Pakistan Journal of Molecular Medicine · PAKISTAN JOURNAL OF MOLECULAR MEDICINE INSTRUCTIONS TO AUTHORS Pakistan Journal of Molecular Medicine (Pakistan J. Mol. Med.) is a quarterly

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,

mailto:[email protected]

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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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

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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



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


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


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.


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


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


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


(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

http://www.nature.com/onc/journal/v27/n32/full/onc200882a.html#bib20


http://cancerres.aacrjournals.org/search?author1=Mikhail+V.+Blagosklonny&sortspec=date&submit=Submit



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





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.

REFERENCES

Abal M, Andreu JM, Barasoain I. Taxanes:

microtubule and centrosome targets and cell

cycle dependent mechanisms of action. Curr

Cancer Drug Targets, 2003; 3: 193–203.

Bacus SS, Gudkov AV, Lowe M, Lyass1 L, Yung

Y, Komarov AP, Keyomarsi K, Yarden Y,

Seger R. Taxol-induced apoptosis depends on

MAP kinase pathways (ERK and p38) and is

independent of p53. Oncogene, 2001; 20:

147-155.

Berndtsson M, Konishi Y, Bonni A, Hagg M,

Shoshan M, Linder S, Havelka AM.

http://www.ncbi.nlm.nih.gov/pubmed?term=Berndtsson%20M%5BAuthor%5D&cauthor=true&cauthor_uid=15907327

http://www.ncbi.nlm.nih.gov/pubmed?term=Konishi%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=15907327

http://www.ncbi.nlm.nih.gov/pubmed?term=Bonni%20A%5BAuthor%5D&cauthor=true&cauthor_uid=15907327

http://www.ncbi.nlm.nih.gov/pubmed?term=H%C3%A4gg%20M%5BAuthor%5D&cauthor=true&cauthor_uid=15907327

http://www.ncbi.nlm.nih.gov/pubmed?term=Shoshan%20M%5BAuthor%5D&cauthor=true&cauthor_uid=15907327

http://www.ncbi.nlm.nih.gov/pubmed?term=Linder%20S%5BAuthor%5D&cauthor=true&cauthor_uid=15907327

http://www.ncbi.nlm.nih.gov/pubmed?term=Havelka%20AM%5BAuthor%5D&cauthor=true&cauthor_uid=15907327


Phosphorylation of BAD at ser-128 during

mitosis and paclitaxel-induced apoptosis.

FEBS Lett., 2005; 579: 3090-94.

Blagosklonny MV, Bishop PC, Robey R, Fojo T,

Bates SE. Loss of Cell Cycle Control Allows

Selective Microtubule-active Drug-induced

Bcl-2 phosphorylation and cytotoxicity in

autonomous cancer cells. Cancer Res., 2000;

60: 3425-28.

Boldt S, Weidle UH, Kolch W. The role of MAPK

pathways in the action of chemotherapeutic

drugs. Carcinogenesis., 2002; 23: 1831-38.

Chakravarthi BVSK, Das P, Surendranath K,

Karande A, Jayabaskaran C. Production of

paclitaxel by Fusariumsolani isolated from

Taxus celebica. J Biosci., 2008; 33: 259-267.

Dziadyk JM, Sui M, Zhu X, Fan W. Paclitaxel-

induced apoptosis may occur without a prior

G2/M-phase arrest. Anticancer Res., 2004;

24: 27-36.

Elavarasi A, Rathna GS, Kalaiselvam M. Taxol

producing mangrove endophytic fungi

Fusarium oxysporum from Rhizophora

annamalayana. Asia Pacific J Trop Biomed.,

2012; 12: 1081-1085.

Engels B, Dahm P, Jennewein S. Metabolic

engineering of taxadiene biosynthesis in yeast

as a first step towards Taxol (paclitaxel)

production. Metab Eng., 2008; 10: 201-206.

Giannakakou P, Robey R, Fojo T, Blagosklonny

MV. Low concentrations of paclitaxel induce

cell type dependent p53, p21 and G1/G2

arrest instead of mitotic arrest: molecular

determinants of paclitaxel-induced

cytotoxicity. Oncogene, 2001; 20: 3806-13.

http://globocan.iarc.fr/. IARC. 2012. Estimated

cancer incidence, mortality and prevalence

worldwide in 2012.

Jailkhani N, Chaudhri VK, Rao KV. Regulatory

cascades of protein phosphatases:

implications for cancer treatment. Anticancer

Agents Med Chem., 2011; 11: 64-77.

Ji Y, Bi Jn, Yan B, Zhu, XD. Taxol-producing

fungi: A new approach to industrial

production of Taxol. Chin J Biotech., 2006;

22: 1-6.

Jordan MA, Wendell K, Gardiner S, Derry WB,

Copp H, Wilson L. Mitotic block induced in

HeLa cells by low concentrations of

paclitaxel (Taxol) results in abnormal mitotic

exit and apoptotic cell death. Cancer Res.,

1996; 56: 816–825.

Jordan MA, Wilson L. Microtubules as a target for

anticancer drugs. Nat Rev Cancer., 2004; 4:

253–265.

Martel C, Ferlay J, Franceschi S. Global burden of

cancers attributable to infections in 2008: a

review and synthetic analysis. The Lancet

Oncol., 2012; 13: 607-615.

Marupudi NI, Han JE, Khan W, Renard VW, Tyler

BM, Brem H. Paclitaxel: a review of adverse

toxicities and novel delivery strategies. John

Hopkins School of Medicine, 2007; 6: 609-

621.

Mcdaid HM, Horwitz SB. Selective potentiation of

paclitaxel (Taxol)-induced cell death by

mitogen-activated protein kinase kinase

inhibition in human cancer cell lines. Mol

Pharmacol., 2001; 60: 290–301.

Miller AV, Hicks MA, Nakajima W, Richardson

AC, Windle JJ, Harada H. Paclitaxel induced

apoptosis is BAK dependent, but BAX and

BIM-independent in breast tumor. PLoS One,

2013; 8: e60685.

Okano JI, Rustgi AK. Prolonged activation of

activating the programmed cell death the

Ras/MEK/ERK pathway independently

paclitaxel induces. J Biol Chem., 2001; 276:

19555-564.

Page M, and Landry N. Bacterial mass production

of taxanes with Erwinia, US Patent 5561055

A, 1 Oct 1996.

Priyadarshini K and Keerthi AU. Paclitaxel against

cancer: A short review. Med chem., 2012; 2:

139-141.

Ross JL, Fygenson k. Mobility of Taxol in

microtubule bundles. Biophys Journals. 2003;

84: 3959-3967.

