International Journal of Clinical Medicine, 2013, 4, 440-450
http://dx.doi.org/10.4236/ijcm.2013.410079 Published Online October 2013 (http://www.scirp.org/journal/ijcm)
Lipocalin: A Novel Diagnostic Marker for Hepatocellular
Carcinoma in Chronic Liver Disease Patients in Egypt
Hoda Aly Abd El Moety1, Rania Mahamed El Sharkawy1, Nevin Abd El Menem Hussein2
1Chemical Pathology, Medical Research Institute, Alexandria University, Alexandria, Egypt; 2Applied Chemistry, Medical Research
Institute, Alexandria University, Alexandria, Egypt.
Received July 13th, 2013; revised August 15th, 2013; accepted September 10th, 2013
Copyright © 2013 Hoda Aly Abd El Moety et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Hepatocellular carcinoma is one of the most prevalent life-threatening human cancers with etiological factors chronic
viral hepatitis B and C infections. Tumor cell dispersion relies on the loss of homotypic cell-cell adhesion. Invasion
through basement membrane and interstitial extracellular matrix is another key event for metastatic progression, which
requires the action of a series of proteolytic enzymes named matrix metalloproteinases, secreted by tumor cells that en-
hance tumor invasiveness and metastasis. TIMPs are dominant inhibitors of MMPs and able to control MMP-mediated
ECM breakdown by binding active forms of MMPs. Lipocalin-2 is known as neutrophil gelatinase associated lipocalin
promotematrix degradation and tumor progression. Aim: To evaluate the importance of lipocalin for diagnosis HCC in Egyp-
tian chronic liver disease patients. Subjects and Methods: 50 patients and 25 controls. (G-1) 25 HCC on top of hepatitis
C. (G-2) 25 hepatitis C. The following done: schistosoma antibodies, ASAM, LKM-1, ANA AKA and CBC. Hepatitis
B surface antigen, Hepatitis C antibodies AFP, Cupper and zinc, Matrix metaloprotinase-9, TIMP-1 and Neutrophil ge-
latinase-associated lipocalin. Results: Median value of MMP-9 level in G-I (206 µg/L) is significantly higher than G-2
(100 µg/L) and G 3 (49 µg/L) p < 0.001. TIMP-9 median value G-1 (48 µg/L) is significantly lower than G-2 (54 µg/L)
and G-3 (113 µg/L) p < 0.001. Lipocalin-2 median levels are significantly higher in G-I (389 ng/mL) than G-2 (166
ng/ml) versus G-3 (60 ng/mL) p < 0.001. Lipocalin-2 associated with increasing lobular inflammation, ballooning &
fibrosis with MMP-9 has an important role in pathogenesis of liver cirrhosis and HCC. Conclusion: We elucidated pre-
dictive value for MMPs, TIMPs, and progression metastasis of hepatocellular carcinoma. Liver cell impairment alters
metabolism of trace metals as zinc and copper, with possible relationship of these changes to pathogenesis of chronic
liver disease. Lipocalin-2 can be used as a future diagnostic marker with better sensitivity and specificity than MMP-9
for the progression of hepatocelluar carcinoma.
Keywords: Lipocalin-2; MMP-9; TIMP-1; Cu; Zn; HCC
1. Introduction
Hepatocellular carcinoma (HCC) is one of the most pre-
valent life-threatening human cancers that is not only
increasing in worldwide incidence in the past decade [1-
4], but also a leading cause of cancer-related deaths world-
wide [3-6]. HCC is an aggressive and enigmatic disease,
which represents approximately 85% of liver cancers
[5,6]. The most prominent etiological factors associated
with HCC consist of chronic viral hepatitis B and C in-
fections [4,7-9], nonalcoholic fatty liver disease [10-12],
and toxin and alcohol exposure [6,9]. The development
and progression of HCC is a multistep and long-term
process characterized by the progressive sequential evo-
lution of morphologically distinct preneoplastic lesions
(formed as a result of chronic liver injury, necro-in-
flammation and regeneration, small cell dysplasia, low-
grade and high grade dysplastic nodules) that culminates
in the formation of HCC [5,13].
