An International Journal for Biomedical Sciences - Biomedicine
An International Journal for Biomedical Sciences - Biomedicine
An International Journal for Biomedical Sciences - Biomedicine
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TRUTH CURES CLING TO TRUTH<br />
<strong>An</strong> <strong>International</strong> <strong>Journal</strong> <strong>for</strong> <strong>Biomedical</strong> <strong>Sciences</strong><br />
Published by:<br />
Indian Association of <strong>Biomedical</strong> Scientists ( IABMS )<br />
www.biomedicineonline.org<br />
(Volume 31, Number 1 (January - March) 2011; Website: www.biomedicineonline.org)<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011<br />
1
Editorial Board<br />
Publication<br />
<strong>Biomedicine</strong> is a quarterly Publication of IABMS. It is indexed in Excerpta Medica, CAB <strong>International</strong> and Ind.Med.<br />
Editorial Office<br />
Dr. Kamakshi Memorial Hospital,<br />
No.1, Radial Road, Pallikaranai, Chennai - 600100.<br />
Tel: 044 - 22469200, 044 - 66300300.<br />
Dr. Ajay Kumar Singh, DRDO,New Delhi.<br />
Dr. M.A. Hussain, Chennai<br />
Dr. S. Karthikeyan, Dr. ALMPGIBMS, Chennai<br />
Dr. R. Sheela Devi, Dr. ALMPGIBMS, Chennai<br />
Dr. Prathima Chatterjee, Salt lake, Kolkata<br />
Dr. D. Sakthisekaran, Dr. ALMPGIBMS, Chennai.<br />
Dr. S. Rajarajan, Presidency College, Chennai<br />
Dr. S. Venkataraman, Chennai<br />
Dr. Victor Rajamanickam, Chennai.<br />
Dr. B.M. Sundaram, Bangaluru<br />
Dr. V. Madhavachandran, Thiruvananthapuram<br />
Dr. A. Subramoniam, Thiruvananthapuram<br />
Dr. Pravati Pal, JIPMER, Puducherry<br />
Dr. D.C. Mathangi, Chettinad Health City, Chennai<br />
Dr. T. Devasena, <strong>An</strong>na University, Chennai<br />
<strong>International</strong> Advisory Council<br />
Dr. T.G. Govindarajan,<br />
Dr. Kamakshi Memorial Hospital,Chennai, India.<br />
Dr. W. Selvamurthy,<br />
Ministry of Defence, New Delhi, India.<br />
Dr. A. Krishnamurti,<br />
<strong>An</strong>namalai University, India.<br />
Dr. S.P. Thyagarajan,<br />
Ramachandra Medical College & R I, Chennai, India.<br />
Dr Sanguansak Rerksuppaphol,<br />
Srinakharinwirot University, Nakhoru Nayok, Thailand<br />
Dr. M. Ramachandran,<br />
Atlanta, United States of America.<br />
Dr. Sashi Bala Singh,<br />
Ministry of Defence, New Delhi, India.<br />
Dr. V. <strong>An</strong>antharaman,<br />
Chennai, India.<br />
Dr. S. Ramakrishnan,<br />
Chennai, India.<br />
Dr. Suprita Gupta,<br />
National Medical College, Birgunj, Nepal.<br />
Dr. <strong>An</strong>ita Dushyanth,<br />
Los <strong>An</strong>geles, Cali<strong>for</strong>nia, United states of America<br />
Editor in Chief<br />
Dr. G. Rajagopal,<br />
Professor of Biochemistry,<br />
Dr Kamakshi Memorial Hospital,<br />
Pallikaranai, Chennai - 600100.<br />
Email: editorbiomedicine@gmail.com<br />
Mobile: 919791078027<br />
Editors<br />
Dr. (Mrs) S. Pushkala,<br />
Prof. & HOD, Dept. of Immunology,<br />
Dr. MGR Medical University, Chennai.<br />
Dr. T. Tirunalasundari,<br />
Prof. of Biotechnology,<br />
Bharatidasan University,<br />
Tiruchirapalli.<br />
Dr. Joseph Dominic,<br />
Medical Oncologist,<br />
Dr. Kamakshi Memorial Hospital.<br />
Dr.K. Ramesh Rao,<br />
Professor & HOD, Dept. of Pathology,<br />
Chettinad Hospital & Research Institute, Chennai.<br />
Managing Editor<br />
Mr. M. Alagusundaram,<br />
Asst. Prof., Department of Microbiology,<br />
Asan Memorial College of Arts & Science, Chennai.<br />
Language Editor<br />
Mrs. J. Aruna,<br />
Asst. Prof., Department of English,<br />
Mohamed Sathak College of Arts & Science, Chennai.<br />
Advisor, Pathology<br />
Dr. S. Rajalakshmi,<br />
Dr. Kamakshi Memorial Hospital, Chennai.<br />
Disclaimer<br />
The journal is not responsible <strong>for</strong> any statements made by authors.<br />
Copy Right:<br />
No part of this journal should be reproduced without written<br />
permission from the Editor in Chief.<br />
Communication Address<br />
All correspondence should be addressed to:<br />
Dr. G. Rajagopal, Editor in Chief, <strong>Biomedicine</strong>.<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011<br />
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<strong>Biomedicine</strong><br />
Vol.31; No.1: (January - March) 2011<br />
Contents<br />
Topics and Authors Page(s)<br />
I. Editorial..................................................................................................................................................... 1<br />
II Former Presidents of IABMS - (1985 - 1987)......................................................................................... 2<br />
III. Review / Special articles<br />
1 Health beneficial effects of black tea.............................................................................................3 - 8<br />
Mahejabeen Fatima and Syed Ibrahim Rizvi<br />
2 Modification of Lifestyle:Hypertension in Obese........................................................................9-12<br />
Tandra Majhi, Geeta Jaiswal<br />
IV. Research Papers<br />
3 Differential effect of Nardostachys jatamansi rhizome extract on acetylcholinesterase<br />
in different regions of brain in rats under chronic stress........................................................13-21<br />
Gloria Karkada, K. B. Shenoy, Harsha Halahalli, K. S. Karanth<br />
4 Heart rate reserve – A useful tool <strong>for</strong> evaluation of chest pain in middle aged........................22-26<br />
Ganashree C P, S M Nataraj, Rajalakshmi R,Vijaya Y Vageesh<br />
5 Serum α1- antitrypsin level and antioxidant status in smokers with Chronic Obstructive<br />
Pulmonary disease.......................................................................................................................27-31<br />
S. Venkata Rao , B.D. Toora, V.S.Ravi Kiran and S. Indira.<br />
6 Cardiovascular autonomic function tests responses in patients<br />
with diabetes mellitus and non diabetics..................................................................................31-38<br />
C.N. Mamatha and Ravipati Sarath.<br />
7 Association between Serum Lipoprotein(a) Concentrations and Serum<br />
Triglycerides in Type 2 Diabetes Mellitus..................................................................................39-44<br />
T. Sharmila krishna, J.N.Naidu, M. Audhisesha Reddy, K.Ramalingam, E. Venkat Rao.<br />
8 Ameliorative effect of Coccinia grandis in streptozotocin induced diabetic rats......................45-52<br />
Bhuvaneswari Palanisamy, Krishnakumari Shanmugasundaram and Rajeswari Paramasivam<br />
9 Testicular toxicity in arsenic exposed albino rats: ameliorative<br />
effects of ascorbic acid and α-tocopherol...................................................................................53-63<br />
Avijit Dey, Arindam Bose & Prabir Kr. Mukhopadhyay<br />
10 Evaluation of antioxidant status in niddm with and without complications.........................64-68<br />
T. Vivian Samuel, S. Smilee Johncy<br />
11 Clinico-Biochemical Profile of Hypokalemic Patients.................................................................69-73<br />
<strong>An</strong>il Kumar Pandey, Varsha Vijay Akhade, M Sri Hari Babu,Y Himabindu<br />
12 <strong>An</strong>tifungal activity of Bacopa Monniera against Dermatophytic Fungus.................................74-77<br />
S.R.Ayyappan, R.Srikumar, R.Thangaraj, R.Jegadeesh, L. Hariprasath<br />
13 Prevalence of Extended Spectrum Beta Lactamase producing<br />
Enterobacteriaceae in Clinical specimens.................................................................................78-83<br />
G.S. Ravi, B.V.S.Krishna, Namratha W.Nandihal,Asha B.Patil and M.R. Chandrasekhar.<br />
14 <strong>An</strong>ti-inflammatory effects of Allium sativum (Garlic) in experimental Rats............................84-89<br />
M.K.Jayanthi and Murali Dhar<br />
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15 Reactive Oxygen Species during hypoxia-reperfusion injury under general anaesthesia..........90-95<br />
Sanjeev Kumar and Ashok kumar<br />
16 Assessment of probiotic properties of strains of L.fermentum and L.reuteri isolated<br />
from human breast milk................................................................................................................96-102<br />
R. Ilayaraja, Radhamadhavan.<br />
17 Perinatal androgen levels and Sexual dimorphic digit ratio in Down syndrome Children........103-108<br />
Suresh Bidarkotimath and S. Viveka<br />
18 Serum Enzymes, Initial and follow- up Lipid profile in Acute Myocardial Infarction...............109-113<br />
Suman.S. Dambal, V. Indumati. and P.B. Desai.<br />
19 Association of serum ferritin levels between rheumatoid arthritic obese and<br />
Non rheumatoid arthritic obese....................................................................................................114-118<br />
Tandra Majhi and A.K. Srivastara<br />
V Case Reports<br />
20 A case of asymptomatic anomalous pancreatico biliary ductal union<br />
of pancreatic - biliary type..........................................................................................................119-121<br />
V.S. <strong>An</strong>andarani.<br />
21 Unusual complication at Feeding Jejunostomy in Boerhaave’s Syndrome.................................122-125<br />
Vithalkumar. M. Betigeri, <strong>An</strong>upama V Betigeri, Nanda Kishore Maroju and<br />
Kasturi Satya Venkata Kumar Subba Rao<br />
VI Letters to the Editor<br />
22 Blood Pressure Changes in menstrual cycle...................................................................................126-127<br />
L. Rajeshwari and D.H. Rajendra<br />
23 Measurement of Thyroid Stimulating Hormone in serum as the initial test<br />
in the assessment of thyroid disorder............................................................................................128-130<br />
V. S. Ravi Kiran, S.Venkata Rao, K. Ambika Devi<br />
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Editorial <strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 1<br />
A Good Food<br />
It is a fermented food of India especially southern<br />
India. Its essential components are parboiled rice<br />
and dehulled black gram dhal. It is nutritionally good<br />
and very hygienic. It is named IDLI. Reference to<br />
Idli is made in Tamil Literature between 100 BC and<br />
300 AD.<br />
Preparation<br />
4 parts of parboiled rice and 1 part of dehulled black<br />
gram dhal are separately kept soaked in drinking<br />
water <strong>for</strong> about one hour. A small quantity of dry<br />
methi seeds are added to the dhal. The dhal is then<br />
ground in a wet grinder <strong>for</strong> 3o minutes with periodical<br />
addition of traces of water <strong>for</strong> soft grinding. The<br />
rice is then ground <strong>for</strong> 30 minutes similarly. Both<br />
are mixed and required salt is added. The mixture is<br />
well mixed and allowed to ferment <strong>for</strong> about 8-10<br />
hours or preferably overnight. It is now ready <strong>for</strong><br />
preparing idli by steaming.<br />
Fermentation<br />
The fermentation of idli batter is due to the leavening<br />
action produced exclusively by the activity of a<br />
lactic acid bacterium, Leuconostoc Mesenteroids.<br />
It is responsible <strong>for</strong> the souring as well as gas<br />
production. In the later stages of fermentation,<br />
growth of streptococcus faecalis and still later<br />
pediococcus cerevisiae becomes significant.<br />
Hygienic Cooking<br />
Idli is made by steaming in an idli vessel. It is<br />
there<strong>for</strong>e a sterile hygienic food. Some may prefer<br />
cooking in a micro-wave oven. This is not correct.<br />
Nutritive Value of Idli<br />
The composition of idli makes it nutritionally good.<br />
Parboiled rice retains the B vitamins unlike the raw<br />
rice. It supplements the dietary essential amino<br />
acid, methionine which is deficient in the black<br />
gram dhal. Black gram dhal, on the other hand,<br />
compensates the deficient amino acids, lysine and<br />
threonine in the rice. Idli there<strong>for</strong>e contains all the<br />
eight dietary essential amino acids which the body<br />
can not synthesize. Besides, while eating idlis, a<br />
side dish, usually a chilli powder made of dhal,<br />
and dry chillies is used to which fresh sesame oil<br />
is added <strong>for</strong> taste. Sesame oil contains the dietary<br />
essential fatty acids, which the body needs. The<br />
calorific value of a 35 gram idli is approximately 70<br />
kilo calories. The digestibility of idli is very good.<br />
Cereals contain phytate which interferes with the<br />
absorption of calcium and iron. But fermentation<br />
produces the enzyme phytase which reduces the<br />
phytate content. Fermentation also enhances the<br />
availability of thiamine and riboflavin. Germination<br />
and fermentation increase the bioavailability of Zinc<br />
and Iron from the food grains.<br />
<strong>An</strong>tioxidant activities<br />
The effect of fermentation of idli batter on the<br />
enhancement of antioxidative activities was reflected<br />
by a higher total phenol (TPC) content. Fermemtation<br />
enhanced > 2.5 fold TPC. The fermented batter was<br />
a better free radical scavenger.<br />
Food <strong>for</strong> the sick<br />
Idli is recommended <strong>for</strong> patients in hospitals due to<br />
its hygienic preparation and good digestibility. The<br />
glycemic response in terms of glycemic index was<br />
determined in patients with type 2 diabetes mellitus.<br />
It was observed that consumption of idlis delayed<br />
the rise in blood glucose level.<br />
<strong>An</strong>y time Food<br />
Idlies can be considered as a fast but ideal and safe<br />
food. It is an ideal breakfast food. However, it is<br />
an anytime food. Idlis may be taken <strong>for</strong> the night.<br />
Idli may be taken with chilli-dhall mix, sambar or<br />
coconut chutney.<br />
G.Rajagopal<br />
Editor in Chief<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011<br />
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Former Presidents of IABMS - 5 (1987-1989) <strong>Biomedicine</strong>; 2011; 31 (1): 2<br />
Dr.B.S.Gajalakshmi<br />
M.B.B.S.,M.Sc.,Ph.D.,MNAMS,FIABMS<br />
Bandi Santhana Krishnan Gajalakshmi(Dr BSG)<br />
was born in Chennai.She is a founder member of<br />
IABMS and a life member from 1979.Dr BSG<br />
had early education in Chennai. She was a student<br />
of <strong>An</strong>dhra Medical college, Visakhapatnam and<br />
obtained MBBS degree in 1957. Her professional<br />
career began in Stanley Medical College, Chennai<br />
in 1959 as tutor in Physiology.Under the guidance<br />
of Dr Sarada Subramaniam, she specialised in<br />
Physiology with an M.Sc. degree in 1966 and<br />
later with Ph.D. degree in 1980.She worked in<br />
several medical colleges of TamilNadu in different<br />
capacities as Assistant Professor, Associate<br />
Professor ,and Professor in Physiology.Sh e retired<br />
as Director and H.O.D. of Physiology from the<br />
Institute of Physiology and Experimental Medicine,<br />
Madras Medical College, Chennai. Dr Gajalakshmi<br />
developed a keen interest in the study of the<br />
pathophysiological effects of scorpion venom of<br />
buthus tamulus in different animals.She investigated<br />
the beneficial effects of Lytic Cocktail therapy in<br />
mitigating the toxic effects of the venom.These<br />
studies enabled her to obtain Ph.D. in Physiology<br />
from the University of Madras. Her contributions<br />
to the clinico physiological problems of scorpion<br />
venom received wide recognition especially from<br />
child specialists.Her other fields of interest are<br />
Diabetes, Stress and Occupational hazards.She has<br />
published several research communications and<br />
scientific papers of topical interest in National and<br />
<strong>International</strong> journals. Dr Gajalakshmi is associated<br />
with the activities of several professional bodies.<br />
She is a founder member of the Indian Association<br />
of Medical Scientists (IABMS), Women Doctors’<br />
Association,and Madras Institute of Magneto<br />
Biology. She is a life member of Association of<br />
Physiologists and Pharmacologists of India(APPI),<br />
Indian Science Congress Association, Altrusa<br />
<strong>International</strong> Inc. and Madras Club Women Service<br />
Organisation. Dr Gajalakshmi has organised several<br />
Scientific meetings, conferences and workshops<br />
under the auspices of IABMS to update in<strong>for</strong>mation<br />
especially to young scientists.She was the first person<br />
to introduce a separate session <strong>for</strong> medical students<br />
as well as other faculties in 1982 during the 3rd<br />
<strong>An</strong>nual Conference of IABMS at Stanley Medical<br />
college, Chennai. She was conferred the Fellowship<br />
title of IABMS, instituted <strong>for</strong> the first time during<br />
the <strong>An</strong>nual Conference held at MMC, Chennai, in<br />
1989.She delivered the prestigious M.K.Nambiar<br />
Oration at the 15th <strong>An</strong>nual Conference of IABMS<br />
in 1994. Dr Gajalakshmi was elected unanimously<br />
as President of the 75th platinum jubilee conference<br />
of the Indian Science Congress held at Pune, in<br />
1988.In 1991, she gave J.N.Maitra memorial oration<br />
at the 18th <strong>An</strong>nual Conference of Physiological<br />
Society of India.In 1999, at the 86th Indian Science<br />
Congress she delivered N.M. Basu memorial<br />
oration. Dr.Gajalakshmi, after retirement from<br />
Government service, has joined BRS Hospital as<br />
Director. She is continuing to work part time at this<br />
hospital. To update knowledge in medical sciences,<br />
she organises CME programmes and Seminars. She<br />
became interested in community service after being<br />
inducted into Altrusa <strong>International</strong> Inc. Madras.<br />
She conducted several medical camps covering<br />
dental,diabetes, speech & learning disability,<br />
eye,and cancer detection in women. She was the<br />
course director <strong>for</strong> Auxilliary Nurse Aid Programme<br />
of one year duration <strong>for</strong> Harijan Adidravida girls<br />
from 1995 till 2000 and 125 girls were trained in<br />
this programme and absorbed by varioius hospitals<br />
and clinics. Dr Gajalakshmi has shifted her interest<br />
now to study Vedanthic literature.She disires to<br />
be a Vanaprastha. <strong>Biomedicine</strong> on behalf of all its<br />
readers sends good wishes to this scientist <strong>for</strong> a<br />
happy retired life.<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011<br />
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REVIEW ARTICLE <strong>Biomedicine</strong>; 2011; 31 (1): 3 - 8<br />
Health beneficial effects of black tea<br />
Mahejabeen Fatima and Syed Ibrahim Rizvi<br />
Department of Biochemistry, University of Allahabad, Allahabad 211002, Uttar Pradesh, India.<br />
(Received 20 th December, 2010; Revised 26 th January, 2011; Accepted 7 th March, 2011)<br />
Corresponding author:<br />
Dr. Syed Ibrahim Rizvi<br />
E-Mail: sirizvi@gmail.com<br />
Abstract<br />
Tea is the most common beverage in the world. It is consumed mostly as green tea, oolong, or black tea.<br />
While there are several in<strong>for</strong>mative reviews on the health benefits of green tea, there are few detailed<br />
articles on black tea. Importantly in India the preferred <strong>for</strong>m of tea is black tea. This review is an attempt to<br />
bring together relevant findings on black tea including chemistry of processing, metabolism and absorption<br />
of black tea polyphenols and health benefits.<br />
Key words: <strong>An</strong>tioxidant, Black tea, Health, Polyphenols<br />
Introduction<br />
Tea is the most popular beverage in the world<br />
which is manufactured from the plant species<br />
Camellia sinensis (family Theaceae). It is<br />
grown around the world specially in China,<br />
Japan, Nepal, India, and SriLanka. Tea is<br />
consumed mostly as green tea and black tea,<br />
approximately 20% of the tea is manufactured<br />
as green tea while 80% of the approximately<br />
2.5 million metric tons of manufactured dried<br />
tea is black tea . A small percentage of tea is<br />
manufactured as oolong tea which is about 2%<br />
and usually consumed in Japan and China. The<br />
effect of tea consumption on human health has<br />
lately received much attention since tea has a<br />
high concentration of polyphenols which are<br />
known to possess antioxidant properties.<br />
Harvested tea leaves are processed in the factory<br />
and accordingly there are two types of tea:<br />
Orthodox, and CTC (Crushing Tearing Curling).<br />
The Orthodox tea is the whole leaf tea whereas<br />
CTC tea is widely popular and processed<br />
through the crush, tear and curl (CTC) method.<br />
Traditionally, tea is classified on the basis of the<br />
method of processing tea in to following three<br />
main types: green tea, oolong tea and black tea.<br />
After plucking, wilting leaves undergo slight<br />
enzymatic oxidation by the enzyme polyphenol<br />
oxidase (PPO), which results in fermentation<br />
of tea though this is not actual fermentation.<br />
Green tea is unfermented or nonoxidised<br />
tea and prepared by destroying the enzymes<br />
of fresh tea leaves by steaming followed by<br />
rolling either by hand or rollers, then fired to<br />
dry. Oolong tea is made by withering and then<br />
putting through a series of light rollings be<strong>for</strong>e<br />
firing, which arrests oxidation, this process<br />
is called as semifermentation and results in<br />
partial oxidation. Black tea is oxidized or fully<br />
fermented <strong>for</strong>m of tea. During the preparation<br />
of black tea, leaves are withered to reduce the<br />
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Beneficial effects of black tea<br />
moisture and then rolled by CTC or tea roller<br />
however during the rolling process oxidation<br />
starts when enzymes come into contact with air,<br />
this causes the change in color of leaves from<br />
green to brown. Finally to stop the oxidation<br />
process tea is passed through hot air dryers.<br />
Chemical composition of green and black tea<br />
The chemical composition of tea includes<br />
polyphenols, amino acids, vitamins,<br />
proteins, carbohydrates, trace elements.<br />
In addition, alkaloids such as caffeine<br />
(1,3,7-trimethylxanthine), theobromine and<br />
theophylline are also natural compounds<br />
present in tea. Tea polyphenols are the crucial<br />
compounds among these that include mainly<br />
catechins also found in red wines, apples, grapes<br />
and chocolate. Tea catechins are composed of<br />
epicatechin (EC), epigallocatechin (EGC) and<br />
their galloyl esters such as gallocatechin (GC),<br />
epicatechingallate (ECG), and epigallocatechin<br />
gallate (EGCG). Among teas, green tea<br />
contains large amount of catechins and esters<br />
of gallic acid. Black tea contains several<br />
polyphenols such as bisflavonols, theaflavins<br />
(TFs) and thearubigins (TRs), whereas<br />
oolong tea contains small amount of catechins<br />
and theaflavins. Theaflavins are mixture of<br />
theaflavin 3-gallate (TF3G), theaflavin3′-gallate<br />
(TF3′G), theaflavin3,3′-digallate (TF3,3′DG).<br />
Black tea extract constitutes approximately<br />
20-30% polyphenols, among them TRs is most<br />
abundant comprising about 10±20% with<br />
relative molecular masses ranging from 700 to<br />
40,000 Da and TFs with 0.3±2% on a dry weight<br />
basis . Together they contribute characteristic<br />
color, strength and body to the tea . TFs give<br />
reddish orange color whereas TRs brownish<br />
color to tea. In recent years, TFs have attracted<br />
considerable interest because of their beneficial<br />
health properties including anti-inflammatory ,<br />
antimutagenic and anticlastogenic effects (1).<br />
The chemistry of black tea processing<br />
Tea catechin flavanols, possess the C6 - C3 -<br />
C6 skeletal structure, correspond to 2-phenylsubstituted<br />
benzopyrans and pyrones , and<br />
precursors incorporated from the common<br />
shikimic and acetate-malonate biosynthetic<br />
pathways.<br />
During the manufacturing of black tea,<br />
oxidation of catechins takes place, TFs and<br />
TRs are <strong>for</strong>med through dimerisation and<br />
polymerization respectively (Fig: 1). TFs are<br />
<strong>for</strong>med through oxidative dimerisation between<br />
catechin derived quinones and gallocatechin<br />
with endogenous enzyme polyphenol oxidase<br />
of tea leaf while TRs are <strong>for</strong>med through<br />
the oxidative polymerization of galloyl<br />
esters of catechins through the <strong>for</strong>mation of<br />
benzotropolone skeleton (2).<br />
Metabolism and absorption of black tea<br />
polyphenols<br />
These large and highly polar molecules cannot be<br />
absorbed after oral ingestion but are hydrolyzed<br />
to their aglycones (nonglycosylated <strong>for</strong>ms)<br />
by bacterial enzymes in the lower part of the<br />
intestine (3) and then be incompletely absorbed<br />
or go through further biotrans<strong>for</strong>mation by<br />
bacteria. Recent studies have investigated that<br />
after tea consumption catechins are metabolized<br />
and trans<strong>for</strong>med as sulfated, methylated, or<br />
glucuronidated derivatives by enzymes such as<br />
sulfotranferase, catechol-O-methyltransferase<br />
and glucuronosyltransferase respectively (4)<br />
and it has been hypothesized that break down<br />
of flavonoids into smaller phenolic acids<br />
takes place within the colon from bacterial<br />
degradation, absorption occurs through the<br />
small intestine. These phenolic acids can be<br />
absorbed in the circulatory system (5).<br />
Health benefits of black tea<br />
Tea plays a significant role in protecting cell<br />
membranes from oxidative damage, improving<br />
intestinal microflora which are beneficial to the<br />
body and also prevents dental caries (6). Possible<br />
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Beneficial effects of black tea<br />
beneficial health effects of black tea polyphenols<br />
are antioxidative, antithrombogenic, and antiinflammatory<br />
(1). In vitro studies by using<br />
animal models suggest that consumption of<br />
black tea contribute in the prevention of some<br />
cancers, cardiovascular diseases and to treat<br />
diabetes in clinical trials (7). Moreover, a<br />
number of antioxidants present in green and<br />
black tea, mainly catechins and theaflavins<br />
have anti-carcinogenic , anti-mutagenic (8)<br />
and neuroprotective (9) properties. The health<br />
benefits of black tea are interesting, but more<br />
research on both animals and humans need to<br />
be conducted.<br />
Black tea: positive effects on mood and<br />
mental per<strong>for</strong>mance<br />
Tea contains a unique amino acid, L-theanine<br />
(5-N-ethyl-glutamine) which is a glutamic<br />
acid analogue, a predominant amino acid<br />
component in tea. It constitutes about 1-2%<br />
of the dry weight of tea leaves and accounting<br />
<strong>for</strong> about 50% of the total free amino acids.<br />
Several studies prove that L-theanine has an<br />
effect on brain electrical activity and appears to<br />
have psychoactive properties and can also act<br />
as a neurotransmitter. It increases the alphabrain<br />
waves during resting state (10) that are<br />
associated with relaxed mental state. By using<br />
animal models such as rats, it has been found<br />
that theanine improved memory and learning<br />
ability by modulating serotonin and dopamine<br />
levels (11).<br />
In addition caffeine (1,3,7-trimethylxanthine)<br />
is associated with stimulatory effects on central<br />
nervous system and cause increase in basal<br />
metabolic rate (BMR) by about 3-4% (12). It<br />
has been shown to stimulate thermogenesis by<br />
inhibiting the enzyme phosphodiesterase and<br />
also by the stimulation of substrate cycles such<br />
as the Cori-cycle and the FFA-triglycreride<br />
cycle (13). Caffeine and theophylline inhibit the<br />
enzyme adenosine 3, 5-cyclic monophosphate<br />
(cAMP) phosphodiesterase due to intracellular<br />
accumulation of cAMP (14), this is associated<br />
with freshness in mood which corresponds to<br />
increased mental alertness (15). It has been<br />
hypothesized that theophylline might inhibit<br />
Raf-1-dependent tumor progression in rats, the<br />
inhibition of theophylline is more than caffeine.<br />
Recent epidemiological studies have shown a<br />
reduced risk of Parkinson’s disease associated<br />
with selective amount of consumption tea (16).<br />
Black tea: Cancer chemoprevention<br />
There is evidence that tumor killing is induced<br />
by black tea polyphenols through apoptosis<br />
in Swiss mouse bearing Ehrlich’s ascites<br />
carcinoma (17). <strong>An</strong>other study reports that<br />
TFs inhibits the mammalian thioredoxin<br />
reductase, which is an antioxidant protein,<br />
regulating cellular functions like cell growth<br />
and apopotosis (18). It has been shown that<br />
green and black tea polyphenols inhibit the<br />
effect of hamster buccal pouch carcinogenesis<br />
by altering the detoxifying enzymes phase<br />
I (cytochromeP450s), phase II (glucuronyl<br />
transferase) and redox status, however black tea<br />
polyphenols were more effective and evidence<br />
indicates that it is more potent inhibitor<br />
<strong>for</strong> development of cancer (19). Moreover,<br />
considering the cancer chemoprevention studies<br />
some researches have shown that TF3,3′DG<br />
inhibits UVB induced activator protein (AP-1),<br />
induction and in the down regulation of EGFR<br />
(epidermal growth factor receptor), which<br />
regulates cell proliferation differentiation and<br />
trans<strong>for</strong>mation, in different mouse cell lines (JB6<br />
C141 and A431 ) (20). Consumption of black<br />
tea increases the plasma antioxidant capacity<br />
significantly in animal models and in humans<br />
thus protecting human red blood cells against<br />
oxidative damage induced by agents such as<br />
reactive oxygen and reactive nitrogen species<br />
(21). Black tea polyphenols may suppress the<br />
free radicals, protect HPF-1 (embryonic human<br />
lung fibroblasts) cells against H 2 O 2 induced<br />
damage (22). TFs are capable of anti cancerous<br />
action by inducing apoptosis as well as efficient<br />
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Beneficial effects of black tea<br />
Figure 1. Polyphenol oxidation during black tea processing.<br />
Figure 2. Metabolism of black tea polyphenols in the human body<br />
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Beneficial effects of black tea<br />
inhibitor of antiapoptotic proteins (Bcl-2<br />
family proteins) (23). Other metabolic effects<br />
of black tea include reducing the oxidation of<br />
low density lipoprotein (LDL) that is associated<br />
with atherosclerosis, improving the plasma<br />
lipid profiles in animals after high fat diet (24),<br />
reducing blood glucose and blood triglyceride<br />
levels in aged rats (25) and antioxidative effect<br />
leading to alcohol intoxication by normalization<br />
of cellular metabolism functionings through<br />
alteration in the antioxidative defence enzymes<br />
(1).<br />
<strong>An</strong>tioxidative properties of black tea<br />
<strong>An</strong>tioxidants have the ability to entrap free<br />
radicals and protect the body from damage<br />
caused by free radical-induced oxidative stress.<br />
Tea contains polyphenolic flavonoids which can<br />
enhance the cellular antioxidant enzyme activity<br />
or antioxidant defence of the body. In biological<br />
systems various environmental sources generate<br />
free radicals and reactive oxygen species which<br />
may oxidize proteins, nucleic acids and lipids.<br />
It has been demonstrated through in vitro and<br />
ex vivo studies that black tea polyphenols<br />
possess strong antioxidant and metal chelating<br />
properties, because of the vicinal dihydroxyl<br />
and trihydroxyl group attached to the flavan-<br />
3-ol, which prevents the generation of free<br />
radicals and may protect cells and tissues<br />
against oxidative damage (26). Researches have<br />
revealed that TF3 possess higher antioxidative<br />
activity than EGCG, which is the strongest<br />
antioxidant among all catechins due to the higher<br />
number of hydroxyl (OH) groups, which are<br />
considered to be necessary <strong>for</strong> exerting radical<br />
scavenging activity (antioxidative properties),<br />
than do catechins (27). Investigations show<br />
that antioxidative properties of theaflavins<br />
change in the following order: TF3>TF2>TF1<br />
(28) depending on the position and number of<br />
hydroxyl group in the molecule (27). Moreover,<br />
regarding the structural analysis theaflavin<br />
gallates illustrate stronger antioxidative<br />
properties compared with free theaflavins due<br />
to the position residues influences (28). There<br />
is increasing evidence to show that free radical<br />
damage contribute to many chronic health<br />
problems such as cardiovascular diseases<br />
,diabetes some cancers etc. Many epidemiologic<br />
and laboratory studies suggest that regular<br />
consumption of black tea polyphenols<br />
significantly reduce the risk of many oral cancers<br />
(29) however more research needs to define the<br />
real extent of tea consumption associated with<br />
them in the mammalian system.<br />
Future prospects<br />
Several epidemiological studies suggest that<br />
theaflavins can prevent risk of disease such as<br />
cardiovascular disease and some cancers. On<br />
the basis of laboratory studies regular intake<br />
of black tea can improve the oxidative stress<br />
biomarkers. From several pharmacological<br />
observations, worldwide abundance and<br />
absence of toxicity, theaflavin supplement can<br />
be used as an important natural therapeutic agent<br />
in chemoprevention studies and in the field of<br />
medicine. Further research is needed to explain<br />
the bioavailability and evaluation corresponding<br />
to the optimal amount of consumption of black<br />
tea in mammalian system.<br />
Acknowledgement<br />
This work was supported by UGC Major<br />
Research Project F 37-392/2009 SR sanctioned<br />
to SIR<br />
References:<br />
1. Khan N, Mukhtar H. Tea polyphenols <strong>for</strong> health promotion.<br />
Life Sci. 2007; 81: 519–533.<br />
2. Griffiths LA, Barrow A. Metabolism of flavonoid compounds<br />
in germ-free rats. Biochem J 1972; 130: 1161-1162.<br />
3. Bokkenheuser VD, Shackleton CHL, Winter J. Hydrolysis<br />
of dietary flavonoid glycosides by strains of intestinal<br />
Bacteroides from humans. Biochem J 1987; 248: 953-956.<br />
4. Lambert JD, Sang S, Yang CS. Biotrans<strong>for</strong>mation of green<br />
tea polyphenols and the biological activities of those metabolites.<br />
Mol Pharmacol 2007; 4: 819-25.<br />
5. Rechner AR, Smith MA, Kuhnle G, et al. Colonic metabolism<br />
of dietary polyphenols: influence of structure on mi-<br />
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crobial fermentation products. Free Radic Biol Med 2004;<br />
36: 212-25.<br />
6. Sakanaka S and Okada Y. Inhibitory Effects of Green Tea<br />
Polyphenols on the Production of a Virulence Factor of<br />
