left carotid artery was similarly cannulated for collection of blood samples (when necessary). Urine samples were obtained by exposing the bladder and col- lecting the contents at the end of the experiment. Total volume was measured and a sample was taken for analysis.

The common bile duct was exposed and cannulated (PE-50 polypropylene tubing) after li- gation of the duodenum end. The bile flow rate was monitored to ensure that it stayed within normal limits [8]. All bile samples were collected in 1.5-cc polypropylene Eppendorf tubes under a stream of argon and in the presence of the free radical scavenger 4-hydroxy-2,2,6,6_tetramethyl- piperidinyloxyl (CHydroxyTempo, Aldrich, Mil- waukee, WI).

Determination of metabolite clearance Clearance of [14,15-3H]leukotriene C, (1 pCi/

kg, 39 Ci/mmol, intravenously) was observed by measurement of total radioactivity in blood, bile and urine using standard liquid scintillation techniques (LKB Model 1219). Urinary recovery was established by combining a 200~~1 aliquot of sample with 5 ml Aquasol (New England Nuclear). In order to observe clearance from the systemic circulation, blood samples (0.5 ml) were obtained at 0.5, 1, 2, 5, 10 and 15 min after [3H]leukotriene C, administration. Due to the high color quench obtained with direct counting of whole blood, the samples (0.5 ml) were centrifuged for 5 min at 3000 ‘pm. A 20-~1 aliquot of plasma was taken and combined with 5 ml Aquasol. Biliary excretion of [ 3H]leukotriene C, was observed by collect- ing 3-min fractions over 60 min. Samples were combined with Aquasol (5 ml) and counted (5 min). Similarly, biliary clearances were also de-

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termined following the intravenous administration of [14,15-3H]leukotriene D4 (1 pCi/kg, 40 Ci/ mmol) [14,15-3H]leukotriene E, (1 pCi/kg, 40 Ci/mmol) and N-acetyl[14,15-3H]leukotriene E, (0.5 pCi/kg, 47 Ci/mmol).

Biliary metabolite profiles

The metabolite profiles were examined by the collection, extraction and chromatography of six consecutive lo-min bile samples commencing im- mediately after the intravenous administration of [ 3 Hlleukotriene C, . The metabolite profile of [ ‘Hlleukotriene D4, [ 3H]leukotriene E, and N- acetyl[3H]leukotriene E, were also examined at two discrete time points. A 50-~1 aliquot at I,,,

and t3/, of the metabolite clearance curves were collected and analysed.

Preparation of N-acetyl[14,15-3H]leukotriene E,

N-Acetyl[14,15-3H]leukotriene E, was prepared by treating a solution of [14,153H]leukotriene E, 925 FCi, 47 Ci/mmol), 1 ml of 0.1 M aqueous sodium carbonate/methanol (1 : 1) with 50 ~1 acetic anhydride (excess) at 0 ’ C for 30 min. The resulting mixture was concentrated under a stream of nitrogen and purified by reverse-phase HPLC (SYSTEM 1). Unlabelled N-acetylleukotriene E, was prepared as described previously [5]. The identity of N-acetyl[3H]leukotriene E, was veri- fied by its coelution with the fully characterized unlabelled standard [5].

Leukotriene extraction procedures Each sample was adjusted to a total volume of

1 ml with 0.9% (w/v) saline and combined with 1 mM 4-HydroxyTempo, 30 ~15 M formic acid and 500 ~1 2-propanol. The solution was vortexed and then allowed to stand for 5 ruin at room temperature. After the addition of 1.5 ml of anhydrous ether and further vortexing, the organic layer was taken, combined with 15 ~1 10 M NH,OH, and the solvent was removed (N2). Extraction ef- ficiencies for all metabolites were similar with a 82.1 + 0.7% (n = 18) recovery of radioactivity from collected samples.

In the case of whole-blood analysis, a 50-~1 aliquot was added to 0.5 ml ice-cold methanol in the presence of 4-HydroxyTempo (1 mM) and let stand for 10 min to facilitate protein precipitation.

The sample was then centrifuged (3000 rpm) and the supematant removed. The pellet was washed with 0.5 ml methanol, centrifuged and the super- natants combined. Solvent was then removed (N,).

