European Journal of Clinical Investigation (1989) 19, 53-60

Analysis of cysteinyl leukotrienes in human urine: enhanced excretion in patients with liver cirrhosis and hepatorenal syndrome" M. HUBERT, S. KASTNERt, J. SCHoLMERICHI, W. GEROKI & D. KEPPLERT, TDeutsches Krebsforschungszentr, Heidelberg and tMedizinische Universitiitsklinik Freiburg, Freiburg, FRG

Received 19 April 1988 and in revised form 7 September 1988

Abstract. The cysteiny: leukotrienes, comprising leuk- otriene C4 and its metabolites, are biologically most active mediators, eliminated from the blood circula- tion by the liver and the kidneys. The urine of normal subjects and of patients with hepatic and/or renal failure was analysed for endogenous cysteinyl leuko- trienes. The leukotriene metabolites were separated by reversed-phase high-performance liquid chromato- graphy and subsequently quantified by radioimmu- noassay.

Leukotreine E4 was detected in all urine samples analysed. Its mean concentration increased from 0-3 nmol l-I in healthy subjects to 0.8 nmol 1-' in patients with liver cirrhosis. In patients with hepatorenal syndrome leukotriene E4 averaged 7-8 nmol I-'; in addition, N-acetyl-leukotriene E4 was detected in an average amount of 1.5 nmol I - ' . The mean leukotriene E&reatinine ratio in urine increased from 0-02 in healthy subjects to 0-1 1 in patients with liver cirrhosis and to 1.2 pmol leukotriene E4 mol-' creatinine in patients with hepatorenal syndrome. These results indicate that cysteinyl leukotrienes may play an impor- tant role in the mediator network responsible for the development of the hepatorenal syndrome in patients with severe liver disease.

Keywords. Hepatorenal syndrome, leukotriene analy- sis, liver cirrhosis, urinary leukotrienes.

Introduction

The cysteinyl leukotrienes LTC4, LTD4, and LTE4 are potent mediators of smooth muscle constriction and plasma extravasation [l-81. They produce severe renal

A preliminary report on parts of this work was presented at the 22nd Meeting of the European Association for the Study of the Liver, Torino, Italy, 3-5 September 1987.

Correspondence: M. Huber, Deutsches Krebsforschungszen- trum, Abt. Tumorbiochemie, Im Neuenheimer Feld 280, D-6900 Heidelberg 1, FRG.

Abbreviations: HRS, hepatorenal syndrome; HTMP, 4-hydroxy- 2,2,6,6-tetramethylpiperidine-I-oxyl; LT, leukotriene; RIA, radioimmunoassay; RP-HPLC, reversed-phase high-performance liquid chromatography.

vasoconstriction with concomitant reduction of renal blood flow and glomerular filtration rate in a number of species [9-121. The liver is most active in the uptake, metabolic inactivation, and hepatobiliary elimination of cysteinyl leukotrienes [ 13-26]. Metabolic inactiva- tion comprises both w-oxidation [22-251 and N- acetylation of LTE4 [26-281. The mercapturate, N- acetyl-LTE4, has been identified as a major leukotriene metabolite in rat bile and faeces [26,27,29]. In pn- mates, excretion into the urine is an important additio- nal pathway for the elimination of these mediators, with LTE4 being the predominant metabolite [18,30].

Analysis of systemic leukotriene production in man by measurements in bile [31] requires endoscopic or surgical interventions. We have therefore developed a method for the analysis of cysteinyl leukotrienes in urine. In the present study urine from patients with liver cirrhosis and portal hypertension, with hepato- renal syndrome (HRS), or with hepatic and renal failure due to other aetiologies was analysed for cysteinyl leukotrienes because one may expect that an impaired uptake, metabolic inactivation, and biliary secretion of cysteinyl leukotrienes by a cirrhotic liver is associated with a diversion to renal excretion [16,32,33]. HRS, a functional renal failure that can develop in patients with severe liver disease, is charac- terized by renal vasoconstriction, a reduction in renal blood flow and glomerular filtration rate, and sodium retention [34,35]. Decreased formation of vasodilator prostaglandins PGEz and PGI2 [36-391 and enhanced production of vasoconstrictor thromboxane AZ [39,40] were reported following analysis of urine from patients with HRS. However, neither infusion of prostaglan- dins [41,42] nor the administration of dazoxiben, a thromboxane synthase inhibitor [43], improved renal function. As stated above, cysteinyl leukotrienes are capable of inducing several of the pathophysiological changes observed in functional renal failure [9-121 and therefore, enhanced concentrations of these potent mediators in the kidney of patients with HRS may play a role in the impairment of renal function. A prelimin- ary report on parts of this work was presented earlier 1441-