Rowinsky EK. The development and clinical utility

of the taxane class of anti-microtubule

chemotherapy agents. Ann Rev Med., 1997;

48:353–74.

Sato RXN, Yanai K, Akiyoshi T, Nagai, Wada J,

Koga K, Mibu R, Nakamura M, katano M.

Enhancement of paclitaxel-induced apoptosis

by inhibition of mitogen-activated protein

http://www.ncbi.nlm.nih.gov/pubmed/15907327


http://cancerres.aacrjournals.org/search?author1=Philippe+C.+Bishop&sortspec=date&submit=Submit

http://cancerres.aacrjournals.org/search?author1=Robert+Robey&sortspec=date&submit=Submit

http://cancerres.aacrjournals.org/search?author1=Tito+Fojo&sortspec=date&submit=Submit

http://cancerres.aacrjournals.org/search?author1=Susan+E.+Bates&sortspec=date&submit=Submit

http://globocan.iarc.fr/

http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx


kinase pathway in colon cancer cells.

Anticanc Res., 2009; 29: 261-270.

Xiao H, Pinard PV, Fuentes NF, Burd B, Angeletti

R, Fiser a, Horwitz SB, Orr GA. Insights into

the mechanism of microtubule stabilization

by Taxol. PNAS., 2006; 103: 10166-10173.

Zhang P, Zhou P, Jiang C, Yu H, Yu LJ. Screening

of Taxol producing fungi based on PCR

amplification from taxus. Biotechnol Lett.,

2008; 30: 2119-2123.

(Received: July 18, 2014: Revised: August 20, 2014)

EFFECT OF NITROGEN SOURCE & NIACIN ON ß-MANNANASE PRODUCTION BY B. BADIUS

14


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



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.


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-


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


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).


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.


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


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.

REFERENCES

Ab-Rashid S, Darah I and Omar I. Utilization of

palm kernel cake for the production of

mannanase by an indigenous filamentous

fungus, Aspergillus niger USM F4 under

solid substrate fermentation. The Internet J

Microbiol., 2001; 9: 1-5

Adebayo-Tayo B, Elelu T, Akinola G and

Oyinloye I. Screening and production of

mannanase by Bacillus strains isolated from

fermented food condiments. Innovative

Romanian Food Biotechnol., 2013; 13: 53-

62.

Ademark P, Varga A, Medve J, Harjunpa AV,

Drakenberg T and Tjerneld F. Softwood

hemicellulose-degrading enzymes from

Aspergillus niger: Purification and

properties of α-mannanase. J Biotechnol.,

1998; 63: 199-210.

Ali SS, Habib I and Riaz T. 2009. Screening and

characterization of alkaliphilic bacteria

from various industrial effluents. Punjab

univ J Zool., 2010; 24 (1-2): 49-60.

Ali SS, Nazir M and Riaz T. 2010. Effect of salt

stress and vitamin B1 on amylase

production potential in locally isolated

bacteria. Punjab Univ J Zool., 2010; 25 (2):

67-80.

Blibech M, Ellouz Ghorbel MR, Fakhfakh I.

Purification and characterization of low

molecular weight of β-mannanase from


21

Penicellium occitanis Po16. Appl Biochem

Biotechnol., 2010; 160:1227-40.

Christgua S, Andersen LN, Kaupinen S, Heldt-

Hansen HP and Dalboege H. Purified

enzyme exhibiting mannanase activity;

Application in oil, paper, pulp, fruit and

vegetable juice industry and in carrageen

extraction. Patent NOVO-Nor disk, 1994;

9425576, 10 Nov 1994.

Dhawan S and Kaur J. Microbial mannanases: an

overview of production and applications.

Crit. Rev. Biotechnol 2007; 27(4): 197–216.

Feng Y, He Z, Leong Ong S, Hu J, Zhang Z, Jern

Nig W. Optimization of agitation, aeration

and temperature conditions for maximum

β-mannanase production. Enzyme and

Microbial Technol., 2003; 32: 282-89.

Ferreira HM, Filho EXL. Purification and

characterization of β-mannanase from

Trichoderma harzianum strain T4.

Carbohydrate Polym., 2004; 57: 23-29

Francoise VC, Ghakis C, Dupont, Morosoll R and

Kluepfel D. Improved production of

mannanase by Streptomyces lividans. Appl

Environ Microbiol., 1996; 63: 56-58.

Gubitz G, Sachslehner A and Haltrich D.

Microbial mannanases: substrates,

roduction and application. Glycosyl

hatdrolase for biomass conversion.

Washington DC, ACS Symposium series,

American Chemical society, 2001; 769:

239-62.

Gubitz GM, Hayn M, Urbanz G and Steiner W.

Purification and properties of an acidic β-

mannanase from Sclerotium rolfsii. J

Biotechnol., 1996; 45: 165-172.

Gubitz GM, Lischnig T, Stebbing D and Saddler

JN. Enzymatic removal of hemicellulose

from dissolving pulp. Biotechnol. Lett.,

1997; 19: 491-95.

Hashimoto Y and Fukumoto J. Studies on the

enzyme treatment of coffee beans.

Purification of mannanase from Rhizopus

niveus and its action on coffee mannan.

Nippon Nogeikagaku Kaishi, 1969; 43:

317–322.

Hogg D, Pell G, Dupree P, Goubet F, Martin-Orue

SM, Armand S and Gilbert HJ. The

modular architecture of Cell Vibrio

japonicas mannanase in glycoside

hydrolase’s families 5 and 26 points to

differences in their role in mannan

degradation. Biochem J., 2003; 37: 1027-

1043.

Jones GH, Ballou CE. Studies on the structure of

yeast mannan J Biol Chem., 1969; 244:

1043-1045.

Kobayashi Y, Echigen R, Mada M and Mutai M.

Effects of hydrolysates of konjac mannan

and soybean oligosaccharides on intestinal

flora in man and rats. In: Intestinal flora

and food factors. (ed. T Mitsuoka) Gakkai

Shuppan Centre, Tokyo, 1987; pp 79–87.

Lin TC and Chen C. Enhanced mannanase

production by submerged culture of

Aspergillus niger NCH-189 using defatted

copra based media. Process Biochem.,

2004; 39: 1103-1109.

Lowery OH, Rosebrough NJ, Farr AL and

Randall RJ. Protein measurement with the

Folin phenol reagent. J Biol Chem.,

1951;193: 265-275.

Mabrouk ME and El-Ahwany AMD. Production

of β-mannanase by Bacillus

amylolequifaciens 10A1 cultured on potato

peels. Afr J Biotechnol., 2008; 7(8): 1123-

1128.

Malik SK, Mukhtar H, Farooqi AA and Haq I.

