TRUTH CURES CLING TO TRUTH

BIOMEDICINE
An International Journal for Biomedical Sciences

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[Vloume 36, Number 1 (January - March) 2016; website: www.biomedicineonline.org]

 BIOMEDICINE
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www.biomedicineonline.org Biomedicine – Vol. 36 No. 1: 2016

 BIOMEDICINE
Vol. 36 No. 1: (January - March) 2016
Contents
Topics and Authors Page(s)

I Editorial message  001

II Editorial  002
III Former President of IABMS  004
IV Review/Special Articles
1. Chemical and Electronic Sensors used as a Diagnosing Tool for Diseases –A Review  005
Bijithra C, Rajan G and Shanmugasundaram P

V Research Papers
2. In-vitro antioxidant and anti-inflammatory activity of ethanolic extracts of Momordica 011
Charantia 
Shobha CR, Prashant V, Parveen D, Akila P, Suma MN, and Basavanagowdappa H
3. Inflammatory and oxidative stressmarkers in Acute myocardial infarction  021
Ashok Prabhu, Sudha K, Kiran Kumar AM, and Rajib Kumar Pandey
4. A study of CVD risk assessment in post-menopausal women in comparison with pre- 026
menopausal women with serum lipid profile and hs CRP as risk markers. 
Yasmeen Fatima, Sreekantha, Ramesh, Sadananjali, Saba, and Pallavi
5. Assessment of serum leptin levels and lipid profile in hypertensive obese cases  033
Keshavamurthy HR, and Sunitha S
6. Urea reduction rate as dialysis adequacy indicator and serum albumin as mortality 039
indicator in hemodialysis patients. 
Shanthala D, Indumati.V, Krishnaswamy D, Vijay V, Rajeshwari V, Ramesh, and Shilpa A
7. Study of Oxidative stress and Apolipoprotein A-I in reduction of Reverse Cholesterol 044
Transport in type 2 Diabetes Mellitus 
Suman D, Shashikant N, Padmaja N, and Vishwanath P
8. Evaluation of serum high molecular weight adiponectin and lipid profiles in predicting 049
the risk of coronary artery disease among cad patients 
Chitra devi M, Chandra sekhar M, Sivasubramaniam P
9. Relationship of BMI and Dental Caries among children in Chennai  057
Visha MG, and Deepa Gurunathan
10. A study on Prevalence of Hypertension in cardiovascular risk factors among adults in 062
Chittoor district population 
Bhavani Yamasani, Khadervali Nagoor, and Raziya Dudekula
11. Lipid peroxidation and antioxidant status in vitiligo patients  072
Pallavi Mishra, Rajkumari Rathore, and Prashant Hisalkar
 12. Effects of threshold inspiratory muscle trainer in bronchial asthma  077
Shiny S James, Rekha K, Anandh V, Chandrasekar L, and Unnikrishnan R
13. Assessment of serum uric acid in type 2 diabetes mellitus in a middle-aged south Indian 083
population 
Jyoti John, RajLaxmi Sarangi, Asha Dinakaran, Umadevi SV, Somanath Padhi, and Nitin
Ashok John
14. A comparative study in search of the best treatment for non- metastatic locally-advanced 090
squamous cell carcinoma of head and neck 
Sumana M, and Siddhartha D
15. Comparison in between subjects of determinants of oxygen uptake during maximal 098
and submaximal exercise –ventilation and O2 pulse, determinants of O2 uptake are less
dependent on load in submaximal exercise at ventilator threshold 
Thiagarajan KA, Vasanthi C, Parikh T, Madhusudhan Rao V, Arumugam S
16. Is autonomic function test helps to assess the severity of metabolic syndrome: A study on 103
comparison of Frequency-Domain recordings of Heart rate variability (HRV) with the
severity of metabolic syndrome 
Bhagyashree. N, Ramaswamy C, Ganesh. M, and Udaya Ganesh. B
17. Antioxidant activity of Phytoformulation 1, a polyherbal formulation, on 109
hyperlipidemicWistar rats 
Vanaja R, and Mercy Jasmine J
18. Comparative effects of different teaching methods in pharmacology in second MBBS 117
medical students 
Baswaraj Munge, Kodandaramu Burli, Sindhura Nagisetty, Mamata Bandhopadhyay, and
Prasad Naidu
19. Effect of electromagnetic radiation exposure on hematological parameters of swiss 121
albino mice and their modulation by high protein diet 
Debajyoti Bhattacharya, Niladri Ghosh, and Mausumi Sikdar (nee) Bhakta
20. Ethanol enhances lamivudine-induced liver toxicity: Investigation on hepatoprotective 128
properties of silibinin-phosphatidylcholine complex in rats 
Balasubramanian Jesudas, Ramanathan Raghu, Ganapathy Bhavani, Devaraj Ezhilarasan,
and Sivanesan Karthikeyan
21. Is Internet use Related to Academic Performance in Medical Students? A study from 138
South Indian Medical College 
Ravi Kishore Polepalli

VI Case Report
22. Raising the index of suspicion for cerebral venous thrombosis: A case report.  141
Renata Mazurek, Naveen Ramesh, Kiran PV, and Avita Johnson
23. Oral Squamous papilloma of hard palate  145
Jamin Joseph, and Karpagavalli S
001 Biomedicine: 2016; 36(1): 001-001

EDITORIAL MESSAGE

Dear Reader,

As the New Editor-in-Chief of the journal

Biomedicine, I am writing to invite you to send
your most important research papers to the journal.
The Journal will not only accommodate traditional,
theoretical and experimental biomedical research,
but also welcomes research that builds molecular
discoveries and medical research that connect to
clinical states within the journal’s domain.
The qualitative and timely publication of Volume
36 of our esteemed international journals has brought
great joy and happiness to the entire fraternity of the
journal and honorable members of the editorial and
advisory board. The rich experience and varied expertise of the board members is providing immense succor in
propelling the journal to meet a comfortable place in areas of medical research and accentuate its visibility. The
aim of the journal is to percolate knowledge in biomedical research with erudition by providing our ecosystem for
budding researchers in India.
Large number of research papers have been handed over to me in February this year. They were reviewed and
the accepted papers are published in this issue. I extend my heartfelt thanks to the reviewers and members of the
editorial board who so carefully perused the papers and carried out justified evaluations in very short time. Based on
their evaluation, we could accept 23 research papers. I am sure that these papers will offer qualitative information
and thoughtful ideas to our accomplished readers. I also thank all those who profusely conveyed their appreciation
and best wishes for future issues. I convey my deep gratitude to the office bearers and members of IABMS who
gave me this opportunity to serve as Editor in Chief. The publication of this journal in the planned time frame was
possible due to untiring help and support by our previous Editor-in-Chief.
I humbly invite all the authors and their professional colleagues to send their research papers for consideration
for publication in our forthcoming issue as per the “Scope and Guidelines to Authors” given in this issue. Immense
effort was put by our team to format the research papers right from abstract to reference. Additionally plagiarism
and grammar check was performed. I am sure that with organized team efforts, the journal will soon prove its name
among best quality national and international journals in biological sciences. I solicit your continuous support,
help, guidance and blessing to take Biomedicine to new heights, particularly, in-terms of visibility in Google
Scholar in increasing its impact factor.

Dr. E. Padmini
Editor-in-Chief
Biomedicine
An International Journal for Biomedical Scientists
A quarterly journal of IABMS, Chennai, India.

The highest honor bestowed upon me was to interact with Bharat Ratna Dr.A.P.J.Abdul Kalam during Golden Jubilee c­ elebrations at
Bharathi Women’s College in April 2015. I pray and seek his blessings for successful completion of my turn as EIC.

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002 Biomedicine: 2016; 36(1): 002-003

EDITORIAL

Xenohormesis and Calorie Restriction

It has long been valued that diets rich in fruits and vegetables promote health, reduce the risk of life killing disease and
correlate with increased longevity. This has typically been attributed to the antioxidant properties of plant-derived foods.
At the molecular level, many phytochemicals exert their effects in various ways. One is by directly interacting with and
modulating specific enzymes or receptors. Polyphenols such as resveratrol (found in grapes) and quercetin (in tea for
example), which are produced by stressed plants, activate sirtuin enzymes and extend the lifespan of fungi and animals,
apparently by mimicking the beneficial effects of caloric restriction. Caloric restriction (CR) is currently the only way to
slow down aging in mammals. It has been well documented that calorie restrictions (CR) delay most diseases of aging
and stress tolerance including cancer, metabolism, atherosclerosis, Type 2 diabetes and even neurodegeneration.
As a result, an intense interest was raised to explore how CR works at the molecular level to find small molecules
that can mimic its effects. The fact that these foreign molecules that are non-nutritive and seemingly unrelated to any
endogenous molecule alter the same biochemical pathways that mediate the response to an energy deficit is an interest-
ing aspect. A possible explanation provided is that the Sirtuin enzymes have evolved to respond to plant stress molecules
as indicators of an approaching deterioration of the environment. The Sirtuin family appears to be first arising in module
eukaryotes, possibly to help them cope with adverse conditions. Sirtuin genes are unique in their activity, and their level
is positively correlated with lifespan. Sirtuins are a class of enzymes that could have beneficial effects on a variety of
human diseases. Sirtuins are found in plants, yeast and animals and may underlie the remarkable health benefits of CR.
The hormesis hypothesis of CR, therefore, is based on the concept that low calorie intake is itself a mild stress which
evokes a general stress response that promote a better health and longer life.  It may entirely or partially account for the
beneficial effects of phytochemicals. Plant molecules induced by the stress such as resveratrol, butein, and fisetin can
evoke defense responses in fungi, flies, fish, and mice, leading to an increased life span. Such molecules are known as
“caloric restriction mimetics”. However, it remains to be clearly understood.
Xenohormesis, defined as an adaptive response in regulating the physiology of an organism to molecular cues that acts
as neither nutritive nor direct stressors, most likely occurs at some level. The term hormesis refers to a process by which
a mild stress provides health benefits by causing the organisms to mount a defense response.  The Xenohormesis Hypoth-
esis can be explained using Resveratrol as an example. Resveratrol is a small polyphenol found in grapes and wine. It is
identified in a screen for compounds that mimic the effects of Caloric Restriction (CR) by activating the sirtuin enzyme
SIRT1. Sirtuins, a family of NAD+-dependent deacetylases, conserved from yeast to humans have been proposed to medi-
ate lifespan extension, that effects Caloric Restriction in lower organisms.
Consistently, resveratrol was found to extend the lifespan of wild type yeast, but not that of mutant strains lacking the
yeast sirtuin, Sir2. It depicts that the beneficial effect of resveratrol on yeast cells is attributed to a very specific manipulation
of a conserved signaling pathway and not due to a general property such as antioxidant capacity or nutritive value. From
this, it was observed that the absence of an endogenous small-molecule activator of Sir2/SIRT1, prompted the suggestion
of a differential way by which plants might influence organisms. It assumes that the evolutionarily conserved survival path-
ways might respond to chemical cues in the environment or the food supply. For growing a yeast cell on a grape, an increase
in the concentration of resveratrol was observed suggesting that it is induced in the plant after injury or infection and serves
as a useful marker that the food supply is about to become limiting. Therefore, the yeast cell might gain a selective advan-
tage by responding to resveratrol and activation of sirtuin enzymes in the same way it would to an actual deficit of calories.
The complex relationship between animals and the plants they consume is yet to be understood clearly. Throughout evolu-
tion, the flux of various phytochemicals in food sources would have provided a wealth of information to an animal capable
of deciphering the signals. The existence of such signals and selective pressure to adapt to environmental changes, like
xenohormesis occurs at some level. To what extent this phenomenon might contribute to the beneficial effects of specific
phytochemicals in fruits and vegetables, remains to be seen. It has been suggested that a seemingly coordinated beneficial
response to a phytochemical such as resveratrol could reflect mimicry of an endogenous signaling molecule. Plants and

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003 Biomedicine: 2016; 36(1): 002-003

animals retain a surprising degree of similarity in certain signaling pathways.

Many neurotransmitters, like acetylcholine, histamine, serotonin, glutamate
and GABA, are made by plants. Similar signaling molecules might exist in
every species. Genistein has been found to interact with the estrogen receptor
in some cases and several other polyphenols have been established as bonafide
estrogen mimics. No endogenous molecules similar to plant polyphenols have
been identified in mammals due to the absence of the biosynthetic pathways
for polyphenols. It is hypothesized that stressed plants may contain a host of
potentially therapeutic molecules that have yet to be recognized. The timing of
therapeutic effects may also play an important role. Direct stresses may induce
protective hermetic responses early in life, but have diminished effects later in
life. Similarly, there may be an optimal therapeutic window in which a protec-
tive Xenohormetic response can be established. Elucidating the mechanism by
which plant foods benefit human health has enormous potential to improve the
quality of life and prevent or treat major diseases. There are also non-poly-
phenolic endogenous modulators waiting to be discovered. Importantly, if they
are discovered, their existence would not rule out Xenohormesis. Any Progress
towards the testing of the CR and Xenohormesis hypothesis will be an impor-
tant challenge in the coming years.

Reference
1. Bazzano, L.A., Serdula, M.K., and Liu, S. Dietary intake of fruits
and vegetables and risk of cardiovascular disease. Curr. Atheroscler.
Rep. 2003; 5: 492–499. 

2. Block, G., Patterson, B., and Subar, A. Fruit, vegetables and cancer prevention: A review of the epidemiological
evidence. Nutr. Cancer. 1992; 18: 1–29. 

3. De Santi, C., Pietrabissa, A., Spisni, R., Mosca, F., Pacifici, GM. Sulphation of resveratrol, a natural product
present in grapes and wine, in the human liver and duodenum. Xenobiotica. 2000; 30: 609–617. 

4. Halliwell, B. Dietary polyphenols: Good, bad, or indifferent for your health? Cardiovasc. Res. 2007; 73: 341–
347. 

5. HemaIswarya, S., and Doble, M. Potential synergism of natural products in the treatment of cancer. Phytother
Res. 2006; 20: 239–249.

6. Lotito, SB., and Frei, B. Consumption of flavonoid-rich foods and increased plasma antioxidant capacity in
humans: Cause, consequence, or epiphenomenon? Free. Radic. Biol. Med. 2006; 41: 1727–1746. 

7. Padmini, E., Chithra, B., and Christina Joseph Mary, S. genistein attenuates oxidative damage in preeclamptic
placental trophoblast. Hyp. Preg. 2016; 35(2): 250-263.

8. Suzuki, M., Wilcox, BJ., and Wilcox, CD. Implications from and for food cultures for cardiovascular disease:
Longevity. Asia. Pac. J. Clin. Nutr. 2001; 10: 165–171. 

9. Treutter, D. Significance of flavonoids in plant resistance and enhancement of their biosynthesis.  Plant Biol
(Stuttg). 2005; 7: 581–591.

10. Westphal, C.H., Dipp, M.A., and Guarente, L. The sirtuins are a group of proteins linked to aging, metabolism
and stress tolerance in several organisms.Trends. Biochem. Sci. 2007; 32: 555–560.

Dr. E. Padmini
Editor-in-Chief, Biomedicine

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004 Biomedicine: 2016; 36(1): 004-004

FORMER PRESIDENT OF IABMS

Prof. G. Victor Rajamanickam the immediate past president of the Indian Association
of Biomedical Scientist (2012-2015) hails from a small village called Usilampatti in
Madurai. He graduated from the Alagappa college and continued his postgraduation
at Annamalai University in Geology. As the eldest son in the family he joined service
as SRF in the CSIR laboratory-National Institute of Oceonography

Prof. Victor as he is known joined the Department of Earth Science at Tamil

University, Thanjavur in 1985. He developed the department with several projects
and research grants from various funding agencies both nationally and internationally. He was the key player
in starting the Ocean Science Technology Cell (OSTC) funded by Department of Ocean Development. He
was recognized as Distinguished Professor of OSTC. Based on his initiatives, Itwas considered to be the best
Earth Science laboratory in Tamil Nadu and was recognized as Centre for Excellence by the Ministry of Ocean
Development.Off-late the Department of Science and Technology has entrusted the National Coordination of
Coastal Resource Management as a Sub-Programme to this Department. During his tenure at Tamil University he
has served at various capacities as HOD, Dean, Senate member and Syndicate member and was helpful to anyone
who approached him to do research. In this process he guided as many as 38 Ph. Ds and 49 M. Phil candidates
in various fields of science ranging from Geology, Geography, Geophysics, Geochemistry, Geomorphology,
Geoinformatics, Geomedicine, Pharmacology, Biochemistry, Siddha Medicine, Disaster Management, Remote
sensing, Agriculture including History of Science making him a supervisor with multidisciplinary expertise.
Accordingly he has several patents (6) journal publications (254) and books (25) to his credit.

After his retirement at Tamil University his quench to do research and motivate young research minds
made him to serve at SASTRA University, Thanjavur as Dean and Coordinator for the Centre for Advanced
Research in Indian System of Medicine (CARISM). He developed a good GLP and NABL Accredited
laboratory with state of the art technology for drug testing including herbomineral drugs. Later he continued
as Director (Research) at Sai Ram group of Institutions and presentlyas Senior Scientist at VelsUniversity. His
enormous teaching and research experience and expertise made him an able administrator too.

He has several awards and distinctions to his credit which includes the DAAD Fellowship (1977),
Visiting Professorship to UK and Germany (1982,1985), Tamilnadu Life Time Achievement award in
Environmental Science (2011), V.V Swaminathan award (2009),BhramiahSastriMemorial Award (2012) and
M.K. Nambiar Oration Award(2015) by the Indian Association of Biomedical Scientists.Even now at the age of
73 he servesthe Central Council for Research in Siddha,Ministry of AYUSH as Expert member for Research.
He is also an expert member of the Research Board in Kerala. He continues to give invited lectures and
radio talks on various aspects of science and technology encouraging multidisciplinary and interdisciplinary
research to unravel the mysteries of creation in a scientific manner. Till date he continues to motivate and guide
all his students and scholars in all walks of life to achieve great things for this country.

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Biomedicine: 2016; 36(1): 005-010 005

Review Article
Chemical and electronic sensors used as a diagnosing tool for diseases—A review
C. Bijithra1, G. Rajan1, and P. Shanmugasundaram1
1
School of Pharmaceutical Sciences, Vels University (VISTAS), Chennai, India.
(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author
P. Shanmugasundaram. Email: samsimahe@gmail.com

Abstract

This article discusses the development of various sensor tools based on chemicals and electronics, which are
finding a place in recent diagnosis of diseases like Tuberculosis, Vibrio cholera, Dengue fever, yellow fever
and Ebola, Cancer, HIV, Liver diseases, Cardio vascular diseases, Skin diseases, Thyroid disease, Depression,
Stomach diseases.

Key words: Brain Sensor, Electronic-Nose, E-Nose, Miniature Sensors, Mini Sensors, Mobile Phone-Sensors,
Sensor Diagnosis, Sensors

Introduction A diagnostic procedure may be performed by vari-

I
dentification of a condition, disease, disorder,
or problem by systematic analysis of the
background or history, examination of the signs or
symptoms, evaluation of the research or test results,
and investigation of the assumed or probable causes.
Effective prognosis is not possible without effective
diagnosis (Business dictionary). A diagnosis, in the
sense of diagnostic procedure, can be regarded as an
attempt at classification of an individual’s condition
into separate and distinct categories that allow medical
decisions about treatment and prognosis to be made.
Subsequently, a diagnostic opinion is often described
in terms of a disease or other condition, but in the case
of a wrong diagnosis, the individual’s actual disease or
condition is not the same as the individual’s diagnosis.
Diagnosis is also the identification of the nature
and cause of a certain phenomenon. Diagnosis is
used in many different disciplines with variations
in the use of logics, analytics, and experience to
determine “cause and effect.” In systems engineer-
ing and computer science, it is typically used to
determine the causes of symptoms, mitigations,
and solutions.
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ous health care professionals such as a physician,
physical therapist, optometrist, healthcare scien-
tist, chiropractor, dentist, podiatrist, nurse practi-
tioner, or physician assistant.
It might be a management-naming or prognosis-
naming exercise. It may indicate either degree
of abnormality on a continuum or kind of abnor-
mality in a classification. It’s influenced by non-
medical factors such as power, ethics and financial
incentives for patient or doctor. Traditional and
conventional diagnostic tools and Chemicals are
very time consuming and costly. With the advent
of various sensors applied to individual diseases
we find a paradigm shift from the traditional sys-
tem of diagnosis to that of the modern system of
diagnostic tools.
A sensor is a device that detects events that occur in
the physical environment (like light, heat, motion,
moisture, pressure, and more), and responds with
an output, usually an electrical, mechanical or opti-
cal signal. A sensor is a transducer whose purpose
is to sense (that is, to detect) some characteristic of
its environments.
Biomedicine – Vol. 36 No. 1: 2016
006
The household mercury thermometer is a simple
example of a sensor—it detects temperature and
reacts with a measurable expansion of liquid.
Sensors are everywhere—they can be found in
everyday applications like touch-sensitive eleva-
tor buttons and lamp dimmer surfaces that respond
to touch, but there are also many kinds of sensors
that go unnoticed by most—like sensors that are
used in medicine, robotics, aerospace and more.
Sensors are divided into two groups: active and
passive sensors. Active sensors (such as photo-
conductive cells or light detection sensors) require
a power supply while passive ones (radiometers,
film photography) do not.
Application of Sensor Devices in Various Diseases
There is an urgent need in the medical diagnos-
tics laboratories for accurate, fast and inexpensive
devices, which can be routinely used. The reliable
and accurate information on the desired biochemi-
cal parameters is an essential prerequisite for
effective healthcare. In this context, biosensors are
considered to provide viable solutions to the prob-
lems posed by the contemporary healthcare indus-
try. This is because these biosensing devices offer
considerable advantages, such as specificity, small
size faster response and cost. It is anticipated that
these bioanalytical tools can be used for frequent
measurements of metabolites, blood cations and
gases, etc. Malhotra and Chaubey in their research
article they made an attempt to highlight some of
the trends that rule the research and developments
of some of the important biosensors that are likely
to accelerate the growth of clinical diagnostics
industry.
Research is going on in electronic nose (e-nose)
devises application in medical diagnosis. Gardner
et al. in their research have applied an e-nose to the
identification of pathogens from cultures and diag-
nosing illness from breath samples. These initial
results suggest that an e-nose will be able to assist
in the diagnosis of diseases in the near future.
Tuberculosis (TB) remains one of the world’s
deadliest communicable diseases. In 2013, an esti-
mated 9.0 million people developed TB and 1.5
million died from the disease, 360,000 of whom
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Bijithra et al.: Chemical and Electronic Sensors …….. Diseases
were HIV-positive. TB is slowly declining each
year and it is estimated that 37 million lives were
saved between 2000 and 2013 through effective
diagnosis and treatment. However, given that most
deaths from TB are preventable, the death toll from
the disease is still unacceptably high and efforts
to combat it must be accelerated if 2015 global
targets, set within the context of the Millennium
Development Goals (MDGs), are to be met.
A wide spectrum of diagnostic technologies and
tools are used to identify the agents causing infec-
tious diseases. In fact, monitoring Volatile Organic
Compounds (VOCs) in the breath may soon become
an interesting supplement (or even an alternative)
to conventional medical diagnostics, thanks to the
rapid advances in the techniques for breath collec-
tion and gas-analysis
The diagnosis of active TB is critical for control-
ling the disease. Diagnosis methods require highly
skilled personnel and specialized laboratory infra-
structure. There are several diagnostic methods
for detection of active tuberculosis (e.g. sputum
smear microscopy, tuberculosis culture from spu-
tum and Xpert MTB/RIF), Although these methods
are widely used for diagnosing TB, they suffer of
specificity and sensitivity limitations and micro-
biological culture takes several weeks to confirm
a clinical diagnosis and at the proof-of-concept
stage a breath test using nanomaterial-based sen-
sors are used.
Recently, several studies have speculated that the
distinguishing features of each breath print do not
arise solely from pathogen metabolism, but are
also due to changes of host VOCs, possibly in con-
junction with the immunological response.
Sensor to detect cholera quickly
Although the bacterium, V. cholerae, cannot be
sensed directly, remotely sensed data can be used
to infer its presence. In the study reported here,
satellite data were used to monitor the timing and
spread of cholera. Public domain remote sens-
ing data for the Bay of Bengal were compared
directly with cholera case data collected in Ban-
gladesh from 1992–1995. The remote sensing data
Biomedicine – Vol. 36 No. 1: 2016
Bijithra et al.: Chemical and Electronic Sensors …….. Diseases
included sea surface temperature and sea surface
height. It was discovered that sea surface tempera-
ture shows an annual cycle similar to the cholera
case data. Sea surface height may be an indicator
of incursion of plankton-laden water inland, e.g.,
tidal rivers, because it was also found to be corre-
lated with cholera outbreaks. The extensive studies
accomplished during the past 25 years, confirming
the hypothesis that V. cholera is autochthonous to
the aquatic environment and is a commensal of
zooplankton, i.e., copepods, when combined with
the findings of the satellite data analyses, provide
strong evidence that cholera epidemics are cli-
mate-linked.
A nano-structured Magnesium Oxide (nanoMgO)
based electrochemical DNA biosensor is created.
It exhibits a sensitivity of 16.80 nA/ng/cm2, faster
than any available sensor, and has a response time
of 2 seconds. It also has a lower detection limit
of 59.12  ng/µL with good reproducibility. It is a
new sensor that can quickly detect Vibrio cholerae,
the cholera-causing bug, in clinical samples. Con-
ventional diagnosis methods of cholera are based
on culture of bacterium, biochemical reactions
and agglutination tests against serogroup-specific
antisera. The whole process of V. cholera isolation
and identification takes at least 2–3 days and dur-
ing this period, the disease can spread explosively.
PCR tests take 3–4 hours and require expertise in
molecular biology. Besides this, false positivity of
PCR has been reported especially in commercial
assays due to cross-reactions. Therefore, rapid and
sensitive diagnosis has been the need to detect the
infectious disease at an early stage.
Paper test quickly detects Ebola, dengue and
yellow fever
Researchers in the US have developed  a silver
nanoparticle-based paper test to simultaneously
detect dengue, yellow fever and Ebola.
The Ebola epidemic in West Africa underscores an
urgent need for rapid diagnostics; quick identifica-
tion and patient isolation can benefit the sick and
the healthy. However, dengue, yellow fever and
Ebola all initially manifest as a fever and head-
ache, so are easily mixed up.
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007
Now, this huge problem has a tiny solution—an
8 × 3 cm lateral flow test. Lee Gehrke and his team
at the Massachusetts Institute of Technology and
Harvard Medical School adapted the traditional
single marker lateral flow test to diagnose several
diseases at once. It costs $2, takes 10 minutes, and
there is no need for a power supply, trained spe-
cialist or expensive equipment.
The test is made from strips of paper containing
antibodies attached to triangular silver nanopar-
ticles of varying size according to the disease they
recognize and bind to. Silver nanoparticles appear
as different colours according to their size, so
when a patient’s serum sample migrates through
the device, distinctive colored lines appear on the
paper to indicate positive results for Ebola, den-
gue or yellow fever. This pattern of lines can be
analysed by eye but the team are also working on
a mobile phone application to aid diagnosis. “An
app could be very useful for diseases that are mos-
quito-spread,” says Gehrke. “It adds a date and
geographical stamp to the test results so the spread
of disease can be followed in real-time.
Sensors to Diagnose Skin Diseases
Color related skin conditions or alterations can
be identified accurately using optical instruments.
While in the past such skin color measurements
were only possible in laboratories with expensive
spectrometers or special measurement equipment,
nowadays doctors can use handhelds based on
multi-spectral color sensors. The demand for pre-
cise color measurement in both the area of analysis
using instruments for medical diagnostics such as
pointof-care (Po Ct) and in dermatological research
and applications, and especially for diagnostic
documentation, is constantly rising. For example,
in pharmacology—skin conditions or changes as a
result of disease or environmental influences can
be determined and documented via optical measur-
ing instruments. The results of these measurements
can be used both for evaluating skin diseases and
for preparing treatment or assessing treatment
success. Gray scale levels, colors and spectral
data followed by a subsequent analysis, color and
spectral algorithms as well as application-specific
Biomedicine – Vol. 36 No. 1: 2016
008
evaluation are the basis for a broad application of
optical measurement methods in the field of diag-
nosis. Color measuring tasks are performed in dif-
ferent ways. Tristimulus sensors are compact and
optimized for large numbers and fast measurement
tasks. Using an RGB filter, they are ideal for color
detection. True color sensors with XYZ filter are
suitable for absolute color measurement based in
the Cie 1931 standard for human eye perception.
Both sensors work according to the colormetric
principle.
Researchers, including one of Indian-origin, are
developing a new wearable vapour sensor that
could offer continuous disease monitoring for
patients with diabetes, high blood pressure, anemia
or lung disease. The new sensor, being developed
at the University of Michigan, can detect airborne
chemicals either exhaled or released through the
skin.
Smart Phone Sensors to Detect Mental Health
Depression is a common, burdensome, often recur-
ring mental health disorder that frequently goes
undetected and untreated. Mobile phones are ubiq-
uitous and have an increasingly large complement
of sensors that can potentially be useful in moni-
toring behavioral patterns that might be indica-
tive of depressive symptoms. Features extracted
from mobile phone sensor data, including GPS
and phone usage, provided behavioral markers
that were strongly related to depressive symptom
severity. While these findings must be replicated in
a larger study among participants with confirmed
clinical symptoms, they suggest that phone sen-
sors offer numerous clinical opportunities, includ-
ing continuous monitoring of at-risk populations
with little patient burden and interventions that
can provide just-in-time outreach.
Stomach Diseases Identification by Sensors
Patel et al. in his article applied a short descrip-
tion of key enabling technologies (i.e. sensor
technology, communication technology, and data
analysis techniques) that have allowed research-
ers to implement wearable systems is followed
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Bijithra et al.: Chemical and Electronic Sensors …….. Diseases
by a detailed description of major areas of appli-
cation of wearable technology. Applications
described in this review paper include those that
focus on health and wellness, safety, home reha-
bilitation, assessment of treatment efficacy, and
early detection of disorders. The integration of
wearable and ambient sensors is discussed in the
context of achieving home monitoring of older
adults and subjects with chronic conditions.
Future work required to advance the field toward
clinical deployment of wearable sensors and sys-
tems is discussed.
Researchers in Australia are developing a new gas-
sensing capsule that can evaluate stomach health
and wirelessly transmit results to a smartphone.
According to the scientists, the new technology
could improve diagnostics and assess gas as a bio-
marker for overall gut health analysis.
A team from the Royal Melbourne Institute of
Technology (RMIT) and Monash University, led
by RMIT professor Kourosh Kalantar-zadeh,
designed the capsule to be swallowed and passed
safely through the digestive system. The device
features biocompatible cladding, a gas-permeable
membrane, and a gas sensor, microprocessor,
and wireless transmitter powered by a battery.
According to an RMIT  press release, the system
is designed to communicate results directly to a
smartphone. Research shows that intestinal gas-
ses share a link with colon cancer, irritable bowel
syndrome, and inflammatory bowel disease.
The RMIT team, which published its findings in
Trends in Biotechnology, indicated that further
study of the gasses could link their presence to
other health issues.
Sensor Detection by Epilepsy
Due to the recent developments in the technology
era, and it is perceived that the technical develop-
ment has improved in every field. Using this devel-
opment, the world has gained a great deal even in
the medical field. In the aware of that, all disorders
have being cured by many technical equipments
and detected effectively before it occurs. In that
case, the Epilepsy disorder can be recognized and
Biomedicine – Vol. 36 No. 1: 2016
Bijithra et al.: Chemical and Electronic Sensors …….. Diseases
makes an alert to the people using an application
in Android smart phones. This kind of application
is already exists but it will send unwanted alert
message. In this paper, we mainly focused on to
avoid the unwanted alert message using brain sen-
sor. It has an in-built function which can trigger
alert messages whenever the brain function goes
abnormal.
The main reason behind the proposed system is
that the brain function of person wills same at ever.
It could vary only at the time of any disease attack
in the neural system. Hence the false identification
can be easily reduced.
A sensor for detecting various fatal diseases in
advance, including HIV, has been developed.
Researchers at the Moscow Institute of Phys-
ics And Technology are of the opinion that the e
sensor—an optical chip—will enable the doctors
to identify tumour markers, whose presence in the
body signals the emergence and growth of cancer-
ous tumours.
The researchers—Dmitry Fedyanin and Yury Ste-
bunov—are of the opinion that the new sensor will
combine high sensitivity with a comparative ease
of production and miniature dimensions, allowing
it to be used in all portable devices, such as smart-
phones, wearable electronics.
The researchers further explained that unlike simi-
lar devices, their sensor has no complex junctions
and can be produced through a standard CMOS
process technology used in microelectronics. The
sensor doesn’t have a single circuit, and its design
is very simple.”
Early therapy conveys a double benefit—not only
to improve the health of individuals but at the same
time by lowering their viral load, reducing the risk
of HIV transmission.
The highly sensitive nanomechanical sensor can
analyse the chemical composition of substances
and detect viral disease markers which appear
when the immune system responds to incurable or
hard-to-cure diseases.
The ultrasensitive sensor can track changes of
just a few kilodaltons in the mass of a cantilever
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009
in real time. One Dalton is roughly the mass of
a proton or neutron, and several thousand Dal-
tons are the mass of individual proteins and DNA
molecules.
So the new optical sensor will allow for diagnos-
ing diseases long before they can be detected by
any other method, which will pave the way for a
new-generation of diagnostics.
One chip, several millimetres in size, will be able
to accommodate several thousand such sensors,
configured to detect different particles or mol-
ecules, said the paper published in the journal
Scientific Reports.
Mobile Health Care is the integration of mobile
computing and health monitoring. It is the appli-
cation of mobile computing technologies for
improving monitoring. It is the application of
mobile computing technologies for improving
communication among patients, physicians, and
other health care workers. As mobile devices have
become an inseparable part of our life it can inte-
grate health care more seamlessly to our every-
day life. It enables the delivery of accurate medi-
cal information anytime anywhere by means of
mobile devices. Recent technological advances in
sensors, low-power integrated circuits, and wire-
less communications have enabled the design of
low-cost, miniature, lightweight and intelligent
bio-sensor nodes. These nodes, capable of sens-
ing, processing, and communicating one or more
vital signs, can be seamlessly integrated into wire-
less personal or body area networks for mobile
health monitoring.
Conclusion
Although the mobile sensor devices have been
shown to be accurate and have clinical utility,
they continue to be underutilized in the healthcare
industry. Incorporating smart wearable sensors into
routine care of patients could augment physician-
patient relationships, increase the autonomy and
involvement of patients in regards to their health-
care and will provide for novel remote monitor-
ing techniques which will revolutionize healthcare
management.
Biomedicine – Vol. 36 No. 1: 2016
010
References
  1. Malhotra, B.D., and Chaubey, A. Biosensors for
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Biomedicine – Vol. 36 No. 1: 2016
Biomedicine: 2016; 36(1): 011-020 011

Research Paper
In-vitro antioxidant and anti-inflammatory activity of ethanolic extracts of Momordica
charantia: 50% ethanolic extract of MC has good antioxidant activity
Shobha C.R.,1 Prashant Vishwanath,1 Parveen Doddamani,1 Akila Prashant,1 Suma M.N.,1 and
Basavanagowdappa H.2
1
Department of Biochemistry, Center of Excellence in Molecular Biology and Regenerative Medicine, JSS
Medical College, JSS University, Mysore, Karnataka, India.
2
Department of Medicine, JSS Medical College, JSS University, Mysore, Karnataka, India.

(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author
Dr. Prashant Vishwanath. Email: drmvps@gmail.com

Abstract

Introduction and Aim: Deep–colored vegetables and fruits including bitter melon are good sources of phe-
nolic compounds which are known to possess antioxidant, antimutagen, antitumor, anti-inflammatory, and
anticarcinogenic properties. To determine the antioxidant and anti-inflammatory activity in different percent-
age of ethanolic extract of Momordica Charantia (EEMC).

Materials and Methods: The paste of Momordica Charantia (MC) fruit was lyophilized using freeze dryer
and the powder obtained was stored at -80°C in airtight plastic container. EEMC was prepared by graded
ethanol fractionation method. The extracts were concentrated, freeze-dried and reconstituted in ethanol for
the assessment of (a) antioxidant activity was determined by total phenols—using Folin–Ciocalteu (F–C), total
flavanoid by Aluminum Chloride Colorimetric Method, ferric reducing antioxidant power (FRAP), 2,2-diphe-
nyl-1-picrylhydrazyl (DPPH) and reducing power assay;(b) anti-inflammatory activity by red cell membrane
stabilisation assay.

Results: The total phenolic content and total flavanoid content of EEMC decreased with increasing ethanol
concentration from 50% to 100%. The 50% EEMC has more potent antioxidant activity when compared to
70% and 100% EEMC by FRAP, DPPH and reducing power assay. The anti-inflammatory activity is more by
50% when compared to 70% and 100%.

Conclusion: The data showed that the 50% EEMC contain a high percentage of phenolic content, flavanoid
content, antioxidant and anti-inflammatory potential than 70% and 100%. Further studies to identifying the
key antioxidants in this extract are under way.

Key words: Anti-inflammatory Activity, Antioxidant, DPPH, FRAP, Momordica charantia.

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012
Introduction
P
henolics are the group of phytochemicals
that account for most of the antioxidant
activity in plants or plant products. It is
estimated that there are about eight thousand
naturally occurring plant phenolics and about half
this number are flavonoids. Phenolic compounds
are mainly synthesized from cinnamic acid, which
is formed from phenylalanine by the action of
L-phenylalanine ammonia-lyase, the branch point
enzyme between primary (shikimate pathway)
and secondary (phenylpropanoid) metabolism.
Phenolic compounds act as radical scavengers,
reducing agents and chelators of metal ions and
flavonoids act as a free radical scavenger, inhibitor
of hydrolytic and oxidative enzymes and also has
anti-inflammatory action (1). The antioxidant
activities of the phenolic compounds are related to
the structure, generally depending on the number
and position of hydroxyl groups and glycosylation
or other substituents.
The antioxidant test models differ in differ-
ent aspects. Therefore, it is difficult to compare
exactly one method to another one. Antioxidants
can deactivate radicals by two major mechanisms,
hydrogen atom transfer (HAT) and single electron
transfer (SET). The end result of both the mecha-
nism is the same, but kinetics and potential for side
reactions differ. HAT-based methods measure the
classical ability of an antioxidant to quench free
radicals by hydrogen donation and SET-based
methods detect the ability of a potential antioxi-
dant to transfer one electron to reduce any com-
pound, including metals, carbonyls, and radicals
(2). The 2,2-Diphenyl-1-picrylhydrazyl (DPPH)
act by both HAT and SET mechanism whereas Fer-
ric Reducing Antioxidant Power (FRAP) by SET
mechanism. Many researchers have used both the
DPPH and the FRAP assays in their plant activity
screening programs, to obtain a better description
of the antioxidant activity. A single method is not
suitable for all and there is no shortcut approach to
determine antioxidant activity.
It is known that deep–colored vegetables and fruits
including Momordica Charantia (MC) are good
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Shobha et al.: In-vitro antioxidant …………. Momordica Charantia
sources of phenolic compounds which are known as
hydrophilic antioxidants, those posses antioxidant,
antimutagen, antitumor, anti-inflammatory, and
anticarcinogenic properties (3,4). Extraction, as the
term is used pharmaceutically, involves the separa-
tion of medicinally active portions of plant or ani-
mal tissues from the inactive or inert components by
using selective solvents in standard extraction pro-
cedures. Different solvents like water, acetone, meth-
anol, ethanol, n-butanol, etc. are used for extraction
of phytochemical constituents from plants (5,6). The
extraction of the phenolic compounds is influenced
by their chemical nature, the extraction method, the
sample size, time, storage conditions and the pres-
ence of interfering substances. The phenolic extracts
of plants are selectively soluble in the different sol-
vent. The use of an alcoholic solution provides sat-
isfactory results for the extraction process (7). With
these references, we proposed to evaluate phytocon-
stituent activities and several biological activities of
MC using alcohol.
Materials and Methods
The study was conducted in Center of Excellence
for Molecular Biology and Regenerative Medicine
(CEMR) Laboratory, Department of Biochemistry,
JSS Medical College, Mysore, Karnataka, India.
Preparation of ethanolic extract of Momordica
charantia (EEMC)
EEMC was prepared by graded ethanol fractionation
method as described earlier (8). Different percentage
(50%, 70%, and 100%) of EEMC was obtained using
50  g of lyophilized powder. The obtained different
percentage solutions of EEMC were then concen-
trated using Rotovapor R-215 (Buchi, Switzerland)
which was later filtered using Whatman’s filter paper
number-1. The concentrated extracts were subjected
for lyophilisation using freeze dryer (Alpha 2-4 LD
Plus from Christ, GmbH) which was done by dehy-
drating all the 50%, 70% and 100% concentrated
extracts completely at reduced pressure after being
frozen at -80°C. Once completely dehydrated, the
concentrated lyophilized powder extracts were pre-
served at -80°C.
Biomedicine – Vol. 36 No. 1: 2016
Shobha et al.: In-vitro antioxidant …………. Momordica Charantia 
For further analysis EEMC stock was prepared by
dissolving the lyophilized powder in Phosphate
buffered saline (PBS) (HiMedia Laboratories).
In vitro biochemical analysis
Different percentages of EEMC were subjected to
various biochemical analyses to determine the total
phenolic content, reducing sugar, antioxidant activ-
ity, anti-inflammatory activity and flavanoids. All
the analyses were done in triplicate, and the reagents
for each analysis were prepared freshly.
The estimation of total phenol content (TPC) was
performed using Folin-Ciocalteu solution and the
gallic acid as standard (9). Percentage Total Phe-
nol (%TP) were obtained by back calculating for
dried powdered plant materials and expressed as
Percentage gram weight (% w/w). The total flava-
noid content (TFC) in the extracts were estimated by
an aluminium chloride colorimetric method using
Quercetin as standard and was expressed as quer-
cetin equivalents (mg of QE/g sample) (10). The
estimation of reducing sugar was performed using
3,5-Dinitrosalicylic acid method using D-glucose
as standard. Percentage Reducing Sugar (%RS) was
obtained by back calculating for dried powdered
plant materials and expressed as Percentage Gram
weight (% w/w).
Identification of reducing sugar in EEMC by
paper chromatography
Paper chromatography was done using Butanol: Ace-
tic acid: Distilled Water in the ratio 12:3:5 as mobile
phase and 1.66% of Phthalic acid in n-Butanol: Dis-
tilled water: Aniline in the ratio 95:4:1 as Visualizing
agent. Rf (Relative fraction) for standards including
extract were determined by the equation: Rf = Dis-
tance travelled by the substance/Distance travelled
by the solvent front. Identification of sugars present
in 50% EEMC was compared with the Rf values of
standards in the same run and was confirmed by a
cascade of colorimetric reactions.
The quantitative estimation of Glucose present in
the EEMC was determined by Glucose Oxidase and
Peroxidase (GOP-POD) Method using D-glucose
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013
as standard. Glucose concentration present in 50%
EEMC was derived from the standard calibration
curve and expressed as the concentration of glucose
in mg/dl.
Preparation of stock of different percentage of
EEMC
Based on the total phenolic content the stock of
50%, 70%, and 100% EEMC was prepared in the
concentration of 80 µg/ml, which was further seri-
ally diluted up to 1.25 µg/ml in ethanol.
Ferric reducing the ability of plasma (FRAP assay)
was used to measure the antioxidant power of the
extracts taking FeSO4 as standard (11). The antioxi-
dant capacity based on the ability to reduce ferric
ions to ferrous form was calculated from the linear
calibration curve and expressed as mmol of FeSO4
equivalents (FRAP units). The free radical scaveng-
ing activity was estimated by 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH) method using ascorbic acid as
standard (12). The radical scavenging activity was
expressed as the percentage inhibition (I%) and cal-
culated as per the equation:
I (%) = (A blank - A sample / A blank) × 100,
where A blank is the absorbance of the blank reagent
with no testing compound, and A sample is the
absorbance of the test sample with all reagents. The
IC-50 value was calculated from the plot of inhibi-
tion (%) against the concentration of the extract.
The estimation of Antioxidant Activity of the
extracts was performed by Reducing Power Assay
using ascorbic acid as standard (13). The anti-inflam-
matory activity of the extracts was determined by
Human Red Blood Corpuscle (HRBC) Membrane
Stabilization Assay (Hypotonicity Induced) using
diclofenac as positive control (14).
The percentage inhibition of haemolysis or
membrane stabilization was calculated using
the following equation:
% Inhibition of haemolysis = (A1 - A2/A1) × 100
Biomedicine – Vol. 36 No. 1: 2016
014
where: A1 =  Absorbance of hypotonic buff-
ered saline and A2 = Absorbance of the test
sample in hyposaline and were compared with
the % inhibition of positive control.
Statistical analysis
All statistical analysis was performed using Graph-
Pad Prism 5 for Windows, version 5.01. Data were
expressed as mean values  ±  SEM. A one-way
ANOVA model was used to compare means between
the groups. Each sample was analyzed thrice. Post
hoc pairwise multiple comparisons were evalu-
ated using the Bonferroni post-test, after ANOVA.
Results were considered significant at p < 0.05
Results
TPC of different percentage of EEMC
The total phenol content in 50%, 70% and 100%
EEMC has been reported in our previous study
(0.029%, 0.0098% and 0.0022% respectively) (8).
Further experiments were planned based on the
total phenol content in each of these extracts.
TFC in different percentage of EEMC
Fig. 1  Percentage of total flavanoid content in 50%, 70% and
100% EEMC.
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Shobha et al.: In-vitro antioxidant …………. Momordica Charantia
50% ethanolic extract had the highest flavanoid content (22%)
when compared to 70% and 100% ethanolic extract. The inset
shows the standard graph with R2 = 0.9985.
The data obtained showed that, the 50% etha-
nolic extract had the highest flavanoid content
(22%) followed by the 70% (14.5%) and 100%
(11%) ethanolic extracts respectively (Fig.
1) (p  =  <0.016) using quercetin as standard
(R 2  =  0.9985). Since a gradient extraction was
followed, most of the water soluble fractions of
the flavanoids were found in 50% extracts than
70% and 100% extracts.
Reducing sugar in different percentage of EEMC
Fig. 2  Percentage of reducing sugar in 50%, 70% and 100%
EEMC.
50% ethanolic extract had the highest percentage of reducing
sugar (2.3%) when compared to 70% and 100% ethanolic
extract. The inset shows the standard graph with R2 = 0.9920.
The data obtained showed that, the 50% ethano-
lic extract had the highest percentage of reduc-
ing sugar (2.3%) followed by the 70% (0.51%)
and 100% (0.06%) ethanolic extracts respectively
(Fig. 2) (p = <0.0001) using D-glucose as standard
(R2 = 0.9920).
Identification of sugar in 50% EEMC by paper
chromatography
Biomedicine – Vol. 36 No. 1: 2016
Shobha et al.: In-vitro antioxidant …………. Momordica Charantia  015

DPPH and reducing power assay

Fig. 4  Antioxidant activity of 50%, 70% and 100% EEMC by

FRAP method.

