IRON HOMEOSTASIS REGULATING FACTORS IN
HEPATITIS C
Atef Amer1 ; Yousri Aboelmagd2 and Mohamed Elshahat3*
1professor of organic chemistry, depatment of chemistry , faculty of science, zagazig
university.
2professor of biochemistry,,department of biochemistry , faculty of medicine, zagazig
university .
3master student ,depatment of chemistry , faculty of science, zagazig university.
*Corresponding author : Elshahat304@gmail.com
Key words :Hepcidin,HCV,Iron,liver,fibrosis,cirrhosis .
ABSTRACT :
Hepcidin is the central regulator of systemic iron homeostasis.
Dysregulation of hepcidin production results in a variety of iron disorders.
Hepcidin deficiency is the cause of iron overload in hereditary
hemochromatosis, iron-loading anemias, and hepatitis C. Hepcidin excess is
associated with anemia of inflammation, chronic kidney disease and ironrefractory
iron deficiency anemia. The main objective of this study is to
elucidate the correlation between Hepcidin and Iron as adiagnostic
biomarkers in Hcv infected patients . infection with hepatitis c virus is
amajor cause of choronic liver disease , many experimental and clinical
studies suggest that excessive iron in CHC is a cofactor promoting the
progression of liver damage and increasing the risk for fibrosis. This study
contain two groups; Group I (healthy subjects): This group included fifteen
healthy persons with ages ranged (33-63) years, they had no history of liver
disease which may interfere with the studied parameters, This group
represents 30%. Group II : This group included thirty five patients infected
with hepatitis C virus, their ages range from 41 to 76 years, This group
represents 70%.All clinical individuals in this study were collected from
Outpatient Clinics of Zagazig University Hospitals. The following
parameters ; Complete blood pictures , Liver functions tests , Kidney
functions tests, Hepcidin levels and Iron levels were performed for all
groups.The obtained result revealed asignificant decrease of hepcidin in
HCV infected patients compared to the control group Also, asignificant
increase in Iron level in infected patients compared to control group .
INTRODUCTION:
Hepatitis C virus (HCV) belongs to genus Hepacivirus, family
Flaviridae, and it is a small positive strand RNA, enveloped virus. HCV is
one of the main etiological agents of progressive liver diseases resulting in
liver cirrhosis and hepatocellular carcinoma. Infection by HCV is the main
cause of chronic liver diseases, mainly fibrosis, cirrhosis and liver failure as
well as hepatocellular carcinoma that affect about 170 million people all
Egypt. J. of Appl. Sci., 35 (11) 2020 174-187
over the world (Flamm et al., 2003). In Egypt, the prevalence of HCV is the
highest worldwide (Frank et al., 2000) with an estimated prevalence of
14.7% among the general population during 2008 (El-Zanaty and Way,
2009).
Accumulation of iron in liver is common in HCV infection results in
liver fibrosis and increased risk of hepatocellular carcinoma (Lambrecht et
al., 2011). HCV Patients have a relative low hepcidin level compared to
uninfected peoples (Fujita et al., 2007) resulting in unrestricted duodenal
iron absorption and iron release from macrophages through the iron
transporter ferroportin (Nemeth and Ganz, 2009).
The human hepcidin gene (HAMP) encodes a precursor of hepcidin
– preprohepcidin that is 84 amino acid proteins. Preprohepcidin is cleaved to
give 60 aa prohepcidin that gives rise to hepcidin. Hepcidin: 25 aa, 22 aa and
20 aa peptide are the three forms of hepcidin. These three forms can be
detected in urine, but hepcidin‐ 25 and hepcidin‐ 20 only present in human
serum (Rossi, 2005). The major form of hepcidin is hepcidin‐ 25, which
contains 8 cysteine residues conjugated by disulfide bonds (Krause et al.,
2000).
So, hepcidin is considered as the master regulator of iron
homeostasis. Generally, it decreases the level of iron in serum. This
mechanism depends mainly on the interactions between hepcidin and
ferroportin. Ferroportin, the only known mammalian cellular iron exporter,
is expressed on the surface of hepatocytes, reticulo‐ endothelial
macrophages.
