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Pakistan Journal of Molecular Medicine (Pakistan J. Mol. Med.)
________________________________________________________________
An official Institute of the Molecular Biology and Biotechnology (IMBB) &
Centre for Research in Molecular Medicine (CRiMM)
The University of Lahore, Lahore-54000, Pakistan
(http://www.pjmm.uol.edu.pk)
E-mail: [email protected]
________________________________________________________________
Editor-in-Chief: Prof. Dr. M.H. Qazi
Editors: Prof. Dr. Syed Shahid Ali
Prof. Dr. Mushtaq A. Saleem
Sub-Editor: Dr. Arif Malik
Editorial Board:
Local Foreign
Dr. M. Arsalan Univ. of Lahore,
Dr. A.R. Shakoori SBS, Univ. of the Punjab
Dr. Richard M. Wilkins Univ. New Castle Upon Tyne, UK
Dr. S. Riazuddin Allama Iqbal Med. College, Lahore,
Dr. Waheed Akhtar SBS, Univ. of the Punjab
Dr. Amir Mahmood Qazi Toronto Cancer Research Lab., Canada
Dr. Syed Amir Gilani Univ. of Lahore,
Dr. Mughees Ahmad UHS, Lahore
Dr. Shamshad Gilani Univ. Med. Dentistry, NJ, USA
Dr. Javed Iqbal Mirza Univ. of Lahore,
Dr. Naveed Wasif Univ. of Lahore,
Dr. Mahmood Rasool King Abdul Aziz Univ., Jeddah, KSA
Dr. A.M. Cheema Univ. of Lahore,
Dr. Saeed A. Malik BZ Univ., Multan,
Dr. S. Taseer Hussain Howard Univ., Washington DC, USA
Dr, Masood Qureshi Univ. of Karachi,
Dr. Javed Qureshi Univ. of Lahore,
Dr. Shams Masood Harvard Univ. Boston, MA, USA
Dr. Ejaz Rasul Univ. of Lahore,
Dr. Ijaz Ahmad Univ. of Lahore,
Dr. David Mantle New Castle Gen. Hospital, UK
Dr. J.I. Qazi Univ. of Punjab,
Dr. Bashir Ahmad Univ. of Lahore,
Dr. Amjad Javed Univ. of Albama, USA
Dr. Akram Muneer Univ. of Lahore,
Dr. Saqib Shahzad Univ. of Lahore,
Dr. Mohd. Zaini Asmawi Univ. Sains, Malaysia
Dr. M. Zameer Ahmad Allama Iqbal Med. College, Lahore,
PAKISTAN JOURNAL OF MOLECULAR MEDICINE INSTRUCTIONS TO AUTHORS
Pakistan Journal of Molecular Medicine (Pakistan J. Mol. Med.) is a quarterly
journal, being published by the Institute of Molecular Biology and Biotechnology
(IMBB), and Centre for Research in Molecular Medicine (CRiMM), The
University of Lahore, Lahore, Pakistan. This journal accepts the articles and
reviews (invited and non-invited) in the disciplines of biochemistry,
biotechnology, environmental sciences, forensic science, medicine, microbiology,
molecular biology, pathology, pharmacology, physiology, toxicology and other
biomedical sciences.
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respects and printed strictly according to the prescribed style should be sent in
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Pakistan Journal of Molecular Medicine, The University of Lahore, Defense
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Some citations as they must appear in manuscript are given below:
In Journals:
Rasgela PG and Kaymak F. Evaluation of genotoxic and cytotoxic effects of
Natamycin in mice bone marrow cells. Pakistan J Zool., 2013; 45 (4):
1103-1112.
Engels B, Dahm P and Jennewein S. Metabolic engineering of taxadiene
biosynthesis in yeast as a first step towards Taxol (Paclitaxel) production.
Metab Eng., 2008; 10: 201-206.
In Books:
Jurenka RA. Biochemistry of female moth sex pheromones. In: Insect
Pheromones Biochemistry and Molecular Biology. (eds. GJ Blomquist,
RG Vogt), Elsevier Academic Press, Oxford, 2003, pp. 53-80.
Boulos L. Medicinal plants of North Africa. Reference Publications Inc.,
Michigan, 1980.
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Page charges: The Editorial committee of Pakistan J. Mol. Med., has decided to
charge Rs. 1500/- for each published article up to six printed pages with black and
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Page-proofs and reprints: One set of proofs will be sent to the corresponding
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Copyright: After publication, the copyright of the article shall be the property of
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from the Editorial committee.
Correspondence: All the correspondence should be addressed to: Dr. M.H. Qazi,
Editor-in-Chief, Pakistan Journal of Molecular Medicine, Institute of Molecular
Biology and Biotechnology (IMBB) , The University of Lahore, Lahore, Pakistan
and also Emailed at [email protected].
Correspondence: All the correspondence should be addressed to: Dr. M.H. Qazi,
Editor-in-Chief, Pakistan Journal of Molecular Medicine, Institute of Molecular
Biology and Biotechnology (IMBB) , The University of Lahore, Lahore, Pakistan
and also Emailed at [email protected].
PAKISTAN JOURNAL OF MOLECULAR MEDICINE
(Pakistan J. Mol. Med.) VOL. 1 (1-2), 2014
Sr # Contents Page #
1 Comparative Study of Widely Used Chemotherapeutic Agent Paclitaxel Using
HeLa and Vero Cell Lines
Sumaira Shaheen, Usman Mirza, Humaira Shoukat and Mahmood Husain Qazi
5-16
2 Effect of Different Nitrogen Sources and Niacin on β-Mannanase Production from
Locally Isolated Bacterial Strain Bacillus badius
Syed Shahid Ali, Bushra Atta and Tanzeela Riaz
17-22
3 Effects of Emamectin Benzoate and its Mixture with Silymarin on Some Enzymatic
Activities of Blood, Liver and Kidney of Chicks
Mushtaq A. Saleem, Sadia Yasin, Muhammad Yousaf, Waleed Javed Hashmi and Rabail
Alam
23-29
4 Assessment of Liver Peroxidation in Diabetic HCV Patients Receiving Interferon
Therapy
Arif Malik, Sahar Javed, Rabail Alam, Zunaira Tahir, Fatima Zahid, Saeed Ismail, Hina
Aataka Riaz, Naveed Shuja and Abdul Manan
30-33
5 Changes in Blood Pressure After Spinal Anesthesia for Caesarean Section in
Labour Class at Lahore
Khurshid Alam, Mushtaq Ahmad Saleem and Faeza Hasnain
34-39
6 Assessment and Correlation of Sialic Acid with Liver Enzymes in Patients Suffering
From Dengue Fever
Arif Malik, Shumaila Arif, Ejaz Rasul, Zunira Aasfa Riaz, Hina Aataka Riaz, Naveed
Shuja, Abdul Manan, Shaista Andlib, Zunaira Tahir, Zunera Tariq and Syed Shahid Ali
40-43
7 Prevalence of Anti-HCV and Frequency of Alloimmunization in Repeatedly
Transfused Thalassemia Major Patients
Mahmood Husain Qazi, Arif Malik, Sahar Javed, Zunera Tariq, Zunaira Tahir, Naveed
Shuja and Abdul Manan
44-47
8 Biochemical and Antioxidative Response of Asthmatic Patients Receiving
Salbutamol
Arif Malik, Hafiz Muhammad Arsalan, Maria Amjad, Naveed Shuja, Abdul Manan,
Saima Zaheer, Amna Mahmood, Sarmad Bashir and Mushtaq A. Saleem
48-53
9 Clinical Insights and Mechanisms Involved During Interferon Induced Thyroditis
in HCV Patients – A Review
Alina Butt, Mahwish Arooj, Arif Mailk, Abdul Manan, Syed Shahid Ali and Mahmood
Husain Qazi
54-60
CHEMOTHERAPEUTIC EFFECT OF PACLITAXEL 5
Pakistan J. Mol. Med., 1(1-2), pp. 5-16, 2014
www.pjmm.uol.edu.pk
Comparative Study of Widely Used Chemotherapeutic Agent
Paclitaxel Using HeLa and Vero Cell Lines
Sumaira Shaheen, Usman Mirza, Humaira Shoukat and Mahmood Husain Qazi*
Center for Research in Molecular Medicine and Institute of Molecular Biology and Biotechnology,
The University of Lahore. 1-kM Defense Road, Off Bhuptian Chowk, Raiwind Road, Lahore, Pakistan.
ABSTRACT
The present study was undertaken to explore the mechanism of paclitaxel induced
apoptosis. Previous studies have shown that paclitaxel stabilizes microtubules and causes cell
cycle arrest. It also acts through MAPK pathway. Moreover, the drug is known to inactivate anti-
apoptotic Bcl-2 family proteins. In this background we have undertaken this study to find out (a)
the exact mechanism by which paclitaxel inhibits MAPK pathway using two separate cell lines
namely HeLa cells, cervical cancer cells and Vero cells, a transformed monkey kidney cell line.
The data obtained show that in HeLa cells paclitaxel acts by blocking MEK and consequently,
inactivating ERK1/2 and inhibiting phosphorylation of BAD at serine 112 through RSK. This
releases BAD from ubiquitination by 14-3-3. These data are shown as western blots of various
proteins involved in this pathway, (b) we are also providing information about the status of
PARP in paclitaxel treated HeLa and Vero cells. These activities of paclitaxel are clear in HeLa
cells as well as in the Vero cells which respond in the same manner and (c) we have also
examined whether paclitaxel can directly bind to the hydrophobic groove of antiapoptotic Bcl-2,
Bcl-xL and Mcl-1 proteins. This has been done through bioinformatics tools. The data indicate
that the drug binds to hydrophobic groove of Bcl-2 and Bcl-xL forming hydrogen bonds with
BH1, BH2 and BH3 domains of these proteins. However, in case of Mcl-1 the drug binds only
with BH3. This, in our opinion, is one important mechanism through which paclitaxel causes
apoptosis in the two cell types and possibly in various cancers for which it is used as therapeutic
agent.
Key words: Apoptosis, antiapoptotic protein, therapeutic effect, HeLa cells, MAPK, MEK,
ERK, RSK, BAD pathway, Bcl-2 and Mcl-2 family of proteins.
INTRODUCTION
Chemotherapy is a conventional approach
along with surgery and radiation therapy to treat
cancer, a major threat to life ((Jailkhani et al.,
2011). It caused 8.2 million deaths in 2012
worldwide; a shocking fact revealed by International
Agency for Research on Cancer (IARC), a sub-
group of World Health Organization (WHO).
_________________________________ * Corresponding author: [email protected]
Among these, more than 60 % deaths occurred in
Asia, Africa and Central America. Furthermore, it
has been predicted by IARC that this death rate may
elevate from 14 million to 22 million within the next
10-20 years, if the tendency towards cancer cure and
treatment remains the same (Globocan 2012, IARC:
Martel et al., 2012).
Most chemotherapy drugs target various
survival signaling mechanisms normally present in
the cell. Among other drugs, Paclitaxel, a bark
extract of Taxus brevifolia (pacific yew tree), is a
commonly used drug regimen for treatment of
CHEMOTHERAPEUTIC EFFECT OF PACLITAXEL 6
various cancers (Priyadarshini and Keerthi, 2012).
Furthermore, other recent studies suggest that some
microorganisms are also capable of producing this
compound (Elavarasi et al., 2012: Zang et al., 2008:
Chakarwati et al., 2008: Engles et al., 2008: Ji et al.,
2006: Page and Landry, 1996). Since its discovery
in 1962, it has been used in war against cancer very
effectively for induction of apoptosis. It was
formulated and sold under the trade name of Taxol
marketed by Bristol-Myers-Squibb Company
(Marupudi et al., 2007: Rowinsky, 1997). It has
been documented that paclitaxel is effective against
a variety of cancers such as ovarian, lung, liver and
breast cancer (Priyadarshini and Keerthi, 2012).
Though paclitaxel is a potent FDA approved
antineoplastic agent but we still lack confirmatory
data on its exact molecular mechanism of action
through which it induces cell death (Miller et al.,
2013). Among various mechanisms, the drug is
known to prevent microtubules disassembly (Xiao et
al., 2006: Ross and Fygenson, 2003) resulting in
arrest of mitosis at G2/M phase of cell cycle
(Dziadyk et al., 2004: Giannakakou et al., 2001).
Furthermore a large number of studies have been
devoted to understanding the mechanism by which
paclitaxel causes apoptosis while acting on the
MAPK (p38, JNK and ERK) proliferative pathway
(Berndtsson et al., 2005: Boldt et al., 2002: Bacus et
al 2001). Yet many questions with regard to the
involvement of ERK/RSK/BAD pathway remain
unanswered.
In view of the above this study has been
undertaken; (a) to find out how paclitaxel
specifically inhibits MEK/ERK/RSK/BAD pathway
(b) to examine the role of PARP, if any in paclitaxel
treated cells and (c) to find out whether paclitaxel
has a role in inducing apoptosis by blocking anti-
apoptotic proteins.
MATERIALS AND METHODS
Cell lines
Two cell lines were used for this comparative
study. HeLa, a cervical cancer cell line was placed
at our disposal by School of Biological Sciences
(SBS), University of The Punjab. The Vero cell line,
originated from African monkey kidney cells was
provided by WTO lab, University of Veterinary and
Animal Sciences, Lahore. Both cell types were
cultured in Dulbecco’s modified eagle medium
(DMEM) as specified below.
Cell culture reagents and accessories Cell culture reagents including DMEM
enriched with high percentage of glucose and
purified heat inactivated fetal bovine serum (FBS),
5000 Units of penicillin and streptomycin, L-
glutamine (200mM), 10X trypsin-EDTA and
phosphate buffer saline (PBS) were purchased from
Invitrogen, Life Technologies, USA. Radio immuno
precipitation assay (RIPA) lysis kit (Invitrogen, life
Technologies, USA) was used for preparation of
whole cell lysates after paclitaxel administration
which was purchased from Tocris Bioscience, USA.
Cell viability assessment was done by using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) reagent from Invitrogen, Life
Technologies, USA.
Cell culture specific (coated and UV
irradiated) vessels and plates were purchased from
specific companies. Flasks of various sizes such as
T-25, T-75 and T-150 were obtained from Cornings,
USA along with 6-well, 96-well and 100mm Petri
plates. Some primary antibodies were obtained from
Invitrogen, Life Technologies, USA and remaining
antibodies were purchased from Santa Cruz, USA
including secondary HRP conjugated anti-rabbit and
anti-mouse antibodies. HRP conjugated 3,3′,5,5′-
Tetramethylbenzidine (TMB) and pre-stained
protein marker (Novex®) were also obtained from
Invitrogen, USA. Lactate dehydrogenase (LDH) kit
was purchased from Human, Germany. The kit was
based on spectrophotometric analysis of
cytotoxicity.
Experimental procedures
Maintenance of cell culture
Continuous culture of growing cells was
maintained by using complete growth medium
comprising of DMEM high glucose, supplemented
with FBS at a final concentration of 10%, 100 Units
of pen-strep solution and 2mM final concentration
of L-glutamine. All preparations and handling
procedures were carried out in Class II biological
safety cabinet (ESCO) to ensure sterility of
solutions. Physical environment of growing cells
CHEMOTHERAPEUTIC EFFECT OF PACLITAXEL 7
was also kept constant by providing uninterrupted
supply of 5% CO2 in a humidified incubator at 37°C
temperature.
Preparation and administration of paclitaxel
Paclitaxel was dissolved in 10mM DMSO to
form 3mM stock solution. A working solution of
100µM was prepared from the stock solution for the
ease of use. It was administered in varying
concentrations (0.1µM, 0.5µM, 1µM, 5µM and
10µM) separately on HeLa and Vero cells for 24
hours.
Anti-proliferation assay Efficacy of paclitaxel was determined by
MTT assay. An equal number of both types of cells
(5 x 103) were seeded in 96-well plates and allowed
to settle down overnight in CO2 incubator at 37°C,
followed by the application of various
concentrations (0.1µM, 0.5µM, 1µM, 5µM and
10µM) of paclitaxel in time dependent manner.
After 24 hours, MTT reagent was added to each
well in 5mg/ml of PBS (1X) concentration and
again subjected to incubation for two hours. This
period of incubation allows live cells to uptake MTT
reagent and converts it into reduced blue colored
formazan crystals. These formazan crystals are
water insoluble. Therefore, a specific buffer
containing 50% DMSO and 20% SDS is added to
each well to solubilize these crystals overnight.
Plate was analyzed by taking absorbance in CODA,
an automated analyzer (BioRad, USA) at 570nm.
Absorbance of treated cells was compared with
untreated cells used as control. Percentage of viable
cells was calculated by using following formula:
Abs. of sample
%viable cells = X 100
Abs. of control
All readings were taken as biological triplicates in
three independent experiments. Similar assay
conditions were designed for both cell lines for
reliable comparison. Moreover, mean values of
percentage cell viability were plotted as bar charts
and graphically with SEM in each group.
Cytotoxicity assay
LDH assay is a well know determinant of cell
killing activities of chemotherapeutic drugs. It is
measured in culture supernatants. A commercially
available kit from Human, Germany was used and
assayed on Microlab 300 chemistry analyzer
according to the protocol provided with kit. Six-well
plates and 100mm Petri plates were seeded with
equal number of cells of both cell lines. After
getting confluent growth, different doses (1µM,
5µM and 10µM) of Paclitaxel were administered for
required time. A plate without treatment of
paclitaxel was also used in parallel to serve as
control. After 24 hour treatment, cells were
harvested and pelleted down. Supernatants for each
concentration of paclitaxel were saved immediately
thereafter for estimation of LDH activity.
RIPA lysis
Cells were plated and treated with paclitaxel
as described above. Cells were pelleted at 1000g in
a refrigerated centrifuge (Sigma). Buffer cocktail for
RIPA lysis was prepared freshly by mixing
individual components that is sodium orthovandate
(Na3VO4), protease inhibitor and PMSF just before
lysis. After washing, the cells were lysed with RIPA
cocktail for 30 minutes on ice. Centrifugation was
carried out at 14000g. Supernatants were used as
protein extract and debris was discarded.
Protein Quantification
Exact concentration of the extracted protein
including control was estimated by Qubit protein
quantification kit by using the standard kit protocol.
SDS-PAGE Electrophoretic separation of proteins was
accomplished by SDS-PAGE. SDS enables all
protein species to unfold and acquire similar
negative charge. In this way proteins resolve on the
gel with similar properties, same force and in the
same direction when provided with continuous
supply of electric field. All protein samples were
mixed with loading dye (2X sample buffer) and
denatured for 4-5 minutes on boiling water. Equal
amount of protein was loaded onto 12% resolving
gel with 4% stacking gel in each well with the help
of Hamilton syringe. Glycerol in the loading dye
provides density to protein samples and prevents
them from mixing with running buffer in the
CHEMOTHERAPEUTIC EFFECT OF PACLITAXEL 8
electrophoretic tank (Mini Tetraprotean®, BioRad,
USA). A pre-stained protein marker was used as
standard in parallel.
Western Blotting
Briefly describing, resolved proteins were
then transferred onto the nitrocellulose membrane
(Millipore) electrophoretically with the help of
towbin buffer containing 20% methanol using 90
volts for 90 minutes. After transfer membrane was
blocked with 3% BSA solution in PBS-T as
blocking buffer. Membrane was then incubated with
selected primary antibody overnight at 4°C. Next
day after washing with PBS-T, incubation of
membrane with HRP conjugated secondary
antibody was done for 1-2 hours at room
temperature. After final washings results were
visualized by using HRP conjugated TMB substrate
as detection reagent. Blue colored bands appeared
when TMB bonded to primary and secondary
antibody complex. Furthermore, results were
captured and saved in Gel Documentation system
(BioRad, USA).