Dispersion of tumor cells from the primary tumor is
considered one of the key events for metastatic progres-
sion. Tumor cell dispersion relies on the loss of homo-
typic cell-cell adhesion, which is largely mediated by E-
cadherin/catenin complex [14]. Invasion through base-
ment membrane and interstitial extracellular matrix is
another key event for metastatic progression, which re-
quires the action of a series of proteolytic enzymes
named matrix metalloproteinases (MMPs) [15,17]. MMPs
are a group of zinc-dependent endopeptidases that share

Lipocalin: A Novel Diagnostic Marker for Hepatocellular Carcinoma in Chronic Liver Disease Patients in Egypt 441
many structural and functional properties but with dif-
ferent substrate specificities [15,18,19].
The ability of cancer cells to degrade the extracellular
matrix and basement membrane is an important initial
step in the process of tumor invasion [20,22]. Matrix
metalloproteinases (MMPs), secreted by tumor cells, are
extracellular matrix-degrading enzymes that enhance
tumor invasiveness and metastasis [23,24]. MMP-9 plays
a key role in the invasive ability and the degradation of
the basement membrane, which is composed of type IV
collagen [21].
The activity of MMPs is regulated by a group of
molecules named tissue inhibitors of MMP (TIMPs) that
reside in the normal tissue and counter react with MMPs
with a 1:1 stoichiometry [15,19,25,26]. There are four
members of the TIMP family designated TIMP-1, -2, -3
and -4 [27]. Tissue inhibitors of matrix metalloproteinase
(TIMPs) are the dominant inhibitors of MMPs and are
able to control MMP-mediated ECM breakdown by bind-
ing active or latent forms of MMPs in a 1:1 molecular
ratio [28].
The expression of MMPs is regulated by various fac-
tors, such as growth factors, cytokines, and proteinases
inhibitors. The endogenous tissue inhibitors of metallo-
proteinses (TIMPs) are specific inhibitors of MMPs. Im-
balance between the MMPs and TIMPs might, therefore,
contribute to degradation or deposition of the ECM
[29,30]. Inhibition of MMP-mediated migration of inva-
sion could thus provide a means of preventing cancer
metastasis [31].
Hepatic fibrosis is the common end point to chronic
injury of variable aetiology. Also it is a bidirectional pro-
cess with significant reversible component. HSC is the
principal cell producing extracellular matrix during fi-
brogenesis and the main source of TIMP-1 which inhibits
the endogenous matrix-degrading activity of the MMPs,
thus promoting scar deposition.
Hepatic macrophages are the master regulator of this
dynamic fibrogenesis-fibrosis resolution paradigm, where
they are rich sources of scar degrading MMPs as MMP-9
which can promote HSC apoptosis which is critical fea-
ture of scar resolution. Loss of the pro-inflammatory pro-
fibrotic signals expressed by microphages during fibro-
genesis might alter local milieu to favour fibrosis resolu-
tion [31] (Figures 1 and 2) [32,33].
Lipocalin-2 (LCN2), also known as NGAL (neutrophil
gelatinase-associated lipocalin), is a 25- KDa transporter
belonging to the lipocalin super family. LCN2 binds
MMP-9 and forms a 115-135-KDa MMP-9-LCN2 com-
plex, preventing the degradation of MMP-9. The
Figure 1. Macrophages: Central regulators of hepatic fibrogenesis and fibrosis resolution [32].

Lipocalin: A Novel Diagnostic Marker for Hepatocellular Carcinoma in Chronic Liver Disease Patients in Egypt 442
Figure 2. Factors that promote fibrosis progression to cirrhosis [33].
MMP-9/LCN-complex is present in the urine of breast
cancer patient and is believed to promote MMP-mediated
matrix degradation and tumor progression [32-36].