53

54 M. HUBER et al.

Table la. Laboratory and clinical data of the patients with liver cirrhosis and HRS

Cirrhosis with

mean (n = 5) Cirrhosis HRS mean (n = 8) (range) (range) Nonnal values

Blood urea, serum (mmol 1 - 1)

Creatinine, serum (pnoll-') Bilirubin, total, serum -01 1-1) AST (U 1-l)

Thromboplastin time ("A) Proteins, total,

Serum amylase serum (g I-')

(u 1-9

Uh? Volume/24 h (ml)

Lcukocytes/field

Rend function Endogenous creatinine clearence (ml min-') Na/K ratio

FENA

Liver function Child A/B/C 1451 Previous ascites Encephafopathy Variceal bleeding

6.2 (1.5-17)

83 (53- 168) 70

(18-222) 35

53 (1 1-68)

(42-61) 73

04-94] 98

(40-184)

1460 (640-3000)

10 (5-20)

84 (31-179)

4.1 (2.3-5.9)

0-6 (0.3-1-5)

IlIl6 6 o f 8 6 0 f 8 4 o f 8

35.0' (18.1-46.1) 488' (327-725) 399

(77-736) 50 (30-70) 32* (21-49) 55 (49-62)

100 (50-220)

720

7 (2-15)

(100-22000)

11' (0.6-20)

0.5* (0.2-0.9)

002' (0~01-0~04)

0/0/5 4 o f 5 4 o f 5 2 o f 5

1.7-7.8

<I15

c 21

< 19

70-100

65-80

10-100

-1500

< 4

90-1 70

2-5

05-1.0

'Values in patients with HRS differed from those with cirrhosis by PI 0.01 (Wilcoxon-Mann-Whitney test for the one-sided problem).

Patients and methods

Patients Leukotriene excretion into urine was studied in seven healthy controls (24-54 years of age; two females and five males), in eight patients (17-62 years of age; three females and five males) with liver cirrhosis (five alcoholic, three posthepatitic), in five patients (43-74 years of age; one female and four males) with liver cirrhosis (four alcoholic, one posthepatitic) and HRS as defined by the criteria of Papper [34] and Fine and Sakhrani [351, and in three male patients (25-65 years of age) with hepatic disease associated with renal failure due to other aetiologies. The laboratory and clinical data of the patients are given in Tables la and lb. Cirrhotic patients with and without HRS were not different with respect to the intake of medications at the time of the study. Cirrhosis of the liver was proven

by histological assessment. Patients with HRS were not receiving non-steroidal anti-inflammatory drugs. All HRS patients underwent volume expansion with- out improvement of renal function. One of these patients survived and was re-examined five months later. He did not show evidence of renal failure at the second examination. Fractional sodium excretion FEN^) had increased from 0.01 to 0.9% and the Na/K ratio in urine from 0.5 to 1.8, while serum creatinine decreased from 725 to 150 pmol 1-'. However, para- meters of liver function were similar (total serum proteins: 56 vs. 49 g 1-I; thromboplastin time: 42 vs. 49%; AST: 52 vs. 29 U 1-*; bilirubin 77 vs. 116 nmol I-'; serum cholinesterase 1394 vs. 1230 U 1-I).

Materials LTE4 was from Biosigma, Miinchen, FRG. [14,15-

CYSTEINYL LEUKOTRIENES IN HUMAN URINE 55

Table lb. Laboratory and clinical data of the patients with hepatic disease and renal failure not due to HRS

~~ ~

Liver cirrhosis Liver cirrhosis, chronic glomerulo- encephalopath y. nephritis, protein- renal failure due uria, hypertension, to hypovolemia. Hepatic and renal renal acidosis and Beforelafter volume failure due to anaemia, secondary substitution Weil's disease hyperpara thyroidism

Blood Urea, stnun (mmol I-') Creatinine, serum @no1 I-') Bilirubin, total, serum @ n 0 1 1 - ~ ) AST (U I-') Throitmplistin time ("41) Proteins, total, serum (g I-') Serum amylase (U 1-1)