Fig. 3  Ascending paper chromatography for qualitative estimation

of reducing sugars in the 50% extract.

The distance moved by the sample corresponds

to that of glucose standard. 50% ethanolic extract had the highest antioxidant
activity compared to 70% and 100% ethanolic
Table 1  Rf value of extract and the standards from extracts respectively by FRAP (p = 0.004). The
chromatography. inset shows the standard graph with R2 = 0.9926.

Compound Rf value Fig. 5  Antioxidant activity of 50%, 70% and 100% EEMC by
Xylulose 0.37 DPPH method

Maltose 0.19
Glucose 0.31
Sample(extract) 0.30
Fructose 0.344

The Rf value of the sample corresponds to that of

glucose standard.

In the paper chromatography the distance moved

by the sample corresponded to that of the glucose
standard (Fig. 3). This was confirmed by calculat-
ing the Rf values which is represented in Table 1.
So the reducing sugar present in the extract may be
Glucose. 50% ethanolic extract had the highest antioxi-
dant activity followed by the 70% and 100%
The presence of glucose was confirmed by GOD- ethanolic extracts respectively by DPPH method
POD method, which showed 332mg/dl of glucose in (p = 0.079). The inset shows the standard graph
the 50% EEMC. with R2 = 0.9644.
Antioxidant activity of EEMC using FRAP,

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016 Shobha et al.: In-vitro antioxidant …………. Momordica Charantia

Fig. 6  Antioxidant activity of 50%, 70% and 100% EEMC by
Reducing Power Assay.
The data obtained from the DPPH method showed
that, the 50% (11.43  µg/ml) EEMC has lowest
IC(50) value than 70% (16.42  µg/ml) EEMC and
100% (175.4 µg/ml) EEMC (Fig. 7).

Anti-inflammatory activity
of EEMC using HRBC membrane
stabilization assay

Fig. 8  Anti-inflammatory activity of 50%, 70% and 100% EEMC.

15
50
70
10
% RBC Protection
100
5
50% ethanolic extract had the highest antioxi-
dant activity followed by the 70% and 100% eth-
anolic extracts respectively by Reducing power 0
assay (p = 0.010). The inset shows the standard
0

10

20

40

graph with R2 = 0.9947.
The data obtained showed that, the 50% ethanolic
extract had the highest antioxidant activity followed
by the 70% and 100% ethanolic extracts respec-
tively by FRAP (p = 0.004), DPPH (p = 0.079) and
Reducing Power Assay (p = 0.010), which may be
attributed to the presence of higher percentage of
phenolic acid and flavanoid content in 50% EEMC
(Figs. 4–6).
Fig. 7  IC(50) of EEMC by DPPH method
200
IC (50) value in mg/ml
80
-5 concentration of EEMC in mg/ml
-10
50% ethanolic extract had the highest anti-
inflammatory activity followed by the 70% and
100% ethanolic extracts respectively (p = 0.001)
using diclofenac sodium as positive control.
The data obtained showed that, the 50% ethanolic
extract had the highest anti-inflammatory activity
followed by the 70% and 100% ethanolic extracts
respectively (Fig. 8) (p  =  <0.001) using diclofenac
sodium as positive control.

150

Discussion
100
Medicinal value of MC has been attributed to its
50 high antioxidant property which is related to phe-
nols, flavonoids, isoflavones, terpenes, anthraqui-
0 nones, and glucosinolates. Based on the previous
references which have stated that ethanol is a bet-
50

0
70

10

Different percentage of EEMC ter solvent for extraction of phytochemicals (6,7),

we have used ethanol as a solvent for extraction to
50% ethanolic extract has lowest IC(50) value evaluate phytoconstituent activities and biological
(11.43 µg/ml) when compared to 70% and 100% activities of MC. Amira et al. have shown that the
ethanolic extract.

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Shobha et al.: In-vitro antioxidant …………. Momordica Charantia 
pure solvents were inefficient extraction media for
antioxidant, and enhanced extraction yields were
obtained from solvent containing a higher concen-
tration of water (3). Koffi et al in their work have
shown that since the vast majority of polyphenols
are not water soluble manufacturers would have to
use extraction solvents with a mixture of suitable
solvents to obtain fractions rich in polyphenols (7).
Based on these studies we have used different per-
centage of ethanol, that is, 50%, 70% and 100% for
extraction and the result obtained in our study goes
in accordance to these references
Phenolic compounds are known to have antioxi-
dant activity, and it is due to the redox properties,
which play an important role in adsorbing and neu-
tralizing free radicals, quenching singlet and triplet
oxygen, or decomposing peroxides (15). Flavonoid
s are the largest group of naturally occurring phe-
nolic compounds, which occurs in different parts
of the plant both in a free state and as glycosides.
The polyphenolic nature of flavanoids acts as an
antioxidant which scavenges injurious free radicals
such as super oxide and hydroxyl radicals. MC is
a good source of phenolic compounds which pos-
sess potent antioxidant activity. Semiz and Sen have
shown that in-vivo treatment of rats with MC fruit
extract enhanced both antioxidant enzyme (AOE)
and Glutathione S-Transferase (GST) activities (16).
Hamissou et al. have shown that MC was 82.05% as
effective as ascorbic acid in inhibiting the free radi-
cal DPPH and has higher total phenolic compounds,
indicating that MC is a high antioxidant containing
vegetable food compared to zucchini, in aqueous
extracts (17). Even though in these studies they have
shown that MC is having phenol content and anti-
oxidant activity, they have used either crude extract
or single percentage of extract. But in our study, we
have used a different percentage of extract and esti-
mated the TPC, TFC, antioxidant activity and anti-
inflammatory activity.
The TPC was estimated using the F-C method
using gallic acid as standard. A Linear response
was observed between 5 and 60  µg/µl and TPC
was expressed as GAE (R2  =  0.9236). The result
obtained in our study showed that the 50% (0.029%)
www.biomedicineonline.org
017
EEMC is having highest phenol content followed
by 70% (0.0098%) and 100% (0.0022%) EEMC.
The TFC was estimated using Aluminum Chlo-
ride Colorimetric Method using quercetin as stan-
dard (R2 = 0.9985). The result obtained in our study
showed that the 50% (22%) EEMC is having high-
est flavanoid content followed by 70% (14.5%) and
100% (11%) EEMC (Fig. 1).
Moure et al. showed that, as a polarity of the sol-
vent increased, extraction yields of total soluble sol-
ids and total extractable polyphenolics were higher
(18). The data obtained in our work agrees with this
study. The result of TPC and TFC shows that the
reactivity of phenolic compounds depends upon
the polarity of the medium, as there is a decrease
in the polarity, reactivity also decreases proportion-
ally showing minimum reactivity with 100% etha-
nol soluble fractions of phenolic contents. Further
phenolic acid profiling using HPLC will be done in
order to specify the key compound present in the
50% EEMC.
The reducing sugar was estimated by DNS method
using D-Glucose as standard (R2  =  0.9920). The
result obtained showed that the 50% (2.3%) EEMC
is having the highest percentage of reducing sugar
followed by 70% (0.51%) and 100% (0.06%) EEMC
(Fig. 2). Since the 50% EEMC has shown the high-
est percentage of reduci ng sugar qualitative estima-
tion of reducing sugar was done by ascending paper
chromatography using glucose, fructose, xylulose
and maltose as standard. The result obtained showed
the presence of glucose in 50% EEMC (Fig. 3 and
Table 1). The presence of glucose was further con-
firmed by the GOD-POD method using D-glucose
as standard, which surprisingly showed 332mg/dl of
glucose in the extract. The GOD-POD method is a
specific method for estimation of D-Glucose, since
the enzyme specifically oxidises only D-Glucose.
There are several methods to determine antioxidant
activity of plants. The antioxidant activity of dif-
ferent percentage of EEMC in our study was deter-
mined using three methods, that is, FRAP, DPPH
and reducing power assay. The phytochemicals
which are responsible for the scavenging activity
Biomedicine – Vol. 36 No. 1: 2016
018 Shobha et al.: In-vitro antioxidant …………. Momordica Charantia
are the phenolic and flavonoid content in the extract
(19). The Higher absorbance of the reaction mixture
indicates higher reductive potential (20). The result
obtained in our study showed that the 50% ethanolic
extract had the highest antioxidant activity followed
by the 70% and 100% ethanolic extracts respec-
tively, in FRAP (Fig. 4) ,DPPH (Fig. 5) and Reduc-
ing Power Assay (Fig. 6), which may be attributed to
the presence of higher percentage of phenolic acids
and flavanoids.
IC(50) value is defined as the concentration of
antioxidant required for 50% scavenging of DPPH
radicals, which is a parameter widely used to mea-
sure antioxidant activity. Smaller the IC(50) value
corresponds to a higher antioxidant activity of the
plant extract (21). The data obtained in our study
showed that the 50% (11.43 µg/ml) EEMC has low-
est IC(50) value than 70% (16.42 µg/ml) EEMC and
100% (175.4  µg/ml) EEMC and attributes to TPC
and TFC (Fig. 7). The DPPH radical scavenging
activity of EEMC increased gradually as the con-
centration was increased. Decrease in absorbance of
DPPH solution indicated by a change in the colour
from purple to yellow which depends on the intrinsic
antioxidant activity of antioxidant and on the speed
of reaction between DPPH and antioxidant present
in the extract.
The anti-inflammatory activity of EEMC was esti-
mated by HRBC Membrane Stabilization Assay
using diclofenac sodium as a positive control. The
erythrocyte membrane is analogous to the lysosomal
membrane and its stabilization by the extract implies
that the extract will stabilize lysosomal membranes
which will, in turn, limit the inflammatory response
by preventing the release of lysosomal constituents.
Cioua et al. and Chao et al. have independently
shown that MC in sepsis-induced mice reduced
expression of proteins associated with inflammation
like Cyclooxygenase-2 (COX-2), Inducible nitric
oxide Synthase (iNOS), and Nuclear factor-kappaB
(NF-kB) and reduced secretions of pro-inflamma-
tory cytokines and other substances, hence reducing
organ damage (22,23). Umukoro et al have shown
that aqueous leaf extract of MC inhibits the late
phase of inflammatory events, namely the release of
www.biomedicineonline.org
chemical mediators and thus suggesting that it may
offer some beneficial effects in the management of
inflammatory conditions (24). In these studies they
have used crude extract or single percentage of
extract and explained about the anti-inflammatory
activity of MC, but in our study we have used dif-
ferent percentage of extract for the anti-inflamma-
tory activity. The data obtained in our study showed
that the 50% ethanolic extract had the highest anti-
inflammatory activity compared to 70% and 100%
ethanolic extracts respectively (Fig. 8). The extracts
might have exhibited membrane stabilization effect
by inhibiting hypotonicity-induced lyses of erythro-
cyte membrane. Thus 50% EEMC is having more
potent anti-inflammatory activity than 70% and
100% EEMC (Fig. 9).
Thus, the data obtained showed that 50% EEMC is
having highest antioxidant activity and anti-inflam-
matory activity when compared to 70% and 100%
EEMC. Further studies will be done to know the
key antioxidant compound in the extract by phenolic
acid profiling using HPLC
Acknowledgments
We thank the Department of Science and technology-
Fund for the improvement of science and technology
infrastructure for funding the Center of Excellence
for Molecular Biology and Regenerative Medicine
(CEMR) laboratory and facilitating the project and
JSS University for funding the project. We would
also like to acknowledge Dr. MVSST SubbaRao,
Associate Professor, Department of Biochemistry,
JSS Medical College, Mysore for his support and
guidance to carry out the biochemical analysis
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Biomedicine: 2016; 36(1): 021-025 021

Inflammatory and oxidative stress markers in acute myocardial infarction

Ashok Prabhu1, K. Sudha1, A.M. Kiran Kumar1, and Rajib Kumar Pandey1
1
Department of Biochemistry, Center For Basic Sciences, Kasturba Medical College, Mangaluru, Manipal
University, Manipal, India.
(Received: Feb 2016   Accepted: Mar 2016)

Corresponding Author
Dr. K. Sudha. Email: sudha.k@manipal.edu

Abstract

Introduction and Aim: The present study aims to correlate oxidative stress markers viz., oxidized LDL,
Ischemia modified albumin (IMA)and inflammatory marker—matrix metalloproteinase 9 (MMP 9) with the
established cardiac markers (CK MB, Troponin T) and to assess their clinical utility in the diagnosis of acute
myocardial infarction.

Materials and Methods: Ninety patients complaining of chest pain for the first time, were divided into 2
groupswhich included 45patients with acute myocardial infarction and 45age and sex matched patients with
normal ECG and normal troponin T, who served as controls. Serum troponin T, CK MB, were determined by
ECLIA, IMA, oxidized LDL spectrophotometrically and MMP-9 was measured by ELISA.

Results: Oxidized LDL and IMA were significantly elevated in MI compared to controls. However, an increase
in MMP 9 in AMI patients was not statistically significant. Both specificity and sensitivity of newer markers
were lower than gold standard markers viz., CK MB and Troponin T. There was a significant positive correla-
tion between standard markers and oxidized LDL (p < 0.001) and an insignificant positive correlation with
other newer markers studied.

Conclusion: It can be concluded that usefulness of IMA and MMP9 as predictive cardiac markers remain
feeble. However, oxidized LDL may be included in the battery of extended lipid profile as well as in the panel
of cardiac markers.

Key words: Cardiac Markers, Ischemia Modified Albumin, Matrix Metalloproteinase

Introduction is oxidation of unsaturated fatty acids of the cell

M
yocardial infarction (MI), happens to
be a prime cause of death and disability
worldwide with an ongoing increase
in incidence. Coronary artery diseases (CAD), is
due to a complex interplay between genetic and
environmental risk factors setting into motion an
inflammatory cascade of monocyte migration, lipid
oxidation and atheromatous plaque formation (1).
The most common event ofmyocardial ischemia
membrane which may induce proinflammatory
pathways, activation of matrix metalloproteinase,
vascular smooth muscle cell proliferation and death,
endothelial dysfunction and lipid peroxidation (2).
Reactive oxygen species have dose dependent action
on the heart while small amounts serve as signaling
molecules and result in cardiac protection, large
amount cause lipid peroxidation and myocardial
damage. ROS can cause reversible or irreversible

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022 Ashok Prabhu et al.: Inflammatory………………Myocardial Infarction

Parameter Control (N = 45) Myocardial Infarction p-Value

(N = 45)
Troponin T (pg/ml) 8.35 ± 3.04 82.13 ± 78.50 <0.001*
CKMB (IU/L) 14.17 ± 5.6 40.2 ± 20.3 <0.001*
MMP9 (pg/ml) 11932 17970* 0.200
OxLDL (µmol/L) 68.8 ± 19.3 95.01 ± 16.02 <0.001*
IMA (Abs unit) 0.55 ± 0.21 0.746 ± 0.34 <0.001*
AIMA (Abs.unit) 0.52 ± 0.2 0.707 ± 0.4 <0.001*
Table 1  Comparison of study parameters among the groups.
N = sample size.

are Troponin I or T and CK-MB, which have high

Parameters R p-Value
myo­cardial tissue specificity as well as high clinical
MMP9 0.060 0.577
sensitivity. Although current markers have greatly
Oxd LDL 0.451* 0.001*
improved the diagnosis and quickened the treatment
IMA 0.133 0.213
of AMI patients, there is still scope for improvement,
ADIMA 0.100 0.350
especially in the area of early detection and treatment
CK-MB 0.526* 0.001*
as these markers assess different pathophysiological
Table 2  Correlation coefficients (r) of TroponinT with Study mechanisms in myocardial ischemia. Hence, the
parameters.
aim of the present study is to correlate serum IMA
and oxidized LDL, the markers of oxidative stress
Parameters R p-Value and MMP9, an inflammatory marker with thewell-
MMP9 0.108 0.313 established markers CK MB and Troponin T (TnT)
Oxd LDL 0.432 0.001* of acute myocardial Infarction.
IMA 0.100 0.35
ADIMA 0.046 0.665 Materials and Methods
Table 3  Correlation coefficients (r) of CK-MB with study
parameter. This study is a case control study comprising of 90
subjects of either sex, of 20–60 age, who presented
modifications of cardiac myofibrillar proteins and with a complaint of chest pain for the first time.
serum proteins like albumin and LDL (3). ROS Patients with previous history of myocardial infrac-
produced due to ischemia damage the metal binding tion, malignancy, patients with statins, known cases
site on the amino terminus of albumin and lower of kidney and liver diseases were excluded. Selected
metal binding capacity for cobalt (4). Ischemia subjects were diagnosed and classified as controls
modified albumin (IMA) increases within minutes and MI patients, depending upon the assessment as
of the onset of ischemia and returns to normal per the clinical signs, symptoms, TnT levels and the
within 6–12  hours. LDL is the most vulnerable ECG findings at the time of admission. Informed
target of oxidation, and oxidative modification of consent was taken from all the subjects and the study
LDL is a key step in the initiation and progression was approved by the institutional ethical and research
of atherosclerosis. Oxidized LDL induces local committee. 5ml of whole blood was collected in red
inflammation in the atherosclerotic plaque, leads to cap vacutainer, serum separated was used for the
endothelial dysfunction and MI (5). Matrix metallo estimation of albumin, CKMB, troponin T, IMA,
proteinase 9 (MMP 9), a gelatinase secreted by MMP9, and oxidized LDL. Estimation of serum
inflammatory cells, macrophages has shown to play TnT was done by ECLIA (7) and that of CK-MB was
a role in the pathogenesis of atherosclerosis and done by immunological UV kinetic method (8) Isch-
MI (6). The currently preferred biomarkersfor MI emia modified albumin was determined by cobalt-
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Ashok Prabhu et al.: Inflammatory………………Myocardial Infarction  023

TnT CKMB MMP9 OxdLDL AIMA IMA

Sensitivity 75.6 77.8 57.8 73.3 71.1 57.8
Specificity 96 89 62.2 80 67 72
PPV 94.4 87.5 60.4 78.5 68 66.6
NPV 79.6 80 59.5 75 69.7 63.8
OR 66.4 28 2.25 11 4.92 3.5
ROC AUC 0.96 0.91 0.567 0.86 0.72 0.651
Cut off 14 25.5 14398 90.1 0.58 0.57
(pg/l) (IU/L) (pg/ml) (µmol/l) (ABSU) (ABSU)
LR+ 18.9 7 1.5 3.66 2.1 2
LR- 0.21 0.25 0.67 0.32 0.55 0.42
Table 4  Efficiency of serum TnT, CKMB, MMP9, OxdLDL, IMA and adjusted IMA (AIMA) as marker of cardiac ischemia.

albumin binding method (9). Adjusted Ischemia

Modified Albumin (AIMA) was calculated by multi-
plying concentration of albumin with the absorbance
of IMA. MMP-9 was assayed by ELISA (10) and
that of oxidized LDL by precipitation method (11).
Data were analyzed using IBM SPSS version17. Stu-
dent’s t-test was used to determine the significance
of variables, p value less than 0.05 was considered
statistically significant. The association of analytical
markers with clinical outcome was compared with
chi-square test. Results of TnT, CKMB, oxdLDL,
MMP9, AIMA and IMA were analyzed for clini-
cal sensitivity, specificity, positive predictive value
(PPV), negative predictive value (NPV), positive
likely hood ratio (LR+) and negative likely hoodratio
(LR-). ROC Curve is generated for individual ana- nin T and CKMB respectively (Tables 2 and 3). The
lytes to find out the cutoff values. sensitivity of biomarkers in the descending order was
CK MB, Troponin T, oxidized LDL, Ad IMA, IMA,
Results MMP-9. The specificity of the cardiac markers in the
descending order was TnT, CK MB, oxidized LDL,
Serum troponin T and CK MB was significantly higher IMA, Ad IMA, MMP-9 (Table 4).
(p < 0.001) in AMI patients compared to patients with
normal ECG. Oxidized LDL values were strikingly Discussion
elevated in patients compared to controls (p < 0.001).
Further, a statistically significant increase in IMA ECG and cardiac troponin are the most widely used-
and AIMA was observed in AMI patients compared cardiac markers though ECG can be misleading in
to controls. However, median values of MMP-9 did 8% of the patients with AMI. Serum proteins mark-
not differ significantly between the groups (Table ers have become increasingly important to improve
1). Correlation studies on IMA, ADIMA, MMP-9 the diagnosis of myocardial infarction, in identifying
did not show any statistical significance with gold people at risk of having an infarct and inothers to
standard markers CK MB and TnT (Tables 2 and 3). predict long term prognosis following MI (12). This
However, oxidized LDL showed a positive correla- study showed significant increase in TnT in patients
tion (r value 0.452, 0.432) and p < 0.001 with Tropo- with AMI, indicating potential relevancy of this

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024 Ashok Prabhu et al.: Inflammatory………………Myocardial Infarction
marker for predicting cardiac tissue damage with
specificity and sensitivity of 96% and 75.6%, respec-
tively and PPV of 94.4 and positive LR of 18.9 which
is in agreement with the findings of Janice et al. (13).
The present study proves the fact that CKMB is
more sensitive (NPV-80) than TnT which is in con-
currence with earlier studies (14,15). Microscopic
study of atherosclerotic plaque (both coronaryand
aortic) in humans had demonstrated excess matrix
degrading activity, suggesting that elevated levels
of matrix metalloproteinase (MMP) might act as a
surrogate marker of atherosclerotic plaque burden.
However, elevated levels of MMP had demonstrated
no utility in evaluation of acute coronary syndrome
patients (ACS) (16). This finding is justified by the
present study. However, Benjamin etal showed that-
MMP-9 was reduced by therapies associated with
favorable outcome in atherosclerosis and thus may
serve as a surrogate biomarker for treatment effi-
ciency (5). Several studies have demonstrated that
oxidative stress markers in plasma are increased in
coronary heart disease and in patients with classical
risk factors (2). Free radicals cause oxidation of sev-
eral plasma proteins such as albumin, ceruloplasmin,
transferrin. Albumin, which is considered a “sacrifi-
cial” antioxidant, via thiol groups, provides tenfold
protection in plasma by capturing oxygen radicals.
ROS produced due to ischemia, damage the metal
binding site on the amino terminus of albumin and
lower metal binding capacity leading to increased
formation of IMA (4). Although our study presented
a significant increase in the serum levels of IMA
and AIMA in the MI cases as compared to the con-
trols, there was no significant correlation observed
with any of the cardiac markers. However, Chawla
et al. (17) reported higher specificity and sensitivity
along with a higher positive and negative predictive
value for IMA as compared to the CK-MB. Sinha
et al. (18) reported that IMA along with either the
ECG or TnT measurements, or both, increased the
sensitivity for the diagnosis ACS to 90%–95%. IMA
increases within minutes of the onset of ischemia
and returns to normal within 6–12 hours, is not spe-
cific to cardiac tissue, hence several earlier studies
have reported contradicting results. Furthermore,
our earlier study on MI showed that advanced oxida-
tion protein products (AOPP), predominantly aggre-
www.biomedicineonline.org
gates of albumin damaged by oxidative stress, was
significantly elevated in plasma. AOPP also act as
inflammatory mediatorsmay serve as prognostic fac-
tor of severe forms of cardiovascular diseases (19).
LDL is the most vulnerable target of oxidation, and
oxidative modification of LDL is a key step in the
initiation and progression of atherosclerosis. Oxi-
dized LDL is a pro-inflammatory mediator highly
correlated with adverse cardiovascular events. The
present study exhibited significant rise in oxidized
LDL level in MI cases compared to the controls,
the rise significantly correlated with the recognized
cardiac markers, TnT and CK-MB. Similar results
have been reported inseveral studies on patients with
acute coronary syndromes (20,21).
The study focuses on the role of newer biomole-
cules as cardiac markers to increase the diagnos-
tic efficiency and to establish the multiple marker
approaches towards investigating acute myocar-
dial infarction and for early patient care. It can
be concluded that though usefulness of IMA and
MMP9 as predictive cardiac markers remain fee-
ble, oxidized LDL may be included in the battery
of extended lipid profile as well as in the panel of
cardiac markers.
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  1. Berenson, G.S., Srinivasan, S.R., Bao, W., New-
man, W.P., et al. Association between multiple
cardiovascular risk factors and atherosclerosis in
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Study. N. Engl. J. Med. 1998; 338: 1690–1692.
  2. Blake, D.R., Allen, R.E., and Lunec, J. Free
radicals in biological systems—a review ori-
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1987; 43: 371–385.
  3. Vanden Hoek, T.L., Becker, L.B., Shao, Z.H.,
Li, C.Q., and Schumaker, P.T. Preconditioning
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541–548.
  4. Naghavi, M., Libby, P., Falk, E., Casscells, S.W.,
Litovsky, S., et al. From vulnerable plaque to
vulnerable patient: a call for new definitions
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tion. 2003; 108: 1664–1672.
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  5. Scirica, B.M. Acute coronary syndrome emerg-
ing tools for diagnosis and riskassessment. J.
Am. Coll. Cardiol. 2010; 55:1403–1415.
  6. Raitakari, O.T., Juonala, M., Kähönen, M., Tait-
tonen, L., Laitinen, T., et al. Cardiovascular risk
factors in childhood and carotid artery intima-
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290: 2277–2283.
  7. Ahmad, M.I., and Sharma, N. Biomarkers in
acute myocardial infarction. J. Clin. Exp. Car-
diol. 2012; 3: E672–E672.
  8. Morrow, D.A., Cannon, C.P., Jesse, R.L., Newby,
L.K., Ravkilde, J., et al. National Academy of
Clinical Biochemistry Laboratory Medicine
Practice Guidelines: clinical characteristics and
utilization of biochemical markers in acute coro-
nary syndromes. Clin. Chem. 2007; 53: 552–574.
  9. Bar-Or, D., Curtis, G., Rao, N., et al. Character-
ization of the Co2+ and Ni2+ binding amino-
acid residues of the N terminus of human albu-
min: an insight into the mechanism of a new
assay for myocardial ischemia. Eur. J. Biochem.
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10. Minematsu, N., Nakamura, H., Tateno, H.,
Nakajima, T., and Yamaguchi, K. Genetic poly-
morphism in matrix metalloproteinase-9 and
pulmonary emphysema. Biochem. Biophys.
Res. Commun. 2001; 289: 116–119.
11. Tsimikas, S., Palinski, W., and Witztum, J.L.
Circulating autoantibodies to oxidized LDL
correlate with arterial accumulation and deple-
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12. Malek, F., Jiresova, E., Dohnalova, A., Kop-
rivova, H., and Space, K.R. Serum copper as
amarker of inflammation in prediction of short
term outcome in high risk patients with chronic
heart failure. Int. J. Cardiol. 2003; 113: 51–53.
13. Janice, Z., Robert, F., Denise, M., et al. Diag-
nostic marker cooperative study for the diagno-
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sis of acute myocardial infarction. Circulation.
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14. Gama, R., Swain, D.G., Nightingale, P.G., and
Buckley, B.M. The effective use of cardiac
enzymes and electrocardiograms in the diagno-
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Postgrad. Med. J. 1990; 66: 375–377.
15. Dekker, M.S., Mosterd, A., Hof, A.W.J., and
Hoes, A.W. Novel biochemical markers in
suspected acute coronary syndrome: systemic
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1001–1010.
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Multiple biomarker use for detection of adverse
events in patients presenting with symptoms
suggestive of acute coronary syndrome. Clin.
Chem. 2007; 53: 874–881.
17. Chawla, R., Goyal, N., Calton, R., and Goyal,
S. Ischemia modified albumin: a novel marker
for acute coronary syndrome. Indian J. Clin.
Biochem. 2006; 21: 77–82.
18. Sinha, M.K., Roy, D., Gaze, D.C., Collinson,
P.O., and Kaski, J.C. Role of “Ischemia Modi-
fied Albumin”, a new biochemical marker of
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Oxidized LDL and malondialdehyde-modified
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21. Holvoet, P., Mertens, A., Verhamme, P.,
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21: 844–848.
Biomedicine – Vol. 36 No. 1: 2016
026 Biomedicine: 2016; 36(1): 026-032

A study of CVD risk assessment in post-menopausal women in comparison with pre-

menopausal women with serum lipid profile and hs CRP as risk markers
Yasmeen Fatima,1 Sreekantha,1 Ramesh,2 Sadananjali,1 Saba,3 and Pallavi4
Department of Biochemistry 1RIMS, Raichur; 2VIMS, Bellary; 3KBN, Gulbarga; 4NMC, Raichur, India.
(Received: March 2016   Accepted: Mar 2016)

Corresponding Author
Yasmeen Fatima. Email: dryasmeenfatima@gmail.com

Abstract

Introduction and Aim: Menopause is the permanent cessation of menstruation. Since the cardio vascular
disease is the leading cause of death among post-menopausal women and hs-CRP is an acute phase reac-
tant involved in the development of atherosclerosis. Thus, addition of hs-CRP to traditional lipid screening
improves the ability to accurately predicting global cardiovascular risk among post-menopausal women.

Materials and Methods: Fifety post-menopausal and 50 pre-menopausal women in the reproductive age
group were selected in the study. We have measured serum lipid profile [total cholesterol (TC), triglyceride
(TG), HDL, LDL, and VLDL] and serum hs CRP level in both the groups. The comparison and correlation of
hs-CRP and lipid profile were done using correlation test and the p-value less than 0.05 was considered signifi-
cant. Lipid profile estimated by enzymatic methods and serum hs-CRP by Turbidimetric method.

Results: Study showed statistically significant higher values of serum TC, TG, LDL, VLDL and hs CRP lev-
els in post-menopausal women compared to pre-menopausal women (p < 0.001). And statistically significant
lower values of HDL in post-menopausal women compared to pre-menopausal women (p < 0.001).

Conclusion: Our study showed dyslipidaemia and high hs-CRP in post-menopausal women, justifying that
post-menopausal women are at an increased risk for CVD. Thus, addition of hs-CRP to traditional lipid screen-
ing improves CVD risk assessment among post-menopausal women. And specific health education is needed
for all women to prevent the emerging CVD risk.

Key words: Cardiovascular Disease, HDL, LDL, Post-menopausal, Pre-menopausal

Introduction CVD will claim 25 million lives annually and

C
ardiovascular Disease (CVD) is an emerging
dominant chronic disease in the world. At
the beginning of the 20th century, CVD
accounted for less than 10% of all deaths worldwide.
At 21st century, CVD accounts for nearly half of
all deaths in the developed world and 25% in the
developing world (1). By 2020, it is predicted that
that coronary heart disease will surpass infectious
disease as the world’s number one cause of death
and disability. This will lead to at least one in every
three deaths due to CVD in developing countries (2).
Framingham Study suggests that female CVD mor-
bidity rates accelerate more quickly than do those
of males after the age of 45  years. Menopause is

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Yasmeen fathima et al.: A study of CVD risk………………markers
defined by The WHO as ‘the permanent cessation of
menstruation as a result of the loss of ovarian activ-
ity’ (3). Menopause develops as the result of low
estrogen and due to the disturbed hormonal cycle of
ovulation (4). It occurs at a mean age of 51 years.
A woman today will live approximately for about a
third of her life beyond the menopause (5).
Many risk factors have been identified as contribu-
tory to the development of CVD. Cholesterol as the
main culprit in atherosclerosis formation, given the
concept of in sudation of lipids as a cause for ath-
erosclerosis (2). Low-Density Lipoprotein (LDL),
has been implicated in the development of CVD (5).
And HDL protects from atherosclerosis formation
by its role in reverse cholesterol transport.
As atherosclerosis is a chronic process where inflam-
mation plays an important role not only in trigger-
ing but also in promoting atherosclerotic plaque
development and complications, C-reactive protein
(CRP) has emerged as a novel and potentially clini-
cally useful marker for increased CVD risk (6). It
suggests the possibility that subclinical states of
atherosclerosis can be associated with an increase
in circulating markers of inflammation before acute
events occur. Based on data of in vitro studies it has
been proposed that CRP is actively contributing to
disease progression, and it should be considered as a
true risk factor and also as a target for intervention.
Studies have also demonstrated a significant asso-
ciation between CRP and future risk of metabolic
syndrome, diabetes and hypertension (7). National
guidelines for measurement of CRP as a marker of
CVD risk have been issued jointly by AHA (Ameri-
can Heart Association) and CDC (Centres for Dis-
eases Control and Prevention). Prospective stud-
ies have shown that single hsCRP measurement is
a strong predictor of myocardial infarction, stroke,
peripheral vascular disease and sudden cardiac
death (7). Standard clinical assays of CRP typically
have a lower detection limit of 3 to 8  mg/l. Thus,
these assays lack sensitivity within the lower normal
range. Hence, detection of hsCRP allows a better
analysis of CRP distribution within the normal range
in general population. Also hsCRP concentrations in
healthy humans are neither subjected to diurnal or
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027
seasonal variations. hsCRP seems to be a stronger
predictor of cardiovascular events than LDL. Fur-
ther hsCRP testing may also have potential prognos-
tic value among low risk groups (8).
Our study aimed to establish differences Cardio vas-
cular disease risk in post-menopausal women com-
pared to young menstruating women estimating and
comparing serum lipid profile and hsCRP in both the
groups. This implies that in order to modify risks of
CVD in older women, addition of hsCRP testing to
traditional lipid screening improves CVD risk pre-
diction and intervention with regard to dyslipidae-
mia should begin early.
Materials and Methods
Study was conducted at Raichur Institute of Medi-
cal Sciences and Rajiv Gandhi Super Speciality
Hospital and Research Centre, Raichur, from Sep-
tember 2014 to September 2015. Women attending
outpatient department and those working as house
keepers in college were included in the study. The
study comprised total 100 women, which included
50 Post-menopausal women who attained meno-
pause at least 1  year before with daily moderate
working habits and without any disease and dis-
order as cases. And 50 healthy pre-menopausal
women of reproductive age group having regular
periodic menses, with daily moderate working hab-
its and without any disease and disorder as controls.
Women with CVD, Hypertension, DM, any sys-
temic disorder, any neoplasia, or any other inflam-
matory disease, and those who are on exogenous
hormones and hypolipidemic drugs were excluded
from the study. And also to minimize the effect of
life style on lipid profile, smokers, alcoholics, sed-
entary women and trained athletes or sports women
were also excluded from the study.
After 12  hours overnight fasting 6  ml of venous
blood samples were collected from both the groups
and the samples were centrifuged for the estimation
of serum total cholesterol (TC) and serum triglycer-
ides (TG), HDL and high sensitive C-reactive pro-
tein (hs CRP). TC level estimated by CHOD-PAP
method (9), TG level by GPO Trinder method (10),
Biomedicine – Vol. 36 No. 1: 2016
028 Yasmeen fathima et al.: A study of CVD risk………………markers

HDL level by phosphotungstic acid methodend

point (11). And LDL, VLDL values were calculated
400 222.86 186.88
by applying Friedwald’s equation (8). 162.38 154.8
200 97.26 90.79
37.3
19.45
VLDL = TG/5 and LDL = TC - (VLDL + HDL) 0
T.CHO TG LDL VLDL
hs CRP estimated by antigen antibody reaction by
the End Point Method (12).
pre-menopusal women post-menopausal women
Statistical analysis
Graph 1  Bar diagram of TC, TG, VLDL, LDLin pre and post-
menopausal women showing—serum TC, TG, LDL and VLDL are
The results of all profiles TC, TG, LDL, VLDL, raised in post-menopausal women compared to pre-menopausal
HDL, and hsCRP of 100 samples were expressed as women and the difference is statistically highly significant.
mean  ±  SD. Comparison between both the groups
done by student unpaired t – test. One way analysis
of variance (ANOVA) was used to compare mean
values in the two groups. Pearson’s correlation was
applied to correlate between the parameters. p-Value
52.14
of less than 0.05 was considered significant. And 100 30.76
p-value less than 0.001 was considered highly sig- 0
nificant. Data was analysed using Microsoft excel, HDL
SPSS 22 version.
pre-menopusal women post-me nopausal women
Results
Graph 2  Bar diagram of HDL level in pre and post-menopausal
Showing-Positive correlation of hs CRP with LDL women showing—The serum HDL values are decreased in
and negative correlation with HDL in pre-meno- post-menopausal women than pre-menopausal women and the
difference is statistically highly significant.
pausal women

Showing-Positive correlation of hsCRP with LDL

and negative correlation with HDLin post-meno-
pausal women.

Discussion

As CVD is the leading cause of death among the

post-menopausal women. We have done this com-
parative study of serum lipid profile and hs CRP as
markers of CVD risk in both pre and post-meno- Graph 3  Bar diagram of hs CRP in pre and post-menopausal
pausal women, so that the early intervention can be women showing—hs CRP is raised in post-menopausal women
taken by educating them. Atherosclerosis is a chronic than pre-menopausal women and the difference is statistically
highly significant.
process with an active and ongoing inflammatory
component. CRP is a marker of inflammation which
We have done the study on postmenopausal women of
can be used to predict the risk of CVD. Detection of
age 58.06 ± 5.88 years and in premenopausal women
hsCRP allows a better analysis of CRP distribution
of age 32.78  ±  5.96  years. Menopause normally
within the normal range in the general population as
occurs between 45 to 50  years of age. In Framing-
hsCRP concentrations in healthy humans are neither
ham cohort study, 2873 women were followed up for
subjected to diurnal or seasonal variations.
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Yasmeen fathima et al.: A study of CVD risk………………markers 029

Serum biochemical Normal values (13) Pre-menopausal Post-menopausal

parameters Women, n = 50 women, n = 50
(mean ± SD) (mean ± SD)
TC (mg/dl) <200 162.38 ± 42.66 222.86 ± 17.84**
TG (mg/dl) <160 97.26 ± 16.05 186.88 ± 13.41**
LDL (mg/dl) <100 90.79 ± 48.26 154.8 ± 20.21**
VLDL (mg/dl) <32 19.45 ± 3.21 37.3 ± 2.68**
HDL (mg/dl) >40 52.14 ± 10.84 30.76 ± 4.86**
hs CRP (mg/l) <1 0.73 ± 0.24 1.68 ± 0.36**
Table 1  Comparison of parameters in both pre and post-menopausal women.
*Significant p < 0.05. **Highly significant p < 0.001.

24 years. Gordon et al. noted an increase in incidence
and severity of CAD after menopause. The incidences
of CAD in postmenopausal women were more than
double than those in pre-menopausal women (14).
In our study the TC, TG, LDL, and VLDL levels
are increased in postmenopausal women than in pre-
menopausal women and is statistically highly signif-
icant (p < 0.001). These findings are in accordance
with the studies done by (3,5,13,15–17). TC is an
independent risk factor for CVD and Razay et al. (18)
showed that 1%, increase in TC is associated with
atleast 2% increase in the incidence of CAD. They
also showed that TC was 19% higher in postmeno-
pausal women compared to premenopausal women.
TC level includes levels of HDL, LDL and VLDL.
After menopause plasma level of LDL and VLDL
increases, but plasma level of HDL decreases so the
increase in plasma LDL and VLDL levels is more
hs CRP LDL
Pearson’s correlation 0.871
p-Value 0.000
Significance HS
Table 2  Pearson’s correlation of hsCRP with lipidprofile, pre-
menopausal women.
hs CRP LDL
Pearson’s correlation 0.565
p-Value 0.000
Significance HS
Table 3  Pearson’s correlation of hsCRP with lipid profile, in post-
menopausal women.
than decrease in HDL level. Hence the net effect is
that plasma level of TC increases after menopause
(16). According to the study done by Razay et al., in
the postmenopausal women, TG was higher by 31%
when compared to premenopausal women.
Circulating estrogen is a regulator of lipoprotein
lipase, it catalyses the hydrolysis of VLDL to form
IDL and later LDL. Hepatic triglyceride lipase
hydrolyses the TG of IDL to produce LDL. After
menopause due to estrogen deficiency, there will be
increased plasma lipoprotein lipase and hepatic TG
lipase activity causing plasma LDL accumulation
and also leads to down-regulation of LDL receptors
(19), as estrogen stimulates the synthesis of LDL
receptors (16).
From our study it is evident that pre-menopausal
women are having high HDL than postmeno-
pausal women and is statistically highly significant
(p < 0.001). These findings are in accordance with
HDL
many studies (3,5,13,15–17). Due to estrogen defi-
-0.68
ciency, postmenopausal women have highest activ-
0.000
HS ity of hepatic lipase and enhances the uptake and
catabolism of HDL thus decreasing plasma HDL
concentration (20). Plasma LCAT activity is greater
in the postmenopausal women suggesting that, cho-
lesterol esterification is accelerated in plasma HDL
HDL (21). In postmenopausal women there is increased
-0.548 LDL accumulation, so more and more HDL gets
0.000 esterified. Hence they have lower HDL compared to
HS premenopausal women. For every 10 mg/dl increase
in HDL, there is a corresponding 50% decrease in
CAD risk (22). So HDL is an independent and better

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030
predictor of CAD risk in women (23).
According to our study the hsCRP values are sig-
nificantly higher in post-menopausal women
1.68  ±  0.36  mg/l than pre-menopausal women.
0.73 ± 0.24 mg/l. These findings are similar to studies
done by Shende et al. (13) and Suguna and Jayara-
jan (24). Joint guidelines from the Centers for Dis-
ease Control and Prevention (CDC) and the Ameri-
can Heart Association (AHA) named hs-CRP as the
inflammatory marker of choice to assess CVD risk.
The guidelines support the use of hs-CRP in primary
prevention and set cut off points according to rela-
tive risk categories: low risk (<1.0  mg/l), average
risk (1.0–3.0 mg/l), and high risk (>3.0 mg/l) (25).
According to our study post-menopausal women are
having increased risk for CVD. Study done by Sug-
una and Jayarajan the hs CRP levels were not statisti-
cally increased among the post-menopausal women
compared to pre-menopausal women. However the
frequency of elevated hsCRP levels (>1.0  mg/dl)
in post-menopausal women was 4.33 times greater
than in Pre-menopausal women.
Plasma CRP levels reflect the amount and activ-
ity of pro inflammatory cytokines such as TNF- α,
IL-1 and IL-6 which are involved in the process of
atherosclerotic plaque formation. CRP synthesis in
the liver is largely under the control of IL-6 which
is secreted from adipose tissue (13). During meno-
pause changes in the concentration of sex hormones,
influence levels of hsCRP, which binds to oxidized
LDL, causing increase expression of adhesion mol-
ecules enhancing the atherogenicity of LDL (24).
In a study on healthy men by Ridker et al., it was
observed that evaluation of CRP added to the predictive
value of lipids on risk of first MI. It was seen that patients
with highest tertile of both TC and CRP had relative risk
of first MI 5.3 times more than that of individuals in
lowest tertile of both parameters (26). In our study also
both TC and hs CRP are significantly increased.
We observed significant positive correlation
between hs CRP with LDL and the negative correla-
tion between hs CRP with HDL level. This suggests
that unfavourable lipid profile may facilitate the for-
mation of foam cells in arterial wall increasing the
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Yasmeen fathima et al.: A study of CVD risk………………markers
inflammatory activity. Ridker et al. in their study did
a comparison of LDL-cholesterol and CRP among
apparently healthy women. They found minimal
correlation (r = 0.08) between LDL cholesterol and
CRP (27), suggesting that each level predicted risk
in different groups (28).
According to the present study post-menopausal
women are having high risk for CVD risk and serum
hsCRP along with lipid profile should be used as
periodic screening for CVD risk assessment.
Conclusion
The current study showed significant dyslipidae-
miaandincreased hsCRP levelin post-menopausal
women compared to pre-menopausal women
clearly indicating that post-menopausal women are
at increased risk for CVD. Addition of hsCRP test-
ing to traditional lipid screening improves CVD risk
prediction. And it should be considered as a routine
biochemical parameter in order to prevent the occur-
rence of CVD and to prolong life in postmenopausal
women. And also intervention should begin in peri-
menopausal state which include specific health
education, regular exercise, healthy diet, periodic
screening of lipid profile and hs CRP, stress free life.
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Biomedicine: 2016; 36(1): 033-038 033

Assessment of serum leptin levels and lipid profile in hypertensive obese cases
Keshavamurthy H.R.1 and Sunitha S.2
Department of Biochemistry, Pariyaram Medical College,
1

Kerala University of Health Sciences, Thrissur, Kerala, India.

Department of Biochemistry, Kempegowda Institute of Medical Sciences,
2

Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka, India.

(Received: Dec 2016   Accepted: Mar 2016)

Corresponding Author
Dr. Keshavamurthy H.R. Email: keshavamurthy.calmness@gmail.com

Abstract

Indroduction and Aim: Obesity is associated with an increased risk of morbidity and mortality as well as reduced
life expectancy. It is well known that obesity is related to hypertension through mechanisms such as sympathetic
overactivity and excess renal sodium reabsorption. Obesity and hypertension are also linked by leptin, a peptide
produced by the adipocytes that is elevated in obese individuals. The purpose of the study was to estimate and
compare Serum Leptin levels and Lipid profile in Hypertensive obese and Normotensive obese subjects.

Materials and Methods: The study was carried out in 45 Hypertensive obese cases, in the age group of
30–60 years and 45 age and gender matched Normotensive obese controls. Fasting blood samples were col-
lected. Serum lipid profile and Leptin were estimated by enzymatic and ELISA methods respectively.

Results: The obese Hypertensive group showed significantly higher values of Leptin (p < 0.05), total choles-
terol (TC) (p < 0.05), TC/HDL ratio (p < 0.05), LDL/HDL ratio (p < 0.05), SBP (p < 0.05) and DBP (p < 0.05).
The obese Hypertensive group also showed significantly lower values of HDL (p < 0.05). Leptin showed a
positive significant correlation with BMI in the obese Hypertensive group.

Conclusion: All the above derangements confirm that Leptin correlates with Hypertension and the mecha-
nisms for development of selective Leptin resistance seem to be the main leading cause for the development
of obesity related hypertension. And the dyslipidemia along with obesity and hypertension places the patient
at a higher risk of metabolic syndrome.