EXPERIMENTAL:
All clinical individuals in this study were collected from
Outpatient Clinics of Zagazig University Hospitals.
Subjects:
Fifty individuals were included in this study. Group I (healthy
control), This group included fifty healthy persons with ages ranged (33-63)
years, they had no history of liver disease, malignant tumors or any other
diseases which may interfere with the studied parameters, This group
represents 30% and the second group, included thirty five patients infected
with hepatitis C virus, their ages range from 41 to 76 years, This group
represents 70%.
Methods:
All groups were subjected to Complete blood pictures ( CBC
),Liver functions tests ( L.F.T), Kidney functions tests (L.F.T), Hepcidin and
Iron levels.Complete blood count (CBC)Was done on automated cell
counter, [ XS 500i (Sysmex, Japan) ] , Liver and kidney functions were
done by using biomed reagents, hepcidin was determined using Human
Hepc25(Hepcidin 25) ELISA Kit supplied by Elabscience Biotechnology
Inc and The quantitative determination of Iron by means of particle-
175 Egypt. J. of Appl. Sci., 35 (11) 2020
enhanced turbidimetric immunoassay using Spectrum Kit supplied by
Egyptian Company for Biotechnology (S.A.E).
RESULTS AND DISCUSSION:
Hepatitis C virus (HCV) is considered as a major health problem
worldwide because HCV patients are at risk of progressive liver disease that
favors the generation of long term complications such as cirrhosis, end stage
liver disease and hepatocellular carcinoma (Spengler and Nattermann,
2007). Egypt has one of the highest prevalence of hepatitis C in the world.
HCV genotype 4 is the most common strain in Egypt followed by HCV
genotype 1 (90% and 10% respectively) (Bazeed et al., 2016)
The liver is the main iron storage organ and it plays a fundamental
role in iron metabolism. The iron transport protein, transferrin, and the major
iron storage protein, ferritin, are both synthesized in the liver. Iron
homeostasis is critical for human because iron is an essential element
necessary for many basic biological processes; however, excess iron may
also be highly cytotoxic. Elevated serum levels of iron represent early
markers for the severity of liver disease.
Hepcidin is synthesized in the liver and it is thought to be a key
regulator for iron homeostasis. It is induced mainly by infection and
inflammation. Hepcidin expression levels in chronic liver diseases were
strongly correlated with either the serum ferritin concentration or degree
of iron deposits in the liver (Farid et al., 2012).
The aim of the present study is evaluation of hepcidin level and
its relation with iron state in HCV infected patients. Serum samples were
collected from control (non HCV) individuals and diseases (HCV
infected patients) for determination of liver function test (Total bilirubin,
Albumin, ALT and AST), kidney function test (Blood urea and
creatinine), CBC (Platelets, WBCs, RBCs and Hb), iron and iron binding
capacity as well as determination of hepcidin level and its relations with
the previous parameters in Table 1 and Fig 1.
Table (1): Statistical analysis of complete blood count (CBC) in
control and diseased samples.
CBC Control (n=15) Diseased (n=35)
Platelets (×106/L) 362.47 ± 11.57 301.69** ± 11.84
WBCs (×109/L) 5.37 ± 1.05 6.03 ± 1.05
RBCs (×1012/L) 4.67 ± 1.01 4.15** ± 1.02
Hemoglobin (g/dL) 13.08 ± 0.21 11.98** ± 0.16
n: Number of examined samples.
Means with ** lables means that HCV group having are highly significant
different than control means (P < 0.01)
** Highly significant
Egypt. J. of Appl. Sci., 35 (11) 2020 176
Bilirubin
dL (Table 2 and Fig. 2).