Incubation of rat blood and plasma with [‘HJ- leukotriene C, in vitro

Rats were anesthetized (sodium pentobarbital, 65 mg/kg intraperitoneally) and the right jugular vein cannulated. Heparin (5000 U/kg in saline) was administered intravenously and 5 min later blood was collected (cardiac puncture) into vacu- miners (Becton Dickenson). Plasma was prepared by centrifugation of the blood at 3000 rpm for 5 min. Both blood and plasma samples (0.5 ml) were then incubated with [3H]leukotriene C, (0.5 PCi, 39 Ci/mmol) at a constant temperature of 38°C. 50-~1 aliquots of each sample were removed at 0.5, 1, 2, 5, 15 and 30 rnin after [3H]leukotriene C, addition. The reactions were quenched by im- mediate extraction as described above.

Chromatography

HPLC separation of the metabolites was achieved by using a Nova Pak Cl8 reverse-phase column (3.9 mm x 15 cm, Waters) with a mobile phase (SYSTEM 1) of 60% methanol in aqueous buffer (0.1% acetic acid (pH 5.4)/l mM EDTA, premixed and run isocratically, 0.5 ml/rnin). Peak identification was carried out on the basis of coinjection with fully characterized synthetic standards in two solvent systems. SYSTEM I and SYSTEM II (30% MeCN in aqueous buffer/O.l% acetic acid (pH 5.4)/l mM EDTA, premixed, isocratic, 0.5 ml/mm.

The fractions eluting off the column were collected at l-mm intervals and a lOO+l aliquot counted in a scintillation counter (LKB model 1219) after addition of Aquasol (New England Nuclear, 5 ml). Peaks were initially identified by retention times of authentic standards in this solvent system (X = 280 nm, range = 0.005 AU). 100 ~1 of the fraction of interest was then coin- jetted with 50 ng at the authentic standard and the eluate coincident with the synthetic standard collected and counted. Identification was con- firmed if the majority of the radioactivity coeluted with the standard.

In the case of bile samples after [ 3H]leukotriene

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C, ad~nistration, the above procedure was car-

ried out for the first experiment while subsequent experiments were based on comparison of reten-

tion times to synthetic standards in SYSTEM I. For blood and bile, identification of separate peaks of radioactivity were carried out as described above for the first experiment. In subsequent experi-

ments, each of the samples were coinjected with 50 ng of each of the synthetic standards and

chromatographed in SYSTEM I.

HPLC equipment used was a model 110 pump (Waters) equipped with a U6K manual injector (Waters). Elution of standards was monitored by a variable ultraviolet wavelength detector (481,

Waters), with data being collected by a Hewlett Packard Integrator (model 3390A). All solvents were HPLC grade or better.

[14,15-3H]leukot~ene C, (39 Ci/mmol), ]14,15- 3H]leukotriene D4 (40 Ci/mmol) and [14,15-

3H]leukotriene E, (40 Ci/mmol) were obtained

from New England Nuclear (Boston, MA). Sodium pentobarbital and xylocaine were obtained from

MTC Pharmaceuticals, Mississauga, Ontario. All statistical observations are described as the

mean * S.E. of the mean, while statistical com- parisons were made by Student’s r-test for unpaired observations.

Results

The intravenous administration of [ 3H]leukotriene C, (2.6. lo-” mol/kg, n = 6) to the anesthetized rat resulted in a rapid elimination of radioactivity from the circulation and a significant time-related biliary excretion of leukotriene metabolites (Fig. 2). Total biliary recovery was 69 + 4.1% over 60 min. Urinary recovery of radioactiv-

ity was 0.9 + 0.3% (n = 3) over the 60-min period. [ 3 Hlleukotriene C, administration showed that the majority of the counts were rapidly eliminated

from the blood stream within 5 min. A profile of the peptido-leukotrienes in the bile

after [3H]leukotriene C, administration is illustrated in Fig. 2. At the early time points, negli- gible amounts of leukotriene C, were observed and leukotriene D4 was the major product. N- Acetylleukotriene E, was the major metabolite at the later time points. In all instance, no significant levels of leukotriene E, were observed. In ad-

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Fig. 2. Top panel: clearance of total radioactivity from the circulation of the anesthetized rat following the intravenous administration of [3H]leukotriene C, (1 nCi/kg, n = 3. Middle panel: percentage recovery of total radioactivity from the bile after [ 3H]leukotriene C, a~nistration (n = 6). Bottom panel: composition of radioactivity recovered from the bile of the rat over time following intravenous [ ‘Hlleukotriene C, administration (n = 3). leukotriene D4, n ; N-acetylleukotriene E,, 0;

and 0, radioactivity remaining at the solvent front.