Key words: Hypertension, Leptin, Lipid Profile, Obesity

Introduction cardio-vascular and cerebrovascular diseases,

O
besity has reached epidemic proportions
in India in the 21st century with morbid
obesity affecting 5% of the country’s
population. Obese patients are more prone to develop
diseases such as Hypertension, Type II Diabetes,
Hyperlipidemia, Cholelithiasis, Arteriosclerosis,
certain type of cancers and osteoarthritis.
Hypertension and obesity are common risk fac-
tors for the development of cardiovascular dis-
eases, along with diabetes and hyperlipidemia.
Almost half of the hypertensive patients are
obese and the prevelance of hypertension in

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034 Keshavamurthy, Sunitha: Assessment of ………………. obese cases
obese individuals is two times higher than in
general population (1).
Leptin, an adipocyte derived hormone regulates
apetite and enhances energy expenditure by acti-
vating sympathetic nerve activity to thermogenic
brown adipose tissue (2). However in common
obesity leptin loses the ability to inhibit energy
intake and increase energy expenditure (3).
Leptin is a possible mediator of obesity related
hypertension since it acts in the hypothalamus by
stimulating sympathetic nervous system (SNS)
centrally, which indirectly causes vasoconstric-
tion and excess renal sodium absorption (4). The
purpose of our study was to estimate and compare
serum Leptin levels and lipid profile in Hyperten-
sive obese and Normotensive obese subjects.
Materials and Methods
The study was carried out on 45 hypertensive obese
cases and 45 age and sex matched normotensive
obese cases in the age group of 30–60  years. The
study was conducted at Kempegowda institute of
Medical sciences and hospital. The diagnosis of
Obesity was fulfilled as per WHO criteria and the
diagnosis of Hypertension was fulfilled as per JNC-
7th report. Patients with Diabetes mellitus, endocri-
nal disorders, family history of hyperlipidemia, car-
diovascular disease, patients taking systemic drugs
especially lipid lowering agents, smoking, alcohol
users and other conditions which may alter lipid
profile were excluded from the study. The institu-
tional ethical committee approved the study pro-
tocol. Informed consent was obtained from all the
participants.
A pre-structured and pre-tested proforma was used
to collect the data. Baseline data including age,
BMI, detailed medical history, clinical examinations
and relevant investigations were included as part of
the methodology. Serum Leptin and serum lipid pro-
file were measured in all participants from morning
blood samples collected after 12 hours of fasting.
Serum Leptin was measured by sandwich ELISA
method (Diagnostic Biochem Canada Inc. Cat. No.
CAN-L-4260; Version; 8.1; August 2009). Serum
Cholesterol was estimated by enzymatic, colori-
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metric method. Serum triglycerides was estimated
by enzymatic colorimetric test. Serum HDL-cho-
lesterol was estimated by homogenous enzymatic
colorimetric test. Serum VLDL cholesterol was
calculated according to the formula VLDL = TG/5.
Serum LDL-cholesterol estimated by homogenous
enzymatic colorimetric assay. TC/HDL and LDL/
HDL ratios were determined. Body mass index
(BMI) was calculated as the ratio of weight (kg) to
height squared (m2).
Statistical analysis
Null hypothesis. There is no significant difference
in the mean value of parameter between the two
groups, that is, mean1  =  mean 2.
Alternate hypothesis. There is significant differ-
ence in the mean value of parameter between the
two groups, that is, mean ≠ mean 2.
Level of significance. α 0.05
SPSS software version 13.0 was used for statisti-
cal analysis.
Statistical test used. t-Test and Mann-Whitney test
were both used. Correlation analysis between Lep-
tin, BMI and lipid profile were done using Pear-
son’s rank correlation.
Decision criterion. If p < 0.05, we reject the null
hypothesis and accept the alternate hypothesis.
Results
Among the total sample of 90, 16 males (36%) and
29 females (64%) were hypertensive obese cases
(Table 1 and Fig. 1). The age ranged from 30 to
60 years and the maximum number of cases were
in the age group of 31–39  years but the mean of
age between the cases (41.64 ± 8.25) and controls
(42.00 ± 7.69) was not statistically significant.
Cases Controls
Gender
N % N %
Male 16 36 16 36
Female 29 64 29 64
Total 45 100 45 100
Table 1  Gender distribution in the two Table groups
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Keshavamurthy, Sunitha: Assessment of ………………. obese cases 035

Case Mean ± SD Control Mean ± SD p-Value

BMI 33.94 ± 2.99 32.62 ± 2.16 0.051
TC 213.07 ± 43.58 191.64 ± 33.30 0.013*
TG 175.07 ± 81.06 160.00 ± 74.23 0.337
LDL 140.84 ± 44.30 127.40 ± 32.47 0.196
VLDL 38.42 ± 13.55 35.47 ± 6.61 0.591
HDL 36.29 ± 4.28 41.44 ± 4.53 ˂0.001*
TC/HDL 5.97 ± 1.45 4.71 ± 1.10 ˂0.001*
LDL/HDL 3.96 ± 1.37 3.14 ± 0.95 0.003*
LEPTIN 46.83 ± 16.31 36.11 ± 12.41 0.001*
Table 2  Comparison of Biochemical results of study subjects
* Denotes significant difference.

The mean of BMI of the case and controls groups (p = 0.001) (Table 2 and Fig. 2). A significant posi-
were 33.94 ± 2.99 and 32.62 ± 2.16, respectively tive correlation between Leptin levels with BMI
which was not found to be statistically significant (ρ = 0.231, p = 0.029), TG (ρ = 0.347, p = 0.001)
(p = 0.051) (Table 2 and Fig. 2). and negative correlation with HDL (ρ  =  0.318,
The mean TC levels in the cases and controls were p = 0.002) has been shown in Table 4.
213.07 ± 43.58 and 191.64 ± 33.30, respectively,
which was found to be statistically significant Parameter p p-Value
(p = 0.013). The mean TC/HDL ratio in the cases BMI 0.231 0.029*
and controls were 5.97  ±  1.45 and 4.71  ±  1.10, TC 0.078 0.468
respectively which was found to be statistically TG 0.347 0.001*
significant (p  <  0.001). There was no significant LDL 0.085 0.424
difference between the case and control group with
HDL − 0.318 0.002*
respect to TG, LDL-C, VLDL-C.
VLDL − 0.114 0.286
The mean HDL-C levels in the cases and controls TC/HDL 0.180 0.090
were 36.29 ± 4.28 and 41.44 ± 4.53 respectively,
LDL/HDL 0.188 0.076
which was found to be statistically significant. The
mean LDL/HDL ratio in the cases and controls Table 4  Correlation between Leptin and other parameters
(Pearson’s Rank Correlation)
were 3.96  ±  1.37 and 3.14  ±  0.95, respectively,
which was found to be statistically significant * Denotes significant correlation.

(p = 0.003).
Higher mean Leptin was recorded in cases com- Discussion
pared to controls and the difference in mean Leptin Since Leptin is an adipose tissue-derived hormone,
between the two groups is statistically significant it is possible that its association with BP is partly
explained by obesity and weight gain, one of the
Age group Cases Controls well-known risk factors for hypertension (5). The
(years) N % N % results from earlier studies showed strong corre-
31–39 20 44 18 40 lation between leptin and hypertension in humans
40–49 16 36 17 38 suggesting that leptin has a significant role in the
50–59 9 20 10 22 pathophysiology of obesity induced hypertension,
but some studies were not able to confirm this
Total 45 100 45 100
association (6). As previous studies revealed that
Table 3  Sample distribution according to age group increased leptin levels combined with decreased

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036 Keshavamurthy, Sunitha: Assessment of ………………. obese cases
NO production and enhanced sympathetic activ-
ity may contribute to blood pressure elevation in
the obese (7). In this study the difference in mean
Leptin between the obese hypertensive and obese
normotensive is statistically significant. A signifi-
cant positive correlation of leptin with BMI is pres-
ent. This was consistent with the study by Vedrana
et al. (1) who showed plasma leptin levels were
significantly higher in hypertensive obese patients
than in normotensive obese patients in both gen-
ders. Duanduan et al. (8), Caroline et al. (9), Cos-
tas et al. (10), Kawaljit et al. (11), also showed
that plasma leptin levels were significantly higher
in hypertensive patients than the normotensive
patients. This study was in contrast with Freddy et
al. (4) who showed that plasma leptin levels were
not significantly higher in hypertensive patients
compared to normotensive patients.
Leptin has no effect on blood pressure in healthy
eutrophic subjects because it has pressor and
depressor effects that are in constant balance in
healthy individuals (12). In obese hypertensives
the balance between those pressor and depressor
mechanisms is damaged. The possible explana-
tion is selective leptin resistance (13) where only
pressor effects take place and depressor effects of
leptin are lost. The mechanism of selective leptin
resistance has still not been identified, but theo-
ries involve intracellular signaling disruption and
saturable transport across blood-brain barrier (14).
Due to selective leptin resistance, the preserved
sympathoactivation causes pressor effects in the
kidneys and blood vessels, thus elevating blood
pressure and leading to further cardiovascular
complications.
It should be noted that some of the hypertensive
patients in our study were treated with antihyper-
tensive medications. Previous studies suggest that
antihypertensive agents have metabolic effects
beside blood pressure lowering such as increas-
ing level of adiponectin and decreasing leptin.
Therefore, the difference between plasma leptin
levels in hypertensive and normotensive patients
might have been greater if there was no influence
of antihypertensive medication (15). Central or
abdominal obesity is typical for males and acting
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through many CHD factors, including disturbances
in plasma glucose, insulin, total cholesterol, low-
density lipoprotein cholesterol and apolipopro-
teins A-I and B, can influence the development of
atherosclerosis (16). Intraabdominal fat may lower
HDL levels by increasing the fractional catabolic
rate of high density lipoprotein particles contain-
ing apo A-I, suggesting a mechanism, by which
central adiposity may be proatherogenic (17).
There was a female preponderance in this study
with 36% of the males and 64% of the females
were obese hypertensives. In our study it is also
shown that the mean BMI is higher in cases than
in controls and the difference between them is not
statistically significant. This was consistent with
the study by Pooja et al. (18) Obesity and dyslipi-
demia are commonly associated with a recognized
risk for the development of Metabolic syndrome.
Metabolic syndrome is a complex of interrelated
risk factors for cardiovascular disease and diabetes
mellitus. These factors include raised blood pres-
sure, elevated glucose, cholesterol and Triglycer-
ide levels and low HDL-C (19). Hypertension is
recognized globally as a major public health prob-
lem. It is also a well-known risk factor for coro-
nary heart disease, type 2 diabetes mellitus and
renal diseases (20). In our study the mean total
cholesterol, TC/HDL ratio and LDL/HDL ratio
was higher in cases than in controls and the dif-
ference between them was found to be statistically
significant and this was consistent with the study
by Bhatti et al. (21). The mean HDL is lower in
cases than in controls and the association is sta-
tistically significant and this was also consistent
with Bhatti et al. This study did not show a sta-
tistically significant difference between cases and
controls regarding Triglycerides (TG), LDL-C and
VLDL-C and this was consistent with the study by
Szczygielska et al. (22).
The parameters such as Leptin, Total Cholesterol,
TC/HDL, LDL/HDL were significantly elevated in
the study group and HDL was significantly lower.
All the above derangements confirm that Leptin
correlates with Hypertension and the mechanisms
for development of selective Leptin resistance
seem to be the main leading cause for the devel-
Biomedicine – Vol. 36 No. 1: 2016
Keshavamurthy, Sunitha: Assessment of ………………. obese cases
opment of obesity related hypertension. And the
dyslipidemia along with obesity and hypertension
places the patient at a higher risk of metabolic
syndrome. Thus follow-up of these patients with
regard to the development of diseases associated
with atherosclerosis may be beneficial.
Because our study consisted of a limited number
of cases and controls from a single population,
further studies with large number of cases will be
beneficial in elucidating the relationship between
Leptin, obesity, Hypertension, Lipid profile and
the atherosclerotic risk factors.
Acknowledgments
I owe my thanks to Dr. C. Yogitha, Associate Pro-
fessor, Department of Medicine, Kempegowda
Institute of Medical Sciences, Bangalore, for her
expert guidance and warm support. I heartily
acknowledge Dr. B.V. Ravi, Professor and Head,
Department of Biochemistry, Kempegowda Insti-
tute of Medical Sciences, Bangalore for his sug-
gestions and support in successful compilation of
this work. My sincere thanks to Mr. Tejasvi T.V,
who helped me in the statistical analysis of this
study and gave it to me on time.
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Biomedicine: 2016; 36(1): 039-043 039

Urea reduction rate as dialysis adequacy indicator and serum albumin as

mortality indicator in hemodialysis patients
Shanthala D.,1 Indumati V.,1 Krishnaswamy D.,1 Vijay Rajeshwari V.,1
Ramesh,1 and Shilpa A1
1
Department of Biochemistry, Vijaynagar Institute of Medical Sciences, Ballari, Karnataka, India.
(Received: Dec 2016   Accepted: Mar 2016)

Corresponding Author
Shanthala D. Email: shanthala.ravikumar@yahoo.com

Abstract

Introduction and Aim: Hemodialysis (HD) provides renal replacement therapy for those with end-stage kid-
ney disease (ESKD). Urea reduction rate (URR) is used as a marker of dialysis adequacy. Hypoalbuminemia
is a major risk factor for mortality and morbidity in ESKD patients. Recent studies have shown that both URR
and serum albumin levels are predictors of mortality in HD patients. Hence present study was undertaken to
assess the achievement of URR as dialysis adequacy predictor and to assess whether serum albumin levels
predicts mortality in ESKD patients on HD.

Materials and Methods: We analyzed Blood Urea levels by Urease method, in pre-dialysis and post-dialysis
blood samples of 35 patients who were on HD. URR was calculated by (predialysis urea − postdialysis urea)
divided by predialysis urea and it was expressed in %. Serum albumin was estimated in post-dialysis blood
sample by Biuret method. The patients were followed up for a period of 1 year and any mortalities during this
period were noted. Data analysis was done by using SPSS, version 17.

Results: There was statistically significant decreased in post dialysis blood urea levels (42.88 ± 9.88 mg/dl)
when compared to pre-dialysis blood urea levels (117.91 ± 26.83 mg/dl) with p-value of <0.001. URR was
found to be 63.65% ± 3.35%. The mean serum albumin of HD patients was 2.95 ± 0.35 g/dl. There was no
significant change in serum albumin levels in HD patients who died during the course of the study.

Conclusion: The National kidney foundation—kidney disease outcome quality initiative (NKF-K/DOQI)
target of the mean urea reduction rate (URR) was almost achieved, thus suggesting the adequacy of the dialy-
sis. Though mean serum albumin is decreased to 2.9 g/dl (<3.5 g/dl), in our study it was not associated with
increased mortality in HD patients.

Key words: Chronic Kidney Disease, Hemodialysis, NKF-K/DOQI, URR

Introduction unwanted solutes, equilibration of desired solutes

D
and also helps in fluid management. The provision
ialysis provides renal replacement therapy
of dialysis requires some assessment of whether
for those with end-stage renal disease
the delivered dose is adequate for the patient or
(ESRD). Dialysis provides removal of
not (1).

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040 Shanthala et al.: Urea reduction ……………………. hemodialysis patients
A number of studies have demonstrated a cor-
relation between the delivered dose of the hae-
modialysis and the patient mortality and morbid-
ity (2–6). These studies have demonstrated that
the mortality among ESRD patients was lower
when sufficient hemodialysis (HD) treatments
were provided. The clinical signs and symptoms
alone were not the reliable indicators of the HD
adequacy (7).
Adequacy of dialysis can be assessed in several
ways. The most common acceptable methods
are—formal urea − kinetic kt/v, urea reduction rate
(URR), natural log kt/v and the Daugirdas second
generation formula (1). The URR was first popu-
larized by Lowrie and Lew in 1991 as a method of
measuring amount of dialysis that correlated with
patient outcome (2).
Of the three methods, that is, urea kinetic model-
ling, kt/v and URR, which are considered appro-
priate by National kidney Foundation—Kidney
Disease Quality Initiative (NKF-K DOQI) for
measuring the delivered dose of HD, URR is the
simplest and most commonly used parameter to
express dialysis dose (8). Urea is a small, readily
dialyzed solute which is the bulk catabolite of the
dietary protein that constitutes 90% of the waste
nitrogen accumulated in body. Although urea is
not only the uremic toxin, all current indices of
dialysis dose are based on urea measurements,
and thus set urea removal as the major goal of
haemodialysis (1). The NKF-KDOQI guidelines
recommend that the adequacy of the dialysis
dose should be measured routinely, typically on a
monthly basis with a target of 65% for URR per
dialysis session, which is delivered three times a
week (9).
Serum albumin is the principal nutritional marker,
used to identify malnutrition in patients with
chronic kidney disease (CKD) (10). Serum albu-
min levels are lower in dialysis patients and are
powerful predictors of mortality (11). It is well
established that albumin levels are decreased in
patients with inflammatory disorders and other ill-
ness. Possible contributory mechanisms include
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down regulated production of albumin mRNA by
the liver, leading to reduced synthesis, increased
catabolism and increased vascular permeability
(12,13).
NKF-K/DOQI guidelines for nutrition recom-
mended, serum albumin to be measured routinely
in long term dialysis patients and to achieve a tar-
get of ˃3.5  g/dl. The guidelines focused on the
inverse association between the serum albumin
and mortality (9).
Hence the present study was undertaken,
1. To estimate URR and Serum albumin in HD
patients.
2. To assess the achievement of URR of 65% as
dialysis adequacy predictor as per NKF-K/
DOQI guidelines.
3. To assess whether Serum albumin levels pre-
dict mortality in CKD patients on HD.
Materials and Methods
Our study included 35 CKD patients who were on
HD. They were randomly selected from HD unit
of Nephrology, VIMS, Ballari, Karnataka, India.
The study was approved by the Institutional ethi-
cal committee. An informed consent was obtained
from all the 35 patients.
Inclusion criteria
CKD patients on HD aged between 25 and 60 years
of age, who were on regular (three times per week)
HD sessions.
Exclusion criteria
Liver diseases, chronic alcoholics, malignancies,
congenital renal disorders.
Dialysis prescription
All patients were hemodialysed using Fresenius
Medical Care (4008S) hemodialyzer machine
through permanent arteriovenous fistulas. They were
dialyzed with polysulphone low flux dialyzer having
Biomedicine – Vol. 36 No. 1: 2016
Shanthala et al.: Urea reduction ……………………. hemodialysis patients
a blood flow rate of 180–200 ml/min and dialysate
flow rate of 200–400 ml/min for 3–4 hours. It was
performed thrice a week.
Laboratory Analysis
Three milliliters of blood sample was collected
before initiation and after termination of haemodi-
alysis during midweek HD session, in each month
for three consecutive months. Blood urea levels
were measured in all the samples using Glutamate
dehydrogenase -Urease method and the mean of
pre-dialysis and post-dialysis urea levels were
calculated. The other parameters measured were
Blood glucose by glucose oxidase and peroxidase
method, Serum Creatinine by modified Jaffe’s
method in pre-dialysis blood sample and Serum
albumin by Bromocresol Green method in post-
dialysis blood sample.
URR was calculated by the following formula:
(pre dialysis urea − post dialysis urea) divided
by pre-dialysis urea and it was expressed in per-
centage. The patients were followed up for a period
of 1 year and any mortality during this period was
noted. Statistical analysis was performed using
SPSS, version 17 using paired two-tailed Student’s
t-test for Urea.
Data are expressed as mean  ±  standard deviation
(SD). A p-value of ˂0.05 was considered statisti-
cally significant. The patients were followed up for
a period of 1 year and any mortalities during this
period were noted.
Results
Among the 35 CKD Patients in our study, 3
were diabetic nephropathy, 4 were hyperten-
Descriptive Statistics
Parameter No. of cases (n)
Pre dialysis urea (mg/dl) 35
Post dialysis urea (mg/dl) 35
URR (%) 35
Table 1  Pre and Post dialysis Urea levels.
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041
Fig. 1  Pre and Post dialysis Urea levels.
sive nephropathy and 28 patients were non dia-
betic CKD. The mean age of the CKD patients in
the study was 38.1  ±  12.5  years. Among the 35
patients, 26 (74.6%) were males and 9 (25.6%)
were females.
There was statistically significant decrease in post-
dialysis blood urea levels (42.88  ±  9.88  mg/dl)
when compared to pre-dialysis blood Urea levels
(117.91  ±  26.83  mg/dl) with a p-value of ˂0.001
(Table 1 and Fig. 1).
The Urea reduction rate (URR) in 35 patients was
63.65% ± 3.35%. The mean Serum Albumin levels
in HD patients were 2.95 ± 0.35 g/dl. The serum
albumin levels in different chronic kidney diseases
are shown in Table 2 and Fig. 2.
The mean blood glucose levels were
105.32 ± 17.61 mg/dl, mean Serum Creatinine lev-
els were 8.70 ± 2.4 mg/dl and mean Serum protein
levels were 5.75 ± 0.66 g/dl in the pre-dialysis blood
sample. During the follow up period of 1 year, three
patients died with a mortality rate of 8.5%.
Discussion
The National Cooperative Dialysis Study (NCDS)
p-value =0.001
Min Max Mean ± SD (g/dl)
84 195 117.91 ± 26.83
24 60 42.88 ± 9.88
56 71 63.65 ± 3.35
Biomedicine – Vol. 36 No. 1: 2016
042
Disease
Non diabetic CKD
Diabetic nephropathy
Chronic glomerular nephropathy
HTN nephropathy
IgA nephropathy
Obstructive nephropathy
Total
Table 2  Serum Albumin levels in different types of Chronic Kidney Disease.
identified the dose of dialysis delivery as a factor
affecting patient morbidity and mortality, and this
led to guidelines for prescribing dialysis dose (14).
A number of studies have identified that the URR
and Serum albumin concentration as powerful pre-
dictors of mortality in patients on HD (15).
In our study URR was 63.65%  ±  3.35% with a
range of 60.3–67%. This shows that URR was
almost achieved as per the recommended guide-
line of NKF-K/DOQI to suggest the adequacy of
the dialysis dose. Similar to our study Sunanda et
al. (16) reported a URR of 66.4%. Brimble et al.
(17) in one study reported URR of 72% and another
study (18) by the same another reported URR of
67.1%. Owen et al. (19) reported that a URR of
55–59% has relative risk of 1.28 compared to a
URR of 65–69%.
Our study showed that the mean Serum albumin
levels were 2.95 ± 0.35 g/dl and it did not reach the
Fig. 2  Serum Albumin levels in different types of Chronic Kidney
Disease.
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Shanthala et al.: Urea reduction ……………………. hemodialysis patients
Frequency Percentage Sr. Albumin (g/dl)
25 80.0 2.94 ± 0.36
03 05.7 2.76 ± 0.46
01 02.9 3.20
04 05.7 3.0 ± 0.29
01 02.9 2.50
01 02.9 3.40
35 100 Mean = 2.95 ± 0.35
target levels (3.5  g/dl) of NKF-K/DOQI. In con-
trast, Colling et al. (4) reported that patients with
Serum albumin levels of less than 3.5 g/dl have a
relative mortality risk of 3.13, compared to albu-
min levels of 4.0 g/dl.
In our study though there was a positive corre-
lation between URR and Serum albumin levels
with a strength of 0.08, it was not statistically
significant, indicating that Serum albumin can-
not be used as a mortality marker. The mortal-
ity rate was only 8.5% in our study, though the
serum albumin levels (2.95 ± 0.35 g/dl) were less
than the target levels of 3.5 g/dl. There was also
no significant difference in serum albumin levels
in the three patients who died when compared to
other patients.
Hertel et al. (15) concluded that high URR
and Serum albumin levels are associated with
improved mortality statistics in the HD patients.
The low albumin levels that are encountered in
incident dialysis patients are likely a consequence
of a combination of protein -calorie malnutrition,
inflammation and plasma volume expansion (20).
Conclusion
The NKF-KDOQI target of the mean Urea reduc-
tion rate (URR) was almost achieved in our study,
suggesting the adequacy of the dialysis dose.
Though the mean Serum albumin levels were
2.95  ±  0.35  g/dl (less than the target of 3.5  g/dl)
in our study, it was not associated with increased
mortality in HD patients.
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Shanthala et al.: Urea reduction ……………………. hemodialysis patients
References
  1. Kerr, P., Perkovic, V., Petrie, J., Agar, J., and
Disney, A. Dialysis Adequacy (HD) Guidelines.
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with Renal Impairment. 2005: 1–7.
  2. Lowrie, E.G., and Lew, N.L. The urea reduc-
tion ratio (URR) a simple method for evaluat-
ing hemodialysis treatment. Contemp. Dial.
Nephrol. 1991; 12: 11–20.
  3. Fernandez, J.M., Carbonell, M.E., Mazzuchi,
N., and Pelruccelli, D. Simultaneous analysis of
the morbidity and mortality factors in chronic
hemodialysis patients. Kidney Int. 1992; 41:
1029–1034.
  4. Colling, A.J., Ma, J.Z., Umen, A., and Kes-
haviah, P. The urea index and other predictors
of the survival of hemodialysis patients. Am. J.
Kidney Dis. 1994: 23: 272–272.
  5. Hakim, R.M., Breyer, J., Ismail, N., and Schul-
man, G. Effects of the dose of dialysis on the
morbidity and the mortality. Am. J. Kidney Dis.
1994; 23: 661–669.
  6. Held, P.J., Port, F.K., Wolfe, R.A., Stannard,
D.C., Carroll, C.E., Daugirdas, J.T., et al. The
dose of the hemodialysis and the patient mor-
tality. Kidney Int. 1996; 50: 550–556.
  7. Delmez, J.A., and Windus, D.W. Hemodialy-
sis prescription and delivery in a metropolitan
community. Kidney Int. 1992; 41: 1023–1028.
  8. European Best Practice Guidelines Expect
Group on Haemodialysis. Section II. Haemo-
dialysis adequacy. Nephrol. Dial. Transplant.
2002; 17(supple 7): S16–S31.
  9. National Kidney Foundation. K/DOQI Clini-
cal practice Guidelines for Haemodialysis
Adequacy, 2000. Am. J. Kidney Dis. 2001;
37(supple 1): S7–S64.
10. Allon, N.F., and Stephen, Z.F. Reassessment of
albumin as a nutritional marker in kidney dis-
ease. J. Am. Soc. Nephrol. 2010; 21: 223–230.
11. Lowrie, E.G., and Lew, N.L. Death risk in
hemodialysis patients: the predictive value of
commonly measured variables and an evalua-
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12. Moshage, H.J., Janssen, J.A., Franssen, J.H.,
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Walace, P.I., Ledingham, I.M., and Calman,
K.C. Increased vascular permeablility: a major
cause of hypoalbuminaemia in disease and
injury. Lancet. 1985; 1: 781–784.
14. Gotch, F.A., and Saegent, J.A. A mechanistic
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McConnell, K.R., and Caruana, R.J. Correla-
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Carlisle, E.J. Comparism of the volume of the
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19. Owen Jr. W.F., Lew, N.L., Liv, Y., Lowrie, E.G.,
and Lazarus, J.M. The urea reduction ratio and
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044 Biomedicine: 2016; 36(1): 044-048

Study of oxidative stress and apolipoprotein A-I in reduction of reverse cholesterol

transport in type 2 diabetes mellitus
Suman Doddamani,1 Shashikant Nikam,1 Padmaja Nikam,1 and Vishwanath Patil2
Department of Biochemistry, Belgaum Institute of Medical Sciences, Belagavi, Karnataka, India.
1

2
Department of Medicine, Karnataka Institute of Medical Sciences, Hubli, Karnataka, India.
(Received: Jan 2016   Accepted: Mar 2016)
Corresponding Author
Shashikant Nikam. Email: nikam31@gmail.com

Abstract

Introduction and Aim: Diabetes Mellitus (DM) refers to a group of common metabolic disorders that share
the phenotype of hyperglycemia. Oxidative stress plays an important role in the development and progression
of diabetes and its complications. The present study has been undertaken to evaluate the effect of oxidative
stress on apolipoprotein A-I in newly detected type 2 DM. 200 participants were enrolled in the study.

Methods: 100 with newly detected type 2 DM were taken as study group and 100 age and sex matched
healthy participants were taken as control group. Biochemical parameters such as serum Malondialdehyde
(MDA), Superoxide dismutase (SOD), Apolipoprotein A-I, Total cholesterol, Triglyceride, HDL, LDL, VLDL
and erythrocyte reduced Glutathione levels were analyzed in all the participants.

Results: The patients with type 2 DM showed increased oxidative stress and decreased apolipoprotein A-I and
HDL levels.

Conclusion: Hence the present study concludes that increased oxidative stress and glycated apolipoprotein
A-I might be responsible for the reduction of reverse cholesterol transport (RCT), which may lead to increased
incidence of atherosclerosis and cardiovascular complications in type 2 Diabetes Mellitus.

Key words: Oxidative stress, Apolipoprotein A-I, RCT, Diabetes Mellitus.

Introduction Diabetes. 50% of diabetic patients’ deaths occur due

D
iabetes Mellitus refers to a group of
common metabolic disorders that share
the phenotype of hyperglycemia (1).
Oxidative stress is a condition in which there is
either increased rate of free radical production or
there is impairment in antioxidant mechanisms (2).
Increased oxidative stress is one of the etiologies
for the development and progression of Diabetes
and its complications (3). Atherosclerosis is one
of the major complications associated with type 2
to cardiovascular disease (4). Low HDL is a strong
risk factor for the development of atherosclerosis.
The cardioprotective role of HDL is related to its
role in Reverse Cholesterol Transport (RCT).
Apolipoprotein A-I is the most abundant protein
in HDL, the concentration of apo A-I is known to
be inversely correlated with cardiovascular risk
(5). HDL associated apo A-I play a crucial role
in cholesterol homeostasis by regulating reverse
cholesterol transport delivering it to the liver (5).

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Suman Doddamani et al.: Study of Oxidative stress … Diabetes Mellitus
The present study was undertaken to study the role of
oxidative stress and apolipoprotein A-I in reduction
of reverse cholesterol transport in type 2 DM.
Materials and Methods
The study group was comprised of 100 newly
detected type 2 diabetic patients in the age group of
30–60  years visiting medicine Out Patient Depart-
ment of BIMS (Belgaum Institute of Medical Sci-
ences) Hospital, Belgaum. The diagnosis of Diabetes
Mellitus was done by senior physician. The diagno-
sis of type 2 DM was confirmed by measuring fast-
ing blood glucose (>126  mg/dl) and 2  hour OGTT
(>200 mg/dl) values on two occasions as per Ameri-
can Diabetic Association’s revised criteria. 100 age
and sex matched healthy participants were taken as
control group. The study was conducted from Decem-
ber 2011 to May 2013 at department of Biochemis-
try, BIMS, Belagavi. All authors hereby declare that
the experiments have been examined and approved
by institutional ethical committee and have therefore
been performed in accordance with the ethical stan-
dards laid down in the 1964 declaration of Helsinki.
After obtaining informed written consent, 10 ml of
12 hours fasting venous blood sample was collected
from diabetic patients and the control participants
under all aseptic conditions. The blood samples were
used for measuring various parameters. Estimation
of apolipoprotein A-I was done by measurement of
antigen-antibody reaction by the end-point method
using immunoturbiditimetry (6). HDL cholesterol
(6) and total cholesterol was measured by CHOD-
POD method (6). Triglyceride estimation was done
by GPO-PAP method (6). VLDL and LDL choles-
terol was calculated by formula (6). Fasting blood
glucose was measured by Glucose Oxidase Peroxi-
dase method (7). Serum MDA levels were measured
by method of Wilbur et al. (8). Serum SOD was
measured by Marklund and Marklund method (9).
Erythrocyte reduced glutathione (GSH) measure-
ment was done by method of Beutler et al. (10).
Exclusion criteria
Patients on hypolipidemic drugs, antioxidant sup-
plements, steroids and oral contraceptives were
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045
excluded. Known cases of hypothyroidism, hyper-
thyroidism, Cushing’s syndrome, kidney diseases,
hepatic diseases, alcoholics, smokers, tobacco
chewers and patients with Type 1 Diabetes Mellitus
were also excluded from the study.
Limitations
The duration of diabetes before the formal diagnosis
was unknown.
Statistical analysis
The results are expressed as mean ± SD. The results
are further subjected to Student’s t-test, differ-
ences between means are considered significant at
p < 0.05.
Results
Study found that serum MDA, LDL, VLDL,
total cholesterol, triglycerides were significantly
increased in type 2 DM when compared to control
group. Erythrocyte reduced glutathione, serum apo-
lipoprotein A-I, SOD and HDL were significantly
decreased in type 2 DM when compared to control
group (Table 1).
Discussion
Oxidative stress is a widely accepted participant in
the development and progression of diabetes and
its complications. Diabetes Mellitus is associated
with hyperglycemia which may lead to cellular
damage; increased extra vascular matrix produc-
tion and vascular dysfunction which have been
implicated in the pathogenesis of vascular disease
in type 2 DM (11, 12). This study shows increased
oxidative stress in diabetics when compared with
the control group.
Present study found that MDA levels were signifi-
cantly (p < 0.05) increased in type 2 DM when com-
pared to the control group (Table 1). These findings
are in agreement with the findings of Mahreen et al.
(13) and Ozdemir et al. (14). Higher blood glucose
level is associated with free radical mediated lipid
peroxidation (15). The peroxidative breakdown of
phospholipids might lead to accumulation of MDA
in type 2 diabetics.
Biomedicine – Vol. 36 No. 1: 2016
046
In healthy individuals, oxidative damage to tissue is
prevented by a system of defence called antioxidant
enzymes. Mechanisms involved in the increased
oxidative stress in diabetes include not only oxygen
free radical generation due to nonenzymatic glyca-
tion, autooxidation of glycation products, but also
changes in the tissue content and activity of antioxi-
dant defense systems (16).
Study found that serum SOD activity and concentra-
tion of erythrocyte reduced glutathione was signifi-
cantly (<0.05) decreased in type 2 DM as compared
to the control group (Table 1). These findings are
in agreement with Abou-Seif and Youssef (17), and
Yoshida et al. (18). Reduced glutathione functions as
a free radical scavenger, thus with increased oxidative
stress the levels of reduced glutathione may be low-
ered. In diabetic patients, the autoxidation of glucose
results in the formation of hydrogen peroxide which
inactivates SOD (19) and this accumulated hydrogen
peroxide may be one of the reactive oxygen species
responsible for lowered activity of SOD and raised
levels of MDA in type 2 diabetic patients. Reduced
activity of the antioxidant enzymes may increase the
susceptibility of diabetic patients to oxidative injury.
This study also found that the levels of apo A-I were
significantly reduced (p < 0.001) in newly detected
type 2 Diabetes Mellitus (Table 1) when compared
Newly detected type 2 DM
Sl. No. Parameters
1 Serum MDA (nmol/ml)
2 Serum SOD (units/ml)
Erythrocyte reduced GSH
3 4.54 ± 0.32*
(micro mol/g of Hb)
4 Apolipoprotein-A-I (mg/dl)
5 HDL (mg/dl)
6 LDL (mg/dl)
7 VLDL (mg/dl)
8 Total cholesterol (mg/dl)
9 Triacylglycerol (mg/dl)
Table 1  Biochemical parameters in newly detected Type 2 DM and Control participants.
n = number of participants, p < 0.05, significant.
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Suman Doddamani et al.: Study of Oxidative stress … Diabetes Mellitus
with the control group. Gugliucci et. Al. concluded
that apo A-I levels are decreased in type 2 DM (4).
Calvo et. al. also showed glycation of apo A-I result-
ing in decreased levels of apo A-I in type 2 DM (20).
In type 2 Diabetes Mellitus there is over production of
active oxygen and this is associated with auto-oxida-
tion of glucose which may result in glycation of apoli-
poprotein A-I. This glycated apo A-I might be respon-
sible for lowered functional capacity of apo A-I.
The levels of HDL cholesterol were significantly
lowered (p < 0.01) in newly detected type 2 Diabe-
tes Mellitus in comparison with the control group.
(Table 1) Smith and Lall (21) concluded that HDL
cholesterol was significantly lower in diabetic sub-
jects as compared to controls. It has been reported
that non-enzymatic glycation of apolipoprotein A-I
in diabetics alters the functional ability of HDL
(22). Recent studies suggest that glycation of apo
A-I adversely affects HDL metabolism (23–25)
leading to decreased levels of HDL. Decreased lev-
els of apolipoprotein A-I may lead to reduction of
reverse cholesterol transport concluded by Dodda-
mani et al. (26).
Thus the present study indicates that lowered HDL
cholesterol might lead to reduction of reverse cho-
lesterol transport, this may result in increased risk of
atherosclerosis in type 2 DM.
Controls (n = 100)
(n = 100)
9.02 ± 1.36* 5.78 ± 0.83*
2.75 ± 0.98* 3.77 ± 0.79*
6.03 ± 0.95*
133.10 ± 24.32* 188.72 ± 19.49*
33.37 ± 4.44* 48.76 ± 16.84*
130.57 ± 36.04* 95.98 ± 39.16*
40.38 ± 17.12* 29.74 ± 19.70*
205.43 ± 35.70* 175.07 ± 39.88*
227.36 ± 106.01* 155.56 ± 107.22*
Biomedicine – Vol. 36 No. 1: 2016
Suman Doddamani et al.: Study of Oxidative stress … Diabetes Mellitus
Conclusion
Present study concludes that increased oxida-
tive stress and glycated apolipoprotein A-I might
cause lowered HDL levels leading to reduction of
reverse cholesterol transport which may lead to
increased incidence of cardiovascular complica-
tions in type 2 DM.
Acknowledgments
Authors are thankful to the Director, BIMS, Bel-
agavi, for funding the project and to all teaching and
non-teaching staff of Biochemistry, General Medi-
cine and Statistics department for their help.
Conflict of Interest
The authors declare that there is no conflict of inter-
est regarding the publication of this manuscript.
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Biomedicine: 2016; 36(1): 049-056 049

Evaluation of serum high molecular weight adiponectin and lipid profiles in predicting
the risk of coronary artery disease among CAD patients

Chitra Devi M.,1 Chandra Sekhar M.,2 and Sivasubramaniam P.3

Government Dharmapuri Medical College, Dharmapuri, India.

1

2
Meenakshi Medical College & Research Institute, Enathur, Kanchipuram, India.
3
Cardiac Care Centre, Dharmapuri, India.

(Received: Feb 2016   Accepted: Mar 2016)

Corresponding Author
Chitra Devi. M. Email: chitrakmc@gmail.com

Abstract
Introduction and Aim: We sought to assess the value of serum High Molecular Weight (HMW) Adiponectin
in predicting the risk of coronary artery disease in angiographically proven CAD patients with and without
Type 2 diabetes mellitus. It has been postulated that lower concentrations of serum adiponectin can be an
effective biomarker which is linked to insulin resistance and endothelial dysfunction in both diabetic and CAD
patients.

Materials and Methods: This analytical cross-sectional study was done to evaluate serum HMW Adiponectin
by Enzyme Linked Immunosorbant Assay (ELISA) in 40 cases of angiographically proven coronary artery
disease (CAD) patients with and without diabetes of both sex, aged 40–60 years.

Results: Lower levels of HMW Adiponectin were observed in CAD patients with type 2 diabetes when
compared to CAD patients without Type 2 Diabetes.

Conclusion: Our study confirms that serum HMW adiponectin levels were much lower in CAD patients with
Type 2 diabetes in South Indians when compared with the western standards. Hence serum adiponectin level
should be considered in the laboratory work-up of Type 2 Diabetic Mellitus patients who are more prone for
the risk of CAD.

Keywords: Adipocytokine, Coronary Artery Disease (CAD), Enzyme Linked Immunosorbant Assay (ELISA),
HMW Adiponectin, Hypoadiponectinemia

Introduction diabetes in developing countries (1). Cardiovascular

D
iabetes is no more considered to be an
epidemic disease as it has turned pandemic
and appears to be a worldwide public health
problem. India is titled as the diabetic capital of
the world according to the international journal of
disease remains the biggest cause of the death
recent days and the combined effect of the diabetes
and CAD has placed a greater strain on the health
care providers to ensure a disease free healthy
population, which has become complicated with
growing sedentary life style habits (2).

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050 Chitra devi: Evaluation of serum …………………………………patients
Diabetes is considered as a CAD risk equivalent
according to the adult treatment panel of the
national cholesterol education program guidelines
which confirms that diabetes patients have a threat
for coronary events similar to that of non diabetic
patients who previously had an event (3).
Adiponectin being a protein hormone synthesised
exclusively from the white adipose tissue has a
number of metabolic functions that includes fatty
acid oxidation (4), glucose regulation and also
reduces inflammation and vascular injury (5,6).
Adiponectin an adipose specific protein belong to
collectin family (7,8) and is present profusely in
circulation, accounting for approximately 0.01% of
total plasma protein (9).
It was documented that adiponectin exists in plasma
as trimer—low molecular weight form, hexamer-
medium molecular weight, and as multimer—high
molecular weight form (10,11) of which the high
molecular weight form (HMW) of adiponectin is
considered to be the most biologically active form,
as it regulates the glucose homeostasis by mediating
insulin sensitization (12,13).
Very few epidemiological studies have investigated
the association of HMW Adiponectin and the risk
of developing CAD in type 2 diabetes. There are
strong evidences which had proved that instead of
total adiponectin, it is the HMW Adiponectin or
the ratio between total and HMW Adiponectin that
is strongly related to the development of type 2
diabetes (14)
Low levels of HMW Adiponectin has been identified
in CAD patients (15) and in type 2 diabetic patients
where the findings has suggested that adiponectin
has both anti atherogenic and antidiabetic properties,
and it acts as an endogenous mediator of both
vascular and metabolic disorders.
It has been reported that adiponectin accumulates
in the damaged vascular walls (16) when the
endothelium of the carotid arteries is damaged by a
balloon catheter in rats, and it is also observed that
adiponectin prevents the attachment of monocytes
to the endothelial cells (17) which is an early event
in atherosclerotic vascular change.
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In this study we investigated the role of serum
HMW Adiponectin in angiographically proven 40
cases of coronary artery disease (CAD) patients
with and without Type 2 diabetes of both sex,
aged between 40 and 60  years and in 20 healthy
controls and their lipid levels to rule out the possible
significant relationship which may contribute to the
atherosclerotic vascular complications in both CAD
and T2DM patients.
Grouping of Subjects
Based on the presence or absence of type 2 diabetes
mellitus, CAD (proven by coronary angiography)
patients were categorised as the follows;
Group I—Control subjects; Group II—CAD patients
with diabetes (CAD+T2DM); Group III—CAD
patients without diabetes (CAD-T2DM). Control
subjects were selected based on the following
criteria. They had no history of T2DM and CAD,
they were non smokers and non consumers of alcohol
and had no history of endocrinal dysfunctions,
hyperlipidaemia, and hypertension and they were
not on any medication.
CAD subjects with T2DM included in the study
were as under:
T2DM diagnosed according to World Health
Organization (2015) criteria and they had undergone
coronary angiography for suspected ischaemic heart
disease with the following:
The subjects had Positive exercise stress testing
in ECG, ECG changes suggesting of CAD, Acute
coronary syndrome patients and patients who were
admitted previously in the hospital for CAD and
who had undergone previous cardiac interventions
like percutaneous coronary intervention (PCI),
Percutaneous transluminal coronary angioplasty
(PTCA), and coronary artery bypass graft (CABG)
surgery. CAD subjects without T2DM included
patients with all the above said criteria and were
non-diabetic.
T2DM Patients were diagnosed when the fasting
blood glucose levels were >126  mg/dl or the
2  hours postprandial blood glucose levels were
>200 mg/dl.
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Chitra devi: Evaluation of serum …………………………………patients
Exclusion Criteria included Patients with Type I
Diabetes, Patients of Chronic alcoholism, Patients who
had Gross obesity and Body Mass Index >35 kg/m2.
Patients with inflammatory diseases, Hypothyroidism,
endocrinopathies, dilated cardiomyopathy, valvular
disease, pre existing hepatocellular disease,
cerebrovascular diseases were excluded. Patients
with renal insufficiency and patients taking
thiazolidinedione drugs were also excluded.
All the patients in each group after the selection
criteria, a detailed history was obtained, followed
with a thorough clinical examination. The protocol
of the study was approved by the members of
institution ethical committee constituted by MMCH
and RI as per ICMR guidelines and Informed consent
was obtained from each of the study subjects.
Blood Sampling and Laboratory
Examinations
All samples were collected by venipuncture about
7  ml of venous blood were withdrawn from each
subject after 12 hours of fasting for the measurement
of HMW Adiponectin, ApolipoproteinA1,
Apolipoprotein B, into plain and EDTA treated
tubes and the samples are divided as follows. Within
30 minutes, blood was analysed for fasting glucose.
4 ml blood was collected and the blood sample was
left to clot, and then centrifuged at 3000/r.p.m for
10 minutes. Serum was separated into two separate
aliquots. The first aliquot was used for laboratory
analysis of lipid profile; the second aliquot was
immediately frozen at -20°C in the deep freezer for
analysis of HMW Adiponectin.
Total cholesterol, triglycerides and high density
lipoprotein cholesterol were measured by
ChemWellÒ 2910 Automated EIA and Chemistry
Analyzer. Low density lipoprotein cholesterol was
estimated indirectly using the Friedewald formula
(LDL cholesterol  =  Total cholesterol  - HDL
cholesterol  +  1/5 Triglycerides), for subjects with
a serum TG concentration of less than 400  mg/
ml. Apolipoprotein A1 and Apolipoprotein B
were measured by enzyme linked immunosorbent
assay after the serum samples were thawed at
room temperature. Serum HMW Adiponectin
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051
concentration was measured by enzyme linked
immunosorbent assay using commercially available
human HMW Adiponectin ELISA kit (Cusabio
biotech Co., Ltd., CSB-E13400h). Apolipoprotein
A1, was measured by using commercially available
human Apolipoprotein A1, ELISA kit (AssayPro,
EA5201-1) and Apolipoprotein B was measured by
using commercially available human Apolipoprotein
B, ELISA kit (AssayPro, EA7001-1).
Anthropometric Assessments
Anthropometric indices including height and weight
were measured while subjects were in the standing
position and wearing light clothing without shoes.
Body weight was measured in kilograms to the
nearest 0.5 kg. Height was measured in centimeters
to the nearest 0.5 cm. Body Mass Index (BMI) was
calculated as the body weight in kilogram divided
by the square of height in meters (kg/m2).
CAD diagnosis criteria included Patients who had
Acute MI based on ECG, (ST elevation of atleast
0.1  mv) in two or more leads, Patients who had
Angina pectoris based on clinical history, ECG,
Coronary Angiography, Patients who had 50% or
greater organic stenosis of at least one major coronary
artery (confirmed by coronary Angiogram). Coronary
angiography findings (occlusion of a main coronary
artery branch with TIMI—Grade flow 0, 1, 2).
Statistical analysis
All data were analyzed and were expressed as
Mean  ±  SE. statistics were performed using SPSS
for windows version 17 software. Significant
differences between groups were compared by one
way analysis of variance (ANOVA) with Duncans
test for post hoc comparisons of each group. Pearson
correlation coefficients were calculated to evaluate
the relationship between serum Adiponectin and all
other study variables. p Value <0.01 was considered
as statistically significant.
Results
The anthropometric and biochemical characteristics
of the patients and control group are presented in
Table 1.
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052 Chitra devi: Evaluation of serum …………………………………patients

Group-II Group-III
Group-I (Control) p Value
(CAD+T2DM) (CAD-T2DM)
Age, yearsNS 48.45 ± 1.57 51.75 ± 1.40 51.10 ± 1.44 0.253
BMI, kg/m2** 22.62c ± 0.35 29.08a ± 0.43 26.62b ± 0.50 0.000
SBP, mmHg** 115.05c ± 0.97 135.85a ± 1.14 132.95b ± 1.51 0.000
DBP, mmHg** 72.35  ± 0.75
c
80.80  ± 1.07
a
75.75  ± 0.64
b
0.000
TGL, mg/dl** 131.10c ± 3.08 207.85a ± 5.81 162.00b ± 5.10 0.000
TC, mg/dl** 179.60  ± 1.96
b
200.50  ± 2.82
a
126.00  ± 1.80
c
0.000
HDL, mg/dl** 48.35a ± 1.21 33.00c  ± 0.59 41.15b ± 1.75 0.000
TC/HDL** 3.76  ± 0.11
b
6.12  ± 0.15
a
3.20  ± 0.19
c
0.000
LDL, mg/dl** 105.65c ± 2.55 143.90a ± 5.70 119.26b ± 4.12 0.000
HMW ADP, µg/ml** 15.70a ± 1.28 7.11b ± 0.75 12.78a ± 1.78 0.000
APO A1, mg/dl** 133.05  ± 1.38
a
102.90  ± 0.79
c
117.70  ± 1.08
b
0.000
APO B, mg/dl** 82.75b ± 1.94 165.10a ± 1.46 167.20a ± 1.75 0.000
APO B/APO A1** 0.62  ± 0.02
c
1.61  ± 0.02
a
1.43  ± 0.02
b
0.000
Table 1  Anthropometric and biochemical characteristics of patients and control subjects.