the Periodontal-Disease-Causing <strong>An</strong>aerobic Bacterium<br />
Porphyromonas gingivalis. J Agric Food Chem 2004; 52:<br />
1688-1692.<br />
7. MacKenzie T, Learye l and Brooks WB. The effect of an<br />
extract of green and black tea on glucose control in adults<br />
with type 2 diabetes mellitus: double-blind randomized<br />
study. Metab. Clin. Exp. 2007; 56: 1340-1344.<br />
8. Gupta S, Saha B, Giri AK. Comparative antimutagenic and<br />
anticlastogenic effects of green tea and black tea: a review.<br />
Mutat Res. 2002; 512: 37-65.<br />
9. Mandel SA, Avramovich-Tirosh Y, Reznichenko L, et al.<br />
Multifunctional activities of green tea catechins in neuroprotection.<br />
Modulation of cell survival genes,iron-dependent<br />
oxidative stress and PKC signaling pathway. Neurosignals<br />
2005; 14: 46-60.<br />
10. Junega LR, Chu D, Okubo T, Nagato Y, Yokogushi H. Ltheanine<br />
- a unique amino acid of green tea and its relaxation<br />
effect in humans. Trends in Food Sci Technol 1999;<br />
10: 199-204.<br />
11. Kim SK, Yum KS, Lee Y. Effect of changes in muscle tension<br />
and brain alpha wave activity induced by functional<br />
beverage on golf per<strong>for</strong>mance. Proceedings of the <strong>International</strong><br />
Symposium on Food, Nutrition and Health <strong>for</strong> the<br />
21st Century, 2001.<br />
12. Unno T, Suzuki Y, Kakuda T, Hayakawa T, Tsuge H. Metabolism<br />
of theanine, gamma-glutamylethylamide in rats. J<br />
Agric Food Chem 1999; 47: 1593-1596.<br />
13. Diepvens K, Westerterp KR, Westerterp-Plantenga MS.<br />
Obesity and thermogenesis related to the consumption of<br />
caffeine, ephedrine, capsaicin, and green tea. Am J Physiol<br />
Regul Integr Comp Physiol 2007; 292: 77-85.<br />
14. Dulloo AG, Geissler CA, Horton T, Collins A, Miller DS.<br />
Normal caffeine consumption: influence on thermogenesis<br />
and daily energy expenditure in lean and postobese human<br />
volunteers. Am J Clin Nutr 1989; 49: 44-50.<br />
15. Rall TW. Drugs used in the treatment of asthma.<br />
In:Goodman-Gilman A, Rall TW, Nies AS, Taylor P, editors.<br />
The pharmacological basis of therapeutics. 8th ed.<br />
New York: Pergamon Press. 1990; 618-37.<br />
16. Webster Ross G, Abbott RD, Petrovitch H, et al. Association<br />
of coffee and caffeine intake with the risk of Parkinson’s<br />
disease. J Am Med Assn 2000; 283: 2674-2679.<br />
17. Bhattacharyya A, Choudhuri T, Pal S, et al. Apoptogenic<br />
effects of black tea on Ehrlich’s ascites carcinoma cell.<br />
Carcinogenesis 2003; 24: 75-80.<br />
18. Du Y, Wu Y, Cao X , et al. Inhibition of mammalian thioredoxin<br />
reductase by black tea and its constituents: Implications<br />
<strong>for</strong> anticancer actions. Biochimie 2009; 91: 434-444.<br />
19. Chandra Mohana KVP, Harab Y, Abrahamc SK, Naginia S.<br />
Comparative evaluation of the chemopreventive efficacy<br />
of green and black tea polyphenols in the hamster buccal<br />
pouch carcinogenesis model. Clin. Biochem. 2005; 38:<br />
879-886.<br />
20. Mizuno H, Cho YY, Zhu F, et al. Theaflavin-3, 3′-Digallate<br />
Induces Epidermal Growth Factor Receptor Down-Regulation.<br />
Mol Carcinog 2006; 45(3): 204-212.<br />
21. Sarkar A and Bhaduri A. Black Tea Is a Powerful Chemopreventor<br />
of Reactive Oxygen and Nitrogen Species:<br />
Comparison with Its Individual Catechin Constituents and<br />
Green Tea. Biochem Biophys Res Commun 2001; 284:<br />
173-178.<br />
22. Yang Z, Tu Y, Xia H, Jie G, Chen X, He P. Suppression<br />
of free-radicals and protection against H2O2-induced oxidative<br />
damage in HPF-1 cell by oxidized phenolic compounds<br />
present in black tea. Food Chem 2007; 105: 1349-<br />
1356.<br />
23. Feng Q, Torii Y, Uchida K, Nakamura Y, Hara Y and Osawa<br />
T. Black Tea Polyphenols, Theaflavins, Prevent Cellular<br />
DNA Damage by Inhibiting Oxidative Stress and Suppressing<br />
Cytochrome P450 1A1 in Cell Cultures. J. Agric.<br />
Food Chem 2002; 5: 213-220.<br />
24. Liu S, Lu H, Zhao Q, et al. Theaflavin derivatives in black<br />
tea and catechin derivatives in green tea inhibit HIV-1<br />
entry by targeting gp41. Biochim et Biophys Acta 2005:<br />
270-281.<br />
25. Luczaj W, Skrzydlewska E. <strong>An</strong>tioxidant properties of<br />
black tea in alcohol intoxication. Food Cheml Toxicol<br />
2004; 42: 2045-2051.<br />
26. Wang C, Li Y. Research progress on property and application<br />
of theaflavins. Afri J Biotechnol 2006; 5: 213-218.<br />
27. Leung LK, Su Y, Chen R, Zhang Z, Huang Y, Chen ZY.<br />
Theaflavins in black tea and catechins in green tea are<br />
equally effective antioxidants. J Nutr 2001; 131: 2248-51.<br />
28. Wang H, Helliwell K. Determination of flavonols in green<br />
and black tea leaves and green tea infusions by high-per<strong>for</strong>mance<br />
liquid chromatography. Food Res Int 2001; 34:<br />
223-7.<br />
29. Lee MJ, Lambert JD, Prabhu, et al. Delivery of tea polyphenols<br />
to the oral cavity by green tea leaves and black tea<br />
extract. Cancer Detect Prev 2004; 13: 132- 7.<br />
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8
Special article <strong>Biomedicine</strong>; 2011; 31 (1): 9 - 12<br />
Modification of Lifestyle:Hypertension in Obese<br />
*Tandra Majhi, **Geeta Jaiswal<br />
*Department of Psychiatry,C.S.M.Medical University, Lucnow- 226003, Uttar Pradesh, India.<br />
**Department of Biochemistry, M.L.N.Medical College, Allahabad -211003, Uttar Pradesh, India.<br />
(Received 29 th June, 2010; Revised 20 th Decemebr, 2010; Accepted 16 th January, 2011)<br />
Corresponding Author<br />
Dr. Tandra Majhi<br />
Email: tandra.majhi@gmail.com<br />
rinku.majhi@gmail.com<br />
Abstract<br />
Obesity is a complex, multifactorial disease that is associated with essential hypertension in Men & Women.<br />
In India, hypertension in the general population is largely undetected, and the available data and study<br />
on the topic is merely a tip of the iceberg. Several large epidemiological studies have documented that<br />
associated between body weight and blood pressure. The age, physical activity, dietary intake and lifestyle<br />
definitely influence on body weight associated with blood pressure. When, lifestyle modifications are<br />
adopted by the obese as a primary prevention strategy in Indian population, it may help to avoid health<br />
problem due to obesity.<br />
Key words: Blood Pressure, Serum electrolytes, Life style & Physical activity<br />
Introduction<br />
Obesity and hypertension are both public health<br />
problems in Western society. Results from<br />
the Framingham study have shown that high<br />
blood pressure(1) and overweight(2) are both<br />
independent risk factors <strong>for</strong> cardiovascular<br />
disease. Hypertension is one of the most<br />
common obesity-related complication, and<br />
about 30% of hypertension individuals can<br />
be classified as being obese(3). In a group of<br />
hypertensive women taken from the Nurses<br />
Health Study(4) , the related risk of fatal and<br />
nonfatal coronary heart disease increased<br />
from the lowest to the highest <strong>for</strong>m of obesity.<br />
Prevalence in India, hypertension in the<br />
general population is largely undetected, and the<br />
available data and study on the topic is merely a<br />
tip of the iceberg. Epidemiological studies show<br />
that hypertension is present in 25% urban and<br />
10% rural population in India. There are 31.5<br />
million hypertension in the rural and 34 million<br />
in the urban population(5). The estimated total<br />
number of people with hypertension in India on<br />
2000 was 60.4 million males and 57.8 million<br />
females and projected to increase to 107.3<br />
million and 106.2 million respectively in 2025.<br />
Obesity and Hypertension<br />
Hypertension is generally defined as a systolic<br />
blood pressure of 140 mm Hg and a diastolic<br />
blood pressure of 90 mm Hg according to the<br />
sixth report of the Joint National Committee<br />
on Prevention, Detection Evaluation and<br />
Treatment of High Blood Pressure(6). Obesity<br />
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Modification of life style: Hypertension in Obese<br />
has shown to be independent risk factor <strong>for</strong> the<br />
development of hypertension. Several large<br />
epidemiological studies have documented the<br />
association between body weight and blood<br />
pressure(7,8). The relationship between body<br />
mass index and blood pressure was studied<br />
in 25-60 years of age, in a sample from urban<br />
population. In this study, BMI & WHR were<br />
significantly associated with systolic and<br />
diastolic blood pressure and was independent<br />
of age, sex, lifestyle, dietary intake and sodium<br />
and potassium excretion. In a recent, updated<br />
evolution of the Nurses Health Study, a longterm<br />
follow-up study of more than 80,000<br />
female nurses, BMIs at 18 years of age and<br />
middle age were positively associated with the<br />
occurrence of hypertension(9). Long-term and<br />
medium term weight loss was associated with<br />
a reduced risk in the group of women with a<br />
high baseline BMI. A central fat distribution is<br />
a better predictor <strong>for</strong> hypertension than overall<br />
fat mass. Earlier studies have shown a good<br />
correlation between the Weight/Height Ratio (<br />
WHR)and blood pressure levels(10,11). Later<br />
studies using computed tomography to measure<br />
to exact amount of visceral adipose tissue<br />
showed strong correlation with systolic and<br />
diastolic blood pressure levels both in European<br />
and African population (12-15).<br />
Life style modification and Physical activity<br />
Importance of lifestyle modification can<br />
be addressed by the fact that most of the<br />
community physicians in India who were<br />
treating hypertension have no time to tell the<br />
patients regarding lifestyle modifications.<br />
The low level of physical activity of individuals<br />
demanded by modern life, there<strong>for</strong>e, has<br />
created a condition that has made dietary<br />
intake a more noticeable component of obesity<br />
risk than it ever was be<strong>for</strong>e. A further lifestyle<br />
change is the daily stress to which human are<br />
exposed. There are different physiological<br />
responses to stress. The so-called “fight or<br />
flight” response involves stimulation of the<br />
10<br />
sympatho-adrenal system, and this response<br />
has been implicated in hypertension. Increase<br />
in aerobic physical activity such as brisk<br />
walking, jogging, swimming or bicycling has<br />
been shown to lower blood pressure. This<br />
reduction is independent of weight loss(16). A<br />
meta-analysis of 54 randomized control trials<br />
show a net reduction of 3.8 mm Hg in systolic<br />
and 2.6 mm Hg in diastolic blood pressure<br />
in individuals per<strong>for</strong>ming aerobic exercise.<br />
Compared to controls, physical activity reduces<br />
systemic vascular resistance due to decreased<br />
sympathetic and nervous system activity. This<br />
is evidence by lower plasma nor-epinephrine<br />
level in exercising individuals as compared to<br />
sedentary people(17).<br />
Dietary salt intake<br />
Food intake in humans is by nature, very poorly<br />
controlled. There has never been any real<br />
evolutionary pressure to suppress the appetite.<br />
In fact, it is quite the opposite. Because man<br />
is a hunter-gatherer and because of the annual<br />
cycles of “feast and famine” the evolutionary<br />
drive has been to seek food constantly, to gorge<br />
when it is readily available, and to be able to<br />
survive periods of famine.<br />
Our studies have indicated in this respect the<br />
total caloric (carbohydrates, fats, protein and<br />
minerals) intake was more than prescribed RDA.<br />
Raised BMI and WHR of the experimental<br />
group there<strong>for</strong>e may be a result of over nutrition<br />
leading to a highly fed state. The total caloric<br />
intake was much high in the Indian obese almost<br />
by 132.21 percent their control counterparts.<br />
There<strong>for</strong>e the of total caloric intake is a major<br />
and main contributor towards the increase in<br />
body weight and hypertension in India. In these<br />
days of increasing economic status it has<br />
resulted in a higher consumption of sugar, salt,<br />
meat and saturated fatty acids amongst Indians<br />
and there<strong>for</strong>e has increased the possibility<br />
of obesity with blood pressure. Dietary salt<br />
intake in associated with blood pressure.<br />
Epidemiological , clinical , experimental and<br />
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Modification of life style: Hypertension in Obese<br />
randomized controlled trials suggest that health<br />
intervention, including government policies and<br />
action to regulate reduction in the salt control of<br />
processed food are cost-effective ways to limit<br />
cardiovascular disease(18-21). This could avert<br />
over 21 million disability –adjusted life-years<br />
per year worldwide(22). The Dietary Approach<br />
to Stop Hypertension (DASH) sodium feeding<br />
study showed that an even lower intake of<br />
sodium (approximately) by both normotensives<br />
and hypertensives(23).<br />
The golden rule is :<br />
Avoid excessive intake of salt in cooking.<br />
Increase intake of green vegetables and fruits.<br />
Avoid foods high in salt.<br />
Avoid junk foods or fast food which are high in<br />
salt as well as fat content.<br />
Obesity and serum electrolytes<br />
Several large epidemiological studies<br />
have documented that associated between<br />
body weight and blood pressure. BMI was<br />
significantly associated with systolic and<br />
diastolic blood pressure and was independent<br />
of age, alcohol intake, smoking habits, sodium<br />
and potassium excretion. Epidemiological and<br />
clinical studies have shown that potassium<br />
intake has an important role in regulating blood<br />
pressure in both the general population and<br />
people with high blood pressure(24). From our<br />
study we found that the mean serum sodium<br />
levels were significantly higher in Indian obese<br />
compared to the non-obese group. However<br />
mean serum potassium levels were significantly<br />
(47.92%) lower. As observed, raised blood<br />
pressure and serum sodium may be attributed<br />
to an increased cardiac output in part due to<br />
the additional blood flow required <strong>for</strong> the extra<br />
adipose tissue. However, blood flow in the heart,<br />
kidneys, gastro-intestinal tract, and skeletal<br />
muscle also increased metabolic demands. As<br />
hypertension is sustained <strong>for</strong> many months and<br />
years, total peripheral vascular resistance may<br />
also be increased.<br />
Management<br />
11<br />
The management of overweight control should<br />
be influenced by the degree of obesity and over<br />
risk status of associated diseases. Management<br />
includes both reducing excess body weight<br />
and instituting other measures to control<br />
accompanying risk factors obesity should be<br />
assessed in terms of BMI, waist circumference<br />
and the patient’s motivation to lose weight.<br />
The initial goal of weight loss therapy is to<br />
reduce body weight to approximately 10%<br />
from baseline. If one can achieve this goal,<br />
further weight loss attempted, indicated through<br />
evolution. Those patients who are unable to<br />
achieve significant weight loss, they may also<br />
need to participate in a weight management<br />
program.<br />
Weight loss strategies<br />
Diet therapy<br />
A diet should create a deficit of 500-1000 kcal/<br />
day. Depending on the patient’s risk status, the<br />
low-caloric diet should be consistent with the<br />
therapeutic life-style changes defined by the<br />
National Cholesterol education Program. Apart<br />
from decreasing saturated fat, total fat should<br />
be 30% or less of total calories (including low<br />
carbohydrates & salts), saturated fat should<br />
not produce weight loss unless the total<br />
caloric intake is also decreased. Fiber diet and<br />
nutritional antioxidants (Vit C & E) should be<br />
encouraged.<br />
Physical activity<br />
<strong>An</strong> increase in physical activity is an important<br />
component of weight loss therapy although<br />
physical activity alone will not usually produce<br />
significant weight loss in obese patients.<br />
Most weight loss occurs because of decrease<br />
in caloric intake. Sustained physical activity<br />
is, however, helpful <strong>for</strong> maintaining a lower<br />
weight. There<strong>for</strong>e the dietary intake and<br />
physical activity should be balanced to maintain<br />
the weight loss.<br />
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Modification of life style: Hypertension in Obese<br />
Conclusion<br />
As the prevalence of overweight and obesity<br />
has increased, so have related health care costsboth<br />
direct and indirect. It must be noted that<br />
the studies hav shown a positive relationship<br />
between obesity and hypertension. It indicated<br />
that age, physical activity, dietary intake<br />
(including salt intake) and life-style definitely<br />
influence body weight and blood pressure levels<br />
in the obese subjects. If life style modifications<br />
are adopted by the obese people as a primary<br />
prevention strategy in Indian population, then<br />
it may help to avoid health problem specially<br />
hypertension related with obesity.<br />
References<br />
1. Kannal WB, Blood Pressure as a cardiovascular risk factor<br />
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evaluation and treatment of high blood pressure.<br />
Arch. Intern. Med 1997;157:2413-2446.<br />
7. Stamler R, Stamler J, Riedlinger WF, Algera G, Roberts<br />
RH. Weight and blood pressure: findings in hypertension<br />
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9. Huang Z, Willett WC, Manson JE at al Body weight-<br />
weigth change, and risk <strong>for</strong> hypertension in women. <strong>An</strong>n<br />
Indian Med. 1998;128:81-88.<br />
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10. Lapidus L, Bengtsson C, Larsson B, Penvert K, Rybo E,<br />
Sjostrom L. contribution of adipose tissue and risk of cardiovascular<br />
death: a 12 year follow up of participation in<br />
the population study of women in Gothenburg. Br Med. J<br />
1984;289: 1257-1261.<br />
11. Larsson B, Svardsudd K, Welin L, Wilhelmsen L, Bjorntorp<br />
P, Tibblin G. Abdominal adipose tissue distribution,<br />
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1913. Br Med. J 1984;288:1401-1404.<br />
12. Peirin AN, Sothmann MS, Hoffmann RG et al. Adiposity<br />
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1989;110: 867-872.<br />
13. Kamai M, Tokunaga K, Fujioka S, Yamashila S, Kameda-<br />
Takemura K, Matsuzawa Y. Decrease in intra abdominal<br />
visceral fat may reduce blood pressure in obese hypertensive<br />
women. Hypertension 1996;27: 125-129.<br />
14. Han TS, Van Leer. EM, Seidell JC, Lean MEJ, Waist circumference<br />
action levels in the indentification of cardiovascular<br />
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Br Med J 1995; 311: 1401-1405.<br />
15. Okosum IS, Forrester TE, Rotimi CN, Osotimetion BO, Muna<br />
WF, Looper RS, Abdominal adiposity in six populations of<br />
west African descent: Prevalence and population attributable<br />
fraction of hypertension. Obes Res. 1999;7: 453-462.<br />
16. He J, Klag MJ, Caballero B et al. Plasma insulin levels<br />
and incidence of hypertension in African Americans and<br />
Whites. Arch. Intern Med 1999;159:498-503.<br />
17. Whetton SP, Chin A, Xin X, He J. Effect of aerobic exercise<br />
on blood pressure: A meta-analysis of randomized,<br />
controlled trials. <strong>An</strong>n Intern Med, 2002;136:493-503.<br />
18. Intersalt cooperative research group. INTERSALT- an international<br />
study of electrolyte excretion and blood pressure:<br />
results <strong>for</strong> 24-hour urinary sodium and potassium<br />
excretion. B, Medical J.1988; 297:319-28.<br />
19. Forte JG, Miguel JM, Miguel MJ et al. Salt and blood pressure:<br />
a community trial. J Hum Hypertens. 1989; 3:179-84.<br />
20. Denton D, Weisinger R, Mundy NI et al. the effect of increased<br />
salt intake on blood pressure of chimpazees. The<br />
Natl Med. J 1995;1009-16.<br />
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pressure of reduced dilteway sodium and diatery approaches<br />
to stop hypertension (DASH) diet. New Eng. J<br />
Med. 2001; 344:3-10.<br />
22. Murray CSL, Lauer JA, Hutubessy RCW et al. Effectiveness<br />
and costs of interventions to lower systolic blood pressure<br />
and cholesterol: a global and regional analysis on reduction<br />
of cardiovascular- disease risk. Lancet 2003; 361:717-25.<br />
23. HeJ, Ogden LG, Vupputuri S et al. Dietery sodium intake<br />
and subsequent risk of cardiovascular disease in over<br />
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Am. Hypertens. 1999; 12: 848-51.<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
Research Paper <strong>Biomedicine</strong>; 2011; 31 (1): 13 - 21<br />
Differential effect of Nardostachys jatamansi rhizome extract on acetylcholinesterase<br />
in different regions of brain in rats under chronic stress<br />
*Gloria Karkada **K. B. Shenoy**** Harsha Halahalli ****K. S. Karanth<br />
*Department of Biotechnology, Nitte Mahalinga Adyanthaya Institute of Technology,<br />
Nitte - 574110, Karnataka, India.<br />
**Department of Applied Zoology, Mangalore University, Mangalore-574119 India.<br />
***Department of Physiology, KS Hegde Medical Academy, Mangalore-575018, India.<br />
****Department of Pharmacology, KS Hegde Medical Academy, Mangalore-575018.<br />
(Received 16 th July, 2010; Revised 6 th December, 2010; Accepted 6 th January, 2011)<br />
Corresponding Author<br />
Dr K. B. Shenoy<br />
Email: bshenoyk@gmail.com<br />
Abstract<br />
Background and objectives: Nardostachys jatamansi, a popular herb in Indian medicine, has been<br />
investigated <strong>for</strong> various effects on central nervous system. However the neurobiological mechanisms<br />
of its actions are not clear. It was hypothesized that N.jatamansi root ethanolic extract (NJE) modulates<br />
acetylcholinesterase activity levels in brain regions relevant <strong>for</strong> learning and memory.<br />
Methods: A factorial study design was adopted to investigate the effects of two experimental interventions<br />
- treatment with NJE and chronic restraint stress <strong>for</strong> 21 days – on AChE activity levels in the hippocampus,<br />
frontal cortex and striatum.<br />
Results: Unstressed animals treated with NJE had significantly higher level of AChE activity in the frontal<br />
cortex (179% higher) and hippocampus( 36% higher) compared to controls. AChE activity was also<br />
significantly higher (by 54 %) in the frontal cortex of stressed animals and concomitant NJE treatment<br />
when compared to untreated stressed group. Chronic restraint stress per se had no effect on AChE levels in<br />
the brain regions studied.<br />
Interpretation and Conclusions: Enhancement of AChE activity in the frontal cortex following NJE<br />
administration may be indicative of positive plasticity of cholinergic pathways to the neocortex. The<br />
implications of this region specific cholinergic modulation <strong>for</strong> the possible development of N.jatamansi<br />
based anti-dementia and nootropic interventions remain to be clarified.<br />
Keywords: Acetylcholinesterase, Cholinergic, Chronic restraint stress, Nardostachys jatamansi, Nootropic<br />
Introduction<br />
Nardostachys jatamansi has been valued <strong>for</strong><br />
its therapeutic properties in the Ayurveda and<br />
Unani traditions of complimentary medicine.<br />
13<br />
The rhizomal extracts of N. jatamansi have<br />
been particularly studied <strong>for</strong> effects on the<br />
central nervous system. N. Jatamansi ethanolic<br />
root extracts (NJE) have been reported to have<br />
anticonvulsant activity in a rat electroshock<br />
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Effect of Nardostachys jatamansi rhizome in brain<br />
seizure model (1), anti-parkinsonism activity<br />
in a rat 6-OHDA model (2), neuroprotective<br />
effect in a rat cerebral ischemia model (3) and<br />
antidepressant activity in tail suspension and<br />
<strong>for</strong>ced swim mouse models of depression (4).<br />
A few studies have investigated possible<br />
neurobiological mechanisms of the action of<br />
N.jatamansi extracts. The anti-parkinsonism<br />
effect of NJE has been correlated with<br />
enhanced anti-oxidant defenses, restoration of<br />
dopamine and its metabolites, enhanced striatal<br />
dopaminergic D2 receptor binding and tyrosine<br />
hydroxylase expression (2). The antidepressant<br />
activity may be related to modulation of levels<br />
of neurotransmitters such as norepinephrine,<br />
dopamine, serotonin, 5-hydroxyindoleacetic<br />
acid and gamma-aminobutyric acid (5). <strong>An</strong>other<br />
study in a mouse model of depression has<br />
suggested altered monoamine oxidase (MAO-A<br />
& MAO-B) activities and GABAergic function<br />
(4). Neuroprotection af<strong>for</strong>ded by NJE in a<br />
model of cerebral ischemia has been attributed<br />
to its anti-oxidant properties (3). While most<br />
studies have focused on therapeutic utility of<br />
NJE in disease models of the CNS, there has<br />
also been an interest in the possible nootropic<br />
effects of N. jatamansi. Learning and memory<br />
as assessed by elevated plus maze and passive<br />
avoidance task in young mice has been reported<br />
to be improved by administration of NJE. NJE<br />
was also shown to reverse age related amnesia<br />
in older mice. The finding of a reversal of<br />
scopolamine induced amnesia by NJE has been<br />
suggested as evidence <strong>for</strong> possible cholinergic<br />
modulation by NJE (6). The importance of<br />
cholinergic function <strong>for</strong> cognitive functions<br />
in general is well recognized and insights into<br />
the pathophysiology of Alzheimer’s disease<br />
has underlined the role of acetylcholine and<br />
its metabolism in cognition (7). Estimation of<br />
acetylcholinesterase (AChE) activity in brain<br />
regions has been used as a marker of cholinergic<br />
function. AChE is localized to cholinergic<br />
synapses and is involved in the hydrolysis of<br />
acetylcholine (ACh) to choline and acetate<br />
and thereby terminating the neurotransmitter<br />
14<br />
action of ACh. A reduction in AChE activity has<br />
generally been found in conditions of impaired<br />
cognition. For instance, AChE activity has been<br />
found to be reduced in the hippocampus and<br />
cortex of patients suffering from Alzheimer’s<br />
disease (8, 9). Evidence from animal models<br />
of learning and memory impairments has also<br />
pointed to a correlation between AChE activity<br />
and cognition. AChE activity levels have been<br />
reported to be decreased in animals subjected to<br />
21 days of chronic restraint stress – an animal<br />
model that has been shown to be consistently<br />
associated with hippocampus dependent<br />
learning and memory impairments (10, 11).<br />
<strong>An</strong>imal models of stress are increasingly being<br />
used to understand neurobiological bases of<br />
neuropsychiatric disorders and therapeutic<br />
strategies. There is significant evidence to link<br />
stress to the etiopathology of affective disorders<br />
and cognitive impairment (12). In this context<br />
a few studies have evaluated the efficacy of<br />
NJE in preventing or reversing the deleterious<br />
effects of stress on the central nervous system.<br />
For instance, pre-treatment with NJE has been<br />
reported to reduce markers of stress such as<br />
elevation of plasma corticosterone, gastric<br />
ulceration and increase in adrenal and spleen<br />
weights in rats subjected to an acute stress of<br />
restraint and cold exposure. These anti-stress<br />
effects were attributed to anti-oxidant actions<br />
of NJE (13). N. jatamansi has been suggested<br />
to have a beneficial impact on learning and<br />
memory and also to be protective against stress.<br />
N. jatamansi is there<strong>for</strong>e a promising candidate<br />
<strong>for</strong> a nootropic agent. However, the mechanism<br />
of action of NJE with respect to its nootropic<br />
effect is not clear. We hypothesized that NJE<br />
has a modulatory effect on the cholinergic<br />
system which may underlie its nootropic<br />
effect. We aimed to investigate the efficacy<br />
of NJE in modulating AChE levels in both<br />
normal control rats as well as rats which were<br />
subjected to chronic restraint stress which has<br />
been previously reported to alter AChE levels.<br />
AChE activity levels were measured in the<br />
hippocampus and frontal cortex – two brain<br />
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Effect of Nardostachys jatamansi rhizome in brain<br />
areas traditionally recognized to be important <strong>for</strong><br />
declarative learning and memory as well as the<br />
striatum which served as a control brain region.<br />
A factorial study design was adopted in which<br />
groups of animals differed systematically in<br />
terms of the interventions – treatment with NJE<br />
and exposure to chronic restraint stress. This<br />
allowed us to apply robust statistical methods<br />
to analyze the effects of NJE treatment on both<br />
normal control rats as well as stressed rats<br />
Materials and Methods<br />
Experimental subjects<br />
The study was conducted at K S Hegde Medical<br />
Academy, Mangalore. The Institutional animal<br />
ethics committee approved the experimental<br />
protocols. All ef<strong>for</strong>ts were made to minimize<br />
both the suffering and the number of animals<br />
used. Inbred Wistar strain male rats from the<br />
institutional animal house, aged between 60-80<br />
days, weighing 180-200gms served as subjects.<br />
The rats were reared in a 12 hour light-dark<br />
environment with ad libitum access to food and<br />
water except during restraint stress procedure.<br />
A factorial study design was adopted to delineate<br />
the effects of two experimental interventions<br />
treatment with NJE chronic restraint stress –<br />
on AChE activity levels in three brain regions.<br />
Thirty two animals were randomized to four<br />
groups (N = 8 <strong>for</strong> each group).<br />
Group 1: Normal control group (NC)<br />
Group 2: N. jatamansi root extract treated group (JM)<br />
Group 3: Chronic restrai nt stress group (RS)<br />
Group 4: Chronic restraint stress + N. jatamansi<br />
root extract treatment group (RS+JM)<br />
Preparation and administration of N.<br />
jatamansi extract<br />
Briefly, botanically identified roots and<br />
rhizomes of N. jatamansi were purchased from<br />
a recognized and licensed supplier (Amsar<br />
Private Ltd-Indore). <strong>An</strong> ethanolic extract was<br />
prepared following standardized methods<br />
15<br />
described earlier (14). They were cut into<br />
small pieces, powdered and refluxed with 95%<br />
ethanol in a Soxhlet extractor <strong>for</strong> 6-8 hours. The<br />
extract was evaporated to dryness under reduced<br />
pressure and temperature using a rotary vacuum<br />
evaporator and the dried residue was stored at<br />
4º C. The yield of dry extract from the crude<br />
powder was about 10%. Based on the previous<br />
studies a dose of 200mg/kg body weight was<br />
decided and the extract was administered orally<br />
<strong>for</strong> 21 days.<br />
Chronic restraint stress<br />
Chronic restraint stress involved placing the<br />
rats in a wire mesh rodent restrainer <strong>for</strong> 6<br />
hours/day <strong>for</strong> 21 days. This <strong>for</strong>m of stress has<br />
been reported to elevate cortciosterone levels,<br />
increase adrenal and spleen weights and cause<br />
gastric ulcers (15, 10).<br />
Corticosterone estimation to establish<br />
effectiveness of stress<br />
The effectiveness of the chronic restraint stress<br />
procedure was established in separate cohort<br />
of six rats of the same mean age and weight as<br />
the study groups. Blood samples were obtained<br />
from each animal on Days 0, 3, 10 and 21 (Day 1<br />
being the start of the stress procedure). For each<br />
collection, animals were lightly anesthetized<br />
with ether and rapidly bled from the retroorbital<br />
sinus into collection tubes containing<br />
sodium citrate as anticoagulant. Samples were<br />
centrifuged at 4º C <strong>for</strong> 15 min at 3000 rpm.<br />
Serum aliquots were aspirated and stored in<br />
sealable polypropylene microcentrifuge tubes<br />
at −70º C until assayed <strong>for</strong> serum corticosterone<br />
concentrations using a Enzyme Immuno Assay<br />
(EIA) kit obtained from Cayman chemicals Inc<br />
USA(16).<br />
Estimation of AChE activity<br />
The frontal cortex (FC), hippocampus (HP)<br />
and striatum (ST) were rapidly dissected<br />
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Effect of Nardostachys jatamansi rhizome in brain<br />
out, weighed and homogenized in 0.1M<br />
Phosphate buffer (pH 8).The homogenates were<br />
centrifuged at 10,500rpm <strong>for</strong> 20 minutes at 4°C<br />
to obtain post mitochondrial supernatant, which<br />
was stored at -70°C till further use. AChE<br />
activity was estimated by Ellman’s method<br />
(17) with modifications as described elsewhere<br />
(11). The esterase activity was measured by<br />
providing the substrate acetylthiocholine iodide<br />
(ATC). Thiocholine released by the cleavage of<br />
ATC by AChE is allowed to react with the -SH<br />
reagent 5,5’-dithiobis-(2-nitrobenzoic acid)<br />
(DTNB), which is reduced to thionitrobenzoic<br />
acid, a yellow colored anion with an absorption<br />
maxima at 412nm. Change in the absorbance<br />
at 412nm was noted every 2 min <strong>for</strong> 10 min at<br />
412 nm using a spectrophotometer. The activity<br />
was calculated and expressed as micromoles<br />
hydrolyzed per min per gram of tissue.<br />
Statistical analysis<br />
AChE activity levels were entered into a threeway<br />
ANOVA with Stress (Levels: No Stress,<br />
Restraint Stress), Treatment (Levels: No<br />
Treatment, NJE Treatment) and Region (Levels:<br />
Frontal Cortex, Hippocampus, Striatum) as the<br />
three factors to assess between-subjects main<br />
effects and the interaction between the three<br />
factors. If the main effects were significant<br />
(p
Effect of Nardostachys jatamansi rhizome in brain<br />
Table 1: AChE activity in the hippocampus(HP), frontal cortex(FC) and striatum(ST)<br />
HP FC ST<br />
NC 2.75+0.68 2.21+0.80 4.55+2.58<br />
JM 3.74+1.41 6.18+1.17 4.17+1.67<br />
RS 1.99+0.11 3.43+1.79 4.92+3.87<br />
RS+JM 3.30+1.10 5.27+2.69 3.48+1.03<br />
Values represent mean+standard deviation of AChE activity. NC: Normal control group, JM: NJE treated group, RS:<br />
Chronic restraint stress group, RS+JM: Chronic restraint stress + NJE treatment group. N = 8 <strong>for</strong> each group<br />
Figure 1: Serum corticosterone levels in the rats subjected to chronic restraint stress <strong>for</strong> 21 days. Values represent mean<br />