Bioinformatics work
Data collection
In order to validate the results by using
chemoinformatics, the three dimensional structure
of human Bcl-2, BAK, BAX, Bcl-xL and Mcl-1
were obtained from protein data bank (PDB ID:
1GJH, 2MIS, 1FI6, 1BXL, 2KBW respectively) and
structure of paclitaxel was obtained from Pubchem
compound database.
Proteins and Ligand preparation
All protein structures from protein data bank
were further processed prior to docking studies. The
protein 3D structures were initially prepared by
removing all the non-standard amino acids, Hetro
atoms and water molecules followed by energy
minimization to remove the bad clashes using
Chimera by applying AMBER ff12SB as force field.
The 2D structure of paclitaxel was converted to
mol2 format by using open babel followed by
energy minimization by applying Hyperchem’s
MM+ force field.
Determination of binding site
Binding sites and active sites of proteins are
usually associated with structural pockets and
cavities harboring the affinity for particular drugs.
For the present study, our aim was to block the
hydrophobic pocket surrounding by BH domains.
The BH domains locations and binding residues in a
sequence were determined from extensive literature
review and Uniprot database (www.uniprot.org)
Molecular docking procedure
All molecular docking studies were
performed using AUTODOCK 4.0v 1.5.6 installed
in a single machine with LINUX (Ubunto) as an
operating system. Automated molecular docking
was performed to locate the particular binding
conformations of paclitaxel on protein structures
one by one according to AUTODOCK 4.0 specified
instructions. For this regard, polar hydrogen atoms
and Kollman charges were assigned to proteins.
Gasteiger partial charges were assigned and non-
polar hydrogens were merged for paclitaxel. The
standard docking protocol for docking was applied
consisted of 100 runs using initial population of
150, a maximum number of 27000 iterations,
mutation rate 0.02 and a crossover rate of 0.80.
Post-docking analysis was performed and best drug
conformation was selected as the docked pose of
paclitaxel, with a lowest binding energy.
RESULTS
Remarkable effect of paclitaxel on HeLa and
Vero cells has been observed through MTT assay.
MTT assay is commonly used to assess the growth
inhibition rate of cells after drug application. Assay
was carried out by following the protocol described
under the section of materials and methods. Effect
of in vitro treatment of increasing concentrations of
paclitaxel (0.1µM, 0.5µM, 1µM, 5µM and 10µM)
was recorded in both HeLa and Vero cell lines after
24 hours and compared with optical density of
untreated cells. Persistent decrease in cell viability
in a dose dependent manner has been observed in
both cell types. A sudden reduction in cell viability
is observed in HeLa cells at 5µM and 10µM
concentration of paclitaxel (Fig. 1). Similar results
have been observed for Vero cells. However, extent
of drug activity is lower in Vero cells as compared
to HeLa cells, which is shown in Figure 2.
CHEMOTHERAPEUTIC EFFECT OF PACLITAXEL 9
Fig. 1. MTT assay on HeLa cells. Bar
chart represents cell viability after treatment
with various concentrations of paclitaxel. Mean
values of cell viability of three independent
experiments are plotted with SEM values.
Prominent effects are seen at 5 and 10µM
concentrations.
Fig. 2. MTT Assay on Vero cells. Bar
chart shows effect of paclitaxel at various
concentrations. Mean values of cell viability of
three independent experiments are plotted with
their standard error of mean (±SEM). Cell
viability is reduced with increasing drug
concentration.
Lactate dehydrogenase assay Cytotoxic effect of paclitaxel was further
confirmed by LDH assay which is a stable enzyme
released in culture medium upon cell membrane
disruption and is directly related to tissue or cell
damage. Measurement of LDH quantity indicates
number of disrupted cells and it acts as a marker for
dead cells. Increased enzyme activity is directly
proportional to high levels of cytotoxicity. HeLa and
Vero cells were plated and treated with various
concentrations of paclitaxel for assay according to
the protocol described under section of materials
and methods. Remarkable increase in LDH activity
has been seen in HeLa cells with increasing dosage.
However, slight increase in enzyme activity has
been observed in Vero cells with similar drug
concentrations. Untreated cells were used as control
(Fig. 3A, B).
A
B
Fig. 3. LDH Assay. (A) Bar diagram
shows measurement of LDH activity in culture
supernatants of HeLa cells. Each bar is drawn
on the basis of mean (n=3) value of LDH
activity at each drug concentration with
standard error of mean (±SEM). Successive
increase in LDH activity predicts increased
number of dead cells in response to increasing
concentrations of paclitaxel treatment. (B) Bar
diagram represents measurements of LDH
activity in culture supernatants of Vero cells.
Little increase in LDH activity shows
comparatively little effect of increasing
concentrations of paclitaxel. Each bar represents
mean (n=3) value of LDH activity with standard
error of mean (±SEM).
0
20
40
60
80
100
120
C 0.1µM 0.5µM 1µM 5µM 10µM
Cell
via
bil
ity
(%
)
Paclitxel Conc.
0
20
40
60
80
100
120
C 0.1µM 0.5µM 1µM 5µM 10µM
Cell
via
bil
ity
(%
)
Paclitaxel Conc.
0
200
400
600
800
1000
1200
C 1µM 5µM 10µM
LDH
act
ivit
y (U
/l)
Paclitaxel Conc.
Vero
CHEMOTHERAPEUTIC EFFECT OF PACLITAXEL 10
Fig. 4. Western Blotting of protein
extracted from two cell types, namely (A) Vero
and (B) HeLa cells after treatment with various
doses of paclitaxel. The doses used are recorded
on the top of each figure. Each protein as
indicated on the left of the figure was separately
resolved on 12% SDS-PAGE and blotted on to
nitrocellulose membrane as described in
materials and methods. Beta actin was used as
loading control in each case.
Western Blot Analysis
In order to explore downstream targets of
paclitaxel, various proteins related to MAPK
pathway have been identified through western blot
technique (Fig. 4) MAPK pathway is a major
signaling pathway that promotes survival and inhibit
apoptosis by phosphorylating pro-apoptotic protein
BAD at serine 112 residue thus causing hindrance to
normally ongoing apoptotic mechanism. We have
observed expression of all these closely related
proteins BAD, pBAD-112, pBAD-136, MEK1/2,
pERK1/2, RSK (P90
) and apoptotic marker cleaved
PARP by protein blotting. Equal number of cells
were plated and subjected to drug treatment. Three
concentrations of paclitaxel were selected from
results of MTT and LDH assays that are 1µM, 5µM
and 10µM. Lysates were prepared and resolved on
12% SDS-PAGE. Specific antibodies were applied
to detect proteins of interest according to the
protocol mentioned in materials and methods.
Results were analyzed with the help of Image
analysis software (BioRad). We found decrease in
pERK1/2 expression in HeLa cells which can be
directly correlated with decreased activity of total
ERK1/2. This is also correlated with decreased
pBAD-112 and increase in total BAD, which we
found in our results. Moreover, decrease in pBAD-
136 was observed in HeLa cells at 10µM
concentration as compared to control (untreated
cells) while no difference was seen in Vero cells.
However, we observed almost persistent expression
of MEK1/2, an immediate upstream molecule of
ERK1/2 and RSK which is a substrate of ERK1/2,
in both HeLa and Vero cells. Most importantly,
elevated levels of cleaved PARP have been
observed at all drug concentrations in both cell
types.
Binding of Paclitaxel with anti-apoptotic and
pro-apoptotic proteins
Molecular dynamic studies by using bio-
informative mythologies and tools are of
fundamental importance for fully understanding the
drug protein interactions. To fulfill this aspect of the
study tertiary structures of three anti-apoptotic
proteins namely Bcl-2, Bcl-xL and Mcl-1 along with
two pro-apoptotic proteins (BAX and BAK) were
downloaded from protein data base in PDB file
format. 1GJH, 1BXL, 2KBW, 2MIS, 1FI6 are
protein entries for selected proteins respectively.
Structure of paclitaxel was downloaded from
Pubchem, a compound database. Pre-processing of
target proteins, ligand preparation and docking were
done by using the procedures shown in materials
and methods section. Hydrophobic groove formed
by BH1, BH2 and BH3 domains are a characteristic
feature of these target proteins. Occupation of this
hydrophobic groove by any ligand (peptide or drug)
may modulate the functions of these proteins.
Using the same bio-informatics tools
paclitaxel was docked on two pro-apoptotic proteins
(Fig. 5). In this case the drug bound to two proteins
but did not occupy the hydrophobic groove which is
a significant binding. The binding of the drug with
these proteins outside the hydrophobic pocket with
energies and amino acid binding are shown in
Table 1.
In case of anti-apoptotic proteins, significant
results have been obtained. Paclitaxel shows strong
binding with Bcl-2, Bcl-xL and Mcl-1 (Fig. 6). The
binding pattern of the drug varies in each
anti-apoptotic protein. In each case however, the
CHEMOTHERAPEUTIC EFFECT OF PACLITAXEL 11
Table 1: Binding of paclitaxel with Bcl-2 family of proteins.
Protein
targets
BH
domain
Amino acid
Position
in sequence
Binding site
Interacting residues
Binding
energy
(Kcal/M)
Hydrogen
bonds
Bond Length
Å
(4 Å Radius)
Hydrophobic
bonds
BAK
BH3 74-88 Arg88
-8.8
1 (Arg88) 3.113
6 BH1 117-136 Ser117, Ser121, Arg127,
Glu120
2 (Ser121) 3.388
BH2 169-184 3.63
BAX
BH3 59-73
-9.7
1 2.011
4 BH1 98-118 Asp158, Trp158
BH2 150-165
Bcl-2
BH3 93-107 Ala100, Asp103, Phe104,
Arg107 -10.1
9 BH1 136-155 Asn143, Gly145 1(Tyr202) 3.153
BH2 187-202 Leu201, Tyr202
Bcl-xL
BH3 86-100 Ala93, Glu96
-10.1
5 BH1 129-148 Gly138, Ala142 1(Tyr195) 1.882
BH2 180-195 Tyr195
Mcl-1
BH3 209-232
-10.2
2(Arg310) 2.076
6 BH1 252-272 Asn260 2.596
BH2 304-319 Trp305, Gln309, Arg310,
Phe318
(A): BAK-Paclitaxel (B): BAX-Paclitaxel
Fig. 5. Docking of paclitaxel on to the pro-apoptotic proteins. Three distinct colors represent different domains
of the hydrophobic groove. Green color is specific for BH1; red for BH2 and yellow is for BH3. (A) Shows paclitaxel
docked onto BAK. It may be seen that paclitaxel is partially bound with BH1 and BH3 domains. (B) Shows docking
of paclitaxel onto BAX. The drug interacts only with BH2 domain.
hydrophobic groove through which the anti-
apoptotic proteins capture the pro-apoptotic proteins
is preoccupied with the drug, thus releasing the pro-
apoptotic proteins for oligomerization. Binding
energies calculated for each receptor protein are also
recorded in Table 1. Both Bcl-2 and Bcl-xL bind
with paclitaxel with energy of -10.1 and covers all
three domains of hydrophobic groove. In Mcl-1
CHEMOTHERAPEUTIC EFFECT OF PACLITAXEL 12
(A): Bcl-2-Paclitaxel
(B): Bcl-xL-Paclitaxel
(C): Mcl-1-Paclitaxel
Fig. 6. Paclitaxel docked onto anti-
apoptotic proteins. (A) Shows paclitaxel
completely occupying hydrophobic pocket
formed by BH1 (Green), BH2 (Red) and BH3
(Yellow) domains. (C) Mcl-1 shows partial
binding of paclitaxel only at BH1 and BH2
domains.
however, the drug is bound only to BH2 and
partially to BH1. The binding energy though is of
the order of -10.2.
From the binding studies it can be concluded
that Bcl-2 and Bcl-xL are the major targets of the
drug, occupying the hydrophobic groove. The
interaction of the drug with amino acids of BH1,
BH2 and BH3 domains is recorded in Table 1. This
table also indicates the strength of bonding with
amino acids in Kcal/M and the length of bond in
angstrom.
DISCUSSION
Several controversies have emerged in
respect of the mechanism by which paclitaxel
induces apoptosis. A few studies have reported that
paclitaxel stabilizes microtubules including spindle
fibers. This may cause arrest of cell cycle at G2/M
phase (Jordan and Wilson, 2004: Abal et al., 2003).
However, activation of MEK followed by ERK, p38
and Janus NH2 terminal Kinase (JNK) has also been
reported (Boldt et al., 2002: Bacus et al., 2001). The
activation of these MAPK pathway proteins induces
apoptosis. This happens independent of arrest of cell
cycle in the G2/M phase (Dziadyk et al., 2004).
Evidence has been provided that phosphorylation of
Bcl-2 (Mcdaid and Horwitz, 2001: Blagosklonny1
et al., 2000 ) is one mechanism which releases the
pro-apoptotic BAK and BAX for oligomerization at
outer mitochondrial membrane resulting in the
release of cytochrome c and subsequent events of
apoptosis through caspase-3. These controversies
have led us to examine the mechanism of apoptosis
induced by paclitaxel in two cell lines separately
exposed to various doses of paclitaxel. Our results
are in agreement with previous reports in as much as
induction of apoptosis is concerned. Yet the data
obtained show variation in the two cell lines used in
this study. Whereas, maximum apoptotic activity
has been observed in HeLa cells at dose levels of
5µM and 10 µM, the apoptotic activity at the
corresponding doses is much less in Vero cells
(Figs. 1, 2).
We have studied another parameter which
involves the estimation of LDH. With the death of
cells, the enzyme is released in the medium. Our
assay results confirm that paclitaxel is more
CHEMOTHERAPEUTIC EFFECT OF PACLITAXEL 13
effective in causing apoptosis in HeLa cells as
compared to Vero cells. In essence the two
parameters MTT and LDH give similar results in the
cell lines used. We are unable to identify similar
data on apoptotic activity of paclitaxel using LDH
activity as a marker (Fig. 3).
Since the anti-apoptotic proteins Bcl-2, Bcl-
xL and Mcl-1 are known to be involved in
promoting cell proliferation, it was considered
necessary to examine whether paclitaxel can bind to
these anti-apoptotic proteins and consequently
releasing BAX and BAK for oligomerization on the
outer mitochondrial membrane.
Western blot studies show a number of
interesting features (Fig. 4). The study was focused
on p-ERK1/2, pBAD-112, pBAD-136, MEK 1 and
MEK2, RSK and cleaved PARP. These proteins
were blotted separately for paclitaxel treated HeLa
cells and Vero cells. In as much as ERK is
concerned we have noted that in HeLa cells there is
some decrease in ERK 1 as compared to ERK2 in
the blotted protein. In Vero cells the reverse is true.
There is marginal increase in ERK1 and
recognizable increase in ERK2 at higher dose levels.
These results are consistent with the previous
studies where paclitaxel induced activation of
RaS/MEK/ERK pathway independent of programed
cell death (Okano and Rustgi, 2001), although
conflicting reports are also available (Sato et al.,
2009). We are interpreting that the decrease of
pERK in HeLa cells is associated with novel
MEK/ERK/RSK/BAD pathway. A few reports have
earlier reported that the normal activation of ERK is
followed by phosphorylation of RSK which in turn
phosphorylates BAD at Serine 112 position. This
results in ubiquitination of ERK through 14-3-3.
Paclitaxel, as our result show decreases the
phosphorylation of ERK and thus spares the pro-
apoptotic BAD for oligomerization at the outer
mitochondrial membrane and release of cytochrome
c. This results in activation of caspases 9 in the
apoptosome and subsequently of executioner
caspases 3. We could not substantiate this evidence
in the Vero cells. Obviously, then, the therapeutic
effect of paclitaxel varies from one cancer cell type
to another. Similarly, pBAD-136 shows substantial
decrease in HeLa cells but remains unchanged in
Vero cells. It is important to note that serine 136 of
BAD is phosphorylated by Akt. This should be
interpreted to mean that paclitaxel acts in three
ways: (a) through arresting the cell cycle at G2/M
phase as demonstrated in other studies (Jordan and
Wilson, 2004: Abal et al., 2003: Jordan et al.,
1996), (b) through MEK/ERK/RSK/BAD pathway
as demonstrated in this report and (c) through Akt
pathway as indicated above.
We were particularly interested in examining
the data on the western blot of PARP. It is known
that cleaved PARP normally undertakes repair of
DNA damage through PARP function. This might
be one reason that cell cycle continues through
G2/M phase however, cleaved PARP is responsible
for causing cell death through apoptosis or necrosis.
We have observed a considerable increase in PARP
at all doses of paclitaxel both in Vero and HeLa
cells. This observation adds to the already known
apoptotic functions of paclitaxel.
It is suspected that paclitaxel may act through
blocking the hydrophobic groove of anti-apoptotic
Bcl-2 proteins. No information is available in
literature about the direct activity of paclitaxel in
respect of blocking the anti-apoptotic Bcl-2 proteins
and release of pro-apoptotic BAX and BAK which
may proceed towards the process of apoptosis
through the well-established pathway involving
release of cytochrome c by mitochondria. However,
indirect evidence is shows that Bcl-2 is
phosphorylated by p38 and prevented from binding
with the pro-apoptotic proteins. In view of this, bio
informative tools have been used in this study to
find out whether paclitaxel can block the
hydrophobic groove of anti-apoptotic proteins (Bcl-
2, Bcl-xL and Mcl-1), like other natural flavonoids.
In this laboratory, we have previously reported the
binding of Curcumin, Deguelin, HA14-1 and
Silibinin with these proteins. The results obtained
with paclitaxel give new information that two
proteins, namely Bcl-2 and Bcl-xl are completely
blocked by this drug. The drug establishes a number
of hydrogen bonds with the amino acids of BH1,
BH2 and BH3 domains (Fig. 6, Table 1).
Additionally, it has been noted that the drug does
not completely occupy the hydrophobic groove of
Mcl-1. It binds partially with BH1 and BH2 domains.
We propose that this is yet another mechanism
through which paclitaxel induces apoptosis. This
CHEMOTHERAPEUTIC EFFECT OF PACLITAXEL 14
Fig. 7. The results obtained in this study are summarized in this figure. It may be observed that activation of
RTK by the activator (ligand) which may include a growth factor in cancer cells like HeLa. This generalized diagram
shows the activation of RaS-MAPK pathway culminating in the activation of ERK. (1) Indicates inhibition of survival
by the drug used resulting in phosphorylation of BAD at serine 112, (2) indicates inhibition of anti-apoptotic proteins
(explored by bio informative tools) and activation of Akt and phosphorylation of BAD at serine 136. The drug inhibits
this pathway, (3) indicates the activation of caspases 9 & 3 which lead to execution of cells after release of
cytochrome c through rupture of mitochondrial membrane.
will require further elucidation after obtaining the
western blots of these proteins in paclitaxel treated
cells.
Taken together we can conclude that our
studies indicate: (a) apoptotic effect of paclitaxel
varies from one cancer cell type to the other, (b) by
and large and at least in HeLa cells it acts through
MEK/ERK/RSK/BAD pathway, (c) Akt pathway
seems to be activated in sympathy with the
activation of RAS/MAPK pathway, (d) cleaved
PARP seems to be an indication of its participation
in cell death through apoptosis and necrosis and (e)
additionally, as reported vide supra, paclitaxel
directly block the anti-apoptotic proteins for
inducing apoptosis (Fig. 7). Obviously further
studies are needed on different cell lines to elucidate
more fully the findings of this study.
