THE AMERICAN JOURNAL OF GASTROENTEROLOGY Vol. 95, No.

1, 2000
© 2000 by Am. Coll. of Gastroenterology ISSN 0002-9270/00/$20.00
Published by Elsevier Science Inc. PII S0002-9270(99)00743-1

Hepatic Tissue Endothelin-1

Levels in Chronic Liver Disease
Correlate With Disease Severity and Ascites
I. Alam, M.D., N. M. Bass, M.D., Ph.D., P. Bacchetti, Ph.D., L. Gee, and D. C. Rockey, M.D.
Departments of Medicine and Biostatistics and the Liver Center, University of California, San Francisco,
California; and the Department of Medicine and Liver Center, Duke University Medical Center, Durham,
North Carolina

OBJECTIVES: Plasma endothelin-1 (ET-1) levels are in-
creased in patients with cirrhosis and ET-1 production is
increased in the liver itself during experimental injury.
These data suggest a possible role for this vasoactive peptide
in intrahepatic microcirculatory changes that contribute to
the pathogenesis of portal hypertension in cirrhosis. There-
fore the aims of this study were to determine whether ET-1
levels were abnormal in the livers of patients with cirrhosis
and to investigate possible clinical correlates of altered
hepatic ET-1 in cirrhosis.
METHODS: Liver specimens were obtained from explants at
the time of liver transplantation in 62 cirrhotic patients; 49
without pretransplantation transjugular intrahepatic porto-
systemic shunt (TIPS) and 13 with pretransplantation TIPS.
The presence of ascites was evaluated by physical exami-
nation and ultrasonography. Control specimens consisted of
livers with normal morphology obtained from patients who
died from nonliver-related causes. Hepatic ET-1 was mea-
sured by enzyme immunoassay.
RESULTS: Hepatic ET-1 levels in cirrhotics without (0.17
pg/mg liver tissue) or with TIPS (0.12 pg/mg) were higher
than in control patients [0.04 pg/mg (p ⫽ 0.02 for ET-1
levels in cirrhotics with or without TIPS vs control)]. In
cirrhotics without ascites who had not had TIPS, ET-1 levels
(0.07 pg/mg [0.04 –1.00]) were similar to those of the con-
trols. In contrast, ET-1 content was increased in cirrhotics
with small (0.11 pg/mg; p ⫽ 0.0002) and moderate-to-large
(0.69 pg/mg; p ⫽ 0.0002) amounts of ascites compared to
patients without ascites. There was a modest correlation
between ET-1 levels and Child-Pugh score (correlation co-
efficient 0.32; p ⫽ 0.03) and ET-1 levels were significantly
higher in patients with Child-Pugh score of 13 or greater
(0.88 pg/mg; p ⫽ 0.02) than in those with Child-Pugh score
of 12 or less (0.16 pg/mg).
CONCLUSIONS: Hepatic tissue ET-1 levels are increased in
the liver of patients with cirrhosis. This increase appears to
be proportional to the severity of both liver disease and
ascites. These data raise a possible role for ET-1 in modu-
lation of intrahepatic resistance in cirrhotic portal
hypertension. (Am J Gastroenterol 2000;95:199 –203.
© 2000 by Am. Coll. of Gastroenterology)
INTRODUCTION
The endothelins compromise a family of potent vasocon-
strictors (1). Three unique endothelin peptides, each con-
sisting of 21 amino acids, have been identified and termed
endothelin-1 (ET-1), endothelin-2 (ET-2), and endothelin-3
(ET-3). The endothelins have been implicated in a wide
variety of normal and pathological processes. Their different
effects are in part related to the receptor to which they bind
endothelin-A (ETA) or endothelin-B (ETB) (2). Their major
function appears to be in the local control of vascular tone
(3). ET-1 and ET-3 appear to induce vasoconstriction
through stimulation of smooth muscle cells and vasodilation
due to effects on endothelial cells, respectively.
Circulating levels of immunoreactive ET-1 have been
reported to be elevated in a number of diseases including
hypertension, atherosclerosis, acute myocardial infarction,
and acute renal failure (1). Current information concerning
plasma endothelin levels in cirrhosis is conflicting. During
the past several years, numerous investigators have shown
that both plasma ET-1 and ET-3 concentrations are in-
creased in cirrhotics with ascites, varices, and hepatorenal
syndrome (4 –14). However, some studies have reported
both normal (15) and reduced (16) plasma levels in cirrhosis.
Data demonstrating that ET-1 is overexpressed in differ-
ent cellular compartments in cirrhotic tissue (17, 18), and
that ET-1 is higher in the hepatic vein than in portal vein in
patients with portal hypertension (19), have led investigators
to postulate that the increased ET-1 levels in patients with
cirrhosis are related to increased production by the diseased
liver. This is supported by the recent demonstration of
increased production of ET-1 (preproET-1 mRNA and pep-
tide) in the liver during experimental injury (18), and in-
creased hepatic levels of ET-1 mRNA and ET-1 in cirrhotic
rats with ascites (20). However, hepatic endothelin levels in
patients with cirrhosis have not been reported. Therefore,
the present study was undertaken to determine whether
200 Alam et al. AJG – Vol. 95, No. 1, 2000