Urine Volume/24 h (ml) Leukocytes/field

Renal function Endogenous creatinine clearance (d min-I) Na/K ratio FENA (%I Liuer function Child A/B/C [45] Previous ascites Encephalopathy Variceal bleeding

23.411 7.0

4421168

2801222

67/65 48/47

85l--

161-

15ojr900 31-

1/41

010.6 <0-01/0.5

C + + -

28.6

551

314

44 41

56

474

3800 -

36

2.5 0.04

- - - -

24.8

645

12

6 57

62

112

1100 I

9

0.7 0.04

A + - -

3H]LTC4, [ 14,15JH]LTD4, and [ 14, 15-3H]LTE4 (1 -48 TBq mmol-' or 40 Ci mmol-I each) were from New England Nuclear/Du Pont, Boston, MA. N-Acetyl- LTE4 and N-acet~l-[~HlLTE~ were synthesized from LTE4 and [3H]LTE4, respectively, as described pre- viously 1261. Reversed-phase high-performance liquid chromatograpy (RP-HPLC) separation [26] was used to control for the purity of the leukotrienes and to purify LTE4 and N-acetyl-LTE4 employed as standards for the radioimmunoassay (RIA). The concentration of unlabelled leukotrienes was determined by absorbance measurements at 280 nm using a molar absorption coefficient of 40 OOO 1 mol-1 cm-'. 4-Hydroxy-2,2,6,6- tetramethylpiperidine- 1-oxyl (HTMP) was from Sigma, St Louis, MO. Centricon-10 Microconcentra- tors were from Amicon, Danvers, MA. The rabbit cysteinyl leukotriene antiserum was kindly donated by Professor BA Peskar, Ruhr-Universitat Bochum, FRG.

Collection and deproteinization of urine samples

Urine was obtained either from spontaneous micturi- tion or was collected from a catheter introduced into the urinary bladder. Aliquots (1 ml) of each urine sample were immediately mixed with 2 ml of ice-cold 90% aqueous methanol, containing 0.5 mmol 1-' EDTA, 1 mmol 1-' HTMP, pH 8-5, and stored at -20°C under argon. Another aliquot was used to measure the urinary creatinine concentration and to count leukocytes in urine.

The urine samples, containing 60% methanol, were filtered through a low-adsorption, hydrophilic mem- brane with a 10000 molecular weight cutoff (Centri- con-10 Microconcentrator) and the filtrate was eva- porated in a centrifuge under low pressure (SpeedVac, Savant, Farmingdale, NY). The dried samples were resuspended in 80% ice-cold aqueous methanol con- taining 1 mmol 1- ' HTMP, and stored at -20°C under

56 M. HUBER et al.

argon. After at least 3 h, the samples were centrifuged for 15 min at 30 000 g at 4°C and the supernatants were evaporated to dryness. After this second deproteiniz- ation step, the samples were carefully redissolved in 300 pl of 30% aqueous methanol.

Similar results were obtained when cysteinyl leuko- trienes were measured in larger urine samples (e.g. 50 ml) using the following stabilization and deproteiniza- tion procedure: 40 mmol L-serine borate, 20 mmol EDTA, 5 mmol HTMP, and 200 mmol KHC03 were added as a powder per litre of fresh human urine. Samples were then stored at - 20°C under argon. SEP- PAK c18 cartridges (Waters, Milford, MA) were pretreated sequentially with (i) methanol, (ii) a solu- tion containing 90% HzO, 10% methanol, and 14 mmol 1-' EDTA, and (iii) H20. 50 ml of the thawed urine samples were mixed with 1 ml of HCl(1 moll- ') and pumped slowly but without delay through the pretreated cartridges. Polar compounds interfering with leukotriene analysis were eluted with 20% aqueous methanol containing 10 mmol I-' EDTA. Cysteinyl leukotrienes were then eluted with 5 ml of 80% aqueous methanol containing 10 mmol 1-' KHC03. The eluates were evaporated to dryness and the samples resuspended in 80% ice-cold aqueous methanol and further processed as described in the paragraph above. Recovery of cysteinyl leukotrienes after deproteinization and HPLC separation, accord- ing to this procedure was about 55%.