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(Received: July 18, 2014: Revised: August 20, 2014)
EFFECT OF NITROGEN SOURCE & NIACIN ON ß-MANNANASE PRODUCTION BY B. BADIUS
14
Pakistan J. Mol. Med., 1(1-2), pp. 17-22, 2014
www.pjmm.uol.edu.pk
Effect of Different Nitrogen Sources and Niacin on
β-Mannanase Production from Locally Isolated Bacterial
Strain Bacillus badius
Syed Shahid Ali,1* Bushra Atta
2 and Tanzeela Riaz
2
1Institute of Molecular Biology and Biotechnology (IMBB),
The University of Lahore, Defense Road Campus, Lahore, Pakistan; 2Biochemistry and Toxicology Lab., Department of Zoology, University of the Punjab, Lahore, Pakistan
ABSTRACT
β-1, 4-Mannanase is a mannan-degrading, glycohydrolase with wide applications in food,
poultry, detergent, oil and natural gas industry. This enzyme is also used in up gradation of
variety of agricultural wastes. In the present study, effect of different nitrogen sources, two
inorganic (ammonium nitrate and sodium nitrate) one organic (soya bean meal) and niacin on β-
1, 4-Mannanase produced by Bacillus badius was investigated using MS-agar plates. Enzyme
production and total soluble proteins were determined using different nitrogen sources (0.5%) in
the medium. Significant increase in enzyme activity (7-9 folds) in soya bean meal containing
media was observed when compared with ammonium nitrate and sodium nitrate (14-24 fold
Enzyme specific activity in crude protein extract also showed 2-3 fold rise in soya bean meal
supplemented media. The enzyme activity and protein contents were maximum in soya bean
meal and minimum in sodium nitrate. Niacin added to the bacterial culture with soya bean meal
as nitrogen source, showed 4.6-9.4 fold increase in enzyme activity, 3.0-3.7 folds increase in
protein content and 1.7-2.5 fold increase in enzyme specific activity, but this increase was
significantly less than their respective controls. It was concluded that among the three nitrogen
sources used in this study, soya bean meal served as the best nitrogen source for β-mannanase
production by B. badius. Moreover, niacin added to the medium also played a significantly
positive role in enhancing the enzyme activity.
Key words: Enzyme activity, glycohydrolase, polysaccharidase, hydrolytic enzyme, enzyme
characterization, nicotinic acid, soluble proteins, enzyme specific activity, vitamin-B3
INTRODUCTION
The most diverse plant cell wall
polysaccharides are those which have 1,4-linked
mannosides. The mannosidic bond of these
polymers is hydrolyzed by β-mannanase (mannan-
endo-1,4-mannosidase; EC 3.2.1.78) enzyme
(McCleary, 1988), which is a member of glycoside
hydrolase (GHs) family (Hogg et al., 2003), and
______________________________________ * Corresponding author: [email protected]
catalyses’ the random cleavage of β-1,4-mannosidic
linkages in the backbone of mannans, glucomannans
and galactomannan (Naughton et al., 2001; Dhawan
and Kaur, 2007), resulting in production of different
oligosaccharides as major products. Hetero-1, 4-β-
D-mannans, one of the main components of
hemicellulose, are hydrolyzed to mannose with the
help of endo-acting β-mannanases (1,4-β-D-
mannan-mannanohydrolase [EC 3.2.1.78])
(McCleary, 1988), and exo-acting β-mannosidases
(β-D-mannopyranoside hydrolase [EC 3.2.1.25]
(McCleary and Matheson, 1983; 1986). These
EFFECT OF NITROGEN SOURCE & NIACIN ON ß-MANNANASE PRODUCTION BY B. BADIUS
15
hydrolases are also classified as hemicellulases
(Shallom and Shoham, 2003).
β-Mannan-based natural polymers are
extensively used in industries, such as, in food
processing and the massive hydraulic fracturing of
oil and gas wells. Treatment of coffee beans by
enzymes (Hashimoto and Fukumoto, 1969) and
Konjac production (Oda et al., 1993a,b) both
involve the β-mannan-based hydrolysis of
oligosaccharides. Soil, water bodies, manure and
animal rumen contain mannanolytic and other
glycolytic microbes which can selectively work in
acidic and alkaline environment (Zakaria et al.,
1998; Ali et al., 2009). In several industrial
processes, for example extraction of vegetable oils
from leguminous seeds, viscosity reduction of
extracts during the production of instant coffee
and manufacture of oligosaccharide, mannanases
have been tested (Gubitz et al., 1996; Ademark et
al., 1998). The enzyme has also been used in textile
industry (Pedersen et al., 1995).
Mannanases can be useful in food, feed as
well as in paper and pulp industries (Kobayashi et
al., 1987; McCleary, 1988; Ademark et al., 1998).
Mannanase has been effectively used in clarification
of fruit juices (Christgua et al., 1994), in
manufacturing of instant coffee, chocolate and
cocoa liquor (Francoise et al., 1996). In addition,
mannanases are potentially applied in the
pharmaceutical industry for the production of
physiologically active oligosaccharides (Lin and
Chen, 2004).
Despite having high practical potentialities,
the use of mannanase is still limited due to low
yields and high-production costs (Zhang et al.,
2000). Various microorganisms have been reported
as mannanase producers (McCleary, 1988). Among
those is Bacillus subtilis which is recommended
because of its safety, fast growth and ability to
secrete a high level of mannanase into the medium
(Mendoza et al., 1994; Zakaria et al., 1998).
In this study we are reporting the production
of β-mannanase enzyme by a locally isolated
bacterial strain B. badius. The objective of the study
also include to explore the ways to increase the
mannanase production potential by metabolite
adjustments by supplementing different nutritional
sources such as nitrogen and niacin (vitamin B3) in
the bacterial culture media.
MATERIALS AND METHODS
Screening of bacterial isolates
Forty five bacterial isolates from this Lab
(Sanaullah and Ali, 2012) were screened for β-
mannanase activity on mineral salt (MS) agar plates,
according to Jones and Ballou (1969) as modified
by adding galactomannan (substrate) as locust bean
gum (LBG) 4g/l; NH4NO3 0.5g/l; MgSO4.7H2O
0.2g/l; FeSO4.7H2O 0.01g/l; CaCl2.2H2O 0.05g/l;
K2HPO4 7.54g/l; KH2PO4 2.32g/l; agar 15g/l; pH
was adjusted at 9 because it showed maximum
growth at this alkaline pH (Sanaullah and Ali,
2012). The medium was autoclaved at 15 lbs
pressure for 15 minutes (Mabrouk and El Ahwany,
(2008). The plates were inoculated by streaking
straight in the middle with inoculating loop
followed by incubation for 24 h at 37±1ºC. β-
mannanase activity was detected on agar plates by
staining the plates with iodine for 15 minutes (Fig.
1). The enzyme activity (galactomannan hydrolysis)
was determined as the ratio of hydrolysis zones
(clear areas) to that of the colony.
Estimation of β-mannanase activity
β-mannanase activity of the bacterial medium
was assayed by measuring amount of reducing
sugars produced in the medium from the substrate
using DNS (di-Nitrosalicylate) method (Miller,
1959) For this purpose the bacterial media were
centrifuged to remove the bacteria as pellet. The
clear supernatant obtained was used as a source of
enzyme and soluble proteins. The substrate-buffer
mixture was prepared by homogenizing 0.5% LBG
in 50mM phosphate buffer (pH 7.0) at 100ºC. The
mixture was stored at room temperature overnight
followed by autoclaving at 15 lbs pressure for 20
min. The insoluble substrate was removed by
centrifugation at 4x103rpm and 4ºC for 30 minutes.
For reaction, the supernatant (1.8ml) as substrate
buffer mixture and crude enzyme (0.2ml) were
mixed and incubated at 50ºC for 5 minutes followed
by addition of 1 ml DNS reagent. The mixture was
allowed to boil in water bath for exactly 15 minutes.
The absorbance of developed red brown color was
measured at 575nm wavelength with spectrophoto-
EFFECT OF NITROGEN SOURCE & NIACIN ON ß-MANNANASE PRODUCTION BY B. BADIUS
16
meter. The β-mannanase activity was measured
from the standard curve prepared by using D-
mannose as a standard. The enzyme activity has
been expressed as units of enzyme activity/ml of the
bacterial medium. One unit in this study has been
defined as the amount of enzyme that liberates 1 µg
of mannose/min/ml of the medium.
Protein estimation
The total soluble proteins produced by the B.
badius in the medium were estimated according to
Lowery et al. (1951) from the supernatant after
pelleting out the bacteria. The soluble proteins were
quantified by preparing the protein standard curve
using BSA.
Effect of nitrogen source on β-mannanase
production
The mineral salt (MS) medium supplemented
with 1% galactomannan (locust bean gum) as a
carbon source was used for this study (Rattanasuk
and Ketudat-Cairns, 2009). Two inorganic nitrogen
sources, i.e., ammonium nitrate, sodium nitrate and
one organic i.e., soya bean meal were used to study
the role of nitrogen source in enzyme production.
Their concentrations in the mineral basal media
were fixed at 0.5% in separate experiments. The pH
of the medium was adjusted at 7.0 throughout the
experimental work. Inoculum (50µl) was transferred
to 100ml flasks containing 50ml of sterilized MS
medium and was allowed to incubate for 24, 48, 72
and 96 h at 37ºC in shaking water bath at 140 rpm.
After the desired incubation period, respective broth
media were removed from water bath and
centrifuged at 10,000 rpm for 15 minutes at 4ºC.
The supernatants containing crude enzyme and
soluble proteins were collected carefully and used
for estimation of enzyme activity and soluble
protein content (Mabrouk and El-Ahwany, 2008).
Effect of niacin on β-mannanase and soluble
proteins of B. badius
Niacin (0.01%) was added to galactomannan
containing MS medium using soya bean ammonium
nitrate as nitrogen source to evaluate its effect on β-
mannanase activity and total proteins of B. badius.
The enzyme activity and medium soluble proteins
were determined at 24, 48, 72 and 96 h incubation.
The procedure used for both analyses has already
been described in the previous section.
RESULTS
Screening of isolates for β-mannanase activity Out of 45 isolates, 18 showed the β-
mannanase activity (clear areas) on galactomannan-
containing agar plates after 24 h incubation. The
activity ratio was determined by measuring areas of
hydrolysis zones around the streaked areas
(Table 1). Bacillus badius, with highest hydrolysis
zone (best enzyme producer) was selected for
further study (Fig. 1).
Fig. 1: Hydrolysis zone indicating the β-
mannanase activity of Bacillus badius after 24 h
incubation on iodine agar plate.
β-mannanase activity of B. badius
β-Mannanase activity of the B. badius was
compared by using three different nitrogen sources
in the bacterial medium i.e., sodium nitrate,
ammonium nitrate and soya bean meal after 24, 48,
72 and 96 h incubations. Significantly high level of
β-mannanase activity (62-152 units/ml) was found
in soya bean meal containing media from 24-96 h
incubation periods as compared to sodium nitrate
(7-11 units/ml) and ammonium nitrate (8-22
units/ml). The activity was maximum at 72 h
incubation period (Table 2). The lowest enzyme
activity was observed in case of sodium nitrate as a
nitrogen source while enzyme activity in case of
EFFECT OF NITROGEN SOURCE & NIACIN ON ß-MANNANASE PRODUCTION BY B. BADIUS
17
ammonium nitrate as nitrogen source was somewhat
higher than sodium nitrate during this 96 h
incubation period. It was also noticed that maximum
activity in all 3 nitrogen sources was at 72 h
incubation period after which the drop in enzyme
activity was observed (Table 2).
Table 1: Activity ratios for β-mannanase produced by
different mannanase producing bacterial
isolates on iodine-agar plates containing LBG
(galactomannan) as a substrate
Sr. # Isolate # *Activity Ratio
1 BCTL-SL-188 22.03
2 BCTL-SL-173 6.23
3 BCTL-SL-164 7.46
4 BCTL-SL-187 7.31
5 BCTL-FL-16 3.87
6 BCTL-FL-20 17.95
7 BCTL-SL-172 16.91
8 BCTL-SL-192 6.69
9 BCTL-SL-195 5.19
10 BCTL-SL-189 6.5
11 BCTL-VL-16 17.39
12 BCTL-VL-17 16.5
13 BCTL-SL-178 9.53
14 BCTL-SL-170 12.0
15 BCTL-SL-167 4.0
16 BCTL-VL-15 12.28
17 BCTL-VL-192 11.43
18 BCTL-SL-166 14.0
Total soluble proteins
The total soluble proteins of the bacterial
media showed almost similar trend in case of three
different nitrogen sources as was found in β-
mannanase activity. The maximum soluble protein
content observed was 50mg/ml in the medium
containing soya bean meal after 72 h of incubation
at 37°C which exhibited about 44% decrease at 96 h
incubation. The overall range of protein content in
this case was from 28-50mg/ml during 24-96 h
incubation.
In case of sodium nitrate and ammonium
nitrate supplemented media, the protein contents
were in the range of 7.41 to 10.22mg /ml and 7.92 to
12.54mg/ml, respectively under similar incubation
and temperature conditions which showed increase
up to 72 h incubation while decreased 3.7 fold and
3.5 fold, respectively at 96 h incubation (Table 3).
Enzyme specific activity
The enzyme activity and total soluble protein
content were used to calculate the specific activity
of the enzyme (enzyme activity/mg of protein).
When specific enzyme activities of soya bean meal-,
ammonium nitrate- and sodium nitrate-
supplemented media were compared, the maximum
activity (2.34-3.06 units/mg of protein) was found in
soya-supplemented medium. The specific activity in
this case showed regular increase from 2.52 units/
mg at 24 h to 3.06 unit/mg of protein up to 72 h
incubation which showed 24% decline following
extending the incubation period up to 96 h. The
enzyme specific activity showed significant
decrease in sodium nitrate (62-64%) and ammonium
nitrate-supplemented (43-59%) media, respectively
when compared with their respective incubation
period from soya bean meal containing media. It
was also noticed that maximum specific activity was
found at 72 h incubation in all nitrogen sources used
followed by decrease at 96 hour duration (Table 4).
Effect of niacin on β-mannanase and total soluble
proteins B. badius
The β-mannanase activity and total soluble
proteins produced by B. badius following
supplementing niacin (0.01%) was studied in the
bacterial medium using soya bean meal as a
nitrogen source. In the control media, β-mannanase
activity was ranged from 57-139 units/mg. The
maximum activity (395.5 units/mg) was observed at
72 h while lowest (106.11 units/mg) was found at 96
h incubation periods. Following supplementing
niacin at 0.01% concentration in the medium, highly
significant increase (1.8 fold, 1.7 fold and 2.85 fold)
in bacterial mannanase activity was noticed at 48,
72 and 96 h incubations, respectively. Similarly
total soluble protein content following addition of
niacin to the bacterial medium showed 19% and
20% rise from their respective controls, at 48 and 72
h incubation periods. The specific activity of the
enzyme in control bacterial media was in the range
of 1.80 to 2.53 units/mg of protein which following
niacin addition to the medium almost showed
similar pattern as exhibited by mannanase activity.
The specific activity exhibited 2-3 fold increase in
this 96 h incubation period with maximum at 72 h
and minimum at 96 h (Table 5).
EFFECT OF NITROGEN SOURCE & NIACIN ON ß-MANNANASE PRODUCTION BY B. BADIUS
18
Table 2: Effect of various nitrogen sources in the medium on β-mannanase production by the bacterial isolate, Bacillus
badius.
Nitrogen source β-mannanase activity ( units/ml)d
24 48 72 96
Ammonium nitrate 12.41±1.78 15.04±1.15 21.95±1.58 b 7.54±0.52 a
Sodium nitrate 7.95±1.22 8.29.05±1.17* 10.65±1.21** 6.69±1.08
Soya bean meal 115.34±2.65*** 125.85±2.24***a 151.75±3.59***b 62.34±2.33***b
The data has been shown is given in terms of Mean ±SEM, Students “t” test, n=3. Significance has been shown in rows as; a, b, c, and in
columns as: *, **, *** with p> 0.05; p > 0.01; p> 0.001, respectively. In rows the data has been compared with ammonium nitrate,
while in columns comparison was made with respective nitrogen source at 24 hour incubation. d Enzyme units; 1 unit enzyme activity= amount of enzyme that liberates 1µg of mannose/ minute/ ml of the bacterial medium.
Table 3: Effect of various nitrogen sources in the medium on total soluble protein content of Bacillus badius.
Nitrogen source Total protein content (mg/ml)
24 48 72 96
Ammonium nitrate 9.31±1.24 10.32±1.51 12.54±1.62 a 7.92±0.94
Sodium nitrate 8.23±1.14 8.64±1.43 10.22±1.35 7.41±1.20
Soya bean meal 45.64±3.15** 46.75±2.73*** 49.46±2.13*** 27.52±2.11**b
The data has been shown as Mean ±SEM, student’s “t” test, n=3. For further details of statistical analysis, see foot note in table 2.
Table 4: Effect of various nitrogen sources in the culture medium, on the enzyme specific activity of Bacillus badius.
Nitrogen source Enzyme Specific activity (units /mg of protein)
24 48 72 96
Ammonium nitrate (Control) 1.33 1.46 1.75 0.95
Sodium nitrate 0.97 0.96 1.04 0.90
Soya bean meal 2.52 2.69 3.06 2.34
Table 5: Effect of niacin (0.01%), added to the Bacillus badius culture medium, on β-mannanase activity, soluble proteins
and enzyme specific activity. Parameters Treatment 24 hours 48 hours 72 hours 96 hours
β-Mannanase activity (units/ml) Control 110.56±2.48 115.04±4.13 138.59±2.58 c 56.78±3.42 c
Niacin 198.51±4.27*** 235.15±4.72***b 395.50±6.68***c 106.11±3.70**c
Total protein content (mg ml) Control 43.67±2.61 48.72±3.23 60.24±3.02 b 31.45±2.13 a
Niacin 44.52±2.72 58.16±3.63**b 72.57±3.42***c 29.08±2.52 b
Enzyme Specific activity
(units/ mg of protein)
Control 2.53 2.36 2.31 1.80
Niacin 4.46 4.03 5.50 3.66
. The data has been shown as Mean ±SEM, Students “t” test; with n value = 3. For other details, see Table 5.
EFFECT OF NITROGEN SOURCE & NIACIN ON ß-MANNANASE PRODUCTION BY B. BADIUS
19
DISCUSSION
Enzymes are important biological catalysts
that increase the rate of biological reactions
(Soetan et al., 2010). Since several decades the
microbial enzymes have been employed for
variety of industrial and biosynthetic purposes.
Bacterial enzymes are also playing an important
role in the up gradation of low cost plant biomass
and waste and biodegradation of toxic organic
pollutants (Gubitz et al., 1997, 2001., Peixoto et
al., 2011). In the current study, Bacillus badius
which demonstrated highest activity ratio (22.03)
qualitatively, was further tested for increased β-
mannanase activity following supplementing the
medium with three different nitrogen sources i.e.,
sodium nitrate, ammonium nitrate and soya bean
meal.
Many workers from various labs have
demonstrated the microbial β-mannanase activity
under various medium conditions (Ferreira and
Filho, 2004; Blibech et al., 2010; Meenakshi et
al., 2010). In a similar study, Phothichitto et al.
(2006) obtained highest mannanase activity ratio
of 6.00 produced by two bacterial isolates NT 6.4
and NT 6.3. Mabrouk and El-Ahwany (2008)
reported the β-mannanase activity-ratio of 1.6
shown by Bacillus amylolequifaciens which was
significantly low than the activity ratio shown by
B. badius in the present study. However,
Adebayo-Tayo (2013) screened 16 Bacillus
strains for mannanase activity for 24, 48 and 72
hour durations using LBG, yeast extract, peptone
containing MS media. Bacillus licheniformis S4d
and S4e exhibited maximum diameter (34mm and
30 mm, respectively) at above three incubation
durations. Almost all 16 strains showed uniform
activity (clear zones) at 24, 48 and 72 hours in this
study. Increase in β-mannanase activity has also
been reported by different labs following
supplementing the microbial culture media with
variety of carbon and nitrogen sources and other
growth factors (Feng et al., 2003; Ab-Rashid et
al., 2011; Adebayo-Tayo et al., 2013). In a study
on six mannanase producing strains of Bacillus,
highest enzyme production and growth was given
by Bacillus megaterium UI strain with 1.561 U/ml
activities (Adebayo-Tayo et al., 2013).