2. Aim
The aim was to evaluate the importance of lipocalin for
diagnosis of hepatocllular carcinoma in Egyptian chronic
liver disease patients.
3. Subjects and Methods
The present study was carried out on 75 adult subjects
their age ranged from 40 to 60 years. The hepatic patients
group (50 subjects) selected from those admitted from
the hepatology and oncology Departments, Medical Re-
search Institute, University of Alexandria from 2010 to
2012 and 25 healthy volunteers were considered as a
control group (with matched age).
The patients were classified into the following groups:
1) Group-1: 25 hepatocellular carcinoma on top of
hepatitis C group.
2) Group-2: 25 hepatitis C group.
3) Group-3: 25 healthy subjects as control group.
All subjects lived in the same rural area (the Abbess
group of villages on the edge of the Alexandria gover-
norate) and were seen at the Internal Medicine and on-
cology Departments of Alexandria University’s Medical
Research Institute Teaching Hospital, in Alexandria,
Egypt. The study protocol was approved by Alexandria
University’s Ethical Committee and all the subjects gave
their written informed consent before participating in the
study.
All subjects were subjected to the following parame-
ters were done: schistosoma antibodies using indirect
haemagglutination test [37]. Antismooth muscle antibod-
ies ASAM, liver kidney microsomal antibodies LKM-1,
antinuclear antibodies ANA and anti-keratin antibodies
AKA by indirect immunoflourescent technique [38].
Complete blood picture on automated cell counter and
verified by smear examination [39]. Hepatitis B surface
antigen [40], Hepatitis C antibodies [41], Serum alpha
fetoprotein by ELISA [41]. Cupper and zinc by atomic
absorption spectrophotometry [42]. Matrix metaloproti-
nase-9 and Inhibitor (TIMP-1) and Neutrophil gelati-
nase-associated lipocalin (NGAL) by ELISA [43-45].
Blood sampling: 5 ml of venous blood were with-
drawn from all patients and control subjects. All blood
samples were collected into sterile tubes (gel separation
tube and EDTA for CBC), gel separation tubes were left
to be clotted then centrifuged at 25˚C for 10 minutes;
serum was stored at −80˚C until used for determination
of serum MMP9, TIMP-1 and NGAL. All other parame-
ters were analyzed immediately with no storage.
4. Results
The present study showed a statistically significant dif-
ference (p < 0.001) between the studied groups for
TIMP-1, MMP-9, Lipocaline-2, Cu, Zn and Cu/Zn ratio
(Table 1).
A significant negative correlation between the MMP-9
and TIMP in the HCC & HCV group (r = −0.0483, p =
0.015) while no significant correlations in the other
grups for all other parameters (Table 2). o

Lipocalin: A Novel Diagnostic Marker for Hepatocellular Carcinoma in Chronic Liver Disease Patients in Egypt 443
Table 1. Comparison between the diffe re nt studied groups accor ding to different par a me te r s.
HCC & HCV(G1) HCV(G2) Control(G3) p
TIMP-1
Mean ± SD 47.44 ± 8.98 58.04 ± 9.52 110.80 ± 9.36
Median 48.0 54.0 113.0
<0.001*
p1 <0.001* <0.001*
p2 0.001*
MMP-9
Mean ± SD 204.64 ± 36.23 99.80 ± 9.80 49.88 ± 3.09
Median 206.0 100.0 49.0
<0.001*
p1 <0.001* <0.001*
p2 <0.001*
LIPOCALIN-2
Mean ± SD 387.68 ± 11.53 168.36 ± 15.36 58.72 ± 7.46
Median 389.0 166.0 60.0
<0.001*
p1 <0.001* <0.001*
p2 <0.001*
Cu
Mean ± SD 1.05 ± 0.23 0.92 ± 0.23 0.46 ± 0.09
Median 1.10 1.0 0.50
<0.001*
p1 <0.001* <0.001*
p2 0.053
Zn
Mean ± SD 0.30±0.05 0.56 ± 0.06 0.67 ± 0.04
Median 0.31 0.56 0.67
<0.001*
p1 <0.001* <0.001*
p2 <0.001*
Cu/Zn ratio
Mean ± SD 3.24 ± 1.17 1.90±0.46 0.68 ± 0.14
Median 3.13 2.04 0.67
<0.001*
p1 <0.001* <0.001*
p2 <0.001*
*: Statistically significant at p ≤ 0.05 between the three studied groups; p: p value for F test (ANOVA) for comparing between the different studied group; p1: p
value for Post Hoc test (Scheffe) for comparing between control with each group; p2: p value for Post Hoc test (Scheffe) for comparing between HCC & HCV
and HCV.
