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Plasma erythropoietin levels in anaemic and non-anaemic
patients with chronic liver diseases
Cosimo Marcello Bruno, Sergio Neri, Claudio Sciacca, Gaetano Bertino, Pietro Di Prima, Danila Cilio, Rinaldo Pellicano,
Luciano Caruso, Raffaello Cristaldi
Cosimo Marcello Bruno, Sergio Neri, Claudio Sciacca, Gaetano
Bertino, Pietro Di Prima, Danila Cilio, Luciano Caruso, Raffaello
Cristaldi, Department of Internal Medicine and Sistemic Diseases,
University of Catania, Italy
Rinaldo Pellicano, Department of Gastro-Hepatology, Molinette
Hospital, Torino, Italy
Correspondence to: Professor C.M. Bruno, Dip. Medicina Interna e
Patologie Sistemiche, Osp. S. Marta, via G. Clementi 36, 95124
Catania, Italy. firstname.lastname@example.org
Telephone: +39-95-7435706 Fax: +39-95-7435706
Received: 2003-10-08 Accepted: 2003-12-24
AIM: To investigate the serum erythropoietin (Epo) levels
in patients with chronic liver diseases and to compare to
subjects with iron-deficiency anaemia and healthy controls.
METHODS: We examined 31 anaemic (ALC) and 22 non-
anaemic (NALC) cirrhotic patients, 21 non- anaemic subjects
with chronic active hepatitis (CAH), 24 patients with iron-
deficiency anaemia (ID) and 15 healthy controls. Circulating
Epo levels (ELISA; R&D Systems, Europe Ltd, Abingdon,
UK) and haemoglobin (Hb) concentration were determined
in all subjects.
RESULTS: Mean±SD of Epo values was 26.9±10.8 mU/mL
in ALC patients, 12.5±8.0 mU/mL in NALC subjects,
11.6±6.3 mU/mL in CAH patients, 56.4±12.7 mU/mL in the
cases of ID and 9.3±2.6 mU/mL in controls. No significant
difference (P> 0.05) was found in Epo levels between
controls, CAH and NALC patients. ALC individuals had higher
Epo levels (P<0.01) than these groups whereas ID subjects
had even higher levels (P<0.001) than patients suffering
CONCLUSION: Increased Epo values in cirrhotics, are only
detectable when haemoglobin was lesser than 12 g/dL.
Nevertheless, this rise in value is lower than that observed
in anaemic patients with iron-deficiency and appears blunted
and inadequate in comparison to the degree of anaemia.
Bruno CM, Neri S, Sciacca C, Bertino G, Di Prima P, Cilio D,
Pellicano R, Caruso L, Cristaldi R. Plasma erythropoietin levels
in anaemic and non-anaemic patients with chronic liver diseases.
World J Gastroenterol 2004; 10(9): 1353-1356
Chronic anaemia is often observed in patients with liver disease,
especially in advanced stages, and an inverse relation has been
reported between haemoglobin (Hb) concentration or hematocrit
value and survivorship. Inapparent gastrointestinal bleeding,
folate and vitamin B12 deficiency, autoimmune haemolysis,
altered oxide-reductive balance and hypersplenism are
underlying mechanisms responsible for the anaemic state[2-4].
Moreover, a reduced proliferation of erythroid precursor
cells has been described in the bone marrow of these patients[4,5].
Erythropoietin (Epo) is an endogenous glycoprotein
stimulating erythrocytosis which interacts with erythroid
progenitor cells to promote their proliferation and maintain
their viability as they differentiate[6,7]. The regulation of
erythropoiesis is a biological feedback loop whereby the degree
of tissue oxygenation sets the amount of Epo production, the
concentration of erythropoietin in turn drives the bone marrow
to produce a level of red cells at which oxygen delivery is
sufficient to lower Epo production. The gene codifying for
this growth factor has been isolated and a regulatory region
involved with oxygen sensing has been defined. Expression in
response to hypoxia is mediated via a DNA-binding complex.
Since literature data[9,10] suggest an emerging role of this
hormone in causing haematological changes in chronic
diseases, suboptimal production of, or response to, Epo might
contribute to the pathogenesis of chronic anaemia in cirrhotics.
Some studies regarding the association between Epo levels
and cirrhosis have appeared in literature[11-16] but reported data
The aim of this study was to investigate the circulating
Epo levels in patients suffering from chronic liver disease of
various degrees, with and without concomitant anaemia,
compared to subjects with iron-deficiency uncomplicated
anaemia as well as to healthy controls, in order to assess the
relationship between serum Epo and Hb concentration.
MATERIALS AND METHODS
We examined 74 patients suffering from chronic liver diseases
(21 chronic active hepatitis [CAH] and 53 cirrhosis), 24 patients
with iron-deficiency (ID) uncomplicated anaemia and 15 healthy
control subjects, comparable to sex and age. Haematology,
albumin concentration, aspartate aminotransferase (AST) and
alanine aminotransferase (ALT) levels, serum bilirubin and
prothrombin time (as percentage of prothrombin activity), were
measured in all patients and controls.