Enzyme production may be dependent on
bacterial cell growth and incubation period.
During present study, highest β-mannanase
producing isolate B. badius showed maximum
enzyme activity at 72 h of incubation. Rattanasuk
and Ketudat-Cairns (2009) reported, 1.70 U/l β-
mannanase activity by Crysobacterium
indolegens, after 24 h and Manjula et al. (2010)
achieved optimum mannanase production
(0.74U/mg) at 44-48 h of incubation by
Paenibacillus sp. Olaniyi et al. (2013) tested
various carbon sources like, cassava peels, yam
peel, potato peel, pineapple peel, orange peel,
copra peel, rice and wheat bran. The maximum
mannanase activity (37.96 U/ml, 4.75 U/ml/hr)
was observed with cassava peel and at 96 h
incubation after which the enzyme activity
decreased sharply upto 99% at 192 h incubation.
The data in the current report showed
maximum enzyme activity and soluble protein
content at 72 h incubation followed by significant
decrease in both components at 96 h incubation.
This decrease may be associated with decrease in
medium nutrients and accumulation of various
byproducts and metabolic waste in the bacterial
medium Meenakshi et al., 2010; Malik et al.,
2010).
Amongst the nitrogen sources used, soya
bean meal proved best with 115-152 units/ml
enzyme in the bacterial medium. The activity
increased gradually from 24-72 h incubations after
which about 60% sharp decline was noticed at 96
h incubation. The enzyme activity in other two
nitrogen sources i.e., sodium nitrate and
ammonium nitrate was 14 and 9 times reduced at
24 h and 14 and 7 times reduced at 72 h
incubations, respectively. The maximum
mannanase activity using sodium nitrate as a
nitrogen source in the medium was 10.65±1.21
units/ ml at 72 h incubation which was decreased
by 37% at 96 h incubation. Ammonium nitrate as
a nitrogen source also behaved similarly with
12.41±1.78 units/ml at 24 h, 21.95±1.15 units/ml
at 72 h and 7.54±0.52 units/ml at 96 h
incubations. Similar results were reported by
Mabrouk and El-Ahwany (2008) by using various
inorganic sources as well as agro-industrial
byproducts, on the production of β-mannanase. In
this report ammonium nitrate was found to be the
best nitrogen source among various inorganic
sources. Amongst the agro-industrial by-products
like, potato peel, cotton seed meal, palm seed
powder and wheat bran, the highest specific
activity was achieved by cotton seed meal
EFFECT OF NITROGEN SOURCE & NIACIN ON ß-MANNANASE PRODUCTION BY B. BADIUS
20
(Mabrouk and El-Ahwany, 2008). Among above
organic and inorganic nitrogen sources,
ammonium nitrate was appeared to be best
nitrogen source allowing the production of highest
mannanase activity. In the current study two
inorganic (sodium nitrate and ammonium nitrate)
and one organic nitrogen source (soya bean meal)
were used which showed highest activity in soya
bean as a whole, while ammonium nitrate showed
maximum activity among the two inorganic
sources but it was significantly low than the soya
bean meal. In another study using various organic
and inorganic nitrogen sources (soya beans, locust
beans, yeast extract, whey, peptone, ammonium
nitrate, ammonium chloride and urea) ammonium
nitrate gave maximum enzyme activity and yield
(Olaniyi et al., 2013). These results are in
contradiction with the present study in which
agro-industrial waste, soya bean meal, produced
highest amount of mannanase i.e., 151.26 units/ml
activity by B. badius after 72 h. The remarkable
differences in the enzyme yields in different
studies may be due to varying amounts of
essential amino acids, peptide, vitamins, trace
elements and other mineral salts in different
organic nitrogen sources which may be produced
in the medium by metabolite adjustments.
Since soya bean meal containing media
produced highest mannanase activity during the
current experiment, the effect of niacin on
mannanase production was evaluated using soya
bean meal as nitrogen a source. Niacin (vitamin
B3) plays an important role in energy producing
mechanism as oxidized cofactors (NAD+ and
NADP+)
and in biosynthetic pathways as reduced
cofactors (NADH and NADPH). In the current
study, addition of niacin in the soya bean meal
containing media resulted in gradual rise in
enzyme activity from 80%, 104% and 186% at 24,
48 and 72 h incubations when compared with their
respective control values. This increase was found
in both control and niacin supplemented media
but significantly higher rise was noticed in niacin
added media as compared to control media, right
from 24 h incubation period. The enzyme activity
dropped sharply from 72 h value, by 60% in
control and 96% in niacin added media on
extending the incubation up to 96 h. This drop in
enzyme activity after 72 h might be due to
depletion of medium nutrients and/ accumulation
of metabolic wastes and other by-products. The
soluble protein content of the media also
increased by 19% and 21% after 48 h and 72 h
incubations, respectively. The specific activity of
the enzyme in the crude bacterial medium showed
71-138% rise following niacin addition. It is
concluded from this study that B. badius is a good
candidate for mannanase production using soya
bean meal as a nitrogen source and niacin as an
additional growth factor. In another similar report
from this lab, thiamine (vitamin B1) at 5mg and
10 mg/l concentration in the bacterial medium
produced significant inhibition of amylase
activity by two Bacillus strains BCTL-X-11 and
BCTL-SL-101 (Ali et al., 2010). These findings
suggest that the concentrations of thiamine used
during this study were fatally higher than the
optimum concentrations of this cofactor required
for the growth of these bacterial strains.
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(Received: February 15, 2014; Revised: May 12, 2014)
EFFECTS OF EMAMECTIN & ITS MIXTURE WITH SILYMARIN ON SOME ENZYME ACTIVITIES IN CHICK
24
Pakistan J. Mol. Med., 1(1-2), pp. 24-29, 2014
www.pjmm.uol.edu.pk
Effects of Emamectin Benzoate and its Mixture with Silymarin
on Some Enzymatic Activities of Blood, Liver and
Kidney of Chicks
Mushtaq A. Saleem, Sadia Yasin, Muhammad Yousaf, Waleed Javed Hashmi and Rabail Alam
Institute of Molecular Biology and Biotechnology,
The University of Lahore, Defense road Campus, Raiwind Road, Lahore, Pakistan.
ABSTRACT
The major objectives of the present experimentations were to determine the effect of
emamectin benzoate on blood, liver and kidney enzyme activities in chicks and to determine the
role of silymarin as an antidote on some enzymatic activities such as alkaline phosphatase
(AKP), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT)
and lactate dehydrogenase (LDH) activities and some macromolecules such as total cholesterol
(TCh), triacylglyceride (TAG) and total protein (TP) contents. For this study, 33 chicks (6 days
old) were taken and divided into three groups i.e., control, treatment and antidote groups with 11
chicks in each. Emamectin was administered to chick at the rate of 3 mg/kg body weight/day
with one drop of ethanol. Antidote was administered with emamectin at the rate of 3 mg/kg body
weight/day with one drop of ethanol and silymarin at the rate of 2.97 mg/chick. Total
cholesterol, triacylglyceride (TAG) and total protein levels showed non-significant changes in
the blood sample. Major changes were seen in TAG (176.00 mg/dl) content of blood and GPT
activity of both liver (58.62 IU/L) and kidney (56.85 IU/L) of chicks. Silymarin restored the
levels to the normal range. Thus, it was concluded that silymarin served as an effective antidote
as it normalized emamectin benzoate disturbed enzyme activities and macromolecules tested in
the present study.
Key Words: Emamectin, silymarin, enzyme activities, macromolecules, Streptomyces
avermitilis.
INTRODUCTION
Emamectin benzoate is an insecticide
which is derived from abamectin (Gacemi and
Guenaoui, 2012). Emamectin is the product of soil
microorganisms Streptomyces avermitilis prepared
by the process of fermentation (Schallman et al.,
1987).
Eight novel avermectin homologues of
emamectin (A1a, A1b, A2a, A2b, B1a, B1b, B2a
and B2b) have been discovered. The most active
component of avermectin is B1 series (Sutherland
and Campbell, 1990). Its physical appearance is off
white powder (Waddy et al., 2007). It is soluble in
water at 25°C (0.024 g/L at pH 7) (Yoshii et al.,
2004). Two homologue compounds as B1a (90%)
and B1b (10%) are major constituents of
emamectin. The structure of B1a and B1b differs
only at Carbon-25 position, having sec-butyl group
and isopropyl groups respectively (Yen and Lin,
2004).
The products of emamectin have been
extensively used throughout the world for the
control of lepidopteron insect pests on vegetable
crops (Gacemi and Guenaoui, 2012). It was
EFFECTS OF EMAMECTIN & ITS MIXTURE WITH SILYMARIN ON SOME ENZYME ACTIVITIES IN CHICK
25
observed that predatory mites have been found
highly susceptible to avermectin which are
beneficial organisms. In recent years the use of
avermectin has been evaluated in an Integrated Pest
Management (IPM) system carefully. Bioassays
conducted with fresh residue of avermectin sprayed
on leaves and left to dry, manifested 100% mortality
of proto-nymphs of predatory mite, Phytoseius
plumifer (Acari: Phytoseiidae) (Nadimi et al., 2009).
Emamectin benzoate received its first global
registration under the trade name Affrim® in Japan
in 1998. Emamectin as an insecticide has been used
as major resort for the reduction of the major losses
caused by infestation of insect pests in various
crops, vegetables and fruit plant (Khan et al., 2012).
Silymarin is an anti-oxidant with diverse range of
effects on biological and pharmaceutics. It is extract
of Silybum marianum belonging to a family of
Asteraceae. It is commonly known as milk thistle.
Jahan et al. (2013) reported that silymarin has an
ability to protect tissues (liver and kidney) from
damage which is caused by administration of
alcohols and some other toxins. The active
components of silymarin are silybin A, silybin B,
isosilybin A and silydianin (Kaur and Agarwal,
2007).
Effects of silymarin have also been indicated
in various diseases of different organs such as
prostate, lungs, central nervous system, kidneys,
pancreas, and skin (Gazak et al., 2007). It was
firstly grown in Europe and was used as liver tonic
(Mayer et al., 2005). It is composed of flavono-
lignans with 3 isomers (silybin, silydianin and
silychristin) as well as flavonoids which also have
two isomers (taxifolin and quercetin) (Shahbazi et
al., 2012). Silibinin is known as the most active
component of silymarin (Polyak et al., 2013). It is
insoluble in water and administrated orally in
encapsulated form (Schandalik et al., 1992). Its
chemical structure has resemblance with estradiol
which allows it to bind with mammalian estrogen
receptor as target cells and lead to the activation of
endoplasmic reticulum (ER) (Miksicek, 1995).
The process of regeneration of cells
stimulated by silymarin ensures that it is useful in
the treatment of toxicity in liver and kidney damage,
liver and kidney cirrhosis and chronic enlargement
of liver related diseases (Saller et al., 2001). The
seed of silymarin plant has been used for the
recovery of liver more than two thousand years ago
(Kren and Waletrova, 2005). Removal of the toxins
from the liver is its major function (Rainone, 2005).
The two enzymes such as superoxide
dismutase and glutathione peroxidase have
antioxidant activity that act as alloxan and cause
pancreatic damage, which is treated by silymarin
(Soto et al., 2003). In addition, some injuries like
liver enlargement, hepatocellular necrosis, fibrosis
as well as carcinomas take place due to the
excessive iron deposition in liver (Zhao et al.,
2005). Liver and kidney show antioxidative and
membrane stabilizing effects due to silymarin. The
metabolism of plasma lipoproteins is regulated by
liver. Advanced studies revealed that silymarin act
as a hypo-cholesterolaemic agent (Skottova and
Krecman, 1998).
In the present experiments, we have evaluated
the effects of emamectin benzoate in chick’s serum,
liver and kidney through estimation of some
enzymatic activities such as alkaline phosphatase
(AKP), glutamate oxaloacetate transaminase (GOT),
glutamate pyruvate transaminase (GPT), lactate
dehydrogenase (LDH), and some macromolecules
such as total cholesterol (TCh), triacylglyceride
(TAG) and total protein (TP) contents. In the course
of trials, we have also studied the effect of silymarin
as an anti-oxidative agent to determine whether or
not it can normalize the affected biochemical
component levels.
MATERIALS AND METHODS
Sample collection procedure
For the collection of blood, liver and kidney
samples from chicks, firstly the birds were kept in
the animal house of the University of Lahore at
25°C. Thirty three healthy chicks (6 days) were
taken with average weight of 170g from the Poultry
Farm at Kasur district for this study. They were
divided into three groups of 11 each i.e., emamectin
treated, silymarin treated and control groups.
Emamectin was administered to first group of
chicks at a dose of 3 mg/kg body weight/ day for
one week. Second group of 11 chicks were treated
with emamectin and silymarin simultaneously along
with water for one week. The third group (11
EFFECTS OF EMAMECTIN & ITS MIXTURE WITH SILYMARIN ON SOME ENZYME ACTIVITIES IN CHICK
26
chicks) served as control group. After the stipulated
period, all chicks were dissected. The blood samples
were collected to separate the serum for biochemical
analysis. Liver and kidney samples were
homogenized in 3.0 ml of 0.89% saline. After
homogenization, the tubes were centrifuged at 3500
rpm for 15 min. The clear supernatant samples were
collected in separate tubes for further biochemical
analyses.
Biochemical analyses
Serum and extracts of liver and kidney were
processed and analyzed for estimation of AKP
activity as described by Hafkenscheid and Kohler
(1986), LDH by Buhl and Jackson (1978), GPT and
GOT by Thefeld and Wallhofer (1974), TP by
Gornall (1945), TCh by Allain (1974) and TAG by
Werner and Gabrielson (1981). Statistical analysis
was conducted by using one way analysis of
variance and means were calculated by Duncan’s
multiple range tests (Wahua, 1999).
RESULTS
In the present experiments, biochemical
effects of emamectin benzoate and silymarin were
analyzed on alkaline phosphatase (AKP), lactate
dehydrogenase (LDH), glutamate oxaloacetate
transaminase (GOT), glutamate pyruvate
transaminase (GPT), total protein (TP), total
cholesterol (TCh) and triglyceride (TAG) in chicks,
and the data were recorded. Description of all the
variables is given in the relevant Tables.
The results in Table 1 reveal that the
administration of emamectin benzoate showed
considerable effect on TCh level in serum (p<0.05),
as it was increased significantly (170mg/dl) than
control whereas silymarin tended to normalize TCh
activity (155mg/dl).
The above results demonstrated that TAG
level was affected by emamectin benzoate in serum
(p<0.05). This represents that administration of
emamectin in chicks lead to increase in TAG level
(176.00 mg/dl) while normalization of TAG activity
is produced by administration of silymarin (142.00
mg/dl).
It was also observed that emamectin benzoate
had remarkable effect on TP level in serum
(p<0.05). Its administration in chicks lead to
significantly decreased TP level (1.6 g/dl) as
compared to control group. On the other hand,
silymarin brought re-adjustment of TP contents by
showing its value as 2.1 g/dl.
The results in Table 2 show that emamectin
benzoate increased the level of GPT (58.62 IU/L)
while it was decreased to 34.19 IU/L in antidote
treatment. The administration of emamectin
benzoate in chicks lead to significant increase of
GOT level in liver i.e., 59.62 IU/L as compared to
control (44.68 IU/L). The LDH activity increased to
175 IU/L in treatment and decreased to 179.00 IU/L
in antidote treated chicks. The activity of ALP was
increased in the liver (81.71 IU/L) following
administration of emamectin whereas silymarin
tended to normalize the enzyme activity (73.66
IU/L).
The results given in Table 3 demonstrate that
emamectin benzoate had significant effect on GPT
level (p< 0.05). It lead to elevate the GPT activity in
kidney (58.62 IU/L) while silymarin normalized the
enzyme activity (41.25 IU/L) in chicks. The GOT
level was increased (46.98IU/L) in insecticide
treatment and decreased to 27.68 IU/L after antidote
administration. Emamectin benzoate produced
significant effect on LDH (p > 0.05) by exhibiting
282.7 IU/L activity. Conversely the administration
of silymarin manifested re-adjustment of LDH
contents (193.06 IU/L). Likewise AKP activity
manifested significant increase (120.0 IU/L) in
kidney of emamectin treated chicks whereas it
demonstrated readjustment (76.26 IU/L) in
silymarin treatment.
DISCUSSION
The main objective of present study was to
analyze the toxic effect of emamectin benzoate and
silymarin as an antidote in emamectin toxicated
chicks. The results in this study supported the pre-
existing findings according to which anti-oxidative
property of silymarin has an ability to combat
against poisonous chemicals and provide protection
to liver and kidney (Samah and Ashmawy, 2012).
Loss of functional integration of cell
membrane of liver occurred due to increase in the
activity of enzymes like transaminases (GOT, GPT)
EFFECTS OF EMAMECTIN & ITS MIXTURE WITH SILYMARIN ON SOME ENZYME ACTIVITIES IN CHICK
27
and alkaline phosphatase. These are involved in the
induction of cellular leakage and loss of the
functional integrity of cell membranes of liver. The
liver function was disturbed by increase in GPT and
Table 1: Biochemical effect of emamectin benzoate and its mixture with silymarin on some biochemical components of
blood of chicks.
Enzymes Control Treatment Antidote Significance
Triacylglyceride 135.8±4.18 176±5.60 142±6.16 P>0.001
Total Protein 2.5±0.09 1.6±0.11 2.1±0.57 P 0.001
Total Cholesterol 150.4±7.70 170±7.67 155.5±3.46 0.001
Table 2: Biochemical effect of emamectin and silymarin on some enzyme activities of chick’s liver.
Enzymes Control Treatment Antidote Significance
Glutamate pyruvate transaminase 31.81±3.00 58.62±2.01 34.19±1.16 0.299
Glutamate oxaloacetate transaminase 44.68±4.09 59.62±3.56 51.92±2.05 0.300
Lactate dehydrogenase 185.73±3.0 175±2.00 179.00±3.00 0.240
Alkaline phosphatase 70.07±2.00 81.71±4.01 73.66±4.33 0.201
Table 3: Biochemical effect of emamectin and its mixture with silymarin on some enzyme activities of chick’s kidney.
Enzymes Control Treatment Antidote Significance
Glutamate pyruvate transaminase 40.28±4.96 56.85±3.99 41.25 ± 3.76 0.301
Glutamate oxaloacetate transaminase 28.15 ± 3.66 46.98±4.12 27.68 ± 4.18 0.198
Lactate dehydrogenase 194.27±8.44 282.7±7.25 193.06±6.55 0.258
Alkaline phosphatase 78.86±2.92 120.0±3.0 76.26±3.89 0.688
GOT activities. Antioxidant ability enables
hepatocytes to counteract oxidative stress (Shaker et
al., 2010). It has been evaluated by the present
experimentation that TAG level was increased (2.60
mmol/L) due to the administration of emamectin but
silymarin tended to restore this level (1.20 mmol/l)
nearly to normal range. Liver and kidney was found
affected by showing high level of TAG (Narayan et
al., 2008).
It was also observed in this study that kidney
damage induced by emamectin is normalized by
silymarin which probably acted as an antidote
against emamectin intoxication. Exposure of
silymarin maintains the normal physiology of
kidney tissues. Silymarin is also known to possess
hepato-protective and anti-carcinogenic activity
(Kang et al., 2004). It was observed during this
study that silymarin treated kidney tissue of chick
demonstrated normal levels of biochemical
parameters. It appears from the present findings that
silymarin provided protection against hazardous
chemicals because it contains anti-oxidant property.
It also plays key role as a free radical scavenger.