± standard error of mean (N = 6). Repeated Measures ANOVA: F(3, 12) = 22.547, p
Effect of Nardostachys jatamansi rhizome in brain<br />
Figure 2: Profile plots showing the effect of NJE on AChE activity levels (µmol/min/g) in the hippocampus (HP),<br />
frontal cortex (FC) and striatum (ST).<br />
(A) Three-way ANOVA analysis with the three factors being Stress (2 levels: No stress, Chronic restraint stress), Treatment (2 levels:<br />
No treatment, NJE treatment) and Brain Region (3 levels: hippocampus, frontal cortex, striatum) as the three factors. Significant main effects of<br />
Treatment [F(1, 84) = 7.585, p < 0.01]; Region [ F(2, 84) = 5.458, p < 0.01]; Interaction effect of Treatment*Region [F(2, 84) = 8.410, p < 0.001].<br />
(B) Bonferroni adjusted pairwise comparisons of AChE activity in HP, FC and ST between Untreated (Normal Control) and NJE treated<br />
(unstressed) groups. *p Untreated.<br />
(C) (C) Bonferroni adjusted pairwise comparisons of AChE activity in HP, FC and ST between Chronic restraint stressed (Untreated) and<br />
NJE treated (chronic restraint stressed) groups. *p Untreated.<br />
Values represent mean ± standard error of mean.<br />
Figure 3: Regional differences in mean AChE activity levels in Untreated and NJE treated animals (Both unstressed<br />
controls and chronic restraint stressed combined <strong>for</strong> comparison). HP: hippocampus, FC: frontal cortex, ST: striatum.<br />
*pHP; #pFC; $pHP; ^pST. Bonferroni adjusted comparisons. Values<br />
represent mean ± standard error of mean.<br />
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18
Effect of Nardostachys jatamansi rhizome in brain<br />
HP (p < 0.01) and FC (p < 0.05). There were<br />
no significant differences between FC and HP.<br />
However, in animals treated with NJE, AChE<br />
levels in the FC were significantly higher<br />
compared to both HP (p < 0.01) and ST (p <<br />
0.05). There were no significant differences<br />
between FC and ST in this group. In the animals<br />
which received NJE treatment, AChE level in<br />
the FC was significantly greater than in those<br />
which did not receive such treatment (p < 0.001)<br />
(Figure 3). No such effect of treatment was seen<br />
in the other brain regions studied.<br />
Thus treatment with NJE was associated with<br />
significantly greater AChE in the frontal cortex<br />
irrespective of whether the animals were<br />
subjected to chronic restraint stress or not.<br />
Discussion<br />
The current study assessed the effects of two<br />
interventions – 21 day treatment with NJE<br />
and 21 days of chronic restraint stress – on<br />
AChE activity in three brain regions, viz.<br />
hippocampus, frontal cortex and striatum. A<br />
factorial analysis revealed that NJE treatment<br />
had a differential effect on AChE activity on<br />
the three brain regions studied in both normal<br />
control animals as well as animals which were<br />
subjected chronic restraint stress. Stress per se<br />
had no effect on AChE activity levels.<br />
To the best of our knowledge, this is the first<br />
report of the modulatory effects of NJE on<br />
regional brain AChE activity. NJE has been<br />
shown to modulate other neurotransmitter<br />
systems in the brain. For instance, 15 days<br />
of NJE administration has been shown to<br />
increase norepinephrine, dopamine, serotonin,<br />
5-hydroxyindoleacetic acid and gammaaminobutyric<br />
acid in animal the rat brain (5).<br />
NJE treatment has also been reported to increase<br />
activity levels of MAO-A and MAO-B thus<br />
possibly influencing catecholamine levels in the<br />
mouse brain (4). However, the effect of NJE on<br />
AChE levels or other indicators of cholinergic<br />
function has not been studied so far. A previous<br />
study reported a reversal of scopolamine<br />
19<br />
induced amnesia by NJE administration. This<br />
is suggestive of a modulatory effect of NJE on<br />
cholinergic neurotransmission. The finding of<br />
increased AChE activity levels in the present<br />
study lends further support to the possibility<br />
that cholinergic modulation is involved in the<br />
nootropic effects of N. jatamansi.<br />
The interpretation of the finding of increased<br />
AChE activity levels in terms of its implications<br />
on learning and memory is complex. Generally,<br />
a decrease in AChE activity has been correlated<br />
with impairment of cognitive functions,<br />
particularly of hippocampal and neocortical<br />
dependent learning and memory. This is<br />
exemplified by the finding of decreased AChE<br />
activity levels in the hippocampus and cortex<br />
of post-mortem brain samples from patients<br />
with Alzheimer’s disease (9). Decreased AChE<br />
activity in the hippocampus has also been<br />
described animal models of impaired learning<br />
and memory. Chronic restraint stress <strong>for</strong> 21<br />
days which causes demonstrable deficits in<br />
hippocampal dependent spatial and reference<br />
memory errors has been associated with<br />
decreases AChE activity (10, 11). Conversely,<br />
interventions which reverse chronic restraint<br />
stress induced learning-memory deficits have<br />
been associated with restoration of AChE<br />
levels in the hippocampus (11). These findings<br />
may be interpreted as evidence <strong>for</strong> a positive<br />
correlation between AChE activity levels in the<br />
hippocampus / neocortex and learning-memory<br />
capabilities of the mammalian brain. AChE<br />
activity levels in the hippocampus has been<br />
shown to correspond to neuronal activity levels<br />
in the septo-hippocampal cholinergic pathway<br />
(18)and lesions interrupting these projections<br />
is associated with decreased AChE activity in<br />
their target regions (18, 19). Thus AChE activity<br />
levels may reflect structural and functional<br />
integrity of cholinergic pathways. It may<br />
there<strong>for</strong>e be speculated that the enhancement<br />
of AChE activity in the frontal cortex following<br />
21 days NJE administration may be indicative<br />
of positive plasticity of cholinergic pathways<br />
to the neocortex. However, it must be noted<br />
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Effect of Nardostachys jatamansi rhizome in brain<br />
that an enhanced AChE activity could result<br />
in accelerated ACh hydrolysis at cholinergic<br />
synapses thus compromising cholinergic<br />
modulation of cognitive functions. Thus the<br />
true implications of the elevated AChE activity<br />
levels in the frontal cortex resulting from NJE<br />
treatment needs to be assessed in conjunction<br />
with its behavioral effects. AChE activity levels<br />
were highest in the striatum followed by the<br />
hippocampus and frontal cortex. This is in<br />
concordance with the findings of Hammond<br />
& Brimijoin (9) in human post-mortem brain<br />
samples. Though the AChE activity levels in<br />
the chronic restraint stress group did not differ<br />
significantly from the normal control group,<br />
mean AChE activity level in the hippocampus<br />
was lower in the stressed group (Table 1).<br />
This supports previous findings of reduced<br />
hippocampal AChE in chronic stress (10, 11).<br />
In the present study, chronic restraint stress <strong>for</strong><br />
21 days did not have significant effect on AChE<br />
activity levels in the hippocampus, frontal<br />
cortex or striatum. This is contrary to some<br />
studies which have reported reduced AChE<br />
activity in the hippocampus and frontal cortex<br />
in similar models of chronic restraint stress<br />
but is in concordance with studies that have<br />
reported that chronic stress does not affect the<br />
basal cholinergic function (20). The absence of<br />
change in AChE activity levels is unlikely to be<br />
due to an ineffectiveness of the stress protocol<br />
as significant elevation of serum corticosterone<br />
during the early period of the chronic restraint<br />
stress was observed as has been described in<br />
earlier studies (15). The interesting finding to<br />
emerge from this study is the elevation in AChE<br />
activity levels in the frontal cortex associated<br />
with NJE treatment in both chronically stressed<br />
rats as well as normal control unstressed rats.<br />
This observation suggests the possibility that N.<br />
jatamansi has a nootropic effect that can enhance<br />
cognitive function even in normal unperturbed<br />
animals. However it remains to be seen whether<br />
the elevated AChE activity translates to an<br />
enhanced behavioral per<strong>for</strong>mance.<br />
Conclusion<br />
20<br />
The novel finding that 21 days of administration<br />
of NJE preferentially elevates AChE activity<br />
levels in the frontal cortex suggests a possible<br />
mechanism <strong>for</strong> a nootropic effect of N.<br />
jatamansi. The implications of this region<br />
specific cholinergic modulation <strong>for</strong> the possible<br />
development of N. jatamansi based antidementia<br />
and nootropic interventions remains<br />
to be clarified.<br />
References<br />
1. Rao V S, Rao A and Karanth K S. <strong>An</strong>ticonvulsant and<br />
neurotoxicity profile of Nardostachys jatamansi in rats. J<br />
Ethnopharmacol 2005; 102(3): 351-356.<br />
2. Ahmad M, Yousuf S, Khan M B, et al. Attenuation by<br />
Nardostachys jatamansi of 6-hydroxydopamine-induced<br />
parkinsonism in rats: behavioral, neurochemical, and<br />
immunohistochemical studies. Pharmacol, Biochem,<br />
Behav 2006; 83(1): 150-160.<br />
3. Salim S, Ahmad M, Khan S Z, Ahmad S A and Islam F.<br />
Protective effect of Nardostachys jatamansi in rat cerebral<br />
ischemia. Pharmacol, Biochem, Behav 2003; 74(2): 481-486.<br />
4. Dhingra D and Goyal P K. Inhibition of MAO and GABA:<br />
probable mechanisms <strong>for</strong> antidepressant-like activity of<br />
Nardostachys jatamansi DC in mice. Ind J Exp Biol 2008;<br />
46(4): 212-218.<br />
5. Prabhu V, Karanth K S and Rao A. Effects of Nardostachys<br />
jatamansi on biogenic amines and inhibitory amino acids<br />
in the rat brain. Planta Medica 1994; 60(2): 114-117.<br />
6. Joshi H and Parle M. Nardostachys jatamansi improves<br />
learning and memory in mice. J Med Food 2006; 9(1):<br />
113-118.<br />
7. Greig N H, Utsuki T, Ingram D K, et al. Selective<br />
butyrylcholinesterase inhibition elevates brain<br />
acetylcholine, augments learning and lowers Alzheimer<br />
beta-amyloid peptide in rodent. Proc Natl Acad Sci U S A<br />
2005; 102(47): 17213-8<br />
8. Fishman E B, Siek G C, MacCallum R D, Bird E D,<br />
Volicer L, and Marquis JK. Distribution of the molecular<br />
<strong>for</strong>ms of acetylcholinesterase in human brain: alterations<br />
in dementia of the Alzheimer type. <strong>An</strong>n Neurol 1986;<br />
19(3): 246-252.<br />
9. Hammond P and Brimijoin S. Acetylcholinesterase in<br />
Huntington’s and Alzheimer’s diseases: simultaneous<br />
enzyme assay and immunoassay of multiple brain regions.<br />
J Neurochem 1988; 50(4): 1111-1116.<br />
10. Sunanda, Rao B S S, Raju T R. Restraint Stress-Induced<br />
Alterations in the Levels of Biogenic Amines, Amino<br />
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Effect of Nardostachys jatamansi rhizome in brain<br />
Acids, and AChE Activity in the Hippocampus. Neurochem<br />
Res 2000; 25(12): 1547-1552.<br />
11. Srikumar B, Raju T and Rao B S S. The involvement<br />
of cholinergic and noradrenergic systems in behavioral<br />
recovery following oxotremorine treatment to chronically<br />
stressed rats. Neuroscience 2006; 143(3): 679-688.<br />
12. McEwen B S. The neurobiology of stress: from serendipity<br />
to clinical relevance. Brain Res 2000; 886(1-2): 172-189<br />
13. Lyle N, Bhattacharyya D, Sur T K, Munshi S, Paul S,<br />
Chatterjee S, et al.Stress modulating antioxidant effect of<br />
Nardostachys jatamansi. Ind J Biochem & Biophy 2009;<br />
46(1): 93-98.<br />
14. Ali S, <strong>An</strong>sari K A, Kabeer and Diwaker G. Nardostachys<br />
jatamansi protects against liver damage induced by<br />
thioacetamide in rats. J Ethnopharmacol 2000; 71(3): 359-<br />
363.<br />
15. Luine V, Martinez C, Villegas M, Magarinos A and McEwen<br />
B S. Restraint stress reversibly enhances spatial memory<br />
per<strong>for</strong>mance. Physiol & Behav 1996; 59(1): 27-32.<br />
21<br />
16. Palacious V G, Marquez R S, Jaime B H, et al. Further<br />
definition of the effect of corticosterone on the sleep wake<br />
pattern in the male rat. Pharmacol. Biochem. Behav 2001;<br />
70: 305-310.<br />
17. Ellman G L, Gourtney D, <strong>An</strong>dres V and Featherstone<br />
R M. A new rapid colorimetric determination of<br />
acetylcholinesterase activity. Biochem Pharmacol 1961;<br />
7: 88-95.<br />
18. Lewis P R, Shute C C and Silver A. Confirmation from<br />
choline acetylase analyses of a massive cholinergic<br />
innervation to the rat hippocampus. J Physiol 1967;<br />
191(1): 215-224.<br />
19. 19. Mellgren S I and Srebro B. Changes in<br />
acetylcholinesterase and distribution of degenerating<br />
fibres in the hippocampal region after septal lesions in the<br />
rat. Brain Res 1973; 52: 19-36.<br />
20. 20. Mizoguchi K, Yuzurihara M, Ishige A, Sasaki H, Tabira<br />
T.Effect of chronic stress on cholinergic transmission<br />
in rat hippocampus. Brain Res 2001; 915(1): 108-1<br />
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22<br />
<strong>Biomedicine</strong>; 2011; 31 (1): 22 - 26<br />
Heart rate reserve – A useful tool <strong>for</strong> evaluation of chest pain in middle aged<br />
*C.P. Ganashree, S M Nataraj, R. Rajalakshmi,Vijaya Y Vageesh<br />
Department of Physiology, JSS Medical College, Mysore - 570015, Karnataka.<br />
* PGIBMS awardee at 31st <strong>An</strong>nual conference of IABMS<br />
(Received 12th November, 2010; Revised 20th December, 2010; Accepted 10th January, 2011)<br />
Corresponding author<br />
Ganashree C P<br />
E-mail: ganaraghu@yahoo.com<br />
Abstract<br />
Background: Chronotropic incompetence (CI) is the inability of heart rate response to meet metabolic<br />
demand. It is an attenuated heart rate response to exercise and is a predictor of all- cause mortality in healthy<br />
populations.<br />
Objectives: To study heart rate reserve and CI in middle aged with non specific chest pain to isotonic<br />
exercise.<br />
Methods: This is a retrospective cross sectional study carried out in the department of Medicine, JSS<br />
hospital. Study group comprised of 43 patients(Males-30 and Females-13) in the age group 40-50 years<br />
with history of nonspecific chest pain but normotensive, nondiabetic and normal resting ECG. Standard<br />
Bruce protocol was followed. Two groups were made-Group A(6 patients with typical angina) and Group<br />
B(37 patients with non typical angina).<br />
Chronotropic incompetence was concluded if derived value is less than 85% of the patient’s heart rate<br />
reserve.<br />
Results: Mean Heart rate reserve was significantly lowered in Group A when compared to Group B (60.4750<br />
vs78.8978, p value
Heart rate reserve in the evaluation of chest pain<br />
events in the next five years than was ST<br />
segment depression(1). Many further studies<br />
have confirmed that chronotropic incompetence<br />
is an independent predictor of risk in patients<br />
with coronary artery disease; although<br />
inferior to myocardial perfusion scanning in<br />
the prediction of cardiac death, it does add<br />
incremental prognostic value to this test(2).<br />
Chronotropic incompetence is also predictive<br />
of adverse cardiac events and total mortality<br />
in apparently healthy individuals even after<br />
adjustment <strong>for</strong> factors such as age, ST segment<br />
shift, physical activity, and traditional coronary<br />
disease risk factors(3). Further confirmation of<br />
the prognostic value of the heart rate response<br />
to exercise has been demonstrated in a study of<br />
about 6000 healthy male Parisian civil servants<br />
who underwent exercise testing between 1967<br />
and 1972(4). A low heart rate response to<br />
exercise proved to be a powerful predictor of<br />
both sudden death and total mortality though<br />
not of death from ‘‘non-sudden myocardial<br />
infarction’’. This finding was particularly<br />
striking as the lack of heart rate response in<br />
these individuals was relative; subjects who<br />
achieved less than 85% of their predicted<br />
maximum peak heart rate were excluded from<br />
the study. Chronotropic incompetence (CI) is<br />
the inability to proportionally increase heart<br />
rate (HR) to meet the increase in metabolic<br />
demand (4). It can refer to the inadequacy of<br />
the sinus node or of the escape pacemaker<br />
in the case of heart block. It can also refer to<br />
the inadequacy of AV nodal regulation of<br />
the supraventricular pacemaker as in atrial<br />
fibrillation. The clinical manifestations of CI<br />
are protean and include the inability to achieve<br />
maximal HR, a delay in achieving maximal<br />
HR, inadequate submaximal or recovering HR<br />
or rate instability with exertion (5,6). It is not<br />
known whether chronotropic incompetence is<br />
independently predictive of all-cause mortality<br />
among patients referred <strong>for</strong> stress testing after<br />
23<br />
accounting <strong>for</strong> myocardial perfusion defects.<br />
The purpose of this study was to examine the<br />
association of chronotropic incompetence with<br />
myocardial perfusion defects and to determine<br />
the ability of chronotropic incompetence to<br />
predict all-cause mortality in a low-risk cohort<br />
of patients referred <strong>for</strong> exercise treadmill test.<br />
Materials and methods:<br />
This is a retrospective cross sectional study<br />
conducted in Treadmill Unit, Department of<br />
Medicine, JSS Hospital, JSS Medical College,<br />
Mysore. Approval from the Ethical committee<br />
was obtained prior to the commencement of the<br />
study.<br />
Subjects:<br />
Study group included Forty three patients<br />
comprising 30 males and 13 females belonging<br />
to the age group of 40-50 years. They came with<br />
a history of non-specific chest pain. They were<br />
normotensive, nondiabetic and had normal<br />
Electrocardiogram (ECG) at rest. Simple<br />
random selection was made. Patients with<br />
evidence of recent myocardial infarction, angina<br />
pectoris of recent onset or with a pronounced<br />
change in the severity or frequency of angina<br />
were excluded from the study. Standard Bruce<br />
Protocol was followed. Subjects were divided<br />
into two groups-Group A (6 patients with<br />
typical angina) and Group B (37patients with<br />
non typical angina).<br />
Clinical data<br />
Be<strong>for</strong>e exercise testing, a structured interview<br />
and chart review yielded data on symptoms,<br />
medications, coronary risk factors, prior cardiac<br />
events and a number of cardiac and noncardiac<br />
diagnosis. Resting hypertension was defined<br />
as a resting systolic blood pressure ≥140 mm<br />
Hg, a resting diastolic blood pressure of ≥90<br />
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Heart rate reserve in the evaluation of chest pain<br />
mm Hg or treatment with antihypertensive<br />
medications(7). Assessment of diabetes was<br />
based on questioning and medication use.<br />
Exercise Testing<br />
Treadmill testing was carried out according to<br />
standard protocols, usually Bruce or modified<br />
Bruce(8,9). To facilitate estimation of exercise<br />
capacity, leaning on handrails during exercise<br />
was explicitly not allowed. During each<br />
stage of exercise, data on symptoms, rhythm,<br />
heart rate, blood pressure (by indirect armcuff<br />
sphygmomanometer), estimated work<br />
load in metabolic equivalents (METs) and ST<br />
segments were collected and entered on-line.<br />
Estimated functional capacity in METs was<br />
estimated from standard published tables(9).<br />
Based on protocol and total time completed in<br />
the final stage; a MET is a measure of oxygen<br />
consumption equal to 3.5 ml/kg/min, which<br />
represents basal, resting metabolic needs. When<br />
ST segments were interpretable, an ischemic<br />
response was considered present if there was<br />
≥1 mm of horizontal or down sloping STsegment<br />
depression of 80 ms after the J-point<br />
or if there was ≥1 mm of additional ST-segment<br />
elevation in leads without pathologic Q waves.<br />
If a patient had more than one treadmill exercise<br />
echocardiogram per<strong>for</strong>med during the study<br />
period, only the first one was considered <strong>for</strong><br />
analysis. Chronotropic incompetence was<br />
first assessed as failure to achieve 85% of the<br />
age-predicted heart rate. This method may be<br />
confounded by effects of age, physical fitness<br />
and resting heart rate, so chronotropic response<br />
was also assessed by calculating the ratio of<br />
heart rate reserve (HRR) used at peak exercise;<br />
this chronotropic index has been described in<br />
detail elsewhere(3,10). In an analogous fashion,<br />
the percent HRR used is:<br />
%HRR used = (HR peak -HR rest ) / (220-age-<br />
HR rest ) x 100.<br />
24<br />
In a group of healthy, non-hospitalized<br />
adults, the ratio of percent HRR used during<br />
exercise was approximate. Thus, chronotropic<br />
incompetence can be defined as a percent HRR<br />
used ratio of
Heart rate reserve in the evaluation of chest pain<br />
Group Statistics<br />
Table 1: Heart rate reserve (in mean±SD) in patients with angina and without angina<br />
<strong>An</strong>gina No.of patients Mean Std. Deviation T-test P value<br />
Positive 6 60.4750 19.26660<br />
Negative 37 78.8978 13.87697<br />
P value
Heart rate reserve in the evaluation of chest pain<br />
capacity, cardiovascular risk factors, and ST<br />
segment depression. Results of this study was<br />
similar to Jouven et al(11) reported that impaired<br />
ability to increase HR during exercise stress<br />
testing is a powerful predictor of the risk of<br />
cardiovascular mortality in 5713 asymptomatic<br />
subjects. However, the underlying mechanisms<br />
are not completely understood.<br />
Chin et al (14) reported that 72% of the patients<br />
with chronotropic incompetence had significant<br />
coronary heart disease. Patterson et al(15).<br />
showed that achieving
27<br />
<strong>Biomedicine</strong>; 2011; 31 (1): 27 - 31<br />
Serum α 1 - antitrypsin level and antioxidant status in smokers with Chronic<br />
Obstructive Pulmonary disease<br />
*S. Venkata Rao , **B.D. Toora, ***V.S.Ravi Kiran and ****S. Indira<br />
*Department of Biochemistry, Katuri Medical College, Chinakondrupadu, Guntur - 522 019,<br />
<strong>An</strong>dhra Pradesh, India.<br />
** Aarupadai Veedu Medical College, Pondicherry ,607402, India.<br />
***Alluri Sitarama Raju Academy of Medical sciences, Eluru-534005, West Godavari Dist.,<br />
<strong>An</strong>dhra Pradesh, India.<br />
**** S.D.M.S.College,Vijayawada - 520010, <strong>An</strong>dhra Pradesh, India.<br />
(Received 12 th August, 2010; Revised 28 th February, 2011; Accepted 8 th March, 2011)<br />
Corresponding author<br />
Dr.S.Venkata Rao,<br />
E-mail: s_vrao11@yahoo.co.in<br />
Abstract<br />
Background & objectives: Smoking induces slow respiratory problems including chronic obstructive<br />
pulmonary disease (COPD). Cigarette smoke contains free radicals and decreases alpha1 antitrypsin levels<br />
in the body. The aim of the present study was to study the relationship between levels of α 1 antitrypsin and<br />
antioxidant status in smokers with COPD.<br />
Methods: A 20 controls without smoking and a total of 80 COPD cases with history of smoking were<br />
studied by spirometry and classified in to four groups according to GOLD. The α 1 antitrypsin, superoxide<br />
dismutase, vitamin E and vitamin C were measured in all the subjects respectively.<br />
Results:Controls had a normal spirometry. Cases showed a decrease in FEV1% with corresponding increase<br />
in the number of cigarettes/day. The α1 antitrypsin, superoxide dismutase, vitamin E and vitamin C were<br />
decreased proportionately with increase in the number of cigarettes/day in cases when compared to the<br />
controls respectively. The difference in the means of each parameter between different case groups and<br />
controls is statistically highly significant.<br />
Interpretation & conclusion:Smoking inhibits α 1 antitrypsin. Cigarette smoke contains free radicals. The<br />
soot attracts neutrophils to the site of which releases more free radicals. Thus there is more elastase and less<br />
protease inhibitor, leading to lung damage. Our study showed decreased α 1 antitrypsin and antioxidant levels<br />
in smokers due to the increased inhibitory and consumption of antioxidants in the body due to increased<br />
production of free radicals respectively.<br />
Introduction<br />
Cigarette smoking is the major global health<br />
hazard and it successfully induces several<br />
respiratory and the major systemic problems.<br />
Cigarette smoke contains more than 5000<br />
different chemicals of which many are oxidants,<br />
including Hydrogen Peroxide, Oxygen free<br />
radical ,Hydroxyl free radical and nitric<br />
oxide(1). Cigarette smoke can be separated into<br />
a gas and a tar phase and both of these contains<br />
abundant oxidants. Iron is present in cigarette<br />
smoke and in the epithelial lining fluid, iron<br />
catalyze the production of free radicals through<br />
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Serum α 1 - antitrypsin in smokers with COPD<br />
the Fenton’s and Haber- weiss reactions. Air<br />
pollutants are another exogenous source of<br />
reactive oxygen species in the lungs (2). Smoking<br />
induced chronic obstructive pulmonary disease<br />
(COPD) is the fourth most common cause of<br />
death in adults (3).The increased oxidative<br />
burden occurs in lungs of patients with chronic<br />
obstructive pulmonary disease and this results<br />
in an imbalance between oxidants and anti<br />
oxidants,(4) leads to pathogenesis of COPD(5)<br />
The other reasons are due to excessive smoking,<br />
occupational hazards, emphysema and air<br />
pollution (6). Oxidative stress is also thought to<br />
play an important role in the diseased aspect of<br />
the lungs with systemic consequences ,such as<br />
muscle dysfunction and weight loss(7).<br />
Global Initiative on Obstructive Lung Disease<br />
(GOLD) guidelines defined smoking induced<br />
COPD as “a disease state characterized by air<br />
flow limitation which is to a greater extent<br />
irreversible. Air flow limitation is progressive<br />
and with an abnormal inflammatory response<br />
of the lungs to noxious particles or gases (8).<br />
In alveolar capillary units the unopposed<br />
actions of proteases and oxidants results in<br />
destruction of the alveoli and Emphysema<br />
appears. Smoking induced COPD comprising a<br />
chronic bronchitis, emphysema, smaller air way<br />
disease. Clinical features are similar exertional<br />
dyspnoea , cough and sputum production in<br />
usually a prolonged period. Pathogenesis is<br />
after cigarette smoke, 90% of all patients with<br />
COPD are smokers, almost 20% of these<br />
smokers develop the condition (9). Persistent<br />
reduction in FEV1 (<strong>for</strong>ced expiratory maneuver)<br />
is the common finding in COPD. Diagnosis is<br />
made by measurement of pulmonary function.<br />
(10). Assessment of FEV1/FVC (FEV1, <strong>for</strong>ced<br />
expiratory volume in one second, FVC, <strong>for</strong>ced<br />
vital capacity ) is the indicator <strong>for</strong> early airway<br />
obstruction. A decline in the pulmonary function<br />
is a good index every year (11). A reduced<br />
FEV1/FVC ratio is characteristic. \<br />
In general the smoking, finally produce<br />
COPD within less than 10 pack /10 years. 1<br />
pack a year is equal to 20 cigarettes/day/1 year.<br />
28<br />
The oxidants are counteracted by enzymatic<br />
antioxidants in airways are superoxide<br />
dismutase, glutathione peroxidase, catalase,<br />
glutathione-S-transferase, xanthin oxidase, etc.<br />
The non-enzymatic antioxidants are Vitamin C,<br />
Vitamin E (Alfa Tocoferol), urate, lipoic acid<br />
and bilirubin(3) RTLF (Respiratory-tract lining<br />
fluid) that covers the respiratory epithelium<br />
contains Vitamin C, reduced glutathione (GSH),<br />
urate, Vitamin E and extracellular superoxide<br />
dismutase (SOD)(9), pulmonary antioxidant<br />
system is excellently adaptive.. Hence the<br />
evaluated parameters in this regard are Alfa 1<br />
<strong>An</strong>titrypsin, SOD, Vitamin E and Vitamin C<br />
in the prognostic study of COPD patients.<br />
Methods<br />
A total of 80 COPD male cases with history of<br />
smoking and 20 age and sex matched healthy<br />
controls with no smoking, attending our hospital<br />
were included in the study. Furthermore 80<br />
COPD cases with history of smoking were<br />
classified in to four groups according to GOLD<br />
and number of cigarettes/day. The study was not<br />
done in the acute phase but in the stable chronic<br />
phase. All subjects were studied by spirometry<br />
and classified into controls and four case groups<br />
according to the Global Initiative <strong>for</strong> Chronic<br />
Obstructive Lung Disease (GOLD)(12) as<br />
follows :<br />
Controls : n = 20, no smoking, normal<br />
spirometry, no risk<br />
Group I : n = 20, 5 – 10 cigarettes/day, FEV1/<br />
FVC ‹ 70%, FEV1 greater than or equal to 80%<br />
predicted, mild COPD.<br />
Group II : n = 20, 11 – 15 cigarettes/day, FEV1/<br />
FVC ‹ 70%, FEV1 greater than or equal to 50%<br />
to ‹ 80% predicted, moderate COPD.<br />
Group III : n = 20, 16 – 20 cigarettes/day, FEV1<br />
greater than or equal to 30% to ‹ 50% predicted,<br />
moderate COPD.<br />
Group IV : n = 20, ≥ 20 cigarettes/day, FEV1/<br />
FVC ‹ 70%, FEV1 ‹ 30% predicted or FEV1 ‹<br />
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Serum α 1 - antitrypsin in smokers with COPD<br />
50%, severe COPD.<br />
The Serum α1 antitrypsin was estimated by<br />
quantitative turbidimetric test kit of Spinreact<br />
Company. The antioxidant status was assessed<br />
by estimating superoxide dimustase,vitamin E,<br />
and vitamin C.<br />
SOD was assayed by the method of Marklund<br />
and Marklund (13) modified by Nandi et al .<br />
(14). This method is based on the ability of<br />
SOD to inhibit autoxidation of pyrogallol under<br />
specific conditions. Reading was taken at 420<br />
nm and expressed as units/ml.<br />
Vitamin E was estimated in serum by the method<br />
of Baker and Frank (15), which is based on the<br />
Table 1.<br />
29<br />
reduction of ferric to ferrous ions by tocopherol,<br />
which then <strong>for</strong>ms a red colored complex with<br />
2,2′,bipyridyl that is read at 520 nm. The level<br />
of vitamin E was expressed as mg/l serum.<br />
Plasma VitaminC was estimated by Aye<br />
Kyaw’s Method(16) where phosphotungstic<br />
acid first deprotienize the plasma and then react<br />
with ascorbic acid to produce blue color.<br />
Statistical analysis<br />
Statistical analysis of data of alpha1 antitrypsin and antioxidant parameters<br />
Statistical analysis was done using SalStat<br />
statistical software. ANOVA test was used<br />
to compare the means between controls and<br />
different case groups at 5% level of significance<br />
Group Control Group-I Group-II Group-III Group-IV p – value<br />
No. of cigarettes/day<br />
Nil 5 – 10 11 – 15 16 – 20 ≥20<br />
n 20 20 20 20 20<br />
Forced<br />
expiratory<br />
volume<br />
(FEV1%)<br />
α1 <strong>An</strong>titrypsin<br />
(mg/dl)<br />
Superoxide<br />
dismutase (U/<br />
ml)<br />
Vitamin C<br />
(Ascorbic<br />
acid) (mg/dl)<br />
Vitamin E<br />
(tocopherol)<br />
(mg/dl)<br />
80.20000<br />
±<br />
3.33404<br />
160.15000<br />
±<br />
22.96972<br />
3.32400<br />
±<br />
0.25270<br />
0.40100<br />
±<br />
0.04471<br />
1.52250<br />
±<br />
0.27376<br />
(Values are expressed in mean ± SD)<br />
65.75000<br />
±<br />
7.35473<br />
85.10000<br />
±<br />
3.44735<br />
3.02000<br />
±<br />
0.57546<br />
0.32500<br />
±<br />
0.05296<br />
1.26450<br />
±<br />
0.32806<br />
63.75000<br />
±<br />
10.06231<br />
72.30000<br />
±<br />
3.77108<br />
2.29300<br />
±<br />
0.29491<br />
0.30200<br />
±<br />
0.06387<br />
1.19600<br />
±<br />
0.22526<br />
38.10000<br />
±<br />
3.87842<br />
36.90000<br />
±<br />
5.91964<br />
2.12850<br />
±<br />
0.40081<br />
0.24200<br />
±<br />
0.04396<br />
0.77700<br />
±<br />
0.09200<br />
22.80000<br />
±<br />
2.33057<br />
45.75000<br />
±<br />
8.76521<br />
1.69500<br />
±<br />
0.22130<br />
0.20800<br />
±<br />
0.03270<br />
0.50100<br />
±<br />
0.10642<br />
0.0000001<br />
0.0000001<br />
0.0000001<br />
0.0000001<br />
0.0000001<br />
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Discussion<br />
In table-1, the values are expressed in terms of<br />
mean ± SD and One-way analysis of variance<br />
(ANOVA) was used to compare the means<br />
between controls and different cases groups<br />
respectively at 5% level of significance. The α1<br />
<strong>An</strong>titrypsin was decreased proportionately with<br />
increasing number cigarettes per day as shown<br />
in the table-1. The difference in the means<br />
between controls and different case groups is<br />
statistically highly significant as p = 0.0000001<br />
as shown in table-1. The antioxidant parameters<br />
like superoxide dismutase, vitamin E and<br />
vitamin C were also decreased proportionately<br />
with increasing number cigarettes per day<br />
respectively as shown in the table-1. The<br />
difference in the means of each parameter<br />
between controls and different case groups was<br />
statistically highly significant as p = 0.0000001<br />
as shown in the table-1.<br />
Conclusion<br />
Our study shows, depending on the severity<br />
of smoking, α1 <strong>An</strong>titrypsin and antioxidant<br />
parameters like superoxide dismutase, vitamin<br />
E and vitamin C were found to be decreased<br />
proportionately . The α1 <strong>An</strong>titrypsin as it<br />
is an acute phase protein, it may increase<br />
in the early phase but later it decreases<br />
respectively. However, the α1 <strong>An</strong>titrypsin<br />
level was studied during stable chronic phase<br />
of COPD. Our findings had a correlations<br />
with other similar studies like Somayajulu<br />
G.L: et al., (17) and Sayyad et al(18)<br />
.<br />
Smoking oxidizes the Methionine (358-residue)<br />
of alpha -1 antitrypsin and thus inactivates<br />
the protein. Hence such alpha-1 antitrypsin<br />
molecule can’t bind to the protease ‘active<br />
elastase’ and thus proteolysis of lung and tissue<br />
damage occurs accelerating the development of<br />
Emphysema. Smoking inhibits α1 antitrypsin.<br />
Cigarette smoke contains free radicals. The<br />
soot attracts neutrophils to the site of which<br />
30<br />
releases more free radicals. Thus there is more<br />
elastase and less protease inhibitor, leading<br />
to lung damage. Our study showed decreased<br />
antioxidant levels in smokers due to the<br />
increased consumption of antioxidants in the<br />
body which in turn due to increased production<br />
of free radicals respectively.<br />
Our study reveals that, along with specific<br />