Sixty patients include 40 subjects of CAD of which
20 subjects had CAD and T2DM, 20 subjects had
CAD without T2DM and 20 control subjects.
Patients who were CAD and T2DM had the highest
BMI, SBP, DBP, total cholesterol, triglycerides;
LDL-C, TC/HDL ratio and they had the lowest
level of HMW Adiponectin, Apolipoprotein A1, and
HDL-C cholesterol. The results revealed that there
was a statistically significant differences in serum
Adiponectin levels between CAD without T2DM
patients and CAD with T2DM patients (12.78 ± 1.78
vs. 7.11 ± 0.75 µg/ml, p < 0.01), and between CAD
without T2DM and control subjects (12.78  ±  1.78
vs. 15.70  ±  1.28  µg/ml, p < 0.01) and between
patients with CAD with T2DM and control subjects
(07.11 ± 0.75 vs. 15.70 ± 1.28 µg/ml, p < 0.01) as
shown in Table 1.
Serum High Molecular Weight adiponectin levels
were significantly higher in men when compared
with women as shown in Table 2.
NS, non significant p  <  0.05; abcmean bearing
different superscript in a row differ significantly
**p < 0.01.
Statistical analysis done by ANOVA analysis (post
hoc test: duncans).
Significance fixed at *p  <  0.05, highly significant
**p < 0.001.

Serum HMW adiponectin levels, µg/ml

Groups p Value
Men, mean ± SE Women, mean ± SE
Group I (Normal) 18.79 ± 1.92 13.18 ± 1.35 0.02459
Group II (CAD+T2DM) 8.05 ± 1.02 5.35 ± 0.72 0.087873
Group III (CAD-T2DM) 14.35 ± 2.68 10.43 ± 1.80 0.292257
Table 2  Serum high molecular weight adiponectin levels between male and female subjects.

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Chitra devi: Evaluation of serum …………………………………patients 053

Description r Value p Value adipocytokines. Adipocytokines secreted by the

adipose tissue are adipsin, aphelin, visfatin, leptin,
Age, years -0.011 0.934
resistin, interleukins, TNF-α, CRP, angiotensin II,
BMI, kg/m2 -0.421** 0.001 estrogen, Plasminogen Activator Inhibitor-1 (PAI-1).
SBP, mmHg -0.470** 0.000 Among all these adipokines secreted, adiponectin is
DBP, mmHg -0.242 0.062 the only adipokine considered to be an anti diabetic
and anti atherogenic and hence not surprising to have
Triglyceride, mg/dl -0.398** 0.002 gained attraction in the midst of other adipokines.
Total cholesterol,
-0.264* 0.041 Adiponectin as a protein hormone modulates glucose
mg/dl regulation and fatty acid oxidation and hence any
HDL-C, mg/dl 0.391** 0.002 rapid change in the adiponectin levels might lead
TC/HDL-C -0.464** 0.000 to insulin resistance and dyslipidemia causing
endothelial dysfunction leading to atherosclerosis.
LDL-C, mg/dl -0.206 0.114 This could be a plausible provoking factor for the
APO-A1, mg/dl 0.512** 0.000 cardiovascular complications in T2DM patients.
APO-B, mg/dl -0.406** 0.001 Our main finding is that there were a significant
APOB/APOA1 -0.460** 0.000 lower levels of serum HMW Adiponectin in CAD
with T2DM patients when compared to CAD
Table 3  Correlati on between serum Adiponectin levels and
various cardiovascular risk factors.
alone and normal subjects. Our study supports the
findings of previous studies that lower levels of
* Correlation is significant at the 0.05 level (2-tailed). serum adiponectin is definitely associated with the
coronary artery disease risk factors in diabetes.
**Correlation is significant at the 0.01 level
Decreased levels of serum Adiponectin in coronary
(2-tailed).statistical analysis was done by pearson
artery disease patients (18,19) concurs with our study
correlation.
and it also coincides with the study of 20. Yaturu
Results revealed that there was a negative et al. (20), who has previously reported that the
correlation between serum HMW Adiponectin levels of serum Adiponectin is lower in both Type II
level and BMI (r  =  -0.421, p  =  0.001), SBP diabetic and CAD patients. The study is in line with
(r = -0.470, p = 0.000), DBP (r = -0.242, p = 0.062), the study of Hotta et al. and Kumada et al., who had
triglycerides (r = -0.398, p = 0.002), total cholesterol also already reported that adiponectin concentration
(r  =  -0.264, p  =  0.041), low density lipoprotein were significantly lower in patients with coronary
cholesterol (r = -0.206, p = 0.114), Apo lipoprotein artery disease when compared to control subjects.
B (r  =  -0.406, p  =  0.001), ApoB /ApoA1 ratio Our study is controversial with the study of
(r = -0.460, p = 0.000), TC/HDL ratio (r = -0.464, Monika et al., who had concluded that there was
p = 0.000). There was a positive correlation between no statistically significant difference found among
serum High Molecular Weight Adiponectin levels diabetic patients with or without CAD. When
and serum HDL cholesterol (r = 0.391, p = 0.002), concerned with systolic and diastolic BP, both were
Apolipoprotein A1 (r = 0.512, p = 0.000), among the found to be significantly higher in CAD patients
study groups. with Type 2 DM and are negatively correlated with
serum adiponectin. Ouchi et al., has reported that
Discussion serum adiponectin is independently correlated with
Advanced research in adipose tissue has given vasodilator response to reactive hyperaemia, and
us strong evidence that it is not just an energy assessing the serum concentration of adiponectin
storage depot, but an active endocrine organ could be an independent parameter for predicting
secreting a variety of biological substances called endothelial dysfunction in CAD patients.

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054 Chitra devi: Evaluation of serum …………………………………patients
There was a significant difference in the Adiponectin
concentration of men and women in control
subjects. Cnop et al. (21), has given a possible
reason that the sex differences in adiponectin levels
may be due to the different numbers and size of
the fat cells distribution in the individual sexes. In
our study women had lower levels of adiponectin
which may be because of the fact of more adipose
tissue deposition. Few studies have reported that
women had a higher serum adiponectin levels
when compared to men which is controversial
with our study. Nishizawa et al. (22), has indicated
that androgen has the capability of decreasing
serum adiponectin level and the androgen induced
hypoadiponectinemia could be related to a high risk
of insulin resistance and atherosclerosis in men.
In our study there was no significant difference
observed among CAD patients with and without
T2DM except in control subjects.
When considering the lipid profile, HDL cholesterol,
Apo A1 were positively correlated with serum
HMW Adiponectin in CAD patients with T2DM
and were negatively correlated with triglycerides,
total cholesterol, LDL cholesterol, Apolipoprotein
B, TC/HDL ratio, Apolipoprotein B, Apo B/ApoA1
ratio and this is in concurrence with the work done
in Japanese patients (23) The strongest correlation
is present between Adiponectin, HDL and Apo
A1. It may be postulated that adiponectin may
directly stimulate the lipoprotein lipase expression,
which might increase the production of HDL
Cholesterol levels and it may also be postulated
that an “adipovascular” axis (24) may contribute to
the increased risk of cardiovascular events in CAD
patients.
The probable reason for the low adiponectin levels
may be related with the reduced expression of Nitric
oxide and increased expression of angiotensin II and
cellular adhesion molecules from the Endothelium
(17) where studies have suggested that adiponectin
modulates the inflammatory vascular response by
inhibiting the expression of adhesion molecules
on endothelial cells (15), and inhibits endothelial
cells nuclear factor kappa B signalling (25) and
suppressing the macrophage function (26).
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Conclusion
The study reports that HMW adiponectin as an
antidiabetic, antiatherogenic adipocytokine regulates
insulin resistance and dyslipdemia. This study
suggests that, presence of lower levels of HMW
adiponectin, Apo A1 and HDL-C could be strongly
associated with endothelial dysfunction. This
mechanism may lead to atherosclerosis progressing
to coronary artery disease in Type 2 DM patients.
Despite the smaller sample size, our study provides
evidence for an inverse association between serum
HMW Adiponectin and the lipid profiles which
might lead to subsequent risk of developing CAD
among Type 2 DM in the age group between 40-
60. Hence individuals with a low level of HMW
Adiponectin, low HDL-C and low levels of Apo A1
are more prone for an increased risk of developing
both CAD and T2DM. This opens a new research
for the underlying mechanism of evaluating HMW
Adiponectin in CAD and in T2DM patients.
Therefore, lower HMW Adiponectin levels cannot
just be a bio-marker but it could also be a causal
risk factor for CAD in T2DM patients and hence
additional measurement of HMW Adiponectin in
laboratory set up might help in identifying a highly
prevalent subgroup that are at an increased risk of
developing Coronary Artery Disease.
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Biomedicine: 2016; 36(1): 057-061 057

Relationship of BMI and dental caries among children in Chennai

Visha M.G.1 and Deepa Gurunathan1
1
Department of Paediatric Dentistry, Saveetha Dental College, Chennai, India.
(Received: Jan 2016   Accepted: Mar 2016)
Corresponding Author
Deepa Gurunathan. Email: drgdeepa@yahoo.co.in

Abstract

Introduction and Aim: Dental caries (tooth decay) is a breakdown of teeth due to the activities of bacteria.
Obesity is a medical condition in which excess fat has accumulated to the extent that it has negative effects
on health. It is measured by BMI. One of the key factors for causing dental caries is diet and obesity is also
associated with dietary intake. Both obesity and dental caries are major health issues in children. To determine
the association of obesity and dental caries among children.

Materials and Methods: The study population consisted of 151 randomly selected children in Chennai popu-
lation. The children were categorised based on the BMI percentage (under weight, normal, over weight and
obese). On co-relation of caries index by Klein, Palmer and Knutson (1938) with BMI (body mass index) of
each child the results were calculated by Metric method.

Results: A total of 151 (88 girls and 63 boys) children were examined and it was found that underweight chil-
dren were 27.2%, 27.8% children were normal, 25.8% were overweight and 19.2% of children were obese.
In 151 children, 22.5% had permanent tooth decay (DT) and 49.0% had primary tooth decay (dt). Based on
the significant BMI groups; underweight children had 7.3% of permanent tooth dental caries and 22.0% had
primary tooth decay. Children with normal BMI had 14.3% of permanent tooth decay and 19.0% had primary
tooth decay. Overweight children had 38.5% of permanent tooth decay and 82.1% had primary tooth decay.
The children subjected to have higher values of BMI being obese had 34.5% of permanent tooth dental caries
and 86.2% had primary tooth decay. The presence of carious lesions was more prevalent in overweight and
obese children when compared to underweight and normal children.

Conclusion: In this study from the statistical analysis of BMI and dental caries it is concluded that there is an
association of obesity and dental caries as the prevalence of dental caries was found to be more in obese and
overweight children than normal and underweight children.

Key words: Body Mass Index, Dental Caries, Obesity

Introduction excess body fat has accumulated to the extent that

C
hildhood obesity is a major public health
problem which is increasing at a high rate
in both developed and developing countries
(1). Obesity refers to a medical condition in which
it may have a negative effect on health, leading to
reduced life expectancy and other health problems
(2,3). An individual is considered obese when their
body mass index (BMI) (4) exceeds 30  kg/m2,

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058 Visha and Deepa : Relationship of BMI……………………..in chennai
with the range 25–30  kg/m2 defined as overweight.
The measurement obtained by dividing a person’s
weight by the square of the person’s height. BMI
categories include: underweight, normal, overweight
and obese. Dental caries is defined as a localized,
progressively destructive disease of the teeth that
starts at the external surface (usually the enamel) with
the apparent dissolution of the inorganic components
by organic acids that are produced in immediate
proximity to the tooth by the enzymatic action of
masses of microorganisms (in the bacterial plaque) on
carbohydrates; the initial demineralization is followed
by an enzymatic destruction of the protein matrix with
subsequent cavitation and direct bacterial invasion; in
the dentin, demineralization of the walls of the tubules
is followed by bacterial invasion and destruction of the
organic matrix. The correlation between dental caries
and BMI consists of various contributing factors
which include biological, genetic, socioeconomic,
cultural, dietary, and environmental and sedentary
lifestyle concern (5). If the highest incidence of caries,
overweight and obesity are found in a particular group
such as those who consume foods and beverages with
a high content of refined carbohydrates (6–11), then it
is possible to establish a relationship between the two
conditions. Although community water fluoridation
has significantly reduced the prevalence of smooth
surface caries, dental caries remains to be a common
chronic childhood disease. Obesity and dental caries
are two highly prevalent health problems in both
children and adolescent. The goal of this study was
in investigating the association between dental caries
and obesity in children.
Materials and Methods
The Study sample consisted of 151 (88 girls and 63
boys) randomly selected children among Chennai
population. Children of varying ages were exam-
ined. A Questionnaire was composed and was used
in the examination of randomly selected children
in Chennai. An informed consent from parents was
obtained. The completed Questionnaire consisted of
demographic data such as Age, Sex (12) and Height,
Weight were recorded and BMI was calculated.
Intra oral examination was done as the second part
of the study. The hard tissue examination consisted
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of DMF(S) and DMF (T) caries index which was
introduced by Klein, Palmer and Knutson in 1938
and modified by WHO. BMI for each individual was
calculated based on the height and weight measured
by Metric method of calculation.
According to WHO, the ranges of BMI are catego-
rised as follows:
1. BMI less than 18.5—Underweight
2. BMI range of 18.5–24.9—Normal
3. BMI range of 25–29.9—overweight
4. BMI range of 30 and above—Obese.
Outcome variables included: (1) measures of den-
tal caries prevalence; and (2) severity of caries in
primary and permanent dentitions; (3) measures of
overweight and obese prevalence.
Descriptive statistics were used to summarize demo-
graphic data. . The collected data was analysed with
SPSS 16.0 version. To A chi-square test was used to
investigate the relationship between Obesity and BMI
at the examination describe about the data descrip-
tive statistics frequency analysis, percentage analy-
sis, mean and S.D. were used. To find the significance
between the variables Pearson’s Chi-Square test was
used. In the above statistical tool, the probability
value 0.05 was considered as significant level.
Results
The study sample consisted of 151 (88 girls and 63
boys) randomly selected children among Chennai
Fig. 1  Represents the distribution of the children based on BMI
percentage.
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Visha and Deepa : Relationship of BMI……………………..in chennai 059

Based on the significant BMI groups; underweight

children had 7.3% of permanent tooth dental caries
and 22.0% had primary tooth decay. Children with
normal BMI had 14.3% of permanent tooth decay
and 19.0% had primary tooth decay. Overweight
children had 38.5% of permanent tooth decay and
82.1% had primary tooth decay. The children sub-
jected to have higher values of BMI being obese had
34.5% of permanent tooth dental caries and 86.2%
had primary tooth decay (Table 1 and Fig. 3).

Fig. 2  Shows the incidence of dental caries in
permanent (DT) and primary tooth (dt).
Based on the collected study sample, on compari-
son with the percentage of caries associated with
the significant BMI categories, it was found that the
prevalence of dental caries is greater in overweight
and obese children.

Discussion

The study of dental caries is always a challenging task

Fig. 3  Represents the association of dental caries and Body mass index.
population. Based on a prepared questionnaire (12) the
required details for this study were documented. The
questionnaires were reviewed and recorded. Among the
151 study sample, underweight children were 27.2%,
27.8% children were normal, 25.8% were overweight,
and 19.2% of children were obese (Fig. 1).
as the onset of carious lesion includes various of fac-
tors such as maintenance of oral cleanliness, diet intake
frequency and composition, socioeconomic status,
salivary immunoglobulins, bacterial load and fluoride
intake (12). Obesity is described by the energy and
metabolism imbalance and is responsible for various
complications (13,14). Dental caries and childhood
obesity are also affected by Socio-economic status.
The interaction between genetic and environmental
factors develops dental caries. The process involves
the destruction of hard tissues by acidic by-products
from bacterial carbohydrate fermentation (15). Sugar
composition in soft drinks and snacks are considered
highly cariogenic.

In 151 children, 22.5% had permanent tooth decay In 2001, a study by Macek and Mitola was published
(DT) and 49.0% had primary tooth decay (dt) (Fig. 2). to decrease and prevent obesity and overweight

Body mass index Permanent teeth Primary teeth

N (%) N (%)
No Decay Decay No Decay Decay
Underweight 38 (92.7) 3 (7.3) 32 (78.0) 9 (22.0)
Normal 36 (85.7) 6 (14.3) 34 (81.0) 8 (19.0)
Overweight 24 (61.5) 15 (38.5) 7 (17.9) 32 (82.1)
Obese 19 (65.5) 10 (34.5) 4 (13.8) 25 (86.2)
Total 117 (77.5) 34 (22.5) 77 (51.0) 74 (49.0)
Table 1  Represents the prevalence of caries based on the BMI categories.

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060 Visha and Deepa : Relationship of BMI……………………..in chennai
as dental professionals have an important role in
influencing eating habits and food choices (16). A
study conducted at the school of Dental Medicine,
University of Pennsylvania had 142 subjects was
reported in a paediatric clinic. BMI percentile was
used instead of BMI for weight classification, and
the number of decayed surfaces on primary and
permanent teeth was used as the caries index. Car-
ies prevalence in that sample was low, and there-
fore, no association was found between obesity and
caries (17). The low prevalence may be due to local
factors such as access to fluoridated water or fluo-
ride programs in the school system. Another study
was done in Brazil that included 2,651 preschool
children which also found no association between
obesity and caries. The study found more caries in
children in public schools vs. private schools which
suggested that SES may be associated with caries
prevalence (18). A study of Dietary determinants of
dental caries suggested that caries risk is greatest if
sugars are consumed at high frequency and are in a
form that is retained in the mouth for long periods.
Therefore, the study concluded that there can be an
association of diet and dental caries (19). Another
study associating obesity and dental caries was con-
ducted at the University of Dammam, Kingdom of
Saudi Arabia found that participants who consumed
fast foods, soft drinks and sweet beverages on a
daily basis were more prevalent to obesity than the
participants who consumed home cooked food for
the daily meal. Hence a correlation exists between
diet and obesity.
Conclusion
It can be concluded that there appears to be an asso-
ciation between obesity and dental caries as the
prevalence of dental caries was found to be more
in obese and overweight children than normal and
underweight children.
Recommendations
Future research with a larger sample and over a
bigger geographical area should be used to asses a
comparison of diet between obese/overweight chil-
dren and underweight/normal children to discover
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whether a direct association between childhood obe-
sity and dental caries does or does not exist.
Acknowledgement
I would like to convey my sincere regards to the par-
ticipants and their parents for their co-operation and
also the statistician for the support.
Potential Conflict of Interest
No.
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062 Biomedicine: 2016; 36(1): 062-071

A study on prevalence of hypertension in cardiovascular risk factors among adults in

Chittoor district population
Bhavani Yamasani,1 Khadervali Nagoor,2 and Raziya Dudekula3
Departments of 1Community Medicine, 2Community Medicine, and
3
Physiology, Sri Padmavathi Medical College (W), SVIMS, Tirupathi, India.

(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author
Dr. Bhavani Yamasani. Email: svims.faculty@gmail.com

Abstract

Introduction and Aim: Socio-demographic patterning of cardiovascular disease risk factors information is
useful for predicting the future course of the epidemic and designing community-based interventions to reduce
risk factors in the rural population.

Materials and Methods: A total of 734 subjects aged 30 years and above of both sexes were selected for the study.

Results: Prevalence of the hypertension was found to be 28.2%. The prevalence was found to be significantly
higher in those with extra intake of salt (66.7%), previous cardiovascular/cerebrovascular events (61.9%), fam-
ily history of hypertension (47.4%), stress (44.2%), current smoking (41.1%), current alcohol intake (39.0%),
age 50 years and above (38.8%), other than unskilled occupation (35.3%), upper and middle socioeconomic
status (34.3%), males (33.2%). Contrastingly significantly higher prevalence was found in those who do phys-
ical exercise regularly (44.8%) and who were taking oils rich in PUFA (41.0%). It is found that the prevalence
of medium and high risk was found to be significantly higher in males (41.8%) than in females (5.5%).

Conclusion: The present study has found a significant proportion of undiagnosed and inadequately treated
hypertension. The cardiovascular risk has been found to be significantly higher in males than females. The
study revealed that there is enormous scope for intervention in the form of reduction of modifiable risk factors
of cardiovascular diseases.

Key words: Alcohol, Cardiovascular Diseases, Hypertension, Stress

Introduction history. Modifiable risk factors include smoking, high

C
ardiovascular diseases (CVD) have been
leading cause of morbidity and mortality in
India. Recent trends indicate that the disease
has escalated to younger age group. It has a significant
presence in males and females in both urban and
rural population (1). The risk factors are classified as
non-modifiable and modifiable. Non-modifiable risk
factors include age, sex, genetic factors and family
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blood pressure, elevated serum cholesterol, diabetes,
obesity, physical inactivity, stress, poor eating habits,
excessive drinking, tobacco chewing (2). Hypertension
is the pressure exerted by the flow of blood on the
walls of blood vessels. Due to increased pressure, the
heart is over burdened, and work load is increased.
Uncontrolled hypertension will lead to cardiovascular
complications such as myocardial infarction, heart
failure, peripheral arterial disease, renal complications
Biomedicine – Vol. 36 No. 1: 2016
Bhavani Yamasani et al.: A study on ……… Chittoor district population 
like chronic renal failure, end-stage kidney disease,
neurological complications like cerebrovascular
accidents such as stroke, haemorrhage, retinal
complications and aortic Aneurysm (3). The present
study was undertaken to provide the data on the
prevalence of hypertension in cardiovascular disease
risk factors among adults aged 30 years and above in
rural Tirupati of Chittor district population.
Materials and Methods
This study was conducted in a rural area of Tirupati
under the jurisdiction of PHC Mangalam. Most of
the people are daily labourers, vegetable and fruit
vendors, dhobis and a majority of the women are
homemakers. A total of 734 subjects in the age group
of 30 years and above age group were selected from
the study areas to estimate the prevalence. Subjects
were included in the cross-sectional study by using
20 cluster sampling technique. Thus, all the sectors,
all the sub-centers within the sectors and all the vil-
lages/habitations within the subcentres are listed in
alphabetical order and cumulative population. The
study subjects have explained the purpose of the
study and informed consent was taken from the sub-
jects. Within each village/habitation, the investiga-
tor went to the centre of the village, and all houses to
the right side are included until the required number
covered. One subject from each house aged 30 years
and above is interviewed who is selected randomly
from those available at home at the time of the
study. On an average, four families were examined
during each day of the visit. Reassurance was given
regarding the confidentiality of their responses. Eth-
ical clearance for this study was accorded by Insti-
tutional ethical committee, Sri Venkateswara Medi-
cal College, Tirupati. Height was measured by using
portable stadiometer Weight was measured with
calibrated weighing machine. Body mass Index was
calculated by using the formula: weight (kg)/height
(m2). Measurement of Blood pressure was carried
out on each participant by using the standard tech-
nique (4). The diagnosis and classification of Hyper-
tension were done according to the JNC-VII report
(Table 1). As per the cardiovascular risk categoriza-
tion by the American Heart Association, 2008 based
on the prevalence of risk factors like age, smoking,
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063
systolic blood pressure treated and not treated, and
body mass index which were given points for car-
diovascular risk was recorded. We have considered
smoking, alcohol, extra salt intake and cooking oil,
stress, physical exercise and some other parameters
to study the prevalence of hypertension in cardio-
vascular risk diseases.
JNC 6 JNC 7
SBP/DBP
category category
Optimal <120/80 Normal
Normal 120–129/80–84
Prehypertension
Borderline 130–139/85–89
Hypertension ≥140/90 Hypertension
Stage 1 140–159/90–99 Stage 1
Stage 2 160–179/100–109
Stage 2
Stage 3 ≥180/110
Table 1 Joint National Committee on Prevention, Detection,
Evaluation and Treatment of High Blood Pressure) report has
introduced a new classification.
Results
The socio-economic scale based on the per-cap-
ita monthly income of B.G. Prasad Classification
(Table 2), it was found that most of them belong
to upper lower category 40.2% followed by lower
middle category 30.9% (5). It was found that
15.7% are known hypertensive. The duration of
hypertension was found to be less than 5 years in
a majority of cases 73.9% followed by 5–10 years
21.7%. Out of the 115 known hypertensive, 106
(92.2%) is receiving treatment (Table 3). It was
found that the prevalence of hypertension was
significantly higher in 50 years and above 38.8%
compared to those aged less than 50 years 23.1%.
Similarly, the prevalence was found to be signifi-
cantly higher in males 33.2% compared to that
in females 23.0%. Although it was found that the
prevalence is higher in those with secondary and
above educational level 28.5% compared to those
educated up to primary level 27.9%, the differ-
ence is found to be not statistically significant
(P  =  0.86; NS). Those subjects with other than
unskilled occupation had significantly higher
Biomedicine – Vol. 36 No. 1: 2016
064 Bhavani Yamasani et al.: A study on ……… Chittoor district population

prevalence of hypertension 35.3% compared to do not do exercise regularly 24.5% (Table 10). It
unskilled occupations 24.0% (Table 4). Higher was found that most of the male subjects belong
prevalence of hypertension was found among to lower category of cardiovascular risk 58.5%
those unmarried/widowed/divorced group 35.8% while there were 98 subjects 26.3% with severe
compared to married group 27.1%, but how- risk and 58 subjects 15.5% with medium risk. It
ever the difference is not statistically significant was found that most of the female subjects belong
(P = 0.08; NS). An insignificantly higher preva- to lower category of cardiovascular risk 94.4%
lence is found in Hindu religion 28.4% compared while there were 14 subjects 3.9% with moderate
to other religions 25.0%. Similarly, a higher prev- risk and 6 subjects 1.7% with severe risk. It was
alence is found in those belonging to other castes found that overall, 76% belong to low risk fol-
and backward classes 29.9% than in scheduled lowed by 14.2% with severe risk and 9.8% with
caste and tribe 22.2%, but the difference is not medium risk. It can be seen that the prevalence
statistically significant (P  =  0.053; NS). A sig- of medium risk was found to be higher in males
nificantly higher prevalence was found in those 15.5% than in females 3.9% (Table 11). Simi-
belonging to upper and middle socioeconomic larly, the prevalence of high risk is also found
status 34.3% than lower socioeconomic status to be higher in males 26.3% compared to that in
group 21.3% (Table 5). A significantly higher females 1.7%, and the differences are also found
prevalence of hypertension was found in those to be statistically significant (P < 0.001; S).
with a positive family history of hypertension
Socio-economic Per capita monthly income
47.4% than those without positive family history
status (range in rupees)
24.6% (Table 6). A significantly higher preva-
lence of hypertension is found in current smokes Upper 5433 and above
compared to non-smokers 41.1% compared to Upper middle 2716–5432
25.6% and current alcoholics 39.0% compared to Lower middle 1630–2715
25.5% (Table 7). With regard to tobacco chew- Upper lower 815–1629
ing however, no association was found with Lower <815
the difference being not statistically significant
Table 2  B.G. Prasad’s socio economic status scale was used to
(P = 0.77; NS). A significantly higher prevalence
classify study subjects based on percapita monthly income.
of hypertension was found in those taking extra
salt 66.7% than those not taking extra salt 25.0%. S. No. of
With regard to the type of cooking oil, however, Parameter Percentage
No. subjects
a significantly higher prevalence was observed in
1 Known hypertension
those taking oils rich in PUFA 41.0% compared
to those taking other type of oils 23.6% (Table (a) Yes 115 15.7
8). It was found that the prevalence of hyperten- (b) No 619 84.3
sion was higher with all grades of Holmes’ cat-
egorization of stress compared to those without 2 Duration of hypertension (N = 115)
stress, and the difference is also statistically sig- Less than
(a) 85 73.9
nificant (P < 0.001; S) (Table 9). The prevalence 5 years
of hypertension was found to be slightly higher (b) 5–10 years 25 21.7
in those subjects with sedentary life style 29.5% More than
(c) 5 4.4
compared to those who were engaged in the mod- 10 years
erate and severe type of work 26.1% but the dif- 3 Treatment of hypertension (N = 115)
ference, however, is not statistically significant (a) Yes 106 92.2
(P = 0.32; NS). Contrastingly, significantly higher
(b) No 9 7.8
prevalence was found in those who do regular
physical exercise 44.8% compared to those who Table 3  Details of Hypertension among subjects (N = 734).

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Bhavani Yamasani et al.: A study on ……… Chittoor district population  065

Prevalence of Odds ratio and Statistical

S. No. Risk factor
hypertension (%) 95% CI significance
1 Age group
(a) 50 and above years 92/237 (38.8) χ2 = 19.5;
2.10 (1.50–2.94)
(b) Less than 50 years 115/497 (23.1) P < 0.001; S

2 Sex
(a) Male 124/373 (33.2) χ2 = 9.52;
1.66 (1.20–2.31)
(b) Female 83/361 (23.0) P = 0.002; S

3 Level of education
(a) Secondary and above 102/358 (28.5) χ2 = 0.02;
1.02 (0.74–1.41)
(b) Up to primary 105/376 (27.9) P = 0.86; NS

4 Occupation
(a) Not unskilled 96/272 (35.3) χ2 = 10.7;
1.72 (1.24–2.39)
(b) Unskilled 111/462 (24.0) P < 0.001; S

Table 4  Relationship between hypertension and various socio-demographic factors (N = 734).

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066 Bhavani Yamasani et al.: A study on ……… Chittoor district population

Prevalence of Odds ratio and

S. No. Risk factor Statistical significance
hypertension (%) 95% CI
1 Marital status
Unmarried/Divorced/
(a) 34/95 (35.8)
Separated 1.50 (0.95–2.36) χ2 = 3.10; P = 0.08; NS
(b) Married 173/639 (27.1)
2 Religion
(a) Hindu 196/690 (28.4)
1.19 (0.57–2.56) χ2 = 0.24; P = 0.62; NS
(b) Others 11/44 (25.0)
3 Type of family
(a) Joint/Extended 81/264 (30.7)
1.20 (0.86–1.68) χ2 = 1.25; P = 0.23; NS
(b) Nuclear 126/470 (26.8)
4 Social status
Other castes and
(a) 171/572 (29.9)
Backward castes
1.49 (0.97–2.30) χ2 = 3.67; P = 0.053; NS
Scheduled Caste and
(b) 36/162 (22.2)
tribe
5 Social economic status
(a) Upper and middle 134/391 (34.3)
1.92 (1.38–2.68) χ2 = 15.2; P < 0.001; NS
(b) Lower 73/343 (21.3)

Table 5  Relationship between hypertension and various socio-demographic factors (N = 734).

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Bhavani Yamasani et al.: A study on ……… Chittoor district population  067

Prevalence of
S. No Risk factor Odds ratio and 95% CI Statistical significance
hypertension (%)
1 Family history of hypertension
(a) Yes 55/116 (47.4)
(b) No 152/618 (24.6) 2.76 (1.80–4.24) χ2 = 25.1; P < 0.001; S
(b) No 194/713 (27.2)

Table 6  Relationship between hypertension and family history of hypertension subjects.

S. No. Risk factor
hypertension (%)
Current smoking
1 (a) Yes
(b) No
Current alcohol
2 (a) Yes
(b) No
Current tobacco chewing
3 (a) Yes
(b) No
Prevalence of
Odds ratio and 95% CI Statistical significance
51/124 (41.1)
2.03 (1.33–3.10) χ2 = 12.3; P < 0.001; S
156/610 (25.6)
57/146 (39.0)
1.87 (1.26–2.78) χ2 = 10.6; P < 0.001; S
150/588 (25.5)
17/70 (24.3)
0.80 (0.43–1.46) χ2 = 0.58; P = 0.77; NS
190/664 (28.6)

Table 7  Relationship between hypertension and current smoking, alcohol and tobacco intake.

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068 Bhavani Yamasani et al.: A study on ……… Chittoor district population

Prevalence of
S. No. Risk factor Odds ratio and 95% CI Statistical significance
hypertension (%)
1 Extra salt intake
(a) Yes 38/57 (66.7)
6.01 (3.26–11.2) χ2 = 45.2; P < 0.001; S
(b) No 169/677 (25.0)

2 Type of cooking oil

(a) Other type of oils 127/539 (23.6)
0.44 (0.31–0.63) χ2 = 21.6; P < 0.001; S
(b) Oil rich in PUFA 80/195 (41.0)

Table 8  Relationship between hypertension and extra salt intake and type of cooking oil.

Holmes Stress category Prevalence of hypertension (%) Statistical significance

Nil 161/630 (25.6)
Mild 39/90 (43.4)
χ2 = 15.6; df: 2; P < 0.001; S
Moderate 5/12 (41.7)
Severe 2/2 (100.0)
Table 9  Relationship between hypertension and Holmes’ stress categories (N = 734).

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Bhavani Yamasani et al.: A study on ……… Chittoor district population  069

Prevalence of Odds ratio and

S. No. Risk factor Statistical significance
hypertension (%) 95% CI
Nature of work
1
(a) Sedentary 133/451 (29.5)
1.18 (0.85–1.65) χ2 = 0.96; P = 0.32;NS
(b) Moderate and severe 74/283 (26.1)
Physical exercise
2
(a) No 147/600 (24.5) 0.40 (0.27–0.59) χ2 = 22.2; P < 0.001; S

Table 10  Relationship between hypertension and nature of work and physical exercise.

Cardiovascular risk category Males (%) Females (%) Total (%)

Low (less than 10%) 217 (58.5) 341 (94.4) 558 (76.0)
Medium (10–20%) 58 (15.5) 14 (3.9) 72 (9.8)
High (Above 20%) 98 (26.3) 6 (1.7) 104 (14.2)
Total 373 (100.0) 361 (100.0) 734 (100.0)
Table 11  Cardiovascular risk categorization compared between male and female subjects.

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070 Bhavani Yamasani et al.: A study on ……… Chittoor district population
Discussion
Studies have shown that a most cost-effective
approach to containing emerging epidemics of
these diseases were to reduce the prevalence of
these modifiable risk factors (2). Prevalence of
hypertension (including those who are known
hypertensives and newly diagnosed hypertensives
whose blood pressure exceeded the cutoff level of
140/90  mm Hg) is found to be 28.2%. A compa-
rable prevalence of 30.0% was reported in Delhi
industry study; rural Bareilly study 27.4% and
Assam study 33.3% (6–8). A higher prevalence
37.3% was reported in Jaipur study (9). A lower
prevalence was reported in a study in Chennai
25.4% and another study in Andhra Pradesh 20.3%
(10,11). Similar lower prevalence was reported in
rural Meerut study (15.7%). A very low prevalence
of 7.2% was reported in a study in rural Maharash-
tra (12,13). A similar lower prevalence of 8.6%
was reported in Tirupati (14). A lower prevalence
of 4.6% was reported in rural north India study
(15). The differences in the prevalence of hyper-
tension among various studies may be linked to
geographical differences, dietary patterns, behav-
ioural factors, differing time periods, urban and
rural differences as well as differing classifications
adopted (16). It was found in the present study that
only 55.6% are aware of their hypertension. Out of
those aware, 92.2% are currently taking treatment.
Out of those treated 57.5% are adequately treated
with blood pressure under control. Thus only 61 out
of 207 hypertensives (29.5%) are diagnosed and
adequately treated. This finding follows the known
principle of the rule of halves in hypertension to a
major extent. In Chennai study also, it was found
that 55.3% of known hypertensives are adequately
treated while Tirupati study also found that only
41.7% are adequately treated (11,14). It was found
that significantly higher prevalence of hyperten-
sion was found in those with extra salt consump-
tion 66.7%, positive family history of hypertension
47.4%, stress 44.2%, 50  years and above 38.8%,
other than unskilled occupation 35.3%, upper and
middle socioeconomic status 34.3%, males 33.2%.
The prevalence of hypertension was also found in
those unmarried/widowed/divorced 35.8%, other
castes and backward classes 29.9%, and sedentary
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life style 29.5%, higher secondary and above edu-
cation 28.5%, Hindu religion 28.4% but the dif-
ferences is not statistically significant. Contrast-
ingly significant level of hypertension was found
in those doing regular physical exercise 44.8% and
those taking oils rich in PUFA 41.0%. This may be
due to the fact that most hypertensives after being
diagnosed start doing regular physical exercise
and consume oils rich in PUFA out of knowledge
acquired by self or from other sources (2). Con-
trastingly lower prevalence was found in those with
tobacco chewing 24.3% which however not statis-
tically significant. The prevalence of high risk is
also found to be higher in males 26.3% compared
to that in females 1.7% and the differences are also
found to be statistically significant which indicat-
ing the cardiovascular risk has been found to be
significantly higher in males than females. Treat-
ing hypertension has been associated with 15%
reduction in the incidence of myocardial infarction
and 40% reduction of stroke (17).
Conclusion
The present study has found a significant pro-
portion of undiagnosed and inadequately treated
hypertension. The cardiovascular risk has been
found to be significantly higher in males than
females. The study revealed that there is enormous
scope for intervention in the form of reduction of
modifiable risk factors of cardiovascular diseases.
Acknowledgments
Authors acknowledge the valuable suggestions
received from Prof. G. Raviprabhu, Department
of Community Medicine, ACSR Medical College,
during this work.
Conflict of Interest
Nil.
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072 Biomedicine: 2016; 36(1): 072-076

Lipid peroxidation and antioxidant status in vitiligo patients

Pallavi Mishra,1 Rajkumari Rathore,1 and Prashant Hisalkar2
1
RKDF Medical College Hospital and Research Center, Bhopal, Madhya Pradesh, India.
2
People’s College of Medical Science & Research Center, Bhopal, Madhya Pradesh, India.
(Received: Nov 2016   Accepted: Mar 2016)

Corresponding Author

Pallavi Mishra. Email: mishra17pallavi@gmail.com

Abstract

Introduction and Aim: Vitiligo is a skin disorder or discoloration disease. When the immune system turns
against itself (autoimmune disease) then immune cells of the body attack the color-producing cells and in reac-
tion to these abnormalities white patches develop on the skin. These white patches are vitiligo skin disorder.
Etiology of vitiligo and cause of melanocyte death are not clear. Three pathogenic mechanisms – immunologi-
cal, neural and biochemical have been suggested.

Materials and Methods: The study included 50 vitiligo cases and 50 controls. P-MDA estimated by Jean C.
D. et al (1983) method, S-SOD estimated by Mishra H.P. & Fridovich I (1972) method and S-Cu estimated by
Sodium diethyldithiocarbamate (Eden and Green, 1940).

Results: P-MDA, S-SOD and S-Cu were found to be significantly higher level (P<0.001) in vitiligo patients as
compared to control. But the S-Zn was found to be significantly lower level in vitiligo patients than controls.

Conclusion: In oxidative stress, there is insufficient antioxidant activity leading to excessive accumulation of
free radicals which damage cellular compounds such as proteins, carbohydrates, DNA and lipids. This result
confirmed that oxidative stress may play an important role in melanocyte death.

Key Words: Antioxidants, Free radicals, MDA, SOD, Vitiligo

Introduction with scalloped margins (1). Vitiligo is classified as

T
he etiology is still unknown, so there are several focal, segmental, acrofacial, generalized, mucosal
hypothesis concerning the pathogenesis of and universal vitiligo (7).
vitiligo: genetic, autoimmune, neural, auto-
In general oxidative stress is caused by an imbal-
cytotoxic, biochemical and oxidative stress theories
ance between the production of reactive oxygen and
(1–5). It presents in childhood or adult life. It often
a biological systems ability to readily detoxify the
involves the hands, feet, wrists, axilla, periorbital,
reactive intermediates or easily repair the resulting
perioral and anogenital skin (6). Patients with
damage (9,10). Reactive oxygen species (ROS) are
vitiligo present with one to several amelanotic
produced as byproducts of melanogenesis in mela-
macules that appear chalky or milky white in color.
nocyte and controlled in the epidermis by several
The macules are round and/or oval in shape often
antioxidant enzymes such as superoxide-dismutase.

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Pallavi Mishra et al.: Lipid peroxidation………………invitiligo patients 073

S. No. Parameters Control Cases t-Value

(Mean ± SD) (Mean ± SD)
1 P-MDA 3.14 ± 0.35 5.4 ± 0.84 17.583*
2 S-SOD 19.67 ± 6.64 33.07 ± 11.99 6.907*
3 S-Cu 114.58 ± 13.31 129.56 ± 18.005 4.728*
Table 1  Comparison of all parameters in cases and control.
*p < 0.001 shown highly significant result.

S. No. Parameters Male cases Female cases t-Value

(Mean ± SD) (Mean ± SD)
1 P-MDA 5.493 ± 0.809 5.233 ± 0.894 1.048**
2 S-SOD 32.582 ± 12.278 33.924 ± 11.785 0.376**
3 S-Cu 131.956 ± 17.613 125.290 ± 18.399 1.264**
Table 2  Comparison between male and female cases.
**p > 0.1 shown not significant result.

Oxidative stress is the initial pathogenic event in
melanocyte destruction. In oxidative stress, there is
insufficient antioxidant activity leading to excessive
accumulation of free radicals which damage cellular
compounds such as proteins, carbohydrates, DNA,
and lipids.
diamine oxidase. Melanin, the normal body pigment
is synthesized from the essential amino acid L-phe-
nylalanine by an enzyme system dependent on cop-
per, vitamin B6, vitamin C.
Materials and Methods

Copper is an essential mineral that is a component
of several important enzymes in the body and is
essential for good health. Copper deficiency leads to
the variety of abnormalities including anemia, skel-
etal defects, degeneration of the nervous system,
reproductive failure, pronounced cardiovascular
lesions, elevated blood cholesterol, impaired immu-
nity and defects in the pigmentation and structure of
hair. More than a dozen enzymes have been found
to contain copper. The best studied are superoxide
dismutase (SOD), cytochrome C oxidase, cata-
lase, dopamine hydroxylase, uricase, tryptophane
dioxygenase, lecithinase and other monoamine and
S. No. Parameters Compared t-Value
groups
1 P-MDA First and second 3.594*
2 S-SOD First and second 0.224**
3 S-Cu First and second 0.211**
Table 3  Comparison between two different duration group.
*p < 0.001 shown highly significant result and **p > 0.1 shown not
significant result.
This study was conducted in the Department of Bio-
chemistry, Gandhi Medical College, Bhopal M.P.
in association with Department of Skin and V.D.
Hamidia Hospital, Bhopal M.P. The study included
50 vitiligo cases and 50 normal healthy subjects.
This study was undergoing according to sex wise
distribution (n  =  32 males and n  =  18 females),
types of vitiligo, duration of vitiligo, a progression
of disease and diet. There were three sub groups in
this study generalized vitiligo (n = 30), localized vit-
iligo (n = 15) and segmental vitiligo (n = 05) occurs.
Vitiligo patients were divided into two groups who
were suffering from vitiligo more than 1  year and
who were suffering from vitiligo less than 1  year.
Patients with vitiligo not undergoing treatment were
considered for the study and patients suffering from
other chronic diseases like renal diseases, liver dis-
eases, heart diseases, diabetes mellitus, hypopig-
mented scars due to injury or burn, leprosy and vit-
iligo undergoing treatment were excluded from the
study. n = 25 progressive vitiligo cases and n = 25
stable vitiligo cases. n  =  27 vitiligo patients were

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074 Pallavi Mishra et al.: Lipid peroxidation………………invitiligo patients

S. No. Parameters Stable cases Progressive cases t-Value

(Mean ± SD) (Mean ± SD)
1 P-MDA 5.031 ± 0.796 5.768 ± 0.726 3.422*
2 S-SOD 32.896 ± 11.648 33.234 ± 12.579 0.098**
3 S-Cu 129.271 ± 20.319 129.842 ± 15.774 0.111**
Table 4  Comparison between stable cases and progressive cases
*p < 0.001 shown highly significant result and **p > 0.1 shown not significant result.

S. No. Parameters Patients taking Veg Patients taking Veg t-Value

diet (Mean ± SD) and Non Veg diet
(Mean ± SD)
1 P-MDA 5.447 ± 0.789 5.344 ± 0.913 0.428**
2 S-SOD 30.764 ± 12.488 35.766 ± 11.057 1.487**
3 S-Cu 133.230 ± 18.683 125.244 ± 16.536 1.587**
Table 5  Comparison between two different diet groups in cases
**p > 0.1 shown not significant result.

taking vegetarian diet and n = 23 cases were taking
vegetarian and non vegetarian diet.
Plasma malondialdehyde (P-MDA) by Jean et al.
(1983), Serum superoxide dismutase (S-SOD) by
Mishra and Fridovich (1972), Serum copper (S-Cu)
by Sodium diethyldithiocarbamate method (Eden
and Green, 1940; Ventura and King, 1951) tests
were performed on the blood samples of patients
and control.
Statistical analysis
According to sex wise distribution when compared
between male cases and female cases the P-MDA,
S-SOD and S-Cu were not found to be statistically
significant. Vitiligo affects both the sex equally.
According to the type of vitiligo, it was divided into
three groups: the first group was generalized vitiligo
cases, the second group having localized vitiligo
cases and third group comprises of segmental vit-
iligo cases. Among all the parameters, P-MDA was
found to be statistically highly significant in gener-
alized vitiligo cases (p < 0.001) (Fig. 1).