From the results, it is observed that emamectin
benzoate has toxic effect while silymarin acts as an
antidote on some biochemical parameters in chick.
Conclusion drawn from above results indicated that
high dose of emamectin benzoate cause liver
damage and toxicity in kidney (nephrotoxicity). The
chick biochemical parameters which are affected in
this work by emamectin benzoate included AKP,
aminotransferase (GOT, GPT), LDH, total protein,
total cholesterol and triacylglyceride levels. The
observations manifested that the administration of
silymarin induced significant normalizing effect on
emamectin benzoate intoxicated chicks.
The numerous studies from different
laboratories suggested that silymarin acts as a free
radical scavenger with other specific properties and
works as a unique herbal product used as hepato-
EFFECTS OF EMAMECTIN & ITS MIXTURE WITH SILYMARIN ON SOME ENZYME ACTIVITIES IN CHICK
28
and nephro-protective agent (Jensen et al., 2003).
It can be concluded from the results of
present study that silymarin may be used as a
promising medicinal agent. In addition silymarin
may make a breakthrough as a new chemical which
protects vital organs such as liver and kidney.
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(Received: March 18, 2014; Revised: May 08, 2014)
LIPID PEROXIDATION IN DIABETIC HCV PATIENTS
30
Pakistan J. Mol. Med., 1(1-2), pp. 30-33, 2014
www.pjmm.uol.edu.pk
Assessment of Liver Peroxidation in Diabetic HCV Patients
Receiving Interferon Therapy
Arif Malik,* Sahar Javed, Rabail Alam, Zunaira Tahir, Fatima Zahid, Saeed Ismail,
Hina Aataka Riaz, Naveed Shuja and Abdul Manan
Institute of Molecular Biology and Biotechnology, The University of Lahore,
Defense Road Campus, Lahore, Pakistan
ABSTRACT
Malondialdehyde (MDA) is highly toxic-product and a marker of lipid-peroxidation. It
plays an important role in the development of complications in diabetes mellitus and liver
disease. Studies have shown that diabetes mellitus and liver disorder are linked diseases. This
study was undertaken to determine the MDA levels in HCV patients with diabetes receiving
interferon therapy. In this study MDA level was determined in 50 subjects classified in to three
groups, i.e., control (10), HCV patients (10) and HCV patients with diabetes (30). A significant
increase (78%) and (86%) was observed in MDA levels in HCV patients and in HCV with
diabetes patients respectively. Similarly, alanine amino transferase was increased 50% in HCV
patients and (70%) in HCV with diabetic patients. The present work reflects increased MDA
levels in both HCV patients and HCV patients with diabetes due to higher level of lipid-
peroxidation in biological system under pathological state.
Key Words: Hepatitis, diabetes mellitus, malondialdehyde, Interferon, lipid peroxidation,
cirrhosis, liver failure.
INTRODUCTION
Hepatitis C virus (HCV) is a positive-
strand RNA virus and the size of RNA is 9.6 kb
consisting of 10 genes on genetic material that are
translated into six non-structural as well as four
structural proteins. RNA virus utilizes both the host
and viral proteins that mainly infect and replicates in
hepatocytes. The gene expressions of host cell
exhibit the processes of apoptosis, inflammation,
mitogenesis and fibrosis that are regulated by some
HCV proteins (Dubuisson, 2007).
Tumor necrosis factor-α (TNF-α) is an
important component in the inflammatory process.
In HCV infection, increase in TNF- α level and
changes in insulin signalling pathways could result
in insulin resistance (Farrell et al., 2003; Kawaguchi
et al., 2004). A combined therapy of interferon-α
__________________________________ * Corresponding author: [email protected]
along with ribavirin has widely been used as a
standard treatment option for those patients who
have chronic hepatitis C virus infection all over the
world (Hoofnag and Seeff, 2006; Ghanay et al.,
2009). Consequently, antioxidant therapy to lower
oxidative stress is being investigated to treat the
HCV infection and its related consequences.
Lipid peroxidation is a free radical activity,
plays a significant role in the development of
complications in diabetes (Greismacher et al.,
1995). It has been observed that HCV patients have
enhanced oxidative stress indicated by increased
free radical production (Hiramatsu and Arimori,
1988). MDA is one of the products of lipid
peroxidation and was commonly used to determine
the oxidant or antioxidant balance in the patients of
diabetes mellitus as it is stable and easily assessable
LIPID PEROXIDATION IN DIABETIC HCV PATIENTS
31
(Pasaoglu et al., 2004).
MDA can initiate the production of protein-
aldehyde adducts (Niemela et al., 1994), which are
seen primarily in the perivenous section and they
correspond with signs of more advanced liver
damage (steato-necrosis, focal inflammation, and
elevated serum transaminases) (Parkkila et al.,
1995). This study was undertaken to determine the
MDA levels in HCV patients with diabetes
receiving interferon therapy.
MATERIALS AND METHODS
Collection of blood
This study was carried out on 50 patients who
were categorized into three groups i.e., control with
10 (5 males and 5 females), HCV patients with 10
subjects (5 males and 5 females) and HCV/diabetes
patients with 30 subjects (17 males and 13 females).
The blood was collected from a cuboital vein of
patients and serum was separated within one hour.
Biochemical analysis
The serum samples were evaluated for
malondialdehyde (MDA) content and alanine
aminotransferase (ALAT) activity to assess the liver
damage and lipid peroxidation by the method of
Ohkawa et al. (1979). The ALAT was measured by
commercially available kit (Human Diagnostics®).
Data was analyzed by statistical package using
SPSS (version 17).
RESULTS
The result showed that HCV patients with
diabetes have high level of MDA confirmed the
initiation of pathological process, the lipid
peroxidation. In this work the MDA level has been
related with age and sex ID which reflect changes
with respect to each other. A significant increase
(78%) in MDA levels was observed in HCV patients
and (86%) in HCV with diabetic patients as
compared to control. In this study sex is an
independent factor as it is statistically non-
significant (p>0.05) while age show a significant
values (p<0.05) among all HCV, HCV with diabetes
and in control groups. Correlation between different
parameters was also calculated such as MDA, age
and sex ID. The level of MDA in this study shows a
direct relationship with age. The ALAT activity was
increased 50% in HCV patients and 70% in HCV
with diabetic patients (Table 1) Table 1: The Malondialdehyde and alaninamino-
transferase level in diabetic and non-diabetic
HCV patients.
Parameter Control
(n=10)
HCV
patients
(n=10)
HCV/Diabetes
(n=30)
MDA
(nmol/ml)
5.37±1.56 24.18±1.46* 39.63±1.64*
ALAT
(IU/L)
31.99±4.06 63.82±1.45* 108±1.66*
a mean ± SEM; * p< 0.05 student’s “t” test
Table 2: Pearson Correlation between MDA, Age and
Sex ID in diabetic and non-diabetic HCV
patients
Sex ID Age ALAT MDA
Sex ID 1 .052
.719
-.083
.567
.127
.380
Age 1 .542**
.000
.279*
.049
ALAT 1 .613**
.000
MDA 1
Pearson’s Correlation * Significant at <0.05; ** Significant at
<0.01
DISCUSSION
In patients with hepatitis C, serum MDA
level was elevated at the peak of the disease, during
early convalescence and during regression. MDA is
the final product of lipid peroxidation and it has
been stated that lipid peroxidation is one of the main
cause of hepatocyte damage (Nagoev et al., 2002).
This was also supported by significant increase in
the activity of ALAT which was an indication of
increased transamination and liver damage.
Oxidative stress effects both glucose and iron
metabolism. In another report, MDA level was
found to be significantly high in hepatitis group and
in diabetes with hepatitis patients than control group
(Bertelsen et al., 2001). Oxidative stress can be
explained as a condition resulted from an
LIPID PEROXIDATION IN DIABETIC HCV PATIENTS
32
insufficiency in the antioxidant system and
uncontrolled increase in free oxygen radicals under
certain pathological states (Younossi et al., 2002).
The patients of chronic hepatitis C had
increased MDA concentrations, and enhanced
superoxide dismutase activity by peripheral blood
mononuclear cells (Deby and Goutier, 1990; Boya
et al., 1999). The virus also infects the peripheral
lymphocytes in the viral hepatitis. Interferon
stimulated healthy cells against viruses and it is
produced by infected lymphocytes (Tilg et al.,
1995). Lipid peroxidation, a free radical-induced
mechanism is associated in the pathogenesis of
numerous acute and chronic human disorders,
involving liver pathology (Farinati et al., 1995). An
inability in the oxidative capacity of the cells or any
exposure to more oxidant stress can lead to
acceleration of peroxidation reactions of certain
cellular molecules including lipids in the cellular
membrane system (Yasa et al., 1999).
The present work was designed to investigate
the concentration of MDA as end product of lipid
peroxidation in HCV patients with diabetes
receiving interferon therapy as compare with control
subjects. The MDA level in HCV patients showed
significant increased level as compared to control
group. The patients with HCV and diabetes have
highest levels of MDA as compared to HCV
patients and control subjects. It has already been
shown that high levels of free radicals can lead to
the damage of cellular organelles, increased lipid
peroxidation with development of complications of
diabetes mellitus (Matsunami et al., 2010).
Higher MDA levels have been related to
various disease processes and play a significant role
in the pathogenesis of diabetic complications
(Matsunami et al., 2010). The most important end
product of free radical reactions is malondialdehyde
(MDA). Its plasma level is usually used as a marker
of lipid peroxidation (Dalgic et al., 2005). Plasma
MDA was elevated in chronic hepatitis C and liver
cirrhosis patients (Malik et al., 2013).
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34
Pakistan J. Mol. Med., 1(1-2), pp. 34-38, 2014
www.pjmm.uol.edu.pk
Changes in Blood Pressure After Spinal Anesthesia for
Caesarean Section in Labor Class at Lahore
Khurshid Alam,1 Mushtaq Ahmad Saleem
2 and Faeza Hasnain
2
1Nawaz Sharif Social Security Hospital, Multan Road, Lahore, Pakistan
2Institute of Molecular Biology and Biotechnology, The University of Lahore, Defense Road Campus, Lahore
ABSTRACT
This study was carried out to observe changes in blood pressure after spinal anesthesia in
parturients along with the management. Eighty parturients were selected in a single group. The
age of all patients ranged from 21-30 years. The preoperative, peroperative and postoperative
vitals were measured. The patients were preloaded with one liter of ringer’s lactate and 500 ml
of haemaccele. The bupivacaine was injected to every parturient intrathecally for spinal
anesthesia. Hypotension was developed as a consequence of spinal anesthesia. Hypotension was
considered when systolic blood pressure was less than 90 mm Hg. Postspinal hypotension was
treated with injection ephedrine intravenously. Postspinal hypotension was observed in 85% of
patients. Ephedrine 5 mg to 10 mg was given to patients to correct hypotension. The number of
patients who received ephedrine after 5, 10, 15 and 20 minutes interval was 18.5%, 63.8%,
61.3% and 20% respectively. Nausea and vomiting was noticed in 38.8 % cases. The data was
noted and statistical analysis was carried out by using SPSS (17.0). It was concluded from this
study that the ephedrine is an effective agent used for prevention and management of
hypotension.
Keywords: Ephedrine, bupivacaine, hypotension, hypertension.
INTRODUCTION
Caesarean section is a common surgical
procedure performed for the delivery of the
newborn. In caesarean section, spinal anesthesia is
mostly used method due to its safety, simplicity and
cost effectiveness. During the spinal anesthesia
there is a decrease in blood pressure (hypotension)
in the parturients. Blood pressure defines the
force/pressure exerted by the blood on the vessel
wall. The quantity of blood pumped by the heart
into the aorta is approx 5 L/min in a healthy adult
person at rest (Guyton and Hall, 2011a). The terms
which are used to explain arterial blood pressure are
systolic blood pressure, diastolic blood pressure,
pulse pressure and mean arterial pressure
(Sembulingam and Sembulingam, 2010).
Changes in blood pressure are seen with
respect to sex and age (Girling, 2002). In each heart
beat oxygenated blood is pushed into the arteries. In
each pulse the systolic blood pressure is 120mm Hg
and diastolic blood pressure is about 80mm Hg in an
adult person. Pulse pressure is determined by the
difference of systolic and diastolic pressure (Guyton
and Hall, 2011b). The rise in systolic and diastolic
blood pressure increases the cardiovascular
morbidity and mortality. Well calibrated
sphygmomanometer measures blood pressure
accurately. In an adult healthy person when mean
arterial pressure becomes less than 60mm Hg,
hypotension develops. Hypotension becomes fatal
when perfusion of the vital organ is comprised e.g.,
myocardial ischemia or oliguria (Hardman, 2007).
Blood pressure is one of the important vital sign of
life. In a healthy person the blood pressure must be
BLLOD PRESSURE CHANGES UNDER SPINAL ANESTHESIA IN CAESAREAN SECTION
35
in normal range. Cardiologists and physicians has
shown great interest for the changes in blood
pressure (Charkoudian et al., 2010). In all kinds of
surgical procedures, anesthesia is a great blessing
for the patient all over the world. All parturients
must be assessed according to the grading of
American Society of Anesthesiologists (ASA)
(Baxendale, 2007).
There are two types of anesthesia i.e., general
and regional anesthesia. In former the induction of
anesthesia is made by intravenous anesthetic agents
and maintenance of anesthesia is achieved by the
inhalational agents. Common inhalational agents,
desflurane, enflurane, sevoflurane and halothane are
used with oxygen and nitrous oxide in the
proportion of 33% and 66% with the machine
(Mushambi and Smith, 2007). There are various
types of regional anesthesia which includes: surface
anesthesia, infiltration anesthesia, intravenous
regional anesthesia, nerve block anesthesia, epidural
anesthesia and spinal anesthesia.
In spinal anesthesia the local anesthetics are
injected into the subarachnoid space (Rang and
Dale, 2009a). Different local anesthetics like
procaine, lidocaine, tetracaine, levobupivacaine and
bupivacaine are used for spinal anesthesia. Local
anesthetic molecules consist of an aromatic part
linked by an ester or amide bond to a basic side-
chain. The local anesthetics block the generation
and conduction of nerve impulses by blocking the
sodium channels (Rang and Dale, 2009b).
According to the duration of action, local
anesthetics are divided into short and long acting
group. In short acting group i.e., procaine and
lidocaine have the duration of action less than 90
minutes while in the long acting group i.e.,
tetracaine, levobupivacain and bupivacaine have the
duration of action more than 90 minutes. In
caesarean section when spinal anesthesia is given to
the patient there is lowering of blood pressure. For
this complication ephedrine is given to increase the
blood pressure. The blood pressure of patients was
controlled with I/V infusion of ephedrine (1mg per
min) immediately after spinal anesthesia without
any side effects (Mekaway, 2012). The objective of
this study was to prevent the hypotensive hazards
for mother and fetus.
MATERIALS AND METHODS
This descriptive study was carried out after
the approval from ethical committee at the
department of gynecology and obstetrics, Nawaz
Sharif Social Security Hospital, Multan Road,
Lahore from September - December 2012. Eighty
parturients were selected randomly in a single
group. In inclusive criteria the parturient aged 20-30
years and ASA Grade I and ASA Grade II were
considered. Parturients below 20 and above 30 years
were placed in the exclusive criteria. It was
performed while the patient was sitting on the
operating table and placing the feet on the stool and
the forearm rested on the thighs. The anesthetist
should wash up properly and wear sterilized gloves
carefully. Blood pressure, heart rate and oxygen
saturation was maintained and electrocardiography
was also recorded as vital signs before spinal
anesthesia was given. Each parturient was given
peroperatively, 500 ml haemaccele. A 25-guage
spinal needle was used for anesthesia and
introduced at the level of lumber 3-4 in subarchnoid
space in sitting position. The 1.8 ml hyperbaric
solution of bupivacaine was injected into the
subarchnoid space and 3 liters of oxygen/min with
the help of face mask was provided to the
parturients.
The blood pressure was noted every 5
minutes until completion of surgery. When
hypotension occurred it was taken into
consideration. Ephedrine 5 mg bolus and 10 mg
bolus was injected when systolic blood pressure was
between 90-100 mm Hg and or when blood
pressure (systolic) was below 90mm Hg,
respectively. Nausea/vomiting were also noted
during the operation. The readings of blood pressure
were noted for 120 minutes at intervals from the
start of surgery.
RESULTS
Eighty patients were selected randomly in a
single group. According to the demographic data the
age of patients ranged from 21-30 years while the
weight of patients ranged from 60-75kg. According
to the American Society of Anesthesiologists (ASA)
status 76.3% parturients were in ASA I and 23.7%
BLLOD PRESSURE CHANGES UNDER SPINAL ANESTHESIA IN CAESAREAN SECTION
36
Parturients were in ASA II. Nausea/vomiting
occurred in 38.8% and 61.2% parturients had
no such complaints. The duration of surgery was
Table 1: The normal preoperative, peroperative and postoperative blood pressure and pulse rate of patients treated at
Social Security Hospital Lahore.
Systolic BP range Patient (%) Diastolic BP range Patient (%) Pulse rate range Patient (%)
Preoperative 106- 116
117- 127
128- 138
139- 149
11.3
82.4
5.0
1.3
60- 65
66- 71
72- 77
78- 83
84- 89
1.3
11.2
28.8
52.5
6.2
79- 84
85- 90
91- 96
97- 112
30
51.3
11.2
7.5
Peroperative 70- 80
81- 91
92- 102
103- 113
36.2
56.3
6.2
1.3
43- 51
52- 60
61- 69
70- 78
1.3
7.5
58.7
32.5
80- 90
91- 101
102- 112
113- 123
12.5
38.7
43.8
5.00
Postoperative 105- 111
112- 118
119- 125
126- 132
2.5
68.7
27.5
1.3
66- 69
70- 73
74- 77
78- 81
5.00
23.7
43.8
27.5
8- 84
85- 89
90- 94
95- 99
7.5
27.4
43.8
21.3
Table 2: Ephedrine therapy to blood pressure patients
during caesarean section treated at Social
Security Hospital Lahore.
Stable
patient (%)
Ephedrine
therapy
Patient
(%)
Systolic BP
70- 91
92- 113
8.75
6.25
1- 15mg
47.50
1.25
16-30mg
36.25
0.00
Diastolic BP
43- 60
61- 78
1.25
13.75
3.75
45.0
3.75
32.5
Pulse rate
80- 100
101- 123
15.00
0.00
25.0
23.75
11.25
25.00
ranging from 27-46 minutes. Hypotension occurred
after the spinal anesthesia in 85% patients and 15%
remained with stable blood pressure. The percentage
distribution of pre-operative, peroperative and
postoperative, systolic, diastolic and pulse rate are
shown in Table 1. The dose of ephedrine was given
to patients to correct hypotension ranged from 5mg
to 30mg. The Table 2 is showing the doses of
ephedrine ranging from 1-15 mg and 16-30 mg
which was given to the patients according to their
peroperative systolic/diastolic BP and pulse rate.