treatment, the COPD cases should be treated with<br />
naturally occurring antioxidants like vitamin E,<br />
vitamin C and beta carotene respectively. This is<br />
important because, there is increased oxidative<br />
stress, decreased antioxidants and decreased<br />
alpha-1 antirypsin activity in smokers with<br />
COPD cases.<br />
Smoking cessation is the single most effective<br />
and cost-efficient intervention <strong>for</strong> reducing the<br />
risk of COPD and stopping its progression,<br />
according to the guidelines. “Even a brief, threeminute<br />
period of counseling to urge a smoker to<br />
quit can be effective, and at the very least this<br />
should be done <strong>for</strong> every smoker at every visit,”<br />
they stress.<br />
References<br />
1. Pryor W A .,Stone K.Oxidants in cigarette<br />
smoke;Radicals,Hydrogen peroxide,Peroxy nitrate, and<br />
Peroxy nitrite,<strong>An</strong>n N Y Acad Science1993.:686;12-<br />
27;(Medicine).<br />
2. Macnee W,Donaldson K,.Environmental factors in COPD.<br />
Voelkel N F ,Mac Nee W.eds.Chronic Obstructive Lung<br />
Disease. London.B C Deekar.,2002;pp.145-160.<br />
3. Maria Szilasi, Tamas D, Zoltan N, Janos S. Pathology of<br />
chronic obstructive pulmonary disease. Pathology Oncology<br />
Research Vol.12, No.1, 2006.<br />
4. Halliwell B . <strong>An</strong>tioxidants in human health and disease.<br />
<strong>An</strong>nu Rev Nutr 1996;16:33-50. CrossRef<br />
5. McNee W. Oxidants/antioxidants and COPD. Chest<br />
2000; 117 5: Suppl. 1, S303–S317.<br />
6. Rogerio RJ, R Lapa E S,Cellular and biochemical bases of<br />
chronic obstructive pulmonary disease. Journ.Bras.Pneumol.2006;32(3):241-8.<br />
7. Langen R C, Korn S H, Wouters E F.ROS in local and<br />
systemic pathogenesis of COPD, Free Radi Biol Med<br />
2003:35:226-235.(Cross ref)(Medicine).<br />
8. Barnes, PJ, Shapiro, SD, Pauwels, RA Chronic obstruc-<br />
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tive pulmonary disease:molecular and cellular mechanisms.<br />
Eur Respir J 2003;22,672-688<br />
9. Frank J.Kelley. Vitamins and respiratory disease: anti<br />
oxidant micronutrients in pulmonary health and disease.<br />
Proceedings of the Nutrition Society,2005, 64,510-526.<br />
10.Kasper Braunwald, Fauci, Harrison Principles<br />
of Internal Medicine,16 th edi,P.No.1548.<br />
11.John E Repine A B,Ida Lankhorst.Oxidative stress in<br />
Chronic Obstructive Pulmonary Disease. Am J Resp Crit<br />
Care Med Vol.156.pp.341-357,1997.<br />
10. The GOLD Workshop Panel. Global Strategy <strong>for</strong> the Diagnosis,<br />
Management, and Prevention of Chronic Obstructive<br />
Pulmonary Disease: NHLBI/ WHO Workshop Report.<br />
Bethesda, Md: National Heart, Lung, and Blood Institute;<br />
2001. NIH Publication No. 2701.<br />
11. Marklund S, Marklund G (1974): Involvement of the superoxide<br />
anion radical in the autoxidation of pyrogallol<br />
and a convenient assay <strong>for</strong> superoxide dismutase, Eur J<br />
31<br />
Biochem, 47: 469-74.<br />
12. Nishal HK, Sharma MP, Goyal RK, Kaushik GG (1998):<br />
Serum superoxide dismutase levels in diabetes mellitus<br />
with or without microangiopathic complications, JAPI,<br />
46(10): 853-5.<br />
13. Baker H, Frank O. Tocopherol. In: Clinical vitaminology,<br />
methods and interpretation, New York Inter Science Publisher,<br />
John Wiley and Sons, Inc. 1968;172–3<br />
14. Aye Kyaw. A simple colorimetric method <strong>for</strong> ascorbic<br />
acid determination in blood plasma.Clin Chim Acta 1978;<br />
Vol.86: 153-7.<br />
15. Somayajulu, G.L; Raja Rao D, Reddy P.P. Serum<br />
α1antitrypsin in smokers and non smokers. Indian Jour of<br />
Clinical Biochemistry 1996/11 70-72<br />
16. A.K.Sayyed, K.H.Deshpande, A.N. Suryakar, R.D, <strong>An</strong>kush<br />
and R.V Katkam. Oxidative Stress and serum<br />
α1antitrypsin in smokers. Indian Jour of Clinical Biochemistry<br />
2008/23(4) 375-377.<br />
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32<br />
<strong>Biomedicine</strong>; 2011; 31 (1): 31 - 38<br />
Cardiovascular autonomic function test responses in patients with diabetes<br />
mellitus and non diabetics.<br />
Mamatha C.N. Ravipati Sarath<br />
Department of physiology.Hassan Intitute of Medical Sceinces, Hassan, Karnataka, India.<br />
Department of physiology. School of Medical <strong>Sciences</strong>,<br />
University Of Hyderabad, <strong>An</strong>dhra Pradesh, India.<br />
(Received 1 st November 2010; Revised 18 th January 2011; Accepted 30 th January 2011)<br />
Corresponding Author<br />
Dr.Mamatha<br />
E-Mail: mamathramesh78@gmail.com<br />
Abstract<br />
Introduction: The present study is designed with the objective of investigating the relation between cardiovascular<br />
autonomic function and diabetes mellitus.Diabetic autonomic neuropathy (DAN) is a serious and<br />
common complication of diabetes and well studied <strong>for</strong>m of DAN.<br />
Materials and Methods: A total 60 diabetic patients who belong to age group 40 – 60 years attending the<br />
diabetic clinic and medical out patient department are selected as test group. 30 non – diabetic patients<br />
matched <strong>for</strong> age and sex as that of test group, attending medical OPD <strong>for</strong> other complaints in the J.S.S.<br />
Hospital, Mysore, are selected as control group. After thorough examination of the subjects as per the<br />
pro<strong>for</strong>ma, the standard autonomic function tests based on cardiovascular reflexes were per<strong>for</strong>med. ECG<br />
and heart rate were recorded at baseline. Deep breathing test ,heart rate variation to Valsalva manoeuvre,<br />
heart rate response to standing ,blood pressure response from supine to standing were per<strong>for</strong>med to assess<br />
parasympathetic functions of the heart. Blood pressure response to sustained handgrip test is per<strong>for</strong>med to<br />
assess sympathetic function.<br />
Results and Discussion: The results obtained were treated statistically by appropriate methods and compared<br />
between the groups.There was a statistically significant reduction in the test group <strong>for</strong> all the cardiovascular<br />
autonomic functions. Both parasympathetic and sympathetic cardiovascular responses were<br />
significantly decreased in diabetes compared to age-matched healthy controls.<br />
Conclussion: Study has been conluded with the association of Cardiovascular autonomic neuropathy in<br />
patients with diabetes as the disease progresses affecting both sympathetic and parasympathetic components.<br />
keywords: Diabetes mellitus; cardiovascular autonomic neuropathy(CAN); cardiovascular reflex tests.<br />
Introduction:<br />
Diabetes mellitus (DM) is a metabolic disorder<br />
characterized by hyperglycemia due to absolute<br />
or relative deficiency of insulin. The increased<br />
morbidity and mortality is due to its complications<br />
like neuropathy, nephropathy, and retinop-<br />
athy etc, especially seen in 40 to 60 years age<br />
group. Diabetic autonomic neuropathy (DAN)<br />
is a serious and common complication of diabetes.<br />
DAN may affect many system throughout<br />
the body Eg: CVS, GIT, Genitourinary system.<br />
Because of its association with a variety of adverse<br />
outcomes including cardiovascular deaths<br />
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Cardiovascular autonomic function test responses in patients in diabetes mellitus<br />
due to silent myocardial ischemia, cardiovascular<br />
autonomic neuropathy (CAN) is the most<br />
critically important and well studied <strong>for</strong>m of<br />
DAN. One of the studies showed CAN results<br />
from damage to the autonomic nerve fiber that<br />
innervate the heart and blood vessels and results<br />
in abnormalities that is heart controle and vascular<br />
dynamics (1).<br />
Previous studies showed that long term poor<br />
glycemic controle can only increase the risk of<br />
developing advanced diabetic neuropathy(2).<br />
But present studies have confirmed the presence<br />
of autonomic neuropathy at presentation<br />
itself (3) . Some manifestations of autonomic<br />
neuropathy may even precede the diagnosis of<br />
diabetes by several years (4).So these evidences<br />
supports that all patients with diabetes regardless<br />
of metabolic controle are at risk <strong>for</strong> autonomic<br />
neuropathy.<br />
In that CAN,a common <strong>for</strong>m of autonomic<br />
dysfunction found in patients with diabetes<br />
mellitus. Cardiovascular disease is the leading<br />
cause of death among patients with diabetes.<br />
The presence of cardiovascular autonomic neuropathy<br />
has been associated with substantially<br />
increased risk of cardiovascular mortality in diabetes.<br />
Quantitative cardiovascular autonomic<br />
function tests are widely used to detect, verify<br />
and quantify the cardiovascular autonomic dysfunction.<br />
The determination of the presence of CAN is<br />
usually based on a battery of autonomic function<br />
tests rather than just on one test. A number<br />
of autonomic function tests considered reliable,<br />
reproducible, simple and quick to carry out and<br />
all of them are non – invasive. Despite its relationship<br />
to an increased risk of cardiovascular<br />
mortality and its association with multiple<br />
symptoms and impairments, the significance<br />
of DAN has not been fully appreciated. So the<br />
present study is undertaken to assess the severity<br />
of adverse effects of diabetes on autonomic<br />
functions of CVS which helps in early detection<br />
of CAN in asymptomatic diabetic and there by<br />
promotes timely diagnostic and therapeutic intervention.<br />
Materials and Methods: A total of<br />
33<br />
60 diabetic patients who belong to age group<br />
40 – 60 years attending the diabetic clinic and<br />
medical out patient department in the J.S.S<br />
Hospital, Mysore, are selected as test group. 30<br />
non – diabetic patients matched <strong>for</strong> age and sex<br />
as that of test group, attending medical OPD <strong>for</strong><br />
other complaints in the J.S.S. Hospital, Mysore,<br />
are selected as control group.<br />
Inclusion criteria <strong>for</strong> study group Cases of already<br />
diagnosed diabetes mellitus since 5<br />
years who are full filling WHO criteria’s are selected<br />
as test group.<br />
Cases who are in the age group of 40 – 60 years<br />
are selected. Exclusion criteria <strong>for</strong> study group<br />
Non-diabetic patients are excluded from the<br />
study<br />
Patients taking medications other than oral hypoglycemics<br />
that could influence the autonomic<br />
functions and drugs those could affect the cardiovascular<br />
functions are excluded. Disease<br />
states like alcoholic neuropathy that could affect<br />
autonomic functions are excluded. Patients<br />
suffering from cardiac disorders are excluded.<br />
Inclusion criteria <strong>for</strong> control groups Subject<br />
must be non-diabetic Subjects matched <strong>for</strong> age,<br />
sex as that of test group are included as controls.<br />
Subjects attending medicine out patient department<br />
<strong>for</strong> various complaints at J.S.S. Hospital<br />
are included.Exclusion criteria <strong>for</strong> control<br />
group Already diagnosed diabetic patients are<br />
excluded Subjects taking medication that could<br />
affect cardiovascular functions and those who<br />
are suffering from cardiac disorders are excluded<br />
as controle group.<br />
Methods<br />
The subjects were instructed not to have coffee,<br />
tea, cola 12 hours be<strong>for</strong>e the tests and were<br />
asked to have light breakfast two hours be<strong>for</strong>e<br />
the tests. The subject was asked to relax in supine<br />
position <strong>for</strong> 30 minutes. The resting heart<br />
rate was recorded on a standard ECG from lead<br />
II, at a paper speed of 25 mm/sec. BP was measured<br />
with sphygmomanometer by the standard<br />
auscultatory RivaRocci method. The cardiovas-<br />
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Cardiovascular autonomic function test responses in patients in diabetes mellitus<br />
cular tests per<strong>for</strong>med are detailed below in the<br />
order of execution. These tests were demonstrated<br />
to the subjects.<br />
Materials:<br />
Autonomic function tests will be carried out by<br />
using Electro cardiograph Sphygmomanometer<br />
Hand grip dynamometer Procedure of autonomic<br />
evaluation. In the early 1970’s five simple non<br />
invasive cardiovascular reflex tests were proposed<br />
that is valsalva manoeuver, HR response<br />
to deep breathing, HR response to standing up<br />
, BP response to standing up are per<strong>for</strong>med to<br />
assess the parasympathetic and BP response to<br />
sustained handgrip to assess the sympathetic<br />
function have been widely used in a variety of<br />
studies(5).<br />
1.Deep breathing test:<br />
In the sitting position subject was asked to<br />
breath quietly and deeply at the rate of 6 breaths<br />
per minute. A continuous ECG was recorded <strong>for</strong><br />
six cycles with marker to indicate the onset of<br />
each inspiration and expiration. The maximum<br />
and minimum R-R intervals were measured<br />
during each breathing cycles and converted to<br />
beats per minute. The result was then expressed<br />
as mean of the difference between maximum<br />
and minimum heart rate <strong>for</strong> six measured cycles<br />
in beats per minute(6).Deep breathing<br />
difference(DBD)=mean of heart rate differences<br />
in 6 breath cycles.A normal response is a<br />
difference of 15 beats/min or more,11-14 beats/<br />
min borderline and less than 10 beats/min was<br />
considered abnormal.<br />
2.Heart-Rate variation to Valsalva Manoeuver:<br />
The subject was seated com<strong>for</strong>tably and was<br />
asked to blow into a mouthpiece connected to a<br />
mercury sphygmomanometer and holding it at<br />
a pressure of 40 mm of mercury <strong>for</strong> 15 seconds,<br />
while a continuous ECG was being recorded.<br />
34<br />
The ECG was continued to be recorded after release<br />
of pressure at the end of 15 seconds <strong>for</strong> 30<br />
seconds. The heart rate changes induced by the<br />
valsalva manoeuver was expressed as the ratio<br />
of the maximal tachycardia during the manoeuver<br />
to the maximal bradycardia after the manoeuver.<br />
This ratio was defined as the Valsalva<br />
ratio and was calculated as the ratio of maximum<br />
R-R interval after the manoeuver to minimum<br />
R-R interval during the manoeuvre(7).<br />
Valsalva ratio(VR)= maximal tachycardia/maximum<br />
bradycardia= maximum R-R<br />
interval/minimum R-R interval.<br />
A value of 1.10 or less is defined as an<br />
abnormal response, 1.11-1.20 as borderline, and<br />
1.21 or more as a normal response(8).<br />
3. Heart rate response to standing - Postural<br />
Tachycardia Index (PTI) :<br />
The subjects were asked to lie on the examination<br />
table quietly while heart rate is being recorded<br />
on ECG. They were then asked to standup<br />
unaided and ECG was recorded <strong>for</strong> 1 minute.<br />
The shortest R-R interval at or around 15th beat<br />
and longest R-R interval at or around 30th beat<br />
was measured. The result was expressed as ratio<br />
of 30/15.<br />
PTI= Longest R-R interval at 30th beat / shortest<br />
R-R at 15th beat. A ratio of 1.00 or less was<br />
defined as an abnormal response, 1.01-1.03 as<br />
borderline and 1.04 as normal response(9).<br />
4. Blood Pressure Response To Standing<br />
(Orthostatic test):<br />
The subject was asked to rest in a supine position<br />
<strong>for</strong> 5 minutes. The resting BP was recorded.<br />
The subject was then asked to stand unaided<br />
and remain standing unsupported <strong>for</strong> 3 minutes.<br />
The BP was recorded at 30 seconds and 3 minutes<br />
after standing up. The difference between<br />
the resting and standing BP levels was calculated.<br />
The fall in systolic BP at 30 seconds on<br />
standing noted. A fall of 30 mm Hg or more was<br />
defined as abnormal, fall between 11-29 mm Hg<br />
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Cardiovascular autonomic function test responses in patients in diabetes mellitus<br />
Table: Cardiovascular autonomic function tests:<br />
Results<br />
Heart Rate response tests<br />
(Parasympathetic function)<br />
Heart rate response to Valsalva<br />
Manoeuver (VR)<br />
Heart rate response during deep<br />
breathing (DBD)<br />
Table 1 : Comparison of DBD in test and control group and results of T-test : values mentioned are mean and<br />
SD in each group.<br />
Group Mean ± SD T P<br />
Test 12.36 ± 4.79<br />
Control 14.96 ± 5.51<br />
-2.306 0.023<br />
Heart rate response to deep breathing were analysed between the two groups. DBD was found to be significantly lesser in the test group when<br />
compared to control group (P < 0.023).<br />
Table 2 : Comparison of VR in test and control group and results of T-test : values<br />
mentioned are mean and SD in each group.<br />
Group Mean ± SD t P<br />
Test 1.16 ± 0.77<br />
Control 1.25 ± 0.98<br />
-4.365 0.00<br />
Heart rate response to valsalva manoeuver were analysed between the two groups. VR was found to be significantly lesser in the test group when<br />
compared to control group (P < 0.00).<br />
Table 3 : Comparison of PTI in test and control group and results of T-test :<br />
values mentioned are mean and SD in each group.<br />
Group Mean ± SD t P<br />
Test 1.02 ± 0.12<br />
Control 1.08 ± 0.87<br />
-2.57 0.01<br />
Heart rate response to PTI were analysed between the two groups. DBD was found to be significantly lesser in the test group when compared to<br />
control group (P< 0.01).<br />
Normal Borderline Abnormal<br />
1.21 or more 1.11-1.20 1.10 or less<br />
15 beats/min<br />
Or more<br />
11-14<br />
beats/min<br />
10 beats/min<br />
or less.<br />
Heart rat response t standing (PTI) 1.04 or more 1.01-1.03 1.00 or less<br />
Blood Pressure (BP) Reponse<br />
tests (sympathetic function)<br />
BP response to standing<br />
(fall in systolic BP)<br />
BP response to sustained hand<br />
grip (rise in diastolic BP)<br />
10 mm Hg<br />
Or less<br />
16 mm Hg<br />
Or more<br />
11-29 mm Hg<br />
11 - 15 mm Hg<br />
30 mm Hg<br />
Or more<br />
10 mm Hg<br />
Or less<br />
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35
Cardiovascular autonomic function test responses in patients in diabetes mellitus<br />
Table 4 : Comparison of fall in SBP (Orthostatic test) in test and control group and results of T-test : values<br />
mentioned are mean and SD in each group.<br />
Group Mean ± SD T P<br />
Test 9.20 ± 4.02<br />
Control 17.73 ± 6.41<br />
as borderline and fall of 10 mm Hg or less was<br />
considered norma(10).<br />
5.Blood Pressure Response To Sustained<br />
Handgrip:<br />
In this test, sustained muscle contraction is<br />
measured by a handgrip dynamometer, causes a<br />
rise in systolic and diastolic blood pressure and<br />
heart rate. The dynamometer is first squeezed<br />
to isometric maximum , then held at 30% maximum<br />
<strong>for</strong> 5 min. if possible, although even 3<br />
minutes may be adequate. Blood pressure was<br />
recorded in the non exercising arm thrice at<br />
1-minute interval during the procedure. The<br />
maximum reading of the diastolic blood pressure<br />
was taken as the final value. Then the<br />
rise in diastolic blood pressure was calculated<br />
by subtracting resting diastolic blood pressure<br />
from this value. A rise in DBP of less than 10<br />
mm Hg was defined as abnormal, 11-15 mm Hg<br />
as borderline and 16 mm Hg or as normal(11).<br />
Statistical <strong>An</strong>alysis of Data:<br />
Following statistical methods were employed<br />
in the present study:<br />
1. Contingency coefficient test: The Crosstabs<br />
procedure <strong>for</strong>ms two-way and multiway<br />
tables and provides a variety of tests and<br />
36<br />
measures of association <strong>for</strong> two-way tables.<br />
The structure of the table and whether categories<br />
are ordered determine what test or<br />
measure to use.<br />
2. Univariate ANOVA:The Univariate procedure<br />
provides analysis of variance <strong>for</strong> one<br />
dependent variable by one or more factors<br />
and/or variables. You can investigate<br />
interactions between factors as well as the<br />
effects of individual factors, some of which<br />
may be random.Independent samples ‘t’<br />
test:The Independent-Samples T Test procedure<br />
compares means <strong>for</strong> two groups of<br />
cases.<br />
Discussion:<br />
6.65 0.00<br />
Blood pressure response to standing were evaluated between the two groups. There was a significant decline in orthostatic test in test group (P <<br />
0.00).<br />
Table 5 : Comparison of rise in DBP in test and control group and results of T-test: values mentioned are<br />
mean and SD in each group.<br />
Group Mean ± SD T P<br />
Test 12.76 ± 4.80<br />
Control 18.20 ± 3.41<br />
-5.52 0.00<br />
Blood pressure (rise in DBP) response to sustained hand grip was evaluated between test group and control group. Blood pressure response to<br />
sustained hand grip was found to be significantly reduced in test group (P < 0.00).<br />
Diabetes mellitus is a strong risk factor <strong>for</strong><br />
cardiovascular disease. DAN is a risk factor<br />
that independently increases cardiovascular<br />
risk in people with diabetes mellitus. Various<br />
mechanisms are included in the impairment<br />
of autonomic function which leads to exercise<br />
intolerance, orthostatic hypotension, painless<br />
myocardial ischemia and increased risk of<br />
mortality. Alternatively, the metabolic and<br />
vascular changes associated with diabetes eg:<br />
dysfunction of the vascular endothelium may<br />
adversely affect cardiovascular autonomic<br />
function.<br />
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Cardiovascular autonomic function test responses in patients in diabetes mellitus<br />
Cardiovascular autonomic function tests are<br />
widely used to detect, verify and quantify the<br />
cardiovascular autonomic dysfunction. They<br />
have been tested <strong>for</strong> their validity and reliability.<br />
Against this background, the present study<br />
was undertaken to evaluate cardiovascular<br />
autonomic function tests in diabetes and match<br />
the data so obtained with healthy non diabetic<br />
individuals as controls.<br />
Heart rate response to deep breathing:-<br />
This evaluated in 90 subjects, 60 of them<br />
diabetics comprising the test group and 30 were<br />
non diabetic patients as control group. The<br />
mean and standard deviation of test group and<br />
control group are shown in Table 1. The mean<br />
and standard deviation of test group and control<br />
group were 12.36 ± 4.79 and 14.96 ± 5.51<br />
respectively.<br />
DBD was significantly reduced in test group<br />
when compared to control group (P
Cardiovascular autonomic function test responses in patients in diabetes mellitus<br />
Blood Pressure response to sustained<br />
handgrip<br />
. The mean and standard deviation of test<br />
group and control group are shown in Table<br />
5. The mean and standard deviation of test<br />
group and control group were 12.76 ± 4.80<br />
and 18.20 ± 3.41 respectively There was a<br />
significant decline in sustained handgrip test<br />
in test group when compared to control. (P <<br />
0.00). In a study it was found that in patients<br />
with type 2 diabetes showed a decline in<br />
isometric exercise induced increase in heart<br />
rate and blood pressure response in subjects<br />
with CAN(14). The main finding of this study<br />
is that, decreased Heart rate variability and<br />
blunted blood pressure responses, as measured<br />
by cardiovascular testing, is associated with<br />
diabetic patients. Hence, it is suggested that<br />
cardiovascular autonomic functions decline in<br />
diabetic patients as the disease progresses.<br />
Conclusion:<br />
Diabetic autonomic neuropathy is a<br />
heterogenous condition that comprises a wide<br />
range of dysfunction and whose development<br />
might be attributed to diabetes per se or to factors<br />
associated with the disease. Both sympathetic<br />
and parasympathetic fibres may be affected<br />
with parasympathetic dysfunction preceding<br />
sympathetic dysfunction.The etio pathology<br />
of Diabetic Autonomic Neuropathy is multifactorial.It<br />
has been postulated that metabolic<br />
consequences of hyperglycemia rather than<br />
the type of diabetes may lead to autonomic<br />
damage. One factor of particular interest is<br />
oxidative stress. This occurs when there is<br />
an increase within cells of certain reactive<br />
molecules containing oxygen. These can react<br />
with components of cells, including nerves,<br />
and cause damage. Alternatively, the metabolic<br />
and vascular changes associated with diabetes<br />
38<br />
may adversely affect almost all the organs of the<br />
body, particularly the cardiovascular system.<br />
References:<br />
1. Schumer MP, Joyner SA, Pfeifer MA. Cardiovascular au- autonomic<br />
neuropathy testing in patients with diabetes. Diabetes<br />
spectrum 1998; 11: 227-231.<br />
2. mustonen J, Vusitipa M, Mantysaari M. Changes on autonomic<br />
nervous function during the 4-year follow-up in<br />
middle-aged diabetic and non-diabetic subjects initially<br />
free of coronary heart disease. J. Intern. Med – 241 1997;<br />
227-235.<br />
3. Gries FA, Spuler M, Lessmann F. Diabetic cardiovascular<br />
autonomic neuropathy multicenter study group, The epidemiology<br />
of diabetic neuropathy. J. Diabetes complications<br />
1992; 6: 49-57.<br />
4. Wcin TH, Albers JW. Diabetic neuropathies. Phys Med<br />
Rehabil Chin N Am. 2001; 12: 307-320.<br />
5. Ewing DJ, Martyn CN, Young RJ, Clarje BF. The value of<br />
cardiovascular autonomic function test, 10 years experience<br />
in diabetes. Diabetes case 1985; 491-498.<br />
6. Piha SJ. Cardiovascular autonomic function tests. Responses<br />
in healthy subjects and determination of age related<br />
reference value. Rehab research centre 1988;1-148.<br />
7. Hirsch JA, Bishop B. Respiratory sinus arrhythmia in humans:<br />
how breathing pattern modulates heart rate. Am J<br />
Physiol 1981;241:H620-H629.<br />
8. Levin AB. A Simple test of cardiac function based upon<br />
the heart changes induced by valsalva manoeuver. Am J<br />
Cardiol 1966;18:90-99.<br />
9. Ewing DJ, Hume L, Campbell IW, et al. Autonomic mechanism<br />
in initial heart rate response to standing. J Appl<br />
Physiol 1980;49:809-814.<br />
10. Piha SJ. Cardiovascular responses to various autonomic<br />
tests in males and females. Clin Auton Res 1993;3(1):15-<br />
20.<br />
11. Piha SJ. Cardiovascular autonomic reflex tests: Normal<br />
responses and age related reference values. Clin Physiol<br />
1991;11:277-290.<br />
12. Moran A, Palmas W, Field L. et al. Cardiovascular autonomic<br />
neuropathy is associated with microalbuminuria in<br />
older patients with type2 diabetes. The American Diabetes<br />
Association. Diabetes Care 2004;27:972-977.<br />
13. Purewal TS, Watkins PJ. Postural hypotension in diabetic<br />
autonomic neuropathy: a review. Diabet Med<br />
1995;12:192–200..<br />
14. Kahn J, Zola B, Juni J, Vinik AI. Decreased exercise heart<br />
rate in diabetic subjects with cardiac autonomic neuropathy.<br />
Diabetes care 1986; 9: 389-394.<br />
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39<br />
<strong>Biomedicine</strong>; 2011; 31 (1): 39 - 44<br />
Association between Serum Lipoprotein(a) Concentrations and Serum<br />
Triglycerides in Type 2 Diabetes Mellitus.<br />
*T. Sharmila krishna, *J.N.Naidu, *M. Audhisesha Reddy, *K.Ramalingam,<br />
**E. Venkat Rao.<br />
*Department of Biochemistry and ** Department of Community Medicine , Narayana Medical<br />
College , Nellore, <strong>An</strong>dhra Pradesh, India.<br />
(Received 5 th October 2010; Revised 7 th December 2010; Accepted 10 th January 2011)<br />
Corresponding Author<br />
Dr. T.Sharmila Krishna<br />
Email: sharmilakrishna01@yahoo.in<br />
Abstract<br />
Introduction: Diabetes mellitus is a chronic metabolic disorder that is often associated with dyslipidemia,<br />
leading to cardiovascular complications. Studies have shown Lipoprotein(a) [Lp (a)] as a new independent<br />
risk factor <strong>for</strong> coronary artery disease . Concentrations of Lp(a) among diabetics remains controversial.<br />
Objectives: This study was aimed to evaluate the Lp(a) levels and its relation with other serum lipids<br />
among type 2 diabetics, first degree relatives (FDRs) with a family history of type 2 diabetes mellitus and<br />
controls.<br />
Materials <strong>An</strong>d Methods: A total of 55 subjects, both males and females were studied (type 2 diabetics n<br />
= 21, FDRs n=19 and controls n = 15). Type 2 diabetics were further sub-grouped into diabetics with < 5<br />
years and those with ≥ 5 years duration. FBS, PPBS, total cholesterol, triglycerides, LDL-c , HDL-c were<br />
estimated by Spectrophotometry. Lipoprotein(a) and HbA1c were estimated by Immnunoturbidimetry.<br />
Results: The mean Lp(a) levels were lower among type 2 diabetics and FDRs compared to controls,<br />
though it was statistically not significant. A significant negative correlation (p value < 0.007) was observed<br />
between Lp(a) and serum triglycerides concentrations among diabetics of ≥ 5 years duration.<br />
Conclusion: Lp(a) concentrations were negatively correlated with triglyceride levels in type 2 diabetics.<br />
There<strong>for</strong>e, our results suggest that the treatment of diabetic dyslipidemia may indirectly affect Lp(a) concentrations.<br />
So, care must be taken while treating dyslipidemia in type 2 diabetics.<br />
Key words : Lipoprotein (a) , Triglycerides , Type 2 diabetes mellitus.<br />
Introduction:<br />
Lipoprotein (a) was first identified by Karl Berg<br />
in 1963 (1). It consists of an LDL-like particle<br />
and the unique glycoprotein, apolipoprotein (a)<br />
[apo (a)] that is covalently linked to apo-B 100<br />
via a disulfide linkage (2). Elevated levels of Lp<br />
(a) are found to be an independent risk factor <strong>for</strong><br />
Coronary artery disease in NIDDM (3,4,5). The<br />
apolipoprotein (a) of Lp(a) shares high sequence<br />
homology (75 – 90% ) with plasminogen,<br />
suggesting Lp(a) inhibits fibrinolysis and<br />
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Serum LP ‘a’ and TG in Diabetes Mellitus<br />
contribute to the thrombotic phenomenon.<br />
Further, LDL-like structure contributes to<br />
atherogenicity leading to Ischemic heart disease<br />
(6, 7). Studies regarding levels of Lp(a) in type<br />
2 diabetics still remains controversial, some<br />
reporting higher levels ( 8,9,10,11 ), some lower<br />
(12, 13), while few have shown no significant<br />
change (14, 15 ,16). Variations in Lp (a) levels<br />
are mainly under genetic control (17). Various<br />
other factors like ethnicity, diet, drugs and<br />
hormones also affect serum Lp (a) levels (18,19,<br />
20 , 21). There are very few studies showing the<br />
influence of various serum lipids on Lp (a) levels<br />
in diabetics. Although an inverse correlation<br />
between serum Lp (a) and serum triglycerides<br />
has been reported in non-diabetic subjects, there<br />
is scanty literature on this relationship among<br />
the diabetic population.<br />
Materials and methods:<br />
This study was conducted among the out patients<br />
attending the General Medicine department<br />
at Narayana General Hospital, Nellore over a<br />
period of 6 months.<br />
A total of 55 subjects were enrolled <strong>for</strong> the study<br />
(type 2 diabetics<br />
n= 21, non-diabetic first degree relatives<br />
(FDRs) n=19 and healthy controls n=15).<br />
Type 2 diabetic subjects were further subdivided<br />
based on duration of diabetes into those with <<br />
5 yrs duration and those with ≥5 yrs duration.<br />
All of them were in the age group of 20-70<br />
years and both sexes were included. In<strong>for</strong>med<br />
oral consent was taken from all subjects.<br />
Inclusion criteria:<br />
Subjects should have been diabetic <strong>for</strong> at least<br />
one year duration.<br />
To avoid possible transient increase of Lp (a)<br />
after starting insulin treatment, we excluded all<br />
patients in whom insulin was initiated during<br />
40<br />
the first two months preceding the study.<br />
Blood samples were collected after 12 hours<br />
of overnight fasting. 5 ml of venous blood<br />
was collected and part of the blood was<br />
transferred into EDTA anticoagulant containing<br />
tube and sodium fluoride containing tube <strong>for</strong><br />
estimation of HbA1c and glucose respectively.<br />
Remaining blood was allowed to clot and the<br />
serum separated was used to analyze lipids<br />
immediately or stored at – 20°C and analyzed as<br />
batch <strong>for</strong> Lp(a). Glucose and lipid profile were<br />
estimated by Automated chemistry analyzer<br />
[ Humastar 300 (Human Gm BH , Germany)]<br />
using available commercial kits. VLDL was<br />
calculated as triglycerides divided by 5.<br />
Lipoprotein (a) (TULIP QUANTIA KIT) and<br />
HbA1c were estimated by Immunoturbidimetry.<br />
The reference value <strong>for</strong> Lp(a) is less than 30 mg/<br />
dl. For adequate quality control, both normal<br />
and abnormal reference control serum solutions<br />
and calibrators were run be<strong>for</strong>e each testing.<br />
Statistical analysis & Results:<br />
Data were analyzed using statistical software<br />
SPSS 12.0 version. Values were expressed<br />
as mean ± standard error of mean (SEM) <strong>for</strong><br />
continuous variables. Correlations between the<br />
different lipids and serum Lp (a) were done<br />
using Spearman σ correlation. A non-parametric<br />
test, Mann –Whitney test was used to compare<br />
the means between the groups of subjects owing<br />
to skewing of the data. A p value of < 0.05<br />
was considered statistically significant. The<br />
average age and standard deviation of type 2<br />
diabetics, non-diabetic FDRs and controls were<br />
52.7 yrs ± 9.15 , 32.4 yrs ± 9.91 and 31.2 yrs ±<br />
7.35 respectively. Table 1 shows the values of<br />
the biochemical parameters in type 2 diabetic<br />
subjects and controls. Table 2 shows the values<br />
of the biochemical parameters in type 2 diabetic<br />
subjects and FDRs. Table 3 shows the values<br />
of the biochemical parameters in FDRs and<br />
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Serum LP ‘a’ and TG in Diabetes Mellitus<br />
Table 1: Comparison of various biochemical parameters in subjects with type 2 diabetes mellitus and the<br />
controls.<br />
Whole group of Diabetics (n=21) Controls (n = 15)<br />
p Value<br />
FBS (mg/dl) 144.6 ± 8.88 81.2 ± 2.84 0.000*<br />
PPBS(mg/dl) 210.0 ± 14.88 96.2 ± 4.05 0.000*<br />
HbA 1 C (%) 6.92 ± 0.30 5.06 ± 0.12 0.000*<br />
T.Cholesterol (mg/dl) 175.7 ± 11.64 146.8 ± 7.79 0.140<br />
S.Triglycerides (mg/dl) 189.5 ± 26.39 120.7 ± 12.86 0.052<br />
S.VLDL (mg/dl) 51.71 ± 14.49 24.1 ± 2.57 0.038*<br />
S.LDL (mg/dl) 96.33 ± 7.42 87.19 ± 5.09 0.511<br />
S.HDL (mg/dl) 41 ± 2.94 35.5 ± 2.78 0.282<br />
Lp(a) (mg/dl) 19.19 ± 3.08 29.6 ± 4.81 0.105<br />