Table 2. Correlation between different parameters in each studied groups.
MMP-9 LIPOCALIN-2 Cu Zn
r p r p r p r p
TIMP −0.483* 0.015 −0.210 0.314 −0.035 0.868 0.225 0.281
MMP-9 0.040 0.849 0.174 0.405 −0.087 0.680
Lipocalin-2 0.086 0.681 −0.114 0.587
HCC & HCV
Cu −0.134 0.523
TIMP −0.056 0.791 −0.052 0.806 −0.298 0.148 0.100 0.636
MMP-9 0.002 0.991 0.097 0.643 −0.349 0.088
Lipocalin-2 0.230 0.269 −0.121 0.563
HCV
Cu −0.313 0.128
TIMP −0.086 0.683 −0.165 0.429 0.148 0.481 −0.193 0.357
MMP-9 0.299 0.147 −0.202 0.333 0.513* 0.009
Lipocalin-2 −0.296 0.151 0.302 0.143
Control
Cu −0.047 0.823
r: Pearson coefficient; *: Statistically significant at p ≤ 0.05.

Lipocalin: A Novel Diagnostic Marker for Hepatocellular Carcinoma in Chronic Liver Disease Patients in Egypt 444
Positive significant correlation is present between
GGT and lipocaline-2 in the HCC & HCV group (r =
0.465) and between AFP and TIMP in the HCV group (r
= 0.505*) and between Zn and MMP-9 in the control
group, while negative significant correlation between
TIMP and GGT in the control group (r = −0.485*) (Table
3).
No significant difference is present between the stud-
ied groups as regards the AFP (p = 0.051) (Table 4).
The sensitivity, specificity, PPV, NPV and accuracy is
calculated for NGAL, MMP-9, TIMP and AFP (Table
5).
Table 3. Correlation between TIMP-1, MMP-9 and LIPOCALIN-2 with different parameters in each group.
TIMP-1 MMP-9 LIPOCALIN-2
r p r p r p
GOT −0.185 0.377 0.330 0.107 0.394 0.051
GPT −0.280 0.175 0.260 0.209 0.341 0.095
GGT 0.055 0.795 0.166 0.428 0.465* 0.019
ALP −0.262 0.205 0.228 0.273 0.261 0.208
AFP 0.095 0.651 −0.229 0.270 0.265 0.201
Cu −0.035 0.868 0.174 0.405 0.086 0.681
Zn 0.225 0.281 −0.087 0.680 −0.114 0.587
HCC & HCV
Cu/Zn ratio −0.139 0.508 0.103 0.624 0.120 0.567
GOT −0.262 0.205 0.120 0.566 −0.002 0.994
GPT −0.210 0.313 0.220 0.290 0.039 0.853
GGT −0.142 0.499 −0.080 0.704 0.149 0.477
ALP 0.174 0.409 −0.185 0.376 0.003 0.988
AFP 0.505* 0.010 0.008 0.969 0.323 0.115
Cu −0.298 0.148 0.097 0.643 0.230 0.269
Zn 0.100 0.636 −0.349 0.088 −0.121 0.563
HCV
Cu/Zn ratio −0.202 0.332 0.119 0.573 0.278 0.179
GOT −0.194 0.352 0.119 0.570 0.247 0.234
GPT −0.018 0.931 −0.183 0.381 −0.154 0.452
GGT −0.485* 0.014 −0.088 0.676 0.015 0.943
ALP −0.090 0.668 0.071 0.735 0.094 0.656
AFP −0.238 0.281 0.084 0.690 −0.141 0.502
Cu 0.148 0.481 −0.202 0.333 −0.296 0.151
Zn −0.193 0.357 0.513* 0.009 0.302 0.143
Control
Cu/Zn ratio 0.210 0.313 −0.347 0.089 −0.357 0.079
r: Pearson coefficient; *: Statistically significant at p ≤ 0.05.