Diagnosis of patients with liver disease, was based on
clinical (medical history, physical examination), instrumental
(ultrasonography, endoscopy) and laboratory (liver function
tests) data. In 47 out of these cases, hepatic damage was confirmed
by liver biopsy (in the remaining subjects the procedure was
not necessary, as diagnosis was clinically evident).
In 6 out of 21 CAH patients, the etiological agent was
hepatitis B virus (HBV) while in the remaining 15, hepatitis C
virus (HCV). Of the 53 cirrhotic subjects, 19 were infected
with HBV, 31 with HCV while 3 had a history of alcohol abuse.
According to accepted criteria, thirty-one cirrhotic
patients had normocromic normocytic anaemia (ALC group,
mean±SD Hb 10.2±0.9 gr/dL) and twenty-two were non
anemic (NALC group, mean±SD Hb 13.6±0.7 gr/dL).
None of anaemic cirrhotics had signs of iron-deficiency
70 µg/dL, serum ferritin
transferrin saturation 30%).
50 ng/mL and
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In agreement with Child-Pugh’s classification, 12 of
NALC patients were in class B and 10 belonged to class C. In
the ALC group, 13 patients were in class B whereas 18 in
Cirrhotic subjects affected by gastrointestinal bleeding in
the previous 3 mo (as confirmed by endoscopy and fecal occult
blood testing) and those with suspected hepatocellular
carcinoma (on the basis of ultrasonography, alpha-foetoprotein
and carcinoembryonic antigen levels performed during the
screening) were excluded from the study.
Diagnosis in patients with iron-deficiency anaemia, was
based on clinical (medical history, physical examination), and
laboratory (Hb concentration <12 g/dL, serum ferritin <40 ng/dL
and transferrin saturation<16%) data. None of them had clinical
or laboratory (C-reactive protein) evidence of inflammatory
All subjects had normal renal function (serum creatinine
<1.2 mg/dL, creatinine clearance
characteristics of our study groups are summarised in Table 1.
The study was conformed to Helsinki Declaration and
informed consent was obtained from the whole study series.
A blood sample was obtained from all subjects and
ethylenediaminetetraacetic acid (EDTA) was added. Samples
were centrifuged and plasma was stored at -25
was determined by a commercial ELISA kit (R&D Systems
Europe Ltd, Abingdon, UK). The sensitivity of assay was less
than 0.6 mU/mL. Non specific binding was <1%. Intra and
inter-assay variability averaged 3.1% and 3.5%, respectively.
Epo concentrations were expressed as mU/mL.
Analysis of variance and Kruskall-Wallis test were used
to compare mean±SD between various groups. Relationship
between continuous variables was investigated by correlation
test or multiple regression test when they were more than two.
Covariance analysis was performed to assess the difference in
Epo values for adjusted Hb, between ALC and ID patients.
The regression lines between Hb concentration and Epo values
in these two groups were compared by t-test, according to
current statistical procedures.
70 mL/min). The main
No significant difference (P>0.05) in Hb concentration was
observed between controls (13.9±0.3 gr/dL), CAH (13.7±0.5
gr/dL) and NALC (13.6±0.7 gr/dL) patients. Subjects suffering
from ALC (10.2±0.9 gr/dL) and ID (10.1±0.8 gr/dL) had values
significantly lower than others (P<0.05), but Hb concentration
was similar in these two groups (P>0.05). Among cirrhotic
subjects, Hb concentration was similar (P>0.05) in Child-Pough
class B and class C patients both in ALC (10.4±0. 7 gr/dL versus
10.1±0.9 gr/dL, respectively) and in NALC (13.8±0.4 gr/dL
versus 13.5±0.9 gr/dL, respectively) groups.
Mean±SD of plasma Epo was 9.3±2.6 mU/mL in controls,
11.6±6.3 mU/mL in CAH patients, 12.5±8.0 mU/mL in NALC
subjects, 26. 9±10.8 mU/mL in ALC patients and 56.4±12.7
mU/mL in ID individuals.
Regarding Child-Pugh related allocation, mean±SD of
plasma Epo was 24.18±9.56 mU/mL for patients in class B
versus 30.71±10.82 mU/mL for those in class C of the ALC
group, 12.3±7.8 mU/mL in class B versus 12.9±8.1 mU/mL
in class C patients of the NALC group, respectively.
Statistical analysis did not show any significant difference
between controls, CAH patients and NALC patients (P>0.05).
ALC subjects had significantly higher Epo levels than these
three groups (P<0.01) and patients suffering from ID had
significantly higher Epo levels than ALC patients (P<0.001).
In both NALC and ALC groups, no significant difference
(P>0.05) was observed between class B and class C patients.
Epo levels were not related (P>0.05) to albumin concentration,
AST and ALT values, serum bilirubin and prothrombine time
in any group of patients with liver disease.
An inverse significant relationship between Epo and Hb
was found in ID patients (r =-0.61, P=0.001) but not in ALC
patients (r=-0.22, P>0.05). Covariance analysis revealed a
significant difference (P<0.001) in Epo values for adjusted
Hb concentration between ALC and ID patients.