This result was in line with many authors; Wahib et al. (2006)
who studied twenty HCV/PCR-RNA positive patients and found that
total bilirubin increased in 7 patients (35%) and Souza et al.,(2006) who
reported that hyperbilirubinemia has been associated with HCV
Albumin is produced only by the liver and it is the major protein,
which circulates in the blood. It is important to maintain the oncotic
pressure in the vascular system. Any decrease in the oncotic pressure as a
result of low albumin, fluid leak from the interstitial spaces into the
peritoneal cavity, causing ascites (Nagao and Sata, 2010). Albumin is
also very important in the transportation of many molecules; such as free
fatty acids, bilirubin, hormones and drugs.
As illustrated in Table 2 and Fig. 3, serum albumin was 3.8 in
control samples, while it was 3.5 in HCV patients. HCV patients were
highly significant different than control samples (P < 0.01). This result
was in accordance with Nagao and Sata (2010) who reported low
albumin level (<4.0 g/dL). A low serum albumin concentration is an
indicator on poor liver function.
Data found in Table 2 and Fig. 4, showed that the level of
aspartate aminotransferase (AST) was 27.67 ± 1.33 in control samples,
while this level elevated in HCV patients (36.66 ± 1.95), therefore, HCV
patients were highly significant different than control samples (P < 0.01).
This result agreed with Wahib et al., (2006) who reported that AST
increased in15% of HCV patients and Bazeed et al. (2016) who reported
an extremely significant increased AST in HCV patients (42.7 U/L).
The level of alanine aminotransferase (ALT) was 23.44 ± 3.55
and 28.84 ± 1.07 in control and HCV patients, respectively (Table 2 and
Fig. 6). This result was in line, who concluded that in HCV patients,
alterations in the liver tissue is reflected by ALT elevation and mainly
associated with periportal bridging/necrosis, viral load and duration of
disease. Reported an elevation in ALT in HCV patients (56 U/L), as well
as Bazeed et al. (2016) who reported an extremely significant increased
ALT in HCV patients (50 U/L), who demonstrated that during recent
HCV infection, higher ALT levels were detected.
This result could be attributed to when parenchymal liver cells are
damaged, aminotransferases leak from the liver into the blood, resulting
in elevated levels of these enzymes in the bloodstream.
177 Egypt. J. of Appl. Sci., 35 (11) 2020
Table (2): Statistical analysis of liver function tests in control and
diseased sampl
Liver function tests Control (n=15) Diseased (n=35)
0.76 ± 0.04 0.79 ± 0.04
Median = 3.8
IQR= 0.40
Median = 3.5**
IQR= 0.50
27.67 ± 1.33 36.66** ± 1.95
23.44 ± 3.55 28.84 ± 1.07
n: Number of examined samples.
Means with ** lables means that HCV group having are highly significant
different than control means (P < 0.01)
** Highly significant
Results illustrated in Table 3 and Fig. 7
patients, respectively (Table 3 and Fig. 8).
Table (3): Statistical analysis of kidney function tests in control and
diseased samples.
kidney function tests Control (n=15) Diseased (n=35)
26.33 ± 1.45 24.53 ± 1.55
0.77 ± 0.02 0.83± 0.01
n: Number of examined samples.
These findings were in line, who announced that HCV infection
increases the rate of progression of CKD in patients with
glomerulonephritis with an increase in serum creatinine (1.3 mg/dl).
Complete blood count (CBC) is one of the most commonly
performed blood tests because it reveals the peripheral blood changes. It
is routinely performed in health examinations, even in asymptomatic
patients. The data present in Table 4 and Fig. 9, showed that platelets
count was 362.47 ± 11.57 ×106/L in control samples, meanwhile this
level decreased in HCV patients to be 301.69 ± 11.84×106/L. The HCV
patients were highly significant different than control samples (P < 0.01).
This result was agreed with what had been reported, who
illustrated that thrombocytopenia occurs in HCV infection and in liver
cirrhosis. This result could be attributed to; HCV patients have low levels
of serum thrombopoietin, which is mostly produced by the liver tissue
before its release into the bloodstream and it is the main regulator of
platelet production.
