Hyperferritinemia Has Multifactorial
Etiology in Naive Patients with Chronic
Ibrahim Ahmad Hussein El Bacha*, Maria Cristina Elias, Ivonete Sandra de Souza e Silva, Ayk Helena Barbosa Martins and Edison Roberto Parise
Departamento de Medicina, Universidade Federal de São Paulo Ibrahim, Brazil
Submission:June 15, 2020; Published:July 10, 2020
*Corresponding author:Ibrahim Ahmad Hussein El Bacha, Departamento de Medicina, Disciplina de Gastroenterologia, Universidade Federal de São Paulo Ibrahim Ahmad Hussein El Bacha, Rua Dona Adma Jafet, 50 cjs. 144/145 – 14º andar – Bela Vista – São Paulo – SP CEP 01308-050, Brazil
How to cite this article:Ibrahim A H E B, Maria C E, Ivonete S d S S, Ayk H B M, Edison R P. Hyperferritinemia Has Multifactorial Etiology in Naive
Patients with Chronic Hepatitis C. Adv Res Gastroentero Hepatol, 2020;15(3): 555914. DOI: 10.19080/ARGH.2020.15.555914.
Background & Aim: Traditionally, elevated serum ferritin is related to the presence of iron accumulation in the body, especially in the liver. However, in chronic hepatitis C several other factors have been alternatively associated with hyperferritinemia, such as liver enzymes, metabolic factors, hepatic steatosis and liver fibrosis. This study aimed to evaluated the exact contribution of these variables to ferritin serum levels in chronic hepatitis.
Methods: Naïve patients with chronic hepatitis C were selected retrospectively and their ferritin serum levels were correlated with demographic data (age and gender), biochemical (AST, ALT, GGT, ALP and glucose by automated methods; insulin by immunofluorimetry), virological (viral load and genotyping) and histological variables(degree of steatosis, inflammation and staging). Statistical analysis were performed by ANOVA complemented by Dunn’s test and logistic regression analysis.
Results: In two hundred and eighty-two patients with mean age of 50 years and a predominance of male patients (56%) and genotype 1 (65%),the mean serum ferritin levels were 561 ng/mL for males and 270 ng/mL for females with a significant difference in ferritin levels between genders, irrespective of age. The mean HOMA-IR was 3.23 Histological examination showed that 23.4% of the patients had liver cirrhosis, 64.2% fatty liver, and 20% hepatic siderosis. The factors independently associated with hyperferritinemia in multivariate logistic regression analysis were ALT (p = 0.037), GGT (p < 0.001), hepatic siderosis (p = 0.002), steatosis (p = 0.027), and HOMA-IR (p = 0.014).
Conclusion: In treatment- naïve patients with chronic hepatitis C, hyperferritinemia has a multifactorial etiology
Hyperferritinemia is a frequent finding in chronic hepatitis C and affects about 20 to 30% of patients; however, only a small percentage of these patients show evidence of iron accumulation in tissue [1,2]. The increase in ferritin associated with iron overload in patients with chronic hepatitis C has been attributed to alterations in the secretion of hepcidin, a hormone secreted by the liver that regulates intestinal iron absorption [3,4] as well as to other concomitant diseases such as hereditary hemochromatosis, hematological diseases, multiple blood transfusions, porphyria cutanea tarda, chronic alcoholism, and hemodialysis. In the absence of siderosis, hyperferritinemia in hepatitis C has also been associated with inflammatory activity such as acute-
phase proteins, tumor growth and, recently, abnormal glucose metabolism especially in the presence of type 2 diabetes mellitus [5-7]. In addition, increased levels of this transport protein are related to the presence of hepatic steatosis and oxidative stress, and the response to antiviral therapy with interferon [8,9].
Considering the large number of factors that have been associated with hyperferritinemia in patients with chronic hepatitis C, the objective of the present study was to evaluate the correlation of serum ferritin levels with demographic, histological and biochemical (liver enzymes and insulin resistance syndrome) parameters in treatment-naïve patients with chronic hepatitis C.
This was a retrospective study that evaluated the records of
patients chronically infected with hepatitis C virus (HCV), followed
up at the Liver Disease Outpatient Clinic of the Department of
Gastroenterology, Unifesp. Patients who met the following criteria
were included: infection with HCV (confirmed by the detection
of HCV- RNA in serum), liver biopsy with a diagnosis of chronic
hepatitis, and determination of blood ferritin levels.
Exclusion criteria were coinfection with HBV and/or HIV,
chronic alcohol consumption (> 20 g/day), presence of debilitating
diseases, use of immunosuppressors, previous antiviral therapy,
a history of multiple blood transfusions, iron replacement
therapy, known hematological disease, known chronic systemic
diseases, current or past gastrointestinal bleeding, hepatocellular
carcinoma, and other known neoplasms.