Optimization of process parameters for the

biosynthesis of cellulases by Trichoderma

viride. Pakistan J Bot., 2010; 42(6): 4243-

4251.

McCleary BV and Matheson NK. Action patterns

and substrate-binding requirements of β-D-

mannanase with monosaccharides and

mannan-type polysaccharides. Carbohydr

Res., 1983; 119: 191–219.

McCleary BV and Matheson NK. Enzymic

analysis of polysaccharide structure. In:

Advances in carbohydrate chemistry and

biochemistry. (eds. RS Tipson and D

Horton), Academic Press Ltd., London,

United Kingdom, 1986: 147–276.

McCleary BV. Carob and guar galactomannans.

Methods in enzymol, 1988; 160: 523-527.

Meenakshi M, Singh G, Bhalla A and Hoondal

GS. Solid state fermentation and

characterization of partially purified

thermostable mannanase from Bacillus sp.

MG-33. Bioresource, 2010; 5(3): 1689-

1701.

Mendoza NS, Arai M, Kawaguchi T, Yoshida T


22

and Joson LM. Purification and properties

of mannanase from Bacillus subtilis. World

J Microbiol Biotechnol.,1988b; 10: 551-

555.

Miller GL. Use of dinitrosalicylic acid reagent for

determination of reducing agents.

Analytical Chem., 1959; 31: 426-28.

Moreira S and Kuhad RC. An over view of

mannan structure and mannan-degrading

enzyme system. Appl Microbiol Biotechnol,

2006; 79: 165-78.

Naughton PJ, Mikkelsen LL and Jensen BB.

Effects of non-digestible oligosaccharides

on Salmonella enteric, serovar,

typhimurium and nonpathogenic Eschericia

coli in the pig small intestine. In vitro Appl

and Environ Microbiol., 001; 67: 3391-95.

Oda Y, Komaki T and Tonomura K. Production of

β-mannanase and β-mannosidase by

Enterococcus casseliflavus FL 2121

isolated from decayed konjac. Food

Microbiol., 1993a; 10: 353-358.

Oda Y, Komaki T and Tonomura K. Purification

and properties of extracellular β-

mannanases produced by Enterococcus

casseliflavus FL2121 isolated from decayed

konjac. J Ferment Bioeng 1993b; 76: 14–

18.

Olaniyi OO, Igbe FO and Ekundayo TC.

Optimization studies on mannanase

production by Trichosporonoides

oedocephalis in submerged state

fermentation. E3 J Biotechnol Pharmaceut

Res., 2013; 4(7) 110-116.

Pedersen G, Hangen HA, Asferg L and Sorensen

E. Removal of printing paste thickener and

excess dye after textile printing. Patent

Novo Nordisk, 1995; A/S 5405414.

Peixoto RS, Vermelho AB and Rosado AS.

Petroleum-degrading enzymes:

bioremediation and new prospect. Enzyme

Res., 2011; pp. 1-7.

Phothichitto K, Nitisinprasert S, Keawsompong S.

Isolation, screening and identification of

mannanase producing microorganisms.

Kasetsart J (Natural Science), 2006; 40:

26-38.

Rattanasuk S and Ketudat-Cairns M.

Cryseobacterium indologenes, novel

mannanase producing bacteria.

Songklanakarin J Sci Technol., 2009; 31:

395-399.

Sanaullah S, and Ali SS. Role of bacterial

mannanase in biodegradation of solid

waste: strain isolation and characterization.

M.Sc. thesis, University of the Punjab,

Quaid-e-Azam Campus, Lahore, Pakistan

Shallom D and Shoham Y. Microbial

hemicellulases. Curr Opin Microbiol 2003;

6: 219–228.

Soetan K, Aiyelaagbe O and Olaiya CO. A review

of the bichemical, biotechnological and

other applications of enzymes. Afr J

Biotechnol 2010; 9(4): 382-393.

Zakaria MM, Yamamoto S and Yagi T.

Purification and characterization of an

endo-1,4- β-mannanase from Bacillus

subtilis KU-1. FEMS Microbiol Lett., 1998;

158: 25–31.

Zhang J, He ZM and Hu K. Purification and

characterization of β-mannanase from

Bacillus licheniformis for industrial use

Biotechnol Lett., 2000; 22: 1375–78.

(Received: February 15, 2014; Revised: May 12, 2014)

EFFECTS OF EMAMECTIN & ITS MIXTURE WITH SILYMARIN ON SOME ENZYME ACTIVITIES IN CHICK

24


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


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.


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


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)


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.


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.


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-


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.

REFERENCES

1

Allain CC, Poon LS, Chan CS, Richmond, W and

Fu PC. Enzymatic determination of total

serum cholesterol. Clin Chem., 1974; 20(4):

470-475

Buhl SN and Jackson KY. Optimal conditions and

comparison of lactate dehydrogenase

catalysis of the lactate- to –pyruvate and

pyruvate- to -lactate reactions in human

serum at 25, 30 and 37 degrees. Clin Chem.,

1978; 24(5): 828-831

Gacemi A and Guenaoui Y. Efficacy of emamectin

benzoate on Tutaabsoluta meyrick

(Lepidoptera: Gelechiidae) infesting a

protected tomato crop in Algeria. Acad J

Entomol., 2012; 5 (1): 37-40.

Gazak R, Walterova D, Kren V, Silybin and

Silymarin - New and emerging applications

in medicine. Curr Med Chem., 2007; 14:

315–338.

Gornall AG, Bardawill CJ and David MM.

Determination of serum proteins by means of

the biuret reaction. J Biol Chem., 1949;

177(2): 751-766.

Hafkenscheid JCM and Kohler BEM. Temperature

conversion factors for four enzymes in

commercial control sera. Clin Chem., 1986;

32: 184-185.

Jahan AN, Rehman K and Nosheen S.

Gemmomodification: An emerging source of

natural antioxidants from Silybum marianum.

Pakistan J Pharm Sci., 2013; 26 (3): 585-591.

Jensen PD, Jensen FT, Christensen T, Nielsen JL

and Ellegarrd J. Relationship between

hepatocellular injury and transfusional iron

overload prior to and during iron chelation

with desferrioxamine: a study in adult

patients with acquired anemia. Blood, 2003;

101(1):91–96.

Kang JS, Jeon YJ, Park SK, Yang KH and Kim

HM. Protection against lipopolysaccharide-

induced sepsis and inhibition of interleukin-1

beta and prostaglandin E2 synthesis by

silymarin. Biochem Pharmacol., 2004;

67:175–181.

Kaur M and Agarwal R. Silymarin and epithelial

cancer chemoprevention: How close we are

to bedside. Toxicol Appl Pharm., 2007;

224:350–359.