The WBCs count was 5.37 ± 1.05×109/L in control samples,
while this count increased in HCV patients (6.03 ± 1.05×109/L) (Table 4
Egypt. J. of Appl. Sci., 35 (11) 2020 178
and Fig. 10). This result agreed with, who reported that HCV infected
group showed significantly higher WBC, lymphocyte, and monocyte
counts compared with control group.
Hemoglobin level in control samples was 13.08 ± 0.21 g/dL but
HCV patients had a lower level of Hb (11.98 ± 0.16 g/dL) (Table 4 and
Fig11&12). A highly significant different was detected between HCV
patients and control samples (P < 0.01).
Table (4): Statistical analysis of iron and total iron binding capacity
in control and diseased samples.
tests Control (n=15) Diseased (n=35)
Iron (Ug/dL) 66.47 ± 2.13 119.43** ± 2.12
TIBC (Ug/dL) 379.78 ± 2.68 295.74** ± 1.47
n: Number of examined samples.
Means with ** lables means that HCV group having are highly significant
differentce than control means (P < 0.01)
** Highly significant
This result agreed, who reported that HCV is associated with low
levels of Hb compared with control group but disagreed, who reported that
HCV infected group showed significantly higher Hb levels (14.7 ± 1.5 g/dl)
compared with control group (13.4 ± 1.8 g/dl)
The liver is the main iron storage organ because a third of the total
iron of the body is deposited in hepatocytes, portal tracts, sinusoidal
mesenchymal cells, and reticuloendothelial cells. Increased serum iron level
and the decreased level of TIBC were correlated with progressive hepatic
parenchymal disease.
Hepcidin hormone is mainly produced by the liver and released in
circulation. When it reaches the circulation, it regulates the metabolism of
iron by controlling iron transport to the duodenal enterocytes and iron export
from the macrophages. So, it is the key regulator of iron metabolism and is a
significant biomarker for systemic inflammatory states. Hepcidin acts by
binding to iron carriers, causing internalization and lysosomal degradation
(Nemeth, 2010) in Table 5.
Table (5): Statistical analysis of hepcidin level in control and
diseased samples
Test Control (n=15) Diseased (n=35)
Hepcidin (ng/mL) 81.49 ± 0.96 29.98** ± 1.03
n: Number of examined samples.
Means with ** lables means that HCV group having are highly significant
differentce than control means (P < 0.01)
** Highly significant
This result agreed with Fujita et al. (2007), Farid et al. (2012)
who reported that hepcidin is significantly lower in HCV patient.
179 Egypt. J. of Appl. Sci., 35 (11) 2020
The results found in Table 6 illustrated the correlation coefficient
of relations between hepcidin and liver function test, kidney function test,
CBC, iron, total iron binding capacity and HCV. A highly significant
correlation was detected between hepcidin and HCV, AST, platelets
count, RBCs count, Hb, Iron and TIBC (P < 0.05). These correlations
were positive between hepcidin and platelets count, RBCs count, Hb and
TIBC (0.42, 0.39, 0.48 and 0.94, respectively), while negative
correlations were detected between hepcidin and HCV, AST and iron (-
0.65, -0.40 and -0.87). Meanwhile, non-significant correlations were
detected between hepcidin and BIL T, ALB, ALT, blood urea, creatinine
and WBCs.
Table (6): Correlation coefficient of relations between hepcidin and
liver function test, kidney function test, CBC, iron, total
iron binding capacity and HCV.
Hepcidin
r P-value
Age -0.05 0.796NS
BIL T -0.09 0.590NS
ALB -0.07 0.656NS
AST -0.40 0.005**
ALT -0.24 0.094NS
B.Urea 0.17 0.241NS
CRET -0.11 0.459NS
PLT 0.42 0.002**
WBCs -0.26 0.073NS
RBCs 0.39 0.005**
HB 0.48 0.000**
Iron -0.87 0.000**
TIBC 0.94 0.000**
HCV RNA by
PCR
-0.65 0.000**
r: correlation coefficient
0 to 0.2 weak relationship
0.3 to 0.6 moderate relationship
0.7 to1 strong relationship
** Highly significant, NS non-significant
These results agreed, but disagreed with Farid et al. (2012) who
observed inverse correlations. These variations could be attributed to the
differences in disease
Egypt. J. of Appl. Sci., 35 (11) 2020 180
Fig. (1): Platelets (×106/L) count in control and diseased samples.