Data analysis was performed using the online t ÔÇô
test calculator with a value of p < 0.001 and p < 0.05
considered highly significant and significant respec-
tively. Comparison of two groups was done by the
Student’sÔÇÖs paired or unpaired t-test. Results are
expressed as mean ± SD.
Results
The P-MDA, S-SOD, and S-Cu were found to be
significantly higher level in vitiligo cases as com-
pared to controls because fresh cases were selected
for the study. Therefore initially oxidative stress
occur to which suppress antioxidant level (S-SOD
and S-Cu) were also increased (Table 1).
According to the duration of vitiligo, among all the
parameters the P-MDA was found to be statistically
highly significant in patients having vitiligo more
than 1 year (Table 3).
According to the progression of the disease, P-MDA
was found to be statistically highly significant in
progressive vitiligo cases. Progression of disease
increases oxidative stress (Table 4).
According to the diet wise comparison between
cases, there was no statistically significant differ-
ence was found in any parameters in patients tak-
ing vegetarian diet and non-vegetarian diet which
shown diet has no role in vitiligo (Table 5).

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Pallavi Mishra et al.: Lipid peroxidation………………invitiligo patients
Fig. 1  Comparison among types of vitiligo cases.
Discussion
The etiology of vitiligo is still unknown. So there are
many hypotheses to explain its pathogenesis. One of
the hypotheses to explain vitiligo is the self-destruc-
tive theory of melanocytes, which suggests a role for
oxidative stress (9–13). The observations and results
show that the P-MDA, S-SOD, and S-Cu were found
to be a significantly higher level in vitiligo patients
as compared to control. This is comparable to the
study of Yildirim et al. (2003), Yousry et al. (2007),
Jain et al. (2008). Recently many studies have
reported accumulation of free radicals (oxidative
stress) in the epidermal layers of the affected skin
(14,15) and blood of vitiligo patients (16). Oxidative
stress could be an important phenomenon leading
to melanocyte death in vitiligo. Damage caused by
free radicals could be a possible pathogenic factor
for vitiligo (17).
This study is also supported by the O. Arican, E.B.
Kurutas, which was found significantly higher lev-
els of SOD activity of erythrocytes in patients with
active localized vitiligo. In addition, high SOD
activities were correlated with high immune com-
petence (18). P.V. Sravani, N. Kishore Babu, K.V.T.
Gopal et al. in his study the levels of SOD in the
vitiliginous skin of vitiligo patients were found to
be higher than the levels of SOD in normal skin
of control. So this study concluded that oxidative
stress plays an important role in the pathogenesis
of vitiigo.
www.biomedicineonline.org
075
Acknowledgments
The authors are thankful to Dr. B.K. Agrawal (Ex-
HOD) Department of Biochemistry and Dr. Anna
Alex (HOD) Department of Skin and V.D. Gandhi
Medical College Bhopal MP for providing facilities
to carry out this work and proper guidance.
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  2. Nordlund, J.J., and Ortonne, J.P. Vitiligo vul-
garis. In The pigmentary system. Physiology
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R.E. Boissy, V.J. Hearing, R.A. King, W.S. Oet-
ting, and J.P. Ortonne (Eds.), Blackwell Scien-
tific, Oxford, 1998; 513–551.
  3. Stephen, O., and Kovacs, M.D. St Louis, Mis-
souri. Vitiligo. J. Am. Acad. Dermatol. 1998;
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  4. Nordlund, J.J., and Majumdar, P.P. Recent inves-
tigation on vitiligo vulgaris: advances in clinical
research. Dermatol. Clin. 1997; 15: 769–787.
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Dermatol. 1993; 31: 621–623.
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  7. Valia, R.G., and Valia, A.R. Pigmentary disor-
ders—vitiligo. InIADVL Textbook of Derma-
tology, 3rd Edn., S. Dhar, P. Datta, and R. Mala-
kar (Eds.), Bhalani Publishing House, Mumbai,
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cell injury. Free Radic. Biol. Med. 2000; 28:
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tive oxygen species, antioxidants and the mam-
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Increased sensitivity to peroxidative agents as
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possible pathogenic factor of melanocytes dam-
age in vitiligo. J. Invest. Dermatol. 1997; 109:
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damage in the pathogenesis of vitiligo.In Vit-
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New York, NY, ; 123–135.
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dative stress in vitiligo. Skin Pharmacol. Appl.
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Beazley, W.D., Peters, E.M., and Marles, L.K.
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Biomedicine – Vol. 36 No. 1: 2016
Biomedicine: 2016; 36(1): 077-082 077

Effects of threshold inspiratory muscle trainer in bronchial asthma

Shiny S. James,1 K. Rekha,1 Vaiyapuri Anandh,2 L. Chandrasekar,2 And Radhakrishnan
Unnikrishnan2
1
Department of Cardio-Pulmonary, Saveetha College of Physiotherapy, Chennai, India.
2
Department of Physical Therapy, Applied Medical Sciences College, Majmaah University, Majmaah, Saudi
Arabia.
(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author
K. Rekha. Email: futurdreams88@gmail.com

Abstract

Introduction and Aim: Many asthma patients develop weakness of their chest and diaphragm muscles, this
further contributes to symptoms like shortness of breath or an impaired capability to exercise. Loss of respira-
tory muscle strength could occur in asthma. If this weakness is substantial, then it is necessary to strengthen
the respiratory muscles to improve their functions. Thereby this study is proposed to strengthen the respiratory
muscles in asthma patients.

Materials and Methods: A randomized placebo-controlled study performed in Bronchial asthma to strengthen
Inspiratory Muscles using Threshold Inspiratory Muscle trainer. Subjects were randomized into 2 groups
experimental and control group. Subjects of both the groups performed FEV1, PEFR and 6 minutes walk test
as a pretest following which subjects were treated with Threshold device with resistance for experimental
group and control group were treated with threshold device without resistance for 30 minutes. Three days a
week for a period of 1 month following which post test was done.

Results: Difference between the groups shows that group A is statistically significant with the p value >
0.0001 for all the variables FEV1, PEFR and 6 min walk test.

Conclusion: Threshold Inspiratory muscle training with resistance proved to be effective for improving the
Inspiratory muscle strength and Endurance.

Key words: Bronchial Asthma, Inspiratory Muscle Training, Threshold Inspiratory Muscle Trainer

Introduction 30 million patients consisting of 2.4% adults and

A
sthma is a global health problem with
raising prevalence rate in the world.
According to WHO 300 million people are
known to have Bronchial asthma. 255,000 have died
due to asthma in 2005. It has been estimated that in
India prevalence rate is about 3% which is around
4%–20% is children (5).
Asthma is defined as a “chronic inflammatory dis-
order of the airways in which many cells and cellu-
lar elements play a role.” The chronic inflammation
causes an associated increased resistance of air-
ways and hyperinflation that leads to recurrent epi-

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078 Shiny James et al.: Effects of threshold……………………bronchial asthma
sodes of wheezing, breathlessness, chest tightness,
and coughing, particularly at night or in the early
morning (3), lung places the respiratory muscles
at mechanical disadvantage (1). Generalized loss
of respiratory muscle bulk occurs in COPD com-
monly, in asthma patients also it occurs due to the
compounded effects of steroid therapy, asthma also
shows relevant respiratory muscle weakness (1).
Airways smooth muscle also increases in size along
with an increase in the numbers of mucous glands.
Other cell types involved include: T lymphocytes,
macrophages, and neutrophils. There may also be
involvement of other components of the immune
system including: cytokines, chemokines, hista-
mine, and leukotrienes among others (2).
Asthma patients can be treated with both medical
and non medical intervention, the aim of the treat-
ment being to prevent recurrent asthma attacks,
optimal lung function and normal breathing pat-
tern and well-tolerated exercise capacity. Chronic
bronchoconstriction in asthmatics is associated
with increased inspiratory muscle work, thereby
strengthening the inspiratory muscles among
asthma could reduce dyspnea and improve exer-
cise tolerance level (3).
Threshold Inspiratory muscle trainers is a device
which was designed to provide resistance during
breathing; that is independent of in Inspiratory flow
rate. The study has concluded that patients trained
with threshold inspiratory muscle trainer were able to
increase their Inspiratory muscle strength and endur-
ance (4). Threshold loading enhances the velocity of
inspiratory muscle contraction and provides a favor-
able alteration for breathing pattern and time (9).
In the previous studies, it has been proved that the
Threshold Inspiratory muscle trainer has trained
both strength and endurances in normal subjects
as well as in patients with COPD quadriplegics
(7), patients with cystic fibrosis (8). Inspiratory
muscle strength training have also been investi-
gated in COPD patients, and evidence states that
the stimulus or load placed on the respiratory
muscles during training was enough to strengthen
the inspiratory muscles as a part of the pulmonary
rehabilitation program and reduced the duration of
hospitalization (12).
www.biomedicineonline.org
Six minute walk test is a simple test easy to admin-
ister, better tolerated and reflective of activities of
daily living than the other walk tests. It is a test
developed to check the physical fitness level of the
healthy individual (13). In this study, we perform
6 minute walk test to assess the exercise tolerance
level of the asthma patients.
Among bronchial asthma, it is known that Inspira-
tory muscles are impaired due to hyperinflation of
lung and diaphragm become flatten and shorten.
Many techniques (breathing exercise, incentive
spirometer, PI flex, etc.) which are used to strength
the Inspiratory muscle are commonly used.
Whereas Threshold Inspiratory muscle trainer
(IMT) device is not commonly used in India due to
lack of awareness, thereby this study aim to create
awareness and make use of the threshold Inspira-
tory muscle trainer device as it provides resistive
breathing which helps in improving the Inspira-
tory Muscle strength and endurance and there by
reduces dyspnea. This study proposed to find out
the effect of Threshold Inspiratory Muscle Trainer
(IMT) in Bronchial Asthma.
Materials and Methods
A placebo controlled study was undertaken at
Saveetha Medical College and hospital with 79
Bronchial asthma subjects. Ethical approval and
informed consent have been obtained prior to the
study. Both males and females subjects with mild
and moderate Bronchial asthma (FEV 79% to 40%)
between 20 and 60 years of age, using bronchodi-
lators were included in the study. Subjects were
excluded if they had status asthmatics, perceptual
disorders, and Unstable cardiovascular condition.
Subjects were asked to rest in a chair for approxi-
mately 10 minutes after arriving outpatient depart-
ment. Forced Expiratory volume in one second
(FEV1) and Peak Expiratory flow rate (PEFR)
were performed and measured using the digital
spirometer, 3 trials were performed, and the best
trial was recorded (14). Following which 6  Min-
ute Walk Distance was monitored (13). Patients
were instructed to walk for 6  minutes in a corri-
dor for a measured distance at their fastest pace
and cover the longest possible distance under the
Biomedicine – Vol. 36 No. 1: 2016
Shiny James et al.: Effects of threshold……………………bronchial asthma 079

Experimental Group (40) Control Group (39)

(Mean ± SD) (Mean ± SD)
Females (14) Males (26) Females (11) Males (28)
Age (years) 36.50 ± 9.26 39.85 ± 9.76 41.91 ± 9.65 39.36 ± 8.01
Height (cms) 158.07 ± 8.77 164.58 ± 8.01 158 ± 4.94 161.89 ± 8.67
Weight (kg) 67.07 ± 6.51 75.77 ± 11 62.55 ± 5.75 71.61 ± 9.41
BMI (kg/m2) 26.8 ± 2.4 28.1 ± 4.7 25.1 ± 3.18 27.2 ± 2.77
Systolic blood pressure (mm Hg) 125.71 ± 8.91 127.04 ± 8.17 127.82 ± 7.44 129.07 ± 7.36
Diastolic blood pressure (mm Hg) 82 ± 4.87 84.19 ± 4.90 80.73 ± 5.55 83.61 ± 5.31
Table 1  Characteristics of Participants.

Outcome measure Mean Std. deviation t-Value p-Value

FEV1 (%) Pretest 64.85 10.85 12.9554 <0.0001
Post test 71.13 9.53
PEFR (%) Pretest 62.58 7.52 16.8910 <0.0001
Post test 69.35 6.57
6 MWD (ft) pretest 267.43 11.74 15.9865 <0.0001
Post test 279.58 11.22
Table 2  Observations of Group A.

Outcome measure Mean Std. deviation t-Value p-Value

FEV1(%) Pretest 60.33 8.31 13.1801 <0.0001
Post test 61.56 8.29
PEFR (%) Pretest 62.08 6.63 4.6679 <0.0001
Post test 63.38 6.11
6 MWT (ft) Pretest 267.13 12.51 14.0000 <0.0001
Post test 268.56 12.39
Table 3  Observations of Group B.

supervision of the physiotherapist (13). And the
distance walked will be recorded, all the tests were
performed at the baseline as a pretest and at the
end of 1 month as a posttest.
Seventy-nine subjects were randomized into two
groups, Group A—Experimental group and Group
B—Control group. Subjects were not aware in
which group they were placed, and subjects were
new to the use of Threshold device. Group A con-
sists of 40 subjects were trained with Threshold
Inspiratory muscle trainer (Respironics product
REF HS730, USA) in sitting position. The sub-
ject was asked place their lips tightly around the
mouthpiece, Asked to inhale (breathe in) through
threshold Inspiratory Muscle trainer against the
www.biomedicineonline.org
spring loaded valve that provides resistance fol-
lowing which subject was asked to Exhale (breathe
out) normally. Threshold Inspiratory muscle
trainer with resistance trained for 5 sets per day
each set consists of 10 repetitions with 2 minutes
rest between each sets, 3  days a week, 1 session
per day, each session consisted of 30  mins inter-
vention period for 1 month. Intensity: 12 cm H2O
(20% of MIP) in the first week, 16 cm H2O (30%
of MIP) in the second week, 20 cm H2O (50% of
MIP) in the third week and 24  cm H2O (70% of
MIP) in the fourth week (21).
Group B consists of 39 subjects they were trained
with Threshold Inspiratory muscle trainer with-
out resistance, subjects were asked to Inhale with
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080 Shiny James et al.: Effects of threshold……………………bronchial asthma

their lips sealed around the mouth piece of Thresh-

old device and exhale normally no resistance was 70
69.35

provided against Inspiration. Subjects were asked

68
to perform inspiration and expiration with that
66
Threshold device for 10 repetitions with 2 minutes 62.58
63.38
Pretest
rest between each set. Five sets with 1 session per 64 62.08
post test

day lasting for 30 minutes, 3 days a week and so 62

on for a total duration of 1 month. 60

58
Group A Group B
Results
Statistical significance of Pretest and posttest data Fig. 2  Comparison of PEFR between Group A and Group B.
for all the outcome measures are analyzed using
the paired t-test within the group and unpaired t
test for between the groups. p-value <0.05 is con- 279.58

sidered to be statistically significant. 280

278
276

Discussion 274
272 268.56 Pre test
270 267.43 267.13
The study shows that Inspiratory muscle strength 268
Post test

was impaired in patients with asthma which con- 266

264
tributed to other factors like increased dyspnea and 262

reduced exercise tolerance. Weiner et al. has demon- 260

Group A Group B
strated that both inspiratory and expiratory muscle
weakness among COPD patients in which he spe- Fig. 3  Comparison of 6 Minute walk Distance between Group A
cifically trained both the group of muscles and con- and Group B.

cluded that improving inspiratory muscle strength

alone improves 6 minute walk distance and reduces cise, and for Group B the device was used without
dyspnea, and he found that no special benefits were resistance dial it does not provide resistance against
observed in the combined treatment of both inspira- breathing. The intervention was given for 1 month
tory and expiratory muscle strength (15). duration 3 times a week with 30  minutes a day.
Group A showed significant differences that Thresh-
In our study we used Inspiratory Threshold Mus- old Inspiratory Muscle training with resistance is
cle trainer, This device had been used for Both the more effective in patients with Bronchial Asthma,
groups but Group A it had been used with the resis- which increased the Inspiratory muscle strength and
tance dial which provides resistive breathing exer- endurance, increased exercise tolerance capacity
by reducing dyspnea while performance. Whereas
group B also showed slight improvement as it was
71.13 also a form of breathing exercise but it did not show
72
70
significance compared to Group A.
Romer et al. stated that inspiratory airflow is pro-
68 64.85
66
64 60.33
61.56 Pretest portional to the velocity of muscle shortening, an
62 post test
inspiratory pressure is proportional to force gen-
60
58
eration, training with high flow loads and resis-
56 tive loads are known to increase maximum static
54
Group A Group B
pressure and maximal inspiratory flow rate (16).
Respiratory muscles could be trained similarly
Fig. 1  Comparison of FEV1 between Group A and Group B. to other skeletal muscles which significantly
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Shiny James et al.: Effects of threshold……………………bronchial asthma
improves respiratory muscle performance. The
study suggests that men could generate maximal
inspiratory and expiratory pressure than women
for a given resistance during breathing (17). Resis-
tance during inhalation comes from constant pres-
sure loading that takes account of inspiratory flow,
the valve allows the load to be altered according to
the desired level of the patient (10).
Turner et al. demonstrated increase in inspiratory
muscle strength following inspiratory muscle train-
ing, the results showed an increase in diaphragm
thickness and hypertrophy of type 1 and type 2
muscle fibers of external intercostals muscles (18)
and increased pulmonary function variables FEV1
and FVC. Hill et al. performed high-intensity
inspiratory muscle training for COPD and demon-
strated that inspiratory muscle strength increased
to the greater extent resulting from the reduction in
respiratory motor output and perception of inspi-
ratory effort (19). Loss of fat free mass causes a
reduction in skeletal muscle mass including inspi-
ratory muscles and it is associated with impaired
inspiratory muscle function. High-intensity inspi-
ratory muscle training produces an increase in
inspiratory muscle function which increases dia-
phragm thickness and lung volumes (20). Martyn
et al. demonstrated that ventilator muscle perfor-
mance allows patients to develop breathing strate-
gies to handle high inspiratory loads. By increasing
loads during inspiration, there was a fall in minute
ventilation, increased oxygen consumption (21).
Inspiratory muscle training and breathing exercises
were effective to promote biomechanical factors
of respiratory muscle function including muscles
of external intercostals for accessory participation
during expiration caused greater thoraco-abdominal
mobility. The study showed that inspiratory muscle
training performed in children with asthma showed
significant improvement by increasing Maximal
Inspiratory Pressure, and Maximal Expiratory Pres-
sure and reduced airway obstruction consequently
improved activities of daily living (22).
As we know Bronchial Asthma had been a Prob-
lem of any age group in recent years, many suffer
due to this problem. Threshold Inspiratory Muscle
training among Bronchial Asthma improved Inspi-
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081
ratory Muscle Strength and Endurance by improv-
ing FEV1, and PEFR and also Improved 6 minute
walk Distance by reducing dyspnea by enhancing
the daily activities of the individual.
Conclusion
Inspiratory muscle training using Threshold device
with the resistance dial had proven to be effective
when compared with the Threshold device without
resistance among the Bronchial Asthma, which had
significant effects in improving the FEV1, PEFR,
6 Minute walk Distance and reduced dyspnea.
Acknowledgement
We thank all the participants for their cooperation
and interest delivered in this study.
Conflict of Interest
None.
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Biomedicine – Vol. 36 No. 1: 2016
Biomedicine: 2016; 36(1): 083-089 083

Assessment of serum uric acid in type 2 diabetes mellitus in a middle-aged south Indian
population
Jyoti John,1 RajLaxmi Sarangi,1 Asha Dinakaran,1 S.V. Umadevi,2 Somanath Padhi,3 and Nitin
Ashok John,2
Departments of 1Biochemistry and 3Pathology, Pondicherry Institute of Medical Sciences, Pondicherry,
India.
2
Department of Physiology, Indira Gandhi Medical College and Research Institute, Pondicherry, India.
(Received: Feb 2016   Accepted: Mar 2016)

Corresponding Author

Dr. Jyoti John. Email: jyotijohn7@gmail.com

Abstract

Introduction and Aim: Several studies in recent past have postulated the role of high serum uric acid levels
in the pathophysiology of the insulin resistance, metabolic syndrome, and type 2 diabetes mellitus (T2DM).
However, controversy persists whether hyperuricemia has a causal association with T2DM; and requires con-
firmation by further studies.

Materials and Methods: In this case-control study, fasting plasma glucose (FPG), serum uric acid (UA),
plasma insulin, glycosylated hemoglobin (HbA1c), HOMA-IR and HOMA- B were measured among 70 T2DM
patients (both males and females, age group; 30–80 years); and compared with 50 healthy age-matched controls.

Results: Compared to controls, patients with T2DM had higher FPG (90.62 ± 6.84 vs. 165.81 ± 61.96 mg/
dl, p < 0.001), HOMA-IR (2.29 ± 1.44 vs. 5.16 ± 4.41, p < 0.001), plasma insulin (10.04 ± 6.03 μU/ml vs.
12.71  ±  10.77 μU/ml) (p  =  0.420) and serum uric acid levels (4.77  ±  1.51  mg/dl vs. 5.33  ±  1.94  mg/dl )
(p = 0.126) but lower beta cell function (131.10% ± 75.76% vs. 59.92% ± 57.13%, p < 0.001). Uric acid corre-
lated negatively with levels of fasting plasma glucose (r = -0.243; p < 0.05) in cases but not controls. The lev-
els of uric acid are correlated positively with the calculated beta cell function (HOMA-B) (r = 0.274; p < 0.05).

Conclusion: Our patients with type 2 diabetes mellitus had significantly higher fasting plasma glucose and
HOMA-IR but significantly lower beta cell function as compared to controls. Uric acid shows a significant
negative correlation with fasting plasma glucose but a significant positive correlation with beta cell function
in our cases.

Key words: Beta Cell Function, HbA1 c, HOMA-B, HOMA-IR, Insulin Resistance, Type 2 Diabetes Mel-
litus, Uric Acid

Introduction converts uric acid into allantoin. As a result, humans

U
ric acid is the end product of purine have higher uric acid levels than most other mammals
metabolism in humans. This is because having the enzyme uricase. An important role of uric
humans lack the enzyme uricase which acid is that it can function as an antioxidant in the

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084 Jyoti John et al.: Assessment of serum uric acid………….. Indian population
plasma (1,2). Urate (the soluble form of uric acid
in the blood) is a scavenger of super oxide and
hydroxyl radical, and can also chelate transition
metals. But at increased concentrations, it can also
act as a prooxidant and may be a marker of oxidative
stress (3). Increased oxidative stress is known to
play a potential role in the development of diabetes
mellitus and its complications (4).
Uric acid has recently been implicated in the patho-
physiology of type 2 diabetes mellitus (T2DM).
There is a complex relationship between uric acid and
hyperinsulinaemia. Increased serum uric acid levels
are seen before the development of insulin resis-
tance and diabetes mellitus (5). Hyperinsulinemia,
in turn, leads to decreased renal excretion of urate
(6,7). While the increased uric acid may be due to the
increased uric acid absorption in the proximal tubule
secondary to hyperinsulinemia, a growing number
of researchers have found that uric acid may predict
the development of metabolic syndrome, obesity, and
diabetes (8–10).
Though increased uric acid levels could be one of
the risk factors for diabetes mellitus, controversy
exists on whether uric acid plays a causal role. There
are studies showing positive association while some
studies show an inverse association. Review of the
available literature shows that there is ambiguity
regarding association between serum urate levels
and diabetes mellitus. Moreover to the best of our
knowledge, very few studies have been done in this
part of South India evaluating the role of uric acid in
diabetes mellitus. With this background, we decided
to carry out the present study. The objectives of the
study were to measure the levels of serum uric acid,
plasma glucose, plasma insulin and glycosylated
haemoglobin in cases of diabetes mellitus and con-
trols and to ascertain whether there is any associa-
tion between serum uric acid and insulin resistance
in patients of diabetes mellitus.
Materials and Methods
This was a cross-sectional study carried out at our
institute between the period January 2014 to July
2014. The study was approved by our Institutional
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Ethical Committee (IEC number: RC/13/128). A
total of 70 patients with type 2 diabetes mellitus
both males and females between the age group
of 30–80 years were included in our study. Those
patients with a history of Hypertension/Gout/
Rheumatoid arthritis/ Kidney disease/Liver dis-
ease/ Acute myocardial infarction/ Stroke/Alco-
holism were excluded from the study. Patients
with any other Acute or Chronic illnesses were
also excluded from the study. We also included 50
healthy controls that were in the same age group.
A single blood sample (5 ml) was drawn from the
subjects and analysed for fasting plasma glucose,
serum uric acid, plasma insulin and glycated hae-
moglobin levels. Diagnostic criterion for cases
was fasting plasma glucose >126 mg/dl or HbA1C
>6.5%. The fasting plasma glucose (FPG) levels
were measured using Cobas Integra 400+ Auto-
analyser (Roche). The fasting plasma insulin lev-
els were measured using Cobas e 411 Autoanalyser
(Roche). The serum uric acid levels were measured
by uricase-peroxidase enzymatic end point method
using semi-autoanalyser (Kit by Accurex). The gly-
cated haemoglobin levels were measured by latex
enhanced Immunoturbidimetry (Kit by Agappe).
The HOMA-IR (homeostasis model assessment
of insulin resistance) and HOMA-B (homeostasis
model assessment of β-cell function) were calcu-
lated based on the formula:
HOMA-IR = fasting plasma glucose (mg/dl) × fasting plasma insulin (µU/ml)
405
HOMA-B = 360 × fasting plasma insulin (µU/ml)
fasting plasma glucose (mg/dl) -63
Statistical analysis
The quantitative data was presented as a mean and
standard deviation. Mann-Whitney U test was done
to assess the statistical significance of the difference
between mean values amongst cases and controls.
Spearman’s correlation co-efficient was used to ana-
lyze the association between biochemical param-
eters. A value of p  < 0.05 was taken as statistical
significance. Analysis was done using IBM SPSS
software (version 20.0).
Observations
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Jyoti John et al.: Assessment of serum uric acid………….. Indian population
Variable Cases (n = 70)
Age (in years) 53.73 ± 11.49
Fasting plasma glucose (mg/dl) 165.81 ± 61.96
Plasma insulin (µU/ml) 12.71 ± 10.77
Serum uric acid (mg/dl) 5.33 ± 1.94
HOMA-IR 5.16 ± 4.41
HOMA-B (%) 59.92 ± 57.13
Table 1  Table showing mean values of age, fasting plasma glucose, plasma insulin, serum uric acid, HOMA-IR and HOMA-B in cases and
controls.
All variables are expressed as mean ± standard deviation (p-value calculated using Mann-Whitney U test).
Results
The mean fasting plasma glucose level in cases
was 165.81 ± 61.96 mg/dl and that in controls were
90.62  ±  6.84  mg/dl. The difference is statistically
significant (p < 0.001). The plasma insulin levels in
cases were 12.71 ± 10.77 μU/ml and that in controls
were 10.04 ± 6.03 μU/ml (p = 0.420). The levels of
serum uric acid were higher in cases (5.33 ± 1.94 mg/
dl) as compared to controls (4.77  ±  1.51  mg/dl)
(p = 0.126). The HOMA-IR in cases was 5.16 ± 4.41
and that in controls were 2.29 ± 1.44. The difference
is statistically significant (p < 0.001). The HOMA-
B in cases was 59.92  ±  57.13 and that in controls
were 131.10 ± 75.76. The difference is statistically
significant (p < 0.001).
In this study, we found that the levels of fasting
plasma glucose correlated negatively with levels of
serum uric acid (r = -0.243; p < 0.05). We also found
that the levels of fasting plasma glucose correlated
negatively with HOMA-B (r = -0.636; p < 0.001).
The levels of fasting plasma glucose correlated posi-
tively with levels of HbA1c (r = 0.573; p < 0.001)
and HOMA-IR (r = -0.359; p = 0.002) in our cases.
We also found that the levels of uric acid correlated
positively with the calculated beta cell function
(HOMA-B) (r = 0.274; p < 0.05).
Discussion
In our study, patients with type 2 diabetes mellitus
had significantly higher fasting plasma glucose and
HOMA-IR indicating insulin resistance in our cases.
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085
Controls (n = 50) p-Value
49.82 ± 13.19 0.106
90.62 ± 6.84 <0.001
10.04 ± 6.03 0.420
4.77 ± 1.51 0.126
2.29 ± 1.44 <0.001
131.10 ± 75.76 <0.001
Moreover, our cases had significantly lower beta cell
function as compared to controls.
Our results also showed higher levels of uric acid in
cases as compared to controls though the difference
is not statistically significant. Our findings similar
to the findings of Bakshi et al. (11). Gill et al. (12)
also found that HbA1C, serum insulin, and serum
uric acid were increased in the patients with Type 2
diabetes mellitus as compared to controls. Wasada
et al. (13) found that elevated serum uric acid is a
feature of insulin resistance syndrome. One study
showed that uric acid was found to have prognostic
significance in type 2 diabetes patients along with an
increased mortality risk (14).
Our results also show that uric acid has a negative
correlation with fasting plasma glucose in patients
with type 2 diabetes mellitus. The difference is sta-
tistically significant (p  <  0.05). Our findings simi-
lar to the findings of other researchers who found
an inverse association between uric acid levels and
type 2 diabetes mellitus (Bhandaru and Shankar
(15), Oda et al. (16–22).
A negative correlation of uric acid was observed
with fasting plasma glucose levels. There is a
complex relationship between uric acid and insu-
lin resistance. Serum uric acid levels are known to
increase before the development of insulin resis-
tance and diabetes mellitus (5). Increased uric acid
may be due to the increased uric acid absorption in
the proximal tubule secondary to hyperinsulinemia.
Once hyperinsulinemia develops, this, in turn, leads
to decreased renal excretion of urate (6,7). Hence,
Biomedicine – Vol. 36 No. 1: 2016
 086

FBS Insulin Uric acid HOMA-IR Beta cell HbA1c Age

Spearman’s FBS Correlation Coefficient 1.000 0.063 -0.243* 0.359** -0.636** 0.573** -0.156

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rho Sig. (2-tailed) . 0.607 0.042 0.002 0.000 0.000 0.197
N 70 70 70 70 70 70 70
Insulin Correlation Coefficient 0.063 1.000 0.189 0.929** 0.678** 0.097 -0.185
Sig. (2-tailed) 0.607 . 0.117 0.000 0.000 0.424 0.125
N 70 70 70 70 70 70 70
Uric acid Correlation Coefficient -0.243* 0.189 1.000 0.094 0.274* -0.016 0.120
Sig. (2-tailed) 0.042 0.117 . 0.439 0.022 0.896 0.324
N 70 70 70 70 70 70 70
HOMA-IR Correlation Coefficient 0.359** 0.929** 0.094 1.000 0.423** 0.232 -0.221
Sig. (2-tailed) 0.002 0.000 0.439 . 0.000 0.053 0.066
N 70 70 70 70 70 70 70
Beta cell Correlation Coefficient -0.636** .678** .274* .423** 1.000 -0.301* -0.016
Sig. (2-tailed) 0.000 0.000 0.022 0.000 . 0.011 0.897
N 70 70 70 70 70 70 70
HbA1c Correlation Coefficient 0.573** 0.097 -0.016 0.232 -0.301* 1.000 -0.051
Sig. (2-tailed) 0.000 0.424 0.896 0.053 0.011 . 0.675
N 70 70 70 70 70 70 70
Age Correlation Coefficient -0.156 -0.185 .120 -0.221 -0.016 -0.051 1.000
Sig. (2-tailed) 0.197 0.125 0.324 0.066 0.897 0.675 .
N 70 70 70 70 70 70 70
Table 2  Spearman’s correlations coefficient in patients of type 2 diabetes mellitus (n = 70).
* Correlation is significant at the 0.05 level (2-tailed).

**Correlation is significant at the 0.01 level (2-tailed).

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Jyoti John et al.: Assessment of serum uric acid………….. Indian population
Jyoti John et al.: Assessment of serum uric acid………….. Indian population
the inverse relationship between serum uric acid and
fasting plasma glucose observed in our diabetic sub-
jects may probably be caused by the increased renal
excretion of uric acid in the presence of hypergly-
caemia.
The hyperglycemia seen in type 2 diabetes mellitus
may lead to generation of free radicals, disturbing
the endogenous antioxidant defense system includ-
ing its effect on uric acid homeostasis. In addition,
hyperuricemia induces reactive oxygen species pro-
duction inside adipose cells (23,24). Since oxidative
stress in adipocytes has been shown to lead to insu-
lin resistance, uric acid-induced oxidative stress in
adipose tissue might play a key role in insulin resis-
tance.
Our study showed that the levels of uric acid cor-
related positively with the calculated beta cell func-
tion (HOMA-B) in our cases, and this was statisti-
cally significant (p < 0.05). In one study by Wei et
al. type 2 diabetic patients with higher serum uric
acid level were found to have better insulin secre-
tion, but their residual beta cell function seemed to
decay more quickly (25). In another study Robles-
Cervantes et al. found that the serum concentration
of uric acid showed a positive relationship with the
total phase of insulin secretion and concluded that
even in states prior to hyperuricemia, uric acid can
play an important role in the function of the beta cell
in patients with type 2 diabetes mellitus (26).
The limitation of our study was that it was a cross
sectional study, and a causal role cannot be assigned
to uric acid. Further, the sample size was small and
further prospective studies are needed to elucidate the
relationship between uric acid and diabetes mellitus.
Conclusion
Our patients with type 2 diabetes mellitus had signif-
icantly higher fasting plasma glucose and HOMA-
IR but significantly lower beta cell function as
compared to controls. Uric acid shows a significant
negative correlation with fasting plasma glucose
in our cases. Though a strong relationship exists
between serum uric acid and diabetes mellitus, the
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087
exact etiopathogenetic mechanism by which uric
acid contributes to this disease are yet to be unrav-
elled. It is clear that further research in this field is
needed to fully comprehend the role of uric acid in
diabetes mellitus.
Acknowledgements
We acknowledge the help received from our col-
league Dr. Lopamudra Ray, Assistant Professor and
Mr. Tamizharasan, Technician, Dept of Biochemis-
try, PIMS and also from Dr. K Ravichandran, Stat-
istician, PIMS.
Conflict of Interest
There is no conflict of interest.
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090 Biomedicine: 2016; 36(1): 090-097

A comparative study in search of the best treatment for non-metastatic locally-advanced

squamous cell carcinoma of head and neck
Sumana Maiti1 and Siddhartha Das2
1
Department of Radio-Therapy, Institute of Post-graduate Medical Education and Research (IPGMER),
Kolkata, West Bengal, India.
2
Department of Physiology, Bankura Sammilani Medical College, Bankura, West Bengal, India.
(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author

Dr. Siddhartha Das. Email: das.siddhartha01@gmail.com

ABSTRACT

Introduction and Aim: Treatment of locally advanced head neck carcinomas has been the matter of dispute
over years. The specific objectives of this study was to compare the efficacy and toxicity profiles of three
different radiotherapy regimens namely, hypo-fractionated Christie regimen, conventional pure accelerated
radiotherapy, and concurrent chemo-radiotherapy.

Materials and Methods: Sixty-nine patients were divided in three arms with 23 patients receiving “Christie”
regimen, 24 patients pure accelerated regimen, and 22 patients concurrent chemo-radiation. The response rates
(using RECIST criteria) and toxicity patterns—both acute and late (by RTOG/EORTC criteria) were assessed
and statistically analyzed.

Results: Overall response rates did not show statistically significant difference amongst three arms. Acute
grade 2 and grade 3 skin and mucosal toxicities were highest in concurrent chemo-radiation arm (p value
0.014 and 0.016 respectively) whereas acute salivary gland toxicity was highest in “Christie” regimen arm (p
value 0.029). However there were no statistically significant differences in late toxicities amongst three arms.
Improved disease free survival (DFS) was observed in concurrent chemo-radiation arm (p value 0.043).

Conclusions: Concurrent chemo-radiation is the best treatment option in terms of loco-regional control and
disease free survival with acceptable increase in acute toxicities in locally advanced squamous cell carcinoma
of head and neck.

Key words: Chemo-Radiation, Christie Regimen, Head Neck Carcinoma.

Introduction Head and neck cancer is best managed in a multi-dis-

C
arcinoma of head and neck is more common
in male than female due to indiscriminate
use of tobacco in various forms and
alcohol. Nearly 60% of newly diagnosed patients
present with locally advanced but non-metastatic
disease (1).
ciplinary setting. Surgery, radiation therapy, chemo-
therapy and, more recently, biologic therapy are often
employed in various combinations in an attempt to
eradicate both clinically apparent and occult diseases.
The goals of treatment include maximizing tumor
control while maintaining function and quality of life.

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Sumana Maiti and Siddhartha Das: A comparative ……….. head and neck
Historically, patients with un-resectable head neck
squamous cell carcinomas used to be treated by
radiotherapy (RT) alone. The radiation dose frac-
tionation has evolved from once daily treatment to
hyper-fractionation and accelerated fractionation
(2). Nonetheless, even the most effective (RT) regi-
mens result in local controls rate of 50%–70% and
disease free survivals of 30%–40%. These circum-
stances lead to the combined modality-concurrent
chemo-radiotherapy as the standard care of treat-
ment for locally advanced head and neck cancer (3).
The optimum treatment time for loco-regional con-
trol is unclear. The possible cause for treatment
resistance could be radiation induced accelerated
proliferation of clonogenic tumour cells. The wors-
ening in the loco-regional control by lengthening
the treatment time has been clinically and biologi-
cally documented (4). Furthermore, several clinical
studies showed reduction in total treatment time has
improved tumour control (5). A shorter treatment
time may be accomplished by applying a higher
dose per fraction or by increasing the weekly num-
ber of radiation fractions.
The benefit of an increased tumour cell kill because
of large fraction size in a short overall treatment
time is counteracted from the radiobiological point
of view, by an increased potential for late side effects
(6). However late toxicity is often less relevant in
patients treated in advanced setting.
The Christie Hospital in Manchester developed a
3-week schedule of RT during World War II when
RT facilities were limited. Results were found not to
be different from the conventional schedules used in
the previous treatment periods in terms of local con-
trol and toxicity (7). This schedule was, therefore,
adopted by Christie hospital and number of other
British cancer centers as a standard RT schedule for
early-stage laryngeal cancer. Many randomized and
non-controlled trials have also shown no difference
in local control between conventional and hypo-
fractionated schedules. Surprisingly, many of these
schedules gave less severe late normal tissue reac-
tion than expected given the short overall treatment
time and the high fraction dose (8).
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091
The effort of this study was to compare the response
patterns in non-metastatic locally advanced head
and neck cancer with squamous cell histology by
three modes of radiotherapy namely: the hypo-frac-
tionated “Christie” regimen; pure accelerated radia-
tion (6 fractions per week); and concurrent chemo-
therapy with conventional radiation.
The specific aim of this study was as follows:
1. To compare the response rates in terms of com-
plete response, partial response and stable dis-
ease in different arms by Response Evaluation
Criteria in Solid Tumors (RECIST) criteria
(9).
2. To determine and compare the acute and chronic
toxicities according to the RTOG/EORTC
(Radiation Therapy Oncology Group/ Euro-
pean Organization for Research and Treat-
ment of Cancer) Acute and Chronic Radiation
Morbidity Criteria.
3. To assess the disease free survival (DFS) rates in
different regimens.
Materials and Methods
This study was done upon ethical clearance by the
Institutional Ethics Committee and as per Helsinki
declaration.
Study design
A single-institutional, prospective, open labeled,
longitudinal, randomized controlled study.
Patients attending the Radiotherapy OPD, Depart-
ment of Radiotherapy, IPGMER, Kolkata were
selected for study.
Inclusion criteria
1. Age 18–70 years
2. Histologically proven non-metastatic stage
III, IVA and IVB Squamous cell carcinoma of
head and neck (staging according to Prognostic
Groups, American Joint Committee on Cancer,
AJCC, 6th edition.)
Biomedicine – Vol. 36 No. 1: 2016
092 Sumana Maiti and Siddhartha Das: A comparative ……….. head and neck

3. No previous history of treatment of cancer Treatment plan

4. Creatinine clearance more than or equal to Treatment was given using megavoltage equipment
60 ml/minute. with Cobalt-60, energy 1.17, 1.33  MeV (average
1.25 MeV). The minimum Source-Surface Distance
Exclusion criteria (SSD) was 80 cm.

1. Histopathology other than squamous cell carci-
noma.
2. Patients with stage I or II diseases
3. Evidence of distant metastases by clinical or
radiological examination.
4. Prior Radiotherapy for any reason to head and
neck region.
5. No other primary malignancies.
Study period
January 2010 to August 2012.
Sample size
Patients in Arm A (Christie Regimen) received
External Beam Radiotherapy(EBRT) with dose of
50  Gy (Gray) in 15 fractions, single fraction per
day, 5 fractions per week, 3.33 Gy per fraction, total
duration 3 weeks.
Arm B received 66 Gy in 33 fractions, 2 Gy per frac-
tion, 6 fractions per week, single fraction per day,
total duration 51/2 weeks.
Arm C received 66  Gy in 33 fractions, 2  Gy per
fraction, single fraction per day, total duration
61/2 weeks with IV inj cisplatin (30 mg/m2) weekly.
Inj Cisplatin was given from the first day of start of
EBRT.

Fifty (50). Patients in arm A and C were assigned five fractions

Detailed procedure of study
Before the inception of the study an application
was submitted to the Institutional Ethics Committee
(IEC); IEC after proper scrutiny and detailed delib-
eration review approved the research proposal.
Pre-treatment assessment
1. Detailed history, clinical examination with ENT
examination
2. Biopsy from primary site and/or FNAC from
lymph node
3. Contrast enhanced CT scan of head and neck
4. Routine investigations—hematological, bio-
chemical, chest skiagram
Radiation therapy—External beam radiation
therapy (EBRT)
per week, given one fraction daily from Monday to
Friday. Patients in arm B were assigned six fractions
per week, one fraction daily, from Monday to Fri-
day, and the sixth fraction given on Saturday.
After completion of treatment
Six to 8  weeks after completion of treatment, a
detailed ENT examination and Contrast enhanced
CT scan of head and neck were done to assess the
response. Biopsies from suspicious clinical and /or
radiological residual disease of primary site and/or
nodal area were performed. Those with residual dis-
ease were sent for surgery if feasible.
The primary end point of the study was loco-
regional control in terms of complete response, par-
tial response, stable disease and progressive disease
after EBRT in three different arms using recently
published “Response Evaluation Criteria in Solid
Tumors” (RECIST) criteria (9).

Instrument used—cobalt 60-ATC-C9 (Picker manu- The acute and late toxicity of the patients in all arms
facture) were assessed during the treatment and during the

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Groups
GR A (n = 23), n % GR B (n = 24), n % GR C (n = 22), n %
Age (years)
40–49 6 (26) 7 (29.2) 5 (22.7)
50–59 8 (34.8) 7 (29.2) 9 (40.9)
60–69 9 (39.1) 10 (41.7) 8 (36.4)
Table 1  Number of patients in the treatment protocol.
ANOVA test p-value ~0.740 (not significant).

Groups
Responses Group A: Christie Group B: Pure accelerated Group C: Cisplatin
regimen, n (%) radiation, n (%) chemoradiation, n (%)
Complete response 11 47.8 11 45.8 11 50.0
Partial response 7 30.4 6 25.0 6 27.3
Stable 3 13.0 5 20.8 2 9.1
Table 2  Response assessment by RECIST, 6–8 weeks post treatment.

Groups
Acute radiation
Group A: Christie Group B: Pure accelerated GROUP C: Cisplatin p Value
induced changes
regimen, n (%) radiation, n (%) chemoradiation, n (%)
Skin
Grade 0 6 26.1 2 8.3 1 4.5 0.014
Grade 1 3 13.0 6 25.0 1 4.5
Grade 2 8 34.8 10 41.7 7 31.8
Grade 3 6 26.1 6 25.0 13 59.1
Mucosal
Grade 0 2 8.7 3 12.5 1 4.5 0.016
Grade 1 3 13.0 8 33.3 2 9.1
Grade 2 11 47.8 9 37.5 7 31.8
Grade 3 7 30.4 4 16.7 12 54.5
Table 3  Radiation induced acute toxicities.

Groups
Salivary gland Group A: Christie Group B: Pure accelerated GROUP C: Cisplatin p Value
regimen, n (%) radiation, n (%) chemoradiation, n (%)
Acute
Grade 1 5 21.7 12 50.0 13 59.1 0.029

Grade 2 18 78.3 12 50.0 9 40.9

Late
Grade 1 9 39.1 19 79.2 13 59.1 0.064
Grade 2 12 52.2 3 12.5 9 40.9
Grade 3 2 8.7 2 8.3 0 0.0
Table 4  Radiation induced Acute and Late Salivary gland changes.