Khan BA, Akhtar N, Rasul A, Khan H, Murtaza G,

Ali A, Khan KA, Zaman S, Jamil A, Waseem

K and Mahmood T. Human skin, aging and

antioxidants. J Medicinal Plants Res., 2012;

6(1): 1-6

Khan, Z., Maddawy, E and Gad, SB. Hepato-renal

protection of silymarin in comparison with

vitamins E in rats. J Pharmacol., 2012; 6(3):

236-244.

Kren VND and Walterova D. Silybin and Silymarin:

New effects and applications. Biomed Paper,

2005; 149(1):29-41.

Mayer KE, Mayer RP and Lee SS. Silymarin

treatment of viral hepatitis: a systematic

review. J Viral Hepatitis, 2005; 12: 559-567.

Miksicek R. Estrogenic flavonoids: Structural

requirements for biological activity. Proc Soc

Exp Biol Med., 1995; 208: 44-50.

Nadimi A, Kamali K, Arbabi M and Abdoli F.

Selectivity of three miticides to spider mite

predator Phytoseiulus plumifer (Acari:

Phytoseiidae) under laboratory conditions.

Agr Sci China,, 2009; 8(3):326–331.

Narayan P, Yadav A, Anirban PA, Karuna SB,

Dyaneshwar U, Bawankule A, Anil K. Gupta

A, Mahendra P, Darohar A, Suman PS and

Khanuja A. Synergistic effect of silymarin

and standardized extract of Phyllanthus

amarus against CCI4-induced hepatotoxicity

in Rattus norvegicus. Phytomed., 2008; 15:

1053-1061

Oda SS and El-Ashmawy IM. Adverse effects of the

anabolic steroid, boldenone undecylenate on

reproductive functions of male rabbits.

International J Exp Pathol., 93(3): 172-178.

2012

Oda SS and El-Ashmawy IM. Protective effect of

silymarin on mercury-Induced acute nephro-


29

hepatotoxicity in rats. Global Veterinatia,

2012; 9(4):376-383.

Olsvik PA, lie KK, Mykkeltvedt E, Samuelsen OB,

Petersen K, Stavrum AK and Lunestad BT.

Pharmacokinetics and transcriptional effects

of the anti-salmon lice drug emamectin

benzoate in Atlantic Salmon. BMC

Pharmacol., 2008; 8: 1-14 pp.

Polyak SJ, Oberlies NH, Pecheur EI, Dahari H and

Frenci P. Silymarin for HCV infection.

Antiviral Therapy, 2013; 18: 141-147.

Rainone F. Silymarin protects tissue by supporting

normal cellular defenses. Am Form

Physician., 2005; 72 (7): 1285-1288.

Saller R, Meier R and Brignoli R. The use of

silymarin in the treatment of liver diseases.

Drugs, 2001; 61: 2035-2063.

Schallman MD, Valentino D, Streicher S and Ruby

C. ‘Streptomyces avermitilis’ mutants

defective in methylation of avermectins.

Antimicrobial Agents and Chemotherapy,

1987; 31(5): 744-747.

Schandalik R, Gatti G and Perucca E.

Pharmacokinetics of silybin in bile following

administration of sillipide and silymarin in

cholecyctectomy patients. Arzneimittel

forschung, 1992; 42: 964-968.

Shahbazi F, Khavidaki SD, Khalili H and Pezeshki

ML. Potential renoprotective effects of

silymarin against nephrotoxic drugs. J Pharm

Pharmaceut Sci., 2012; 15(1): 112-123.

Shaker E, Mahmoud H and Mnaa S. Silymarin, the

antioxidant component and Silybum

marianum extracts prevent liver damage.

Food and Chem Toxicol., 2010; 48: 803-806.

Skottova N and Krecman V. Silymarin as a potential

hypocholesterolaemic. Drug Physiol Res.,

1998; 47: 1-7

Soto C, Recoba R, Barron H, Alvarez C and Favari

L. Silymarin increases antioxidant enzymes

in alloxan-induced diabetes in rat pancreas.

Comp Biochem Physiol C Toxicol

Pharmacol., 2003; 136: 205–212.

Sutherland IH and Campbell WC. Development,

pharmacokinetics and mode of action of

ivermectin. Acta Leidens, 1990; 59: 1-2.

Waddy, SL, Merritt, VA, Gibson, MNH and Aiken,

DE. Effects of emamectin benzoate on the

molt cycle of ovigerous American lobsters

Homarus americanus is influenced by the

dosing regimen. Aquat Biol., 2007; 11: 47-52.

Wahua TAT. Applied Statistics for Scientific

Studies. Africa Link Books. Aba Nigeria

1999, p.365.

Wallhofer H, Schmidt E and Schmidt UFW.

Synopsis Der Leberkrankheiten. (ed G

Thieme) Verlag Stuttgart, Clinical and

Laboratory Standard Institute. 1974

Werner M, Gabrielson DG and Eastman J.

Ultramicro determination of serum

triglycerides by bioluminescent assay. Clin

Chem., 1981; 27(2): 268-271

Yen TH and Lin JL. Acute poisoning with

emamectin benzoate. J Toxicol Clin Toxicol.,

2004; 42: 657-661.

Yoshii K, Ishimitsu S, Tonogai Y, Arakawa K,

Murata H and Mikami H. Simultaneous

determination of emamectin, its metabolites,

milbemectin, ivermectin and abamectin. J

Health Sci., 2004; 50(1): 17-24.

Zhao YLIH, Gao Z and Xu H. Effects of dietary

baicalin supplementation on iron overload-

induced mouse liver oxidative injury. Eur J

Pharmacol., 2005; 509(2–3):195–200.

(Received: March 18, 2014; Revised: May 08, 2014)

LIPID PEROXIDATION IN DIABETIC HCV PATIENTS

30


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



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.


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


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).

REFERENCES

Bertelsen M, Naggard EE and Carrier MJ. Oxidative

stress impairs insulin internalization in

endothelial cells in vitro. Diabetologia, 2001;

44: 605-613.

Boya P, Pena A, Beloqui O, Larrea E, Conchillo M

and Caselruiz Y. Antioxidant status and

glutathione metabolism in peripheral blood

mononuclear cells from patients with chronic

hepatitis C. J Hepatol., 1999; 31: 808-814.

Dalgic B, Sonmez N, Biberoglu G, Hasanoglu A

and Erbas D. Evaluation of oxidative stress in

Wilson's disease and non-Wilsonian chronic

liver disease in childhood. Turk J

Gastroenterol., 2005; 16: 7-11.

Deby C and Goutier R. New perspectives on the

biochemistry of superoxide anion and the

efficiency of superoxide dismutases. Biochem

Pharmacol., 1990; 39: 399-405.