Table 4. Comparison between the different studied groups according to AFP.
HCC & HCV HCV Control p
AFP
Mean ± SD 1014.87 ± 2817.24 46.54 ± 50.85 1.77 ± 1.55
Median 250.0 15.0 1.20
0.051
p1 0.117 0.096
p2 0.995
*: Statistically significant at p ≤ 0.05 between the three studied groups; p: p value for Kruskal Wallis test for comparing between the different studied group; p1:
p value for Mann Whitney test for comparing between control with each group; p2: p value for Mann Whitney test) for comparing between HCC & HCV and
HCV.

Lipocalin: A Novel Diagnostic Marker for Hepatocellular Carcinoma in Chronic Liver Disease Patients in Egypt 445
Table 5. Agreement (sensitivity, specificity and accuracy) for NGAL, MMP-9, Inhibitor and AFP with HCV and HCC &
HCV.
HCV HCC & HCV Sensitivity Specificity PPV NPV Accuracy
≤200 25 0
NGAL
>200 0 50
100.0 100.0 100.0 100.0 100.0
≤117 21 5
MMP-9
>117 4 20
80.0 84.0 83.33 80.77 82.0
>48 23 12
Inhibitor
≤48 2 13
52.0 92.0 86.67 65.71 72.0
AFP >103 22 1 96.0 88.0 88.89 95.65 92.0
5. Discussion
Liver cancer is the fifth common diagnosed cancer
worldwide and the third leading cause of cancer death.
Hepatocellular carcinoma primarily develops in cirrhosis
resulting from chronic infection by hepatitis B and C
viruses, alcoholic injury and to a lesser extent from ge-
netically determined disorders such as heamochromatosis
[46].
This associations is alarming due to globally high
prevalence of this conditions and may contribute to the
rising incidence of HCC which is increasingly commonly
in the united states with an age adjusted incidence rising
from 1.5 to 4.9 per 100,000 Individuals in the past 30
years. This worrisome trend has been primarily attributed
to the high prevalence of chronic hepatitis C in this
population, so there is an urgent need to develop and
validate noninvasive tests that can accurately reflect the
full spectrum of hepatic inflammation and fibrosis in
chronic hepatitis C patients [47-49].
Liver fibrosis is traditionally viewed as a progressive
pathological process involving multiple cellular and mo-
lecular events that lead ultimately to deposition of excess
matrix proteins in the extracellular space. When this
process is combined with ineffective regeneration and
repair, there is increasing distortion of the normal liver
architecture, and the end result is cirrhosis. The role of
hepatic stellate cells (HSCs) in this process has led to
important validity of this “progression-only” model [50].
Current evidence indicates that liver fibrosis is dy-
namic and can be bidirectional involving phases of pro-
gression and regression, that in addition to increased ma-
trix synthesis, this pathological process involves major
changes in the regulation of matrix degradation [51].
In the extracellular space, matrix degradation occurs
predominantly as a consequence of the action of a family
of enzymes called the matrix metalloproteinases (MMPs).
These are secreted from HSC cells into the extra-cellular
space as proenzymes, which are then activated by a
number of specific, usually cell surface-associated, cleav-
age mechanisms.
The active enzymes are in turn inhibited by a family of
tissue inhibitors of metalloproteinases (TIMP-1 to -4).
By this combination of mechanisms, extracellular matrix
degradation is closely regulated, which prevents inad-
vertent tissue damage. The three most relevant MMPs are
gelatinase A (MMP-2), gelatinase B (MMP-9), and stro-
melysin (MMP-3).