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094 Sumana Maiti and Siddhartha Das: A comparative ……….. head and neck

Late radiation Groups

induced changes Group A: Christie Group B: Pure accelerated GROUP C: Cisplatin p Value
regimen, n (%) radiation, n (%) chemoradiation, n (%)
Skin
Grade 0 3 13.0 3 12.5 1 4.5 0.4.35
Grade 1 10 43.5 14 58.3 11 50.0
Grade 2 6 26.1 6 25.0 9 40.9
Grade 3 4 17.4 1 4.2 1 4.5
Mucosal
Grade 0 6 26.1 2 8.3 0 0 0.381
Grade 1 10 43.5 15 62.5 15 68.2
Grade 2 5 21.7 6 25.0 4 18.2
Grade 3 2 8.7 1 4.2 3 13.6
Table 5  Radiation induced late toxicities.

follow-up period using clinical status, laboratory
tests and the grade according to the RTOG/ EORTC
(Radiation Therapy Oncology Group/European
Organization for Research and Treatment of Cancer)
Acute and Chronic Radiation Morbidity Criteria.
Statistical analysis was done using SPSS version 17.
The ANOVA test was used for comparing baseline
profiles, the response rates and toxicities among
patients of three treatment arms, with p value <0.05
as significant. Disease free survival (DFS) was mea-
sured from the date of completion of treatment to
the date of first relapse (loco-regional or distant
metastasis) or death. The disease free survival was
determined using the Kaplan Meier survival analy-
sis with Log Rank test for comparing the DFS.
Result and Analysis
The study was designed as single institutional, pro-
spective, open labeled, longitudinal, randomized con-
trol study, conducted from January 2010 to July 2012.
Sixty-nine patients were randomly selected after
fulfilling the eligibility criteria with 23 patients in
“Christie Regimen” (group A), 24 in “Pure accel-
erated radiotherapy” (group B) and 22 patients in
“Concurrent chemo-radiation” (group C).
Comparison of demographic profiles amongst dif-
ferent treatment arms showed no statistically signifi-
cant difference.
ANOVA analysis showed no statistical difference in
Overall response (CR+PR) rates among the three3
treatment arms (p value ~ 0.945)analysis showed no
statistical difference in Overall response (CR+PR)
rates among the three3 treatment arms (p value ~
0.945) treatment arms (p value ~0.945).
Sixty-nine patients were evaluated for response at
stipulated 6–8 weeks post treatment using RECIST
criteria. Overall response rates (CR+PR) were
78.2% in Arm A—Christie arm, 70.8% in Arm B—
pure accelerated radiotherapy schedule and 77.3%
in arm C—concurrent chemoradiation with ANOVA
analysis showed no significant statistical difference
(p value ~0.945).
The skin toxicity assessed by RTOG Acute Morbid-
ity Scoring was significantly high in chemo-radia-
tion arm showing highest grade 2 and 3 toxicities
of 90.9% versus 66.7% in pure accelerated regi-
men arm and 60.9% in “Christie regimen” arm with
ANOVA test df 2.66, f 4.560 and p value 0.014 (sig-
nificant). (Table 3)
The acute mucosal toxicity assessed by RTOG
Acute Morbidity Scoring was significantly high in
chemo-radiation arm showing highest grade 2 and 3
toxicities of 86.3% versus 54.2% in pure accelerated
radiation arm and 78.2% in “Christie regimen” arm,
with ANOVA test df 2.66, f 4.376 and p value 0.016
(significant) (Table 3). The acute and late radiation
induced toxicities of salivary gland were more in

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Sumana Maiti and Siddhartha Das: A comparative ……….. head and neck 095

Groups Median
Estimate Std. error 95% Confidence interval
Lower bound Upper bound
Group A: Christie regimen 8.000 1.239 5.572 10.428
Group B: Pure accelerated radiation 12.000 2.276 7.538 16.462
Group C: Cisplatin chemoradiation 13.000 1.460 10.138 15.862
Overall 11.000 1.293 8.465 13.535
Table 6  Medians for Disease Free Survival Time (in months).
Estimation is limited to the largest survival time if it is censored.

Chi-square Df Sig. therapy and radiation may improve the local control
Log Rank 6.294 2 0.043 and survival rate because of the additive or synergis-
tic effect of chemo-radiation. Altered fractionation
(Mantel-Cox)
improves loco-regional control in patients with con-
Table 7a  Disease Free survival Comparisons. traindication for chemotherapy (11).

Chi-square Df Sig. This study aims to compare the outcome of three

Log Rank 6.544 2 0.038
(Mantel-Cox)
Table 7b.  Disease free survival comparisons.
Adjusted for Stage. Test of equality of survival distributions for the different
levels of GROUPS.
Christie radiation arm than other arms with the acute
ones significantly higher (Table 4).
In spite of high toxicities in all the arms, there were
no statistical difference in late effects in any of the
treatment arms (Table 5).
At a median disease free survival of 11 months, the
disease free survival using Kaplan Meier survival
analysis with adjustment for stage, was significantly
better in chemo-radiation arm with Log Rank analy-
sis Chi Square 6.544, df 2 and p value 0.043 (Tables
6 and 7).
Discussion
Locally advanced head and neck cancer is usually
associated with a poor prognosis because of high
recurrence rate despite aggressive management with
surgery followed by post-operative radiation (10).
In an attempt to improve the prognosis, concurrent
chemo-radiation was introduced, chemotherapy act-
ing as a radio-sensitizer. The combination of chemo-
different radiotherapy regimens—hypo-fractionated
“Christie regimen,” conventional pure accelerated
radiotherapy and concurrent chemo-radiation using
weekly inj cisplatin (30 mg/m2). No such compara-
tive study is available in the existing literature. Very
few study are available in literature using “Christie
regimen” in definitive settings.
The present study results corroborates with previous
studies with regard to the response rates along with
significantly increased acute skin toxicities and acute
mucosal toxicities in cases treated with concurrent
chemo-radiotherapy using weekly cisplatin (10,12).
But head and neck cancer patients commonly carry
excessive co-morbidities due to chronic tobacco
and alcohol use and many of them would not meet
the basic eligibility criteria for extensive chemo-
radiotherapy with existing co-morbid conditions
like renal failure, hearing difficulties or neuropathy.
Instead, pure accelerated radiation and hypo-frac-
tionated “Christie regimens” are possible treatment
options.
The well documented relationship between acceler-
ation by 1 week and improved local control has also
been established in the present study, where over-
all response rate is better, though the toxicities were
high but could be managed conservatively and there
were no incidence of treatment break.

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096 Sumana Maiti and Siddhartha Das: A comparative ……….. head and neck
The results of our study showed insignificant sta-
tistical difference between the responses and late
toxicities among the three treatment arms (Tables 2,
4, and 5), despite significantly increased acute skin
and mucosal toxicities in concurrent chemo-radia-
tion arm and acute salivary gland toxicity in Christie
regimen arm.
Whithers et al. reported that the total dose of RT
needed to control 50% of head and neck carcino-
mas progressively increased with time whenever
radiation therapy treatment was prolonged beyond
1 month (4). This increase was attributed to acceler-
ated repopulation, and it was estimated to ~0.6 Gy/
day, but may be as high as 1 Gy/day. Extension of
overall duration of therapy results in decrease in
tumour control rate by 1%–2% each day after the
lag period of 4 weeks (13).
Biological Equivalent Dose (BED) corrected for
time in concurrent chemo-radiation arm (treat-
ment duration of 45 days) is 58.4 Gy (α/β = 10 Gy)
and for pure accelerated arm is 61.6  Gy assuming
α/β  =  10  Gy, total treatment duration of 38  days
(14). Net results should show that maximum benefit
would be seen in pure accelerated arm when com-
paring only radiation.
However, addition of chemotherapy with its addi-
tive effects (cytotoxicity) and supra additive effects
(described by 5th “R” of radiobiology, i.e., radio-
sensitivity), the resultant BED is increased by
9–11  Gy BED equivalent. So from radiobiological
point of view, concurrent chemo-radiotherapy is
always a better option. The benefit of chemotherapy
was seen in improved disease free survival.
It was also well established that fraction size would
be the dominant factor in determining late effects;
overall treatment time has little influence. However,
fraction size and overall treatment time both deter-
mine the response of acutely responding tissues (15).
Despite highest acute salivary gland toxicity in
Christie regimen, no statistically significant differ-
ence in chronic toxicity was observed amongst three
arms (Table 4). Possible explanation may be shorter
follow up period or salivary glands received more
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than maximum tolerance dose (TD5/5 = 26 Gy) in
all three arms due to conventional planning of appli-
cation.
However, there were several pitfalls of the study.
1. Our sample size was small, so any statistical
data was to be interpreted with caution.
2. Locally advanced head and neck cancer in Indian
population is probably different from those of
the Western World, establishing the basic con-
cept of pharmaco-genomics and its impact on
cancer.
Conclusion of the Study
Concurrent chemo-radiation is the best option in
terms of loco-regional control and disease free sur-
vival with acceptable increase in toxicity in case of
locally advanced squamous cell carcinoma of head
and neck.
With co-morbidities limiting the aggressive use of
chemotherapeutic agents in many patients, altered
radiation fractionations, that is, pure accelerated
and hypo-fractionated “Christie” radiation regimens
remain viable alternatives to chemo-radiation, with
similar response rates and acceptable toxicities, but
definitively inferior disease free survival.
Further studies with larger sample size and longer
follow up period are required for establishing these
observations.
References
  1. Edward, C., et al. Perez and Brady’s Principles
and practice of radiation Oncology, 5th Edn.,
Lippincott Wolter Kluwer, Philadelphia, 2008;
817.
  2. Fu, K.K., Pajak, T.F., and Trotti, A. Radiation
Therapy Oncology Group (RTOG) phase III
randomized study to compare hyperfraction-
ation and two variants of accelerated fraction-
ation to standard fractionation radiotherapy for
head and neck squamous cell carcinomas first
report of RTOG 9003. Int. J. Radiat. Oncol.
Biol. Phys. 2000; 48: 7–16.
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  3. Fu, K.K. Combined modality therapy for head
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  8. Corry, J.F., et al. The ‘Quad Shot’, a phase II
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  9. Therasse, P., Arbuk, S.G., Eisenhauer, E.A., et
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review. Cancer Control. 2002; 9: 387–399.
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12. Nguyen, N.P., and Sallah, S. Combined chemo-
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13. Overgaard, J., et al. Five compared with six
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098 Biomedicine: 2016; 36(1): 098-102

Comparison in between subjects of determinants of oxygen uptake during maximal

and submaximal exercise: ventilation and O2 pulse, determinants of O2 uptake are less
dependent on load in submaximal exercise at ventilatory threshold
Thiagarajan K.A.,1 Vasanthi C.,1 Parikh T.,1 Madhusudhan Rao V.,1 and Arumugam S.1
1
Department of Arthroscopy and Sports Medicine, Sri Ramachandra Medical College and Research Institute,
Sri Ramachandra University, Porur, Chennai, India.

(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author

V. Madhusudhan Rao. Email: madhuoxon@hotmail.com

Abstract

Introduction and Aim: Stroke volume (SV), heart rate (HR), (a - v)O2 and ventilation (Ve) determine the
rate of Oxygen uptake (VO2). These, in turn, are determined by chemical, non-chemical, psychological fac-
tors and load (work rate). The present study was undertaken to study if load determined factors determining
O2 uptake viz minute ventilation (Ve) and O2 pulse (an indicator of SV and (a - v)O2) during maximal and
submaximal exercise. Also examined was the relationship between VO2 and Ve, O2 pulse to elucidate influ-
ences other than load acting on Ve and O2 pulse.

Materials and Methods: The study conducted on young healthy male volunteers. Load at maximal exercise
was computed from the load at VO2 max and load at submaximal exercise was computed from the load at ven-
tilatory threshold (50%–80%VO2max). Data was collected from 15 out 25 subjects studied, excluding data
which showed artifacts, incomplete data and spurious values. Subjects exercised breathing through a mask
on a treadmill to exhaustion as the load was increased by changing incline and speed every 2.5 minutes. Data
analysed by Oxycon Pro analyser connected to a computer. Load, O2 uptake, O2 pulse, minute ventilation
(Ve), EqO2 and dO2/dW were computed for maximal and submaximal exercise. Correlation coefficients were
calculated for maximal and submaximal exercise for VO2 vs O2pulse, VO2 vs Ve and O2 pulse vs Load, Ve
vs Load and VO2 vs Load. Paired t-test was done for EqO2 and dO2/dW.

Results: VO2 was correlated with O2 pulse and Ve for both maximal and submaximal exercise. O2 pulse,
Ve and VO2 (max) were correlated with Load for maximal exercise. There was less correlation of O2
pulse, Ve with load at submaximal exercise. EqO2 was significantly different. dO2/dW was not signifi-
cantly different.

Conclusion: Cardiac output (O2 pulse-SV and (a - v)O2) and ventilation (Ve) are less determined by the load
during submaximal exercise, chemical factors being important in driving ventilation and central command
independent of the load for increasing cardiac output. In contrast, cardiac output and ventilation are deter-
mined by Load during maximal exercise. However dO2/dW is similar, and Load correlated with VO2 during
both indicating that load has a role.

Key words: EqO2, Load, Oxygen Pulse, Oxygen Uptake, Workrate

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Thiagarajan et al.: Comparison in between ………… ventilatory threshold
Introduction
T
he determinants of O2 uptake (VO2) are
ventilation and cardiac output. It has been
found that work rate (load) and O2 uptake
(VO2) show a linear relationship at all intensities
(1). However, it is not known what the relationship
is between load and determinants of VO2 between
subjects. The present study was conducted to
examine between subjects the relationship between
load (work rate) and determinants of VO2 viz Ve
and O2 pulse which is discussed next. From work
on hypoxia it has been found that pulmonary gas
exchange since PaO2 is lower and cardiac output,
since SV and HR are lower than normal, contribute
to VO2 in normals (2). The factors that determine
pulmonary gas exchange and cardiac output are
ventilation (Ve) and O2pulse. Ventilation determines
the supply of O2 and O2 pulse (O2/HR) derived
from the Fick equation, which is a measure of SV
and (a - v)O2 (3,4) determines cardiac output and
extraction of O2. These viz Ve and O2 pulse are the
determinants of VO2 and are controlled in turn by
chemical, nonchemical, psychological factors (5)
and load.
It is possible to study the effect of the load during
incremental exercise on Ve, O2 pulse and VO2 at
ventilatory threshold (VT) for submaximal exercise
and at VO2max which provides data for maximal
exercise. VT was chosen to study submaximal exer-
cise as it enables comparison between subjects for
submaximal exercise (VT) and data at VO2max
provide a comparison between subjects for maximal
exercise (ME).
The present study also examined the relationship
between VO2 on one hand and Ve andO2 pulse on
the other to elucidate influences other than load act-
ing on Ve and O2 pulse.
Materials and Methods
The study was carried out on normal young healthy
male volunteers performing incremental exercise
on a treadmill. Ethical clearance and consent were
obtained for the study. Subjects breathed through
a mask and exercised to exhaustion on a tread-
mill while incline and speed were increased every
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099
2.5  min. Data were analysed by Oxycon Pro anal-
yser connected to a computer. Load (work rate),
VO2 (oxygen uptake), O2 pulse (O2/HR), Ve (ven-
tilation), EqO2 (a ventilatory equivalent of O2
uptake  -  Ve/VO2)and dO2/dW were computed at
VT (ventilatory threshold 50%–80% VO2max) (6)
for submaximal and at VO2max for maximal exer-
cise. Data were studied from 15 out of 25 subjects
excluding data with artefacts, incomplete data and
spurious values.
Results
The data are tabulated in Table 1. As indicated EqO2
is significantly less (p  =  0.0000) at VT indicating
that Ve is relatively lower at VT. Ve is less corre-
lated with load at VT (r = 0.5 at VT and r = 0.74 at
ME) and O2 pulse is less correlated with load at VT
(r = 0.63 at VT and 0.73 at ME) indicating that the
determinants of VO2 viz Ve and O2 pulse are less
dependent at VT on load which acts through corti-
cal influences and that load is maximally effective
at maximal exercise. VO2at VT as %VO2 max is
63.8 ± 10.4 (CV -16%) indicating that cortical influ-
ences are variable at VT. VO2 is correlated with load
both at VT and ME (r = 0.67 at VT and 0.74 at ME)
This is consistent with the result that dO2/dW is not
significantly different at VT and ME (p  =  0.068)
indicating that load also has a role.
Despite the fact that at VT , Ve and O2 pulse are
less correlated with load at submaximal exercise ,
both during submaximal as well and maximal exer-
cise, VO2 is correlated with Ve (r = 0.87 at VT and
r = 0.57 at ME) and with O2 pulse (r = 0.67 at VT and
r = 0.82 at ME) indicating that chemical influences
for Ve and non load dependent central command
activation of sympathetic for O2pulse are important
at VT given that cortical influences through load at
submaximal exercise are submaximal. At maximal
exercise load is maximally active in determining Ve
and O2 pulse.
Discussion
Load as determinant of Ve and EqO2 vs chemical
influences.
If ventilation is matched with O2 uptake (VO2)
throughout an incremental exercise, one should
Biomedicine – Vol. 36 No. 1: 2016
 O2/

100
Age, BM, Ht, dO2/ Load, Ve, l/ VO2, VT%
Name EqO2 HR,
years kg cm dW W mi ml VO2 max
ml
VT M VT M VT M VT M VT M VT M

MM 22 53 172 20.6 31 10.5 10 51 148 23 65 7.8 10.7 947 1898 48

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LR 18 60 175 25.6 45 10.5 10 171 313 64 143 12.7 13.8 2283 2988 76

AJ 19 65 172 28.3 38 7.9 7.7 186 258 64 107 11.7 13.3 2134 2646 80

SG 19 51 171 22.8 32.6 11.9 11.1 90 200 40 95 9.7 13.3 1564 2709 80

GG 19 74 172 18.4 28 8 9.8 135 296 35 106 12.7 17.7 1768 3593 49

MD 19 80 183 23.4 29 9.7 11.1 147 232 55 101 14.4 17 2166 3301 65

SM 21 91 189 21 35.8 8.87 8.63 172 367 46 139 15.9 19 2086 3727 55

KR 21 76 176 23.3 38.2 7.2 9.86 220 308 54 137 12.7 18.8 2167 3620 59

ZA 19 50 165 24.6 31.5 6.23 7.63 196 240 43 77 10.2 14.3 1589 2266 70

PH 19 50 178 23.3 35.7 6.96 9.13 139 196 42 92 11.9 15.6 1589 2406 66

ASK 26 77 180 22.1 33.6 6.48 7.63 223 391 47 124 16.4 20.5 2015 3551 56

TS 19 63 177 28.1 35.6 15.4 15 112 183 65 118 11.8 15.6 2084 3098 67

YS 19 60 172 23.2 36.2 8.52 9.1 169 237 50 103 12 14.5 1966 2688 73

SKT 28 70 161 18.5 32.5 7.15 9.8 130 200 33 92 10 16 1624 2665 60

RV 19 68 175 23.1 36 8.28 9.4 125 270 44 124 12.4 15.8 1749 3258 53

Table 1  Data from 15 subjects. VT—data at Ventilatory Threshold (Submaximal Exercise); M—data at VO2max (Maximal Exercise).

Biomedicine – Vol. 36 No. 1: 2016

Thiagarajan et al.: Comparison in between ………… ventilatory threshold
Thiagarajan et al.: Comparison in between ………… ventilatory threshold
find that EqO2 (a ventilatory equivalent of O2
uptake  - Ve/VO2) is unchanged. However EqO2
is lower at VT, also noted in an earlier study (7)
indicating that Ve is relatively less at submaxi-
mal exercise than at maximal exercise. Since Ve
is poorly correlated with load (r = 0.5) at VT and
since load acts through cortical mechanisms it
suggests that psychological factors are lower in
submaximal exercise. It is possible that load is
not great enough and that chemical influences are
more important in driving ventilation below VT.
Inadequate supply of O2 leads to lactic acid accu-
mulation by anaerobic metabolism. H+ ions drive
ventilation which increases disproportionately to
O2 uptake(VO2) beyond VT (8). However, there
is a correlation between load (work rate) and VO2
as well at submaximal exercise (r  =  0.67) and at
maximal exercise (r  =  0.74) indicating that load
has a role.
Load as a determinant of O2 pulse
The other factor that determines O2uptake is the
cardiovascular response indicated by O2 pulse (O2/
HR) which is a measure of stroke volume (SV) and
(a  -  v)O2. SV depends on EDV and to a greater
extent on inotropic effect and (a -  v)O2 depends
on muscle metabolic activity. Cortical influence
through Central Command seemingly only in part
activated by the load during exercise increases
inotropic and chronotropic effects by stimulation
of sympathetic increasing SV and HR. O2 pulse
which indicates SV is less dependent on load in
submaximal exercise since O2 pulse is less corre-
lated with load (r = 0.63) than in maximal exercise
(r  =  0.73). This suggests that cortical influences
are not maximally active during submaximal exer-
cise. Increase in (a - v)O2 due to muscle metabo-
lism along with higher SV could account for the
greater O2 pulse at higher intensities of exercise.
Mechanism of action of load
Since load acts through cortical mechanisms,
the findings suggest that psychological factors
in submaximal exercise are not uniform between
subjects. The finding that CV is large-16%, for
VO2 at VT expressed as % of VO2max also
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101
indicates that the effect of load is variable. In
contrast in maximal exercise, the psychological
effort exerted is maximal and dependent on load
between subjects. This is analogous to the phe-
nomenon that HR varies between individuals in
submaximal exercise, but HR max is about the
same for all (9). Since the load is determined by
body mass and psychological effect is maximal
at VO2max, along with chemical influences, psy-
chological effort multiplied by load leads to the
finding that Ve and O2 pulse, the central com-
mand being maximally activated are correlated
with load at maximal exercise.
Determinants of VO2
Despite the fact that Ve and O2 pulse are less cor-
related with a load in submaximal exercise, VO2
is correlated with both. This shows that VO2 is
matched with a supply of O2 and extraction of
O2 throughout an incremental exercise. As men-
tioned earlier Ve and O2 pulse are determined by
chemical and nonchemical influences. Periph-
eral chemoreceptors have been implicated in
ventilatory response (6). Non-chemical control
viz cortical outflow (motivation) and proprio-
ceptor stimulation for ventilatory response and
central command activation independent of load
to stimulate sympathetic activity resulting in an
increase in cardiac output through an increase in
SV and HR seem to be important influences in
both submaximal and maximal exercise. Opinion
is divided the relative contribution of SV and HR
to increase in cardiac output. The contribution of
SV reportedly levels off (10) or increases (11) till
VO2max. This is relevant while considering the
relative contributions of SV and (a - v)O2 to O2
pulse which increases throughout an incremental
exercise. It has been found that after training, an
increase in VO2 is due to increase in cardiac out-
put not (a - v)O2 (12).
Conclusion
The determinants of VO2 (oxygen uptake) viz Ve
and O2 pulse (cardiac output) are less dependent
on load at submaximal exercise. In contrast, load
determines Ve and O2 pulse at maximal exercise.
Biomedicine – Vol. 36 No. 1: 2016
102 Thiagarajan et al.: Comparison in between ………… ventilatory threshold
However, VO2 is correlated with Ve and O2 pulse
both at submaximal and maximal exercise. This
shows that cortical influences act through two
mechanisms viz load independent and load depen-
dent and that chemical influences contribute to
driving ventilation especially at submaximal exer-
cise leading to a ventilatory threshold.
References
  1. Muniz-Pumares, D., Godfrey, R., Pedlar, C., and
Glaster, M. The relationship between O2 uptake
and work rate below and above lactate threshold.
Br. J. Sports Med. 2013; 47: e4.
  2. Calbet, J.A.L., Boushel, R., Radegran, G., Son-
dergard, H., Wagner, P.D., and Saltin, B. Deter-
minants of maximal O2 uptake in severe acute
hypoxia. Am. J. Physiol. 2003; 284: 291–303.
  3. Hopker, J.G. Controversies in the physiological
basis of ‘anaerobic threshold’ and their impli-
cations for clinical cardiopulmonary testing.
Anaesthesia. 2011; 66: 111–123.
  4. Klainman, E., Fink, G., Lebzelter, J., Krel-
baumm, T., and Kramer, MR. The relationship
between left ventricular function assessed by
multigated nuclear test and cardiopulmonary
exercise test in patients with ischemic heart dis-
ease. Chest. 2002; 121: 841–845.
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  5. Jenkins, S.P.R. Sports Science Handbook, Vol.
1. Multi-Science Publishing, United Kingdom,
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  6. Plowman, S.A., and Smith, D.L. Exercise Physi-
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liams and Wilkins, Philadelphia, 2014.
  7. Solberg, G., Robstad, B., Skjonsberg, O.H., and
Borschenius, F. Respiratory gas exchange indi-
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  8. Kenny, W.L., Wilmore, J.H., and Costill, D.L.
Physiology of Sports and Exercise, 5th Edn.
Human Kinetics, Champaign, IL, 2012.
  9. McArdle, W.D.., Katch, F.L., and Katch, V.I.
Exercise Physiology, 3rd Edn. Lea and Febiger,
Philadelphia, PA, 1991.
10. Rowland, T.W., and Green, G.M. Circulatory
response to exercise: are we misreading Fick?.
Chest. 2005; 127: 1023–1030.
11. Gledhill, N., Cox, D., and Jamnik, R. Endurance
athlete’s stroke volume does not plateau: major
advantage in diastolic function. Med. Sci. Sports
Exerc. 1994; 26: 1116–1121.
12. Basset, D.R., and Howley, E.T. Limiting factors
for maximum oxygen uptake and determinants
of endurance performance. Med. Sci. Sports
Exerc. 2000; 32: 70–84.
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Biomedicine: 2016; 36(1): 103-108 103

Is autonomic function test helps to assess the severity of metabolic syndrome: A study
on comparison of Frequency-Domain recordings of Heart rate variability (HRV) with
the severity of metabolic syndrome
Bhagyashree N.,1 C. Ramaswamy,2 Ganesh M.,3 and Udaya Ganesh B.4
1
Assistant Professor, ACS Medical College & Hospital, and Ph.D scholar, Saveetha University, Chennai, India.
2
Ph.D Guide, Saveetha University, Chennai, India.
3
ACS Medical College & Hospital, Chennai, India.
4
Sri Jayendra Saraswathi Ayurveda College & Hospital, Chennai, India.

(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author
Mrs. Bhagyashree N. Email: bhagyashivanugraha@gmail.com

Abstract

Introduction and Aim: Metabolic syndrome constitutes a bouquet of three or more of any five components
like obesity, hypertension, diabetes, hypertriglyceridemia and low-HDL concentration in an individual and
cardiovascular disease which is being the center. To find out the relationship between HRV parameters with
the severity of the metabolic syndrome.

Materials and Methods: A total of ninety patients were divided into 3 groups as a group I with those hav-
ing only 3 components of the metabolic syndrome, group II with more than 3 components of the metabolic
syndrome and group III with less than three components of the metabolic syndrome. The group I served as
normal MS people, the group II as severe MS and group III as a control group. The Frequency-domain HRV
parameters like TP, LF, HF and LF/HF ratio were recorded in those subjects and were analyzed.

Result: The result showed that group II has high LF and low HF than the group I and III. The LF/HF ratio of
group II was higher among them. All these parameters of group 1 and 3 were comparable. The TP among the
groups showed no significant difference.

Conclusion: Once metabolic syndrome set in, cardiac reflexes are activated and proportionate to severity, the
sympathetic activity increases, and the parasympathetic activity decreases causing reduced cardiac autonomic
control. The LF/HF ratio increases proportionately with the severity of the diseases. Hence, the LF/HF ratio
may be a better prognostic tool towards the management of metabolic syndrome.

Key words: Cardiac Autonomic Modulation, Frequency–Domain, HRV, Metabolic Syndrome

Introduction glucose tolerance and central obesity (1). The

M
etabolic syndrome (MS) also known
as syndrome X is characterized by
hypertriglyceridemia, low concentration
of high density lipoprotein (HDL) cholesterol
(dyslipidemia), elevated blood pressure, impaired
metabolic syndrome carries an increased risk for
cardiovascular disease and diabetes (2) is associated
with alterations in the function of numerous elements
of the cardiovascular system. The autonomic nervous
system plays a central role in the cardiovascular

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104 Bhagyashree et al. Is autonomic function ....................... metabolic syndrome
regulation (3). Cardiovascular disease is the leading
cause of death and disability worldwide (4). Heart
rate variability (HRV) represents cardiac autonomic
control (5,6) and it has emerged as a practical,
noninvasive tool to quantitatively investigate
cardiac autonomic dysregulation and it has been
proposed as a predictor of increased risk for cardiac
mortality (4). Reduced HRV significantly increases
cardiovascular mortality (5). The high-frequency
component (HF, 0.15–0.4 Hz) of the HRV spectrum
reflects vagal modulation of the sinus node; the low-
frequency component (LF, 0.04–0.14 Hz) represents
either sympathetic modulation or a combination of
sympathetic and vagal influences. The ratio between
LF and HF components (LF/HF ratio) is considered
to reflect sympathovagal influences on heart rate
control (6). Although many attempts have been done
to study the cardiac autonomic functions with each of
the individual components of metabolic syndrome,
the relationship between severities of the metabolic
syndrome with the cardiac autonomic dysfunction
is not available in the literature. Hence, the present
study was conducted to find out the cardiac autonomic
changes in subjects with the number of metabolic
abnormalities that constitute metabolic syndrome
and we hypothesized that significant difference in
cardiac autonomic dysfunction is seen in subjects
with more number of metabolic abnormalities.
Objective
To find out the relationship between the frequency–
domain parameters of HRV with the severity of met-
abolic syndrome proportionately indicated by the
number of constellation of diseases that constitute
metabolic syndrome.
Materials and Methods
The study was carried out among 90 subjects in three
groups, each comprising 30 subjects. Age-matched,
sex-matched subjects were recruited for the study.
Group I: Subjects with any three components of
metabolic syndrome (normal MS group)
Group II: Subjects with more than three components
of metabolic syndrome (severe MS group)
www.biomedicineonline.org
Group III: Subjects with less than three components
of metabolic syndrome (Control group)
The diagnosed metabolic syndrome patients were
recruited for the present study. The diagnostic cri-
teria for metabolic syndrome were according to
National Cholesterol Education Program Adult
Treatment Panel III (NCEP ATP III) criteria (≥3 of 5
risk factors) (7) and with the inclusion and exclusion
criteria as detailed below.
Inclusion criteria
Subjects with –
 Waist circumference ≥102 cm (40 inches) in
men and ≥88 cm (35 inches) in women.
 Triglyceride ≥150  mg/dl or history of drug
consumption for hypertriglyceridemia.
 HDL ≤40  mg/dl in men and ≤50  mg/dl in
women or history of drug consumption.
 Systolic BP ≥130  mmHg and diastolic BP
≥85  mmHg or history of antihypertensive
drug consumption.
 Fasting blood glucose ≥100 mg/dl, history of
diabetes mellitus or using antidiabetic drug.
Exclusion criteria
Pregnant women, patients suffering from the neuro-
logical defect, patients suffering from the urological
defect, patients having cancer, patients with acute
coronary syndromes within 3 months, patients with
atrial fibrillation, subjects with the past/present his-
tory of RS, CVS disorders.
The study was carried out in the department of
Physiology, A C S Medical College and Hospital,
Chennai. The participants were from A C S Medi-
cal College and Hospital, Saveetha Medical College
and Hospital, Chennai. The study commenced after
getting the approval from the Institutional Human
Ethical Committee (IHEC), Saveetha University,
Chennai.
The data were collected from the participants after
giving a detailed explanation about the procedure
Biomedicine – Vol. 36 No. 1: 2016
Bhagyashree et al. Is autonomic function ....................... metabolic syndrome 105

and their cooperation and willingness were obtained
with informed consent. A detailed clinical history
of all the subjects was taken. Relevant past history,
family history, any drug history, personal history
like smoking, alcoholism etc. was taken.
The ECG recording was taken for 5  minutes after
10 minutes of seated rest, under standardized condi-
tions to minimize artifacts. ECG signal was obtained
using limb lead II and analysis was performed using
HRV analysis software version 1.1, Biomedical Sig-
nal Analysis Group, Department of Applied physics,
University of Kuopio, Finland. The recording was
done in the morning hours between 9 AM and 11
AM. The subjects were instructed to avoid coffee or
alcohol 24 hours prior to testing and to avoid food
two hours prior to testing. While recording, the sub-
jects were instructed to close the eyes, and to avoid
talking, coughing, moving of hands, shaking the
legs and body, and sleeping during the test.
The data is analyzed between the groups statistically
by using Student’s t-test and the p < 0.05 was con-
sidered as significant.

Result

Parameters Group I Group II p-Value

Total power (ms2) 684.03 ± 1694.69 542.03 ± 1308.58 0.717 NS
LF (%) 50.34 ± 30.10 70.76 ± 29.95 0.010*
HF (%) 16.79 ± 12.95 8.39 ± 8.16 0.003*
LF/HF 4.11 ± 3.74 11.18 ± 6.95 <0.0001#
Table 1  Comparison of Frequency-domain parameters of HRV among Group I and Group II (Values expressed as mean ± SD).

NS, not significant; *p = <0.05 (significant); #p = <0.001 (highly
significant).
The result of the present study (Table 1) showed
that there is no significant difference with the
total power (TP) spectral component of HRV
between group 1 and II. But, the LF and HF per-
centage difference was significant between group
1 and II (p<0.05) and the ratio between LF and
HF was highly significant between group 1 and II
(p = <0.0001).

Parameters Group II Group III p-Value

Total power (ms ) 2
542.03 ± 1308.58 364.16 ± 1079.95 0.568 NS
LF (%) 70.76 ± 29.95 53.91 ± 32.51 0.041*
HF (%) 8.39 ± 8.16 16.70 ± 13.34 0.005*
LF/HF 11.18 ± 6.95 4.03 ± 3.09 <0.0001#
Table 2  Comparison of Frequency-domain parameters of HRV among Group II and Group III (Values expressed as mean ± SD).

NS, not significant; *p  =  <0.05 (significant);
#
p = <0.001 (highly significant).
The result of the present study (Table 2) showed that
there is no significant difference with the total power
spectral component of HRV between group II and
III. But, the LF and HF percentage difference was
significant between group II and III (p < 0.05) and
the ratio between LF and HF was highly significant
between group II and III (p = <0.0001).

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106 Bhagyashree et al. Is autonomic function ....................... metabolic syndrome

Parameters Group I Group III p-Value

Total power (ms )2
684.03 ± 1694.69 364.16 ± 1079.95 0.386 NS
LF (%) 50.34 ± 30.10 53.91 ± 32.51 0.660 NS
HF (%) 16.79 ± 12.95 16.70 ± 13.34 0.978 NS
LF/HF 4.11 ± 3.74 4.03 ± 3.09 0.928 NS
Table 3  Comparison of Frequency-domain parameters of HRV among Group I and Group III (Values expressed as mean ± SD).

NS, not significant. maximum effect in group I and least in group III.
This indicates that though autonomic modulation
The result of the present study (Table 3) showed no
takes place in metabolic syndrome people, over-
significant difference with TP, LF, HF and LF/HF
all changes evident in both sympathetic and para-
between group I and III.
sympathetic activity more or less nullified each
Discussion other variations. Further, in this study the LF val-
ues which reflects mainly the sympathetic activ-
Metabolic syndrome constitutes three or more
ity and also parasympathetic activity via barore-
among the constellation of diseases comprising
ceptor reflex was 50.34  ±  30.10; 70.76  ±  29.95
obesity, hypertension, diabetes mellitus, hypertri-
and 53.91 ± 32.51 respectively of group I; II and
glyceridemia and low HDL concentration. Though
III. This indicates that the sympathetic activity
metabolic syndrome people are treated, tool is not
increases with the severity of the metabolic syn-
available to determine the prognosis. As the cardiac
drome and also as group I showed less LF val-
diseases invariable form the importance among the
ues than even control group (group III) probably
metabolic syndrome, attempt was made in this study
because metabolic syndrome initially affect baro-
to link the measurement of HRV (heart rate vari-
receptor activity followed by proportionate with
ability) with metabolic syndrome and its severity. In
the severity increasable affects the other cardiac
this study, the groups were made in such a way that
reflexes which enhance sympathetic activity as in
the subjects with any of the three components men-
group II. This argument gets supports from the HF
tioned above were grouped as group I (normal MS
values which are associated with the vagal activ-
group); patients with more than three components
ity obtained in this study. The HF value was less
as group II (severe MS group) and people with less
in group II whereas the HF value between group
than three components as group III and considered
I and III showed no significant difference though
as control group. The frequency-domain spectrum of
HF value is bit more in group I (metabolic syn-
HRV which includes TP (total power spectrum), LF
drome group) than group III (control) and the LF/
(low frequency), HF (high frequency) and LF/HF
HF ratio was more in group II in this study. Assou-
ratio were recorded on these people and the results
mou et al. (8), Chang et al. (9) showed that there
were analyzed.
were no differences in frequency domain measures
In the present study, the TP which studies the of HRV until at least three Metabolic syndrome
cardio-stabilizing reflex activities, though statis- risk factors were present is in agreement with the
tically not significant among the groups, showed above result.

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Bhagyashree et al. Is autonomic function ....................... metabolic syndrome
Thus, the results clearly suggest that in metabolic
syndrome people, among the cardiac reflexes, the first
one which is affected may be the activities of barore-
ceptor and then proportionate to severity other car-
diac reflexes which enhance sympathetic activity. So
the LF/HF ratio increases proportionate to the sever-
ity of metabolic syndrome may serve as a predictable
tool to gauze the degree of severity of the metabolic
syndrome. Chang et al. (9), Min et al. (10), Min et al.
(11) have shown that in a general population, HF and
LF were reduced in subjects with metabolic syndrome
(MetS+) compared to subjects without metabolic syn-
drome (MetS-). Min et al. (11) reported reductions in
HRV with increasing number of metabolic syndrome
components which is in agreement with the result of
the present study. Contrary to these results, Brunner
et al. (12) have shown that, in a smaller cohort of
middle-aged men (aged 45–63 years) TP, LF and HF
were reduced with no differences in LF/HF whereas,
Koskinen et al. (6) have studied metabolic syndrome
and short-term heart rate variability in young adults
and showed that young MetS+ women had reduced
HF nu and increased LF nu and LF/HF, but there was
no difference in men also supports the present result
though that study showed gender differentiation
unlike our study which did not show neither gender
nor age specific.
Conclusion
Metabolic syndrome adversely affects cardiac auto-
nomic control at first through baroreceptor activity
followed by other cardiac reflexes which are enhanc-
ing the sympathetic activity and deteriorating para-
sympathetic activity causing abnormal cardiac auto-
nomic control. So, with the severity of metabolic
syndrome, cardiac autonomic dysfunction is more
pronounced. Hence, the LF/HF ratio may be a better
prognostic tool towards the management of meta-
www.biomedicineonline.org
107
bolic syndrome. Mehmet et al. (13) have quoted that,
since HRV measurement is a noninvasive and rela-
tively cheap technique that may be used for the early
diagnosis of cardiovascular diseases, and valuable
data may be obtained with short recording times.
So, early identification and screening of population
with the metabolic syndrome for cardiac autonomic
function by HRV can be effective step towards the
management of metabolic syndrome which helps to
reduce the cardiovascular disease burden too. Fur-
ther, in-depth studies are needed in this direction to
confirm the findings of the study.
Acknowledgments
Authors would like to deliver sincere thanks to the
Department of Physiology, ACS Medical College &
Hospital and Saveetha Medical College & Hospi-
tal, Chennai for providing the facilities to carry out
research work.
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M.P., and Haffner, S.M. The metabolic syn-
drome as predictor of type 2 diabetes: the San
Antonio heart study. Diabetes Care. 2003; 26:
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  3. Vinik, A.I., Maser, R.E., and Ziegler, D. Neurop-
athy: the crystal ball for cardiovascular disease?
Diabetes Care. 2010; 33: 1688–1690.
  4. Falcone, C., Colonna, A., Bozzini, S., Matrone,
B., Guasti, L., Paganini, E.M., Falcone, R., and
Pelissero, G. Cardiovascular risk factors and
sympatho-vagal balance: importance of time–
domain heart rate variability. J. Clin. Exp. Car-
diol. 2014; 5: 2.
  5. Park, L.E., Choi, J., Park, J., and Gi, C. Heart
rate variability and metabolic syndrome in hos-
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pitalized patients with schizophrenia. J. Korean
Acad. Nurs. 2011; 41: 788–794.
  6. Koskinen, T., Kahonen, M., Julat, A., Mattsson,
N., et al. Metabolic syndrome and short-term
heart rate variability in young adults. The Car-
diovascular Risk in Young Finns study. Diabet.
Med. 2009; 26: 354–361.
  7. Alberti, K.G.M.M., Eckel, R.H., Grundy, S.M.,
Zimmet, P.Z., Cleeman, J.I., Donato, K.A.,
Fruchart, J.-C., James, W.P., Loria, C.M., and
Smith Jr., S.C. Harmonizing the metabolic syn-
drome: a Joint Interim Statement of the Interna-
tional Diabetes Federation Task Force on Epide-
miology and Prevention; National Heart, Lung,
and Blood Institute; American Heart Associa-
tion; World Heart Federation; International Ath-
erosclerosis Society; and International Associa-
tion for the Study of Obesity. Circulation. 2009;
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  8. Assoumou, H.G.N., Pichot, V., Barthelemy, J.C.,
Dauphinot, V., Celle, S., Gosse, P., et al. Meta-
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Yeh, T., et al. Altered cardiac autonomic func-
tion may precede insulin resistance in metabolic
syndrome. Am. J. Med. 2010; 123: 432–438.
10. Min, J.Y., Park, D., Cho, S.I., and Min, K.B.
Exposure to environmental carbon monoxide
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Biomedicine: 2016; 36(1): 109-116 109

Antioxidant activity of Phytoformulation 1, a polyherbal formulation, on

hyperlipidemic Wistar rats
R. Vanaja1 and J. Mercy Jasmine2
1
Department of Biochemistry, Shri Sathya Sai Medical College and Research Institute, Nellikuppam, India.
2
Institute of Biochemistry, Madras Medical College, Chennai, India.
(Received: Feb 2016   Accepted: Mar 2016)

Corresponding Author

J. Mercy Jasmine. Email: jasmine.mercy@gmail.com

Abstract

Introduction and Aim: The antioxidant activity of Phytoformulation 1, on the hyperlipidemic Wistar rats was
examined.

Materials and Methods: Hyperlipidemia was induced by atherogenic diet. The induced animals were segre-
gated into control and treatment groups. The control group was treated with Atorvastatin (10 mg/kg) and the
treatment groups were administered with 250 and 500 mg/kg of Phytoformulation 1 respectively. The level
of antioxidants such as Super Oxide Dismutase (SOD) (serum), Catalase (CAT) (hemolysate), Monaldehyde
(MDA), Vitamin E, Vitamin C (plasma) was measured in the hyperlipidemic rats.

Results: Animals treated with atorvastatin showed increased levels of catalase, SOD, vitamin E and vitamin
C. There was a decrease in the lipid peroxidation when compared with the hyperlipidemic group. Similarly,
there was an increase in the antioxidant activity among the groups treated with 250 and 500 mg/kg phytofor-
mulation 1 with an increased level of catalase, SOD, vitamin E, vitamin C and decreased lipid peroxidation.

Conclusion: The increased SOD and CAT levels expressed in Phytoformulation 1 treated groups showed the
antioxidant activity of polyherbal formulations.

Keywords: Antioxidant activity, Hyperlipidemia, Polyherbal formulations

Introduction the cholesterol biosynthetic pathway and as a result

H
yperlipidemia is a condition characterized
by abnormal elevation of lipids (triglyceride
and cholesterol) and lipoproteins (LDL,
VLDL) levels in the blood (1). Human HMGR (E.C
1.1.1.34) is a 97-kDa, transmembrane glycoprotein,
situated on the endoplasmic reticulum and
peroxisomes (2) and is responsible for catalyzing the
NADPH-dependent, two-step reduction of HMG-
CoA to mevalonate. The inhibition of this enzyme
results in the significant decrease in cholesterol
levels (3,4). This is a highly regulated process within
an attractive target for intervention in the treatment
of hypercholesterolemia. HMG-CoA Reductase
blockers known as statins prevent the synthesis of
cholesterol at the mevalonate and provide significant
protection against coronary artery disease (5,6).
Role Of Oxidative Stress In
Hyperlipidemia
Consumption of high-cholesterol diet (HCD) and
reduced physical activity to dissipate the energy

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110 Vanaja and Mercy Jasmine: Antioxidant activity …………….. Wistar rats

Group I (Normal Control) Normal diet

Group II (Hyperlipidemic Control)
Group III (Atorvastatin 10 mg/kg)
Group VII (Phytoformulation
1250 mg/kg)
Group VIII (Phytoformulation 1
500 mg/kg)
Atherogenic diet containing 1% cholesterol
Atherogenic diet was given for 8 weeks to induce hyperlipidemia
and treated with Atorvastatin (10 mg/kg) for 4 weeks
Atherogenic diet was given for 8 weeks to induce hyperlipidemia
and treated with Phytoformulation 1 (250 mg/kg) for 4 weeks
Atherogenic diet was given for 8 weeks to induce hyperlipidemia
and treated with Phytoformulation 1 (500 mg/kg) for 4 weeks

leads to hyperlipidemia (7). Oxidative stress in
hyperlipidemia is thought to be an important fac-
tor in the development of atherosclerotic plaques
(8). Accumulation of lipid peroxides generated by
free radicals, from fatty acids lead to atherosclerosis
and coronary heart disease (9). Hypolipoproteinae-
mia alters lipid composition of cell membranes and
extracellular matrix and are thus prone to oxidation
by free radicals (10). Free radicals are highly reac-
tive, short-lived molecules that have one or more
unpaired electrons and can damage lipids, proteins,
carbohydrates and DNA (11). They are constantly
generated in our body because of oxidative stress,
through leakage of electrons from the electron trans-
port chain and by the activities of oxidoreductaseen-
zymes (12). It is suggested that antioxidants could
be used as new, preventive and therapeutic agents
to prevent oxidative stress-related disorders (13,14).
Hence, there has been an increased interest in the
development of functional foods and nutraceuticals
to prevent oxidative stress (15) and the oxidation of
lipids (16).
Constituents Normal diet High fat diet
(%) (%)
Carbohydrates 67.3 57.3
Protein 22 22
Fat 5 15
Minerals 4.5 4.5
Vitamin mix 1 1
Cholesterol — 1
Table 1  Composition of atherogenic diet.
Plant based compounds as HMG-CoA reductase
inhibitors
Many plant-derived products have shown to have
potent inhibitory properties towards HMG -CoA
Reductase enzyme. Policosanol safely down-regu-
lates HMG-CoA Reductase – potential as a compo-
nent of the Esselstyn regimen (17). The tocotrienols
derived from barley are widely distributed in the
plant kingdom and differ from tocopherols (Vitamin
E) only in three double bonds in the isoprenoid chain
which appears to be essential for the inhibition of
cholesterogenesis (18). It is currently accepted that
the consumption of fruit-derived antioxidants such
as Vitamin C, Carotenoids, and Flavonoids provides
a preventive effect against cardiovascular disease.
Kiwifruit has potential cardiovascular protective
properties in vitro (19). The tetralin derivatives and
salts were proved to inhibit HMG-CoA Reductase
and so inhibit cholesterol biosynthesis.
Phytoformulation 1 is a polyherbal formulation that
contains extracts of plants with renowned hypo-
lipidemic and hyper antioxidant properties such as
Terminalia arjuna (20,21), Emblica officinalis (22),
Plectranthusbarbatus (23), Allium sativum (24). The
phytochemicals present in the plant extracts con-
tribute to the protective effect of the plants towards
a broad spectrum of clinical conditions. Thus, the
development of novel and sophisticated screening
processes can be used to recognize the numerous
applications of natural products and the introduction
of natural product chemicals for treating disease
could result in life-saving drugs.