Dubuisson J. Hepatitis C virus proteins. World J.

Gastroenterol, 2007; 13: 2406–2415.

Farinati F, Cardin R and Maria DE, Lecis NPE,

Libera G, Burra P, Marafin C, Srurniola GC

and Naccarato R. Zinc, iron and peroxidation

in liver tissue. Biol Trace Elem Res., 1995;

47: 193-199.

Farrell GC, Hui JM and Sud A. Insulin resistance is

associated with chronic hepatitis C virus

infection and fibrosis progression.

Gastroenterol., 2003; 125(6): 1695-1704.

Ghanay MG, Strader DB, Thomas DL and Seeff

LB. Diagnosis, management and treatment of

hepatitis C. Hepatology, 2009; 49: 1335–

1374.

Griesmacher A, Kindhauser AM, Andert SE,

Schreiner W, Toma C and Knoebl P.

Enhanced serum levels of thiobarbituric acid

reactive substances in diabetes mellitus. Am J

Med., 1995; 98: 469-475.

Grover JK, Vats V and Rathi SS. Anti-

hyperglycemic effect of Eugenia jambolana

and Tinospora cordifolia in experimental

diabetes and their effects on key metabolic

enzymes involved in carbohydrate

metabolism. J Ethnopharmacol., 2000; 73:

461–470.

Hiramatsu K and Arimori S. Increased superoxide

production by mononuclear cells of patients

with hypertriglyceridemia and diabetes.

Diabetes, 1988; 37: 832–837.

Hoofnagle JH and Seeff LB. Peg-interferon and

ribavirin for chronic hepatitis C. N Engl J


33

Med., 2006; 355: 2444-2451.

Hwang SJ. Hepatitis C virus infection. J Clin

Microbiol Immunol Infect., 2001; 34:227-234.

Kawaguchi T, Yoshida T and Harada M. Hepatitis C

virus down-regulates insulin receptor

substrates 1 and 2 through up-regulation of

suppressor of cytokine signalling 2004; Am J

Pathol., 2004; 165(5): 1499–1508.

Losada M and Alio JL. Malondialdehyde serum

concentration in type 1 diabetic with and

without retinopathy. Doc Ophthalmol., 1996;

93: 223-9.

Malik A, Qureshi MS, Manan A, Saleem S, Munir

M, Fatima A, Arooj M and Qazi MS.

Assessment of oxidative stress in hepatitis C

patients receiving interferon therapy.

Pakistan J Med Dent., 2013; 2(4): 10-15.

Maritim AC, Sanders RA and Watkins JB. Diabetes,

oxidative stress, and antioxidants. J Biochem

Mol Toxicol., 2003; 17(1): 24-38.

Matsunami T, Sato Y, Sato T and Yukawa M.

Antioxidant status and lipid peroxidation in

diabetic rats under hyperbaric oxygen

exposure. Physiol Res., 2010; 59: 97-104.

Nagoev BS, Abidov MT and Iyanova MR. LPO and

free radical oxidation parameters in patients

with acute viral hepatitis. Exp Biol Med.,

2002; 289(134): 557-58.

Ngaiza JR and Doenhoff MJ. Blood platelets and

schistosome egg excretion. Exp Biol Med

1990; 193(1): 73-79.

Niemela O, Parkkila S, Herttuala S, Halsted C,

Witztum JL, Lanca A and Israel Y. Covalent

protein adducts in the liver as a result of

ethanol metabolism and lipid peroxidation.

Lab Invest 1994; 70: 537-546.

Ohkawa H, Ohishi N and Yagi N. Ohkawa H, N

Ohishi and Yagi K. Assay for lipid peroxides

in animal tissues by thiobarbituric acid

reaction. Anal Biochem., 1979; 95: 351-358.

Parkkila S, Herrtuala S, Villanueva J, Niemela O,

Ruebner B and Halsted CH. Sequential

acetaldehyde production, lipid peroxidation,

and fibrogenesis in micropig model of

alcohol-induced liver disease. Hepatology,

1995; 22: 1208-1214.

Pasaoglu H, Sancak B and Bukam N. Pasaoglu H, B

Sancak and N Bukam (2004). Lipid

peroxidation and resistance to oxidation in

patients with type 2 diabetes mellitus. Tohoku

J Exp Med., 2004; 203: 211-218.

Rosser BG and GJ Gores. Liver cell necrosis:

cellular mechanisms and clinical

implications. Gastroenterology, 1995; 108:

250-275.

Salonen JT, Tuomainen TP and Nyyssenen K.

Relation between iron stores and non-insulin-

dependent diabetes in men: case-control

study. Br Med J., 1999; 317: 727-730.

Tilg H, Vogel W and Dinarello CA. Interferon-α

induced circulating tumor necrosis factor

receptor p55 humans. Blood, 1995; 85: 433-

435.

Yasa MH, Kacmaz M, Ozturk HS and Durak I.

Antioxidant status of erythrocytes from

patients with cirrhosis.

Hepatogastroenterology, 1999; 46: 2460-

2463.

Younossi ZM, Diehl AM and Ong JP. Younossi

ZM, AM Diehl and JP Ong. Nonalcoholic

fatty liver disease: an agenda for clinical

research. Hepatology, 2002; 35: 746-752.

(Received: May 14, 2014; Revised: July 04, 2014)

BLLOD PRESSURE CHANGES UNDER SPINAL ANESTHESIA IN CAESAREAN SECTION

34


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


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.


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%


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


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.

REFERENCES

Barar FSK. Adrenoreceptors Stimulants

(Sympathomimetics). In: Essentials of

Pharmacotherapeutics. 4th edn., S. Chand &

Company Limited, New Delhi, pp.362-349,

2007.

Baxendale BR. Preoperative assessment and

premedication. In: Text book of Anesthesia.

(eds. AR Aitkenhead, G Smith and DJ

Rowbotham). 5th edn., Churchill Livingstone,

Edinburgh, pp.280.

Caritis SN, Abonleish E, Edelstone DI and Muller-

Heubach E. Fetal acid-base state following

spinal or epidural anesthesia for cesarean

section. Obstet Gynecol., 1980; 56 (5): 610-

615.

Charkoudian N, Gusman E Joyner, MJ, Wallin BG

and Osbora J. Integrative mechanisms blood

pressure regulation in humans and rats: cross-

species similarities. Am J Physiol., Regu.

Integr Comp Physiol., 2010; 298: 755-759.

Clark VA. Sharwood-Smith GH and Stewart AVG,.

Ephedrine requirements are reduced during

spinal anaesthesia for caesarean section in

preeclampsia. Int J Obstet Anesth., 2005; 14:

9-13.