HCV-RNA was investigated and quantified in serum by the
polymerase chain reaction (PCR) using the Cobas Amplicor Test
(Roche Diagnostic System, USA), with a detection limit of 50 IU/
mL. Genotyping of HCV was performed by sequencing of the
5’UTR region. Serum levels of alanine aminotransferase (AST),
aspartate aminotransferase (ALT), gamma-glutamyl transferase
(GGT), alkaline phosphatase, and glucose were measured by
an automated kinetic method. Serum ferritin was determined
by chemiluminescence, with the maximum normal limit of the
central laboratory being 400 ng/mL for men and 150 ng/mL
for women, regardless of age. These values were used as cutoffs
for the categorization of ferritin. Altered blood glucose was
defined as levels ≥ 100 mg/mL. Serum insulin was determined
by an immunofluorimetric assay (Perkin Elmer BR-CS). The
homeostasis model assessment of insulin resistance (HOMAIR)
index was calculated using the formula HOMA-IR = [glucose
(nmol/L) * insulin (mU/mL) / 22.5]  and an index > 2.5 was
considered to be altered .
Biopsy specimens were routinely submitted to hematoxylineosin
staining, silver staining of reticulin fibers (Gomori method),
and staining with Masson’s trichrome. The classification of
necroinflammatory activity and staging of chronic hepatitis were
performed using the criteria of the Brazilian Societies of Pathology
and Hepatology . The histological variables were categorized
as the presence or absence of steatosis and liver siderosis (0 x 1,
2 and 3). Inflammatory activity was classified according to the
periportal activity (PPA) as absent or mild versus moderate or
intense (PPA 0 and 1 x PPA 2, 3 and 4) and structural alterations
according to the presence or absence of advanced fibrosis (F0-F2
x F3,F4). For the semi-quantitative analysis of iron, the liver tissue
fragments were stained with Prussian blue (Perls method). This
analysis included the grading of liver siderosis using a system
ranging from 0 to 4, where grade 0 (absence of iron deposits), grade
1 (iron granules visible at 400x magnification), grade 2 (discrete
iron granules visible at 100x magnification), grade 3 (iron granules
visible at 25x magnification), and grade 4 (mass of iron granules
visible at 10x magnification or with the naked eye) (13). Using this
system, the patients were classified as having absent or mild liver
siderosis (grades 0 and 1) or intense siderosis (grades 2 to 4).
Statistical analysis was performed using the SPSS 10 program.
Mean values of the continuous variables were compared by
analysis of variance (ANOVA), complemented by Dunn’s test.
The c2 test was used to compare frequencies and Spearman’s
coefficient to evaluate the correlation between the variables
studied. Binary logistic regression analysis was performed after
categorization of the variables. The liver enzyme cut-offs were
determined from receiver operating characteristic (ROC) curves.
A total of 282 patients with chronic hepatitis C were included
in the study. The demographic, biochemical, virological and
histological data of the patients are shown in Tables 1 & 2. The
patients with chronic hepatitis C were divided into four groups
according to gender and age (Table 3). Significant differences in
ferritin levels were observed between men and women, regardless
of age. However, there was no significant difference in ferritin
between patients < 50 and ≥ 50 years in the same gender.
The differences observed justify the use of different cut-off
values for gender, but not for the age groups considered, permitting
logistic regression analysis to determine the factors associated
with serum ferritin levels. The results of uni- and multivariate
binary logistic regression analysis are shown in Table 4. The
following variables were significantly associated with ferritinemia
in univariate analysis (Table 4): gender, platelet count, AST, ALT,
GGT, HOMA-IR, body mass index, histological stage, periportal
activity, steatosis, and liver siderosis. However, only ALT, GGT,
HOMA-IR, steatosis and siderosis were independently associated
with serum ferritin levels.
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The levels of ALT in these patients were associated with
periportal activity upon liver histology in univariate (score
13.984; p<0.001) and multivariate analysis [1.364 (1.030-1.805);
p=0.030), confirming the relationship of this enzyme with
inflammatory activity in the disease. On the other hand, liver
steatosis was associated with age > 50 years (OR 1.029, p=0.008),
HCV non-genotype 1 (OR 1.901, p=0.035), and elevated ferritin
levels (OR 2.092, p=0.0015) (Table 5).
Epidemiological studies have indicated renal failure,
hematological diseases, liver diseases, abusive alcohol
consumption, neoplasms, and inflammatory diseases as the most
common causes of hyperferritinemia in clinical practice [14,15].
In chronic hepatitis C, the prevalence of hyperferritinemia is
highly variable depending on the method and cut-off value used
and the population selection criteria. Regarding the latter factor,
comparison of ferritin values according to age and gender (Table
1) showed that in fact gender and not age was the determinant
factor of serum ferritin concentration. In the present series,
considering the cut-off values adopted, 48% of the patients had
elevated ferritin levels. This percentage agrees with the study
of Lange et al,  who also used gender-specific cut-offs but is
much higher than that reported by other authors [17,18]. Part of
this prevalence can be attributed to the larger number of patients
with liver cirrhosis (23.5% of patients) in our sample.
Despite the bias of a retrospectively analysis, with limitations
in the clinical and laboratory assessment of patients such as
the lack of genetic study for hemochromatosis, the sample of
282 patients is robust and we selected criteria that permitted
homogeneous evaluation of the factors involved in the elevation
of serum ferritin levels in patients chronically infected with HCV.