Values are expressed as Mean ± SEM. n=number of subjects , *p < 0 .05 Significant<br />
Table 2: Comparison of various biochemical parameters in subjects with type 2 diabetes mellitus and the FDRs.<br />
Whole group of Diabetics<br />
(n=21)<br />
FDRs (n = 19)<br />
p Value<br />
FBS (mg/dl) 144.6 ± 8.88 86.4 ± 2.0 0.000*<br />
PPBS (mg/dl) 210.0 ± 14.88 101.7 ± 2.96 0.000*<br />
HbA 1 C (%) 6.92 ± 0.30 5.25 ± 0.127 0.000*<br />
T.Cholesterol (mg/dl) 175.7 ± 11.64 181 ± 11.3 0.655<br />
S.Triglycerides (mg/dl) 189.5 ± 26.39 165 ± 18.5 0.616<br />
S.VLDL (mg/dl) 51.71 ± 14.49 33.1 ± 3.7 0.473<br />
S.LDL (mg/dl) 96.33 ± 7.42 103.4 ± 6.8 0.297<br />
S.HDL (mg/dl) 41 ± 2.94 44.8 ± 2.9 0.238<br />
Lp(a) (mg/dl) 19.19 ± 3.08 20.7 ± 4.5 0.818<br />
Values are expressed as Mean ± SEM. n=number of subjects , *p < 0 .05 Significant.<br />
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41
Serum LP ‘a’ and TG in Diabetes Mellitus<br />
Table 3: Comparison of various biochemical parameters in FDRs and controls.<br />
FDRs(n=19) Controls(n=15) p Value<br />
FBS (mg/dl) 86.4 ± 2.0 81.2 ± 2.84 0.138<br />
PPBS (mg/dl) 101.7 ± 2.96 96.2 ± 4.05 0. 263<br />
HbA 1 C (%) 5.25 ± 0.127 5.06 ± 0.12 0. 314<br />
T.Cholesterol (mg/dl) 181 ± 11.3 146.8 ± 7.79 0. 24<br />
S.Triglycerides (mg/dl) 165 ± 18.5 120.7 ± 12.86 0.071<br />
S.VLDL (mg/dl) 33.1 ± 3.7 24.1 ± 2.57 0.07<br />
S.LDL (mg/dl) 103.4 ± 6.8 87.19 ± 5.09 0.077<br />
S.HDL (mg/dl) 44.8 ± 2.9 35.5 ± 2.78 0.031<br />
Lp(a) (mg/dl) 20.7 ± 4.5 29.6 ± 4.81 0.191<br />
Values are expressed as Mean ± SEM. n=number of subjects, *p < 0 .05 Significant.<br />
Figure 1: Scattered diagram showing Correlation between Serum Lp(a) &Serum Triglycerides in diabetics of ≥ 5<br />
years duration.<br />
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42
Serum LP ‘a’ and TG in Diabetes Mellitus<br />
controls. The mean (± SEM) Lp (a) levels were<br />
lower in type 2 diabetics (19.1± 3.8), compared<br />
to FDRs (20.7± 4.5) and controls (29.6 ±<br />
4.8) and also they were lower in FDRs when<br />
compared to controls, though statistically not<br />
significant.<br />
Figure 1 shows a significant negative correlation<br />
(p value 0.007) between serum Lp(a) and serum<br />
triglycerides among type 2 diabetic subjects of<br />
≥ 5 yrs duration . No significant correlation was<br />
observed between serum Lp (a) and other lipids<br />
among type 2 diabetics of < 5 yrs duration, nondiabetic<br />
FDRs and controls.<br />
Discussion:<br />
To the best of our knowledge this is the first<br />
study in India done on the relationship between<br />
serum Lipoprotein (a) and other serum lipids<br />
in type 2 diabetes mellitus. Our study revealed<br />
that Lp (a) concentrations were lower in type 2<br />
diabetics compared to non-diabetic FDRs and<br />
controls. Calmarza et al and Ali AlBahrani et<br />
al (22, 23 ) noticed lower Lp (a) levels among<br />
type 2 diabetics compared to controls, similar<br />
to our findings. We also observed that FDRs<br />
had lower Lp (a) concentrations compared to<br />
controls. But this is in contrast to the study<br />
done by Agathoklis Psyrogiannis et al (24) who<br />
noticed that normoglycemic offspring of type 2<br />
diabetes mellitus had higher Lp (a) levels than<br />
those without family history of type 2 diabetes<br />
mellitus. The present study revealed a significant<br />
negative correlation ( p value 0.007) between<br />
serum Lp (a) and serum triglycerides among<br />
type 2 diabetics of ≥ 5 years duration. Similar<br />
finding was noted by Hernandez et al (25 ) using<br />
a different method <strong>for</strong> Lp (a) measurement. Low<br />
levels of Lp (a) have been detected in patients<br />
with lipoprotein lipase deficiency by Sandholzer<br />
et al (26) and it is tempting to speculate that<br />
diabetic subjects have some degree of functional<br />
lipoprotein lipase deficiency that is implicated in<br />
43<br />
the pathogenesis of hypertriglyceridemia. The<br />
inverse relationship between Lp (a) and serum<br />
triglycerides is due to the fact that apo (a) is also<br />
present in Triglyceride rich particles (TRPs).<br />
Apo (a) containing - TRPs, in parallel with<br />
chylomicron remnants , would be rapidly taken<br />
up by the liver through the remnant receptor<br />
pathway, thus the lower levels of lipoprotein (a)<br />
in patients with elevated triglycerides could be<br />
the result of rapid catabolism of TRP apo (a)<br />
compared with the slower apo (a) catabolism<br />
in the LDL density range (23, 25). Supportive<br />
evidence to our findings was reported by Ko et<br />
al in their study that improving insulin resistance<br />
by rosiglitazone (insulin sensitizing agent ) was<br />
associated with lowering of triglycerides and<br />
increase in lipoprotein (a) concentrations (27) .<br />
In conclusion, we observed a significant inverse<br />
correlation between serum Lp (a) and serum<br />
triglycerides in type 2 diabetics of ≥ 5 years<br />
duration. This study highlights that the serum<br />
Lp (a) concentrations depend upon other lipid<br />
parameters. There<strong>for</strong>e, Lipoprotein (a) should<br />
be estimated and its interpretation should be<br />
cautiously exercised and care must be taken<br />
while treating dyslipidemia in diabetics.<br />
More prospective studies should be conducted<br />
in order to substantiate the findings.<br />
Limitations of our study :<br />
Firstly, the present study was per<strong>for</strong>med on a<br />
population confined to a particular area; the<br />
results do not necessarily apply to other racial<br />
groups. Secondly, the small sample size and<br />
further sub grouping of type 2 diabetic subjects<br />
combined with sample storage and method<br />
of estimation might have affected the results.<br />
Lastly, due to lack of funding from external<br />
agencies larger studies could not be per<strong>for</strong>med.<br />
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Serum LP ‘a’ and TG in Diabetes Mellitus<br />
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with Lipoprotein Lipase Deficiency and with Type<br />
III hyperlipidaemia. No evidence <strong>for</strong> triglyceride rich precursor<br />
lipoprotein (a). J.Clin. Invest. 1992 ; 90(5):1958<br />
– 1965.<br />
27. Ko SH, Song KH, Ahn YB , Yoo SJ, Son HS, Yoon K H.<br />
The effect of rosiglitazone on serum Lp(a) levels in Korean<br />
patients with type 2 diabetes. Metabolism 2003 ; 52(6) :<br />
731 – 4.<br />
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<strong>Biomedicine</strong>; 2011; 31 (1): 45 - 52<br />
Ameliorative effect of Coccinia grandis in streptozotocin induced diabetic rats<br />
Bhuvaneswari Palanisamy, Krishnakumari Shanmugasundaram and Rajeswari<br />
Paramasivam<br />
Department of Biochemistry, Kongunadu Arts and Science College, Coimbatore – 641 029, Tamilnadu, INDIA.<br />
(Received 30 th September 2010; Revised 25 th December 2010; Accepted 10 th January 2011)<br />
Corresponding author<br />
Dr. S. Krishnakumari,<br />
E-mail: krishnashanmugambc@gmail.com<br />
Abstract<br />
Background & Objectives: Diabetes, a life long progressive disease has become one of the most<br />
challenging health problems of the 21 st century. India faces a grave health care burden due to the high<br />
prevalence of type-II diabetes and its sequalae. In the ongoing search <strong>for</strong> more effective and safer plant<br />
drugs, we have evaluated the hypoglycemic effects of methanolic extract of Coccinia grandis by analyzing<br />
the key enzymes of glycolysis, gluconeogenesis and Citric acid cycle in liver.<br />
Methods: Four groups of six wistar albino rats of 150-200 g each were grouped into control, diabetic,<br />
diabetic treated and plant treated. After treatment <strong>for</strong> 20 days the animals were sacrificed and the<br />
biochemical parameters like blood glucose, serum insulin, hemoglobin, glycosylated hemoglobin, liver<br />
glycogen, hexokinase, glucose-6-phosphatase, fructose-1, 6-diphosphatase, succinate dehydrogenase and<br />
malate dehydrogenase levels were measured.<br />
Results: Blood glucose and glycosylated hemoglobin levels were siginificantly increased whereas the<br />
serum insulin and hemoglobin levels were siginificantly decreased in diabetic rats and reverted to near<br />
normal in treated groups. Glucose-6-phosphatase and fructose-1, 6-diphosphatase levels were siginificantly<br />
increased whereas the liver glycogen, hexokinase, succinate dehydrogenase and malate dehydrogenase<br />
levels were siginificantly decreased in diabetic rats and reverted to near normal in treated groups.<br />
Interpretation & Conclusion: From the above results it is evident that the methanolic extract of Coccinia<br />
grandis have a potential effect to control hyperglycemia in streptozotocin-nicotinamide induced diabetic<br />
rats.<br />
Key words: enzymes, gluconeogenesis, citric acid cycle, streptozotocin, Coccinia grandis<br />
Introduction<br />
Diabetes mellitus in all its heterogeneity has<br />
taken the center stage as one of the ultimate<br />
medical challenges. It is a group of metabolic<br />
diseases characterized by hyperglycemia<br />
resulting from defects in insulin secretion,<br />
insulin action or both (American Diabetes<br />
Association, 2003). The prevalence of diabetes<br />
45<br />
in India is very rapidly rising and it is estimated<br />
that by the year 2010, 20% of all type II patients<br />
in the world would be contributed from India.<br />
Indians are also susceptible to the major<br />
complications related to diabetes like coronary<br />
artery disease, nephropathy and retinopathy.<br />
Prevalence of the complications is higher in<br />
low socio-economic groups due to lack of good<br />
control of glycaemia and hypertension and<br />
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Ameliorative effect of Coccinia grandis in diabetic rats<br />
also due to behavioral factors. The direct and<br />
indirect costs involved in the treatment of the<br />
chronic disease especially when associated with<br />
the vascular complications are enormous. There<br />
is an urgent need to implement preventive<br />
measures to reduce the high morbidity and<br />
mortality and also to reduce the cost burden to<br />
the patients and to the society (Ramachandran<br />
et al., 2002) Synthetic hypoglycemic agents<br />
can produce serious side-effects that endanger<br />
the life of the diabetic patients. This leads to<br />
increasing demand <strong>for</strong> natural products with<br />
antidiabetic activity and less side-effects.<br />
WHO has suggested the evaluation of plant<br />
potentials as effective therapeutic agents,<br />
especially in areas where we lack safe modern<br />
drugs. In modern medicine no satisfactory<br />
single effective therapy is still available to cure<br />
diabetes mellitus. Also, some of the world’s<br />
richest nations are driving the hardest bargains,<br />
despite the fact that the benefits of treatment are<br />
global and by advising that to develop the much<br />
needed research capacity, developing countries<br />
should no more rely on the industrialized world,<br />
but find their own specific solutions (Iaccarino,<br />
2004). <strong>An</strong> in-depth research into the plants<br />
growing around us will reveal the goldmine<br />
lying unexposed within plants. By undertaking<br />
this type of investigation, it is possible to provide<br />
alternate raw materials <strong>for</strong> the ever growing<br />
pharmaceutical industry and it play a vital role<br />
in providing medicines need to be efficacious,<br />
selective, specific in their action and safe with<br />
minimal side effects. Increasing evidence from<br />
both experimental and clinical studies suggests<br />
that oxidative stress plays a major role in the<br />
pathogenesis of diabetes mellitus. Free radicals<br />
are <strong>for</strong>med disproportionately in diabetes by<br />
glucose oxidation, non-enzymatic glycation<br />
of proteins, and the subsequent oxidative<br />
degradation of glycated proteins (Mehta et al.,<br />
2006). Abnormally high levels of free radicals<br />
and the simultaneous decline of antioxidant<br />
defence mechanisms may lead to the damage<br />
of cellular organelles and enzymes, increased<br />
lipid peroxidation, and development of insulin<br />
46<br />
resistance. These consequences of oxidative<br />
stress may promote the development of<br />
complications of diabetes mellitus.<br />
Diabetes is a state of increased oxidant stress<br />
and there is evidence that oxidation may play<br />
a role in the genesis of complications. Chronic<br />
hyperglycemia is a major initiator of diabetic<br />
microvascular complications (e.g., retinopathy,<br />
neuropathy, and nephropathy). Glucose<br />
processing uses a variety of diverse metabolic<br />
pathways, hence chronic hyperglycemia can<br />
induce multiple cellular changes leading to<br />
complications. It is well established now that<br />
increased oxidative stress plays a major role<br />
in the development of diabetic complications<br />
(Maritim et al., 2003 and Soliman, 2008).<br />
Further, lipid peroxidation and antioxidant<br />
enzymes in blood have been cited as markers<br />
<strong>for</strong> vascular injury/microangiopathy in diabetes<br />
mellitus in several studies (King, 2008).<br />
Material and Methods<br />
This study was conducted in the Department<br />
of Biochemistry, Kongunadu Arts and Science<br />
College, Coimbatore, Tamilnadu.<br />
Collection of plant material and preparation<br />
of plant extract:<br />
The fresh leaves of Coccinia grandis<br />
(Linn.) Voigt (Family: Cucurbitaceae)<br />
Syn. Coccina indica (Wight & Arn) were<br />
collected from Karur district, Tamilnadu,<br />
India. Taxonomic authentication was done<br />
by Dr.V.S.Ramachandran, Taxonomist,<br />
Department of Botany, Bharathiar University,<br />
Coimbatore, Tamilnadu, India. The leaves were<br />
washed with water, shade dried and powdered.<br />
The powdered material (10g) was extracted with<br />
100ml of methanol using Soxhlet apparatus<br />
and filtered. The filtrate was concentrated and<br />
dried under reduced pressure and controlled<br />
temperature.<br />
Chemicals:<br />
The chemicals and solvents used in the study<br />
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Ameliorative effect of Coccinia grandis in diabetic rats<br />
were of highest purity and analytical reagents<br />
grade. They were purchased from SD Fine<br />
Chem., Himedia and Qualigens, India.<br />
Selection of animals:<br />
Female albino rats of Wistar strain weighing<br />
150 200g were purchased from animal breeding<br />
centre of Kerala Agricultural University,<br />
Mannuty, Thrissur, Kerala India. <strong>An</strong>imals were<br />
provided with standard pellet diet (AVM feeds,<br />
Coimbatore) and water was provided ad-libitum<br />
and maintained under standard laboratory<br />
conditions. The animals were allowed to get<br />
acclimatized to the laboratory conditions <strong>for</strong><br />
one week. The experiments were conducted<br />
according to the ethical norms approved by<br />
ministry of social justices and empowerment,<br />
Government of India and Institutional<br />
<strong>An</strong>imal Ethics committee Guide (Approval No:<br />
659/02/a/CPCSEA).<br />
After one week acclimatization period, the<br />
animals were divided into four groups with six<br />
animals in each.<br />
Group I : Control rats fed with standard<br />
pellet diet and water.<br />
Group II : Rats induced with 110 mg/<br />
kg b.wt Nicotinamide<br />
followed by 65 mg/kg<br />
b.wt Streptozotocin intra<br />
venous.<br />
Group III : Diabetic rats treated with<br />
methanolic extract of<br />
Coccinia grandis 200<br />
mg/kg b.wt <strong>for</strong> 20 days<br />
orally<br />
Group IV : Rats treated with methanolic<br />
extract of Coccinia<br />
grandis 200 mg/kg b.wt<br />
<strong>for</strong> 20 days orally<br />
Sample Collection:<br />
After the experimental regimen, the animals<br />
were sacrificed by cervical dislocation under<br />
47<br />
mild chloro<strong>for</strong>m anesthesia. Blood was collected<br />
by an incision made in the jugular veins and<br />
the serum was separated by centrifugation at<br />
2000 rpm <strong>for</strong> 20 minutes. The liver was excised<br />
immediately and thoroughly washed in ice cold<br />
physiological saline. A 10% homogenate of the<br />
washed tissue was prepared in 0.1M Tris HCl<br />
buffer (pH 7.4) in a potter homogenizer filled<br />
with a Teflon plunger at 600 rpm <strong>for</strong> 3 mins.<br />
Blood glucose was done by the method<br />
of Beach and Turner(1958), serum insulin by<br />
the method of <strong>An</strong>derson (1993), hemoglobin<br />
by the method of Drabkin and Austin (1932),<br />
glycosylated hemoglobin by the method of<br />
Sudhakar and Pattabiraman (1981), liver<br />
glycogen by the method of Morales et al (1973),<br />
hexokinase by the method of Branstrup (1957),<br />
glucose – 6 – phosphatase by the method<br />
of Koide and Oda (1959), fructose – 1, 6 –<br />
diphosphatase by the method of Ganccedo and<br />
Ganccedo (1971), succinate dehydrogenase by<br />
the method of Slater and Bonner (1952) and<br />
malate dehydrogenase by the method of Mehler<br />
et al (1948) levels were estimated. Statistical<br />
analysis was per<strong>for</strong>med using SPSS package,<br />
version 6.0. The values were analyzed by one<br />
- way analysis of variance (ANOVA) followed<br />
by Least Significant Difference (LSD). All the<br />
results were expressed as mean ± SD <strong>for</strong> six<br />
rats in each group p
Ameliorative effect of Coccinia grandis in diabetic rats<br />
The treatment with the plant extract caused a<br />
significant decrease in the elevated blood glucose<br />
and increase in the lowered insulin and glycogen<br />
levels to near normal glycemic concentration<br />
in streptozotocin induced diabetic rats, which<br />
is an essential trigger <strong>for</strong> the liver to revert<br />
its normal homeostasis during experimental<br />
diabetes. Normal animals treated with the<br />
plant extract at a dosage of 200 mg/kg body<br />
weight showed a slight reduction in the blood<br />
glucose which may be due to the hypoglycemic<br />
property of Coccinia grandis while insulin and<br />
glycogen levels remained normal. The levels<br />
of total hemoglobin and HbA 1c in control and<br />
experimental animals are depicted in Table 2.<br />
The diabetic rats showed a significant decrease<br />
in the level of total hemoglobin and increase in<br />
the level of HbA 1c when compared with normal<br />
control rats. The level of total hemoglobin<br />
was significantly increased and HbA 1c was<br />
significantly decreased by the administration of<br />
plant extract in diabetic rats. Normal animals<br />
treated with the plant extract at a dosage of 200<br />
mg/kg body weight did not show any significant<br />
change in hemoglobin and HbA 1c levels.<br />
The activity of the enzymes involved in<br />
carbohydrate metabolism such as hexokinase,<br />
glucose – 6 – phosphatase and fructose- 1,<br />
6- diphosphatase in the liver of normal and<br />
treated rats were shown in Table 3. A significant<br />
decrease in hexokinase activity was observed in<br />
the liver of diabetic rats, whereas the activities of<br />
hepatic glucose-6-phosphatase and fructose-1,<br />
6-bisphosphatase were significantly increased<br />
compared with normal rats. Treatment with the<br />
plant extract to the diabetic rats resulted in a<br />
significant increase in hexokinase activity and<br />
decrease in gluconeogenic enzymes activity in<br />
the liver of diabetic rats. Oral administration of<br />
plant extract to normal rats resulted in a nonsignificant<br />
decrease in hepatic gluconeogenic<br />
enzymes and the activity of hexokinase was not<br />
altered significantly compared with normal rats.<br />
Table 4 depicts the effect of methanolic extract<br />
of Coccinia grandis on malate dehydrogenase<br />
and succinate dehydrognase in the liver of<br />
48<br />
control and experimental animals. In the<br />
present study, significant decrease in malate<br />
dehydrogenase and succinate dehydrogenase<br />
activities in the liver of diabetic animals was<br />
observed and the levels increased significantly<br />
in the liver of treated diabetic animals. There<br />
was no significant change in the levels of<br />
SDH and MDH in normal animals which<br />
were administered with the plant extract. <strong>An</strong><br />
increase in succinate dehydrogenase and malate<br />
dehydrogenase activities in treated animals<br />
indicates better utilization of energy yielding<br />
intermediates by TCA cycle.<br />
Discussion<br />
Reduction of glucose transporter expression to<br />
prevent cytotoxic effect of STZ (Thulesen et<br />
al., 1997) and decrease in β cell sensitivity to<br />
glucose due to long term hyperglycemia (Curry,<br />
1986) is believed to be the possible causes of<br />
high level of glucose in the blood stream of<br />
STZ – induced diabetic rats. The increased<br />
glucose level in the diabetic animals might be<br />
due to the destruction of the pancreatic cells<br />
caused by the streptozotocin induction and the<br />
glucose level was found to be near normal in the<br />
treated animals which may be due to the insulin<br />
like activity of Coccinia grandis leaf extract<br />
by activating the glucose uptake by the cells.<br />
Lowered blood glucose levels in the diabetic<br />
rats might be due to increasing glycogenesis,<br />
inhibiting gluconeogenesis in the liver or<br />
inhibiting the absorption of glucose from the<br />
intestine. From the results of clinical studies by<br />
Knekt et al., 2002, it is evident that the reduction<br />
of hyperglycaemia is the most important factor<br />
in the prevention of chronic microvascular<br />
complications of diabetes mellitus (retinopathy,<br />
nepropathy, neuropathy and diabetic foot) as<br />
well as in the prevention of the accelerated<br />
atherosclerosis-related conditions (myocardial<br />
infarction, stroke). The mode of action of the<br />
active compound(s) of the plant material is<br />
probably mediated through enhanced secretion<br />
of insulin from the β-cells of Langerhans or<br />
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Ameliorative effect of Coccinia grandis in diabetic rats<br />
Results<br />
Table 1: Effect of methanolic extract Coccinia grandis on blood glucose, serum insulin and liver glycogen in<br />
control and experimental rats<br />
Parameters Blood glucose Serum insulin Liver glycogen<br />
(mg/dl) (IU/ml) (mg/g tissue)<br />
Group I 105.50±15.37 10.32±0.28 39.56±0.44<br />
Group II 406.00±15.16a * 4.24±0.31a * 21.70±0.44 a *<br />
Group III 268.83±36.59b * 6.38±0.20b * 37.13±0.39 b*<br />
Group IV 83.50±12.88c ns 10.94±0.24 c ns 39.72±0.30c ns<br />
Values are expressed as mean±SD (n=6)(p
Ameliorative effect of Coccinia grandis in diabetic rats<br />
through extrapancreatic mechanism (Akhtar et<br />
al., 2007). The STZ – induced diabetes causes<br />
the destruction of β cells of the Islets, which<br />
leads to a reduction in insulin release and high<br />
blood glucose level namely hyperglycemia.<br />
The observed increase in the levels of serum<br />
insulin indicates that Coccina grandis extract<br />
stimulates insulin secretion by the closure of<br />
K + -ATP channels, membrane depolarization<br />
and stimulation of Ca 2+ influx, an initial key<br />
step in insulin secretion from the remnet β-<br />
cells or from regenerated β-cells. Previous data<br />
shows that, ferulic acid a phenolic compound<br />
increases insulin release in clonal β- cells RIN-<br />
5F (Nomura et al., 2003). The treatment with<br />
the Coccinia grandis extract has enhanced the<br />
rate of glycogenesis as indicated by higher<br />
amounts of hepatic glycogen in the diabetic<br />
treated group. Glycogen content of normal<br />
animals in fasting stage was only slightly higher<br />
than the diabetic animals and this may be due to<br />
the degradation of glycogen to maintain normal<br />
blood glucose levels, whereas glycogen levels<br />
in diabetics were found to be very low despite<br />
high blood glucose levels possibly due to lower<br />
levels of glycogen synthase activity (Singh,<br />
2001). Accumulation of glycogen in liver of<br />
treated animals is somewhat similar to that<br />
reported during insulin therapy. The glycogen<br />
content is decreased in liver muscles of diabetic<br />
rats (Grover et al., 2000). Similar observations,<br />
i.e. hypoglycaemic activity and improved levels<br />
of hepatic glycogen, were reported by Kedar<br />
and Chakrabarti (1982) in diabetic animals<br />
with the treatment of Momordica charantia<br />
<strong>for</strong> 3 weeks. Pectin from ivy gourd (Coccinia<br />
grandis) has shown significant hypoglycemic<br />
activity in rats, stimulating glycogen synthetase<br />
activity and reducing phosphorylase activity<br />
(Grover et al., 2002). The excess of glucose<br />
present in the blood during diabetes, which<br />
react with hemoglobin and <strong>for</strong>m glycosylated<br />
hemoglobin. The various proteins including<br />
hemoglobin undergo an enzymatic glycation<br />
in diabetes. Glycosylated hemoglobin was<br />
found to be increased in diabetes mellitus and<br />
50<br />
the amount of increase is directly proportional<br />
to that of fasting blood glucose level (Sheela<br />
& Augusti, 1992). Lowered levels of total<br />
hemoglobin were observed in diabetic rats<br />
which might be due to the increased <strong>for</strong>mation<br />
of HbA 1c . Hyperglycemia is the clinical<br />
hallmark of poorly controlled diabetes, which<br />
is known to cause glycation, and also known as<br />
non-enzymatic glycosylation. HbA 1c was found<br />
to increase in patients with diabetes mellitus<br />
and the increase was directly proportional to the<br />
fasting blood glucose levels (Alberti, 1982).<br />
In experimental diabetes, enzymes of glucose<br />
metabolism are markedly altered. One of the<br />
key enzymes in the catabolism of glucose is<br />
hexokinase, which phosphorylates glucose and<br />
converts it into glucose-6-phosphate (Laakso et<br />
al., 1995). Hexokinase insufficiency in diabetic<br />
rats can cause decreased glycolysis and decreased<br />
utilization of glucose <strong>for</strong> energy production<br />
(Gancedo & Gancedo, 1971). Hexokinase is<br />
significantly reduced in diabetic rats; this may<br />
be the reason <strong>for</strong> the diminished consumption of<br />
glucose in the system and increased blood sugar<br />
levels. The gluconeogenic enzyme, glucose-<br />
6-phosphatase is a crucial enzyme of glucose<br />
homeostasis because it catalyses the ultimate<br />
biochemical reaction of both glycogenolysis<br />
and gluconeogenesis (Mithievre et al., 1996).<br />
Increased glucose-6- phosphatase activity in<br />
diabetic rats provides hydrogen, which binds<br />
with NADP + in the <strong>for</strong>m of NADPH and enhances<br />
the synthesis of fats from carbohydrates (i.e.<br />
lipogenesis) (Bopanna et al., 1997) and finally<br />
contributes to increased levels of glucose in the<br />
blood. Increased hepatic glucose production in<br />
diabetes mellitus is associated with impaired<br />
suppression of the gluconeogenic enzyme<br />
fructose-1, 6-bisphosphatase. Activation of<br />
gluconeogenic enzymes is due to the state<br />
of insulin deficiency, because under normal<br />
conditions, insulin functions as a suppressor of<br />
gluconeogenic enzymes. In diabetic condition,<br />
hexokinase activity has been decreased,<br />
which may be due to loss of insulin receptors<br />
(Garvey et al., 1985). Previous studies showed<br />
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Ameliorative effect of Coccinia grandis in diabetic rats<br />
that the caffeic acid seemed to suppress the<br />
hepatic glucose output by enhancing hepatic<br />
glucose utilization and inhibiting glucose over<br />
production in db/db mice (Jung et al., 2006).<br />
Administration of the plant extract to the<br />
diabetic rats significantly reduced the levels of<br />
plasma glucose, which activates hexokinase and<br />
increase glycolysis and utilization of glucose<br />
<strong>for</strong> energy production. Similar effects of<br />
mangiferin, in streptozotocin induced diabetic<br />
rats were reported (Sellamuthu et al., 2009). The<br />
hepatic gluconeogenic enzymes (glucose-6phosphatase<br />
and fructose-1, 6-bisphosphatase)<br />
were significantly increased in diabetic state<br />
(Shulman, 2000). The activities of the two<br />
enzymes may be due to the increased synthesis<br />
of enzymes contributing to the increased glucose<br />
production during diabetes by the liver (Pari et<br />
al., 2005). In diabetic rats treated with the extract<br />
modulates and regulates the activities of these<br />
enzymes, either through regulation by cyclic<br />
AMP or by metabolic activation or inhibition<br />
of glycolysis and gluconeogenesis. In previous<br />
reports, para-methoxycinnamic acid markedly<br />
reduced hyperglycemia in STZ-induced diabetic<br />
rats by increasing glycolysis and inhibiting<br />
gluconeogenesis (Adisakwattana et al.,<br />
2005). When Coccinia indica and Momordica<br />
charantia extracts were administered to diabetic<br />
rats, the results indicated that there was lowering<br />
of blood glucose by depressing its synthesis<br />
through depression of the key gluconeogenic<br />
enzymes glucose-6-phosphatase and fructose-<br />
1,6-biphosphatase and also by enhancing<br />
glucose oxidation by the shunt pathway through<br />
activation of its principal enzyme, G6PDH<br />
(Shibib et al.,1993).<br />
Malate dehydrogenase plays an<br />
important role in the citric acid cycle by<br />
providing oxaloacetate <strong>for</strong> the <strong>for</strong>mation of<br />
citrate with acetyl-CoA <strong>for</strong> generating malate<br />
which can feed the cytosolic gluconeogenic<br />
pathway. These enzymes are reported to be<br />
inhibited in tissues of diabetic animals in several<br />
studies (Lemieux et al., 1984). From our results,<br />
we can conclude that the lowered insulin level<br />
51<br />
plays an important role in the pathogenesis of<br />
diabetes and its complication thereby increasing<br />
glucose level in blood. Since the methonolic<br />
extract of Coccinia grandis has the capacity to<br />
activate the glucose uptake into the cells and<br />
enhanced reduction in gluconeogenesis they<br />
act as an antihyperglycemic agent and since it<br />
increases the utilization of the energy yielding<br />
intermediates by TCA cycle it is insulin mimetic.<br />
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1971; 76:132-38.<br />
13. Garvey WT, Olefsky JM and Marshall S. (1985). Insulin<br />
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14. Grover JK, Vats V and Rathi SS. (2000) <strong>An</strong>tihyperglycemic<br />
effect of Eugenia jambolana and Tinospora cordifolia in<br />
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15. Grover JK, Yadav S and Vats V (2002). Medicinal<br />
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16. Iaccarino M. (2004). Mastering science in the South.<br />
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17. Jung UJ, Lee MK, Park YB, Jeon SM and Choi MS.<br />
(2006). <strong>An</strong>tihyperglycemic and antioxidant properties of<br />
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:105-163.<br />
18. Kedar P and Chakrabarti CM (1982). Effects of bitter<br />
gourd (Momordica charantia) seed and Glibenclamide in<br />
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19. King GL. (2008).The role of inflammatory cytokines on<br />
diabetes and its complications. J Periodontol,79: 1527 –<br />
1534.<br />
20. Knekt P, Kumpulainen J, Järvinen R, Rissanen H,<br />
Heliövaara M, Reunanen A, Hakulinen T and Aromaa A.<br />
(2002). Flavonoid intake and risk of chronic diseases. Am<br />
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21. Koide H and Oda T. pathological occurrence of glucose<br />
phosphatase in serum in liver diseases.Clin. Chim. Acta.<br />
1959;4:554.<br />
22. Laakso M, Malkki M and Deeb SS. (1995). Amino acid<br />
substituents in hexokinase II among patients with NIDDM.<br />
Diabetes, 44:330–334.<br />
23. Lemieux G, Aranda MR, Fournel P and Lemieux C. (1984).<br />
Renal enzymes during experimental diabetes mellitus<br />
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and ketoacidosis. Canadian <strong>Journal</strong> of Physiology and<br />
Pharmacology, 62:70–75.<br />
24. Maritim AC, Sanders RA and Watkins JB (2003). Diabetes,<br />
oxidative stress, and antioxidants: a review. J Biochem<br />
MolToxicol, 17: 24-38.<br />
25. Mehler A, Komberg A, Grisolia and Ochoa S. 1948. The<br />
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malate or isocitrate and pyruvate. J. Biol. Chem. 174,961.<br />
26. Mehta JL, Rasouli N and Sinha AK and Molavi B. (2006).<br />
Oxidative stress in diabetes: A mechanistic overview of its<br />
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27. Mithievre G, Vidal G, Zitovn C, Miriasian C, (1996).<br />
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Diabetes, 45:891–896.<br />
28. Morales MA, AJ Jabbagy and HF Terenzi, 1973. Mutations<br />
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31. Ramachandran A., Snehalatha C. and Vijay V. (2002).<br />
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32. Sellamuthu PS, Muniappan BP, Perumal SM and<br />
Kandasamy M. (2009). <strong>An</strong>tihyperglycemic effect of<br />
mangiferin in streptozotocin induced diabetic rats, J.<br />
Health Sci, 55:206–214.<br />
33. Sheela CG and Augusti KT, 1992. <strong>An</strong>tidiabetic effects of<br />
S-allyl cysteine sulphoxide isolated from garlic Allium<br />
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30:523–526.<br />
34. Shibib BA, Khan LA and Rahman R (1993). Hypoglycemic<br />
activity of Coccinia indica and Momordica charantia in<br />
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enzymes glucose-6-phosphatase and fructose-1,6biphosphatase<br />
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35. Shulman GI. (2000).Cellular mechanisms of insulin<br />
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36. Singh SN, Vats P, Suri S, Shyam R, Kmaria MML,<br />
Ranganathan S and Sridharan K. (2001). Effect of an<br />
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Ethanopharmacol, 76: 253-262.<br />
37. Slater EC and Bonner WD. 1952, Succinate dehydrogenase.<br />
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38. Soliman GZA. (2008).Blood lipid peroxidation<br />
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39. Sudhakar NS pattabiaman, TN. A new colorimetric method<br />
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40. Thulesen J, Orskov C, Holst JJ and Poulsens S. (1977).<br />
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www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 53 - 63<br />
Testicular toxicity in arsenic exposed albino rats: ameliorative effects of ascorbic<br />
acid and α-tocopherol<br />
*Avijit Dey, **Arindam Bose,Prabir Kr. *Mukhopadhyay<br />
*Department of Physiology, Presidency College, Kolkata-700 073, India<br />
**Department of Microbiology, Immunology & Molecular Biology, Choitram Hospital & Research<br />
Center, Manikbagh Road, Indore, Madhya Pradesh - 452014, India.<br />