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dition, there was a significant amount of radioactivity which accumulated over time at the solvent front. The identity of this radioactivity is currently unknown, though rechromatography in more polar

reverse-phase solvent systems established that it did not represent tritiated water.

Intravenous administration of [ 3H]leukotriene

D4 (2.5 . lo-” mol/kg), [3H]leukotriene E, (2.5 . 10-l’ mol/kg) and N-acetylleukotriene E, (2.1 .

lo-” mol/kg) also showed significant biliary

elimination of total radioactivity, giving 61.8 f 7.5% (n = 4), 52.2 + 1.5% (n = 4) and 36.9 _t 4.6%

(n = 5) recoveries, respectively (Figs. 3-5). Extensive biliary metabolism was also observed

with these metabolites (Figs. 2-5). Analysis of

aliquots of bile collected at two time points (t,,, (11 min) and t 3,4 (21 min)) after [3H]leukotriene D4 (Fig. 3) showed a similar profile to that ob-

tained after leukotriene C, administration. leukotriene D4 was the major metabolite in bile collected at T,,, (11 min), with N-acetylleukotriene E, being present in smaller but significant amounts. At t 3,4 (21 min), leukotriene D4 had been further transformed, with N-acetylleukotriene E, being present in approximately equal

amounts. Again, there were no significant amounts of leukotriene E, observed. The unidentified radioactivity at the solvent front is significant (15.6%

of observed radioactivity at t,,, and 25.6% at t,,, and, as was observed following the administration of leukotriene C,, appeared to be time-related. There is an extraction artifact evident in the HPLC profiles of all the biliary metabolites. It was identified as such by the extraction and HPLC of authentic standards added to bile. This artifact was not present in extractions from either saline, whole blood or plasma.

Further studies with [ 3H]leukotriene E, (Fig. 4) showed that the only major metabolite identified was N-acetylleukotriene E, (t,,, = 12 min, t,,, = 21 mm). In addition, the intravenous administration of N-acetyl[ 3H]leukotriene E, (Fig. 5) showed an almost identical pattern to that of leukotriene

E, (t,,, = 11 min, t,,, = 24 min). Again, substantial amounts of radioactivity were detected at the solvent front in both cases.

The metabolism of [3H]leukotriene C, was also examined in rat plasma and whole blood in vitro and the results are illustrated in Fig. 6. The only

LTD.,

Fig. 3. Top panel: percentage recovery of total radioactivity from the bile of the anesthetized rat following the intravenous

administration of [‘Hlleukotriene D4 (1 pa/kg n-4). Middle panel: HPLC profile of bile collected at t,,, (11-14 min) after [ 3H]leukotriene D4 administration. Bottom panel: HPLC pro-

file of bile collected at t,,, ( 20-23 min) after [ ‘Hlleukotriene

D4 administration. * Extraction artifact, see text. LT, leukotriene.

LTE, N-AC LTE.q

Fig. 4. Top panel: percentage recovery of total radioactivity from the bile of the anesthetized rat following the intravenous administration of [3H]leukotriene E, (1 pCi/kg, n = 4). Mid- dle panel: HPLC Profile of bile collected at f,,z (11-14 min) after [‘Hlleukotriene E, administration. Bottom panel: HPLC profile of bile collected at tj,4 (19-22 min) after [ 3H]leukotriene E, administration. * Extraction artifact, see

text. LT. leukotriene.

Fig. 5. Top panel: percentage recovery of total radioactivity from the bile of the anesthetized rat following the intravenous administration of N-acetyl[ ‘H]leukotriene E, (0.5 ~Ci/kg, n = 5). Middle panel: HPLC profile of bile collected at t,,, (15-18 min) after N-acetyl[ 3H]leukotriene E, administration. Bottom panel: HPLC profile of bile collected at t3,4 (23-26 min) after N-acetyl[‘H]leukotriene E, administration. * Ex-

traction artifact, see text. LT, leukotriene.