High-performance liquid chromatography

Metabolite separation was performed on a C18-Hyper- sil column ( 4 . 6 ~ 2 5 0 mm, 5-pm particles, Shandon, Runcorn, UK) with a C18-precolumn (Waters, Mil- ford, MA). The mobile phase consisted of methanol, water, and acetic acid (65: 35:O.l by volume), pH 5.6 (solvent I). A second mobile phase was used to confirm the identity of urinary LTE4 and N-acetyl-LTE4: acetonitrile, water, acetic acid (30:70:0-l), pH 5.8 (solvent 11). EDTA (1 mmol 1-I) was present in both mobile phases; the pH was adjusted with ammonium hydroxide. The flow rate was 1 ml min-I. Using these HPLC separations, small amounts of protein remain- ing in the samples were eluted from the column within the first minutes, and thereby separated from cysteinyl leukotrienes. HPLC fractions for RIA were adjusted to pH 7-4 with 1 M K2C03 immediately after elution from HPLC and stored at - 20°C under argon for up to 24 h.

Radioimmunoassay for cysteinyl leukotrienes Radioimmunological analysis in 600 pl aliquots of the HPLC fractions were performed after neutralization and evaporation to dryness using the protocol recently described [18]. In this assay t3H]LTE4 competes with the respective cysteinyl leukotrienes for the antibody binding sites.The lower detection limit for LTE4 was at about 25 fmol. The relative percent cross-reactivities of

LTE4, N-acetyl-LTE4, LTD4, and LTC4 at 50% bind- ing were 100, 280, 260, and 90, respectively [18].

Recovery Leukotriene recovery was controlled by the addition of small amounts of [3HJleukotrienes to the urine samples prior to deproteinization and HPLC separation. After chromatographic separation of the samples 330 pl aliquots of each neutralized 1-min HPLC fraction were counted for radioactivity. The total recovery for LTE4 and N-acetyl-LTE4 averaged 65% (SD 8, n = 24) and 67% (SD 12, n = 24), respectively. Data were corrected for recoveries. Replicate determinations of LTE4 and N-acetyl-LTE4 in urine samples differed by less than

I ,Deocetylation of LTE,NAC!

1 I

1 I I I I 0 10 20 30 40

Retention time (rnin)

F i i 1. RIA of cysteinyl leukotrienes after HPLC separation of the urine from a patient with hepatorenal syndrome. The deproteinized sample was separated in solvent system I and the RIA performed as described under Patientsandrmolyticalmetho&. Arrows indicate the retention times of 13H]leuk0~~e standards added to each urine sample in amounts not interfering with RIA. Non-peak areas were calculated as LTE& immunoreactivity. LmNAc: N-acetyl-LTQ. Upper punel: Pattern of cysteinyl leukotrienes in urine. The dotted and dashed fractions were collected for chemical N-acetylation and enzymatic deacetylation, resptctively. Early HPLC fractions (- - -) contain leukotriene metabolites not detected in the RIA. M W e punel: HPLC and RIA of the LT& fractions shown in the upper panel after chemical N-acetylation. Lower panel: HPLC and RIA of the N-aatyl-LTb fractions shown in the upper panel after enzymatic deacetylation using penicillin amidase f26).

CYSTEINYL LEUKOTRIENES IN HUMAN URINE 57

Table 2a. Cysteinyl leukotrienes in the urine of patients with liver cirrhosis, of patients with HRS, and of normal subjects

Cirrhosis with

mean (n = 5) Cirrhosis HRS Normal subjects mean (n = 8) (range) (range) (range)

mean (n = 7)

L m (nmol I-') 0.8 7.8. 0.3 (0.1-2.6) (4.1- 14) (0.145)

N-ACetyl-LTE, <0.1 1.5* <0.1

L'W 0.1 1** 1.2* 0.02

N-Aatyl-Lm < 0-02 0.22* <0.01

(nmol I-') (0.3-3-3)

pmol/mol creatinine (0.01-0.23) (0.6-1.9) (0.0 14.03)

pmol/mol creatinine (0.03-0.37)

*Values in patients with HRS differed from those with cirrhosis and from values in normal subjects by P10@05 (Wilcoxon-Mann-Whitney test for the one-sided problem).

**Values in patients with cirrhosis differed from values in normal subjects by PjO.01 (Wilcoxon-Mann-Whitney test for the one-sided problem).

10% when separate deproteinizations and HPLC fractionations, using either solvent system I or 11, with subsequent RIA were compared.