(Received 30th August 2010; Revised 25th November 2010; Accepted 30th December 2010)<br />
Corresponding author<br />
Dr. Prabir Kr. Mukhopadhyay<br />
E-mail: p.mukherjeepresi@gmail.com<br />
Abstract:<br />
Backgound and objective: The study was designed to evaluate the protective effect of combined<br />
administration of ascorbic acid and α-tocopherol on the arsenic induced testicular toxicity.<br />
Methods: The histoarchitecture of testis along with sperm number, viability and motility were counted to<br />
study the testicular gametogenic toxicity. The levels of malondialdehyde (MDA) and conjugated dienes in<br />
testicular tissue were measured <strong>for</strong> evaluation of oxidative stress and the activities of testicular superoxide<br />
dismutase (SOD) and catalase were also measured.<br />
Results: Arsenic trioxide treatment (3 mg/kg body weight/ day) in a single dose <strong>for</strong> 30 consecutive days<br />
caused increase in seminiferous tubular luminal space coupled with reduced accumulation of spermatozoa<br />
and disarray in cellular organization. Other significant changes were decrease in sperm count, viability and<br />
motility. This treatment was also associated with significantly high levels of MDA and conjugated dienes<br />
along with significant inhibition of the SOD and catalase activities in the testicular tissue. All these changes<br />
were reversed with ascorbic acid and α-tocopherol co-administration. Supplementation of ascorbic acid<br />
(200 mg/kg body weight/day) and α-tocopherol (400 mg/kg body weight/day) along with same arsenic<br />
exposure caused partial restoration of normalcy. All these sperm physiological changes and altered gonadal<br />
features, both histomorphometric and histological observations were found significantly ameliorated. The<br />
other studied changes were significantly reversed.<br />
Interpretation and conclusion: Results of this study propose that co-treatment of ascorbic acid and<br />
α-tocopherol to arsenic-exposed rats protects the testicular antioxidant system and causes effective recovery<br />
from the toxic effects of arsenic on testes.<br />
Key words: arsenic, testis, oxidative stress, ascorbic acid, α-tocopherol.<br />
Introduction<br />
It has become evident that increasing human<br />
activities have modified the global cycle of<br />
heavy metals and metalloids, including the<br />
toxic non-essential elements like arsenic,<br />
53<br />
As(1). Among these metals, arsenic exhibits a<br />
complex metabolism and is possibly the most<br />
abundant and potential carcinogen (2). Arsenic<br />
is available to the human population through<br />
sources that include drinking water, food and<br />
air. Population exposed to arsenic contaminated<br />
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Effects of ascorbic acid and α-tocopherol in arsenic toxicity<br />
drinking water includes those in Taiwan,<br />
Chaina, Europe, United States, Bangladesh and<br />
India (3, 4). Studies done in September 2006 by<br />
the School of Environmental Studies, Jadavpur<br />
university, Kolkata, <strong>for</strong> the assessment of<br />
ground water arsenic contamination in 19<br />
districts of West Bengal, India have revealed<br />
a gruesome picture which depicts that out of<br />
80.2 million 4.6 million people in West Bengal<br />
are at risk of drinking arsenic contaminated<br />
water containing arsenic > 50 µg/L, where<br />
the WHO recommended maximum arsenic<br />
contaminant level (MCL) is 10 µg/L (5).<br />
Consumption of arsenic containing drinking<br />
water has been associated with cancers (lung,<br />
bladder, skin etc.) and other chronic diseases<br />
such as dermal, cardiovascular, neurologic,<br />
and diabetes (6). A lot of potential toxic effects<br />
are also observed on the reproductive system<br />
also. In female rats, the associated noticeable<br />
changes are the reductions of ovarian weight<br />
along with associated decrease of plasma levels<br />
of LH, FSH and the steroidogenic enzymes<br />
like ∆ 5 -3β-hydroxysteroid dehydrogenase (∆ 5<br />
-3β-HSD), 17β- hydroxysteroid dehydrogenase<br />
(17β-HSD) (7). On male reproduction, the<br />
studied effects of arsenic toxicity include the<br />
reduction of the weight of testes and accessory<br />
sex organs (8), inhibition of androgenic and<br />
gametogenic potentials of testis (8). Arsenic<br />
exposure has shown to depress the functions of<br />
antioxidant defense system leading to oxidative<br />
damage to cellular macromolecules (9).<br />
According to the recent report, arsenic can also<br />
alter the cellular functions (10) and can also<br />
cause DNA damage (10). Studies suggest that<br />
arsenic compounds may also exert their toxicity<br />
through the generation of reactive oxygen<br />
species (ROS), such as superoxide radical,<br />
hydroxyl radical, hydrogen peroxide and nitric<br />
oxide during their metabolism in the cells (9, 11)<br />
and thus may cause cellular damages (12). ROS<br />
attacks and causes peroxidation of sperm plasma<br />
membrane polyunsaturated fatty acids (PUFA)<br />
resulting in the <strong>for</strong>mation of malondialdehyde<br />
(MDA) (13), conjugated diene (CD) radicals<br />
54<br />
(14). Male infertility is reflected by low sperm<br />
count, low sperm motility and bad quality of<br />
sperms (8). Seminiferous tubular diameter is<br />
reported to show a dose and duration dependent<br />
reduction (15). Degeneration and loss of germ<br />
cells, marked atrophy of seminiferous tubules<br />
and Leydig cell are also reported (15).<br />
Recent studies have shown that ascorbic acid<br />
is able to block the arsenic induced impairment<br />
of male reproductive functions and testicular<br />
oxidative changes in mice (16) and could also<br />
protect the liver and kidney of rats from arsenic<br />
induced oxidative stress (17). Administration<br />
of ascorbic acid with α-tocopherol exhibits<br />
profound effects in preventing protein<br />
oxidation and DNA damage in rats (6). The<br />
same supplementation could also prevent lipid<br />
peroxidation and protect the antioxidant system<br />
in arsenic-intoxicated rats (18). Our present<br />
study is an attempt to investigate whether the<br />
combined oral application of ascorbic acid<br />
and α-tocopherol can ameliorate the arsenic<br />
induced oxidative stress mediated alterations of<br />
testicular histoarchitecture, sperm physiology<br />
and testicular antioxidant defense system by<br />
their cumulative antioxidant actions.<br />
Materials and Methods:<br />
<strong>An</strong>imal selection and drug treatment: 24 adult<br />
male albino rats of the Wister stain weighing<br />
130±10 mg were selected <strong>for</strong> this experiment.<br />
The animals were maintained under standard<br />
laboratory conditions (14 hrs light: 10 hrs dark,<br />
25±2° C) with free access to food and water. All<br />
animal experiments were per<strong>for</strong>med according<br />
to the ethical guidelines suggested by the<br />
Institutional <strong>An</strong>imal Ethics Committee (IAEC)<br />
guided by the Committee <strong>for</strong> the purpose of<br />
Control and Supervision of Experiments on<br />
<strong>An</strong>imals (CPCSEA), Ministry of Environment<br />
and Forest, Government of India. For the<br />
experiments, rats were randomly selected into<br />
three groups consisting eight rats in each:<br />
group I, control; group II, arsenic treated; and<br />
group III, arsenic+ ascorbic acid+ α-tocopherol<br />
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Effects of ascorbic acid and α-tocopherol in arsenic toxicity<br />
supplemented. The animals of group I and II<br />
were provided with a control diet composed of<br />
71% carbohydrate, 18% protein, 7% fat, and 4%<br />
salt mixture and vitamins (19). For chronic oral<br />
exposure to arsenic a dose was selected (3 mg /<br />
kg body wt / day), which is within the range of<br />
LD 50 of 70 kg body wt. human (1-4 mg /kg) and<br />
lesser than one thirteenth of LD 50 value of rats<br />
(40mg/kg) (20). Accordingly, rats of group II<br />
and III were orally treated with aqueous solution<br />
of arsenic trioxide, 3mg /kg body wt/ day <strong>for</strong><br />
30 days. The rats of group III, in addition, were<br />
treated with ascorbic acid (200mg / kg body wt<br />
/ day, dissolved in water) and α-tocopherol (400<br />
mg / kg body wt / day, dissolved in olive oil)<br />
by oral gavage <strong>for</strong> once a day. To overcome<br />
the impact of any altered food intake, control<br />
group (group I) rats were pair-fed with other<br />
experimental groups, II and III.<br />
Food and water intake and body wt. of<br />
the rats were monitored throughout the 30 days<br />
of the experiment.<br />
Rats were sacrificed 24 hrs after<br />
the last treatment by light ether anesthesia. The<br />
testes were dissected, weighted and one testis<br />
of each rat was used <strong>for</strong> histological study and<br />
the other, <strong>for</strong> the assessment of levels of free<br />
radicals and scavenger enzyme activities.<br />
Study substance: Arsenic trioxide was used <strong>for</strong><br />
this experiment, purchased from Loba Chemical<br />
Pvt. Ltd., Mumbai, India. α-tocopherol was<br />
purchased from Himedia, Mumbai, India and<br />
L-ascorbic acid was purchased from SRL Pvt.<br />
Ltd., Mumbai, India.<br />
1. Permanent slide preparation <strong>for</strong><br />
histological study: Bouin’s fixed testes were<br />
embedded in paraffin wax and blocks were<br />
prepared. 5μm thin sections were cut from the<br />
middle portion of the testes with a high precision<br />
microtome (IEC Microtome, USA) and stained<br />
with Hematoxylin and Eosin (H/E).<br />
2. Sperm viability count: Immediately after the<br />
sacrifice cauda portion of the epididymis was<br />
cut. The cauda was kept in 1 ml diluent (21).<br />
55<br />
This was kept <strong>for</strong> 5 mins at 37°C. It was then<br />
taken out, and an incision was given through<br />
the cauda and sperms were dispersed in the<br />
fluid. 40 μl of this spermatozoal suspension was<br />
transferred to an eppendrof. This was stained<br />
with EosinY and Nigrosin (40 μl each). This<br />
was mixed gently and 25 μl from the mixture<br />
was taken on a grease free slide and a smear<br />
was drawn and dried. The number of viable<br />
and non-viable sperms was counted under light<br />
microscope (OLYMPUS, CH 20i TR).<br />
3. Sperm count: From the dispersed<br />
spermatozoal suspension, 25 μl was charged<br />
on a Neubauer Haemocytometer (22) and the<br />
numbers of sperms were counted and calculated<br />
using WBC chambers.<br />
4. Sperm motility count: The cauda epididymis<br />
from all three groups was obtained as mentioned<br />
earliar and each cauda was kept in 1 ml PBS<br />
0.2mol, pH 7.4, at 37°C (23). 25 μl of this was<br />
taken on a clean slide covered with a cover slip<br />
and was observed under a microscope. Total<br />
numbers of motile sperms per 100 sperms<br />
were counted. The readings were taken at the<br />
beginning of 1 st hr, 2 nd hr, 3 rd hr of the experiment<br />
(the temperature of spermatozoal suspension<br />
was maintained at 37°C throughout the process).<br />
5. Testicular conjugated dienes measurement:<br />
Testicular conjugated dienes were measured<br />
according to the method of Slater (1984) (24).<br />
Testicular tissue was homogenized in 0.1mol<br />
phosphate buffer (pH 7.4) containing 0.1mol<br />
Na 2 HPO 4 and 0.1mol anhydrous NaH 2 PO 4 at<br />
a tissue concentration of 50 mg/ml. Lipids<br />
of the homogenizing mixture (0.5 ml) were<br />
extracted with chloro<strong>for</strong>m-methanol (2:1)<br />
mixture. It was then centrifuged at 1000g <strong>for</strong> 5<br />
minutes at room temperature. Chloro<strong>for</strong>m was<br />
evapourated and the lipid residue was dissolved<br />
in 1.5 ml cyclohexane. The absorbance of<br />
this dissolved conjugated dienes was read<br />
spectrophotometrically at 233 nm.<br />
6. Testicular malondialdehyde measurement:<br />
Malondialdehyde (MDA) levels in the<br />
testicular tissues from rats of all groups were<br />
measured biochemically following the method<br />
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Effects of ascorbic acid and α-tocopherol in arsenic toxicity<br />
of Ohkawa et al. 1979 (25). Tissue samples<br />
were homogenized in 0.1mol phosphate buffer<br />
(pH7.4) at tissue concentration of 50 mg/ml.<br />
The homogenizing mixture (0.5 ml) was mixed<br />
with 0.5 ml 0.9% saline and 2ml of TBA-TCA<br />
mixture( 0.392 gm thiobarbituric acid in 75 ml<br />
of 0.25 N HCl with 15 gm trichloroacetic acid,<br />
volume upto 100ml by 95% ethanol) and boiled<br />
<strong>for</strong> 10 minutes. The mixture was then cooled<br />
to room temperature and centrifuged at 4,000<br />
rpm <strong>for</strong> 10 minutes. The whole supernatant was<br />
taken in spectrophotometric cuvette and read at<br />
535 nm.<br />
7. Testicular catalase activity: The testicular<br />
catalase activity was measured from all groups<br />
following the method of Beers and Sizer (1952)<br />
(26). Testicular tissue was homogenized in ice<br />
cold medium containing 0.05 mol Tris Hcl<br />
buffer( pH- 7.0) at a tissue concentration of 20<br />
mg/ml. The mixure was centrifused at 10000g<br />
<strong>for</strong> 20 minutes at 4°C. In a spectrophotometric<br />
cuvette 0.5 ml hydrogen peroxide solution (100<br />
μl 30% H 2 O 2 + 99 ml double dist water) and 2.5<br />
ml double distilled water were mixed well and<br />
read at 240 nm. Then 40 μl of the supernatant<br />
was added, mixed well and six readings were<br />
taken at 30 second intervals.<br />
8. Testicular superoxide dismutase (SOD)<br />
activity: Testicular SOD was measured<br />
according to the method of Martin et al (1987)<br />
(27). To measure the testicular SOD activity<br />
the tissue was homogenized in an ice cold<br />
medium containing 0.1mol phosphate buffer<br />
(pH 7.2) at a tissue concentration of 50mg/ml.<br />
The homogenate was centrifuged at 10,000g <strong>for</strong><br />
20 min at 4ºC. In a spectrophotometer cuvette<br />
3ml of 0.05M phosphate buffer with EDTA<br />
was added and then 50µl of haematoxylin was<br />
added and checked OD at 30 second intervals<br />
with sample and without sample at 560 nm.<br />
9. Statistical analysis: The data were expressed<br />
as mean ± SEM (Standard error of mean).<br />
For statistical analysis, the quantitative data<br />
of each parameter from the different groups<br />
were analyzed by Student’s “t” test. The<br />
mean ± SEM was calculated <strong>for</strong> each group<br />
56<br />
and the corresponding level of significance<br />
was calculated. Statistical software system<br />
Minitab version 2009 was employed in the<br />
entire methodologies. p
Effects of ascorbic acid and α-tocopherol in arsenic toxicity<br />
Table 1: Effect of ascorbic acid and α-tocopherol co-administration on the arsenic trioxide induced alterations<br />
in spermatozoal status of mature albino rats.<br />
Parameter<br />
Studied<br />
Control<br />
Group-I<br />
(Mean<br />
±SEM)<br />
1)Sperm count (10 6 /cauda) 102.07<br />
±9.71<br />
2)Sperm viability(%) 28.06<br />
±0.49<br />
3)Sperm motility(%) Ist hr. 87.75<br />
±0.99<br />
2nd hr. 81.0<br />
±0.75<br />
3rd hr. 73.37<br />
±0.78<br />
Treated<br />
Group-II<br />
(Mean<br />
± SEM)<br />
61.76<br />
±0.45<br />
14.80<br />
±0.98<br />
58.87<br />
±2.89<br />
54.87<br />
±1.12<br />
41.0<br />
±1.57<br />
Percentage<br />
decreased<br />
Significance<br />
level<br />
I vs II<br />
39.49 p
Effects of ascorbic acid and α-tocopherol in arsenic toxicity<br />
Fig. 4: Effect of ascorbic acid & α-tocopherol co-administration on arsenic induced alterations in sperm count (values are expressed in<br />
mean ±SEM).<br />
10^6/cauda<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Fig. 5: Effect of ascorbic acid & α-tocopherol co-administration on arsenic induced alterations in sperm viability (values are expressed in<br />
mean ±SEM).<br />
percentage<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
Group-I Group-II Group-III<br />
Group-I Group-II Group-III<br />
Fig. 6: Effect of combined application of ascorbic acid & α-tocopherol on arsenic induced alterations in sperm motility (values are expressed<br />
in mean ±SEM).<br />
Percentage<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
Group-I Group-II Group-III<br />
Ist hr.<br />
2nd hr.<br />
3rd hr.<br />
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58
Effects of ascorbic acid and α-tocopherol in arsenic toxicity<br />
Fig. 7: Effect of ascorbic acid & α-tocopherol co-administration on arsenic induced alterations in testicular conjugated dienes level (values<br />
are expressed in mean ±SEM).<br />
Fig. 8: Effect of ascorbic acid & α-tocopherol co-administration on arsenic induced alterations in testicular MDA level (values are expressed<br />
in mean ±SEM).<br />
‘<br />
nmole/ mg of tissue<br />
45<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
nmole/mg of tissue<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
Group-I Group-II Group-III<br />
Group-I Group-II Group-III<br />
Fig. 9: Effect of ascorbic acid & α-tocopherol co-administration on arsenic induced alterations in testicular CAT activity (values are<br />
expressed in mean ±SEM).<br />
200<br />
180<br />
160<br />
140<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Fig. 10: Effect of ascorbic acid & α-tocopherol co-administration on alterations in testicular SOD activity induced by arsenic (values are<br />
expressed in mean ±SEM).<br />
unit/ mg of tissue<br />
mmole H2O2<br />
consumption/mg of<br />
1.6<br />
1.4<br />
1.2<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
tissue/min<br />
1<br />
0<br />
59<br />
Group-I Group-II Group-III<br />
Group-I Group-II Group-III<br />
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Effects of ascorbic acid and α-tocopherol in arsenic toxicity<br />
1a. Section of control testis showing normal features x 10<br />
1b. Section of one seminiferous tubule of control testis showing normal features x 40<br />
2a. Section of arsenic treated testis showing increases in luminal areas and reduced spermatozoal mass x 10<br />
2b. Section of arsenic treated one seminiferous tubule showing disintegrated cell membrane and disorganized cellular orientation x 10<br />
3a. Section of antioxidant vitamins supplemented testis showing features towards normalcy x 10<br />
3b. Section of antioxidant vitamins supplemented one seminiferous tubule showing almost normal features x 40<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011<br />
60
Effects of ascorbic acid and α-tocopherol in arsenic toxicity<br />
values nearer to that of control (table-1, fig.6).<br />
The mean ± SEM (Standard error of mean) was<br />
calculated <strong>for</strong> each groups and the corresponding<br />
level of significance was calculated by using the<br />
previous statistical analysis.<br />
3. Testicular conjugated dienes: Testicular<br />
conjugated dienes level was significantly<br />
increased (p
Effects of ascorbic acid and α-tocopherol in arsenic toxicity<br />
radicals and thus the tocopheroxyl radical, α TO .<br />
is <strong>for</strong>med (36) which is regenerated to α-TOH<br />
by ascorbate present in the aqueous phase (37).<br />
This reduction of α-TO . by ascorbate prolongs<br />
the life span of this very important antioxidant,<br />
α-tocopherol and the resulting dehydroascorbate<br />
which is having little antioxidant activity is again<br />
reduced back to ascorbate by GSH dependent<br />
reductase. There<strong>for</strong>e the group III rats which<br />
received the combined application of ascorbic<br />
acid and α-tocopherol with arsenic treatment<br />
showed minimal level of alterations in the<br />
studied parameters suggesting the free radical<br />
mediated toxicity in testicular gametogenic<br />
activity caused by arsenic.Alterations of<br />
membrane potentials, loss of cellular functions<br />
and macromolecular damages caused by arsenic<br />
toxicity occur be<strong>for</strong>e the initiation of cell damage.<br />
This co-administration of antioxidant vitamins<br />
possibly prevents the cellular disorganization<br />
by maintaining the antioxidant defence system<br />
in the seminiferous tubules (fig.3a & 3b).<br />
Previous studies on arsenic induced subjects<br />
with the same antioxidant vitamins have also<br />
showed that these have the modulatory activity<br />
on arsenic induced apoptosis by improving<br />
the cellular antioxidant status and scavenging<br />
the free radicals (38). Here the co-treatment<br />
of ascorbic acid and α-tocopherol shows its<br />
efficacy in the prevention of testicular toxicity<br />
by restoring the antrioxidant status of the<br />
testicular tissue. Thus our observations indicate<br />
that the dietary supplimentation of ascorbic acid<br />
and α-tocopherol could ameliorate the arsenic<br />
induced testicular toxicity.<br />
Conclusion:<br />
The study reveals that the detrimental effects<br />
on gametogenic functions of rat testis during<br />
the chronic arsenic exposure can be reversed<br />
by simultaneous administration of antioxidant<br />
vitamins like ascorbic acid and α-tocopherol,<br />
as the loss of antioxidant defense system is one<br />
of the major causes behind the arsenic induced<br />
damages. Clinical trials with these vitamins<br />
62<br />
may be undertaken to find an easy and effective<br />
strategy to combat the detrimental effects on the<br />
reproductive system of the human population<br />
exposed to this silent environmental toxicant<br />
which is gradually increasing day by day and<br />
imposing a serious threat to people worldwide.<br />
Acknowledgement:<br />
The authors gratefully acknowledge the<br />
financial assistance from the Minor Research<br />
Project [No.F.PSW-074/09-10 (ERO)] provided<br />
by the University Grants Commission, New<br />
Delhi, India.<br />
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of ascorbic acid and α-tocopherol on arsenic induced<br />
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<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 64 - 68<br />
Evaluation of antioxidant status in Type 2 Diabetes Mellitus with and without complications<br />
*T. Vivian Samuel, **S. Smilee Johncy<br />
*Department of Biochemistry, **Department of Physiology, J J M Medical College, Davangere,<br />
Karnataka - 577004, India.<br />
(Received 21 st June 2010; Revised 11 th December 2010; Accepted 02 th January 2011)<br />
Corresponding Author<br />
Dr. T. Vivian Samuel,<br />
Email : tviviansamuel@yahoo.co.in<br />
Abstract<br />
Background & objectives:Oxygen free radicals have been implicated in pancreatic beta cell damage<br />
and genesis of diabetes as well as in pathogenesis of its complications like nephropathy, retinopathy and<br />
neuropathy. The present study was aimed to evaluate lipid peroxidation and antioxidant status in Noninsulin<br />
dependent diabetes mellitus (NIDDM) and to assess the same between NIDDM subjects with and<br />
without complications.<br />
Methods: 35 control subjects and 89 NIDDM were studied. Lipid peroxidation in terms of serum<br />
malondialdehyde (MDA), by thiobarbituric acid method, erythrocyte superoxide dismutase (SOD), reduced<br />
glutathione (GSH) and serum vitamin C were estimated using spectrophotometer. Fasting blood glucose<br />
was also measured.<br />
Results : The mean MDA levels in controls, NIDDM without complications and NIDDM with complication<br />
were 3.83 ± 0.26 nmol/ml, 4.73 ± 0.51 nmol/ml and 5.64 ± 0.50<br />
nmol/ml and Vitamin C levels were 1.58 ± 0.10 mg/dl, 0.89 ± 0.20 mg/dl and 0.67 ± 0.07 mg/dl respectively.<br />
Lipid peroxidation was significantly raised in NIDDM with complications than in NIDDM without<br />
complications and in control subjects. The mean SOD, GSH, Vit C levels were significantly lowered in<br />
NIDDM.<br />
Interpretion & Conclusion: The results suggest that the antioxidant deficiency and excessive peroxidation<br />
damage appear very early in NIDDM, well be<strong>for</strong>e the development of complications. Therapeutic measures<br />
to increase antioxidants and control lipid peroxidation are warranted <strong>for</strong> effective control of its complications.<br />
Keywords: Ascorbic acid, Malondialdehyde, Reduced glutathione, Superoxide dismutase.<br />
Introduction:<br />
In diabetes mellitus there is an imbalance in<br />
the antioxidant protective mechanism, leading<br />
to oxygen stress in the cells. Toxic oxygen<br />
derived products are generated in all aerobic<br />
cells which include superoxide radical (O –<br />
2 ),<br />
hydrogen peroxide (H O ) and hydroxyl radical<br />
2 2<br />
(OH – ), the latter being the most lethal1 . All<br />
64<br />
biomolecules may be attacked by free radicals,<br />
but lipids are the more susceptible. The human<br />
cells are abundant and rich sources of PUFA,<br />
hence are readily attacked by oxidizing radicals<br />
by a process known as lipid peroxidation to<br />
<strong>for</strong>m lipid peroxides. This is a self perpetuating<br />
chain reaction and highly damaging 2 . One of<br />
the most important intracellular antioxidant<br />
substances is an enzyme, superoxide dismutase<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
which scavenges free radicals by converting the<br />
harmful superoxide ion into stable hydrogen<br />
peroxide. Ascorbic acid’s role as an antioxidant<br />
is indicated by its known free radical scavenging<br />
action. As a reducing and antioxidant agent, it<br />
directly reacts with O – and OH and various lipid<br />
2<br />
hydroperoxides. As an antioxidant, Vitamin C<br />
exerts a sparing effect on the antioxidant actions<br />
of Vitamin E and selenium. 3 Glutathione in<br />
erythrocyte exists in reduced <strong>for</strong>m (GSH). GSH<br />
serves as both a nucleophile and an effective<br />
reductant by interacting with numerous<br />
electrophilic and oxidizing compounds such as<br />
H O , O 2 2 – and OH. Reduced glutathione readily<br />
2<br />
interacts with free radicals, notably hydroxyl<br />
and carbon radicals, by donating a hydrogen<br />
atom. Such reactions can provide protection by<br />
neutralizing reactive OH, which is considered<br />
a major source of free radical damage 4 . Tare<br />
RS and co-workers illustrated the prevalence<br />
of oxidative stress in diabetes by a highly<br />
significant increase in concentration of MDA<br />
and diminished activity of SOD in comparison<br />
to the control subjects. MDA exhibited a<br />
positive correlation with fasting blood glucose<br />
and glycosylated hemoglobin emphasizing the<br />
phenomenon of antioxiditive glycosylation<br />
of proteins 5 . Hyperglycemia and oxidative<br />
stress has been recognized as cardinal features<br />
of diabetes. Hyperglycemia causes glucose<br />
dependent chemical alterations in body proteins<br />
(non-enzymatic protein glycation) responsible<br />
<strong>for</strong> changes in protein structure and function,<br />
contributing to the pathophysiology of diabetes<br />
and its complications. The present study aims<br />
to assess the lipid peroxidation and antioxidant<br />
status in non-insulin dependent diabetes mellitus<br />
(NIDDM) and also their correlation to glycemic<br />
control and development of complications.<br />
Materials and methods :<br />
The present study was conducted on NIDDM<br />
patients and control subjects. 35 control<br />
subjects who were healthy non-smokers and<br />
non-alcoholics at the time of study and 89<br />
65<br />
NIDDM patients who were on treatment were<br />
studied. The selected subjects included both<br />
males and females in the age group of 40-65<br />
years. Among the NIDDM patients, 49 were<br />
without any complications and 40 were with<br />
microvascular complications like Retinopathy,<br />
Peripheral neuropathy, Nephropathy and both<br />
Retinopathy and Peripheral neuropathy. NIDDM<br />
patients with infectious diseases, smokers and<br />
alcoholics were all excluded from this study.<br />
Overnight fasting blood sample was collected<br />
<strong>for</strong> estimation of fasting blood glucose, serum<br />
MDA, serum ascorbic acid, erythrocyte SOD<br />
and reduced glutathione. Fasting blood glucose<br />
was estimated by O-Toluidine method6 . Serum<br />
MDA was estimated by Thiobarbituric acid<br />
(TBA) method, in which one molecule of MDA<br />
reacts with two molecules of TBA and yields a<br />
pink crystalline pigment which is measured at<br />
535 nm7 . Serum ascorbic acid was estimated by<br />
2,4 – dinitrophenyl hydrazine (DNPH) method<br />
in which ascorbic acid is oxidized by copper<br />
to <strong>for</strong>m dehydroascorbic acid, which when<br />
treated with DNPH and sulfuric acid <strong>for</strong>ms<br />
orange colour which is measured at 520 nm8 .<br />
Blood reduced glutathione was estimated by<br />
5,5 dithiobis – 2 – nitrobenzoic acid (DTNB)<br />
method. DTNB is readily reduced by<br />
sulphydryl compounds, <strong>for</strong>ming a highly<br />
coloured yellow anion. Optical density is<br />
measured at 412 nm. 9 Superoxide dismutase<br />
in hemolysate was estimated using Nitroblue<br />
Tetrazolium (NBT). Illumination of riboflavin<br />
in the presence of oxygen and electron donors<br />
like methionine or EDTA generates superoxide<br />
anion. The reduction of nitroblue tetrazolium<br />
– by O was followed at 560 nm using a<br />
2<br />
spectrophotometer10 .<br />
Statistical analysis :<br />
All the values are expressed as their Mean ± S.D.<br />
Data were subjected <strong>for</strong> analysis using unpaired<br />
‘t’ test, <strong>for</strong> comparison between two groups and<br />
One way ANOVA (F-test) <strong>for</strong> multiple group<br />
comparison.<br />
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Table – 1 : Comparison of FBS, MDA, Vit C, GSH and SOD between controls, NIDDM with complications and<br />
NIDDM without complications<br />
Groups No<br />
FBS<br />
(mg/dl)<br />
MDA<br />
(nmol/ml)<br />
Vit. C<br />
(mg/dl)<br />
GSH<br />
(mg/dl)<br />
SOD<br />
(U/ml)<br />
Controls (I) 35 97.7 ± 5.7 3.83 ± 0.26 1.58 ± 0.10 54.6 ± 3.25 5.16 ± 1.03<br />
NIDDM without<br />
complications(II)<br />
NIDDM with<br />
complications(III)<br />
49 207.0 ± 21.2 4.73 ± 0.51 0.89 ± 0.20 48.6 ± 2.49 3.91 ± 0.49<br />
40 258.1 ± 20.7 5.64 ± 0.50 0.67 ± 0.07 44.9 ± 1.81 3.48 ± 0.27<br />
I versus II p < 0.001 p < 0.001 p < 0.001 p < 0.001 p< 0.001<br />
I versus III p < 0.001 p < 0.001 p< 0.001 p< 0.001 p < 0.001<br />
II versus III p < 0.001 p < 0.001 p< 0.001 p < 0.001 p< 0.001<br />
Table – 2 : Comparision of FBS, MDA, Vit. C, GSH and SOD between NIDDM without complications and<br />
NIDDM with different complications<br />
Groups No.<br />
NIDDM without<br />
complications (II)<br />
NIDDM with retinopathy<br />
(III a)<br />
NIDDM with Neuropathy<br />
(III b)<br />
NIDDM with<br />
Nephropathy(IIIc)<br />
NIDDM with retinopathy<br />
Neuropathy (III d)<br />
FBS<br />
(mg/dl)<br />
MDA<br />
(nmol/ml)<br />
Vit. C<br />
(mg/dl)<br />
GSH<br />
(mg/dl)<br />
SOD<br />
(U/ml)<br />
49 207.0 ± 21.2 4.73 ± 0.51 0.89 ± 0.20 48.6 ± 2.49 3.91 ± 0.49<br />
10 255.8 ± 13.7 5.65 ± 0.21 0.71 ± 0.06 45.2 ± 1.59 3.56 ± 0.25<br />
11 249.6 ± 17.2 5.60 ± 0.19 0.67 ± 0.08 45.8 ± 1.73 3.63 ± 0.22<br />
10 250.3 ± 12.1 5.73 ± 0.20 0.68 ± 0.07 44.8 ± 1.51 3.49 ± 0.29<br />
9 279.9 ± 25.1 5.59 ± 1.02 0.61 ± 0.06 43.5 ± 1.89 3.21 ± 0.21<br />
II versus III a p < 0.001 p < 0.001 p < 0.05 p < 0.001 p < 0.05<br />
II versus III b p < 0.001 p < 0.001 p < 0.01 p < 0.001 N.S.<br />
II versus III c p < 0.001 P < 0.001 p < 0.01 p < 0.001 p < 0.05<br />
II versus III d p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001<br />
Table – 3 : <strong>An</strong>alysis of MDA, Vit. C, GSH, SOD in NIDDM patients depending on the glycemic control<br />
Groups No. of cases<br />
MDA<br />
(nmol/ml)<br />
Vit. C (mg/dl) GSH (mg/dl)<br />
SOD<br />
(U/ml)<br />
A 18 4.29 ± 0.33 1.04 ± 0.27 50.7 ± 1.7 4.17 ± 0.41<br />
B 45 5.18 ± 0.48 0.78 ± 0.12 46.8 ± 2.2 3.71 ± 0.44<br />
C 26 5.66 ± 0.59 0.65 ± 0.05 44.5 ± 1.6 3.43 ± 0.23<br />
F – value 41.7 43.3 54.0 19.4<br />
p – value < 0.001 < 0.001 < 0.001 < 0.001<br />
Groups A – FBS < 200 mg/dl B – FBS 200 – 250 mg/dl C – FBS > 250 mg/dl<br />
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66
Results<br />
It is evident from the Table 1, that there is highly<br />
significant (p < 0.001) increase in FBS and MDA<br />
levels in NIDDM patients with complications<br />
than in NIDDM patients without complications<br />
and in controls. There is highly significant (p <<br />
0.001) decrease in vitamin C, GSH and SOD<br />
in NIDDM patients with complications than<br />
in NIDDM patients without complications<br />
and in control subjects. It is evident from the<br />
Table 2, that there is highly significant (p <<br />
0.001) increase in FBS and MDA in NIDDM<br />
patients with more than one complications<br />
when compared with NIDDM patients with<br />
one complication and NIDDM patients without<br />
any complications. There is highly significant<br />
(p < 0.001) decrease in Vitamin C, GSH and<br />
SOD in NIDDM patients with more than one<br />
complication when compared with NIDDM<br />
patients with one complication and NIDDM<br />
patients without any complications. As shown<br />
in Table 3, it is evident that in NIDDM patients,<br />
the mean MDA level increases with poorly<br />
controlled blood glucose level. On the other<br />
hand the mean Vitamin C, GSH and SOD levels<br />
decrease with uncontrolled blood glucose level.<br />
Discussion :<br />
Free radical mediated cytotoxic process of<br />
lipid peroxidation appears to have a role in<br />
development of the multifactorial disease,<br />
diabetes mellitus. The role of oxygen free<br />
radicals in diabetes and its complications is<br />
being propagated and the weight of supporting<br />
evidence is steadily increasing. In diabetes<br />
mellitus there is an increase in the rate of<br />
production of free radicals and H 2 O 2 due to the<br />
autoxidation of glucose. It is known that chronic<br />
hyperglycemia leads to glycosylation of proteins<br />
like Hb which could also lead to the <strong>for</strong>mation<br />
of free radicals because of autoxidation of<br />
the Amadori products. In poorly controlled<br />
diabetes mellitus, glucose oxidation through<br />
67<br />
the pentose phosphate pathway leads to an<br />
excessive <strong>for</strong>mation of NADPH, which in turn<br />
can promote lipid peroxidation in the presence<br />
of cytochrome P-450 system. Oxyhemoglobin<br />
in erythrocytes could act like cytochrome<br />
P-450 in the presence of NADPH and this could<br />
induce increased lipid peroxidation 11 .<br />
According to Ranjini K. Sundaram et al.,<br />
lipid peroxidation was significantly raised<br />
within the first 2 years of diagnosis. MDA was<br />
significantly elevated in NIDDM and appeared<br />
to be associated with the multiplicity of<br />
complications 12 . In our study, mean MDA level<br />
in NIDDM patients increased progressively<br />
with the increase in blood glucose level. MDA<br />
levels have a positive correlation with increase<br />
in blood glucose levels. Similar findings have<br />
been reported by many workers, Sushil K. Jain<br />
et al., Gallou G.et al 13 . The mean MDA level was<br />
elevated in NIDDM patients with complication<br />
when compared to NIDDM patients without<br />