Fig. (2): WBCs count (×109/L) in control and diseased samples.
Fig. (3): RBCS count (×1012/L) in control and diseased samples.
181 Egypt. J. of Appl. Sci., 35 (11) 2020
Fig. (4): Hemoglobin (g/dL)in control and diseased samples.
Fig. (5): Total bilirubin (BIL T)
Fig. (6 in control and diseased samples
Egypt. J. of Appl. Sci., 35 (11) 2020 182
Fig. (7): Alanine
samples.
Fig. (8
Fig. (9
183 Egypt. J. of Appl. Sci., 35 (11) 2020
Fig. (10): Iron (Ug/dL) in control and diseased samples
Fig. (11): Total iron binding capacity (Ug/dL) in control and diseased
samples.
Fig. (12): Hepcidin level (ng/mL) in control and diseased samples .
Egypt. J. of Appl. Sci., 35 (11) 2020 184
CONCLUSION:
Infection by HCV is a major health problem, threats many peoples
all over the world. The liver is the main detoxifying organ of many
metabolites; also it plays an important role in synthesis of proteins and
production of biochemicals essential for digestion and growth in addition
to its role in iron storage and iron metabolism. For iron homeostasis,
hepcidin is synthesized in the liver and it is the key regulator for iron
homeostasis.
REFERENCES :
Bazeed, F.B. ; E.H. Elsayed and A.A. Abd El-Aziz (2016): Evaluation
of serum aminotransaminases and bilirubin in different
treatment regimens for chronic hepatitis C virus. IOSR-JPBS.;
11(4): 20-26.
El-Zanaty, F. and A. Way (2009): Egypt Demographic and Health
Survey 2008. Egyptian: Ministry of Health. Cairo: El-Zanaty
and Associates and Macro International; p.31. Available
from: http://dhsprogram.com/pubs/pdf/FR220/FR220.pdf.
Farid, S. ; S. Mohamed ; L. Rashid and D. Sbry (2012): A Key
Regulator For Iron Homeostasis in chronic hepatitis C. The
Egyptian J of Hospital Med., 49: 615– 627.
Flamm, S. ; A.M. Di Bisceglie ; A.C. Lyra ; M. Schwartz ; R.K.
Reddy and P. Martin (2003): Hepatitis C-related
hepatocellular carcinoma in the United States: influence of
ethnic status. Am J Gastroenterol.; 98(9): 2060–3.
Frank, C. ; M.K. Mohamed and G.T. Strickland (2000): The role of
parenteral antischistosomal therapy in the spread of hepatitis C
virus in Egypt. Te Lancet; 355: 887–891.
Fujita, N. ; R. Sugimoto ; M. Takeo ; N. Urawa ; R. Mifuji and
Tanaka H. (2007): Hepcidin expression in the liver: relatively
low level in patients with chronic hepatitis C. Molecular Med.;
13(1–2):97–104.
Krause, A. ; S. Neitz ; H. Mägert; A. Schulz ; W. Forssmann and P.
Schulz-Knappe (2000): LEAP-1, a novel highly disulfidebonded
human peptide exhibits antimicrobial activity. FEBS
Lett.; 480: 147–150.
Lambrecht, R.W. ; R.K. Sterling; D. Naishadham; A.M. Stoddard;
T. Rogers and C. Morishima (2011): Iron levels in
hepatocytes and portal tract cells predict progression and
185 Egypt. J. of Appl. Sci., 35 (11) 2020
outcomes of patients with advanced chronic hepatitis C.
Gastroenterol.; 140(5):1490–500.
Nagao, Y. and M. Sata (2010): Serum albumin and mortality risk in a
hyperendemic area of HCV infection in Japan. Virol J.; 7: 375.
Nemeth, E. and T. Ganz (2009): The role of hepcidin in iron
metabolism. Acta Haematol.;122:78–86.