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Vanaja and Mercy Jasmine: Antioxidant activity …………….. Wistar rats 111

S. Group SOD (units/ CAT (µmolH2O2/ MDA (nmol/ml) Vit. E (µg/ml) Vit. C (µg/ml)
No. ml) min/ml)
1. Normal control 5.13 ± 0.09 75.56 ± 0.55 8.18 ± 0.16 6.11 ± 0.03 0.95 ± 0.02
2. Hyperlipidemic 3.01 ± 0.04a**** 37.62 ± 0.40a**** 17.04 ± 0.25a**** 3.14 ± 0.04a**** 0.31 ± 0.09a****
control
3. Atorvastatin 5.09 ± 0.07b**** 71.25 ± 0.35b**** 8.38 ± 0.13b**** 6.08 ± 0.10b**** 0.92 ± 0.02b****
(10 mg/kg)
4. Phytoformulation 4.68 ± 0.12b**** 48.41 ± 0.46b**** 11.98 ± 0.15b**** 5.09 ± 0.11b**** 0.77 ± 0.10b****
1 (250 mg/kg)
5. Phytoformulation 5.05 ± 0.04b**** 58.20 ± 0.55b**** 9.71 ± 0.18b**** 6.00 ± 0.02b**** 0.89 ± 0.01b****
1 (500 mg/kg)
Table 2  Findings observed in the levels of antioxidants [SOD (serum), CAT (hemolysate), MDA, Vit. E, Vit. C (plasma)].
Values are mean ± SD; Number of animals in each group = 6; “a” as compared to normal control; ”b” as compared to hyperlipidemic control. One way ANOVA
followed by Dunnett’s multiple comparison tests. p-value ****<0.0001, ***<0.001, **<0.01, *<0.05.

Materials ad Methods Animals

Plant material Healthy Wistar albino rats both male (200–230  g)

Fresh plant/plant parts were purchased, taxonomic
identities were confirmed and the voucher speci-
men numbers of the plants were deposited at Phy-
topharma testing lab, T. Stanes Company Ltd.
Coimbatore. Fresh plant material was washed under
running tap water, air dried and then homogenized
to fine powder and stored in airtight bottles.
Plant extraction procedure
Ten grams of air-dried powder of the plant constitu-
ents was added to distilled water [1:10] and boiled
on slow heat for 2  h and centrifuged at 5000  rpm
for 10 mins. The supernatant was collected and the
above procedure was repeated thrice. After 6 hours,
the supernatant collected at an interval of every
2  hours were pooled together and concentrated to
make the final volume one-fourth of the original
volume (25). It was then autoclaved at 121°C and
15 lbs pressure and stored at 4°C. The resulted pow-
der was collected for every individual plant. These
powders in equal proportions were mixed, homog-
enized in distilled water [1:20] at 60°C to form a
concoction and used for the experiment.
and female (140–150  g) were used for the experi-
ment. They were housed in plastic cages with filter
tops under controlled conditions of a 12 h light/12 h
dark cycle, 50% humidity and 28°C. The animals
were fed standard rats chow and water ad libitum.
The experiment was conducted after obtaining the
approval of the institutional animal ethical commit-
tee clearance (IAEC approval No: 110/PHARMA/
SCRI, 2011). The composition of atherogenic diet
used during the study was given in Table 1.
Experimental design
In order to induce hyperlipidemia, the method
reported by Bopanna et al. was followed (26).
Animals were divided into six groups, 3 male and
3 female rats each and they received the following
diets with or without treatment for 12 weeks.
Bodyweight and the food intake of the animals were
monitored once a week throughout the experiment.
At the end of the 12th week, the rats were fasted
overnight and blood was drawn from retro-orbital
plexus and the animals were sacrificed by cervical
decapitation. Serum, hemolysate and plasma were

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112 Vanaja and Mercy Jasmine: Antioxidant activity …………….. Wistar rats
prepared for biochemical analysis.
The assay of superoxide dismutase is devised on the
method followed by Mustafa et al. (27). The extent
of lipid peroxidation was estimated according to the
method of Rotruck et al. (28), Okhawa et al. (29).
Catalase activity was assayed following the method
of Kaur et al. (30). Vitamin C was analyzeds by the
spectrophotometric method described by Omaye et
al. (31). Tocopherol was estimated as reported by
Baker et al. (32).
Statistical analysis
One way analysis of variance (ANOVA) followed by
Dunnett’s multiple comparison tests was performed
using GraphPad Prism version 6.00. The limit statis-
tical significance was set at p ≤ 0.05.
Results and Discussion
Changes in antioxidant levels after treatment
with Atorvastatin and phytoformulation 1
The high fat diet increased the free radicals level
which was indicated by the decreased catalase, SOD,
vitamin E, vitamin C levels and increased level of
lipid peroxidation as evidenced by the increased
levels of MDA in comparison to the animals with
a normal diet. Atorvastatin treated group showed
an increase in the level of catalase, SOD, vitamin
E and vitamin C. There was a decrease in the lipid
peroxidation when compared with the hyperlipid-
emic group. Similarly, there was an increase in the
antioxidant activity among the groups treated with
250 and 500  mg/kg phytoformulation 1 with an
increased level of catalase, SOD, vitamin E, vitamin
C and decreased lipid peroxidation.
The previous study by Gesquiere et al. (33), showed
that the free radicals released due to the oxidative
stress cause cellular cholesterol accumulation by
increasing the cholesterol biosynthesis. Hence, the
elevation in the cholesterol levels monitored in the
study may be due to the accelerated cholesterol
biosynthesis or reduction in the cholesterol esters
hydrolysis. A high-fat diet affects the antioxidant
defense mechanism against the process of lipid
www.biomedicineonline.org
peroxidation. Oxidative stress in hyperlipidemia as
shown by reduced levels of antioxidant enzymes
was thought to be a factor in the development of ath-
erosclerotic plaques (34). Antioxidant activity was
reported in the extracts of medicinal plants that con-
tain alkaloids (35), catalase activity predominated in
Piper longum Linn. (36). Epidemiological studies
have shown that the consumption of foods and bev-
erages rich in phenolic content can reduce the risk
of heart disease (37). Phytochemicals can directly
react with superoxide anions and lipid peroxyl radi-
cal and consequently inhibit/break the chain of lipid
peroxidation (38).
High cholesterol in cell membrane and plasma lipo-
protein render them highly susceptible to peroxida-
tion mediated by reactive oxygen species (ROS).
Membrane lipids are susceptible to oxidation due
to their high polyunsaturated fatty acid content. The
enzymatic and non-enzymatic systems associated
with the membrane lipids generate free radicals.
Free radicals oxidise the membrane lipoproteins
to produce by-products such as malondialdehyde
(MDA), which is the end product of lipid peroxida-
tion and is a marker for free radical mediated dam-
age and oxidative stress (39). Lipid peroxidation
is a free radical related process that causes cellular
damage as a result of oxidative stress (40). Increased
levels of MDA served as a marker for accelerated
lipid peroxidation in the plasma caused by the high-
fat diet. Superoxide dismutase (SOD) and catalase
(CAT) are inherent antioxidant defense enzymes
that scavenge the superoxides and lipid peroxides. It
was reported by Lu and Chiang (41) that cholesterol
feeding decreased the activity of SOD and CAT,
enhancing the lipid peroxides which corroborated
well with the present study. A decreased activity of
these enzymes may make the cell predisposed to
free radical damage (42). The increased SOD and
CAT levels expressed in the Decholestrate and phy-
toformulation 1 treated groups showed the antioxi-
dant activity of polyherbal formulations.
Vitamin E is the main lipid-soluble antioxidant in
the body. Vitamin E prevents the propagation of free
radical reactions in the cell membrane (43). Hyper-
lipidemic condition results in the increased LDL
Biomedicine – Vol. 36 No. 1: 2016
Vanaja and Mercy Jasmine: Antioxidant activity …………….. Wistar rats
levels and there by increased transport of LDL parti-
cles into the artery wall, which were prone to oxida-
tive damage (44). Vitamin E, mainly α-tocopherol,
is the major fat-soluble antioxidant present in the
LDL particle. On average, 5–9 vitamin E molecules
are carried by each LDL particle and are believed to
protect LDL from oxidative damage. In vivo, free
radicals generated by endothelial cells of the arte-
rial wall and activated macrophages are thought to
oxidize LDL particles (45). Before being completely
oxidised the LDL particles undergo some modifica-
tions. Initially, the LDL particles contain one intact
polypeptide- apolipoprotein B-100 (apo B-100),
enriched with poly unsaturated fatty acids (PUFA)
and antioxidants. Then it was minimally oxidised
resulting in the formation of oxidized phospholipids
on the surface (46). α-Tocopherol is confined to the
lipid phase of LDL and undergoes oxidation to form
α-Toc-Oo (Tocopheroxy radical) at the surface of the
particle by peroxyl radicals that are confined to an
aqueous phase. α-Toc-Oo reacts with lipid hydroper-
oxide (LOOH), initiating the tocopherol-mediated
peroxidation. Vitamin C can interrupt α-Toc-O o by
exporting the radical from the lipid phase back to
the aqueous phase. The oxidized LDL particles are
recognized by macrophage scavenger receptors and
taken up by the macrophages, forming lipid-laden
foam cells in the fatty streak lesions. Vitamin E
supplementation has been reported to suppress mac-
rophage uptake of oxidized LDL in human arterial
lesions (47). Thus, the increased LDL concentration
causes increased lipid peroxidation. Animals treated
with phytoformulation 1 showed reduced LDL thus
proving the antioxidant potential of the drug.
The depletion of SOD, Catalase, LPO, Vitamin
E and Vitamin C level in the hyperlipidemic con-
trol group might be due to a depressed antioxidant
defence system. Decreased levels of vitamins C
and E were observed in the atherogenic rats group.
Reduced vitamin C level may be due to increased
utilization to trap the reactive oxygen species (ROS)
or could be due to the possible reduction in the con-
centration of glutathione reductase (GSH) that regu-
lates the vitamin C levels. Reduced vitamin E levels
may be due to the increased utilization in scaveng-
ing the radicals or could be due to the decreased
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113
vitamin C because there is a well-established syn-
ergism between vitamin C and E (48). Buettner (49)
suggested that vitamin E and vitamin C cooperate to
protect lipids against peroxidation. Vitamin C repairs
the tocopheroxyl radical of vitamin E, thereby per-
mitting vitamin E to function again as a free radical
chain breaking antioxidant. The polyherbal formula-
tion phytoformulation 1 has the plant extracts such as
Terminalia arjuna, Plectranthusbarbatus, Curcuma
longa], Piper nigrum, Allium sativum with proven
potent antioxidant activity (24,50–53). This further
substantiates the free radical scavenging activity of
the test drugs.
Conclusion
Considering the results of the present study, it may
be concluded that the aqueous extract of phytofor-
mulation 1 exhibit possible protective mechanism
against the development of coronary heart disease.
It also prevents hyperlipidemic complications due to
lipid peroxidation and failure in antioxidant systems.
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Biomedicine: 2016; 36(1): 117-120 117

Comparative effects of different teaching methods in pharmacology in second MBBS

medical students
Baswaraj Munge,1 Kodandaramu Burli,1 Sindhura Nagisetty,1 Mamata Bandhopadhyay,1 and
M. Prasad Naidu2
1
Department of Pharmacology, Maharajah’s Institute of Medical Sciences (MIMS), Vizianagaram, Andhra
Pradesh, India.
Department of Biochemistry, Narayana Medical College and Hospital, Nellore, Andra Pradesh, India.
2

(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author

M. Prasad Naidu. Email: m.prasadnaidu@ymail.com

Abstract

Introduction and Aim: A good teaching involves a good communication. It is a complex process and has five
main components that are sender (source/teacher), receiver (audience/students), message (content/lecture), chan-
nel (medium/chalk and talk, PPT, practical application, etc.) and feedback. The superiority of these aids over one
another has been proven partially. The present study was conducted to evaluate the impact of the chalkboard,
PPT and therapeutic problem-based lectures in pharmacology teaching on second MBBS medical students.

Materials and Methods: A cross-sectional study was conducted at Department of Pharmacology, Mahara-
jah’s Institute of Medical Sciences, Nellimarla, Vizianagaram, Andhrapradesh in 2015. One hundred twenty-
seven second year MBBS medical students (n = 127) were divided into two groups.

Results: Shows, there is no significance difference between pre and post test scores in the first group with PPT
teaching methods (p value 0.22). And Comparison and assessment of pre and post test scores of Therapeutic
problem (Practical based learning) in the chalk and board group. In our study all the three types of teaching
shows significant improvement in the similar extent.

Conclusion: All teaching modules are equally effective, lack of regular reading habit of students may be a
cause for retaining of knowledge and most of the students preferred combination methods.

Key words: Pharmacology and Medical Students, Teaching Methods

Introduction The superiority of these aids over one another has

A
good teaching involves a good communication.
It is a complex process and has five main
components that are the sender (source/
teacher), receiver (audience/students), a message
(content/lecture), channel (medium/chalk and talk,
PPT, practical application, etc.) and feedback.
been proven partially. The present study was con-
ducted to evaluate the impact of the chalkboard, PPT
and therapeutic problem-based lectures in pharma-
cology teaching on second MBBS medical students.
Chalkboard aid is inexpensive, easy to clean and
reuse, allows students to keep pace with the teacher

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118 Baswaraj Munge et al.: Comparative effects…………………. students

n (sample size) M (mean) SD (standard deviation) p-Value

Pretest score 64 0.63 0.58 <0.0001
Posttest score 64 3.92 1.11
Table 1  Comparision and assessment of pre and post test scores with chalk and board teaching method.

n (sample size) M (mean) SD (standard deviation) p-Value

Pretest score 63 0.55 0.31 <0.0001
Posttest score 63 3.6 1.46
Table 2  Comparision and assessment of pre and post test scores with PPT (Powerpoint presentation) teaching method.

n (sample size) M (mean) SD (standard deviation) p-Value

Chalk board 64 3.9 1.11 0.22
Posttest score
PPT Posttest 63 3.6 1.46
score
Table 3  Comparision and assessment of post test scores in the two groups with two teaching methods (chalk board and PPT).

n (sample size) M (mean) SD (standard deviation) p-Value

Pretest score 60 0.55 0.83 0.001
Posttest score 60 4.23 1.66
Table 4  Comparision and assessment of pre and post test scores of Therapeutic problem (Practical based learning) in the chalk
and board group.

n (sample size) M (mean) SD (standard deviation) p-Value

Pretest score 65 0.27 0.41 0.001
Posttest score 65 3.5 1.77
Table 5  Comparision and assessment of pre and post test scores of therapeutic problem (Practical based learning) in PPT group.

n (sample size) M (mean) SD (standard deviation) p-Value

Posttest score 60 4.23 1.66 0.02
Posttest score 65 3.5 1.77
Table 6  Comparison and assessment of post test scores of Therapeutic problem (Practical based learning) in the chalkboard and
PPT group.

n (sample size) M (mean) SD (standard deviation)

Posttest score of Chalk and board 64 3.92 1.11
Pretest score of Therapeutic problem 60 0.55 0.83
Table 7  Comparision and assessment of post test of Chalk board and pre test of Therapeutic problem (Practical based learning).

n (sample size) M (mean) SD (standard deviation)

Posttest score of PPT 63 3.6 1.46
Pretest score of Therapeutic problem 65 0.27 0.41
Table 8  Comparison and assessment of post test PPT score and pre test of Therapeutic problem (Practical based learning) score.

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Baswaraj Munge et al.: Comparative effects…………………. students 119

and is not dependent on electricity. But it is time-

consuming; one cannot go back to what has been
erased and is not so effective for a large number of
students.

PPT has the advantage of using colours, fonts, dia-

grams and animation. Its disadvantage is that dim
light causes loss of eye contact; note-taking is dif-
ficult, has a tendency to overload information and
needs electricity.

Problem based teaching gives the clinical application

of knowledge acquired and makes the student write
a rational prescription for a particular case and helps
Fig. 1  Types of teaching modalities preferred by students.
solving problems in real therapeutic situations. Dis-
advantages are until proper basic knowledge about
giving their choice on different modules by grading
drug’s uses, and adverse effects are understood, the
them in 6 categories and result analysed.
application cannot be done.
The difference in the marks obtained in the three
The present study is planned to compare the effect
groups was analyzed by independent Student’s t-test
of each type in pharmacology teaching by conduct-
using the Statistical Package for Social Sciences
ing pre and post tests by allotting scores for each
(SPSS) version 17.
question.
Results
Materials and Methods
To evaluate the effectiveness of lectures by Chalk-
A cross-sectional study was conducted at Depart-
board, Powerpoint presentation (PPT) and practical
ment of Pharmacology, Maharajah’s Institute of
application by solving therapeutic problems. This
Medical Sciences, Nellimarla, Vizianagaram,
study conducted at Department of Pharmacology,
Andhra Pradesh in 2015. One hundred twenty-seven
Maharajah’s Institute of Medical Sciences, Nelli-
second year MBBS medical students (n = 127) were
marla, Vizianagaram, Andhra Pradesh in 2015.
divided into two groups. First group consisting of 64
students, to whom 1-hour lecture was delivered on Table 3 shows, there is no significance difference
Insulin by using chalk and board followed by a sec- between pre and post test scores in the first group
ond group of 63 students to whom the same content with PPT teaching methods (p-value 0.22).
was taught in 1 hour by using a PowerPoint. Pre-
tested objective type questionnaire consisting of 6 Above table shows most of the students preferred
questions each having one mark was given for evalu- combination teaching method.
ation in both pre and post test. There after two didac-
tic lecture classes were taken for the students on a Discussion
treatment of diabetes mellitus, before the teaching of
a therapeutic problem. While assessing on problem- Chalkboard—(Table 1): The CB method gives a
based learning, all students were dealt together, and significant improvement in scores from pre to post
all faculty members helped as facilitators. A similar which correlates with Dhaliwal (1), Banerjee et al.
pre and post test was conducted for evaluation by (2), Bandyopadhyay (3) Dantu Padmasree (4) stud-
giving 7 similar questions to all students. At the end, ies. PPT—(Table 2): There is significant improve-
the students were given one separate question for ment in post-test scores compared to pretest scores

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120 Baswaraj Munge et al.: Comparative effects…………………. students
with PPT aiding the lecture which correlates with
Galvis et al. (5) Savoy et al. (6), Erdremir (7), Seth et
al. (8) studies. Chalkboard/PPT—(Table 3): Post-test
scores of PPT and CB classes did not show any sta-
tistical significance, correlates with Ricer et al. study
(9). Problem-based learning (Therapeutic problem).
There is no evidence available to evaluate the impact
of a problem-based learning process. In our study, all
the three types of teaching show significant improve-
ment in the similar extent (Tables 4–6).
Most of the students preferred combination meth-
ods, which correlates with Kumar et al. (10) and
Mohan et al. (11) studies. CB Group and PPT group
could not retain knowledge to solve the therapeu-
tic problem may be due to lack of regular reading
(Tables 7 and 8).
Conclusion
All teaching modules are equally effective, lack of
regular reading habit of students may be a cause for
retaining of knowledge and most of the students pre-
ferred combination methods.
Acknowledgements
Cooperation with Department of Pharmacology,
Maharajah’s Institute of Medical Sciences, Nelli-
marla, Vizianagaram, Andhra Pradesh.
Conflict of interest
None declared.
References
  1. Dhaliwal, U. A prospective study of medi-
cal students’ perspective of teaching-learning
media: reiterating the importance of feedback.
J. Indian Med. Assoc. 2007; 105: 621–663.
  2. Banerjee, I., Jauhari, A.C., Bista, D., Johorey,
A.C., Roy, B., and Sathian, B. Medical stu-
dents view about the integrated MBBS course:
a questionnaire based cross-sectional survey
from a Medical College of Kathmandu Valley.
Nepal J. Epidemiol. 2011; 1: 95–100.
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  3. Bandyopadhyay, D. A study on the evaluation
of perception of teaching-learning methods of
pharmacology among the 2nd MBBS students
in Burdwan Medical College, West Bengal,
India. Rev. Prog. 2013. Available from: http://
reviews of progress.org/ArchiveArticleList.
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  4. Padmasree, D., Ujwala, U., and Parhi, K.K.
Teaching-learning methods in Biochemistry:
first MBBS students’ preferences and expecta-
tions. IJHR MIMS. 2015; 2: 26–30.
  5. Galvis, A.T., Ishee, J.H., and Schultz, S. A com-
parison of computer assisted instruction and
traditional classroom to introduce the occupa-
tional adaptation therapy. Int. J. Allied Health
Sci. Pract. 2011; 3: 1–6.
  6. Savoy, A., Proctor, R.W., and Salvendy, G.
Information retention from PowerPoint and
traditional lectures. Comput. Educ. 2009; 52:
858–867.
  7. Erdremir, N. The impact of PowerPoint and
Traditional Lecture on Students’ Achievement
in Physics. J. Turk. Sci. Educ. 2011; 8: 176–
189.
  8. Seth, V., Upadhyaya, P., Ahmad, M., and
Moghe, V. PowerPoint or chalk and talk: Per-
ceptions of medical students versus dental stu-
dents in a medical college in India. Adv. Med.
Educ. Prac. 2010; 1: 11–16.
  9. Ricer, R.E., Filak, A.T., and Short, J. Does a
high tech (computerized, animated, Power-
Point) presentation increase retention of mate-
rial compared to a low tech (black on clear
overheads) presentation. Teach. Learn. Med.
2005; 17: 107–111.
10. Kumar, A., Singh, R., Mohan, L., and Mani,
K.K. Student’s views on audio visual aids used
during didactic lectures in a medical college.
Asian J. Med. Sci. 2013; 4: 36–40.
11. Mohan, L., Sankar, P.R., Kamath, A., Man-
ish, M.S., and Eesha, B.R. Students’ attitudes
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Diagn. Res. 2010; 4: 363–368.
Biomedicine – Vol. 36 No. 1: 2016
Biomedicine: 2016; 36(1): 121-127 121

Effect of electromagnetic radiation exposure on hematological parameters of Swiss

Albino mice and their modulation by high protein diet
Debajyoti Bhattacharya,1 Niladri Ghosh,2 and Mausumi Sikdar (nee) Bhakta1
1
Physiology Unit, Department of Biological Sciences, Presidency University, Kolkata, India.
2
Department of Physiology, University of Calcutta; Kolkata, India
(Received: March 2016   Accepted: Mar 2016)

Corresponding Author:

Dr. Mausumi Sikdar (nee) Bhakta. Email: mausumi.dbs@presiuniv.ac.in

Abstract

Introduction and Aim: Research suggests that mobile phone radiation has adverse effects on haematological
parameters such as blood cell count, haemoglobin concentration, etc. The purpose of this study is to find out
whether high protein diet can reverse the ill effects caused by mobile phone radiation.

Materials and methods: For this study, 24 male Swiss Albino mice were divided into 4 groups (A, B, C, D).
Gr A animals were fed normal diet (5% Casein). Gr B and C animals were exposed to microwave radiation
emitted with average whole body Specific Absorption Rate (SAR) 0.1790 W/kg daily for 3 hours per day for
60 days. In addition to the radiation doses, Gr B animals received normal diet containing 5% casein whereas
Gr C animals received a high protein diet containing 20% casein. Gr D animals were given only High protein
diet (HPD) containing 20% casein. All animal experiments were performed according to the ethical guidelines
of the Institutional Animal Ethics Committee (IAEC) of Presidency University and Committee for the Purpose
of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Culture, Government of India.
After 60 days, blood samples were drawn and various hematological parameters were studied.

Results: Microwave exposure from mobile phone resulted in a significant decrease in (p < 0.05) in total R.B.C.
count, haemoglobin content, whereas, total W.B.C. count, eosinophil, neutrophil and monocyte percentage
increased significantly (p < 0.05). SEM studies demonstrated the presence of distorted R.B.C. structure in
microwave exposed group animals. Signs of improvement in the haematological parameters were recorded in
Group C animals, which were supplemented with HPD.

Conclusion: The study concludes that electromagnetic exposure from mobile phone has adverse effects on
haematological parameters which may be ameliorated by supplementation with HPD containing casein.

Key words: High Protein Diet, Electromagnetic Waves, Haemoglobin, Lymphocytes

Introduction only used for making and receiving calls, but it

I
ncreased use of Electromagnetic (EM)
principles for domestic and industrial purposes
proves that EMF plays an important role in
our daily life. Nowadays, mobile phones are not
www.biomedicineonline.org
also has many other applications, such as banking
transactions, social networking, and web browsing.
The world economic boom also benefitted from
these technologies. According to International
Telecommunication Union (ITU), the number of
Biomedicine – Vol. 36 No. 1: 2016
122 Debajyoti Bhattacharya et al.: Effect of electromagnetic radiation

dant activity is not lost with dephosphorylation or

proteolysis of casein . Casein thus acts as an anti
stress factor (3). But there is no such evidence where
casein acts in vivo as an antioxidant agent.

It has been observed that long term use of micro-

Fig 1  Calibration methodology of power emitted from
mobilephone during exposure (5)
mobile phone subscribers in the world was estimated
to about 5.6 billion in 2014, and it is expected to reach
6 billion by the end of 2015. This increased usage
and growing popularity of wireless technologies in
RF (Radio Frequency) EMF range represents one of
the fastest growing environmental influences (1).
Electromagnetic radiation (EMR) from artifi-
cial sources like power distribution networks, cell
phones in daily life exceeds the natural electromag-
netic fields by thousand folds. Use of cell phones by
the public is responsible for “Electropollution.”
At high power density levels, with high micro-
wave energy deposition rates, thermal effects are
observed. Some of these effects can be explained on
the basis of heat induced thermal stress mechanisms.
EMR have been found to cause cellular heat–stress
responses far more easily than other kinds of stress
including stress caused by heat (2).
Casein, found in milk is a first class protein, con-
taining all the essential amino acids. In vitro stud-
ies done to evaluate the role of casein and casein
derived peptides reveals its role as an antioxidant
agent. Casein and its peptides inhibit enzymatic
and non enzymatic lipid peroxidation. The antioxi-
wave devices, such as mobile phones has negative
impact on haematological parameters in mammals
. Several studies highlight the role played by reac-
tive oxygen species (ROS) in mediating the effects
of microwave radiation emitted by mobile phones
(4, 5, 6).
Researches on health effects of RF EMF are
quite numerous and diverse. It cuts across many
disciplines of engineering, physics, biology and
medicine (7). Not only animals, the electromag-
netic irradiation can also affect the growth of
plants and microorganisms. Therefore, the pres-
ent investigation has been undertaken to study the
effects of microwave exposure on haematological
parameters of Swiss albino mice and the possible
modulatory role of High Protein diet against these
induced changes.
Materials and Methods
Selection of Animals
Twenty-four Swiss Albino male mice (Mus muscu-
lus) weighing 20 ± 10 g were used. They were main-
tained in a 12 hours light/dark cycle at 25°C to 27°C.
All animal experiments were performed according to
the ethical guidelines suggested by the Institutional
Animal Ethics Committee (IAEC) of Presidency
University and Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPC-
SEA), Ministry of Culture, Government of India.

Name of the group No. of animals Treatment

Group A 6 Animals of group A were given only normal diet.
Group B 6 Animals of group B were given normal diet exposed to mobile
phone radiation at a frequency of 0.9 GHz.
Group C 6 Animals of group C were given High Protein Diet exposed to
mobile phone radiation ay same frequency (0.9 GHz)
Group D 6 Animals of group D were given only High Protein Diet.

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 Debajyoti Bhattacharya et al.: Effect of electromagnetic radiation 123

Selection of dose of radiation Differential count (DC)

GSM like frequencies of 0.9  GHz was used for A drop blood was taken on a clean glass slide and
3 hours continuously per day on the exposed groups smear was drawn. It was stained with Leishman
and connected to a directional antennas (8,9). solution, and DC was done under the compound
microscope (13).
Preparation of diet
Sample preparation for scanning electron
All the animals were fed ad libium on two series of microscopy
diets. One hundred grams of first series of diet con-
tained 5 g casein, 38.5 g wheat meal, 46.5 g chick Blood was collected from the middle caudal vein of
pea, 5 g corn oil, 1 g vitamin mixture, 4 g salt mix- the animals, and a drop of it was placed on the cover
ture and it was referred to the normal diet. One hun- slip and gold coated. Then it was examined in SEM
dred grams of second series of diet contained 20 g (ZEISS EVO-MA 10 scanning microscope) (14).
casein, 39 g wheat meal, 31 g chick pea, 5 g corn oil,
1 g vitamin mixture, 4 g salt mixture and this diet is Statistical analysis
considered as High Protein Diet (HPD) (10).
All data was expressed in terms of mean  +  SD
Experimental design (n = 6). Multiple co-relation and One Way ANOVA
was performed to determine the level of significance.
All the animals were divided randomly into four p-Values less than 0.05 were considered as moder-
groups (group A-D) according to the following table: ately significant and 0.01 were as highly significant.
At the end of the 60 days, blood was drawn from all
Results
the animals from each group (A-D) and the follow-
ing parameters were studied: In the study the effect of electromagnetic radiation
on different blood parameters were studied:
Hemoglobin estimation and total RBC and WBC
count Table 1 and Figure 2 depicts that there was signifi-
cant decrease in the total R.B.C. count (p < 0.05) in
Hemoglobin was estimated by Sahli’s hemoglobinom-
the animals exposed to mobile phone radiation (Gr
eter, total RBC, and WBC were counted with Neuber’s
B) in comparison to the control group (Gr A).
Hemocytometer kit with Hayem’s fluid composed of
0.5% (w/v) Sodium Chloride, 0.25% (w/v) Sodium In Group C animals (treated with High Protein diet
Sulphate, 0.25% (w/v) Mercuric Chloride (11,12). along with exposure to mobile phone radiation)

Total count of R.B.C.(million/cu. mm) Hb concentration in g% of four groups

7
5.8±0.089 11.058±0.01 11.083±0.02
5.7±0.141 7 10.078±0.03 10.107±0.02 7
6
4.95±0.138 12 2 2
5
3.437±0.071 10
4
8
3
6
2
4
1
2
0
0
Group A Group B Group C Group D
Group A Group B Group C Group D

Fig  4  
Fig 2 Fig 3

Group  A  =  Control,  Group  B  =  Normal  diet  +  radiation,  Group  C  =  High  Protein  +  radiation,  Group  D  =  High  Protein  diet    

 
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Fig  1  
Table 1 and Fig. 2 depicts that there was significant decrease in the total R.B.C. count

(p<0.05) in the animals exposed to mobile phone radiation (Gr B) in comparison to the

control group (Gr A).

124 Debajyoti Bhattacharya et al.: Effect of electromagnetic radiation

Parameters GR A (Control) GR B (Nomal GR C (High GR D (Only High

diet+radiation) casein+radiation ) casein diet)
exposure
TRBC million/to mobile phone radiation) there
5.7 ± 0.141 was no significant 4.95 ± 0.13**
3.437 ± 0.071* increase in Haemoglobin
5.8 ± 0.089***
cu-mm
concentration with respect to Gr B and there was no significant difference (p>0.05)
W.B.C. 5.05 ± 0.029 6.323 ± 0.092** 5.815 ± 0.80** 5.467 ± 0.103***
between
thousandsthe /mm control group (Gr A) and the group treated with only High Protein diet (Gr
3

Hb % g/dl 11.05 ± 0.017 10.07 ± 0.032** 10.11 ± 0.022** 11.08 ± 0.027***

D).
Table 1  Effect of high casein diet on total count of R.B.C., W.B.C. and Hemoglobin percentage in Albino
mice exposed to mobile phone radiation.
2. are
All values Table 2: Effect
expressed of high casein diet on Differential count of W.B.C.in mice exposed to
as mean ± S.D.
Significance level * = highly significant differences at p < 0.01.
**Significant differences at p < 0.05.
mobile phone radiation:
***Nonsignificant differences at p > 0.05.

Group of animals
Group of Eosinophil% Neutrophil%
Eosinophil,% Neutrophil,%Basophil% Lymphocyte%
Basophil, Monocyte%
% Lymphocyte,% Monocyte, %
animals
Gr AGr A 25.8
25.8 29.03
29.03 3.2 3.2 22.5 22.5 19.4 19.4
Gr B 33.3 35.4 0 8.3 22.9
Gr CGr B 33.3
22.42 35.4
33.64 0 0 8.3 25.23 22.9 18.7
Gr D 29.03 22.58 0 22.58 22.58
Gr C 22.42 33.64 0 25.23 18.7
Table 2  Effect of high casein diet on Differential count of W.B.C. in mice exposed to mobile phone radiation.

Gr Deos, eosinophil, mono,

neu, Neutrophil; 29.03 22.58 baso, basophil.
monocyte; lymp, lymphocyte; 0 22.58 22.58

neu   neu  
neu   neu  
eos   eos   eos   eos  

mono   mono   mono   mono  

lymp   lymp   lymp   lymp  
baso   baso  
baso   baso  
Fig:5.1 GROUP A Fig:5.2 GROUP B Fig:5.3 GROUP C Fig:5.4 GROUP D

neu=
  Neutrophil, eos= Eosinophil,
  mono= Monocyte,
  lymp= lymphocyte,   baso= Basophil

there was significant increase (p < 0.01) in R.B.C. there was significant decrease (p < 0.01) in W.B.C.
As evident from Table 2 and Fig.5, there was
count and it reached almost to the control value. But no significant
count anddifference
it reachedin percentage
almost of
to the control value. But
there was no significant difference (p  >  0.05) the there was no significant difference (p  >  0.05) the
numberscount
R.B.C. of between
monocytes the and basophil.
control group (GrButA)there
and wasW.B.C.
significant
count increase
between thein neutrophil
control group (Gr A) and
the group treated with only High Protein diet (Gr D). the group treated with only High Protein diet (Gr D).
percentage in Gr B Animals (exposed to electromagnetic radiation) (35.4%) in comparison to
Table 1 and Figure 3 depicts there was significant Table 1 and Figure 4 depicts there was significant
Gr A animals, eosinophil percentage is also
increase in the total W. B.C. count (p < 0.01) in the much higher in inGrtheBhaemoglobin
decrease animals (33.3%) in
concentration (p < 0.05)
animals exposed to mobile phone radiation (Gr B) in in the animals exposed to mobile phone radiation (Gr
comparison to
comparison to the
Gr control
A animals. Animals
group (Gr A).in Gr C have almostB) insame eosinophil
comparison to thepercentage like(Gr A). In Group
control group
C animals (treated with High Protein diet along with
GrGroup
In A. There was very
C animals low with
(treated number
HighofProtein
lymphocytes
diet is exposure
present in Gr B animals
to mobile (8.3%) in
phone radiation) there was no sig-
along with exposure to mobile phone radiation) nificant increase in Haemoglobin concentration with
comparison to Gr A (22.5%). The animals in Gr C (treated with high casein diet exposed to
www.biomedicineonline.org Biomedicine – Vol. 36 No. 1: 2016
mobile phone radiation) have almost back to the normal lymphocyte percentage.
 3. Fig.6

Debajyoti
3 EffectBhattacharya et al.: Effect
of high casein diet of
onelectromagnetic
R.B.C. surface radiation
structure exposed to mobile phone radiation: 125

6A 6B

6C 6D

Fig. 6A.  Scanning Electron Microscopy of normal R.B.C. structure (GR A animals).
Fig 6B.  Scanning Electron Microscopy of deformed structure of R.B.C. in presence of radiation (GR B animals).
Fig 6A Scanning Electron Microscopy of normal R.B.C. structure (GR A animals)
Fig 6C.  Scanning Electron Microscopy of R.B.C.in animals exposed to radiation and treated with High Protein diet (GR C animals).
Fig 6D.  Scanning Electron Microscopy of R.B.C. treated with only High Protein diet (GR D animals).
Fig 6B Scanning Electron Microscopy of deformed structure of R.B.C. in presence of radiation (GR B animals)
respect to Gr B and there was no significant difference cells are seen. The number of cells present in the
Fig 6C Scanning Electron Microscopy of R.B.C.in animals exposed to radiation and treated with High Protein
(p > 0.05) between the control group (Gr A) and the film is also normal.
group treated with
diet (GR C animals) only High Protein diet (Gr D).
Figure 6B depicts the electron microscopic image of
AsFig
evident from Table
6D Scanning Electron2 Microscopy
and Figureof5,R.B.C.
theretreated
was no with onlythe R.B.C.
High Proteinof mice
diet exposed
(GR D animals)to mobile phone radia-
significant difference in the percentage of numbers tion treated with normal diet. The cells are distorted
of monocytes and basophil. But there was a signifi- in shape, are clumped, total number of cells are also
cant increase in neutrophil percentage in Gr B Ani- less in the film in comparison to group A.
Fig6A
mals depicts
(exposed to the scanning electron
electromagnetic microscopic
radiation) (35.4%)image of the R.B.C. of mice treated with
in comparison to Gr A animals, eosinophil percent- Figure 6C depicts the electron microscopic structure
normal diet. The normal bi concave shape
age is also much higher in Gr B animals (33.3%) in and size of the cells
of the are seen.
R.B.C. Theexposed
of mice number to ofmobile
cells phone radia-
comparison to Gr A animals. Animals in Gr C have tion treated with High Protein diet. The amount of cell
present in the film is also normal. distortion is much less in comparison to group B ani-
almost same eosinophil percentage like Gr A. There
was a very low number of lymphocytes is present in mals. The arrangements of the cells are also normal.
GrFig
B animals (8.3%)
6B depicts the in comparison
electron to Gr Aimage
microscopic (22.5%).of the R.B.C. of mice exposed to mobile
The animals in Gr C (treated with high casein diet Figure 6D depicts the electron microscopic image
exposed to mobiletreated
phone radiation phonewith
radiation) haveThe
normal diet. almost of the R.B.C.
cells are distorted of mice
in shape, aretreated
clumpedwith High Protein diet.
, total
back to the normal lymphocyte percentage. The cell shows normal size and shape. No structural
number of cells are also less in the film in comparison to deformities
group A. are seen.
Effect of high casein diet on R.B.C. surface structure
exposed to mobile phone radiation. Discussion

Figure 6A depicts the scanning electron microscopic
image of the R.B.C. of mice treated with normal
diet. The normal bi concave shape and size of the
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Result indicates that the total number of R.B.C. is
decreased, and deformed structure is seen in ani-
mals exposed to EMR receiving normal diet (Gr B
animals) in comparison to the control group. There
Biomedicine – Vol. 36 No. 1: 2016

7
 

 
126
may be some apoptotic pathway is responsible for
the decreasing number of R.B.C. The mechanistic
details of apoptosis of R.B.C. caused by EMR are
not known, but studies indicate that prooxidant mol-
ecules released from the R.B.C. might be respon-
sible for such changes. It accelerates the destruction
of R.B.C. which correlates well with poor anti-
oxidant protection within R.B.C. and higher rate
of conversion of haemoglobin to methemoglobin.
Formation of methemoglobin within R.B.C. trig-
gers the development of oxidative stress and causes
structural membrane defects (15,16). As casein has
antioxidant property, it prevents the conversion of
haemoglobin to methemoglobin and development of
oxidative stress. So the total R.B.C. count and struc-
ture is restored in animals receiving high casein diet
along with radiation.
The total haemoglobin concentration was signifi-
cantly decreased in microwave radiation exposed
group (GR B) animals, which may be due to low
RBC count in the same group. This could be attrib-
uted to the interaction between the iron of haem and
the electromagnetic field produced due to mobile
phone induced radiation exposure in all the vital
organs like spleen, bone marrow etc. (17) As casein
plays an important role in cell signalling pathways
that enhance the growth and division of cells rel-
evant to cell proliferation, so in High Protein diet
Group (Gr C animals), the R.B.C. count was restored
towards normal (18). But in radiation exposed ani-
mals, the electromagnetic radiation exposure may
have induced certain structural changes in haemo-
globin which it cannot be restored by High Protein
diet. It may be possible that the new R.B.C. formed
in the group of animals exposed to mobile phone
radiation, but fed a High Protein diet (Gr C), may
have haemoglobin with deformed structure or in
lesser amounts.
From the present study, it can be said that increased
eosinophil and neutrophil count indicates that there
may be certain stress induced tissue damage in the
body caused by electromagnetic radiation (19).
Stress induced by radiation activates caspase 9 and
induces apoptosis of lymphocytes, as a result of
which, the percentage of lymphocytes decreases in
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Debajyoti Bhattacharya et al.: Effect of electromagnetic radiation
the blood (20). Casein is a major component in the
High Protein diet, which has some antioxidant prop-
erties that can ameliorate the ill effects of radiation
and the altered W.B.C. count can be restored back
towards normal.
The SEM images of R.B.C. of the exposed group
showed that the R.B.Cs have distorted shape and stick
next to each other to form a rouleaux. In HPD treated
group the R.B.C. showed normal biconcave shape.
Conclusion
From the present study, it can be concluded that
mobile phone radiation-induced changes in blood
parameters can be reduced by High Protein diet
(High Casein). High Protein diet may have some
protective action which can reduce the ill effects of
mobile phone radiation on blood parameters.
Acknowledgement
Authors acknowledge the FRPDF grant of Presi-
dency University to the corresponding author.
Abbreviations Used
HPD = High Protein diet
Rad = Radiation
SEM = Scanning electron microscopy
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128 Biomedicine: 2016; 36(1): 128-137

Ethanol enhances lamivudine-induced liver toxicity: Investigation on hepatoprotective

properties of silibinin-phosphatidylcholine complex in rats
Balasubramanian Jesudas,1 Ramanathan Raghu,1 Ganapathy Bhavani,1 Devaraj Ezhilarasan,1
and Sivanesan Karthikeyan1
1
Department of Pharmacology and Environmental Toxicology, Dr. ALM PGIBMS, University of Madras,
Taramani Campus, Chennai, Tamil Nadu, India.
(Received: Feb 2016   Accepted: Mar 2016)

Corresponding Author

Dr. Sivanesan Karthikeyan. Email: karthik48y@yahoo.co.in; hepatotoxicology.lab@gmail.com

Abstract

Introduction and Aim: Lamivudine (2’-deoxy-3’thacytidine, 3TC), when advocated with several anti-ret-
roviral drugs for the treatment of HIV infections in human is reported to induce liver toxicity. There is a
paucity of data on the influence of ethanol (EtOH) intake (alcoholism) on 3TC alone induced toxic insults
of the liver. This study evaluates the time-course onset of 3TC alone, EtOH alone and EtOH + 3TC-induced
toxic insults of the liver and the mitigating properties of Silibinin-Phosphatidylcholine complex (SPC) against
EtOH + 3TC-induced hepatotoxicity in rats.

Materials and Methods: Control rats (Group I) received saline from day 1 till day 14 and from day 15 till day
60, they were treated propylene glycol (vehicle). Group II rats (3TC alone Group) were given saline from day 1
till day 14 and from day 15 till day 60, they received 3TC alone (100 mg/kg; p.o.). Group III rats (EtOH alone
Group) were administered EtOH from day one till day 14 at 4 gm/kg/day (p.o.) and its dose was reduced to 2 gm/
kg/day from day 15 till day 60, to maintain alcoholic status. Group IV and V rats received EtOH on par with
Group III. Additionally, Group IV rats (EtOH + 3TC Group) were treated 3TC (100 mg/kg/day; p.o.) along with
EtOH, from day 15 till day 60. Similarly, Group V rats (EtOH + 3TC + SPC Group) received 3TC (100 mg/kg/
day; p.o.)  + SPC (100 mg/kg/day; p.o.) along with EtOH from day 15 till day 60. Group VI rats (SPC alone
Group) received saline from day 1 till day 14 and from day 15 till day 60, they were treated SPC alone (100 mg/
kg/day; p.o.). Marker enzymes of liver toxicity (AST, ALP, γ-GT, ASAL), bilirubin (BIL) and protein were inves-
tigated in serum; and various lipid parameters (TL, TG, CHO, PL, FFA) were evaluated in plasma, on days 15,
30, 45 and 60.

Results: 3TC alone, EtOH alone as well as EtOH + 3TC administrations caused a highly significant (p < 0.001)
progressive increase in marker enzymes (AST, ALP, γ-GT, ASAL), BIL and protein from day 30 till day 60 of
their respective treatments. These toxicants also caused significant (p < 0.001) progressive elevation of all the
lipid parameters (TL, TG, CHO, PL, FFA) investigated in plasma. Hepatotoxicity and hyperlipidemia were
more pronounced in rats receiving EtOH + 3TC treatments as compared to rats receiving 3TC alone and EtOH
alone. Simultaneous administration of SPC in rats receiving EtOH + 3TC, protected against the hepatotoxicity
and hyperlipidemia induced by EtOH + 3TC treatments.