Progelatinase B (MMP-9) is found in liver, but the
principal cellular source is the Kupffer cell. Progelatinase
B expression is significantly increased with Kupffer cell
activation, but the majority is secreted in the proenzyme
form. Progelatinase B can be activated by plasmin and
stromelysin [52,53].
HSC-mediated activation of progelatinase A and B is
significantly induced in the presence of collagen type I
(the principal matrix protein found in fibrotic liver).This
could contribute to further degradation of the normal
liver matrix, leading to Increased activation of HSCs and
increased synthesis of type I collagen, this positive feed-
back loop would theoretically promote progression of
liver fibrosis [52-54].
A non-invasive means of assessment of disease activ-
ity and stage would be very helpful, particularly in moni-
toring patients with chronic hepatitis C virus (HCV) in-
fection overtime. An ideal test would be simple, readily
reliable, inexpensive, accurate, and sensitive.
In the present study it is evident that median value of
the MMP-9 level in G-I was (206 µg/L)) which was sig-
nificantly higher than G-2 (100 µg/L) and G 3 (49 µg/L)
where p < 0.001. Also TIMP-9 median value in G-1 (48
µg/L) was significantly lower than G-2 (54 µg/L) and
G-3 (113 µg/L) where p < 0.001 (Table 1).
G-1 showed negative significant correlation between
MMP-9 and TIMP- I were (r = −0.483 p = 0.015), but G-
2 and G-3 only showed negative correlation were (r =
−0.056 p = 0.791) and (r = −0.086 p = 0.683) respec-
tively (Table 2).
MMP-9 in: G1 was positively correlated with GOT,

Lipocalin: A Novel Diagnostic Marker for Hepatocellular Carcinoma in Chronic Liver Disease Patients in Egypt 446
GPT, GGT, ALP and Cu (r = 0.330/p = 0.107, r = 260/p
= 0.209, r = 0.166/p = 0.428, r = 0.228/p = 0.273, r =
0.174/p = 0.405), while it was negatively correlated with
AFP and Zn (r = −0.224/p = 0.270, r = −0.087/p = 0.680)
respectively (Table 3).
G-2 was positively correlated with GOT, GPT, AFP,
Cu (r = 0.120 p = 0.566, r = 0.290 p = 0.039, r = 0.008 p
= 0.969, r = 0.097 p = 0.643). While it showed negative
correlation with GGT, ALP, Zn (r = −0.080 p = 0.704, r
= −0.185 p = 0.376, r = −349 p = 0.088) respectively
(Table 3).
TIMP-1 in group G-1 was inversely correlated with
GOT, GPT, ALP and Cu (r = −0.185 p = 0.377, r =
−0.280 p = 0.175, r = −262 p = 0.205, r = −0.325 p =
0.868) respectively. While it showed positive correlation
with GGT, AFP and Zn (r = 0.055 p = 0.795, r = 0.095 p
= 0.651, r = 0.225 p = 0.281) respectively (Table 3).
While it showed negative correlation with GOT, GPT,
GGT and Cu (r = −0.262 p = 0.205, r = −210 p = 0.313, r
= 0.142 p = 0.499, r = −0.298 p = 0.148) and positive
correlation with ALP, Zn (r = 0.174 p = 0.409, r = 0.100
p = 0.636) and a positive significant correlation with
AFP (r = 0.505 p = 0.010) respectively in G-2 (Table 3).
These results were in accordance with Ralf L et al. [55]
who stated that several biochemical indicators have been
discussed as a potential non-invasive serum markers of
fibro-proliferation. Among them the matrix metallopro-
teinase (MMPs) and their tissue inhibitors (TIMPs) have
been shown to correlate with the development of hepati-
tis C induced cirrhosis, as a result of an imbalance be-
tween enhanced matrix synthesis and diminished break-
down of connective tissue proteins, the net result of
which is increased deposition of extracellular matrix.