Metabolite identiJication Leukotriene metabolites were identified by cochroma- tography with synthetic 3H-labelled leukotrienes in two different HPLC systems and subsequent RIA. In addition, endogenous LTE4 was characterized by chemical N-acetylation followed by HPLC and RIA of the product N-acetyl-LTE4 [26]. Endogenous N-acetyl- LTE4 was enzymatically deacetylated [26] with sub- sequent HPLC separation and RIA of the product LTE4. The complete N-acetylation of LTE4 and the complete deacetylation of N-acetyl-LTE4 was verified by internal 3H-labelled standards. The recovery of LTE4 from N-acetyl-LTE4 was only 40% due to the additional formation of 1 1-trans-LTE4.

Results

Analysis of urinary cysteinyl leukotrienes in hepatorenal syndrome Substantial amounts of cysteinyl leukotrienes were detected in urine of patients with HRS. LTE4 was the major and N-acetyl-LTE4 the minor metabolite with mean concentrations of 7.8 and 1-5 nmol l-l, respect- ively (Fig. 1, upper panel; Fig. 2, upper panel; Table 2a). These cysteinyl leukotrienes cochromatographed with the respective tritiated standard in both solvent system I and 11. The identity was further confirmed by chemical N-acetylation of LTE4 and enzymatic de- acetylation of N-acetyl-LTE4 (Fig. 1). Taking into account their relative cross-reactivities in the RIA, LTC4 and LTD4 concentrations were below 0.1 and 0.03 nmol I-', respectively. The concentrations of

urinary cysteinyl leukotrienes were significantly enhanced in patients with HRS when compared to normal subjects (Table 2a).

Cysteinyl leukotrienes in the urine of patients with liver cirrhosis In the urine from cirrhotics LTE4 was measured in a mean concentration of 0.8 nmol I - ' (Fig. 2, middle panel; Table 2a). Only minimal amounts of N-acetyl- LTE4 were detected in these patients ( ~ 0 . 1 nmol I-'). LTC4 and LTD4 Concentrations in urine were below 0.1 and 0-03 nmol 1-', respectively. When the patient who survived HRS was reexamined 5 months later, urinary leukotriene excretion was within the range of cirrhotic patients without renal failure. In this urine LTE4 amounted to 0-5 nmol I-' (during HRS: 7-0 nmol 1-'); N-acetyl-LTE4 was below 0.03 nmol 1- ' (during HRS: 0-3 IUIIO~ 1-l).

Urinary cysteinyl leukotrienes in patients with hepatic disease and renal failure not due to HRS LTE4 concentrations between 0.1 and 1.3 and N- acetyl-LTE4 concentrations up to 0.7 nmol 1-' were measured in the urine of patients with hepatic disease and renal failure not due to HRS (Table 2b). Such concentrations were also found in cirrhotic patients without renal failure.

Cysteinyl Ieukotrienes in the urine of normal subjects Small but detectable amounts of LTE4 were found in normal subjects with an average concentration of 0.3 nmol 1-' (Fig. 2, lower panel; Table 2a). A good correlation was found between the creatinine concen- tration and the LTE4 concentration in normal urine (r =0-93). This suggests that the urinary leukotriene

58 M. HUBER et at.

Table 2b. Cysteinyl leukotrienes in the urine of patients with hepatic disease and renal failure not due to HRS

LT& ( n m ~ l l - ~ ) N- ACetyl-Lm (nmoll-1) LT& pmol/mol creatinine

junol/mol creatinine N- Amtyl-Lm

Liver cirrhosis, encephalopathy , renal failure due to hypovolemia. Beforelafter volume substitution

~~

1.310.3 0-7/0-1

0.31/0.05

0.17/0.01

- a c 3 .- L

T -

- I 1 I I I

LTE, 5 - I Hepotorenol syndrome. 1 t

4 -

3-

2 - LTC4 L T E ~ N A c LTD4

l -

0 10 20 30 40 Retention time (min)

Figure 2. HPLC separation and RIA of cysteinyl leukotrienes in human urine. Samples were prepared as described in Figure 1 and in Parienrs and analytical methorls. Arrows indicate the retention times of internal ['Hlleukotriene standards. Note the different ordinate scales in the three chromatograms. LThNAc: N-acetyl-LT&.

concentration in normal subjects is mainly dependent on diuresis. As in patients with cirrhosis, only minimal amounts of N-acetyl-LTE4 were measured; LTC4 and LTD4 were below 0-1 and 0.03 nmol 1-I , respectively.