Corke BC, Datta S, Ostheener GW and Weiss Jb-

Alper MH. Spinal anesthesia for cesarean

section. The influence of hypotension of

neonatal outcome. Anaesthesia, 1982; 37:

658-62.

Coventry DM. Local anesthetic techniques. In:

Textbook of anesthesia (eds. AR Aitkenhead,

G Smith and DJ Rowbotham). 5th edn.,

Churchill Livingstone, Edinburgh, pp. 324-

326, 2007

Elton CD, May A and Buggy DJ. Obstetric


38

anesthesia and analgesia. In: Textbook of

Anesthesia. (eds. AR Aitkenhead, G Smith

and DJ Rowbotham). 5th

Edn., Churchill

Livingstone, Edinburgh, pp. 645. 2007

Girling KJ. Peripheral circulation and control of

cardiac output and arterial pressure. In:

Textbook of Anesthesia. (eds. AR Aitkenhead,

and G Smith), 4th edn., Churchill Livingstone,

Edinburgh, pp. 61-62. 2002

Guyton AC and Hall JE. Overview of the

circulation; Biophysics of pressure, flow, and

resistance. In: Textbook of Medical

Physiology. 12th edn., Elsevier, Saunders

Sciences, pp. 160-162. 2011a,

Guyton AC and Hall JE. Vascular dispensability and

functions of arterial and venous system. In:

Textbook of Medical Physiology. 12th Ed.

Elsevier, Saunders Sciences, pp. 168. 2011b

Hardman JG. Complications during anesthesia. In:

Textbook of anesthesia (eds. AR Aitkenhead,

G Smith and DJ Rowbotham), 5th edn.,

Churchill Livingstone, Edinburgh, pp. 382,

2007

Kestin IG. Spinal anaesthesia in obstetrics. Br J

Anesth., 66: 596-607. 1991

Loubert C. Fluid and vasopressin management for

cesarean delivery under spinal anaesthesia:

Continuing Professional Development. Can J

Anesth., 2012; 59(6): 604-619.

Mekawy NM. Comparative study between

ephedrine infusion vs. CO/post loading of

fluids for prevention of hypotension in

emergency cesarean section under spinal

anesthesia. Egyptian J Anaesth., 28:193-198.

2012.

Mishriky BM and Habib AS. Metoclopramide for

nausea and vomiting prophylaxis during and

after caesarean delivery: A systematic review

and meta-analysis. Br J Anaesth., 2012; 108:

374-383.

Pan PH and Moore CH. Intraoperative antiemetic

efficacy of prophylactic ondansetron versus

droperidol for cesarean section patients under

epidural anesthesia. Anesth Analg., 1996; 61:

982-986.

Ralston DH, Shnider SM and Delorimier AA.

Effects of equipotent ephedrine, metaraminol,

mephentamine and methoxamine on uterine

blood flow in the pregnant ewe.

Anesthesiology, 1974; 40: 354-370. 1974.

Rang HP, Dale MM, Ritter JM and Flower RJ..

Local anesthetics and other drugs affecting

sodium channels. In: Rang and Dale’s

Pharmacology, 6th edn., Churchill

Livingstone, Elsevier, London, pp. 639- 643,

2009a,b.

Sembulingam K and Sembulingam P. Arterial blood

pressure in Cardiovascular System In:

Essential of Medical Physiology. 5th edn.,

Jaypee Brothers Medical Publishers Limited,

India, pp. 580-581, 2010

Turkoz A, Togal T, Gokdeniz RI, Topraks H and

Esroy O. Effectiveness of intravenous

ephedrine infusion during spinal anaesthesia

for caesarean section based on maternal

hypotension, neonatal acid-base status and

lactate levels. Anaesth Intensive care, 30:

316-20. 2002.

Van De Velde M. Spinal anesthesia in the obstetric

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


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



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.


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


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).

REFERENCES

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–


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–

85

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

phenol reagent. J Biol Chem., 1951; 193: 265.

Mcsherry JA. Some medical aspects of the Darien

schema: was it dengue? Scot Med J., 1982;

27: 183-184

Nurden AT, George JN and Philips DR. Platelet

membrane glycoproteins: their structure,

function, and modification in disease.

Biochemistry of Platelets, 1986; 2: 160-212.

Okumura CL and Jameieson GA. Platelet

glycocalicin. II. Purification and

characterization, J Biol Chem., 1976; 251:

5950–5955.

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;

115: 1823-1834.

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

Trop Med Hyg., 2006; 100: 608-614.

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:

Fundamentals and Applications in

Biochemistry, Wiley Publishers. New

York, 4: 477, 1995.. Taniuchi K, Chifu K, Hayashi N, Nakamachi Y,

Yamaguchi N and Miyamato Y. Taniuchi,

K., Chifu, K., Hayshi, N., Nakamachi, Y.,

Yamaguchi, N. and. Miyamato, Y. A new

enzymatic method for the determination of

sialic acid and its application as a marker of

acute phase reactants. Kobe J Med Sci.,

1981; 27: 91-102.

Thornhill WB, Wu WB, Jiang X, Wu X,

Morgan PT and Margiotta JF.

Expression of Kv1.1 delayed rectifier

potassium channels in Lec mutant

Chinese hamster ovary cell lines

reveals a role for sialidation in channel

function. J Biol Chem., 1996; 271:

19093-19098. Varki A, Cummings R, Esko J, Freeze H, Stanley P,

Bertozzi C, Hart G and Etzler M. Essentials

of Glycobiology, Cold Spring Harbor

Laboratory Press, New York, 2008.

(Received: January 23, 2014; Revised: April 14, 2014)

PREVALENCE OF ANTI-HCV & ALLOIMMUNIZATION IN THALASSEMIA MAJOR PATIENTS

44


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


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).


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


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


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).

REFERENCES

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

multitransfused Egyptian patients with β-

thalassemia. Arch Pathol Lab Med., 2014;

138(5): 684-8.

Rehman M, Akhtar GN and Lodhi Y.

Seroprevalence of hepatitis C antibody in

blood donors. Pakistan J Med Sci., 2002; 18:

193-196.

Rosse WF, Gallagher D and Kinney TR.

Transfusion and alloimmunization in sickle

cell disease. Blood, 1990; 76(7): 1431-1437.

Schonewille H, Haak HL and Van zij AM.

Alloimmunization after blood transfusion in

patients with hematologic and oncologic

diseases. Transfusion, 1999; 39: 763-71.

Schonewille H, Van de Watering LM and Brand A.

Additional red blood cell alloantibodies after

blood transfusion in a non-hematological

allimmunized patient cohort: is it time to take

precautionary measures? Transfusion, 2006;

46: 630-5.