The dose of ephedrine given at the time intervals of
5, 10, 15 and 20 minutes are shown in Table 3. Table 3: Blood pressure patients treated at Social
Security Hospital Lahore receiving ephedrine
at various time intervals
Time interval No dose given
(%)
Dose given
(%)
5 min 81.2 18.8
10 min 36.3 63.7
15 min 38.7 61.3
20 min 80 20.00
DISCUSSION
This study was carried out to observe the
changes in blood pressure and at the same time
management of hypotension in C- section after the
induction of spinal anesthesia. It is evident from this
study that hypotension was produced in 85%
parturients when spinal anesthesia was given to
them. A study similar to this was conducted by
Turkoz et al., (2002). The parturients was ranging
from 21-30 years, with weights (normal) and
fulfilling ASA grade I and II criteria. Another study
was conducted by Clark et al., (2005), in which
patients who were pre-eclamptic and had no
hypotension complication when spinal anesthesia
was given to them. The risk factors such as age and
BLLOD PRESSURE CHANGES UNDER SPINAL ANESTHESIA IN CAESAREAN SECTION
37
obesity of the mother, and smoking must be taken
into consideration before giving the spinal
anesthesia (Elton et al., 2007). During the study
nausea/vomiting occurred in 38% parturients as the
effect of hypotension after spinal anesthesia. In
another study (Pan et al., 1996) explained that
ondansetron has equal effects as droperidol for the
correction of the nausea and vomiting in C-section
after spinal anesthesia. Another study also showed
that ondanssetron and metoclopramide have equal
effects in C-section for nausea and vomiting
(Mishriky and Habib, 2012). Nausea and vomiting
was developed peroperatively in spinal anesthesia
may be due to anxiety, arterial hypotension, CNS
hypoperfusion, and movement of abdominal organs
and use of opiates (Kestin, 1998). In this study
vasopressin i.e., ephedrine is used for the correction
and management of hypotension for C-section after
spinal anesthesia. In another research phenylephrine
was given and showed no fatal effects on the mother
and newborn (Loubert, 2012). The phenylephrine is
also a good vasopressin agent. Ephedrine showed no
adverse effects on the mother and fetus in
hypotensive cases (Cartis et al., 1980; Corke et al.,
1982). Ephedrine has effects both on α-adrenergic
and β-adrenergic receptors, either directly or
indirectly whereas phenylephrine has effect directly
on alpha receptors (Barar, 2007). Ephedrine is
proved to be drug of choice as a vasopressin agent,
in the management and treatment of hypotensive
cases when spinal anesthesia is given. Ephedrine has
less vasoconstrictive effect on the uterus and
placental blood vessels (Ralston et al., 1974).
During this study vital signs remained stable
pre and post operatively. A fall of blood pressure
i.e., systolic and diastolic blood pressure and
changes in pulse rate were noticed peroperatively.
There was a gradual lowering of blood pressure
after spinal anesthesia and was treated with 1st,2
nd
and 3rd
bolus doses of ephedrine at time intervals of
5, 10, 15 and 20 minutes. The maximum number of
patients received the dose of ephedrine at the
interval of 10 minutes and 15 minutes. From the
results of our study it is evident that ephedrine is a
good vasopressin agent for the management of post
spinal hypotension. These results are further
supported by Velde (2006).
From the results of present study, it was
concluded that the spinal anaesthesia is most
common technique used for cesarean section. It is a
safest and most economical method as compared
with general anaesthesia. The use of crystalloids
(ringer lactate) preoperatively and colloids
(haemaccele) peroperatively play a key role for the
prevention of hypotension. The vasopressor agent
i.e., ephedrine may be administered for the
management of post spinal hypotension.
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patient: prevention and treatment of
hypotension. Acta Anaesth Belg., 57: 383-
386, 2006.
(Received February 25, 2014; Revised: May 28, 2014)
SIALIC ACID AND HEPATIC DYSFUNCTION IN DENGUE FEVER
40
Pakistan J. Mol. Med., 1(1-2), pp. 40-43, 2014
www.pjmm.uol.edu.pk
Assessment and Correlation of Sialic Acid with Liver
Enzymes in Patients Suffering From Dengue Fever
Arif Malik*, Shumaila Arif, Ejaz Rasul, Zunira Aasfa Riaz, Hina Aataka Riaz, Naveed Shuja,
Abdul Manan, Shaista Andlib, Zunaira Tahir, Zunera Tariq and Syed Shahid Ali
Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore,
Defense Road campus, Lahore, Pakistan
ABSTRACT
The dengue has become severe problem in Pakistan since last few years. The people having
weak immune system and with subsequent dengue attacks are more prone to develop dengue
hemorrhagic fever. Sialic acid is considered as biomarker of acute-phase alterations in biological
systems. It is also one of the vital parameters, in diagnosis of dengue infection. The main
objective of this study was to assess and correlate the sialic acid concentration with liver
enzymes in patients suffering from dengue fever to diagnose the disease. 171 blood samples
were collected, out of which 50 were taken as control and 121 were dengue infected individuals.
The present study shows the significantly low values of sialic acid with significant increase in
total protein in dengue patients. The correlation analysis of these parameters shows the negative
correlation with all parameters like total protein, GPT, GOT and ALP while total protein only
shows the negative association with GOT and ALP. The positive correlation of GPT and GOT
with ALP was observed by the analysis of correlation as compared to controls.
Key Words: Hemorrhagic fever, biochemical pathology, liver function, dengue infection, sialic
acid, diagnostic marker.
INTRODUCTION
Dengue is a mosquito-borne human viral
disease and responsible for the hemorrhagic fever in
the patients. During the severe febrile phase, this
may be predominantly in the urban and periurban
areas (Igarashi, 1997). The Chinese called the
dengue infection as water poison because the
disease spread by the flying insects that was related
with water. Eruption of illness in the French West
Indies in 1635 and in Panama in 1699 could also
have been dengue (McSherry, 1982; Halliwel and
Gutteridge, 1990).
Severe abnormalities in various blood
components have been reported by various workers
during dengue illness (Onlamoon et al., 2010). The
20% elevated level of packed cell volume is a
significant parameter in the diagnosis of the loss
________________________________ * Corresponding author: [email protected]
fluid from the vascular compartment in dengue
infection (Cohen and Halstead, 1964). Moreover,
the levels of white blood cells, red blood cells and
platelets are altered from the first day of infection
up to 14th day of infection (Onlamoon et al., 2010).
The major class of macromolecules (sugar
chains, oligosaccharides and glycans) was
considered inadequately during the molecular
biology revolution of the last few decades, (Sharon
and Lis, 1982). For glycoproteins, sugar chains
forms are essential basic components both
structurally and functionally (Nurden et al., 1986).
The glycoproteins function and their age have been
affected by sialic acid, the terminal molecule is
sugar moiety (Thornhill et al., 1996), which is a
family of monosaccharides in advanced eukaryotes
and certain bacteria. Sialic acid is a determinant of
many useful sugar chains of glycoproteins that play
SIALIC ACID AND HEPATIC DYSFUNCTION IN DENGUE FEVER
41
vital roles in various physiological and pathological
processes (Varki et al., 2008).
Different forms of sialic acids play a
significant role in the regulation of vascular
permeability. High concentration of sialic acid is
found in the vascular endothelium, hence, in severe
dengue illness the endothelium is damaged that
indicates the association for its shedding into the
circulation (Taniuchi et al., 1981). Glycans have
emerged from historical obscurity generating a
specialized field of glycobiology (Rademacher,
1988) which refers to the molecular and cellular
biology and physiology of glycans. There are
numerous potential explanations for the decline in
sialic acid concentrations (Drickamer and Taylo,
2006).
Hepatic dysfunction is common in dengue
infection, and is attributed to a direct viral effect on
liver cells or as a consequence of days -regulated
host immune responses against the virus (Cohen et
al., 1964). Other contributing factors include race,
diabetes, hemoglobinopathies, pre-existing liver
damage and the use of hepatotoxic drugs
(Seneviratne et al., 2006). This main objective of
the study was to assess the correlation of sialic acid
with hepatic enzymes in dengue patients.
MATERIALS AND METHODS
Total 171 blood samples were collected for
this study. Out of these 121 were from dengue
infected individuals and 50 from normal persons
taken as a control group. The blood was collected
from cuboital vein to obtain the serum sample for
biochemical analysis. The chemicals used were
purchased from Sigma-USA.
Biochemical analysis The blood samples were analyzed for sialic
acid, total proteins (TP) and liver function enzymes
i.e., alkaline phosphatase (ALP), glutamic pyruvate
transaminase (GPT) and glutamic oxaloacetate
transaminase (GOT) using the method of Aminoff,
1961; Lowry et al., 1951 and Human Diagnostics
kits, respectively. Data was statistically analyzed by
using software, SPSS (Windows, version-16).
RESULTS
Sialic acid was decreased 86% with high
significant in dengue patients. Amongst the LFT,
ALP activity showed highest increase 88% followed
by GPT (62%) and GOT (50%). Inverse relationship
was found between sialic acid and other testing
parameters such as total protein (-0.149) and GOT
(-0.093) while the significant negative correlation
with GPT values of 0.23 with significant values
(p=0.018) and ALP which has more significant
values (p=0.000) with correlation value (-0.430).
The TP shows the negative correlation with GOT
(-0.036) and ALP (-0.016) with non-significant
values. But it shows the positive correlation with
GPT. The GPT shows the positive correlation with
GOT (0.0675**) and ALP (0.276**) with high
significance (p=0.000 and 0.004) correspondingly.
The parameters showed the marked
differences in dengue patients when compared with
control data. The levels of sialic acid residues, total
proteins, GPT, GOT and ALP of dengue patients
and controls are given in (Table 1). The GPT
activity showed 62% decrease. Table 2 represents
the significant positive correlation of GPT (0.018*)
and ALP (0.000**).
Table 1: Levels of sialic acid, total protein and liver
enzymes in patients suffering from dengue
fever.
Parameters Control (n=50) Dengue (n=121)
Sialic acid (µmol/L) 271.10 ±58.34a 29.19 ±2.04*
Total protein (mg/dl) 5.21 ±0.54 8.37 ±0.25*
GPT (IU/L) 26.60 ±1.74 70.27 ±2.64*
GOT ( IU/L ) 23.80 ±2.31 47.80 ±1.71*
ALP (IU/L) 29.60 ±2.73 247.03 ±7.50*
a Mean ± SEM, student’s “t” test * P<0.05
DISCUSSION
Sialic acid is considered as biomarker of
acute-phase alterations in biological system
(Chrostek et al., 2014). During this phase
glycoprotein with sialic acid as a constituent of the
oligosaccharide side chain being formed by the
liver, stimulated by pro-inflammatory cytokines
such as interleukin-6, and tumor necrosis factor α
SIALIC ACID AND HEPATIC DYSFUNCTION IN DENGUE FEVER
42
(Juffrie et al., 2001). Cytokines were observed to be
associated with disease severity as well as increased
in dengue infection (Chatuverdi et al., 2000). Table 2: Correlations between blood parameters of
patients suffering from dengue fever.
Sialic acid TP GPT GOT ALP
Sialic
acid
1 -0.149 -0.230* -0.093 -0.430**
0.096 0.018 0.341 0.000
TP 1 0.002 -0.036 -0.016
0.985 0.714 0.870
GPT 1 0.675** 0.276**
0.000 0.004
GOT 1 0.173
0.076
ALP 1
Pearson’s correlation (r2) is significant at; * < 0.05 level;
** < 0.01 level; r2 values above 0.5 show strong relationship
wither positive or negative
Reactive oxygen species (ROS) might
specifically chop terminal sialic acid residues in
sialoglycoproteins (Halliwell and Gutteridge, 1990).
Liver dysfunction seen in dengue viral disease
might lead to decreased synthesis and leakage of
proteins during acute phase. Vascular leakage and
coagulation of proteins might also escort to loss of
serum protein into third space (Halstead, 1988). In
DHF/DSS, increased vascular permeability is
suggested because of dysfunction of endothelium
rather than structural destruction (Kurane and
Takasaki, 2001).
Sialic acid has been used as a vital parameter,
in diagnosis of dengue infection (Rajendiran et al.,
2008). The platelet membrane contains many
glycoproteins that show significant roles in platelet
function (Nurden et al., 1986). N-acetyl neuraminic
acid (NANA, a sialic acid residue) is present in
platelet proteins (Okumura and Jamieson, 1976). In
case of circulatory proteins, loss of sialic acid
moiety targets them for degradation by liver cells
that have sialo-glycoprotein receptors (Stryer and
Freeman, 1995).
Studies have also observed that vascular
permeability is maintained by molecules of sialic
acid. An elevated concentration of sialic acid was
found in vascular endothelium. Any damage during
severe dengue infection related to endothelium, may
responsible for its increased levels in blood
circulation (Taniuchi et al., 1981). The findings in
the present study on dengue patients also showed
elevated sialic acid level as compared to controls.
Assessment and correlation of sialic acid with liver
enzymes in patients suffering from dengue fever
was designed to investigate the effect of alteration
of level of sialic acid as biomarker. Different
parameters are being used to diagnose this disease
and to find the exact reason for prognosis of
treatment. In this study evaluation of sialic acid,
total proteins, GPT, GOT and ALP were carried out
for the prognosis of treatment. Significantly low
level of sialic acid (p=0.016) and total proteins
(p=0.015) were observed during this study. On the
other hand, significantly increased values of GPT,
GOT and ALP were also noticed. The correlation
study showed an inverse relationship between the
sialic acid and total proteins (r=-0.149) and GOT
(r=-0.093). GPT (r=0.230) with p=0.018 and ALP
which has more significant (p=0.000) negative
correlation value of -0.430 (p=0.000).
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Aminoff D. Methods for the quantitative estimation
of N-acetylneuraminic acid and their
application to hydrolysates of sialomucoids. J
Biochem, 1961; 81: 384–392.
Chatuverdi UC, Agarwal R, Elbishbishi EA and
Mustafa AS. Cytokine cascade in dengue
hemorrhagic fever: implications for
pathogenesis. FEMS Immunol Med
Microbiol., 2000; 28: 183–188.
Chrostek L, Cylwik B, Gindzienska-Sieskiewicz
E, Gruszewska E, Szmitkowski M and
Sierakowski S. Sialic acid level reflects the
disturbances of glycosylation and acute-phase
reaction in rheumatic diseases. Rheumatol
Int., 2014; 34: 393-399.
Cohen SN and Halstead SB. Shock associated with
dengue infection I: clinical and physiologic
manifestations of dengue hemorrhagic fever
in Thailand. J Pediatr., 1964; 68: 448-456.
Drickamer K and Taylor M. Introduction to
Glycobiology. 2nd edn. Oxford University
Press., Oxford. 2006;
Haistead SB. Pathogenesis of dengue. Challenges to
Molecular Biology Science, 1988; 239: 476–
SIALIC ACID AND HEPATIC DYSFUNCTION IN DENGUE FEVER
43
481.
Halliwell B and Gutteridge JM. Role of free radicals
and catalytic metal ions in human disease: an
overview. Methods Enzymol., 1990; 186: 1–
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Igarashi AS. Impact of dengue virus infection and
its control. FEMS. Immunol Med Microbiol.,
1997; 18: 291-300.
Juffrie M, Meer GM, Hack CE, Hasnnot K, Sutaryo
X, Veerman M and Thijs LG. Inflammatory
mediators in dengue virus infection in
children: interleukin-6 and its relation C-
reactive protein and secretoy phospholipase
A2. J Trop Med Hyg., 2001; 65: 70-75.
Kurane I and Takasaki T. Dengue fever and dengue
haemorrhagic fever: challenges of
controlling an enemy still at large. Rev Med
Virol., 2001; 11: 301–311.
Lowry OH, Rosebrough NJ, Farr AL and Randall
RJ. Protein measurement with the Folin
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Mcsherry JA. Some medical aspects of the Darien
schema: was it dengue? Scot Med J., 1982;
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Nurden AT, George JN and Philips DR. Platelet
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Biochemistry of Platelets, 1986; 2: 160-212.
Okumura CL and Jameieson GA. Platelet
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Onlamoon N, Noisakran S, Hsiao HM, Duncan A,
Villinger F, Ansari AA, and Perng GC.
Dengue virus–induced hemorrhage in a
nonhuman primate model. Blood, 2010;
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Rademacher TW. Glycobiology. Annu Rev
Biochem., 1988; 57: 785–838.
Rajendiran S, Lakshamanappa HS, Zachariah B, and
Nambiar S. Desialylation of plasma proteins
in severe dengue infection: possible role of
oxidative stress. Am J Trop Med Hyg., 2008;
79(3): 372-377.
Seneviratne SL, Malavige GN and De Silva HJ.
Pathogenesis of liver involvement during
dengue viral infections. Trans Royal Soc
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Sharon N and Lis H. Glycoproteins: research
booming on long-ignored ubiquitous
compounds. Mol Cell Biochem., 1982; 42:
167-187.
Stryer L and Freeman WH. Biocatalysis:
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Yamaguchi, N. and. Miyamato, Y. A new
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Thornhill WB, Wu WB, Jiang X, Wu X,
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(Received: January 23, 2014; Revised: April 14, 2014)
PREVALENCE OF ANTI-HCV & ALLOIMMUNIZATION IN THALASSEMIA MAJOR PATIENTS
44
Pakistan J. Mol. Med., 1(1-2), pp. 44-47, 2014
www.pjmm.uol.edu.pk
Prevalence of Anti-HCV and Frequency of Alloimmunization
in Repeatedly Transfused Thalassemia Major Patients
Mahmood Husain Qazi,1 Arif Malik, Sahar Javed, Zunera Tariq, Zunaira Tahir,
Naveed Shuja and Abdul Manan 1Centre for Research in Molecular Medicine, The University of Lahore, Lahore;
Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan
ABSTRACT
Thalassemia is among the most common genetic disorders in the world. The risk of blood
transfusion-associated infections like hepatitis, iron overload, alloimmunization as well as HIV
is increased among the patients of thalassemia major due to multiple blood transfusions. The
study is aimed to investigate the development of red blood cell (RBC) alloimmunization with
alloantibodies complication through transfusion therapy in multiply transfused thalassemia
patients. Data was obtained from 200 beta-thalassemia major patients receiving regular blood
transfusions at a major transfusion center in Lahore Pakistan. Clinical data and laboratory results
were subsequently evaluated. Anti HCV were found reactive in 41% of cases and alloantibodies
were found in 7.5% of thalassemia patients. Descriptive statistics were used to elaborate results.
It is recommended to develop programs that provide antigen-matched RBC transfusions to all
thalassemia patients to prevent alloimmunization to RBC antigens.
Key Words: Alloimmunization, red cell alloantibodies, thalassemia, transfusion transmitted
infection.
INTRODUCTION
Thalassemia major is a congenital
hemolytic anemia caused by the defect in β-globin
chain synthesis. This is an autosomal recessive
disease prevalent in Pakistan. The carrier rate ranges
between 4 and 5.5% in different regions and racial
groups. The management of thalassemia major
essentially comprises of regular “safe blood
transfusion” and a lifelong iron-chelation therapy.
Regular blood transfusions are essential to maintain
growth and development during childhood and also
to sustain good quality of life during adulthood
(Spanos and Karageorga, 2003). Alloimmunity is a
condition in which the body gains immunity, from
another individual of the same species, against the
foreign cells. Antibodies must be identified in the
recipient’s serum before each transfusion so that
________________________________
* Corresponding author: [email protected]
Compatible blood can be provided. Several factors
might have contributed to this finding, such as the
heterogeneity of the population, the difference in the
number of studied patients, the differences in age at
first transfusion, antigenic differences between the
blood donor and the recipient; the recipient's
immune status and immunomodulatory effects of
the allogenic blood transfusions on the recipient's
immune system and splenectomy.
Alloimmunization i.e., development of
alloantibodies against the foreign RBC is one of the
important complications of blood transfusions in
multiple transfused thalassemia patients
(Schonewille et al., 1999; Eder and Chambers,
2007). Alloimmunization further complicates the
transfusion therapy due to difficulty in getting
compatible blood, increased incidence of additional
alloantibody and autoantibody (antibody against self
RBC antigens) development, delayed hemolytic
PREVALENCE OF ANTI-HCV & ALLOIMMUNIZATION IN THALASSEMIA MAJOR PATIENTS
45
transfusion reaction (DHTR) and life-threatening
hyperhaemolysis syndrome (Zumberg et al., 2001;
Schonewille et al., 2006).
Unfortunately, patients, even those managed
at relatively better management centers, are prone to
develop both types of complications, i.e., those
transmitted through blood transfusion (particularly
hepatitis C) as well as sequelae of transfusion
siderosis. Hepatitis B has a declining trend,
probably as a result of regular pre-transfusion
screening for HBsAg, use of hepatitis B vaccination
and improved public awareness about the disease.