Discussion The predominant hepatobiliary elimination of cystei- nyl leukotrienes relative to renal elimination is indi-

Liver cirrhosis, chronic glomerulo- nephritis, protein- uria, hypertension renal acidosis and Hepatic and renal

failure due to anaemia, secondary Weil's disease hyperparathyroidism

0.2 0- 1 <0.1 < 0.1

0.32 0-0 1

< 0.0 I <0.01

cated by tracer studies in the monkey where about 40% from intravenous [3H]LTC4 were recovered as metabo- lites in bile and about 20% in urine within 5 h [18]. On the other hand, intravenously administered rH]LTC4 was metabolized to urinary ['HILTEd in human volun- teers [30]. Analysis in urine of primates provides therefore an alternative to the analysis of systemic cysteinyl leukotriene production in bile, at least under some conditions. Reduction of the functional liver mass in cirrhotic patients may increase the amounts of LTE4 in urine not only because of a reduced uptake of cysteinyl leukotrienes by hepatocytes but also because of a reduced conversion of LTE4 to w-oxidized meta- bolites.

For the analysis of cysteinyl leukotrienes it was necessary to deproteinize the urine samples and to separate leukotriene metabolites by RP-HPLC in order to remove interfering material and to detect leukotriene metabolites with their individual cross- reactivities in the subsequent RIA. The appearance of LTE4 in urine is in agreement with the rapid degrada- tion of LTC4 to LTE4 in the circulating blood [16- 18,20,21,46]. The urinary excretion of small amounts of LTE4 in healthy subjects points to its synthesis under physiological conditions.

The increased concentration of LTE4 in the urine of patients with HRS (Table 2a), together with the decreased concentrations of prostaglandins [36-391 and the elevated concentration of thromboxane [39,40], provide an explanation for some of the functional renal abnormalities which characterize the HRS. Urinary cysteinyl leukotrienes in HRS may be increased not only because of portocaval shunts and reduced liver function but also because of a pathologi- cally increased production. Bacterial endotoxin [15,29], viral infections [47l, and several types of tissue trauma [17] are stimuli which are capable of eliciting enhanced leukotriene production. Moreover, treat- ment with non-steroidal anti-inflammatory drugs can shift arachidonate oxidation to leukotriene production under some conditions [48]. The possible sources of

CYSTEINYL LEUKOTRIENES IN HUMAN URINE 59

0.5

(Normal

0

0 0 -: ! A :

c cn

Figure 3. Comparison of urinary cysteinyl leukotrienes in normal subjects, patients with cirrhosis, and patients with hepatorenal syndrome. Normal subjects had a rather constant urinary LTG excretion of 0.02 pmol mol- ' creatinine. Asterisks indicate values from the patient who survived HRS. LThNAc: N-acetyl-LTE+

urinary leukotrienes include not only extrarenal tissues and cells, such as mast cells, eosinophils, and mono- nuclear phagocytes, releasing these mediators into the blood circulation [2,17,18], but also cells within the kidney and its blood vessels [lo].

The excretion rate of LTE4 amounted to 10 pmol h-' in normal subjects (range 4-18, n=7), to 40 pmol h-' in patients with liver cirrhosis (range 4-131, n = 8), and to 350 pmol h-' in patients with HRS (range 33- 658, n = 5) . The excretion rate in HRS is, however, influenced in addition by the impaired renal function. In patients with HRS, the LTE&reatinine ratio and the N-acetyl-LTE&reatinine ratio in urine is manifold increased (Table 2a, Fig. 3). Most important, however, the biological activity of these mediators in the kidney depends on their concentrations (Table 2a).

Our results provide evidence for a mediator role of cysteinyl leukotrienes in HRS. They may not only contribute to the development of functional renal failure, but in addition, may be determinants for the survival of patients with HRS. Cysteinyl leukotriene concentrations were only slightly enhanced in the urines from patients with hepatic disease associated with renal failure due to other aetiologies (Table 2b). Thus, a massive increase in urinary cysteinyl leuko- triene excretion was associated only with hepatorenal syndrome. LTD4/LTE4 receptor antagonists and/or inhibitors of leukotriene biosynthesis may thus be considered as a rational and new approach to improve the survival rate of patients with hepatorenal syn- drome.

Acknowledgment

This work was supported by the Deutsche Forschungs- gemeinschaft, Bonn, through SFB 154, Freiburg and the Fonds der Chemischen Industrie, Frankfurt, FRG.

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