Spanos T and Karageorga M. Red cell

alloantibodies in patients with thalassemia.

Nathan and Oski's Hematology of Infancy

and Childhood, Philadelphia; Saunder, 2003;

6: 878-96.

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


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,

_________________________


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



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.


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


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


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


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.

REFERENCES

Aebi H. Catalase in Bergmeyer HU (eds.). Methods

in Enzymatic Analysis: Academic Press. New

York, 1984; pp. 276-286..

Alexander S, Dodds L and Armson BA. Perinatal

outcomes in women with asthma during

pregnancy. Obstet Gynecol., 1998; 92: 435-

40.

Barnes P. A new approach to the treatment of

asthma. N Engl Med., 1984; 321: 1517-27.

Benzie IFF and Strain JJ. The ferric reducing ability

of plasma (frap) as a measure of antioxidant

power: the frap assay. Anal Biochem., 1996;

239: 70-76.

Cao G and Prior RL. Comparison of different

analytical methods for assessing total

antioxidant capacity of human serum Clin

Chem., 1998; 44: 1309-1315.

Comhair SA, Ricci KS, Arroliga M, Lara AR,

Dweik RA, Song W, Hazen SL, Bleecker ER,

Busse WW and Chung KF. Correlation of

systemic superoxide dismutase deficiency to

airflow obstruction in asthma. Am Respir Crit

Care Med c, 2005; 172: 306-313.

Douwes J, Gibson P, Pekkanen J and Pearce N. Non

eosinophilic asthma: importance and possible

mechanisms. Thorax, 2002; 57(7): 643-648.

Drazen JM, Silverman EK and Lee, TH.

Heterogeneity of therapeutic responses in

asthma. Br Med Bull., 2000; 56(4): 1054-70.

Goldstein DS, Eisenhofer G and Mccarty R.

Catecholamines: Bridging basic science with

clinical medicine, 1998; pp. 1-1084.

Gordon M, Niswander KR, Berendes H and Kantor

AG. Fetal morbidity following potentially

anoxiogenic conditions. VII. Bronchial

asthma. Am J Obstet Gynecol., 1970; 106:

421-429.

Hernandez E, Angeli, CS and Johnson JCW.

Asthma in pregnancy-current concepts.

Obstet Gynecol., 1980; 55: 739-744.

Hoffman BB, Hardman JG, Limbird LE.

Catecholamines, sympathomimetic drugs, and

adrenergic receptor antagonists. In: The

pharmacological basis of therapeutics, 12th

edn., 2001; pp. 215-268.

Kakkar P, Das B and Viswanathan PN. A modified

spectrophotometer assay of superoxide

dismutase. Indian J Biochem Bio., 1984; 21:

130-132.

Kattan M, Keens TG, and Mellis, CM. The response

to exercise in normal and asthmatic children.

J Pediatr., 1978; 92: 718-21

Martinez FD, Wright, AL and Taussig LM. Asthma

and wheezing in the first six years of life. N

Engl J Med., 1995; 332(3): 133-138.

Masoli M, Fabian D, Holt S and Beasley R. Global

Initiative for Asthma. Global Burden of

Asthma. Wellington: Medical Research

Institute of New Zealand, 2004.

Mcfadden ER. Improper patient techniques with

metered dose inhalers: clinical consequences

and solutions to misuse. J Allergy Clin

Immunol., 1995; 96: 278-83

Moron MS, Depierre J, Mannervik B. Levels of

glutathione, glutathione reductase and

glutathione-S-transferase activities in rat

lung and liver. Biochem Biophys Acta, 1979;

582: 67-78.

Ohkawa H, Ohishi N and Yagi K. Assay for lipid

peroxides in animal tissues by thiobarbituric

acid reaction. J Anal Biochem., 1979; 95:

351-358.

Okudaira H. Why atopic diseases prevail in

developed countries. ACI Int 1998; 10: 110-5.

Popa V. Respiratory pharmacology: β-adrenergic

drugs. Clin Chest., 1986; 7:313-29.

Rabe KF, Adachi M and Lai C. Worldwide severity

and control of asthma in children and adults:

The global Asthma Insights and Reality

surveys. J Allergy Clin Immunol., 2004;

114(1): 40-47.

Rau JL. Sympathomimetic bronchodilators. In:

Respiratory care pharmacology. (ed. JL Rau


53

Jr.) 4th edn. St. Louis: Mosby, 1994; pp.

129-60.

Strachan D, Sibbad B and Weiland S. Worldwide

variations in prevalence of symptoms of

allergic rhino conjunctivitis in children. The

International Study of Asthma and Allergies

in Children (ISAAC). Pediatr Allergy

Immunol., 1997; 8: 161-176.

Svedmyr N, Witek TJ and Schacter EN. Adrenergic

agents. Pharmacology and therapeutics in

respiratory care. Philadelphia 1994; pp. 135-

162.

Szefler S, Martin R and King T. Significant

variability in response to inhaled

corticosteroids for persistent asthma. J

Allergy Clin Immunol., 2002; 109: 410-418.

Turner ES, Greenberger PA and Patterson R.

Management of the pregnant asthmatic

patient. Ann Intern Med., 1980; 93: 905-918.

(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

________________________________


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



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


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


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


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.

REFERENCES

Biron C, Knipe D, Howley P, Griffin D, Lamb R,

Martin M, Straus S and Sen GC. Interferons

and other cytokines. Fields virology.

Lippincott Williams & Wilkins, Philadelphia,

2001; pp. 321-351.

Carella C, Mazziotti G, Morisco F, Manganella G,

Rotondi M, Tuccillo C, Sorvillo F, Caporaso

N and Amato G. Long-term outcome of

interferon-induced thyroid autoimmunity and

prognostic influence of thyroid autoantibody

pattern at the end of treatment. J Clin

Endocrinol Metab., 2001; 86: 1925–1929.

Carella C, Mazziotti G, Morisco F, Rotondi M,

Cioffi M, Tuccillo C, Sorvillo F, Caporaso N

and Amato G. The addition of ribavirin to


59

interferon-alpha therapy in patients with

hepatitis C virus-related chronic hepatitis

does not modify the thyroid autoantibody

pattern but increases the risk of developing

hypothyroidism. Eur J Endocrinol., 2002;

146(6): 743-9.

Chi HC, Chen CY, Tsai MM, Tsai CY and Lin KH.