any complications and in control subjects.<br />
MDA concentration was significantly higher in<br />
NIDDM than in control and also increased in<br />
patients macroangiopathy than patients with no<br />
vascular complications 14 .A likely explanation<br />
<strong>for</strong> a lower Vitamin C status in diabetics is that<br />
ascorbic acid is actively transported into the cells<br />
in its partially oxidized <strong>for</strong>m as dehydroascorbic<br />
acid, which is promptly converted to ascorbic<br />
acid within the cell. The carrier of ascorbic<br />
acid transport serves also to transport glucose<br />
and is inhibited in transporting ascorbic acid<br />
by the hyperglycemia of diabetes. The carrier<br />
number is also subject to increase by insulin and<br />
is believed to be low in diabetes 15 . Increased<br />
antioxidative glycosylation of hemoglobin may<br />
lead to imbalanced generation of free radicals<br />
like superoxide, thereby causing depletion of<br />
SOD which quenches it. Diminished activity<br />
of SOD points out to an exhausted antioxidant<br />
reserve which further exacerbates the oxidative<br />
stress. Excessive peroxidation is associated<br />
with reduced SOD activity in diabetes. Loss<br />
of SOD activity in the erythrocytes appear<br />
to be a function of the duration of diabetes.<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
SOD, inhibited by glycosylation, is lowered<br />
in poorly controlled diabetes mellitus. Due to<br />
the absence of protein synthesizing machinery<br />
in the erythrocytes, the inactivation SOD by<br />
glycosylation may be dominant factor in the loss<br />
of SOD activity observed. SOD deficiency is seen<br />
within 2 years of the detection of NIDDM and<br />
further these development of complications 16 .<br />
In diabetic subjects, the increased sorbitol<br />
synthesized caused NADPH depletion, which<br />
when deficient, limits the reduction of GSSG<br />
to GSH. There<strong>for</strong>e, a major decrease in GSH<br />
may profoundly impair free radicals scavenging<br />
activity, resulting in exacerbated cell damage<br />
after exposure to free radical generated by<br />
glucose autoxidation. The levels of glutathione<br />
are regulated by glutathione peroxidase and<br />
glutathione reductase. The decreased activity of<br />
glutathione reductase in diabetics together with<br />
the decreased transport rate of GSSG indicates<br />
that regeneration and transport systems, which<br />
decrease intracellular GSSG, are impaired in<br />
diabetics, when erythrocytes are exposed to<br />
oxidative stress 17 .<br />
Conclusion :<br />
Therapeutic measures to increase antioxidants<br />
and control lipid peroxidation are warranted<br />
<strong>for</strong> effective control of complications. From<br />
the observations reported, it may be concluded<br />
that antioxidant deficiency and excessive<br />
peroxidative damage appear very early in<br />
NIDDM, well be<strong>for</strong>e the development of<br />
complications.<br />
Acknowledgement:<br />
Authors are grateful to the Head of the<br />
Department Biochemistry, Principal, J J M<br />
Medical College,Davengere <strong>for</strong> their support<br />
and encouragement. Authors are deeply<br />
indebted to all volunteers who participated in<br />
this study.<br />
References<br />
68<br />
1) Halliwell B. Oxygen radicals : A commonsense look at their<br />
nature and medical importance. Lancet 1984; 1:1328-9.<br />
2) Dormandy TL. Free radical reactions in biological systems.<br />
<strong>An</strong>n R Coll Surg Engl 1980; 62(3):188-94.<br />
3) Byung PY. Cellular defenses against damage from reactive<br />
oxygen species. Physiol Rev 1994; 74(1):139-61.<br />
4) Burk RF. Protection against free radical injury by<br />
selenoenzymes. Pharmacol Ther 1990; 45(3):383-5.<br />
5) Tare RS. Role of hyperglycemia and protein glycation in<br />
aggravating oxidative stress associated with diabetes. Med<br />
J of West Indies 1999; 27: 56-9.<br />
6) Carl AB, Edward RA. Tietz text book of clinical chemistry.<br />
3 rd ed. Philadelphia : WB Saunders Company 1999; 965-6.<br />
7) Dillard CJ, Kunert KJ, Tappel AL. Effects of vitamin<br />
E, ascorbic acid and mannitol on alloxan –induced lipid<br />
peroxidation in rats. Arch Biochem Biophys 1982;<br />
216(1):204-12.<br />
8) Omaye ST, Turnbull JD, Sauberlich HE. Selected methods<br />
<strong>for</strong> the determination of ascorbic acid in animal cells,<br />
tissues and fluids. Methods Enzymol 1979; 62: 1-11.<br />
9) Ernest B, Olga D, Barbara Mk. Improved methods <strong>for</strong> the<br />
determination of blood glutathione. J Lab Clin Med 1963;<br />
61(5):882-8.<br />
10) Beauchamp C, Fridonch I. Suproxide dismutase. Improved<br />
assays and an assay applicable to acylamide gels. <strong>An</strong>al<br />
Biochem 1971; 44:276-87.<br />
11) Jain SK. Hyperglycemia can cause membrane lipid<br />
peroxidation and osmotic fragility in human red blood cells.<br />
The <strong>Journal</strong> of Biological Chemistry 1989; 264:21340-5.<br />
12) Ranjini KS, <strong>An</strong>usha B, Selvamani V, Moopil V, Rema M,<br />
Kalathinkal RS. <strong>An</strong>tioxidant status and lipid peroxidation<br />
in type II diabetes mellitus with and without complications.<br />
Clinical Science 1996; 90:255-60.<br />
13) Sushil KJ, Robert M, John D, John JH. Erythrocyte<br />
membrane lipid peroxidation and glycosylated hemoglobin<br />
in Diabetes. Diabetes 1989;38: 1539-43.<br />
14) Gallou G, Ruelland A, Legras B, Maugendra D, Allannic<br />
H, Cloarec L. Plasma malondialdehyde in type I and type<br />
II diabetic patients. Clin Chem Acta 1993;214:227-34.<br />
15) Kapeghain JC, Verlangieri AJ. The effects of glucose<br />
on ascorbic acid uptake in heart endothelial cells :<br />
possible pathogenesis of diabetic angiopathies. Life Sci<br />
1984;34(6):577-84.<br />
16) Peuchant E, Delmes MCB, Couchouron A, Dubourg L,<br />
Thomas MJ, Peromat A et al. Short term insulin therapy and<br />
normoglycemia. Effects on erythrocyte lipid peroxidation<br />
in NIDDM patients. Diabetes Care 1997;20(2):202-7.<br />
17) Murakami K, Kondo T, Ohtsuka Y, Fujiwara Y, Shimada<br />
M, Kawakami Y. Impairment of glutathione metabolism<br />
in erythrocytes from patients with diabetes mellitus.<br />
Metabolism 1989;38(8):753-8.<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
Clinico-Biochemical Profile of Hypokalemic Patients<br />
69<br />
<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 69 - 73<br />
*<strong>An</strong>il Kumar Pandey, **Varsha Vijay Akhade, *** M Sri Hari Babu,****Y Himabindu<br />
*Department of Physiology, GSL Medical College & General hospital,<br />
Rajahmundry - 533294, <strong>An</strong>dhra Pradesh, India.<br />
**Department of Physiology Bidar Institute of Medical <strong>Sciences</strong>, Bidar, Karnataka<br />
***Department of General Medicine, GSL Medical College,<br />
Rajahmundry - 533294, <strong>An</strong>dhra Pradesh, India.<br />
****Department of Obstetrics & Gynaecology, GSL Medical College,<br />
Rajahmundry - 533294, <strong>An</strong>dhra Pradesh, India.<br />
(Received 21 st September,2010;Revised 20 th December,2010; Accepted 3 rd March,2011)<br />
Corresponding Author:<br />
Dr. <strong>An</strong>il Pandey,<br />
E-mail: drpandeyak@yahoo.co.in<br />
Abstract<br />
Background and Objectives: Hypokalemia is a commonly encountered electrolyte disturbance with<br />
diverse range serious manifestations involving cardiovascular & neuromuscular systems. The kidney<br />
determines potassium homeostasis and excess potassium is excreted in the urine. The objective of the<br />
study was to find out clinico-biochemical spectrum of hypokalemia with special reference to preventable<br />
causative factors, so as to help early detection and appropriate management of such cases.<br />
Materials & Methods: Etiology of hypokalemia was assessed in patients admitted to Govt. Medical<br />
College & New Civil Hospital, Majura Gate, Surat over a period of 9-12 month. This was correlated with<br />
the clinical outcome and other clinical variables. Serum potassium was estimated by ion selective electrode<br />
method. During this period 896 investigations <strong>for</strong> electrolytes were carried out.<br />
Results: Out of these 378 investigations showed dyselectrolytemia. Hypokalemia was present in 76 patients.<br />
In majority of the cases hypokalemia was found to be associated with inadequate and/or inappropriate<br />
administration of fluid and/or drugs to the patients. However, patients receiving diuretics were at the highest<br />
risk as 39 out of 87 patients belonged to this category. Hypokalemia was observed in both the young as well<br />
as elderly patients.<br />
Conclusion: Hypokalemic patients with ECG changes had significant lower potassium levels than patients<br />
without ECG changes.<br />
However, the absolute correlation between the degree of potassium deficiency and adverse side effects<br />
was not found, possibly because the occurrence of side effects is related to both the degree and duration of<br />
potassium deficiency and underlying disease process.<br />
Keywords: Hypokalemia, Electrolyte, Diuretics.<br />
Introduction<br />
Hypokalemia is recurrently encountered in<br />
clinical medicine and has been anticipated to<br />
occur in approximately 20% of patients admitted<br />
to general medical and ICU wards(1). It may<br />
result from inadequate diet, transcellular shift<br />
(movement of potassium from serum into cells)<br />
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Clinico - Biochemical Profile of Hypokalemic Patients<br />
and medications (2), Renal & Gastrointestinal<br />
losses and medications especially diuretics<br />
(13). Kypokalemia is also seen <strong>for</strong> 24 hours<br />
after Insulin injection (3). Patients may be<br />
asymptomatic, identified only on routine<br />
electrolyte screening, or may have serious<br />
manifestations like muscular paralysis, cardiac<br />
arrhythmias and cardiac arrest (4-6). Untreated<br />
hypokalemia is associated with morbidity<br />
and mortality. As timely intervention yields<br />
encouraging results, early detection with high<br />
of suspicion is critical to survival.<br />
Most cases of hypokalemia occur in the setting of<br />
specific disease states. Patients receiving thizide<br />
and loop diuretics are at the highest risk, with as<br />
many as 50% developing serum potassium level<br />
of less than 3.5 meq/L (7).Thiazide diuretics are<br />
more likely to cause hypokalemia than loop or<br />
osmotic diuretics. Individuals with secondary<br />
hyperaldosteronism, whether due to CCF,<br />
hepatic insufficiency, or nephritic syndrome may<br />
also exhibit hypokalemia. Though laboratory<br />
tests are the gold standard test <strong>for</strong> diagnosing<br />
changes in the serum electrolyte concentration,<br />
there may be delays in obtaining the results.<br />
The electrocardiogram (ECG) may be a useful<br />
diagnostic tool, if the clinician is aware of the<br />
possible changes resulting from abnormalities<br />
in the serum potassium concentration (10).<br />
In view of varied manifestations and etiological<br />
factors, an ef<strong>for</strong>t was made to study the clinicobiochemical<br />
spectrum of hypokalemia with<br />
special reference to preventable causative<br />
factors, so as to help early detection and<br />
appropriate management of such cases.<br />
Materials and Methods<br />
A prospective hospital based study was<br />
conducted at New Civil Hospital and Govt.<br />
Medical College, Surat during the period from<br />
April 2003 to August 2004. Laboratory reports<br />
of in-patients were screened to identify cases of<br />
hypokalemia. Relevant history including that of<br />
treatment, mode of presentation, investigation<br />
results, etiological diagnosis, clinical outcome<br />
70<br />
and other clinical variables were recorded.<br />
Serum potassium and sodium were estimated<br />
by ion selective electrode method on Instalyte,<br />
Transasia. The hypokalemia was graded as<br />
mild (3-3.5), moderate (2-3 meq/L) and severe<br />
(
Clinico - Biochemical Profile of Hypokalemic Patients<br />
deficits like quadriparesis, paraparesis and<br />
monoparesis being more commonly seen with<br />
severe hypokalemia. The extent of neuromuscular<br />
manifestations were significantly<br />
related to degree of hypokalemia.<br />
The various etiologies of hypokalemia included<br />
severe gastrointestinal losses from diarrhea<br />
and vomiting, renal tubular disorders, diabetic<br />
ketoacidosis, dialysis or inadequate replacement<br />
during prolonged parenteral nutrition or due to<br />
administration of nephrotoxic drugs or laxatives<br />
( Table I).<br />
Two cases of post – insulin infusion also<br />
presented with signs of hypokalemia. One<br />
patient admitted with loose motions was<br />
normokalemic on admission but was found<br />
to be having raised blood sugar. Accordingly,<br />
insulin was administered. Next day, the serum<br />
potassium level dropped to 0.9 meq/L. <strong>An</strong>other<br />
patient of ALL on chemotherapy developed<br />
acute pancreatitis. Investigations reveals<br />
normokalemia but random blood sugar was 92.<br />
mg/dL, <strong>for</strong> which insulin was administered. Next<br />
day the investigations reveledhypokalemia.<br />
These two patients had normal levels os<br />
sodium, while other patients of hypokalemia<br />
also had hyponatremia.<br />
A patient of aplastic anemia with skin infection<br />
was being treated with amphotericin B. Initially<br />
the patient was normokalemic but during the<br />
course of 12-15 days of treatment , gradually<br />
developed hypokalemia. This was because of<br />
toxic effects of drugs on renal tubules. A total<br />
six patients died during the period of study.<br />
Discussion<br />
Hypokalemia has diverse clinical manifestations.<br />
Mild hypokalemia is usually asymptomatic,<br />
while moderate hypokalemia results in<br />
confusion, disorientation, weakness and<br />
discom<strong>for</strong>t of muscles. On occasion, moderate<br />
hypokalemia causes cramps during exercise.<br />
<strong>An</strong>other symptom of moderate hypokalemia<br />
is a discom<strong>for</strong>t in the legs that is experienced<br />
while sitting still. The patient may experience<br />
71<br />
an annoying feeling that can be relieved by<br />
shifting the positions of the legs or by stamping<br />
the feet on the floor. Severe hypokalemia<br />
results in extreme weakness of the body and<br />
on occasion, in paralysis. The paralysis that<br />
occurs is flaccid paralysis or limpness. Paralysis<br />
of muscles of the lungs results in death. In the<br />
present study, 5 severely hypokalemic patients<br />
manifested with severe weakness with inability<br />
to stand and walk. Out of 5 patients, 4 patients<br />
developed severe hypokalemia gradually over a<br />
period of seven days.<br />
<strong>An</strong>other dangerous outcome of severe<br />
hypokalemia is abnormal heart beat (arrhythmia)<br />
that can lead to death from cardiac arrest. Several<br />
prospective studies shown that hypokalemia<br />
predisposes the patients to the development of<br />
a variety of ventricular arrhythmias, including<br />
a fatal ventricular fibrillation. However, cardiac<br />
arrhythmia was not detected in the present. Since<br />
hypokalemia may cause arrhythmia, the ECG<br />
is usually carried out in these patients. In our<br />
study ECG was carried out in 37 out of aq total<br />
of 76 patients of hypokalemia. The ECG in 14<br />
patients showed changes due to hypokalemia,<br />
while the remaining 23 patients did not show<br />
any ECG changes. This result is in con<strong>for</strong>mity<br />
with an earlier report (9). The probable reason<br />
<strong>for</strong> the inconsistency could be that the ECG<br />
reflects tissue potassium level rather than the<br />
serum potassiums.<br />
Diuretics are used to treat a number of medical<br />
conditions, including hypertension, congestive<br />
cardiac failure, liver disease and kidney disease.<br />
However, diuretic treatment can produce<br />
hypokalemia as a side effect and is the most<br />
common cause of hypokalemia in the elderly<br />
patients. The use of furosemide and thiazide,<br />
the two commonly used diuretic drugs were<br />
also the majaor cause of hypokalemia (n = 29)<br />
(38%) in the present study.<br />
More than 98% of the total body potassium<br />
is present in the intracellular compartment,<br />
predominantly in the skeletal muscle cells,<br />
enabling small changes in the distribution<br />
of potassium to alter the extracellular<br />
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Clinico - Biochemical Profile of Hypokalemic Patients<br />
concentration markedly. Certain hormones<br />
such as, insulin activates Na + -K + ATPase,<br />
which results in active potassium uptake.<br />
Active insulin administration produces rapid<br />
potassium shift from from the extracellular to<br />
intracellular space, resulting in hypokalemia.<br />
Our study revealed two such cases of post<br />
insulin hypokalemia. One patient presented<br />
with loose motion and the other was a case<br />
of ALL. Both the cases were normokalemic<br />
on admission. Routine investigations of the<br />
first patient revealed very high blood sugar<br />
<strong>for</strong> which insulin was administered. The<br />
patient of ALL was put on chemotherapy who<br />
developed acute pancreatitis during the course<br />
of his illness and blood sugar was found to be<br />
raised. Accordingly, insulin was administered.<br />
Following day of insulin administration,<br />
the investigation of both patients revealed<br />
hypokalemia strongly suggesting that the drug<br />
had caused their hypokalemia. The condition of<br />
both the patients improved and the serum levels<br />
changed to normokalemia following potassium<br />
supplementation subsequently.<br />
Other common causes of hypokalemia are<br />
excessive diarrhea or vomiting. Diarrhea results<br />
in various abnormalities, such as dehydration<br />
(loss in body water), hyponatremia and<br />
hypokalemia. Little potassium is excreted in<br />
the stool under normal conditions because of<br />
a low stool volume and a low stool potassium<br />
concentration. However in case of diarrhea,<br />
substantial amount may be excreted by this<br />
route. In our study the presenting clinical feature<br />
of loose motions was present in 9 cases. All the<br />
patients had concomitant hyponatremia also.<br />
Vomiting or nasogastric suctioning, can lead to<br />
hypokalemia due to prolonged loss of gastric<br />
contents. A small part of this potassium loss<br />
is direct because these body fluids contain<br />
5-8 meq/L potassium. More importantly,<br />
concomitant alkalosis and intravascular volume<br />
depletion contribute to renal potassium loss.<br />
Metabolic alkalosis results in bicarbonateuria,<br />
which increases potassium excretion both<br />
72<br />
directly and, as a cation to balance the negative<br />
charge of bicarbonate ions, and indirectly,<br />
through stimulation of urinary sodium excretion,<br />
leading to worsening of intravascular volume<br />
depletion (13). All the seven hypokalemic<br />
patients who presented with vomiting in our<br />
study had concomitant hyponatremia.<br />
Hypokalemia is known to be associated<br />
with adverse outcomes and may have fatal<br />
consequences (10). Six of our patients died<br />
during the hospital stay mostly due to effect<br />
of underlying disease. Despite frequent<br />
measurement of serum potassium levels in the<br />
hospital patients, hypokalemia is a relatively<br />
common occurrence. Potassium deficiency<br />
alters the function of several organs and most<br />
prominently affects the heart and neuromuscular<br />
system(12,14). These effects ultimately<br />
determine the morbidity and mortality related to<br />
this condition. Although hypokalemic patients<br />
with ECG changes had significant lower<br />
potassium levels than patients without ECG<br />
changes. However, the absolute correlation<br />
between the degree of potassium deficiency<br />
and adverse side effects was not found, possibly<br />
because the occurrence of side effects is related<br />
to both the degree and duration of potassium<br />
deficiency and underlying disease process.<br />
The accurate treatment of hypokalemia requires<br />
correct identification of the cause. Hypokalemia<br />
can be associated either with normal or decreased<br />
total body potassium content. Normal total<br />
body potassium with hypokalemia is a result of<br />
potassium redistribution from the extracellular<br />
to the intracellular space (10). All the 3 patients<br />
put on the insulin therapy had shown ECG<br />
changes. The hypokalemia in these patients is<br />
a result of redistribution of potassium. Total<br />
body potassium depletion can result from either<br />
renal or extrarenal potassium losses (13). We<br />
suggest that the clinician evaluating a patient<br />
with hypokalemia consider two broad groups<br />
of etiologies; redistribution and true potassium<br />
deficit (table I).<br />
Initial treatment of hypokalemia is often<br />
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Clinico - Biochemical Profile of Hypokalemic Patients<br />
inadequate, and sometimes treatment is delayed.<br />
Hospital-acquired hypokalemia was found to<br />
result from inadequate and / or inappropriate<br />
administration of fluids and / or drugs to patients.<br />
Patients receiving diuretics were at highest risk<br />
with 29 patients developing serum potassium<br />
levels of less than 3.5 meq/L. fifty percent of<br />
such patients have shown ECG changes as 9<br />
out of 17 patients in which ECG was carried<br />
out had changes related to hypokalemia (10).<br />
Hypokalemia was observed in both the young<br />
and elderly. It was associated with adverse<br />
outcomes. It may have fatal consequences, but<br />
timely intervention yields encouraging results<br />
(11).<br />
Conclusion<br />
A total of 976 investigations on electrolytes<br />
were carried out. In all 421 investigations<br />
had electrolyte imbalance. Hypokalemia was<br />
present in 76 patients in whom a total of 287<br />
investigations were carried out. ECG was done<br />
in 37 patients, out of which the findings were<br />
consistent with hypokalemia in only 14 patients.<br />
Remaining 23 patients did not show ECG<br />
changes. However, the absolute correlation<br />
between the degree of potassium deficiency and<br />
adverse side effects was not found, possibly<br />
because the occurrence of side effects is related<br />
to both the degree and duration of potassium<br />
deficiency and underlying disease process.<br />
References<br />
73<br />
1. Gary GS, and Brenner BM. Fluid and electrolyte disturbance.<br />
In: Harrison’s Principles of Internal Medicine. Mc-<br />
Graw-Hill, New York,14 th edi.272-4.<br />
2. Cohn JN, Kowey PR, Welton PK. New guidelines <strong>for</strong> potassium<br />
replacement in clinical practice: a contemporary<br />
review by the national council on potassium in clinical<br />
practice. Arch Intern Med 2000; 160 (16); 2429-2436.<br />
3. Massmi M, Akikatsu N and Yohei T. Electrolyte disorder<br />
following massive insulin overdose in a patient type II<br />
Diabetes. Intern Med 2000;39:55-57.<br />
4. Knochel JP. Neuromuscular manifestation of electrolyte<br />
disorders.Am J med 1982; 72: 521-35.<br />
5. Helfant RH. Hypokalemia and arrhythmias. Am J Med<br />
1986; 80 (suppl 4-A): 13-22.<br />
6. Cannon P. Recognizing and treating cardiac emergencies<br />
due to potassium imbalance. J Cardiovascular Med 1983;<br />
4:467-76.<br />
7. shilliday IR. Loop diuretics in the management of acute<br />
renal failure. A prospective , double blind, placebo control,<br />
randomized study. Nephrol Dial transplant 1997; 12:2592.<br />
8. Mandal AK. Hypokalemia and hyperkalemia. Med Clin<br />
North Am 1997;81:611-39.<br />
9. Singhi S, Marudkar A. Hypokalemia in a pediatric intensive<br />
care unit. Indian pediatr 1995; 33:9-14.<br />
10. A Webster, W Brady, F Morris Recognising signs of danger:<br />
ECG changes resulting from an abnormal serum<br />
potassium concentration.Emergency Medicine <strong>Journal</strong><br />
2002;19:74-77; doi:10.1136/emj.19.1.74<br />
11. Kamel KS: Disorder potassium homeostasis: <strong>An</strong> approach<br />
based on pathophysiology. Am j Kidney Dis 1994; 24:597,.<br />
12. Latronico N, Shehu I, Seghelini E. Neuromuscular sequelae<br />
of critical illness. Curr Opin Crit Care 2005;11(4):381-90.<br />
13. Assadi F. Diagnosis of hypokalemia: a problem-solving approach<br />
to clinical cases. Iran J Kidney Dis 2008;2(3):115-22.<br />
14. Guideline of ECC Committee, Subcommittees and Task<br />
Forces of the American Heart Association. 2005 American<br />
Heart Association Guidelines <strong>for</strong> Cardiopulmonary Resuscitation<br />
and Emergency Cardiovascular Care. Circulation<br />
2005;112(24)(Suppl-IV),1-203.<br />
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74<br />
<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 74 - 77<br />
<strong>An</strong>tifungal activity of Bacopa monniera against Dermatophytic Fungus<br />
*S.R.Ayyappan, **R.Srikumar, ***R.Thangaraj, ****R.Jegadeesh, ****L. Hariprasath<br />
* Department. of Microbiology,Sathyabama University Dental College & Hospital<br />
Jeppiaar Nagar, Old Mahabalipuram Road,Chennai 600 119, India<br />
** Department of Microbiology,Bharathidasan University College <strong>for</strong> Women,<br />
Orathanadu-614625,Thanjavur, Tamilnadu, India<br />
*** Ecoscience Research Foundation,East Coast Road, Palavakkam, Chennai 600041<br />
Tamil Nadu, India.<br />
**** Center <strong>for</strong> Advance Studies in Botany, University Of Madras,<br />
Guindy Campus Chennai- 600 025, Tamil Nadu, India.<br />
(Received 12 th June, 2010; Revised 10 th December, 2010; Accepted 20 th February, 2011)<br />
Corresponding Author<br />
Dr.R.Srikumar,<br />
E-mail: rsrikumar_2003@yahoo.co.in<br />
Abstract<br />
Background & Objectives: Dermatophytes are fungi that can cause infections in keratin tissue constitute<br />
a serious problem, especially in tropical and subtropical countries. <strong>An</strong>timicrobials of plant origin are effective<br />
in the treatment of infectious diseases while simultaneously mitigating many side effects that are often<br />
associated with synthetic antimicrobials. The lack of data on antifungal activity of Bacopa monniera makes<br />
this study unique.<br />
Methods: In the present study crude aqueous and ethanolic extract of B. monniera were prepared and their<br />
antifungal effects were assessed against the dermatophytic fungi namely Aspergillus niger, Aspergillus<br />
flavus, Trichophyton rubrum and Microsporum.<br />
Results: Result showed ethanolic extract had high inhibitory action against the studied fungus when<br />
compared to aqueous extract. In both ethanolic and aqueous extract the maximum inhibition activity was<br />
observed against T. rubrum 80% followed by Microsporum, A. flavus and A. niger. In addition this study<br />
also showed ethanolic extract is an ideal solvent.<br />
Conclusion: The present study revealed that B. monniera have antifungal activity. Among the extracts<br />
evaluated in this study, ethanolic extract showed highest antifungal activity.<br />
Key words: Bacopa monniera; Dermatophyte; <strong>An</strong>tifungal activity<br />
Introduction<br />
Dermatophytes are fungi that can cause<br />
infections of the skin, hair, and nails due to their<br />
ability to utilize keratin. The organisms are<br />
transmitted by either direct contact with infected<br />
host by direct or indirect contact with infected<br />
exfoliated skin or hair. These infections, which<br />
constitute the most frequent fungal diseases in<br />
human, are widespread in tropical countries.<br />
Due to the increasing development of drug<br />
resistance in human pathogens as well as the<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
appearance of undesirable effect of certain<br />
antimicrobial agents, there is a need to search <strong>for</strong><br />
new agents. Plants are among the most important<br />
and common sources of potentially valuable<br />
new drugs. Medicinal plants are widely used <strong>for</strong><br />
treatment of diseases all over the world. Herbal<br />
medicines have been used in treatment of the<br />
various diseases and <strong>for</strong> maintenance health (1).<br />
In addition antimicrobials of plant origin are<br />
effective in the treatment of infectious diseases<br />
while simultaneously mitigating many side<br />
effects that are often associated with synthetic<br />
antimicrobials (2). B. monniera belongs to the<br />
family Scrophulariaceae, commonly called<br />
as Brahmi, Neer brahmi etc. B. monniera is a<br />
small creeping herb with numerous branches,<br />
small oblong leaves and light purple or small<br />
and white flowers, with four or five petals. B.<br />
monneri is reported <strong>for</strong> its tranquilizing (3),<br />
sedative (4), stabilization of mast cells (5),<br />
anti-inflammatory activity via inhibition of<br />
prostaglandin synthesis (6), anticancer effect by<br />
inhibiting the DNA replication in cancer cells<br />
(7). The present study describes the antifungal<br />
potential of aqueous and ethanolic extract of B.<br />
monniera against the dermatophytic fungi.<br />
Materials and Methods<br />
Plant Material<br />
B. monniera was collected from the <strong>An</strong>na<br />
Sidha College, Chennai, Tamil Nadu, India and<br />
taxonomically identified by, the Department of<br />
Botany, University of Madras, Chennai, India,<br />
and specimens were deposited at an herbarium<br />
(No: CASBH12).<br />
Aqueous Extract<br />
The entire plant of B. monniera was grounded<br />
into fine powder, maintained at 60 ºC <strong>for</strong> 3 hr in<br />
sterile distilled water. The resulting suspensions<br />
were filtered and evaporated <strong>for</strong> dryness at 60°C<br />
in vacuo.<br />
Ethanolic Extract<br />
75<br />
B. monniera (coarse powder) was put in a<br />
soxhlet extractor containing 70% of ethanol.<br />
The resulting extract was preserved at 5 o C in an<br />
airtight bottle until further use.<br />
Assay of <strong>An</strong>tifungal Activity<br />
The fungus A. niger, A. flavus, T. rubrum<br />
and Microsporum used in this study were<br />
maintained by culturing on Sabouraud dextrose<br />
agar (SDA) at 28 °C. The antifungal test was<br />
per<strong>for</strong>med by employing the method described<br />
by Yongabi with slight modification (8). In<br />
briefly 500mg of each extract was diluted with<br />
the assay media (100 ml). A uni<strong>for</strong>m portion of<br />
the test fungi was removed using a 5mm steel<br />
borer and aseptically placed on the assay media.<br />
All plates were carefully sealed all around with<br />
a masking tape to avoid any aerial contaminants<br />
and carefully incubated at 28°C <strong>for</strong> 7 days. The<br />
rate of mycelia growth was measured in mm on<br />
8 th day. Ketoconazole (50mg/ml) was used as an<br />
experimental positive control and water served<br />
as the negative control.<br />
Calculation<br />
Percentage of Mycillial inhibition = [(dc-dl)/dc]<br />
x 100<br />
dc = colony diameter in negative control, d1<br />
colony diameter in extract treatment<br />
Results<br />
The studied aqueous and ethanolic crude<br />
extracts had antifungal activities against the<br />
studied fungi, but the activity of inhibition<br />
varied <strong>for</strong> the fungi with respect to the type of<br />
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Table. 1 Effect of aqueous and ethanolic extracts of B. monniera on the growth rate of A. niger, A. flavus, T.<br />
rubrum and Microsporum sp (inhibition expressed in percentage).<br />
extract (Table. 1). In aqueous extract maximum<br />
inhibition activity was observed against T.<br />
rubrum (40%) followed by Microsporum<br />
(38%), A. niger (31.4%), and A. flavus (31.2%).<br />
In ethanolic extract the maximum inhibition<br />
activity was observed against T. rubrum (50%)<br />
followed by Microsporum (47.6%), A. niger<br />
(42.8%) and A. flavus (40.6%). Overall result<br />
showed ethanolic extract had high inhibitory<br />
action against the studied fungus with respect to<br />
the aqueous extract.<br />
Discussion<br />
Plant Extract A. niger A. flavus T. rubrum Microsporum<br />
Aqueous extract of B.<br />
monniera<br />
Ethanolic extract of B.<br />
monniera<br />
The emergence of anti-fungal resistant strain<br />
of various fungi particularly dermatophyte<br />
has prompted research into developing new<br />
strategies <strong>for</strong> fighting fungal infections which<br />
may be less toxic to man (9). Both the aqueous<br />
and ethanolic extracts of B. monniera showed<br />
inhibitory activity against the studied fungus<br />
(Table. 1). Ethanolic extract showed high<br />
inhibitory action against the studied fungus<br />
when compared to aqueous extract. Most of<br />
the phytochemcials already identified in herbs<br />
are reportedly aromatic or saturated organic<br />
molecules which make ethanol as an ideal<br />
solvent this might be the reason <strong>for</strong> a better<br />
antifungal activity observed in the ethanolic<br />
extract (10). The inhibitory action of both the<br />
31.4 31.2 40 38<br />
42.8 40.6 50 47.6<br />
Ketoconazole Control 71.4 75 85 76.1<br />
76<br />
extracts observed in the present study might be<br />
due to rupture of the cytoplasmic membrane of<br />
the fungal cell which may leads to the damage<br />
of intracellular components (11).<br />
Conclusion<br />
This study evidently concludes that B. monniera<br />
have anti-fungal activity. The exact mechanism<br />
behind the antifungal activity of B. monniera<br />
warrants further studies.<br />
Acknowledgement<br />
The authors are thankful to Dr.R.Sheela Devi,<br />
Associate Professor, Department of Physiology,<br />
University of Madras, Chennai, Tamilnadu,<br />
India.<br />
References<br />
1. Sahito SR, Memon MA, Kazi TG, Kazi GH. Evaluation<br />
of mineral contents in medicinal plant Azadirachta indica<br />
(neem). J Chem Soc Pak 2003; 25: 139-143.<br />
2. Kokosha L, Polesny Z, Rada V, Nepovim A, Vanek<br />
T. Screening of some Siberian medicinal plants <strong>for</strong><br />
antimicrobial activity. J Ethnopharmacol 2002; 82: 51-53.<br />
3. Aithal HN and Sirsi M. Pharmacological investigation of<br />
Herpestis monneri. Indian J Pharmacol 1961; 23: 2-5.<br />
4. Malhotra CL and Das PK. Pharmacological studies of<br />
Herpestis monneri. Indian <strong>Journal</strong> of Medical Research<br />
1959, 47: 244-305.<br />
5. Samiulla DS, Prashanth D, Amit A. Mast cell stabilizing<br />
activity of Bacopa monnieri. Fitoterapia 2001; 72: 284-285.<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
6. Jain P, Khanna NK, Trehan T, PendseVK and Godhwani<br />
JL. <strong>An</strong>tiinflammatory effects of an Ayurvedic preparation,<br />
Brahmi Rasayan, in rodents. Indian J Exp Biol 1994; 32:<br />
633-636.<br />
7. Elangovan V, Govindasamy S, Ramamoorthy N,<br />
Balasubramaanian K. In vitro studies on the anticancer<br />
activity of Bacopa monnieri. Fitoterapia 1995; 66: 211-215.<br />
8. Yongabi KA, Dukku UH, Agho MO, Chindo IY. Studies<br />
on the antifungal properties of Urtica dioica, Urticaceae. J<br />