Nemeth, E. ; S. Rivera; V. Gabayan ; C. Keller; S. Taudorf ; B.K.
Pedersen and T. Ganz (2004): IL‐ 6 mediates hypoferremia of
inflammation by inducing the synthesis of the iron regulatory
hormone hepcidin. J Clin Invest.; 113: 1271‐ 1276.
Rossi, E. (2005): Hepcidin the iron regulatory hormone. Clin Biochem
Rev.; 26: 47‐ 49.
Souza, R.M. ; L.A. Freitas ; A.C. Lyra ; C.F. Moraes ; E.L. Braga
and L.G. Lyra (2006): Effect of iron overload on the severity
of liver histologic alterations and on the response to interferon
and ribavirin therapy of patients with hepatitis C infection. Braz
J Med Biol Res.; 39: 79–83.
Spengler, U. and J. Nattermann (2007): Immunopathogenesis in
hepatitis C virus cirrhosis. Clinical science; 112(3): 141-155.
Wahib, A.A. ; El-Nasr M.S. ; A.M. Mangoud ; A.M. El-Shazly
and A.T. Morsy (2006): The liver profile in patients with
hepatitis C virus and/or fascioliasis. J Egypt Soc Parasitol.;
36(2): 405-40.
العوامل المنظمة لإت ا زن الحديد في التهاب الکبدي س
عاطف عامر 1 ، يسري ابوالمجد 2 ، محمد الشحات
-1 استاذ الکمياء – کمية العموم – جامعة الزقازيق 3
-2 استاذ الکمياء الحيويو – کمية الطب – جامعة الزقازيق
-3 قسم الکمياء – کمية العموم – جامعة الزقازيق
تيدف ىذه الد ا رسو الي ايجاد العلاقو بين الييبسيدين والحديد والقدره الاجماليو عمي
ارتباط الحديد في المرضي المصابين بفيروس سي .
وقد اشتممت هذه الد ا رسة عمى الآتي:
فى البداية تم تجميع عينات سيرم من 33 مريض مصاب بالإلتياب الکبدي الوبائي سي
مع أو بدون مضاعفات الکبد وأيضاً 13 عينة لأشخاص سميمة غير مصابة بالإلتياب الکبدي
الوبائي سي من کلا الجنسين من مستشفى الباطنة الجامعى، جامعة الزقازيق.
Egypt. J. of Appl. Sci., 35 (11) 2020 186
تم فحص عينات السيرم کيميائياُ لمع رفة تأثير الإصابة بفيروس سي عمي وظائف الکبد
والکمي وصورة الدم وترکيز الحديد والقدرة الإجمالية عمى الإرتباط بالحديد. بالإضافة إلي تعيين
ترکيز الييبسدين بواسطة الالي ا ز ومقارنة النتائج بالمجموعة السميمة الغيرمصابة.
أظيرت النتائج أن نسبة الحديد قد ا زدت في العينات المصابة بفيروس سي بينما قمت
قدرة إرتباط الحديد الکمية في ىذه العينات بالمقارنة مع المجموعة السميمة.
أوضحت نتائج تحميل العينات بإستخدام الإلي ا ز إنخفاض ممحوظ في کمية ىرمون
الييبسيدين في العينات المصابة بالإلتياب الکبدي الوبائي سي مقارنة مع المجموعة الضابطة.
کما تؤدي الإصابة بالالتياب الکبد الوبائي سي إلي انخفاض مستوى ىرمون الييبسيدين
نتيجة لحالة الالتياب المزمنة التي يحدثيا الفيروس مما أدى إلى ارتفاع الحديد في الدم. لذلک ،
خمصت ىذه الد ا رسة إلى أن الييبسيدين يمعب دو اً ر ميمًا في تنظيم توازن الحديد في الجسم بينما
تؤدي الإصابة بالالتياب الکبد الوبائي سي إلي إحداث خمل في ىذا الإت ا زن
.
187 Egypt. J. of Appl. Sci., 35 (11) 2020
View on SCiNiTO