Molecular Functions of Thyroid Hormones

and Their Clinical Significance in Liver-

Related Diseases. BioMed Res Internat.,

2013; pp. 1-16. doi:10.1155/2013/601361

Czarnywojtek A, Zgorzalewicz SM, Wasko R,

Czepczynski R, Szczepanek PE, Waligorska-

Stachura J, Kurdybacha P, Bereszynska I,

Florek E, Stangierski A, Zdanowska J,

Nikisch E, Sowinski J and Ruchala M.

Patients with chronic hepatitis type C and

interferon-alpha-induced hyperthyroidism in

two-year clinical follow-up. Neuro

Endocrinol Lett., 2013; 4(2): 154-161.

Davies TF, Ando T, Lin RY, Tomer Y and Latif R.

Thyrotropin receptor associated diseases:

from adenomata to Graves disease. J Clin

Invest., 2005; 115: 1972-1983.

Devendra D and Eisenbarth GS. Immunological

endocrine disorders. J Allergy Clin Immunol.,

2003; 111: 624-636.

Dimitry AC. Immunogenetics of Hashimoto’s

thyroditis. J Autoimmune., 2005; 2: 1-21

George SL, Bacon BR, Brunt EM, Mihindu-

kulasuriya KL, Hoffmann J and Bisceglie

AM. Clinical, virologic, histologic, and

biochemical outcomes after successful HCV

therapy: a 5-year follow-up of 150 patients.

Hepatol., 2009; 49: 729-738.

Gianoukakis AG, Khadavi N and Smith TJ.

Cytokines, Graves’ Disease, and Thyroid-

Associated Ophthalmopathy. Mary Ann

Liebert., 2008; 18: 953-958.

Heller B and Rehermann T. Acute Hepatitis C: a

multifaceted disease. Semin Liv Dis., 2005;

25: 7-17.

Jonasch E and Haluska FG. Interferon in

oncological practice: review of interferon

biology, clinical applications, and toxicities.

Oncologist, 2001; 6(1): 34-55.

Karanikas G, Schuetz M, Wahl K, Paul M, Kontur

S, Pietschmann P, Kletter K, Dudczak R and

Willheim M. Relation of anti-TPO

autoantibody titre and T-lymphocyte cytokine

production patterns in Hashimoto's

thyroiditis. Clin Endocrinol., 2005; 63(2):

191-196.

Land KJ, Moll JS, Kaplan MH, Seetharamaiah GS.

Signal transducer and activator of

transcription (Stat)-6-dependent, but not

Stat4-dependent, immunity is required for the

development of autoimmunity in Graves'

hyperthyroidism. Endocrinol., 2004; 145(8):

3724-30.

Masood N, Ghori R, Memon A, Memon S, Memon

KI, Memon I, Jaffri M and Baloch GH.

Frequency of thyroid disorders during

interferon and ribavirin therapy in chronic

hepatitis C infection. J Coll Physic Surg

Pakistan, 2008; 18(6): 347-51.

Massard J, Ratziu V, Thabut D, Moussalli J, Lebray

P, Benhamou Y and Poynard T. Natural

history and predictors of disease severity in

chronic hepatitis C. J Hepatol., 2006; 44: 19-

24.

Mazziotti G, Premawardhana LDKE, Parkes AB,

Adams H, Smyth PPA, Smith DF, Kaluarachi

WN, Wijeyaratne CN, Jayasinghe A, de Silva

DGH and Lazarus JH. Evolution of thyroid

autoimmunity during iodine prophylaxis-the

Sri Lankan experience. Eur J Endocrinol.,

2003; 149: 103-110.

Mazziotti G, Sorvillo F, Carbone A, Cioffi M,

Morisco F and Carella C. Is the IFN-alpha-

related thyroid autoimmunity an

immunologically heterogeneous disease? J

Intern Med., 2002; 252(4): 377-378.

Parmar S and Platanias LC. Interferons:

mechanisms of action and clinical

applications. Curr Opin Oncol 2003; 15(6):

431-439.

Poynard T, Yuen MF, Ratziu V and Lai CL. Viral

Hepatitis C. Lancet, 2003; 362: 2095-2100.

Prummel MF and Laurberg P. Interferon-alpha and

autoimmune thyroid disease. Thyroid, 2003;

13(6): 547-51.

Samuel CE. Antiviral actions of interferons. Clin

Microbiol Rev., 2001; 14: 778-809.

Santantonio T, Sinisi E, Guastadisegni A, Casalino

C, Mazzola M, Gentile A, Leandro G and


60

Pastore G. Natural course of acute hepatitis

C: a long-term prospective study. Dig Liver

Dis., 2003; 35: 104-113.

Sinclair D. Clinical and laboratory aspects of

thyroid autoantibodies. Ann Clin Biochem,

2006; 43: 173-183.

Tomer Y, Blackard JT and Akeno N. Interferon

alpha treatment and thyroid dysfunction.

Endocrinol Metab Clin North Am., 2007;

36(4): 1051-66.

Troisi A, Novati P, Sali L, Colzani M, Monti G,

Cardillo C and Tesauro M. Graves’

thyrotoxicosis following Hashimoto’s

thyroiditis. Res Rep Endocr Dis., 2013; 3: 13-

15.

Vanderpump MP and Tunbridge WM.

Epidemiology and prevention of clinical and

subclinical hypothyroidism. Thyroid, 2002;

12: 839-847.

Wanzhu H, Guangpi X, Hanjie W and Jeffrey G.

Treating autoimmune disease with Chinese

medicine. Edinburgh; New York: Churchill

Livingstone/Elsevier, 2011; pp. 137-156.

Watanabe M, Yamamoto N, Maruoka H, Tamai H,

Matsuzuka F, Miyauchi A and Iwatani Y.

Independent involvement of CD8+ CD25+

cells and thyroid autoantibodies in disease

severity of Hashimoto's disease. Thyroid,

2002; 12(9): 801-808.

Wiese M, Grüngreiff K, Güthoff W, Lafrenz

M, Oesen U and Porst H. Outcome in a

hepatitis C (genotype 1b) single source

outbreak in Germany-a 25-year multicenter

study. J Hepatol., 2005; 43: 590-598.

William R. Global challenges in liver disease.

Hepatol, 2006; 44: 521-526.

Wong V, Fu AX, George J and Cheung NW.

Thyrotoxicosis induced by alpha-interferon

therapy in chronic viral hepatitis. Clin

Endocrinol., 2002; 56: 793-798.

Yoshida H, Tateishi R, Arakawa Y, Sata M,

Fujiyama S, Nishiguchi S, Ishibashi H,

Yamada G, Yokosuka O, Shiratori Y and

Omata M. Benefit of interferon therapy in

hepatocellular carcinoma prevention for

individual patients with chronic hepatitis C.

Gut, 2004; 53: 425-430.

(Received: April 17, 2014; Revised: June 12, 2014)

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