Decreased activity of ECM-removing MMPs is mainly
due to an over expression of their specific inhibitors
(TIMPs) [56].
Sheng Zhang et al. [57] stated that the expressive bal-
ance between matrix metalloproteinase-9 (MMP-9) and
its tissue inhibitor of metalloproteinase-1 (TIMP-1) plays
a critical role in maintaining the degradation and synthe-
sis of extracellular matrix. Loss of such balance is asso-
ciated with invasion and metastasis of tumors and predict
poor prognosis.
Zu-huaGao et al. [58] also stated that well-differenti-
ated hepatocellular carcinoma and to less differentiated
tumors was associated with a gradual decrease in tissue
inhibitors of metalloproteinase, but higher levels of
MMPs. These data suggest that tissue expression of
MMPs and their inhibitors could be useful markers to
predict the progression and metastasis of hepatocellular
carcinoma.
The lipocalins (NEGAL) constitute a broad but evolu-
tionarily conserved family of small proteins, which share
a high similarity in their tertiary structures in spite of a
low degree in amino-acid sequence identity: three highly
conserved sequence motifs form a funnel-like β barrel
which encloses a hydrophobic pocket for the internal
ligand binding.
Individual lipocalins have been implicated in diverse
physiological roles ranging from odor recognition by
binding proteins like retinol-binding protein, purpurin,
α-1-microglobulin, β-lactoglobulin to the regulation of
cell homeostasis and functions as carrier apolipoprotein
D which transports sterols and steroid hormones [59,60].
Harald T et al. [61] also stated that human neutrophil
lipocalin (HNL), a member of the large family of lipo-
calins that exhibit various physiological functions, is
coexpressed in granulocytes with progelatinase B (MMP-
9). Part of it is covalently bound to the proenzyme and
therefore may play a possible role in the activation proc-
ess of promatrix metalloproteinases and can exert an en-
zyme-activating effect in the regulation of inflammatory
and pathophysiological responses of MMP-9 activation.
Our present study was in accordance with the above
documented results where Lipocalin-2 median levels
were significantly higher in G-I (389 ng/mL) than G-2
(166 ng/ml) Versus G-3 (60 ng/mL) where p < 0.001
(Table 1).
Lipocalin -2 (Negal) also in the present study showed
positive correlations with MMP-9 in G-1, G-2 & G-3
where (r = 0.040 p = 0.849, r = 0.002 p = 0.991, r = 0.299
p = 0.147 respectively). But lipocalin-2 showed negative
correlations with TIMP-1 in G-1, G-2 and G-3 were (r =
−0.210 p = 0.314, r = −0.052 p = 0.804, r = −0.165 p =
0.429) respectively (Table 2).
Lipocalin-2 (NEGAL) in G-1 it showed positive sig-
nificant correlation with GGT (r = 0.465* p = 0.019), it
was positively correlated GOT, GPT, ALP, AFP, Cu (r =
0.394 p = 0.051, r = 0.341 p = 0.095, r = 0.261 p = 0.208,
r = 0.265 p = 0.201, r = 0.086 p = 0.681) respectively.
While it was negatively correlated with Zn (r = −0.114 p
= 0.587).
In G-2 it showed negative correlation with GOT and
Zn (r = −0.002 p = 0.994, r = −0.121 p = 0.563), while it
was positively correlated with GPT, GGT, ALP, AFP,
Cu (r = 0.039 p = 0.853, r = 0.149 p = 0.477, r = 0.003 p
= 0.988, r = 0.323 p = 0.115, r = 0.230 p = 0.269) respec-
tively (Table 3).
Lipocalin-2 was associated with increasing lobular in-
flammation, ballooning & fibrosis together with matrix
metalloproteinase (MMP-9) has an important role in the
pathogenesis of liver cirrhosis (LC) and hepatocellular
carcinoma (HCC) [62].