ET-1 levels were abnormal in the livers of patients with Table 1. Clinical Characteristics of the Study Group
cirrhosis, and to establish potential clinical correlates of Without TIPS With TIPS Controls
altered hepatic ET-1 in cirrhosis. Variable (n ⫽ 49) (n ⫽ 13) (n ⫽ 8)
Median age (yr) [range] 49 [28–68] 43 [29–61] 50 [27–60]
Gender (%)
MATERIALS AND METHODS Male 69 77 100
Female 31 23 0
Liver specimens were obtained consecutively from explants Etiology (%)
at the time of orthotopic liver transplantation in 62 patients Alcohol alone 12 31
HCV ⫾ alcohol 43 31
with cirrhosis during the period July 1993 to August 1995. HBV 16 8
Thirteen of these patients had undergone a prior transjugular Cholestatic 20 20
intrahepatic portosystemic shunt (TIPS) procedure. The di- Others 9 10
agnosis of cirrhosis was based on histological findings. Child-Pugh class (%)
Patients with alcoholic liver disease had a longstanding B 57 23
C 43 77
history of alcohol abuse, i.e., a consumption exceeding 50 Ascites (%)
g/day for ⬎5 yr. They had all abstained from alcohol use for None 24 38
at least 6 months before transplantation. Patients with Small 37 31
chronic liver disease secondarily to hepatitis C virus (HCV) Moderate–large 39 31
and hepatitis B virus were anti-HCV antibody and hepatitis Patients with alcoholic liver disease had a long-standing history of alcohol abuse,
(⬎50 g/day for ⬎5 yr), but had abstained from alcohol use for at least 6 months before
B surface antigen positive, respectively. Patients who had transplantation. Ascites was evaluated by physical examination and confirmed (and
nonalcoholic, nonviral cirrhosis were classified as choles- quantitated) by ultrasonography. HCV ⫽ hepatitis C virus; HBV ⫽ hepatitis B virus;
TIPS ⫽ transjugular intrahepatic portosystemic shunt.
tatic (secondary to primary sclerosing cholangitis or primary
biliary cirrhosis; with a total bilirubin of ⬎3 mg/dl) and
others, which included genetic hemochromatosis and auto- interassay variability for determination of ET-1 were each
immune hepatitis. The presence of ascites was evaluated by routinely ⬍10%.
physical examination and roughly quantitated (none, small, Because the distribution of ET-1 levels were skewed and
and moderate-to-large) by ultrasonography or CT before included several high level outliers, we reported ET-1 levels
transplantation. The cirrhotic patients did not have hepato- by using the median and range. Furthermore, statistical
cellular carcinoma, as assessed by ultrasound or CT and differences between subgroups were tested using the non-
histology. All cirrhotic patients were on a low salt diet and parametric Wilcoxon two-sample and Kruskal-Wallis tests.
the majority were receiving oral diuretics. Patients were Exact p values for these tests were calculated whenever one
excluded if they had no pretransplantation ultrasound or CT, of the groups being compared contained fewer than five
or if complete clinical data were not available. Indications subjects. Spearman rank correlations were calculated to
for TIPS were variceal hemorrhage (38%), refractory ascites measure and test the association between ET-1 and Child-
(54%), and hepatorenal syndrome (8%). Pugh score (21). A p value of ⬍0.05 was considered sta-
Normal hepatic tissue was obtained from patients who tistically significant.
had died from nonliver-related causes (motor vehicle acci-
dent or in whom liver tissue adjacent to cancers was ob-
tained) (n ⫽ 8). There was no evidence of liver abnormality
RESULTS
in the control patients, as determined from histological Epidemiological and clinical characteristics of the study
examination. This study was approved by the Committee on group are shown in Table 1. The median (range) age of
Human Research of the University of California, San Fran- cirrhotic patients without pretransplant TIPS was 49 yr
cisco and fulfilled criteria for research as put forth in the (28 – 68 yr); 31% were women; 43% were Child-Pugh class
Declaration of Helsinki. C and 43% had hepatitis C virus ⫾ alcoholic liver disease.
Hepatic tissue ET-1 (picograms per milligram) was mea- The median (range) age patients with pretransplantation
sured by enzyme immunoassay (Peninsula Laboratories, TIPS was 43 yr (29 – 61 yr); 23% were women; 77% were
Belmont, CA). In brief, tissue samples from flushed livers Child-Pugh class C and 31% had hepatitis C virus ⫾ alco-
were snap frozen in liquid nitrogen. From these samples, holic liver disease. The median age of the controls was 50
250 –500 mg were individually sonicated in 0.1 mol/L hy- yr (27– 60 yr) and all were men. There was no evidence of
drochloric acid (HCl), boiled for 10 min at 1000°C and then diffuse parenchymal liver disease in any control subject as
centrifuged at 7000 g for 10 min at 40°C. Tris-HCl (1 assessed by routine histological examination of livers.
mol/L; 2:1 volume) was added to supernatants, which were Hepatic ET-1 levels in the patient groups and controls are
then subjected to enzyme immunoassay as described by the shown in Table 2. ET-1 levels were significantly higher in
manufacturer. Known concentrations of ET-1 were used to cirrhotics without (0.17 pg/mg [0.04 –2.9]) or with TIPS
generate a standard curve, which was used to determine (0.12 pg/mg [0.04 – 0.7]) than in the control patients (0.04
peptide concentrations in unknown samples. The intra- and pg/mg [0 – 0.1]; p ⫽ 0.02 for both comparisons). ET-1 levels
AJG – January, 2000 ET-1 Levels and Disease Severity and Ascites 201