Regression analysis identified serum ALT and GGT levels, HOMAIR,
steatosis and liver siderosis as factors that were independently
associated with hyperferritinemia.
Whereas almost half the patients had hyperferritinemia, iron
deposition in the liver was detected histologically in only 20%
of cases in sinusoidal cells and/or hepatocytes, as reported in
other studies. Silva et al. , studying 96 patients with chronic
hepatitis C including 27% with elevated ferritin levels, found
stainable iron upon histology in only 15.6%. The authors observed
a significant correlation between this finding and hepatic iron
concentration. It should be noted that elevated serum iron in
tissue was only observed in 5% of the cases of that study , in
agreement with Thorbun et al,  who identified elevated hepatic
iron concentrations in only 3% and 10% of patients with chronic
hepatitis C, respectively. As tissue iron can not be responsible for all
observed cases of elevated serum ferritin levels, factors other than
liver siderosis seem to be associated with hyperferritinemia in
chronic hepatitis C . Numerous other factors have been related
to the increase in serum ferritin, including insulin resistance of
type 2 diabetes mellitus [21-23], metabolic syndrome, hepatic
steatosis [10,24,25], increased oxidative stress [3,26,27], liver
fibrosis stage [28,29] and inflammatory activity [20,26].
In the present study, an association was observed between
ALT and ferritin, as also reported by other authors [30-32]. This
independent relationship was even found in the presence of
periportal activity and disease stage, suggesting that ALT overlaps
the histological finding. However, sub-analysis showed that
necroinflammatory activity expressed as periportal activity was
indeed the main factor associated with the increase in ALT levels.
This association of ALT with ferritin supports the concept of this
parameter of iron metabolism as an inflammatory acute-phase
Another factor associated with serum ferritin levels was GGT
activity. Given that multiple factors are associated with the activity
of this enzyme in hepatitis C [28,33], among our patients insulin
resistance and disease stage were found to be independently
associated with the activity of this enzyme (data not shown).
The relationship between serum GGT levels and the degree of
liver fibrosis has been recognized by several authors [17,34].
Elevated GGT in patients with steatosis and insulin resistance can
be attributed to the presence of oxidative stress since this enzyme
participates in the metabolism of conjugates of glutathione, the
main antioxidant agent in the liver. This would also explain its
relationship with ferritinemia, another marker of oxidative stress
It should also be remembered that patients with chronic
hepatitis C are a high-risk population for developing diabetes
[37,38]. Using data from the Third National Health and Nutrition
Examination Survey (NHANES-III), Shaheen et al.  found
hepatitis C to be associated with insulin resistance (measured by
HOMA- IR) and elevated ferritin levels. Serum level of this iron
transporter were a strong predictor of insulin resistance and future development of type 2 diabetes, in agreement with our
findings showing that hyperferritinemia in hepatitis C was directly
related to the HOMA-IR index in both univariate and multivariate
analysis. Supporting this relationship, in another study using
multivariate logistic regression analysis, insulinemia and ferritin
were also independently associated with HCV . Lecube et al
, studying 634 non-cirrhotic patients with chronic hepatitis
C, also found diabetes mellitus to be the main factor associated
with ferritin elevation. Furthermore, the relationship between
insulin resistance and ferritinemia has been described in other
populations [6,7,40]. The installation of insulin resistance is the
link between HCV infection and a higher risk of type 2 diabetes
in patients without other risk factors. This insulin resistance
would result from the direct action of the viral core protein and/
or release of inflammatory cytokines and may regress after viral
The prevalence of steatosis of 65% found in this series is
similar to that reported in studies conducted at the same service
or in a population of the same urban center [11,17]. The factors
associated with hepatic steatosis in this study were ferritinemia,
insulin resistance and non-genotype 1, which are known to be
related to this histological alteration. Several studies have found
an association between elevated serum ferritin and the presence
and degree of steatosis in patients with hepatitis C and with liver
diseases of other etiologies [8,25,45-47], demonstrating a causal
relationship independent of the presence of HCV. It is believed
that ferritinemia is related to an increase in oxidative stress,
which favors alterations in glucose metabolism and hepatic
steatosis in this group of patients because of the interference with
the secretion of apolipoprotein B by hepatocytes .
In this study, we were unable to establish a direct relationship
between fibrosis and ferritin, as observed by other authors
[16,49], probably because three other factors associated with
the degree of liver fibrosis may have masked the importance of
this structural alteration in the logistic regression analysis, i.e.,
insulin resistance, siderosis and serum GGT activity. It should
be remembered that insulin resistance is associated with the
progression of liver disease in chronic HCV infection [1,44].
In conclusion, hyperferritinemia has a multifactorial etiology
in treatment-naïve patients with chronic hepatitis C and is related
to liver siderosis, insulin resistance and hepatic steatosis, in
addition to serum GGT and ALT activity. In this respect, the hepatic
enzymes probably indirectly reflect the inflammatory activity
(represented by ALT levels) and, to a lesser extent, the changes
in hepatic structure (represented by GGT levels). Oxidative stress
may be the common factor linking these conditions.
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