Conclusion: SPC administration mitigates hepatocellular necrosis, cholestasis and hyperlipidemia induced by
EtOH + 3TC treatments and this beneficial effect could be attributed to enhanced bioavailability of silibinin

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Balasubramanian Jesudas et al.: Ethanol enhances ……. in rats
brought about by the carrier polysome, i.e., phosphatidylcholine and the inherent hepatoprotective, antioxi-
dant and anti-hyperlipidemic properties of silibinin.
Key words: 3TC, Ethanol, Hepatotoxicity, Lamivudine, Silibinin-phosphatidylcholine Complex
Introduction
L
amivudine (2'-deoxy-3'-thiacytidine, 3TC) is
a synthetic cytidine 2',3'-dideoxynucleoside
analogue and it belongs to nucleoside
reverse transcriptase class of antiretroviral drugs. It
was initially approved by FDA for the treatment of
chronic hepatitis-B viral infections in adult patients
and later its use was extended for the therapy to
improve asymptomatic and symptomatic human
immune deficiency virus (HIV) infections, either
alone or in its combination with other antiretroviral
drugs, including zidovudine (1). One of the most
common toxic manifestations of 3TC in humans
is the development of hepatotoxicity, which is
shown by elevation in the marker enzymes of liver
toxicity, bilirubin, hepatocellular necrosis and other
degenerative changes in the liver. Hepatic necrosis
is reported to occur in 6% to 31% of recipients of
3TC when administered with other antiretroviral
drugs and hence modifications in dosage or
discontinuation of therapy is recommended to
reduce its liver toxicity (2,3).
Several workers have reported on the problems of
chronic alcohol consumption in HIV patients even
under medical care (4). Ethanol (EtOH) consump-
tion in HIV-positive patients accelerated liver dam-
age leading to injury, inflammation, alcoholic steato-
hepatitis, liver fibrosis and cirrhosis. Though, alcohol
drinking is reported to have an impact on liver fibro-
sis and on the activities of aspartate aminotransferase
enzyme, some investigators have concluded that the
problem of alcohol abuse in HIV-positive patients
is not adequately addressed and the mechanism by
which HIV infection effects liver remains somewhat
elusive (5,6). It should be emphasized that reports
regarding the toxic potentials of 3TC monotherapy
in the liver are not well documented. Moreover, data
on the hepatotoxic potentials of 3TC in alcoholics
are scanty and remedial measures employed to coun-
teract this adversity is poorly defined.
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129
Silibinin a standardized extract obtained from the
seeds of Silybum marianum (L.) Gaertn (Carduus
marianus L., Asteraceae), is an effective antioxi-
dant, membrane stabilizing and hepatoprotective
agent against several drugs and toxins-induced
liver damage (7,8). It is stated that silibinin has
low bioavailability and hence its absorption from
the gastrointestinal tract is limited by the lipid-rich
outer membrane of the small intestine (9). It is pro-
posed that the bioavailability of silibinin could be
enhanced when it is admixed with phosphatidyl-
choline as silibinin-phosphatidylcholine-complex
(SPC). Pharmacokinetic studies have reported that
SPC polysomes enhance the absorption of silib-
inin by five times, in the bile as well as in plasma,
which increases the hepatoprotective ability of
SPC. Hence, recent studies recommend usage of
SPC complex or its polysomes for enhancing their
hepatoprotective, antioxidant and anti-hyperlip-
idemic properties of silibinin against drugs and
toxins-induced liver damage (10). In view of these
reports, in the current study, we investigated hepa-
toprotective potentials of SPC treatment against
EtOH  +  3TC-induced time-course onset of toxic
insults of the liver in rats.
Materials and Methods
Animals
Wistar albino rats of both sexes weighing
175 ± 20 gms, procured from Institutional animal
house facility were used in this study. They were
housed in polypropylene cages under standard
conditions (temp: 27°C ±  2°C; relative humidity;
50-70%; 12  h light/dark cycle) and fed standard
pellet and water ad lib. The experimental proto-
col was approved by Institutional Animal Ethi-
cal Committee (IAEC no. 01/27/2012), Govt. of
India. Constituted for the purpose as per CPCSEA
guidelines.
Biomedicine – Vol. 36 No. 1: 2016
130
Drugs and chemicals
Silibinin, phosphatidylcholine, argininosuccinately-
ase disodium salt and triolein were purchased from
Sigma-Aldrich Chemicals Pvt. Ltd., USA. Lami-
vudine (3TC) was procured as gratis from Central
Drug Research Laboratory (CDRL), Chennai. All
other chemicals used for the study were of analyti-
cal grade and was purchased locally.
Drug treatments and experimental design
Control rats (Group I) received saline from day 1
till day 14 and from day 15 till day 60, they were
treated propylene glycol (vehicle). Group II rats
(3TC alone Group) were given saline from day 1 till
day 14 and from day 15 till day 60, they received
3TC alone (100 mg/kg; p.o.). Group III rats (EtOH
alone Group) were administered EtOH from day 1
till day 14 at 4 gm/kg/day (p.o.) as described pre-
viously (11) and its dose was reduced to 2 gm/kg/
day from day 15 till day 60, to maintain EtOH-
induced liver damage, without producing mortal-
ity of rats. Group IV and V rats received EtOH on
par with Group III. Additionally, Group IV rats
(EtOH + 3TC Group) were treated 3TC (100 mg/
kg/day; p.o.) along with EtOH, from day 15 till day
60. Similarly, Group V rats (EtOH  +  3TC  +  SPC
Group) received 3TC (100 mg/kg/day; p.o.)  + SPC
(100 mg/kg/day; p.o.) along with EtOH from day
15 till day 60. Group VI rats (SPC alone Group)
received saline from day 1 till day 14 and from
day 15 till day 60, they were treated SPC alone
(100 mg/kg/day; p.o.).
3TC was dissolved in saline and was prepared fresh
before use. Silibinin- Phosphatidylcholine Complex
(SPC) was prepared by taking equal quantities of
silibinin and phosphatidylcholine at a ratio of (1:1)
and suspending them in propylene glycol. The vol-
ume of above administrations was maintained at 0.3
to 0.5 ml/100 gm b.w. of the rat.
Collection of serum and plasma
Serum and plasma samples were collected sepa-
rately from all the treatment Groups on days 15, 30,
45 and 60. Blood was withdrawn by the retro-orbital
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Balasubramanian Jesudas et al.: Ethanol enhances ……. in rats
puncture in the overnight fasted rats under mild
ether anesthesia. For separation of serum, blood was
collected (1 to 1.5  ml) in a clean tube and it was
allowed to clot for 20 min at cold (8°C). Serum was
subsequently separated by centrifuging the clotted
blood at 2,500 rpm for 20 min. The clear superna-
tant serum was separated and stored in vials under
refrigerated condition (8°C to 10°C) until further
analysis. For plasma separation, whole blood (0.75
to 1 ml) was collected in clean tubes which were
previously coated with 1% EDTA to prevent clotting
and was centrifuged subsequently at 2,500 rpm for
20 min. The clear supernatant plasma was separated
and stored in vials under refrigerated condition (8°C
to 10°C) until further analysis. All the biochemical
evaluations were performed within 48 h after sam-
ple collection.
Assay of markers of hepatotoxicity in serum
Serum aspartate transaminase (AST) activity was
quantified as described by Reitman and Fran-
kel (12). Briefly, the enzyme liberates pyruvate
in the presence of the substrate DL-aspartic acid
and α-ketoglutaric acid upon reaction with 2,
4-dinitrophenyl hydrazine and sodium hydroxide
(NaOH) to form brownish-orange colored hydra-
zone derivative, whose intensity was measured at
540 nm using the spectrophotometer. Phenol liber-
ated by the enzymatic hydrolysis in the presence
of the substrate disodium phenyl phosphate was
reacted with Folin-Ciocalteu’s phenol reagent and
NaOH to yield blue color, whose optical density
was measured at 640 nm, for the assay of alkaline
phosphatase (ALP) as described by King (13). The
p-nitroaniline liberated by the enzyme γ-glutamyl
transpeptidase (γ-GT) in the presence of the sub-
strate L-γ-glutamyl-p-nitroanilide was made to pro-
duce a yellow color, whose intensity was measured
spectrophotometrically at 420  nm as described
by Rosalki and Rau (14). The enzyme arginino-
succinic acid lyase mediates the degradation of
argininosuccinic acid to arginine and fumaric acid.
The arginine released from the substrate sodium
argininosuccinate was allowed to react with sodium
hypochlorite to form a pink color, whose intensity
was measured at 515 nm for assay of ASAL activ-
Biomedicine – Vol. 36 No. 1: 2016
Balasubramanian Jesudas et al.: Ethanol enhances ……. in rats
ity (15). For the assay of total bilirubin (BIL), the
serum was initially diluted with water, and metha-
nol was added in sufficient quantity to permit pre-
cipitation of protein and to allow the bilirubin to
react with diazo reagent (by Vanden-Berg reaction)
to form a purple compound i.e., azobilirubin, and
its intensity was measured at 540 nm against blank
using spectrophotometer as described by Malloy
and Evelyn (16). The total protein was estimated
spectrophotometrically by the standard method of
Lowry et al. (17).
Assay of plasma lipid profiles
The plasma triglyceride (TG) was estimated as
described by Varley et al. (18). Briefly, the phospho-
lipids were separated using a mixture of isopropanol
and alumina and was subjected to saponification reac-
tion with potassium hydroxide, to liberate glycerol
which was subsequently reacted with acetyl acetone
to yield a yellow color, whose intensity was quanti-
fied spectrophotometrically at 405 nm. For assay of
phospholipids (PL), the protein was initially precip-
itated using trichloro aceticacid and digested with
perchloricacid to liberate phosphorus as described
by Ziversmit and Davis (19). This liberated phos-
phorous was treated with molybdate reagent to form
phospho-molybdic acid and subsequently, reduce
with 1-amino-2-naphthol-4-sulphonicacid, to give
a blue coloured complex (20) and its intensity was
measured against the blank at 797 nm using spectro-
photometer. The total cholesterol (CHO) in plasma
was estimated according to the method of Varley
(21) by subjecting the samples to a reaction mixture
containing acetic acid, ferric chloride and sulphuric
acid to yield pink color and its optical density was
read at 560  nm using spectrophotometer. The free
fattyacids (FFA) were extracted using stable cop-
per reagent to form fattyacid-copper soap (22), and
was coloured with sodium diethyldithio carbamate
(23) whose intensity was measured spectrophoto-
metrically at 437 nm against blank. The total lipid
(TL) was estimated by treating the sample with van-
illin, sulphuric acid and phosphoric acid to yield
pink color and its intensity was measured at 540 nm
using spectrophotometer as described by Frings and
Dunn (24)
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131
Statistical analysis
Experimental results are expressed as mean ± S.D.
The data was subjected to one-way ANOVA and post-
Hoc multiple comparisons were done by Tukey’s test
to evaluate the significance of difference between
treatment groups at various time points, using SPSS
software (Version 16.0). Statistical significance was
considered when p-value was <0.05.
Results
Data on time-course evaluation of various marker
enzymes of hepatotoxicity, BIL and protein in serum
of rats are presented in Fig. 1 (a to f). Administration
of 3TC alone (Group II), EtOH alone (Group III),
as well as EtOH + 3TC (Group IV) produced a pro-
gressive and highly significant increase (p < 0.001)
in the activities of all the marker enzymes of hepato-
toxicity (AST, ALP, γ-GT, ASAL) and BIL in serum
of rats when their respective values were compared
to the vehicle treated control (Group I) from day
30 till day 60, indicating the progression of hepa-
totoxicity. The degree of hepatotoxicity was more
than two-times the normal levels of control in rats
receiving EtOH as well as EtOH + 3TC treatments.
The liver toxicity was much more pronounced in
the later group as compared to the former. The total
protein in serum was elevated highly significantly in
EtOH + 3TC as well as EtOH alone groups on days
45 and 60 and their values were almost near nor-
mal in 3TC alone treated rats. SPC post-treatment in
rats receiving EtOH + 3TC (Group-V) significantly
(p < 0.001) protected against all the above adversities
on all the days of evaluation, revealing its protective
property against EtOH + 3TC-induced toxic insults
of the liver. Administration of SPC alone (Group VI)
did not produce any change in the status of all the
parameters evaluated in serum and their respective
values were on par with the vehicle-treated control
(Group I).
3TC alone, EtOH alone and EtOH + 3TC treat-
ments produced a highly significant (p < 0.001)
progressive increase in TL, TG, CHO, PL and
FFA from day 30 till day 60 in plasma of rats. The
disruption in lipid metabolism and its homeostasis
Biomedicine – Vol. 36 No. 1: 2016
132 Balasubramanian Jesudas et al.: Ethanol enhances ……. in rats

a*
b* a*
a*
a* a*
b* b*

a*
a* a*
a*

a* a*
b* a,b*

a* a*
a*

b*
a*
b*

Fig. 1  Effect of SPC treatment on EtOH + 3TC-induced time-course alteration on markers of hepatotoxicity and protein in
serum of rats.
Data represents mean ± S.D. of 8 nos. of rats in each group. Treatment schedule for each group is described under section materials and methods.
Data was subjected to one-way ANOVA and post-Hoc multiple comparison was done by Tukey’s test. a—Group I compared to Groups II to VI.
b—Group IV compared to Group V. *p < 0.001.

observed in EtOH + 3TC treated rats (Group IV) II) shows mild elevations in most of the above
was on par with EtOH alone treatment (Group III) lipid parameters on days 30 to 60. Derangement
on days 45 and 60. 3TC alone treatment (Group in lipid homeostasis seen in EtOH + 3TC (Group
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Balasubramanian Jesudas et al.: Ethanol enhances ……. in rats 133

a* a*
b* a*
a*
b*

a*
a* a*
b* a*
a* b*
b*

a*
a*

a*
b*

Fig. 2  Effect of SPC treatment on EtOH + 3TC-induced time-course alteration on lipid parameters in serum of rats.
Data represents mean ± S.D. of 8 nos. of rats in each group. Treatment schedule for each group is described under section materials and
methods. Data was subjected to one-way ANOVA and post-Hoc multiple comparison was done by Tukey’s test. a—Group I compared to
Groups II to VI. b—Group IV compared to Group V. *p < 0.001.

IV) treated rats was significantly mitigated and
reversed back towards normalcy in rats post-
treated SPC simultaneously with the above hepa-
totoxic agents and these results show the (Group
V), anti-hyperlipidemic activity of SPC against
EtOH + 3TC-induced hyperlipidemia. SPC alone
treatment (Group VI) did not produce any change
in all the lipid profiles investigated in plasma
and their respective values were comparable to
those of vehicle-treated controls (Group I). Data
on alterations in plasma lipid profiles described
above are presented in Fig. 2(a–e).

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134
Discussion
3TC is believed to have a low frequency of liver
toxicity. However, some studies have recorded ele-
vations in AST, ALT and BIL in patients receiving
3TC (25,26). The two-fold elevations observed in
the status of AST and BIL in this study are in accor-
dance with these reports. In the current investiga-
tion, 3TC treatment enhanced the activities of ASAL
and γ-GT from day 30 till day 60 and similar studies
have not been reported previously. Chronic alco-
holism is well demonstrated to enhance the activi-
ties of transaminases in serum, ALP and γ-GT in
experimental animals and human (27–29) and our
present results are in agreement with these findings.
Data on the status of protein in serum of 3TC alone
treated rats are scanty. Previous studies conducted
in rats have shown that EtOH treatment interferes
with protein metabolism due to its astringent activ-
ity (30) and this could be the probable reason for
the fall in protein observed in serum of rats in this
study. We have shown 2 to 4-fold increase in the
activities of all the marker enzymes of liver toxic-
ity in EtOH + 3TC treated rats as compared to 3TC
alone and EtOH alone treatments (Fig. 1-a–e) and
these results clearly demonstrates that alcoholism
enhances 3TC-induced toxic insults in liver of rats.
Transaminases and ALP are sensitive indicators of
hepatocellular injury and their elevation is said to
occur consequent to hepatocellular necrosis, lead-
ing to their leakage into serum upon toxins-induced
liver injury (31). Elevations of ALP, BIL and pro-
tein are shown to occur during intra and extra
hepatic cholestasis, disruption of protein metabo-
lism and metabolism of non-essential aminoacids
(32). Administration of several antiretroviral drugs
including zidovudine have been documented to
induce cholestatic injury associated with elevation
of transaminases (33). γ-GT is the most sensitive
indicator of hepatobiliary disease and its elevation is
a known marker of EtOH-induced liver injury (30).
The enzyme ASAL is primarily located in paren-
chymal cells and is not present in any other cells of
the liver. Its elevation in serum is considered as the
precise indicator of hepatitis, cholestasis and liver
cell damage (15,34). We have previously shown ele-
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Balasubramanian Jesudas et al.: Ethanol enhances ……. in rats
vation in the activity of ASAL in the antiretroviral
drug zidovudine alone treated rats (33). In the cur-
rent investigation, we have demonstrated elevations
in the activities of marker enzymes of liver toxicity
(AST, ALP, γ-GT, ASAL), BIL and protein in serum
of 3TC alone, EtOH alone and EtOH + 3TC treated
rats and cumulatively these results reveal that these
toxicants induce cholestasis, hepatocellular damage
and disruption of protein metabolism in liver of rats.
Previous studies have demonstrated the develop-
ment of lactic acidosis and hepatic lesions, charac-
terized by microvesicular or macrovesicular fatty
liver upon administration of antiretroviral drugs in
human. However, abnormalities of lipid metabo-
lism in plasma consequent to administration of 3TC
alone treatment has not been reported previously.
In the current investigation, we observed that 3TC
alone treatment caused hyperlipidemia and disrup-
tion of lipid homeostasis. The mechanisms of 3TC-
induced hyperlipidemia could not be explained due
to paucity of previous reports. It should be high-
lighted that administration of antiretroviral drugs
cause mitochondrial damage, which could result
in focal necrosis, cholestasis, proliferation of bili-
ary ducts and deposition of mallory bodies in the
liver (35), and this could be the probable reason for
the observation of 3TC-induced hyperlipidemia in
plasma of rats. Incidentally, induction of mitochon-
drial damage upon 3TC administration has been
documented earlier (36). It is proved that chronic
alcoholism cause accumulation of lipids and affects
lipid metabolism by elevating CHO, TG and PL in
liver and plasma, leading to micro and macrove-
sicular hepatic steatosis (30,37,38). In agreement
with these reports, in the current study, there was
an a highly significant increase in the status of
all the lipid parameters (TL, TG, CHO, PL, FFA)
investigated in plasma of EtOH alone as well as an
EtOH + 3TC treated rats (Fig. 2-a–e).
In this study, elevations in the activities of marker
enzymes of liver toxicity BIL, protein and various
lipid parameters in rats receiving EtOH + 3TC, was
significantly (p < 0.001) mitigated and reversed back
almost towards normalcy in SPC post-treated rats
(Fig. 1,2). These results demonstrate its hepatopro-
Biomedicine – Vol. 36 No. 1: 2016
Balasubramanian Jesudas et al.: Ethanol enhances ……. in rats
tective and anti-hyperlipidemic properties of SPC
against EtOH  +  3TC-induced hepatocellular dam-
age, cholestasis and hyperlipidemia. It is hypoth-
esized that flavanoid molecules like silibinin (which
has poor water solubility and oral absorption), when
mixed with phosphatidylcholine (a carrier phos-
pholipid moiety), a bond is presumed to be formed
between these two molecules that makes it merge
into lipid phase of outer biological cell membranes
(7–10,39). It is likely that such a mechanism would
have contributed for the greater bioavailability of
silibinin and enhanced the mitigating properties of
SPC against EtOH  +  3TC-induced toxic insults of
the liver that is observed in the current study. The
hepatoprotective, antioxidant and free radical scav-
enging properties of silibinin against drug-induced
hepatotoxicity are well documented in the previous
literature (7,8,40). Similarly, several studies have
reported that phosphatidylcholine treatment has
clinical efficacy for protecting various liver diseases
including alcoholic hepatic steatosis, drug-induced
liver damage and hepatitis (8,10,39).
In conclusion, our current results demonstrate that
SPC post-treatment mitigates EtOH + 3TC-induced
hepatic necrosis, cholestasis and hyperlipidemia in
rats. This hepatoprotective ability could be attrib-
uted to the enhanced bioavailability of silibinin. Our
results suggest that SPC could be a better candi-
date for the treatment of ethanol and 3TC-induced
liver damage. However, it is cautioned that in-depth
Phase-III clinical studies are warranted before
implementation of SPC in clinical practice.
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138 Biomedicine: 2016; 36(1): 138-140

Is Internet use related to academic performance in medical students? A study from

South Indian Medical College
Ravi Kishore Polepalli1
1
Department of Biochemistry, MVJ Medical College and Research Hospital, Hoskote, Bangalore, India.
(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author

Dr. Ravi Kishore Polepalli. Email: writekishore@gmail.com; writekishore@yahoo.co.in

Abstract

Introduction and Aim: Indian Medical students spend a substantial amount of time accessing the internet for
curricular and non-curricular purposes. However, the effect of internet use on academic performance is not
clear. The purpose of this study is to evaluate the relationship between internet use and academic performance
in Ist year Medical students in India.

Materials and Methods: One hundred thirty-two students studying in Ist year undergraduate medical course
at MVJ Medical College and Research Hospital, Bangalore participated in the study. Data related to internet
use and academic performance was captured using a pretested questionnaire. Statistical analysis was per-
formed using ANOVA test.

Results: Time spent accessing the internet for overall (curricular and non-curricular) use and curricular use
did not significantly differ between low, mid-and high-level academically performing groups (p = 0.67 and
p = 0.64). However high performers spend significantly less time chatting/messaging as compared to low- and
mid-level performers (p = 0.006).

Conclusion: Students with high-level academic performance tend to spend less time on chatting/messaging
using applications like Whatsapp/Snapchat when compared to mid-level performers. However, there was no
difference between high, mid- and low-level groups in overall and curricular use of the internet.

Key words: Academic Performance, Internet Use, Medical Students

Introduction evidence about the relationship between internet

T
he Internet has become a source of recent use and academic performance in Indian Medical
advances, a platform for innovative, students. The objective of this study is to examine
simplified and self-paced learning methods the relationship between internet use and academic
and tool to independently assess oneself in Medical performance in Ist year Medical students in India.
knowledge. Medical students have been shown to
spend significant time in accessing the internet both Materials and Methods
for curricular and non-curricular (communication,
A cross-sectional design was employed in this
entertainment, etc.) purposes (1–3). However,
study in which Internet usage and its relationship
to the best of our knowledge, there is a lack of

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Ravi Kishore: Is internet use related ……………college
Academic Time spent on Internet for chatting/
performance groups messaging in hours per week, Mean (sd)
Low (n = 44)
Mid (n = 41)
High (n = 45)
Table 1  One way ANOVA analysis of relationship between academic performance and Internet usage (chatting /messaging) in Ist year
Medical students in India.
*Significant p-values (p < 0.05 considered significant).
with the academic performance was evaluated in Ist
year Medical students of MVJ Medical college and
Research Hospital, Bangalore. This study was con-
ducted at Biochemistry department, MVJ Medical
College and Research Hospital, Bangalore. Ethical
approval for the study was obtained from Institu-
tional Ethical Review board. One hundred thirty-
two students of Ist year medical undergraduate
course participated in the study. Informed consent
was obtained from the participants. Pretested anon-
ymous Questionnaire consisting of multiple choice
and open ended questions was used to record the
data. Self-reported scores of Internal assessment
examination in Biochemistry was taken as a mea-
sure of academic performance. Based on the aca-
demic performance in Internal assessment exami-
nation in Biochemistry with a maximum score of
60 marks, students were divided as low perform-
ers (less than 20 marks), mid- performers (21–30
marks) and high performers (41–60 marks). Statisti-
cal analysis was performed using SPSS version 17.
Statistical analysis was performed using one way
ANOVA test.
Results
Out of 132 students in age group 17–21  years,
males constituted 46.2% (n = 61). Overall internet
use included curricular and non-curricular com-
ponents. The mean duration of weekly internet
use (overall) was 11.1(standard deviation  =  7.7)
hours, with 2.9 (standard deviation  =  2.4) hours
spent for the curricular purpose. Statistical analy-
sis using one way ANOVA test showed that there
was no significant difference (p = 0.67 and 0.64)
in internet use for both overall and curricular
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139
F-statistic Statistical
Significance
(p-value)
5.6 (4.7)
8.5 (7.5) 5.275 0.006*
4.8 (3.9)
components between different groups based on
academic performance. However, it was found
that there was a significant difference (p = 0.006)
in internet time for chatting/messaging (part of
a non-curricular component) between groups as
shown in Table 1.
Discussion
The purpose of this study is to evaluate the rela-
tionship between internet use and academic per-
formance in Ist year Indian Medical students. The
mean duration of Internet use (curricular and non-
curricular) was 11.1 hrs per week which was sub-
stantially higher when compared to previous studies
by Unnikrishnan et al. and Maroof et al. where it
was less than 3 hours per week (1,2). To the best of
our knowledge, there have been no studies on the
relationship between internet use and academic per-
formance in Medical students in India. In a study
of Malaysian Medical students, it was found that
higher academic performance was associated with
higher internet usage (4). However, such a relation-
ship was not observed in our study. One plausible
explanation is that hardcopies of reference textbooks
may have served as an exclusive source of prepara-
tory material for exams. A novel finding in our study
was that low-and mid-level performers spent more
time on Internet chatting/messaging on applications
like Whatsapp/Snapchat than high performers, hint-
ing at the distraction caused by frequent messaging/
chatting (5). An unusual finding was that internet
chatting time for low performers was only margin-
ally but significantly higher than high performers;
a possible reason could be low performers could
be indulging in other non-curricular activities (like
Biomedicine – Vol. 36 No. 1: 2016
140
television) leading to low performance. A limita-
tion of this study was that though we related per-
formance in Biochemistry exams to curricular inter-
net use, it should be emphasized that this curricular
use was for all the preclinical subjects (Anatomy,
Physiology and Biochemistry) put together, since it
may be difficult to delineate accurately internet use
among above subjects. After considering the above
findings, it may be prudent to guide students in the
appropriate use of the internet for achieving best
academic outcomes.
Conclusion
Ist year Medical students spend a substantial amount
of time using the internet for curricular and non-curric-
ular purposes. Students with high-level academic per-
formance tend to spend less time on chatting/messag-
ing using applications like Whatsapp/Snapchat when
compared to mid-level performers. However, there
was no difference between high, mid- and low-level
groups in overall and curricular use of the internet.
Acknowledgement
I would like to thank Mr Suresh, Assistant Professor
and Statistician, Department of Community Medi-
cine for helping with Statistical analysis.
www.biomedicineonline.org
Ravi Kishore: Is internet use related ……………college
References
  1. Unnikrishnan, B., Kulshrestha, V., Saraf, A.,
Agrahari, A.C., Prakash, S., Samantaray, L.,
and Parida, A. Pattern of computer and inter-
net use among medical students in coastal south
India. South East Asian J. Med. Educ. 2008; 2:
18−25.
  2. Maroof, A.K., Pawan, P., and Rahul, B. How
are our Medical students using computer and
internet? A study from a medical college in
North India. Nig. Med. J. 2012; 53: 89−93.
  3. Yang, H., Chen, Y., Zheng, L., Xu, X., and Cao,
X. Analysis of internet use behaviors among
medical students in China. BMC Med. Educ.
2014; 14: 67.
  4. Siraj, H.H., Salam, A., Hasan, A.N., Jin, H.T.,
Roslan, B.R., and Othman, N.M. Internet usage
and academic performance: A study in a Malay-
sian Public University. Int. Med. J. 2015; 22:
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  5. Kuss, J.D., and Griffiths, D.M. Online Social
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2011; 8: 3528−3552.
Biomedicine – Vol. 36 No. 1: 2016
Biomedicine: 2016; 36(1): 141-144 141

Case Report
Raising the index of suspicion for cerebral venous thrombosis: A case report
Renata Mazurek,1 Naveen Ramesh,2 Kiran P.V.,3 and Avita Johnson2
1
Medical School for International Health, Ben-Gurion University of the Negev, Be’er Sheva. Israel.
Departments of 2Community Health and 3Emergency Medicine,
St John’s Medical College, Bangalore. Karnataka, India.
(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author
Dr. Naveen Ramesh. Email: drnaveenr@gmail.com

Abstract

Cerebral venous thrombosis (CVT) is an important form of stroke in India that if missed can lead to ther-
apeutic mismanagement and devastating consequences whereas recognition typically follows with good
prognosis. Predominantly occurring in younger patients, known associations include dehydration, the peri-
partum period, infection, other hypercoagulable states and risk factors such as alcohol and OCP use. Unrec-
ognized CVT may result in neurologic impairment, recurrence, brain oedema and herniation. However,
at onset, CVT often lacks classic signs, as with arterial stroke. While neurologic signs may present, non-
specific signs and symptoms are most common, making consideration of CVT in the differential diagnosis
critical in the emergency setting. A 43-year-old patient presented to our department with an acute progres-
sion of a headache, nausea, vomiting, hemiparesis and unresponsiveness. Despite initial symptom relief,
her worsening condition and altered mental status (AMS) prompted hospital arrival. History was significant
only for levothyroxine for hypothyroidism and long-term OCP use for dysmenorrhoea. Brain MRI diag-
nosed CVT, initiating treatment. During recovery, further possible underlying causes were investigated.
Recognition and appropriate referral for CVT are crucial for managing outcomes and addressing causes.
Knowledge of risk factors and an index of suspicion when evaluating younger adults can prevent oversight
of this critical diagnosis.

Key words: Altered Mental Status, Cerebral Venous Thrombosis, Hemiparesis, Oral Contraceptive Pill.

Introduction Furthermore, CVT may be secondary to infection,

C
erebral venous thrombosis (CVT) accounts
for up to 1% of all strokes overall, yet
represents 10-20% of strokes in young
people in India (1), and can result in significant
residual morbidity or mortality. Though not an
uncommon medical presentation, CVT can be
easily misdiagnosed if not considered. Headache
is the most prevalent symptom (1,2), and other
common symptoms, such as vomiting or seizures,
as well as neurologic signs, paresis, speech
disturbance, or confusion, are not specific to CVT.
www.biomedicineonline.org
making it important to distinguish and rule out
the presence of a venous thrombus if symptoms
emerge, even in a backdrop of infection. Besides,
thyroid conditions, hypercoagulable disorders, and
puerperal association should be taken into account
when assessing for CVT. While history may provide
important clues to its development, it is also critical
to evaluate for CVT under the differential diagnosis
of its non-specific signs and symptoms, particularly
in young adults to middle-aged adults. Here we
describe a case of a patient brought to the emergency
room following a three-day history of deteriorating
Biomedicine – Vol. 36 No. 1: 2016
142 Renata Mazurek et al.: Raising the index ……………….. thrombosis
neurologic function.
Case
The patient is a 43-year-old female with a history
of hypothyroidism and dysmenorrhoea, who acutely
developed a severe, pounding headache over the
whole head associated with nausea and relieved by
a high dose of an unknown medication and sleep.
The next day, she began to have vomiting and right-
sided upper and lower extremity weakness such that
she could not use her right arm and dragged her
right leg. Her family also noted that she became less
responsive, using few words, with slurred speech
and inappropriate emotional expression. By the fol-
lowing day, her speech had reduced to monosyllabic
responses and then further to the lack of speech; she
required force to move; she refused to eat or drink,
and she was agitated and disturbed in sleep. Sleep-
ing medication was given to her at home and she was
brought in by her family members. On initial sur-
vey at presentation, the patient was drowsy but afe-
brile. She had a blood pressure of 140/90, heart rate
100 bpm, respiratory rate 20 breaths/min, and oxy-
gen saturation at 99%. No headache, head injury, loss
of consciousness, seizure activity, fever, chills, rash,
chest pain, cough, dyspnoea, constipation, dysuria,
or evidence of bleeding were noted. The patient’s
history was negative for toxins and showed no per-
sonal or family history of the neurologic, hemato-
logic, or systemic disorder. Her only medications
were levothyroxine for hypothyroidism and an OCP
for dysmenorrhoea. On examination, the patient’s
alertness and orientation could not be assessed; the
rest of her general exam was unremarkable other
than for pallor. Of note, she had good skin turgor and
moist mucous membranes, her pupils were equally
round and reactive, and there was no appreciable
thyroid gland enlargement. Cardiac, pulmonary,
and abdominal exams were normal. Her extremities
were warm and well-perfused with palpable pulses,
and non-oedematous. On primary neurological eval-
uation, her GCS score was 11. Given her otherwise
stable condition, a brain MRI was done and dem-
onstrated a left frontal hemorrhagic infarct sugges-
tive of cerebral venous thrombosis with evidence
of extension into the anterior aspect of the superior
sagittal sinus and a midline shift of 2.5mm. Treat-
www.biomedicineonline.org
ment began with mannitol. Corticosteroids were
added for control of intracranial pressure (ICP), and
additionally Levipil (Levetiracetam) started as sei-
zure prophylaxis, along with Clexane (Enoxaparin)
for anticoagulation; OCP medication was stopped.
Upon further neurological exam, her extraocular
movements were intact; facial nerve weakness was
found, along with reduced power in the right upper
extremities compared to all other extremities despite
normal tone and normal deep tendon reflexes. Right
hand grip was absent, and right plantar movement
was decreased. Coordination could not be assessed
but the patient localized to pain. ECG monitoring
was consistent only with sinus tachycardia. CBC,
BMP, and LFTs did not show any abnormalities;
VBG results were within normal limits. A coagula-
tion panel was taken, including ANA, APLA, and
Proteins C and S, along with tests for homocysteine,
vitamin B12, and TSH, and pending results revealed
to be normal. The patient showed expressive aphasia
in the early course of CVT management, however,
all of her neurological signs improved during treat-
ment in the emergency ICU, and she was referred
to the neurology department upon stabilization for
further work-up till discharge.
Discussion
Altered mental status (AMS) in a young patient may
be due to some causes that include neurologic or met-
abolic disorders though cerebrovascular involvement
should not be overlooked. Even without a history of
the cardiovascular or hematologic disease, an acute
presentation with focal neurological defects pre-
cipitated by a severe headache may suggest stroke.
Wasay et al. have reported that the age of stroke onset
is less in South Asian countries, including India (3).
Additionally, it is important to denote the significance
of venous stroke. Although stroke overall is much
less common in younger populations as compared
to older populations, CVT is a critical diagnosis that
should be ruled out especially in younger patients.
The puerperal period has been commonly cited in
association with CVT (1,3,4) and a presentation of
a headache and seizure in a postpartum patient may
signify CVT and would be the indication for evalua-
tion. Symptoms associated with CVT may often not
Biomedicine – Vol. 36 No. 1: 2016
Renata Mazurek et al.: Raising the index ……………….. thrombosis
immediately or obviously suggest the diagnosis and
require raising an index of suspicion. Headache and
vomiting are the most common symptoms (2,4) while
neurological deficits or seizures are not necessarily
present or may take longer to develop. Dehydration
accounts for a major proportion of CVT cases (1,4),
and can compound on the relatively hypercoagula-
ble state of pregnancy in the immediate postpartum
period or other hypercoagulable states. Despite the
patient’s pallor, probably due to her acute lack of food
and drink, dehydration was not evident upon assess-
ment at arrival. However, in non-puerperal patients,
OCP use in women and alcohol use in men have more
recently been identified as important causes for CVT
(1,2,4). At another tertiary care centre, Patil et al.
found OCP consumption to be the most common risk
factor in patients with CVT in the superior sagittal
sinus (1), the most frequent location of CVT (2,4) and
the main sinus affected in our patient. In the evalua-
tion of our patient, long-term OCP use for dysmenor-
rhea was considered to be the most likely cause for
her CVT. After further investigation, other diagnostic
tests performed in the metabolic, autoimmune, and
coagulation panels returned normal. CVT has also
been reported both in patients with hypo- or hyper-
thyroidism, and thyrotoxicosis, though a rare cause,
can be associated with a hypercoagulation (5,6). With
our patient’s history of hypothyroidism, thyroid func-
tion tests were performed as a confirmation and were
within normal limits as expected for patients con-
trolled with thyroid medication. Less evident causes,
including autoimmune disorders, hypercoagulable
disorders such as factor V Leiden mutation or Protein
C or S deficiencies, and homocystinuria are also pos-
sible underlying causes (1,4) and should be accounted
for in the diagnosis and management of CVT, even
if other identifiable causes are present. Infection has
also previously been reported as a major cause of
CVT (1) and may be preceded by headache and vom-
iting. Suspecting CVT in the setting of infection with
AMS may be particularly challenging. Here, imaging
is needed to clarify the presence of neurologic infec-
tion and CVT. With our patient, the absence of fever,
elevated cell counts, and lack of sick contacts made an
infectious cause less plausible. However, in all cases,
a definitive diagnosis can be established best by an
MRI-venogram (MRV) showing the thrombosed
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143
venous sinus. If large enough or late presentation, a
brain MRI (as in this case) may be able to detect the
distribution of the venous infarct, as can a head CT
in very severe cases. The decision to send a patient
directly for MRV is case-dependent on CVT being the
primary differential diagnosis. Our patient underwent
MRI to rule out the possibility of other causes. How-
ever, an MRV would have been done upon a negative
MRI to look for CVT. As CVT cannot be diagnosed
only on clinical grounds, it is a diagnosis that can be
easily missed if not thought of, particularly in hos-
pitals and centres without imaging modalities. Given
the prevalence of CVT in India, it is important at all
levels of the healthcare system to consider CVT in
younger populations when presented with symptoms
and possible risk factors, and to send for imaging or to
refer to a higher centre for imaging if suspected. The
treatment for CVT involves the initiation of fluids to
prevent and treat dehydration. Anticoagulation has
been under some debate due to concerns with hemor-
rhagic strokes. However the benefit of anticoagula-
tion on morbidity and mortality observed, even after
resolution of the CVT, is determined to be greater
than the risk in patients with hemorrhagic stroke (7).
Elevated intracranial pressure (ICP) may accompany
CVT and can be detected on the fundoscopic exam
in cooperative patients. Mannitol is a recommended
therapy for patients presenting with acute deteriora-
tion as this suggests worsening brain oedema and the
use of other ICP-lowering treatments varies by clini-
cal scenario (7). Due to the state of our patient and
her deterioration, mannitol was started to relieve the
suspected elevation of ICP, and corticosteroids were
added. Our patient’s intake of sleeping medication
was unlikely to account for the extent of her drowsi-
ness, and a midline shift on MRI, though minimal,
provided clinical evidence towards reducing ICP
both therapeutically and prophylactically against fur-
ther worsening. Antiseizure medication is given to
patients who have a history of seizure or who pres-
ent with seizure, but may also be given routinely for
CVT (7) or otherwise to patients with neurological
signs on presentation, as was our case. The question
of continuing anticoagulation has favoured the use of
a low-molecular-weight heparin over unfractionated
heparin (8) as a bridge to warfarin. The long-term use
of anticoagulation should depend upon the underly-
Biomedicine – Vol. 36 No. 1: 2016
144 Renata Mazurek et al.: Raising the index ……………….. thrombosis

ing cause of CVT, such as in patients with chronic Hospital for guidance in the evaluation of the case
hypercoagulable states (7). Of note in patients on and diagnosis.
OCPs for medical conditions, such as dysmenorrhea
in our patient, it is necessary to evaluate and provide References
alternative treatment beyond initially stopping OCPs
1. Patil, V.C., Choraria, K., Desai, N., and Agrawal,
in the hospital. Recurrence is uncommon, but the
S. Clinical profile and outcomes of cerebral
risk is higher if patients resume OCPs (4). Sagittal
venous sinus thrombosis at tertiary care center.
sinus thrombosis and multiple sinus involvements are
J. Neurosci. Rural Pract. 2014; 5: 218–224.
associated with the highest mortality rate (1). CVT
2. Anadure, R.K., Nagaraja, D., and Christopher,
mortality and morbidity is related to irresolution of
R. Plasma factor VIII in non-puerperal cerebral
elevated ICP resulting in herniation or brain oedema,
venous thrombosis: a prospective case-control
complications due to infection or underlying causes,
study. J. Neurol. Sci. 2014; 339: 140–143.
recurrence and residual neurological deficits (1,4).
3. Wasay, M., Khatri, I.A., and Kaul, S. Stroke in
Therefore, early recognition remains imperative in
South Asian countries. Nat. Rev. Neurol. 2014;
the course of CVT management. Our patient’s neu-
10: 135–143.
rologic presentation markedly improved upon admin-
4. Narayan, D., Kaul, S., Ravishankar, K., Sury-
istration of ICP-lowering agents and she recovered
aprabha, T., Bandaru, V.S., Mridula, K.R.,
with treatment in our department before continued
Jabeen, S.A., Alladi, S., Meena, A.K., and Bor-
management in the neurology department. Overall,
gohain, R. Risk factors, clinical profile, and
it is important to be aware of both the modifiable
long-term outcome of 428 patients of cerebral
and non-modifiable risk factors in deciding further
sinus venous thrombosis: insights from Nizam’s
consultations; however imaging, preferably MRV,
Institute Venous Stroke Registry, Hyderabad
should not be delayed in confirming a diagnosis, as
(India). Neurol. India. 2012; 60: 154–159.
this allows for initiating the appropriate treatment. In
5. Srikant, B., and Balasubramaniam, S. Grave’s
hospitals lacking imaging modalities, prompt refer-
disease with transverse and sigmoid sinus
ral to a higher centre upon suspicion of CVT is criti-
thrombosis needing surgical intervention. Asian
cal. Anticoagulation, even if available, should not
J. Neurosurg. 2013; 8: 162.
be blindly started without imaging confirmation of
6. Rau, C.S., Lui, C.C., Liang, C.L., Chen, H.J.,
venous thrombosis. While the prognosis is affected
Kuo, Y.L., and Chen, W.F. Superior sagittal sinus
by causes of CVT and time to treatment, diagnostic
thrombosis induced by thyrotoxicosis. J. Neuro-
confirmation allows the best chance in any case for
surg. 2001; 94: 130–132.
influencing outcomes towards prevention of mortal-
7. Nagaraja, D., and Sarma, G.R. Treatment of
ity, worsening morbidity, and recurrence, as well as
cerebral sinus/venous thrombosis. Neurol. India.
addressing underlying factors.
2002; 50: 114–116.
Acknowledgments 8. Misra, U.K., Kalita, J., Chandra, S., Kumar, B.,
and Bansal, V. Low molecular weight heparin
We would like to thank the patient, and to thank versus unfractionated heparin in cerebral venous
Dr. Shakuntala Murty in the Department of Emer- sinus thrombosis: a randomized controlled trial.
gency Medicine at St. John’s Medical College Eur. J. Neurol. 2012; 19:1030–1036.

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Biomedicine: 2016; 36(1): 145-147 145

Oral squamous papilloma of hard palate: A case report

Jamin Joseph1 and S. Karpagavalli1
1
Department of Oral Medicine and Radiology, Saveetha Dental College and Hospitals, Saveetha University,
Chennai, India.
(Received: Jan 2016   Accepted: Mar 2016)

Corresponding Author

Jamin Joseph. Email: jaminpj2@yahoo.com

Abstract

Oral squamous papilloma is a benign epithelial proliferation which results in exophytic papillary growth. Most
of the cases are induced by human papilloma virus, and some are not. These lesions appear as pedunculated,
white or normal coloured cauliflower like projections that arise from the mucosal surface. Conservative sur-
gical excision is the treatment of choice. Here we present a case of the benign squamous papilla of the hard
palate in a 16 years old male. An excisional biopsy was performed and diagnosed as squamous papilloma after
a histological investigation.

Key words: Excisional Biopsy, Human Papilloma Virus, Squamous Papilloma

Introduction Recurrence is uncommon except for lesions in

O
ral squamous papilloma (OSP) is a benign patients infected with human immunodeficiency
proliferation of the stratified squamous virus (HIV) (4).
epithelium, which results in papillary
or verrucous exophytic mass induced by human Case Report
papilloma virus (1). It is the fourth most common
A 16 year old male visited the Department of Oral
oral mucosal mass and found in 4 of every 1,000 and
Medicine and Radiology with a chief complaint of
accounts for 3-4% of all biopsied oral tissue lesions
painless growthon the hard palate since 6 months.
(2). These are common lesions of the oral cavity with
History revealed that the growth was initially small
a predilection for the mucosa of hard and soft palate
and had gradually increased to attain the present
including the uvula and the vermillion border of lips
size. The patient was notsuffering from any other
(3). Its pathogenesis is related to human papilloma
systemic diseases. The patient had not undergone
virus types 6 and 11. The occurrence of these lesions
any dental procedures. The patient was calm and
influenced by smoking, co-existing infections,
quite. There was no history of pain and paraesthesia
dietary deficiencies, and hormonal imbalance (4).
associated with the growth. Intra-oral examination
These lesions are exophytic, papillary mass, usually
revealed the presence of a solitary, well-defined exo-
pedunculated and soft in texture. Lesions of the
phytic growth on the hard palate (Fig. 1). The lesion
condyloma acuminatum, verruca vulgaris, focal
had a cauliflower-like appearance which measures
epithelial hyperplasia and verrucous carcinoma may
2  ×  2  cm in size (Fig. 2). The overlying mucosa
mimic lesions of squamous papilloma. Surgical
was normal in appearance without any secondary
excision is the preferred modality of management.
changes. On palpation, the mass was pedunculated,

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146 Jamine Joseph and Karpagavalli: Oral squamous ………………hard palate

firm in consistency, non-tender and no discharge.

A provisional diagnosis of oral papilloma was
made, and the patient advised special investigation
of ELISA. The ELISA test was negative. Surgical
excision of the lesion was performed without any
post-operative complications. The excised lesion
was sent for histological examination, and diagnosis
of squamous papilloma is confirmed. Post-surgical
follow up has done. The one week follows up shows
adequate healing. Six months follow up shows no
evidence of recurrence

Histopathological examination shows para kera- Fig 1.  Squamous papilloma on hard palate

tinised stratified squamous epithelium of variable

thickness with few areas of broad reteridges. There
is evidence of few areas of parakeratin plugging.
Many long thin fingers like projections extend above
the surface of the mucosa. Correlating the history,
clinical and histological features the final diagnosis
squamous papilloma was made.

Discussion

Oral squamous papilloma is a generic term used

for papillary and verrucous growth composed of
benign epithelium and minor amounts of connec-
tive tissue (4). The squamous papilloma is seemed
Fig 2.  pedunculated mass
to be associated with papilloma virus, the one com-
monly incriminated as causative in skin warts (2).
The association of HPV and squamous cell lesions
at the various site of the body including oral cav-
ity was first described by Syrjanen et al. (5). Squa-
mous papilloma is the fourth most common oral
mucosal mass and forms 3-4% of all biopsied oral
soft tissue lesions (6). It has been shown that the
class of HPV’s is very large and that individually
these viruses are associated with many conditions
of squamous papilloma (7). Human Papilloma virus
is most commonly associated with the lesion with
HPV 6 and 11 in squamous papilloma and condy-
loma acuminatum, while HPV 2 and 57 were more
Fig 3.  Excised squamous papilloma lesion
prevalent in verruca vulgaris lesions (6). Squamous
papilloma are classified as two types: Isolated soli-
Squamous papilloma is an exophytic growth made
tary and multiple recurring. Isolated solitary is
of numerous, small finger like projections which
usually found in adult’s oral cavity while recurring
result in a lesion with a roughened, verrucous or
occurs commonly in children (3). The present case
cauliflower like surface. It is nearly always a well
was an isolated, solitary exophytic lesion.
circumscribed pedunculated tumour, occasionally
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Jamine Joseph and Karpagavalli: Oral squamous ………………hard palate
Fig 4.  Presence of finger like projections
suggestive of squamous papilloma
Fig 5.  Post Excision area of palate
sessile. It is painless, usuallywhite but sometimes
pink in colour (2). The present case had the same
clinical picture which was pink in colour.
Intraorally it is found most commonly on the tongue,
lips, buccal mucosa, gingiva and palate, particularly
that area adjacent to the uvula (2). The papilloma is
usually solitary and enlarges rapidly to a maximum
size of about 0.5 cm, with little or no changes there-
after. However lesions as large as 3.0 cm in greatest
diameter have been reported (7). In the present case,
a lesion was present on the hard palate which mea-
sures 2 cm in diameter.
Histologically these lesions present as many long,
thin fingers like projections extending above the
mucosal surface. Each finger like projections lined
by stratified squamous epithelium and connective
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147
tissue centrally. The spinous cell proliferates in
a papillary pattern. Koilocytes-HPV altered cells
may be observed. The upper epithelial layer shows
pynknotic nuclei, often surrounded by edematous or
optically clear zone, the so called koilocytic cell (4).
The differential diagnosis of oral squamous papil-
loma includes verruciform xanthoma, papillary
hyperplasia, condyloma acuminatum, proliferative
verrucous leukoplakia (3,6). Often squamous papil-
loma may be clinically and microscopically indis-
tinguishable from verruca vulgaris which is virus
induce focal papillary hyperplasia of the epidermis
(6).
Surgical removal is the treatment of choice for oral
squamous papilloma, either by surgical or electro-
cautery excision, cryosurgery, intralesional injec-
tions of interferon or lase ablasion. The recurrence
rate is low for the solitary type compared with mul-
tiple lesions (1,3,4).
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