Table 2. Hepatic Endothelin-1 (ET-1) Levels in Cirrhotics and

Controls
Hepatic ET-1 Levels
(pg/mg)
Patient Group Median Range
Control (n ⫽ 4) 0.05 0.04–0.1
Cirrhotics without TIPS (n ⫽ 49) 0.17*† 0.04–2.9
Cirrhotics with TIPS (n ⫽ 13) 0.12* 0.04–0.7
Liver tissue was snap frozen immediately after explant removal at the time of surgery
and ET-1 was measured by EIA as described in methods. Values in parentheses
indicate the number of patients in each group.
* p ⫽ 0.02 vs controls; † p ⫽ 0.5 vs cirrhotic patients with pretransplantation
TIPS. TIPS ⫽ transjugular intrahepatic portosystemic shunt.

were lower in cirrhotic patients with TIPS compared with

those without TIPS, although this difference did not reach
statistical significance (p ⫽ 0.5).
In cirrhotics without ascites who had not had TIPS, ET-1
levels (0.07 pg/mg [0.04 –1.00]) were similar to those of the
controls (Table 3). In contrast, ET-1 content was increased
in cirrhotics with small (0.11 pg/mg [0.04 – 0.96]; p ⫽
0.0002) and moderate-to-large (0.69 pg/mg [0.04 –2.9]; p ⫽
0.0002) amounts of ascites compared to patients with no
ascites (Table 3 and Fig. 1).
In cirrhotics without TIPS, there was a significant direct
correlation between hepatic tissue ET-1 levels and Child-
Pugh score (Spearman r ⫽ 0.32; p ⫽ 0.03) (Fig. 2). Fur-
thermore, ET-1 levels were significantly higher in patients
with Child-Pugh score ⱖ13 than in those with Child-Pugh
score ⱕ12 (0.88 [0.48 –2.9] vs 0.16 [0.04 –1.26]; p ⫽ 0.02)
(Table 4).
DISCUSSION
A number of studies have demonstrated that systemic ET-1
levels are increased in patients with cirrhosis (4 –14). How-
ever, ET-1 appears to be rapidly cleared from the circulation
and plasma and serum levels are much lower than those that
mediate biological effects, suggesting little physiologic role
for circulating ET-1 (22). Moreover, experimental data have
shown that ET-1 messenger RNA or peptide levels are
increased in the liver after injury, implicating a paracrine or
Figure 1. Hepatic endothelin-1 (ET-1) levels and ascites in cirrhot-
ics without a transjugular intrahepatic portosystemic shunt (TIPS).
Liver tissue was snap frozen after removal of the explant at the
time of surgery and ET-1 was measured by enzyme immunoassay
as described in methods. ET-1 levels were higher in cirrhotics with
small (p ⫽ 0.0002) and a moderate-to-large (p ⫽ 0.0002) amount
of ascites than in patients with no ascites.
autocrine action for ET-1 (17, 18, 20, 23). For this reason,
we have investigated hepatic, rather than systemic, ET-1.
Our findings, in which we demonstrate increased concen-
trations of hepatic ET-1 in patients with cirrhosis, provide
further support for the postulate that ET-1 is overproduced
in the liver after injury.
In this study we demonstrated that hepatic ET-1 levels
were much higher in patients with a Child-Pugh score of 13
or greater than in those with a Child-Pugh score of 12 or
less. A modest but statistically significant correlation was
found between hepatic ET-1 levels and Child-Pugh score.