HIV infection, fortunately, is uncommon in our
setup. Last decade has witnessed a tremendous
increase in the sero-prevalence of hepatitis C
amongst almost all the major cities of Pakistan.
However, it has been observed that amongst blood
donors belonging to different socioeconomic strata,
this sero-prevalence hepatitis is variable. The
percentage is much less amongst young college
students (0.7%) and non-remunerated donors (1.3%)
as compared to 11.8% in factory workers (Rehman
et al., 2002). Furthermore, amongst the major
transfusion transmitted infection (TTI) markers, the
overall HCV sero-prevalence is high (4.1%). Life-
long red blood cell (RBC) transfusion remains the
main treatment for severe thalassemia (Singer et al.,
2000). The use of regular blood transfusion and of
chelation therapy with deferoxamine has led to the
transfusion of thalassemia major from a fatal disease
in early childhood to a chronic illness associated
with prolonged survival (Thuret, 2001; Rehman et
al., 2002).
In Pakistan, an elaborate data on various
blood group systems, particularly the groups other
than ABO and Rh ‘D’ e.g., Kell, Duffy, Kidd, MNS,
Lewis, etc. is lacking. Therefore the prevalence rates
of these groups and any statistical differencess
amongst various racial groups are not known. A
wide discrepancy in the distribution of certain blood
group antigens between donor and recipient can be
responsible for a higher risk of alloimmunization in
multiple transfused patients. However, many of the
antigens present on the erythrocytes infrequently
give rise to alloimmunization even when injected
into patients lacking the antigen. The rate of
immunization ranges from 70% for Rh ‘D’ antigen
to as low as 0.5% for the Duffy antigens. It is also
interesting to observe that the ability to react to
alloantigens varies greatly from person to person.
Some individuals will not become immunized to
any antigen despite repeated transfusion, whereas
others will become immunized, when transfused, to
many of the antigens that they lack (Rosse et al.,
1990).
MATERIALS AND METHODS
Two hundred patients of thalassemia major
were selected through their clinical history for this
study. The blood samples of thalassemia patients
were collected from different hospitals and clinical
labs of Lahore-Pakistan which includes Sundas
Foundation, Fatimeed Foundation and Children
Hospital Lahore. Thalassemia major patients treated
with ten or more transfusions were included in the
study. Diagnosis of thalassemia was confirmed by
standard hemoglobin electrophoresis (Usman et al.,
2011) Clinical data of patients who fulfilled the
criteria were collected and entered in the Performa,
with special reference to current age, age at
diagnosis, frequency of transfusion, present clinical
status, any increase in transfusion requirements. The
sera were separated using standard blood bank
method and stored in labeled tubes at -20°C which
were later on analyzed for detection of anti-HCV
and red cell alloantibodies.
Anti HCV screening was performed on Stat
of the art instrument Architect (i1000 SR) Chemi-
iluminescence (CMIA) Technique. Antibody
Screening and identification was performed by
using Diamed Gel System (Dia-med-ID,
Switzerland. Initially 3 cell panel was used if
antibody screening was positive then an extended 11
cell panel was used to identify the antibody.
RESULTS
Of the total 200 cases studied 176 were male
and 24 were female. The patient’s age was ranged
from 1 to 18 years (mean age of 7.8±4.4 years). The
age on first transfusion was 3 to 48 months. Among
200 patients 41% were reactive for anti-HCV with
age range of 2 to 18 years with mean age of 8.5
years.
The mean age at 1st transfusion was 8.7
PREVALENCE OF ANTI-HCV & ALLOIMMUNIZATION IN THALASSEMIA MAJOR PATIENTS
46
months. Among them 28 having blood group O+ve,
25 with B+ve, 18 with A+ve, 8 with AB+ve and 3
were A-ve. Alloantibodies were detected in 7.5%
patients with age range of 4 to 17 years with mean
age of 9.8 years. Among them 26.6% were Anti-E,
20% were Anti-K, 13.3% Anti-D, 13.3% were Anti-
E+C, 13.3% were Anti-C, 6.6 % was Anti-Kpa and
6.6% were Anti-Le.
Table 1: Alloantibodies detected in thalassemia major
patients.
Alloantibody Number of patients Percentage (%)
Anti E 04 26.66%
Anti K 03 20%
Anti D 02 13.33%
Anti E+c 02 13.3%
Anti c 02 13.3%
Anti Kpa 01 6.66%
Anti Le 01 6.66%
Total 15 100%
Table 2: Anti HCV reactive and non-reactive
thalassemia patients.
Anti HCV Number of Patients Percentage%
Reactive 82 41%
Non.Reactive 118 59%
Total 200 100%
DISCUSSION
The present study was conducted to find out
the rate of alloimmunization and transfusion
transmitted infection. The studies have shown that,
the rate of alloimmunization is increased after
splenectomy (Hussein et al., 2014). The reason put
forward is that the spleen effectively filters many
antigens that may probably cause production of
antibodies. Regular transfusion of blood in
thalassemia major alleviates the harmful aspects
related to anemia and helps in prolong survival. It is
particularly so in patients who are fortunate enough
to receive an adequate, regular iron chelation
therapy, and are therefore protected from organ
damage by iron overload.
HCV infection has gained importance
particularly as one of the major complications in
multiply transfused patients during the last 10 years.
This is especially true for countries where HCV is
more prevalent in general population and therefore
also amongst blood donors. The prevalence rate of
seropositivity increases with the number of
transfusions (Eder and Chambers, 2007). This post
transfusion hepatitis has significantly contributed to
morbidity in thalassemia. Present study reflects a
high prevalence of HCV seropositivity 41%,
although our patients were usually transfused at the
thalassemia management center of Sundas
Foundation, Children Hospital and Fatmeed
Foundation where pre-transfusion screening of the
transfused is regularly performed. However, it was
guessed during interview of the patients’ parents
that in almost all instances, the patients did get
transfused with blood from some other centers,
where pre-transfusion HCV antibody screening was
not guaranteed. Furthermore, the parents of about
60% of patients were not aware of the importance of
HCV antibody screening of transfused blood. In
many previous studies, the prevalence of HCV
antibodies was observed to be reduced after the
institution of a regular HCV screening before
transfusion (Rehman et al., 2002).
Our data concur with results of many studies
that the overall prevalence of red cell
alloimmunization is 7.5%. To prevent
alloimmunization to RBC antigens there has been
recommendations to develop programs that provide
antigen-matched RBC transfusions to all
thalassemia and sickle cell disease patients
(Zumberg et al., 2001). It is obvious that providing
antigen matched blood will effectively prevent
alloimmunization. However, the cost effectiveness
of establishment of these programs for chronically
transfused patients has been a debatable and
controversial. It is difficult also to establish and
maintain a donor pool for each patient. The
complications of alloimmunization that may occur
are many; antibody production, which may lead to
delayed hemolytic transfusion reactions. They may
even present with a delayed transfusion reaction that
may go unrecognized and/or be masked by features
of their underlying disease. Also some patients may
present as emergency and may have multiple
alloantibodies, making difficult to find compatible
blood, and the difficulty increases when these
PREVALENCE OF ANTI-HCV & ALLOIMMUNIZATION IN THALASSEMIA MAJOR PATIENTS
47
patients present to some hospitals for the first time
with no previous records. Leucocyte poor blood can
be provided for these patients, as a number of
studies have shown the role of leuco-depletion in
preventing alloimmunization and autoimmunization
(Spanos and Karageorga, 2003).
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Eder AF and Chambers LA. Noninfectious
complications of blood transfusion. Arch
Pathol Lab Med., 2007; 131: 708-18.
Hussein E, Desooky N, Rihan A, Kamal A.
Predictors of red cell alloimmunization in
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Rehman M, Akhtar GN and Lodhi Y.
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Rosse WF, Gallagher D and Kinney TR.
Transfusion and alloimmunization in sickle
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Schonewille H, Haak HL and Van zij AM.
Alloimmunization after blood transfusion in
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Schonewille H, Van de Watering LM and Brand A.
Additional red blood cell alloantibodies after
blood transfusion in a non-hematological
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Spanos T and Karageorga M. Red cell
alloantibodies in patients with thalassemia.
Nathan and Oski's Hematology of Infancy
and Childhood, Philadelphia; Saunder, 2003;
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Usman M, Moinuddin M, and Ahmed SA. Role
of iron deficiency anemia in the
propagation of beta thalssemia gene.
Korean J Hematol., 2011; 46(1): 41-44. Zumberg MS, Procter JL and Lottenberg R.
Autoantibody formation in the alloimmunised
red blood cell recipient and laboratory
implications. Arch Intern Med., 2001; 22:
285-90.
(Received May 23,2014; Revised July 10, 2014)
ANTI-OXIDATIVE RESPONSE IN ASTHMATIC PATIENTS RECEIVING SALBUTAMOL
48
Pakistan J. Mol. Med., 1(1-2), pp. 48-53, 2014
www.pjmm.uol.edu.pk
Biochemical and Antioxidative Response of Asthmatic Patients
Receiving Salbutamol
Arif Malik, Hafiz Muhammad Arsalan, Maria Amjad, Naveed Shuja, Abdul Manan, Saima Zaheer,
Amna Mahmood, Sarmad Bashir and Mushtaq A. Saleem
Institute of Molecular Biology and Biotechnology, Defense Road Campus, The University of Lahore-Pakistan
ABSTARCT
Asthma is a complicated, heterogeneous airway disease. Chronic asthma is often caused due
to exercise. Another common class of asthma is allergic asthma which is caused by allergens.
Ninety percent asthma cases in children and fifty percent in adults are induced by allergens. Over
production of reactive oxygen species in asthmatics lead to the alterations in antioxidants and
affect the concentration of electrolytes. Salbutamol (β2-adrenoreceptor agonists) is the most
effective bronchodilators used in the bronchial asthma. The objective of this study was to
investigate the electrolytes (Na+, K
+) disorders, anti-oxidative status malondialdehyde (MDA)
levels and their role in hepatic function and lipid profile in male asthmatic patients receiving
salbutamol. The study was carried out on sixty two asthmatic patients already receiving
salbutamol. The MDA level was significantly elevated (72%) while SOD and GSH
concentration were decreased by 92% and 26%, respectively in the asthmatic patients. Serum
potassium level was significantly decreased (61%) while sodium level increased (20%). Liver
function test like ALT, AST, ALP and total bilirubin increased prominently. Among lipid profile
triglycerides and LDL level was raised by 31% and 27%, respectively while HDL showed 32%
decrease. Present study suggests that increase in the serum MDA and sodium levels and decrease
in potassium GSH and SOD levels triggers the asthma.
Key words: Asthma, β2-adrenoreceptor, malondialdehyde, anti-oxidants.
INTRODUCTION
Asthma is a complicated, heterogeneous
disease which is not easy to define and classify. It is
often diagnosed by specific symptoms i.e.
wheezing, tightness of chest, breath shortness,
coughing or inflammation in the airways (Masoli et
al., 2004; Rabe et al., 2004). Asthma is common in
the children of different ages, with transient early
wheeze which remits before the age of six and late
onset of wheeze which starts in the children after
three years old and known as persistent wheeze
(Martinez et al., 1995). For a long time it was
considered that asthma is caused by the
inflammation produced by eosinophils (Barnes,
_________________________
* Corresponding author: [email protected]
1989). In recent studies it was found that non-
eosinophilic inflammation also exist which is often
caused by neutrophils due to which it is related to
asthma (Douwes et al., 2002). Remarkable
variations present in asthmatic patients that elicit
airway narrowing and symptoms of disease, severity
of disease and effectiveness of different modes of
therapies (Drazen et al., 2000; Szefler et al., 2002;
Rabe et al., 2004).
Asthma can be classified in various classes
like exercise induced asthma caused due to physical
exertion or over exertion, asthma occurring at night
also called night time asthma may led to
seriousness. Chronic asthma often caused due to
exercise. Another class of asthma known as allergic
asthma is a common type of asthma caused due to
ANTI-OXIDATIVE RESPONSE IN ASTHMATIC PATIENTS RECEIVING SALBUTAMOL
49
allergens. Ninety percent asthma cases in children
and fifty percent in adults are caused by various
allergens. Different types of allergens are found in
the atmosphere (pollen, mites, molds, smoke and
sprays) and are present everywhere. In case of
allergic asthma, airways are sensitive to the allergen
and able to sensitize the allergen. When the allergen
enter into the airway, they become inflamed and
thick layer of mucus is filled in the airways due to
which coughing, wheezing, shortness of breath and
chest tightening occurs. Irritants causing asthma
also stimulate asthma attack. In case of exercise
induced asthma, breathing may occur through the
mouth instead of nasal passage (Kattan et al., 1978).
In industrialized countries, prevalence of
asthma is continuously increasing and in developing
and low income countries, consistent data is also
available (Starchan et al., 1997 and Okudaira,
1998). Another type of asthma known as bronchial
asthma that often induced during pregnancy about
1-7% in all pregnancy cases (Alexandar et al., 1998;
Gordon et al., 1970). In two percent cases, asthma is
considered the life threatening disorder but this
figure is not accurate (Hernandez et al., 1980). If
asthma is not treated during pregnancy then it may
lead to adverse effects on mother and fetus (Turner
et al., 1980). Epinephrine or non-epinephrine targets
on the adrenergic receptors and these receptors are
considered as important components in sympathetic
nervous system for homeostasis maintenance
against disease. Adrenergic receptors belong to the
cell surface receptors super-family in which signals
are carried out through coupling to guanine-
nucleotide binding proteins also called g-proteins.
Nine subtypes of adrenergic receptors are present in
humans (α1A, α1B, α1Dar, α2A, α2B-, α2Car, β1, β2
and β3AR). Different classical pathways present for
coupling of different receptors (coupling of α1AR
occur through stimulation of phospholipase C
pathway (Gq), coupling of α2AR through inhibition
of adenylyl cyclase (Gi) and coupling of βAR occur
through stimulation of adenylyl cyclase (Gs)
(Hoffman et al., 2001; Goldstein et al., 1998).
β2-adrenoreceptor agonists (β2-agonists) are
the drugs which are used in the bronchial asthma
and are considered the most effective
bronchodilators. For ancient times, adrenergic
activity containing plants were used in medicine but
in modern researches, adrenergic pharmacology
started with the isolation of epinephrine from
adrenal glands at the end of 19th century (Rau, 1994;
Svedmyr et al., 1994 and Popa, 1986) and efficient
aerosolization devices also introduced (McFadden,
1995) and also found that β2-agonist used for asthma
treatment in three different ways i.e., selection of
receptor for reduce side effects produced from
activation of non-target receptors, 2nd
way is the
direct tissue delivery to overcome side effects
produced from activation of target receptor in non-
target tissues and third way is to increase the
duration of action to increase convenience and
eliminate night time awakening.
DNA, lipids as well as protein damage is
caused by the oxidative stress and in the progression
and development of many diseases i.e., asthma and
oxidative stress play a key role. In this present
research, different biomarkers of oxidative stress
were measured in the asthmatic patients i.e.,
superoxide dismutase (SOD), malondialdehyde
(MDA), glutathione (GSH) and catalase (CAT).
High production of reactive oxygen species (ROS)
is controlled by the mechanism called as anti-
oxidant detoxifying mechanism. Two types of
mechanisms, one is called as enzymatic (SOD and
CAT) and other is called as non-enzymatic (GSH)
(Benzie and Strain, 1996 and Cao and Prior, 1998).
The objective of this study was to investigate the
electrolyte disorders, anti-oxidative status, liver
function and lipid profile in in male asthmatic
patients receiving salbutamol.
MATERIALS AND METHODS
Blood collection Blood samples (5ml) were taken from 62
asthmatic patients and 10 normal subjects was
processed for the estimation of reduced glutathione
(GSH), catalase (CAT), superoxide dismutase
(SOD), malondialdehyde (MDA), electrolytes
sodium (Na+) and potassium (K
+) concentration.
Lipid (total cholesterol, TCh; triglycerides, TG;
Low-density lipoprotein, LDL; and high-density
lipoprotein, HDL) and hepatic profile (Alanine
aminotransferase, ALT; aspartate aminotransferase,
AST; Alkaline phosphatase, ALP and total
bilirubin) were estimated by commercially available
ANTI-OXIDATIVE RESPONSE IN ASTHMATIC PATIENTS RECEIVING SALBUTAMOL
50
diagnostic kits.
The activity of SOD was measured by
spectrophotometric method of Kakkar et al., (1984).
MDA was measured by the method of Ohkawa et
al., (1979). The CAT activity was measured by
spectrophotometric method of Aebi, (1984). GSH
was measured by the method of Moron et al., 1979.
The concentration of sodium and potassium was
measured by the flame photometer. Independent
student t-test was applied for the determination of
significant difference between the two groups
(P<0.05).
RESULTS
The results presented in Table 1 reflect the
hepatic function, antioxidative status, lipid as well
as electrolyte profile in asthmatic patients receiving
salbutamol shows highly significant differences
when compared to normal subjects.
Amongst transaminases the ALAT level
showed 24% increase as compared from control
(31.75±19.73 U/L) value but it was statistically non-
significant (P<0.05). The ASAT was also elevated
(39%) in the asthmatic patients from the control
(20.25±5.21U/L) and it was statistically significant
(P<0.05). The ALP level in the asthmatic patients
was remarkably increased (67%) from the control
(55.83±6.28U/L). Total bilirubin of the asthmatic
patients showed 47% increase from its respective
control (1.01±0.07 mg/dL) values (Table 1).
In the present study on lipid profile (TCh, Tg,
LDL, HDL) of the asthmatic patients, it was
observed that the level of the TCh was raised (21%)
from the healthy controls (4.44±0.37mg/dL). The
TG level was also increased (31%) in the patients
suffered from asthma. The LDL level of the
asthmatic patients was raised (27%) from the
control which is 2.31±0.15mg/dL. Contrary to LDL,
the HD level of the asthmatic patients was decreased
(32%) non-significantly from the healthy
individuals with 1.73±0.17mg/dL HDL value
(Table 1).
Hepatic profiles of asthmatics patients also
showed significant changes the activity of ALAT
and ASAT were increased by 24% and 31%
respectively. The activity of ALP showed highly
significant rise of 67% and 47% respectively while
bilirubin showed 32% increased. Both electrolytes
(Na+, K
+), the serum potassium showed (61%)
significant decrease. Moreover, sodium (177.78±
2.88) concentration was increased 21% from control
value (143.15±8.26mEq/L). On the other hand, the
levels of antioxidants (SOD and GSH) were
decreased (92% and 26%) respectively in asthmatics
while MDA level was increased as compared to
control (1.36±0.03). Like antioxidants, potassium
(1.99±0.03) level was also decreased by 72%.
DISCUSSION
Several lines of evidence suggest that
environmental pollutants and oxidants induce
oxidative damage in mitochondria of in airway
epithelial cells. Exposure of oxidants and allergens
induces airway inflammation, resulting in the
release of pro-inflammatory mediators, including
histamine and leukotrienes (Comhair et al., 2005).
Different factors have been reported to play
role in the progression of asthma, such as chemicals
pollutants, radiations and the genetic makeup by
variety of molecular mechanisms. Two important
factors are involved in DNA damage which are
reactive oxygen species (ROS) and reactive nitrogen
species (RNS). Reactive oxygen species and free
radicals have long been known to be mutagenic.
This damage called oxidative stress or oxidative
damage, depends not only on ROS/RNS levels but
also on the body’s defense mechanisms against
them mediated by various cellular antioxidants
(Benzie and Strain, 1996; Cao and Prior, 1998).