Suzuki K et al. [63] stated that biochemical and nutria-
tional roles of trace elements is widely recognized, since
metals are found as constituent components of many
metalloproteins and metalloenzymes. Some trace ele-
ments such as copper act as cofactors particularly in the

Lipocalin: A Novel Diagnostic Marker for Hepatocellular Carcinoma in Chronic Liver Disease Patients in Egypt 447
biosynthesis of collagen. As the disease progress from
chronic hepatitis to liver cirrhosis, serum zinc concentra-
tions decrease, while the copper concentration increases.
In the patients with hepatocellular carcinoma, serum
concentrations of trace elements are similar to those of
liver cirrhosis. In the patients with acute hepatitis, serum
zinc concentrations decrease, while copper concentra-
tions decrease. These trace element abnormalities may
reflect such pathological conditions as liver dysfunction,
cholestasis, hepatic fibrosis or liver regeneration. Also
Copper accumulation in fibrotic livers caused by chronic
hepatitis C may contribute to hepatic injury [64].
We observed significant changes in the trace element
serum levels of patients having hepatocellular carcinoma,
relative to those of healthy controls (p < 0.001), where
Cu median value in G-1 (1.10 µg/L) was significantly
higher than G-2 (1 µg/L) than G3(0.5 µg/L). In contrast,
Zn median value in G-I (0.31 µg/L) was significantly
lower than G-2 (0.56 µg/L) than G-3 (0.67 µg/L) where p
< 0.001 (Table 1).
Also there was negative significant correlation be-
tween Cu and Zn in the three different groups where G-I
(r = −0.134 p = 0.523), G-2 (r = −0.313 p = 0.128) and
G-3 (r = −0.047 p = 0.823). (Table 2)
Moreover, we found significantly elevated median
value of Cu: Zn ratios in G-1 (3.13) patients having
hepatocellular carcinoma than G-2 (2.04) than G-3 (0.67)
where (p < 0.001).
Cu/Zn ratios were also positively correlated with
MMP-9 in G-1 and G-2 (r = 0.103 p = 0.624, r = 0.119 p
= 0.573) also with NEGAL in G-1 and G-2 (r = 0.120 p =
0.567, r = 0.278 p = 0.179) respectively. (Table 3)
Our findings imply that the levels of some trace ele-
ments, such copper, zinc and Cu: Zn ratios, might serve
as biomarkers for the increased severity of viral hepatic
damage.
These results suggested that changes in liver cell pa-
thology compounded by functional impairment may alter
the metabolism of trace metals, in particular, zinc and
copper, with possible relationship of these changes to the
pathogenesis of chronic liver disease [65-68]
The present study revealed that the AUROC for lipo-
calin-2 at a cut-off >200 ng/ml was 1.000, where p <
0.0001, while for MMP-9 at a cut-0ff >117 µg/L was
0.962, where p = 0.023 and for TIMP-1 at a cut off >48
µg/L was 0.771, where p = 0.001 (Figure 3).
Also the diagnostic performance for lipocalin-2 in G-1
was 100% while MMP-9 in the same group showed a
sensitivity of 80% with PPV of 83.33% and specificity of
84% with NPV of 80.77% with an accuracy of 82%.
Whereas the diagnostic performance for TIMP-1 in G-1
showed sensitivity 52% with PPV 86.67% and specificity
of 92% with NPV 65.71% with an accuracy of 72%.
020 406080100
100
80
60
40
20
0
100-Specificity
Sensitivity
Negal
MMP-9
Inhibitor
AFP
Figure 3. ROC curve for AFP, NGAL, MMP-9 and Inhibi-
tor with HCV and HCC & HCV.
6. Conclusions
We elucidated the predictive value for evaluation of MMPs,
TIMPs, and the progression metastasis of hepatocellular
carcinoma.
Liver cell pathology compounded by functional im-
pairment alters the metabolism of trace metals as zinc
and copper, with a possible relationship of these changes
to the pathogenesis of chronic liver disease.
Lipocalin-2 can be used as a future diagnostic marker
with better sensitivity and specificity than MMP-9 for the
progression of hepatocelluar carcinoma.
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