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Discussion

TIME WIN)

%

TIME (MIN)

Fig. 6. Time course of the metabolism of [ 3H]leukotriene C,

(0.5 pa/O.5 ml) in rat plasma (top panel) and whole blood

(bottom panel) in vitro (n = 3). 0, Leukotriene C,; 0;

leukotriene D,; 0, leukotriene E,.

metabolites observed under these conditions were leukotrienes C,, D4 and E,. As shown (upper panel, Fig. 6) within 5 min there was relatively little metabolism of leukotriene C, in plasma (22.9

k 0.7%), with leukotriene D4 (16.1 k 1.9%) and leukotriene E, (6.8 + 1.4%) comprising the identified metabolites. In contrast, in whole blood (lower panel, Fig. 6) there was a slightly faster rate of metabolism of leukotriene C, (31 + 2.2% at 5 mm), with the leukotriene E, levels being significantly higher (15.2 + 1.6%). This suggests that the dipeptidase activity is significantly greater in whole blood than in plasma.

These results show that in the anesthetized rat the intravenous administration of 3H-labeled leukotriene C,, leukotriene D4, leukotriene E, and N-acetyl leukotriene E, results in their rapid clearance from the circulation. The major route of elimination was identified to be via biliary excretion. It was observed that leukotriene D4 was the major biliary metabolite at early time points after either leukotriene C, or leukotriene D4 administration. Of note was that very little leukotriene C,

was detected following leukotriene C, administra-

tion, which implies that the breakdown of

leukotriene C, to leukotriene D, is quite fast, while leukotriene D4 metabolism is comparatively

much slower. Further examination of the metabolism of

leukotrienes C, or leukotriene D4, showed no evidence of biliary excretion of leukotriene E,. Also, the intravenous administration of leukotriene E, showed no evidence of biliary excretion of leukotriene E,, with the majority of the radioactivity in the bile being confined to N-acetylleu-

kotriene E,. This suggests that N-acetylation of leukotriene E, is quite efficient in this species. In

light of this fast conversion, it is interesting that leukotriene E, and N-acetylleukotriene E, showed significantly different biliary recoveries after intravenous administration (P < 0.05, unpaired Stu-

dent’s r-test). This would further suggest that for optimal elimination the acetylation process should not occur until after the metabolite is well ad-

vanced into the hepatic system. It is generally considered that hepatic N-acetylation takes place in the cytosol of the liver [6].

The metabolism of [3H]leukotriene C, with rat whole blood and plasma in vitro showed a time-related conversion of leukotriene C, to leukotrienes D4 and E,, with no evidence of N-acetylation or of the formation of other products. In whole blood the main metabolite is leukotriene E, but, interest- ingly, its rate of formation is comparatively slow when compared to the rapid clearance of leukotriene C, from the circulation (Fig. 2). This would therefore indicate that leukotriene C, metabolism in whole blood or plasma is of limited physiological importance in the intact animal.

Of further interest in these studies was the

consistent observation of radioactivity eluting at

the solvent front of each of the HPLC systems. This radioactivity (which comprised up to 30% of the total radioactivity in each case) appeared to be time dependent and reached a maximum at the later time points. This more polar material(s) probably represents further metabolism of the peptido-leukotrienes. Studies directed towards the identification of these products is currently in progress.

In summary, the peptide leukotrienes are

rapidly cleared from the systemic circulation of the anesthetized rat. The major route of excretion

is via hepatic detoxification, with subsequent biliary elimination. Urinary excretion is minimal.

The anesthetized rat appears to be a good species to study the in vivo production of peptide leukotrienes.

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References

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Paoletti, R. and Ramwell, P., eds.), Vol. II, pp. 11-13, Raven

Press, New York

Ford-Hutchinson, A.W. and Letts, L.G. (19d6) Biological

Actions of Leukotrienes. Hypertension Supplement II, Vol.

8, H-44-11-49

Oming, L., Norin, E., Gustafsson, B. and Hammarstra, S.

(1986) J. Biol. Chem. 261, 766-771

Hagmann, W., Denzlinger, C., Rapp, S., Weckbecker, G.

and Keppler, D. (1986) Prostaglandins. 31, 239-252

Foster, A., Fitzsimmons, B. and Letts, L.G. (1986) Pros-

taglandins 31, 1077-1086

Williams, R.T., (1959) Detoxification Mechanisms, 2nd Edn.,

Wiley, New York

Holme, G. and Piechuta, H. (1981) Immunology 42, 19-24

Klassen, C.D. and Watkins, J.B. (1986) Pharmacol. Rev. 36,

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Metabolism and excretion of peptide leukotrienes in the anesthetized rat

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