The findings during this work revealed that
the levels of several hepatic and lipid enzymes and
metabolites were elevated in asthmatics. Moreover,
sodium concentration was increased as compared to
control. On the other hand, the level of antioxidants
such as SOD and GSH were significantly decreased
in asthmatic patients while MDA level was
increased (Table 1).
In the recent report on mitochondrial
dysfunction and oxidative stress of asthmatic
peripheral cells and tissues, it was observed that
asthmatic patients have low level of anti-oxidative
enzymes. Comhair et al. (2005) measured various
antioxidants in asthmatic patients. The increase in
nitric oxide in the asthmatic patients due to increase
ANTI-OXIDATIVE RESPONSE IN ASTHMATIC PATIENTS RECEIVING SALBUTAMOL
51
in inflammatory cytokines, macrophage
inflammatory protein-1 in the epithelial lining fluid,
Table 1: Biochemical and antioxidative response of asthmatic patients receiving salbutamol.
Variables Control (n=20) Asthmatics (n=62) % increase/ decrease (P<0.05)
ALAT (IU/L) 24.00±5.69a 31.75±1.97 24.40 0.028
ASAT (IU/L) 20.25±5.21 29.50±3.37 31.35 0.011
ALP (IU/L) 55.83±6.28 169.16±1.35 66.99 0.023
Total bilirubin (mg/dL) 1.01±0.07 1.89±0.02 46.56 0.041
Total cholesterol (mg/dL) 4.44±0.37 5.60±0.41 20.71 0.004
Total triglycerides (mg/dL) 1.24±0.15 1.81±0.11 31.39 0.030
LDL (mg/dL) 2.31±0.15 3.18±0.52 27.35 0.007
HDL (mg/dL) 1.73±0.17 1.18±0.04 -31.79 0.001
MDA (µM/ml) 1.36±0.03 4.78±1.70 71.54 0.000
SOD (ng/mL) 0.73±0.025 0.06±0.05 -91.78 0.016
GSH (mg/dL) 9.77±1.17 7.24±0.94 -25.89 0.005
CAT (µM/mol of protein) 4.27±0.73 2.77±0.83 -35.12 0.033
Na+ (mEq/L) 143.15±8.26 177.78±2.88 19.56 0.013
K+ (mEq/L) 5.07±1.02 1.99±0.03 -60.74 0.000
a Mean ± SEM; Student’s `t’ test, P< 0.05 was considered as significant.
eosinophilic infiltrate in bronchoalveolar lavage
fluid and biopsy specimens have been reported.
It was also observed that extracellular glutathione
peroxidase (eGPx) was higher in the airways of
adult asthmatic subjects than in those of healthy
controls and that the source for the increased eGPx
was bronchial epithelial cells (Comhair et al., 2005).
Oxygen free radicals, which are generated
through several enzymatic and non-enzymatic
biological reactions in aerobic organisms, attack a
wide variety of macromolecules such as lipid,
protein, carbohydrate and DNA. Oxidative stress
conditions are characterized by an increase in the
concentration of free radicals, and the damage they
can cause at different levels of the cellular
organization. Either the increase in the rate of free
radical generation and or the decrease in antioxidant
levels lead to oxidative stress conditions.
In the present report MDA and SOD showed
a significant (P˂0.05) negative correlation (-
0.831*). Inverse correlation also exist between
MDA vs GSH, CAT, and potassium (K+) with r
values -0.371*, -0.129* and -0.698* respectively
which are also statistically significant (P˂0.05).
Strong, statistically significant (P˂0.01) positive
correlation exist between SOD vs., CAT, GSH and
potassium (K+) with r values 0.671**, 0.178** and
0.599** respectively (Table 2).
The findings in this study provide an
association of asthma with SOD, GSH and serum
potassium. The asthmatic patients had found a
significantly lower serum level of SOD, GSH and
potassium levels as compared to the healthy
subjects. The level of SOD in the asthmatic patients
was significantly lower as compared to the healthy
individuals. The extent of lipid peroxidation in the
asthmatic patients was remarkably increased.
Table 2: Spearman’s correlation (r) of different
variables in asthematic patients receiving
salbutamol
Parameters r value
MDA Vs SOD -0.831*
MDA Vs CAT -0.129*
MDA Vs GSH -0.371ns
SOD Vs CAT 0.671**
SOD Vs GSH 0.178ns
MDA Vs K+ -0.698*
SOD Vs K+ 0.599*
Significance has been shown as *p<0.05; **p<0.01
The present study showed a relationship
between oxidative stress, electrolyte balance and
asthma. Biochemical study of the asthmatics
showed that oxidative stress and electrolyte balance
play a key role in the progression of asthma. The
ANTI-OXIDATIVE RESPONSE IN ASTHMATIC PATIENTS RECEIVING SALBUTAMOL
52
asthmatic patients had remarkably high lipid
peroxidation due to which the level of MDA was
increased remarkably while the level of SOD, CAT
and GSH was decreased. Moreover, hepatic and
lipid profile were also statistically significant
between the studied groups. It can be suggested
from this study that increased MDA and sodium
levels and decreased potassium, SOD and GSH
levels of the asthmatic patients may contribute to the
progression of asthma.
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(Received: May 29, 2014; Revised: June25, 2014)
INTERFERON INDUCED THYRODITIS IN HCV PATIENTS
54
Pakistan J. Mol. Med., 1(1-2), pp. 54-60, 2014 MINI-REVIEW
www.pjmm.uol.edu.pk
Clinical Insights and Mechanisms Involved During Interferon
Induced Thyroditis in HCV Patients – A Review
Alina Butt, Mahwish Arooj,1 Arif Mailk,* Abdul Manan, Syed Shahid Ali and
Mahmood Husain Qazi1
Institute of Molecular Biology and Biotechnology, The University of Lahore, Defense Road Campus, Lahore,
Pakistan; 1Centre for Research in Molecular Medicine, The University of Lahore
Defense Road Campus, Lahore, Pakistan.
ABSTRACT
Interferon produces autoimmune endocrine disorder of thyroid, when given to the hepatitis
C patients. Two distinct mechanisms are known to be involved in thyroid disorder by interferon
therapy. These are, i) autoimmune interferon induced thyroditis and, ii) non-autoimmune
interferon induced thyroditis. Current review will highlight the clinical manifestation and
immunological processes involved during the development of interferon induced thyroditis so
that a better understanding of the mechanisms can optimize current therapies.
Key word: Interferon, autoimmune, endocrine, hepatitis C, thyroid, immunological, thyroditis.
INTRODUCTION
Hepatitis C virus (HCV) is a small (~55-65
nm), spherical, enveloped, positive-stranded RNA
virus that is responsible for most liver associated
morbidity and mortality. It is characterized by
inflammatory liver damage and causes both acute
and chronic hepatitis in humans. Persistent chronic
HCV infection can gradually evolve into hepatic
fibrosis, cirrhosis, end stage liver diseases and
hepatocellular carcinoma (Poynard et al., 2003,
Heller et al., 2005, William et al., 2006). Hepatitis
C, also known as the silent disease infects
approximately 3% of the world’s population, which
represents 170 million people. Every year 3-4
million individuals are exposed to HCV
(Santantonio et al., 2003, Wiese et al., 2005,
Massard et al., 2006).
Since the discovery of the hepatitis C virus,
great efforts have been made in the development of
an antiviral therapy. Interferon alpha (IFNα) is a
type I interferon that has been extensively used as a
therapeutic agent against HCV (Fig. 1). IFNα is a
________________________________
* Corresponding author: [email protected]
HCV Patients
Auto Immune
Thyroditis
Non Auto Immune
Thyroditis
Graves’ DiseaseHashimoto’s
Thyroditis
Destructive
Thyrotoxicosis
T3 ↑
T4 ↑
TSH ↓
T3 ↓
T4 ↓
TSH ↓
T3 ↑ ↓ normal
T4 ↑ ↓ normal
TSH↓ ↑ normal
Non
autoimmune
hypothyroidism
T3 ↓
T4 ↓
TSH ↑
Fig. 1. Distributed thyroid hormone levels
in HCV patients associated with IFNα treatment
potent cytokine that mediates its effect by binding to
IFNα receptor which leads to activation of many
signaling pathways finally blocking the replication
of virus (Jonasch and Haluska, 2001, Parmar and
Platanias, 2003). IFNα is also an essential mediator
of the innate antiviral immune response which
INTERFERON INDUCED THYRODITIS IN HCV PATIENTS
55
creates its effect by effecting the cellular
physiology, especially the cells of the immune
system (Biron and Sen, 2001; Samuel, 2001). Such
antiviral treatment therapies have been shown to
improve liver histology and decrease the prevalence
of hepatocellular carcinoma in chronic HCV
patients (Yoshida et al., 2004, George et al., 2009).
However, 50% of the patients with chronic hepatitis
C, treated with IFNα did not achieve sustained
clearance of hepatitis C virus. Moreover, there is
evidence that IFNα when used therapeutically in
chronic hepatitis C patients can participate or
exacerbate autoimmune endocrine diseases,
especially of thyroid gland. The consequence of this
side effect has lead to reduction of dose in up to
40% of patients or sometimes discontinuation of
IFNα treatment in up to 14% of those patients who
develop thyroid dysfunction.
Epidemiology of interferon induced thyroditis
(IIT) Two distinct mechanisms develop in case of
thyroid disorder during IFNα therapy of HCV
patients that is autoimmune IIT and non-
autoimmune IIT. Autoimmune IIT include
Hashimoto’s thyroditis (HT) and Graves’ disease
(GD). Non-autoimmune IIT includes destructive
thyroditis (DT) and non- autoimmune
hypothyroidism.
Hashimoto’s thyroditis (HT)
Hashimoto's thyroiditis (Fig. 2) is one of the
most frequent human autoimmune diseases
(Vanderpump and Tunbridge, 2002). It is an organ-
specific T-cell mediated chronic disease typically
characterized by enlargement and dense
lymphocytic infiltration of the thyroid gland. This
disorder has an incidence rate of 2% in general
population. It is ten times more frequent in women
than in men and is considered responsible for high
morbidity rate in women. The prevalence rate of HT
peaks in middle age (Troisi et al., 2013).
Hashimoto’s thyroditis is usually
asymptomatic, and may be found from routine
screening of thyroid function. Onset of disease may
be insidious and may progresses over time.
Symptoms of hypothyroidism are nonspecific and
may be seen at the first consultation. It includes
more commonly fatigue, cold intolerance, and
dryness of skin, weight gain, anorexia, menstruation
disturbance, voice huskiness, abnormal reflexes and
pressure symptoms in the neck from thyroid
enlargement. In very rare cases it has been observed
that a period of temporary thyrotoxicosis lasting
weeks or months is also encountered (Troisi et al.,
2013).
Diagnosis of HT is mainly on the basis of
history and examination of the patient. Serum levels
of thyrotropin (TSH) thyroxine (T4) and
triiodothyronine (T3) are checked. Detection of
serum auto-antibodies generated during the disease
is also examined. Histological and cytological
features of HT include loss of colloid in thyroid
follicles dense thyroidal accumulation of
lymphocytes including numerous plasma cells
(Devendra and Eisenbarth, 2003).
HT manifests itself through subclinical
hypothyroidism that is characterized by an elevated
level of serum TSH whilst serum T4 and T3
remained normal. The predominant features of the
disease are destruction of thyroid follicular cells
(thyrocytes), hypothyroidism, goiter and production
of circulating auto-antibodies. There are two types
of autoantibodies that are mainly produced during
HT that are thyroglobulin (Tg-Ab) and thyroid
peroxidase (TPO-Ab) (Sinclair, 2006).
The TPO-Ab is generated against the enzyme
thyroid peroxidase and is found in association with
Tg-Ab. Thyroid peroxidase is responsible for
iodination of thyroglobulin. TPO-Ab may work
either directly by initiating the process of apoptosis
by enhancing the production of T-cells producing
Th/Tc1 cytokines (Karanikas et al., 2005). TPO-Ab
may also have an indirect effect in which these
autoantibodies interfere with the catalytic function
of enzyme thyroid peroxidase (Carella et al., 2001;
Mazziotti et al., 2003). IFNα treatment may also
provoke a slight defect in the thyroidal
organification of iodide, thus further impairing
thyroid hormone synthesis. All these processes
contribute in development of hypothyroidism.
Immune mediated mechanism
Failure of autoimmune system in interferon
induced HT can be due to multiple factors that may
involve role of certain genes or environmental
INTERFERON INDUCED THYRODITIS IN HCV PATIENTS
56
aspects (Dimitry, 2005). IFNα administration in HT
accelerates the process of thyroid autoimmunity.
Antigen presenting cells (APC) on presentation of
Fig. 2. Immune mediated mechanism in Hashimoto’s thyroditis leading to apoptosis of thyrocyte induced by
IFNα.
antigen accumulates in the thyroid and triggers a
process that involves both TH1 and TH2 (T-helper
cells) elements. IFNα creates its effect by
stimulating increased production of self reactive T
cells. IFNα also increases the expression of major
histocompatibility class (MHC) class I and II
antigens on thyroid cells. The self reactive TH1 and
TH2 cells will recruit cytotoxic T lymphocytes
(CTL) and B cells into the thyroid cell. Auto
reactive B cells will produce antibodies where as the
CTL’s will produce cytokines such as IFN-g, IL-2,
and tumor necrosis factor (TNF-α) that initiates the
process of apoptosis (Watanabe et al., 2002). These
cytokines promote Fas-mediated (trans-membrane
protein) apoptosis through the induction of enzymes
known as caspases. Both autoimmune antibodies
and thyroid-specific CTL’s are responsible for auto-
immune thyrocyte depletion.
Graves’ disease (GD)
GD is characterized by clinical
hyperthyroidism, positive TSHR antibodies (Fig. 3)
and goiter (Davies et al., 2005). The prevalence of
this disorder is less frequent as compared to other
thyroid disorders and has an incidence rate of 0.1-
0.5% in general population. Retrospective studies
have shown that administration of IFNα to HCV
patients leads to the development of GD in already
predisposed people (Wong et al., 2002).
Predisposition of GD include living in an area of
high iodine intake and female sex the incidence of
GD in women is 7:1 as compared to men (Wong et
T3 ↓
T4 ↓
Thyrocyte
APC
THO
TH
1 TC
IFN- α
TH
2 B P
MHC
Class I
MHC
Class II
TNF-α,
INF-γ, IL-2
Apoptosis/
Necrosis
IL-12
Accelerates
Inflammation
Auto
Reactive
Hashimoto’s
Thyroditis
INTERFERON INDUCED THYRODITIS IN HCV PATIENTS
57
al., 2002).
Fig.3. Positive antigen presenting cell and IFNα leads to production of cytokines and TSI. IL-4 and IL-10
activates anti-apoptotic protein leading to survival of thyrocyte in GD.
Signs and symptoms of GD include weight
loss, diarrhea, proximal myopathy, increased
appetite, tremor, tachycardia and palpitation. Other
prominent features of GD are hyperplasia and
hypertrophy of the endocrine glands. It has been
observed that approximately 10% of patients with
GD develop a severe form of Graves’
ophthalmopathy (Protrusion of eye ball and eye lid).
This causes eye lid retraction due to inflammation of
tissue in retro-orbital space. In even more severe
cases constriction of optic nerve may develop,
which can lead to visual impairments (Gianoukakis
et al., 2008).
Diagnosis of GD can be carried out on the
basis of elevated levels of thyroid hormones T4 and
T3 and a characteristic suppressed level of TSH.
Thyroid receptor antibody (TRAb) are detected in
GD. Uptake of iodine is increased in patients with
GD (Davies et al., 2005).
As compared to HT, lymphocytic infiltration
is less florid, lymphoid follicles formation is less
common and destruction of thyrocytes was not seen.
In most of the reported cases it has been observed
that development of GD during IFNα therapy failed
to resolve after completion of therapy and patients
have to undergo further treatment (Wong et al.,
2002).
Immune mediated mechanism
Graves’ disease is generally believed to be a
TH2 (T-helper) cell mediated disease (Land et al.,
2004, Mazziotti et al. (2002) IFNα induces its effect
in GD by release of other cytokines, such as IL-6
and IL-10. It is seen that thyrocytes have specific
T3 ↑
T4 ↑
Thyrocyte
APC
IFN-α
TH2
B
P
TSHR MHC
Class II
Cell Survival
cytokines
Accelerates
Inflammation
TSH reactive B cell
Graves’ Disease
TSI
IL-4, IL10
MHC
Class II
cFLIP and BCL-XL
upregulated
INTERFERON INDUCED THYRODITIS IN HCV PATIENTS
58
binding sites for IL-6, which lowers TSH-mediated
iodine uptake, TSH-mediated expression of thyroid
peroxidase mRNA, and TSH-mediated thyroid
hormone release in vitro. Activated TH2 cells in
thyroid tissue produces cytokines such as IL-4 and
IL-10. Both these cytokines have stimulatory effect
on B cells and switch the production of thyroid
stimulating immunoglobulins (TSI) which mimics
the TSH and binds to (TSHR) receptor, thus
blocking the receptor site for TSH which leads to
increased thyroid hormone production ultimately
causing hyperthyroidism. In GD IL-4 and IL-10
production may also protect thyrocytes from
apoptosis by up regulating cellular FLICE/caspase-8
(cFLIP) inhibitory protein and Bcl-XL an anti-
apoptotic protein (Wanzhu et al., 2011).
Non-autoimmune interferon induced thyroditis
(IIT)
Interferon induced thyroditis can also
manifest itself as non-autoimmune thyroditis.
Approximately 50% of the cases of IIT have been
reported of non-autoimmune thyroditis. Non-
autoimmune thyroditis can either be destructive
thyroditis or non-autoimmune hypothyroidism
(Prummel et al., 2003; Tomer et al., 2007).
Non-autoimmune hypothyroidism
Non-autoimmune hypothyroidism has been
more frequently reported as compared to DT
(Carella et al., 2002; Masood et al., 2008). It can
manifest as both clinical and subclinical
hypothyroidism. The prominent feature of this
disease is that hypothyroidism occurs without the
production of thyroid antibodies. Although transient
hypothyroidism is mostly observed but permanent
hypothyroidism along with production of thyroid
antibodies has also been reported. This implies that
immunological factors play an important role in
shifting temporary hypothyroidism to permanent
hypothyroidism (Wong et al., 2002). It has been
reported that non-autoimmune hypothyroidism may
be caused due to mutation in genes encoding for
proteins involved in TSH pathway. This cause TSH
receptor impairment that leads to suppressed
production of thyroid hormones (Chi et al., 2013).
Destructive thyroditis (DT)
Destructive thyroditis is a self limited
inflammatory condition of thyroid gland that usually
occurs during the first week of IFNα treatment. It
has been suggested that destructive thyroditis is
caused by non immunological mechanisms (Wong
et al., 2002). DT is a three stage disease i.e., first a
phase of hyperthyroidism comes that leads to a
second phase of hypothyroidism. In the third and
last phase thyroid function become normal again. In
a case reported recently relapse of hyperthyroidism
is seen after normal functioning of the thyroid gland
(Czarnywojtek et al., 2013).
It is evident that thyroid dysfunction is the
most common endocrine disease to develop during
IFNα therapy in HCV patients. The likely reason is
IFNα treatment that promotes autoimmune
reactions. HCV itself plays a significant role in
disturbing the thyroid environment thus contributing
to thyroid dysfunction. Since its incidence is quite
high in patients receiving IFNα for HCV it is
essential that physicians treating patients with IFNα
should be aware of the clinical manifestation of IIT.
The study has also highlighted the immunological
and non immunological factors responsible for
various types of IITD. In autoimmune diseases
IFNα alter the balance between TH1 and TH2 cells.
In HT both TH1 and TH2 cells play a predominant
role where as in GD TH2 cells play an important
role. In non-autoimmune diseases non
immunological factors seems to play an essential
role in development.
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(Received: April 17, 2014; Revised: June 12, 2014)