Table 3. Hepatic Endothelin-1 (ET-1) Levels in Controls and

Cirrhotics (without transjugular intrahepatic portosystemic shunt)
With Varying Amounts of Ascites
Hepatic ET-1 Levels
(pg/mg)
Amount of Ascites Median Range
Controls (n ⫽ 4) 0.05 0.04–0.1
None (n ⫽ 12) 0.07 0.04–1.0
Small (n ⫽ 18) 0.11* 0.04–0.96
Moderate–large (n ⫽ 19) 0.69* 0.04–2.9 Figure 2. Relationship between hepatic endothelin-1 (ET-1) levels
and Child-Pugh score in cirrhotic patients without a transjugular
Liver tissue was immediately snap frozen after explant removal at the time of surgery intrahepatic portosystemic shunt (TIPS). ET-1 was measured by
and ET-1 was measured by EIA as described in methods. The presence of ascites was
evaluated by physical examination and confirmed by ultrasonography before trans-
enzyme immunoassay in snap frozen explanted tissue as described
plantation. Values in parentheses indicate the number of patients in each group. * p ⫽ in methods. The Spearman correlation coefficient for a relationship
0.0002 vs cirrhotic patients with no ascites. between ET-1 level and Child-Pugh score was 0.32 (p ⫽ 0.03).
202 Alam et al.
Table 4. Child-Pugh Score and Hepatic Endothelin-1 (ET-1)
Levels in Cirrhotics Without Transjugular Intrahepatic
Portosystemic Shunt
Hepatic ET-1 Levels (pg/mg)
Child-Pugh Score Median Range
Controls (n ⫽ 4) 0.05 0.04–0.1
ⱕ12 (n ⫽ 45) 0.16 0.04–1.26
ⱖ13 (n ⫽ 4) 0.88* 0.48–2.9
Liver tissue was snap frozen after explant removal at the time of surgery and ET-1 was
measured by EIA as described in methods. The severity of cirrhosis was classified
according to Pugh’s modification of Child’s classification (21). Values in parentheses
indicate the number of patients in each group. * p ⫽ 0.02 vs cirrhotic patients with
Child-Pugh score ⬍12.
Therefore, these results suggest that the increase in intrahe-
patic ET-1 level is, at least in part, related to the underlying
severity of cirrhosis. A similar observation has been previ-
ously reported by Tsai et al. (6) for plasma ET-1 levels. In
their study, plasma ET-1 concentrations in Child class C
patients were much higher than in Child class A or B
patients, and there was a significant positive correlation
between plasma ET-1 levels and Child-Pugh score.
Potential mechanisms responsible for increased ET-1 pro-
duction in chronic liver disease include endotoxemia (12,
24) and shear stress in response to altered blood flow (25,
26). Endotoxemia is common in patients with cirrhosis (27,
28) and Lin et al. (28) recently demonstrated that plasma
endotoxin levels progressively increased in relation to the
severity of cirrhosis as assessed by Child-Pugh score. En-
dotoxin has also been shown to enhance ET-1 production
from cultured thoracic endothelial cells and hepatic endo-
thelial cells (24, 29). It has also been suggested that hemo-
dynamic forces have important effects on the structure and
function of vascular endothelial cells. Shear stress stimu-
lates ET production (25, 26) and in the setting of a hyper-
dynamic circulation, which is most prominent in those with
advanced disease (30, 31) and could contribute to increased
production of ET-1 in patients with cirrhosis.
We found that hepatic ET-1 levels in cirrhotic patients
with ascites were higher than in patients with cirrhosis
without ascites. The increase in hepatic ET-1 content in
cirrhotics with ascites compared to those without ascites is
consistent with the finding that ET-1 levels were increased
in proportion to the severity of cirrhosis. The 14-fold rela-
tive increase in hepatic ET levels observed in patients with