Finally, the regression lines between Epo and Hb in ALC
and ID patients, were compared and a significant difference
was found (ALC patients: slope -2.47, ES 2.02, y-intercept
52.39; ID patients: slope -9.79, ES 2.7, y-intercept 155.48;
t=2.23, fd=53, P=0.03).
Anaemia is a multifactorial complication of liver cirrhosis[2-5].
Papers regarding abnormalities of circulating Epo in patients
with liver diseases were few and results were contradictory[11-18].
Some authors have reported higher Epo levels in cirrhotic
patients when compared to healthy controls. An inverse relation
between Epo values and the haematological indices has been
described by some groups.
We assessed plasma Epo levels in anaemic and non-anaemic
patients with chronic liver disease compared to healthy controls
and iron-deficiency anaemic subjects.
Our results showed that non-anaemic patients with liver
disease and controls had similar Epo values, in cirrhotic
patients, circulating Epo was not related to Child-Pugh score.
Moreover, multiple regression test documented that Epo levels
were not related to concentration of albumin, bilirubin, ALT
or AST values and prothrombin time in any of these three
groups of patients with liver disease.
This was in part conflicting with the findings of other
authors[12,13,15] and suggested that liver damage itself,
independent of the degree of dysfunction, was not able to alter
circulating Epo levels.
On the other hand, we detected a significant increase in
levels of this hormone both in ALC patients and in ID patients.
Table 1 Main characteristics of investigated subjects
Controls CAH patients
Age (yr, mean ±SD)
Albumin (g/ dL)
AST (U/ L)
ALT (U/ L)
Bilirubin (mg/ dL)
Prothrombin time (%)
However, a significant inverse correlation between Epo and
Hb was found in ID group (r=-0.61, P<0.05) but not in ALC
group (r=-0.22, P>0.05).
Nevertheless, increased Epo level in anaemic patients versus
healthy controls was not enough to assess Epo production.
In fact, the definition of defective Epo production has relied
on a low Epo value in comparison to reference patients with
similar Hb. Consequently, circulating Epo cannot be simply
compared with normal values and levels found must be
evaluated in relation to the degree of anaemia.
As it has been widely accepted that adequate Epo production
occur in patients with iron-deficiency anaemia, we chosen
ID patients as reference group.
Mean±SD of Hb values was similar in the two groups of
anaemic patients, but mean±SD of Epo concentrations in ALC
group was much lower than that in ID patients.
Analysis of covariance showned a significant difference
in Epo values between the two groups (P<0.001). Moreover,
we also compared the regression lines between Epo and Hb in
ALC and ID patients. The slope and y-intercept of two regression
lines (Figure 1) were significantly different (P<0.05).
Figure 1 Comparison between regression lines of Epo and Hb
in ALC and ID patients.
Therefore, although circulating Epo levels in ALC patients,
were higher than that in non anaemic individuals with liver
disease, they were significantly lower in healthy subjects, than
in ID patients. The reason of this finding is unclear. It seems
that an altered Epo clearance did not result in the difference
because normal metabolism of this hormone was maintained
Cazzola et al. described an inverse relationship between
red blood precursor mass and serum Epo. As in cirrhosis, a
reduction of erythroid cells has been reported[4,5], the finding
of lower Epo values in our ALC patients than in ID patients
was not due to an increased utilization by precursor cells.
Therefore, lower Epo values in ALC subjects, are likely
provoked by an impaired synthesis rather than by an increased
Consequently, we think that Epo production occurred in
different quantitative patterns in these two groups, and other
factors besides Hb concentration, could affect its output in
This can explain why no relationship was found between
Hb and Epo in our ALC patients.
In adults, Epo has been found to be synthesised by the
kidney and to a lesser extent by the liver. Thus, liver failure
could endanger the hepatic residual share of Epo synthesis,
even though in this study, the absence of relation between Epo
values and investigated indices of liver dysfunction did not
support this hypothesis. Alternatively, a reduced sensitivity
of renal cells to hypoxic stimuli could be hypothesized.
Furthermore, the inhibitory action of reactive oxygen species
and nitric oxide as well as malnutrition of cirrhotic patients
might contribute to reduction in Epo synthesis[23-26].
Finally, inflammatory cytokines, namely interleukin-1,
tumor necrosis factor and transforming growth factor, are
enhanced in liver diseases and have been found to inhibit
hypoxia-induced erythropoietin production in vitro and in
Whatever the cause of blunted and unsuitable rise in
circulating Epo is the levels of this hormone in anaemic
cirrhotics, appear defective and inadequate with regard to the
degree of anaemia.
Even though Epo deficiency is not only the cause of
anaemia in cirrhotics, its insufficient concentration could play
a role in the persistence of anaemic status, worsening the
outcome of cirrhotic patients.
In conclusion, non-anaemic patients with chronic liver
disease have normal Epo levels. During cirrhosis, elevated Epo
values are detectable in patients with Hb concentration lesser
than 12 g/dL and are not related to the degree of both Hb
concentration and severity of liver dysfunction. In addition,
such an increase is lower than that observed in patients with
iron-deficiency anaemia and appears blunted and inadequate
in comparison to the degree of anaemia.
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Edited by Wang XL and Xu FM
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