moderate-to-large ascites as compared with healthy subjects
is greater than that reported in the plasma of cirrhotic
patients with ascites studied by Asbert et al. (10) and Uchi-
hara et al. (12). The higher value in our study may be due
to the fact that we measured intrahepatic levels, whereas the
latter studies measured circulating levels (and did not quan-
titate the amount of ascites). Recently, Leivas et al. (20) also
reported a selective increase in ET production in the liver of
cirrhotic rats with ascites (but not in animals without as-
cites), indicating a possible link between ET and develop-
ment of ascites. The mechanism that accounts for increased
hepatic production of ET-1 in cirrhotics with ascites is
AJG – Vol. 95, No. 1, 2000
unknown, but could be related to factors that stimulate ET-1
production. For example, cirrhotic patients with ascites have
high concentrations of endotoxin, angiotensin II, and anti-
diuretic hormone (32) and elevated ET levels could be
related to an increased intrahepatic synthesis promoted by
these substances (24, 33). Finally, we cannot exclude the
possibility that ET-1 contributes to ascites formation, such
that patients with high intrahepatic ET-1 levels are more
likely to develop ascites.
Although statistical differences could not be detected
between hepatic ET-1 levels in patients with and without a
previous TIPS procedure, there was a trend toward a de-
crease in ET-1 levels in patients with a TIPS. Parallel
findings were reported by Martinet et al. (34), who reported
that shunting for refractory ascites induced a marked de-
crease in ET-1 and big ET-1 in portal and renal veins. The
mechanism by which TIPS could lead to decreased produc-
tion of endothelin is not fully understood, but may be related
to a reduction in pressure within the liver. Future work
including study of increased numbers of patients should
provide a clearer understanding of the complex nature and
effect of TIPS on ET-1 production after portal decompres-
sion with TIPS.
The data presented in this study have important implica-
tions for the pathogenesis of portal hypertension. Because
ET acts principally as a local paracrine or autocrine agent
(35), it is possible that locally produced ET contributes to
increased intrahepatic vascular resistance and concomitant
portal hypertension typical of patients with cirrhosis. Cur-
rent evidence indicates that hepatic stellate cells may con-
tribute to increased intrahepatic resistance by perisinusoidal
contraction and constriction of the sinusoid (36, 37). Given
data demonstrating that all hepatic cell types (hepatocytes,
endothelial cells, Kupffer cells, and stellate cells) express
ET receptors, but are most abundant on stellate cells (38)
and that ET-1 induces potent contraction of stellate cells
(36), it appears that stellate cells are a major target of ET
(37, 39). Therefore, overproduced ET after hepatic injury
and cirrhosis may play an important role in the genesis of
increased intrahepatic resistance.
ACKNOWLEDGMENTS
This study was supported by grants to I.A. from the National
Institutes of Health, Bethesda, MD, (T 32 DK 07007), to the
UCSF Liver Center (P30 DK26743) and to DCR (DK 50574
and DK 02124). We thank Tamara Ryan for assistance in
collecting the liver tissue specimens used in this study.
Reprint requests and correspondence: Don Rockey, M.D., Liver
Center Laboratory, Duke University Medical Center, Sands Build-
ing, Room 336, Research Drive, Box 3083, Durham, NC 27710.
Received Nov. 9, 1998; accepted July 7, 1999.
AJG – January, 2000
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