Phytomedicine and Therapeutic 2000; 5: 39-43.<br />
77<br />
9. Patterson TF, Revankar SG, Kirkpatrick WR, Dib O,<br />
Fothergill AW, Redding SW, Sutton DA, Rinaldi MG. Simple<br />
method <strong>for</strong> detecting fluconazole-resistant yeasts with<br />
chromogenic agar. J Clin Microbiol 1996; 34: 1794-1797.<br />
10. Cowan MM. Phytochemicals: health protective effects.<br />
Can J Diet Pract Res 1999; 60: 78-84.<br />
11. Chuang P, Lee C, Chou J, Murugan M, Shieh B, Chen H. <strong>An</strong>tifungal<br />
activity of crude extracts and essential oil of Moringa<br />
oleifera Lam. Bioresour Technol 2007; 98: 232–236.<br />
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78<br />
<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 78 - 83<br />
Prevalence of Extended Spectrum Beta Lactamase producing Enterobacteriaceae<br />
in Clinical specimens<br />
*G.S. Ravi, **B.V.S.Krishna, ***Namratha W.Nandihal,***Asha B.Patil and<br />
****M.R. Chandrasekhar.<br />
* Department of Microbiology, PES Institute of Medical <strong>Sciences</strong> and Research,<br />
Kuppam,Chittoor - 517425, <strong>An</strong>dhra Pradesh, India.<br />
**Department of Microbiology & Infection Control,University Hospital of North Staf<strong>for</strong>dshire<br />
NHS Trust,Central Pathology Laboratories,Hartshill Road, Stoke on Trent ST4 7PX,<br />
United Kingdom.<br />
***Department of Microbiology,Karnataka Institute of Medical <strong>Sciences</strong>,<br />
Hubli - 580022, Karnataka, India.<br />
*** Department of Microbiology, Karnataka Institute of Medical <strong>Sciences</strong>,<br />
Hubli - 580022, Karnataka, India.<br />
****Department of Microbiology,Belgaum Institute of Medical <strong>Sciences</strong>,<br />
Belgaum - 590001, Karnataka, India.<br />
(Received 1 st November, 2010; Revised 2 nd January, 2011; Accepted 20 th January, 2011)<br />
Corresponding Author<br />
Dr. Ravi. G. S<br />
Email: ravinavilehal@yahoo.com<br />
Abstract<br />
Background and objectives: Extended spectrum beta lactamases (ESBLs) are broad spectrum β-lactamase<br />
enzymes found in variety of Enterobactriaceae. Most strains producing these enzymes become highly<br />
effective at inactivating various β- lactam antibiotics and are frequently resistant to many other classes<br />
of antibiotics. The incidence of infection due to ESBL producing Enterobacteriaceae has markedly<br />
increased in recent years. The present work was undertaken to study the prevalence of ESBL producing<br />
Enterobactriaceae in clinical specimens at a tertiary care hospital.<br />
Methods: A total of 313 culture isolates of Enterobactriaceae obtained from different clinical specimens<br />
were included. All the isolates were subjected to screening tests <strong>for</strong> ESBL production & later <strong>for</strong> confirmation<br />
by phenotypic confirmatory combination disc diffusion test.<br />
Results: Out of 313 strains tested, 248 were short listed as potential ESBL producer by screening test and<br />
200(63.89%) were confirmed to be ESBL producers. ESBL rates were high among Klebsiella oxytoca<br />
(89.47%), Klebsiella pneumoniae (71.87%), and Escherichia coli (62.19%). They were commonly isolated<br />
from blood (92.30%), CSF (78.57%) and Urine (65.21%). The multidrug resistance was significantly higher<br />
among ESBL producers than in non-ESBL producers (p value of
Enterobacteriaceae in Clinical specimens<br />
Introduction<br />
Extended spectrum b-lactamases (ESBLs)<br />
are enzymes that confer resistance to and<br />
hydrolyze expanded spectrum cephalosporins<br />
like ceftazidime, cefotoxime, monobactam<br />
-azteronam and related oxyimino b-lactams as<br />
well as to older penicillins and cephalosporins<br />
(1,2). They arise from mutations in the genes<br />
<strong>for</strong> common plasmid mediated b-lactamases<br />
especially TEM and SHV enzymes, that alter the<br />
configuration of the enzyme near its active site<br />
to increase the affinity and hydrolytic ability of<br />
the b-lactamase <strong>for</strong> oxyimino compounds while<br />
simultaneously weakening the overall enzyme<br />
efficiency. Wide spread use of third generation<br />
cephalosporins and aztreonam is believed to<br />
be the major cause of the mutations leading to<br />
emergence of ESBLs (3).<br />
The first bacterial isolate capable of hydrolyzing<br />
extended spectrum cephalosporins producing<br />
an extended spectrum b-lactamase, SHV-2 was<br />
identified in Klebsiella ozaenae in Greece in<br />
1983 (4). There after, within one year there was<br />
wide spread dissemination of ESBL producing<br />
organisms in hospitals of Europe (5). The<br />
prevalence of ESBLs among clinical isolates<br />
varies from country to country and from<br />
institution to institutions. The first reported<br />
outbreak of ESBL producing organisms<br />
occurred in France in 1985(1, 6). More recently,<br />
outbreaks have occurred worldwide, including<br />
several cities in the United Sates (4, 7, 8, 9 ).<br />
ESBLs were initially associated with nosocomial<br />
outbreaks caused by a single enzyme producing<br />
strains. Recently more complex situations with<br />
a significant increase in community isolates<br />
producing ESBLs have been reported. It is<br />
necessary to identify the prevalence of these<br />
ESBL strains in hospitals and to characterize<br />
their epidemiology to control the spread of<br />
these strains (10). This upward trend in the<br />
prevalence of pathogens producing ESBLs is<br />
of increasing clinical concern. Infections with<br />
79<br />
these ESBL producing organisms continue to<br />
be associated with higher rates of mortality,<br />
morbidity and health care costs (11). Also<br />
ESBL mediated resistance poses problems <strong>for</strong><br />
invitro susceptibility testing and reporting (2).<br />
Prevalence of ESBLs in India varies from 6<br />
to 87% (12). With this background, the present<br />
study was undertaken to know the prevalence<br />
of ESBL producers among Enterobacteriaceae<br />
isolates and to compare antimicrobial<br />
susceptibility pattern among ESBL producers &<br />
Non ESBL producers, and to suggest appropriate<br />
therapeutic options.<br />
Materials and Methods<br />
The study was conducted at Microbiology<br />
laboratory, KIMS Hospital Hubli, Karnataka.<br />
Clinical Isolates: A total of 313 consecutive<br />
non repeat culture isolates Enterobacteriaceae<br />
were obtained from 280 different specimens<br />
from different specialty wards of Surgery,<br />
Orthopaedics, Otolaryngology and Neonatal<br />
intensive care unit (NICU) over a period of one<br />
year. The isolates were identified on the basis of<br />
conventional microbiological procedures.<br />
<strong>An</strong>timicrobial susceptibility test<br />
<strong>An</strong>timicrobial susceptibility testing was<br />
determined by Kirby-Bauer disc diffusion<br />
method as per NCCLS recommendations<br />
(13). <strong>An</strong>timicrobial disc used were<br />
Ampicillin(10µg), Co-trimoxazole(1.25/23.7<br />
5µg),Amoxycillin-clavulanicacid(20/10µg),T<br />
etracycline(30µg), Gentamicin(10µg),Amika<br />
cin(30µg),Netilmicin(30µg),Ciprofloxacin(5<br />
µg),Cephalexin(30µg),Cefuroxime(30µg),C<br />
ephotaxime(30µg),Ceftriaxone(30µg),Cefop<br />
erazone(30µg),Celftazidime (30µg), Cefixime(<br />
30µg),Cefpodoxime(30µg), Imipenem (10µg).<br />
<strong>An</strong>timicrobial susceptibility patterns of ESBL<br />
producers and non producers were compared.<br />
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Enterobacteriaceae in Clinical specimens<br />
Screening test <strong>for</strong> ESBLS<br />
As per the NCCLS guidelines, isolates<br />
showing inhibition zone size of ≤22mm<br />
with Ceftazidime(30µg), ≤25 mm<br />
with Ceftriaxone(30µg) and ≤27 mm<br />
Cefotaxime(30µg) were identified as potential<br />
ESBL producers and short listed <strong>for</strong> confirmation<br />
of ESBL production. NCCLS Phenotypic<br />
confirmatory combination disc diffusion test<br />
<strong>for</strong> detection of ESBL was per<strong>for</strong>med (13, 14).<br />
This test requires the use of third generation<br />
cephalosporin antibiotic disc alone and in<br />
combination with clavulanic acid. In this study,<br />
a disc of Ceftazidime (30 µg) alone and a disc<br />
of Ceftazidime +clavulanic acid (30µg/10µg)<br />
were used. Both the discs were placed at a<br />
distance of 25 mm apart, centre to centre on<br />
Mueller Hinton Agar (MHA) plate inoculated<br />
with a bacterial suspension of 0.5 McFarland<br />
turbidity standards and incubated overnight at<br />
37 O C. Difference in zone diameters with and<br />
without clavulanic acid were measured.<br />
Interpretation<br />
<strong>An</strong> increase in inhibition zone diameter of<br />
5mm <strong>for</strong> a combination disc of ceftazidime<br />
+ clavulanic acid disc versus the inhibition zone<br />
diameter around the ceftazidime disc alone,<br />
confirms ESBL production.<br />
Quality control<br />
Escherichia coli ATCC 25922. A 2mm increase<br />
in zone diameter <strong>for</strong> ceftazidime clavulanic acid<br />
disc compared with ceftazidime disc as negative<br />
control. Klebsiella pneumoniae ATCC 700603.<br />
A 5mm increase in ceftazidime clavulanic acid<br />
disc zone diameter as positive control.<br />
The plates <strong>for</strong> the susceptibility tests were<br />
prepared in the laboratory. Mueller – Hinton<br />
Agar (Dehydrated, Hi-Media) was used <strong>for</strong><br />
80<br />
all antimicrobial susceptibility tests. All the<br />
antibiotic discs were obtained from Hi-media<br />
laboratories Pvt. Limited, Mumbai, India.<br />
Statistical <strong>An</strong>alysis<br />
Chi-square test was used with appropriate<br />
correction <strong>for</strong> the observation. Where the cell<br />
frequency was less than five, Fisher exact test<br />
was applied to see the significance of difference<br />
between the resistance levels of various drugs in<br />
ESBL producer strains and non-ESBL producer<br />
strains using EPI 6 software.<br />
Results<br />
A total of 313, non-duplicate isolates belonging<br />
to the family Enterobacteriaceae recovered<br />
from 280 different clinical samples of 279<br />
patients (183Male/96 Female) submitted <strong>for</strong><br />
microbiological analysis from different clinical<br />
specialties, were studied. The major source of<br />
bacterial isolates were pus(215), blood(39),<br />
urine(23) and CSF(14), followed by Ear swab(8),<br />
Sputum(5), Ascitic fluid(3), Pleural fluid(3),<br />
Bile(2) and Throat swab(1) (Table 1). Genus<br />
Klesbsiella was the most common pathogen<br />
isolated from the tested samples, constituting<br />
115/313(36.74%) of the total isolates, Klebsiella<br />
pneumoniae represented 96/313(30.67%) and<br />
Klebsiella oxytoca19/313(6.07%). Followed<br />
by Escherichia coli 82/313(26.17%). Of the<br />
313 isolates, subjected <strong>for</strong> screening test, 248<br />
were short listed as potential ESBL producers<br />
with initial NCCLS screening method. NCCLS<br />
Phenotypic confirmatory identified 200 out of<br />
248 as ESBL producers, indicating a prevalence<br />
of 63.89% of ESBL production amongst all<br />
Enterobacteriaceae.<br />
Distribution of ESBL producers varied among<br />
different species of Enterobacteriaceae. Rates<br />
were high among Klebsiellae oxytoca(89.47%),<br />
Klebsiellae pneuminae(71.87%), Escherichia<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
Enterobacteriaceae in Clinical specimens<br />
coli(62.19%), Citrobacter freundii(62.50%),<br />
Proteus mirabilis(61.11%), Proteus<br />
vulagaris(50.00%) and Providencia<br />
species(44.00%) which were the major isolates<br />
in the group. Even among the organisms detected<br />
in less numbers, ESBLs were detected in 30% of<br />
Citrobacter koseri and in 100% of Enterobacter<br />
species and Morganella morgagni (Table 2).<br />
ESBL producers were more commonly isolated<br />
from blood 36/39(92.30%) followed by CSF<br />
11/14 (78.57%), urine 15/23(65.21) and pus<br />
131/215(60.93%) (Table1). The multi drug<br />
resistance was significantly higher among ESBL<br />
producers than in non-ESBL producers with<br />
a p value of
Enterobacteriaceae in Clinical specimens<br />
the usage of carbapenem antibiotics as the<br />
therapeutic alternative to β lactam antibiotics<br />
as indicated in other studies (22). This is<br />
especially important when treating serious<br />
infections. Misuse and overuse of carbapenems<br />
can lead to emergence of metallobetalactamases<br />
production among Enterobacteriaceae, which<br />
can still worsen the therapeutic options, as<br />
carbapenemases already exist and we have been<br />
contributing the growing types of them.<br />
Conclusion<br />
There is high prevalence of ESBL producers<br />
among Enetrobacteriaceae isolates, more so<br />
in Klebsiella species & Escherichia coli. Not<br />
just the blood & urine samples are the major<br />
source; even the other clinical samples should<br />
also be actively screened <strong>for</strong> ESBL producers<br />
to address the high prevalence rate of ESBL<br />
isolates. ESBL producing organisms often<br />
show co resistance to non β lactam antibiotics,<br />
resulting in limitation of therapeutic options.<br />
Use of a proper screening & confirmatory<br />
test <strong>for</strong> detection of ESBL producers, along<br />
with proper monitoring and judicious usage<br />
of extended spectrum cephalosporins, periodic<br />
surveillance of antibiotic resistance patterns,<br />
existence & effective functioning of Infection<br />
Control committee of doctors and nurses<br />
(microbiologists should lead the role of Infection<br />
control), Education of all grades of doctors<br />
about the dangers of antibiotic use, and having<br />
effective control over antibiotic prescription in<br />
the community and in hospitals, would prevent<br />
the increase occurrence of ESBL producers.<br />
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NCCLS 2002; M 100-S12<br />
14. Livermore DM. b Lactamases in laboratory and clinical<br />
resistance. Clin Microbiol Rew 1995; 8(4): 557-584.<br />
15. Spanu T, Luzzaro F, Perilli M, Amicosante G, Toniolo<br />
A, Fadda G et. al. Occurrence of Extended-Spectrum<br />
b-Lactamases in member of family Enterobacteriaceae<br />
in Italy. Implications <strong>for</strong> resistance to b-lactams and other<br />
antimicrobial drugs. <strong>An</strong>timicrobial Agents chemother<br />
2002; 46 (1):196-202.<br />
16. Jabeen K, Zafar A, Hasan R. Comparison of double disc<br />
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Enterobacteriaceae in Clinical specimens<br />
and combined disc method <strong>for</strong> detection of Extended<br />
Spectrum b Lactamases in Enterobacteriaceae. J Pak Med<br />
Assoc 2003; 53(1):534-536.<br />
17. Venezia SN, Munz OH, Scwartz D, Turner D, Kuzmenko<br />
B, Carmeli Y. Occurrence and Phenotypic characteristics<br />
of Extended Spectrum b Lactamases among the members<br />
of Enterobacteriaceae, Evaluation of Diagnostic tests. J<br />
Clin Microbiol 2003; 41(1):155-158.<br />
18. Datta P, Thakur A, Mishra B, Gupta V. Prevalence of<br />
clinical strains Resistant to various b-Lactamas in a<br />
Tertiary Care Hospital in India. Jpn J Infect Dis 2004;<br />
57:146-149.<br />
19. Mathur P, Kapil A, Das B, Dhawan B. Prevalence of<br />
Extended Spectrum b Lactamase producing GNB in a<br />
83<br />
tertiary care hospital. Indian J Med Res 2000; 15:153-157.<br />
20. Singhal S, Mathur T, Khan S, Upadhyay DJ, Chug S,<br />
Govind R, Rattan A. Evaluation of methods <strong>for</strong> Amp C b<br />
lactamases in Gram Negative clinical isolates from Tertiary<br />
care hospitals. Ind J Clin Microbiol 2005; 23(2):120-124.<br />
21. Jain A, Roy I, Gupta MK, Kumar M and Aggarwal SK.<br />
Prevalence of Extended Spectrum b Lactamases producing<br />
Gram negative bacteria in septicemic neonates in a tertiary<br />
care hospital. J Med Microbiol 2003; 52:421-425.<br />
22. Hansotia JB, Aggarwal B, Pathak AA, Saoji AM. Exended<br />
spectrum b lactamase mediated resistance to third<br />
generation cephalosporins in Klebsiella pneumoniae in<br />
Nagpur, Central India, Ind J Med Res 1997; 105:158-161.<br />
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84<br />
<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 84 - 89<br />
<strong>An</strong>ti-inflammatory effects of Allium sativum (Garlic) in experimental Rats<br />
*M.K.Jayanthi and **Murali Dhar<br />
*Department of Pharmacology and ** Department of Community Medicine, JSS Medical College,<br />
(A constituent college of JSS University), Mysore-570015, Karnataka, India.<br />
(Received 21 st September, 2010; Revised 15 th December, 2010; Accepted 10 th January, 2011)<br />
Corresponding Author<br />
Dr Jayanthi M.K.<br />
Email: dr_jmgatti@yahoo.com<br />
Abstract<br />
Background and Objectives:Non-steroid anti-inflammatory drugs (NSAIDs) are known to cause<br />
adverse effects. On the other hand, garlic is believed to have hypolipidaemic, antibacterial, anti-rheumatic<br />
and anti-inflammatory properties. Hence present study was undertaken to know the <strong>An</strong>ti-inflammatory<br />
effect of allium sativum in Albino Rats.<br />
Materials and Methods: A total of 36 rats were randomized into two groups of 18 each to be utilized<br />
by acute (Carrageenin induced paw edema) and chronic (Cotton pellet induced granuloma) inflammatory<br />
models. Further within each group the animals were randomly allocated to the control, standard (Piroxicam)<br />
and test (Garlic) drug groups. The rats were fed respective drugs orally 1 hour prior to experimentation.<br />
Reduction in paw edema size was recorded at four hours and in dry granuloma weight on 8 th day after<br />
sacrificing the animal. To compare the reductions among three groups, one-way ANOVA was per<strong>for</strong>med.<br />
Results: The effect of the test drug in terms of percent inhibition was found to be about 50% in acute and<br />
21% in chronic models. Also, the acute effect was similar to standard drug. Conclusion: In view of the side<br />
effects of NSAIDs and corticosteroids, it may be envisaged that garlic can be used as an adjuvant in the<br />
inflammatory disorders.<br />
Key words: Allium sativum, <strong>An</strong>ti-inflammatory, Carrageenin, Edema, Granuloma.<br />
Introduction<br />
Inflammation is the characteristic response<br />
of mammalian tissue to injury. Whenever<br />
tissue is injured, there follows at the site of<br />
injury a series of events that tend to destroy<br />
or limit the spread of the injurious agent(1).<br />
The introduction of acetyl salicylic acid,<br />
cortisone, gold salts and phenylbutazone <strong>for</strong><br />
the treatment of inflammatory disorders is an<br />
important milestone in the development of<br />
clinically useful anti-inflammatory agents (2).<br />
Currently available anti-inflammatory agents<br />
are associated with their own side effects. It<br />
has been estimated that about 34 to 46 percent<br />
of the users of NSAIDs will sustain some<br />
gastrointestinal damage due to the inhibition<br />
of the protective cyclooxygenase enzyme in<br />
gastric mucosa (3). There<strong>for</strong>e, researchers<br />
have aimed at identifying and validating<br />
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<strong>An</strong>ti-inflammatory effects of Allium sativum<br />
plant derived substances <strong>for</strong> the treatment of<br />
various diseases. The added advantages of<br />
indigenous medicinal treatment would include<br />
its complementary nature to the conventional<br />
treatment making latter safer, well tolerated<br />
and economical remedy <strong>for</strong> inflammatory<br />
conditions. Allium sativum (Garlic) is a hardy<br />
perennial bulbous scapigerous herb, with a flat<br />
stem. The lower portion of the plant <strong>for</strong>ms a<br />
bulb, which consists of several smaller buds<br />
called cloves, surrounded by a thin white or<br />
pinkish sheath. The leaves are flat, narrow<br />
green; the heads bear small white flowers and<br />
bulbils. Since ancient days, it has been cultivated<br />
throughout India, Pakistan, Bangladesh, and<br />
most tropical countries (4). Garlic has shown<br />
various activities like hypoglycemic, antiinflammatory,<br />
anti-cancerous, anti-rheumatic,<br />
anti-platelet, anti-helmenthic, hypolipidaemic<br />
and radio protective effects on various animal<br />
models (5-9). There<strong>for</strong>e, present study was<br />
conducted with the aim of investigating antiinflammatory<br />
activity of garlic powder. Specific<br />
objectives of the study were, a) to estimate<br />
the anti-inflammatory effects of garlic powder<br />
in acute and chronic inflammatory animal<br />
models, and b) to compare the effects with that<br />
of established anti-inflammatory drug, namely,<br />
Piroxicam.<br />
Material and Methods<br />
Preparation of powder:<br />
Garlic powder was made from chopped<br />
garlic cloves that were oven-dried and then<br />
pulverized. The fine powder thus obtained<br />
was kept in the air tight containers. The yield<br />
of garlic powder from the garlic bulbs after<br />
the processing was found to be 250 gm per kg<br />
of garlic bulbs . A pilot study was done with<br />
different doses (5 mg/kg, 50 mg/kg, 100 mg/<br />
kg and 200 mg/kg) to estimate the dose <strong>for</strong> the<br />
study. The ant-inflammatory activity was found<br />
to be significant at the dose of 100 mg per kg of<br />
body weight and hence the same dose was used<br />
in the study.<br />
85<br />
Chemicals: Piroxicam, Carrageenin (Sigma)<br />
and all other chemicals were of analytical grade.<br />
<strong>An</strong>imals: <strong>An</strong>imals used were albino rats of<br />
either sex, weighing between 120-160 Grams.<br />
The animals were procured from animal research<br />
laboratory, National Institute of Mental Health<br />
and Neuro <strong>Sciences</strong> (NIMHANS), Bangalore<br />
and housed in the animal house of the institute<br />
in groups of 3, at an ambient temperature of<br />
25±1°C with ad libitum access to food and<br />
water. The study protocol was approved by<br />
Institutional <strong>An</strong>imal Ethics Committee.<br />
Methods<br />
To study the anti-inflammatory effect of the<br />
drugs, two models, namely, Carrageenin<br />
induced rat paw edema <strong>An</strong>imal Model and<br />
Cotton pellet induced Granuloma <strong>An</strong>imal<br />
Model were utilized.<br />
A total of 36 rats were divided into two groups<br />
of 18 each to be utilized <strong>for</strong> the two models.<br />
Further, within each model, animals were<br />
randomly allocated to the 3 groups of 6 rats<br />
each; I group: Control (1 ml of Vehicle, 2%<br />
Gum acacia suspension); II group: Standard<br />
drug (Piroxicam 100 mg/kg); III group: Test<br />
drug (Garlic 100 mg/kg).<br />
Carrageenan induced rat paw edema <strong>An</strong>imal<br />
Model<br />
This is an established animal model to screen<br />
the acute anti-inflammatory activity of the drugs<br />
(10, 11). The animals were pretreated with<br />
drugs orally one hour be<strong>for</strong>e the experiment.<br />
Carrageenan (0.05 ml, 1%) was injected<br />
aseptically into the subplantar surface of right<br />
hind paw of each rat. Paw edema was measured<br />
by Mercury Plethysmograph (UGO Basile,<br />
Italy) immediately (i.e., at ‘0’ hour) and at the<br />
end of ‘4’ hours. The difference between the<br />
measurements at 0 and 4 hours was taken as the<br />
actual inhibition of edema.<br />
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<strong>An</strong>ti-inflammatory effects of Allium sativum<br />
Cotton pellet induced Granuloma <strong>An</strong>imal<br />
Model<br />
This is an established animal model to screen the<br />
chronic anti-inflammatory activity of the drugs<br />
(12). Four sterile cotton pellets weighing 10 mg<br />
each were implanted subcutaneously in both the<br />
axilla and groin of each rat. Rats were fed with<br />
the respective drug daily <strong>for</strong> 7 days along with<br />
free access to water and food ad libitum. Later<br />
the animals were sacrificed on the 8 th day and<br />
the cotton pellets with granulation tissue were<br />
removed, cleaned of the extraneous tissue and<br />
dried in a hot air oven to a constant weight and<br />
the dry granuloma weight was determined. The<br />
dry weight of the granuloma (i.e. the amount of<br />
actual granulation tissue <strong>for</strong>med) was calculated<br />
by noting the difference in the dry weight of<br />
the cotton pellets recorded be<strong>for</strong>e and after<br />
implantation.<br />
Statistical <strong>An</strong>alysis:<br />
The effects of standard and test drugs were<br />
estimated by working out mean and standard<br />
deviation of paw edema size (in cm) and dry<br />
granuloma weight (in mg), followed by the<br />
estimation of percent inhibition applying the<br />
<strong>for</strong>mula given below. Percentage inhibition<br />
by the drug = (W d -W c ) / W c X 100, where<br />
W d is the effect in intervention group and W c<br />
is the effect in the control group. One-way<br />
analysis of variance (ANOVA) was per<strong>for</strong>med<br />
to test the significance of differences in mean<br />
paw edema and mean dry granuloma weight<br />
among the three groups followed by Scheffe’s<br />
post-hoc test. All the tests of significance<br />
were interpreted at 5%, 1% or 0.1% level of<br />
significance. In order to quantify the effects of<br />
the drugs, we calculated the inflammation in<br />
the drug groups relative to the control group<br />
and presented the same using bar diagram. Two<br />
relative measures were calculated as following.<br />
Relative effect of a drug = (W d /W c ) X 100<br />
Results<br />
86<br />
Acute anti-inflammatory effect The acute<br />
anti-inflammatory effect of garlic powder<br />
and standard drug was studied by looking at<br />
the changes in Carrageenin-induced Rat Paw<br />
Edema. This model suggested a statistically<br />
significant anti-inflammatory effect of both<br />
the garlic powder and the Piroxicam drug. The<br />
induced rat paw edema was found to be about<br />
half in the two drug groups compared to the<br />
control group. Chronic anti-inflammatory effect<br />
The chronic anti-inflammatory effect of<br />
garlic powder and standard drug was studied<br />
by looking at the changes in Cotton pellet<br />
induced dry granuloma weight. This model<br />
also suggested a statistically significant antiinflammatory<br />
effect of both the garlic powder<br />
and the Piroxicam drug. The induced granuloma<br />
in standard and test drug groups was found to<br />
be respectively 65 and 80 percent of the control<br />
group. Test drug versus standard drug As <strong>for</strong> as<br />
the comparison of the test drug with the standard<br />
drug is concerned; the two showed a similar<br />
effect according to paw edema model. However,<br />
there was stastically significant difference in the<br />
effect of two drugs according to chronic model.<br />
In this model, the inhibition was about 21%<br />
due to the test drug compared to 35% due to<br />
the standard drug. Thus, although the test drug<br />
had a significant effect, quantitatively however,<br />
it was not as much as the standard drug had.<br />
Discussion<br />
The introduction of drugs like Aspirin, Cortisone,<br />
Indomethacin and others has revolutionized the<br />
treatment of rheumatic and musculoskeletal<br />
disorders. The amazing efficacy of cortisone<br />
and phenylbutazone in these inflammatory<br />
disorders has paved the way <strong>for</strong> the introduction<br />
and use of newer anti-inflammatory agents.<br />
However, the safety factor in respect of both the<br />
steroidal and nonsteroidal anti-inflammatory<br />
drugs has been rather intriguing and hence a<br />
definite need is visualized <strong>for</strong> the introduction<br />
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<strong>An</strong>ti-inflammatory effects of Allium sativum<br />
Table 1: Mean and standard deviation (SD) of paw edema (in cm) in the three groups under study along with<br />
the results of analysis of variance (ANOVA) and post-hoc tests<br />
Group<br />
No. of<br />
subjects<br />
Paw edema (Cms)<br />
Mean SD<br />
87<br />
Inhibition (%)<br />
Control 6 3.85 0.166 0.0<br />
Standard drug* 6 1.92 0.128 50.2<br />
Test drug* 6 2.00 0.139 48.1<br />
Note: ANOVA significant (p
<strong>An</strong>ti-inflammatory effects of Allium sativum<br />
of safer anti-inflammatory drugs having no<br />
troublesome adverse effects. There are different<br />
kinds of garlic oils, garlic powder capsules,<br />
garlic pills with enteric coatings, garlic pills<br />
with other herbs added, like cayenne and<br />
aged garlic extract. Garlic powder used in the<br />
above study, as the powder <strong>for</strong>m gets absorbed<br />
readily in the stomach and duodenum. It also<br />
contains the sulfur containing compounds alliin,<br />
ajoene, diallylsulfide, dithiin, S-allylcysteine,<br />
and enzymes, vitamin B, proteins, minerals,<br />
saponins, flavonoids.<br />
One of the most biologically active compounds,<br />
allicin (diallyl thiousulfinate or diallyl disulfide)<br />
does not exist in garlic until it is crushed or cut;<br />
injury to the garlic bulb activates the enzyme<br />
allinase, which metabolizes alliin to allicin.<br />
In addition allicin is further metabolized to<br />
vinyldithiines. Ajoene, is another chemical<br />
constituent thought to be most important to<br />
health .<br />
Carrageenin induced rat-paw edema model has<br />
gained greater importance and support over the<br />
years, because edema induced by carrageenin<br />
is reported to have been inhibited by majority<br />
of the steroidal and the non-steroidal antiinflammatory<br />
drugs. Moreover, the lesions<br />
induced by Carrageenin are said to resemble<br />
histologically those of rheumatoid arthritis<br />
in human being atleast to a certain extent .<br />
These observations have justified the use of<br />
Carrageenin as the prime oedemogen. It has a<br />
biphasic effect. The first phase is due to release<br />
of histamine and serotonin (5-HT) (0-2 hours),<br />
plateau phase is maintained by a kinin like<br />
substance (3 hours) and second accelerating<br />
phase of swelling is attributed to PG release (>4<br />
hours)<br />
In our study Garlic powder 100 mg/kg, per orally<br />
significantly reduced edema induced by the<br />
carrageenin. The results obtained in this model/<br />
method suggest that the anti-inflammatory<br />
activity of garlic powder is almost equal to that<br />
of the standard drug, namely, piroxicam.<br />
88<br />
Cotton pellet induced granuloma is a method<br />
<strong>for</strong> testing the proliferative phase i.e. granuloma<br />
<strong>for</strong>mation, provoked by the subcutaneous<br />
implantation of compressed cotton pellets.<br />
After few days histologically giant cells and<br />
undifferentiated connective tissue can be<br />
observed along with fluid infiltration. The<br />
amount of newly <strong>for</strong>med connective tissue<br />
can be measured after removal and weighing<br />
the dried pellets. This model serves as an<br />
example <strong>for</strong> chronic inflammatory models.<br />
Here, the underlying principle of assaying<br />
the anti-inflammatory activity is based on the<br />
<strong>for</strong>mation of <strong>for</strong>eign body granuloma, following<br />
subcutaneous implantation of cotton-pellets.<br />
Several authors have used this model <strong>for</strong> noting<br />
the anti-granuloma activity of newer agents and<br />
interestingly, the time-intervals, at which the<br />
implanted cotton-pellets (with the granuloma)<br />
have been removed from the sacrificed animals,<br />
have varied considerably, from a few days to a<br />
few weeks (13).<br />
In the present study the implanted cottonpellets<br />
(with the granuloma) were removed on<br />
the eighth day after the experimental procedure<br />
and the dry weight of granuloma served as<br />
the criterion <strong>for</strong> assaying anti-inflammatory<br />
(<strong>An</strong>ti-granuloma) activity. The results observed<br />
are suggestive of modest and very low antigranuloma<br />
activity in respect of piroxicam and<br />
garlic powder, respectively.<br />
In an earlier study, aqueous extracts of fresh<br />
garlic (5, 12.5, 25 and 50 mg/ml) were shown<br />
to inhibit the synthesis of the prostanoids in<br />
a dose dependent manner (14). In another<br />
research, the effects of aqueous extract of raw<br />
garlic and boiled garlic on cyclo-oxygenase<br />
activity in rabbit tissues were studied (15).<br />
Raw garlic inhibited cyclooxygenase activity<br />
non-competitively and irreversibly. There<strong>for</strong>e<br />
the probable mechanism of action of garlic is<br />
by inhibiting cyclooxygenase activity. Allium<br />
sativum yields allicin, a powerful antibiotic. It<br />
has been claimed that it can be used as a home<br />
remedy to help speed recovery from strep throat<br />
or other minor ailments because of its antibiotic<br />
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<strong>An</strong>ti-inflammatory effects of Allium sativum<br />
properties. Recent publications indicate that<br />
garlic extract has broad-spectrum antimicrobial<br />
activity against many genera of bacteria and<br />
fungi. The active component (allicin) has been<br />
isolated and characterized (16).<br />
The standardization of the extracts,<br />
identification and isolation of active principles<br />
and pharmacological studies of these principles<br />
may be considered <strong>for</strong> further detail studies.<br />
Conclusion<br />
In general, the medicinal importance of garlic<br />
preparations has already been substantiated<br />
by the very availability of “garlic pearls” <strong>for</strong><br />
human use. Present study has shown that Garlic<br />
has promising anti-inflammatory activity that is<br />
comparable with that of piroxicam, especially<br />
in the carrageenin induced paw-edema<br />
animal model. These findings are valuable<br />
<strong>for</strong> identifying lead compounds <strong>for</strong> antiinflammatory<br />
drugs, keeping in mind the side<br />
effects of NSAIDs and corticosteroids. In the<br />
light of observations made it may be envisaged<br />
that garlic can be used as an adjuvant in the<br />
inflammatory disorders.<br />
Acknowledgement:<br />
Authors are thankful to Dr. Vijayaraghavan,<br />
Former Professor, Department of Pharmacology,<br />
Govt. Medical College, Mysore <strong>for</strong> the guidance<br />
and support and also to Mr. M. Ganapathy,<br />
Spicex Limited, Mysore <strong>for</strong> the processing of<br />
Garlic powder <strong>for</strong> this study.<br />
References<br />
1. Rang HP, Dale MM, Ritter JM and Flower RJ. <strong>An</strong>tiinflammatory<br />
and immunosuppressant drugs. In: Rang and<br />
Dale’s Pharmacology. 6 th ed. Elseveir Publications. 2008:<br />
226-245.<br />
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2. Nadkarni KM. Indian materia medica. Revised and<br />
enlarged by Nadkarni AK. 3 rd ed (1). Mumbai: Popular<br />
Prakashan. 1976:6.<br />
3. Singh UP, Prithiviraj B, Sarma BK, Singh M and Ray AB.<br />
Role of garlic (Allium sativum L.) in human and plant<br />
diseases. Indian J Exp Biol 2001; 39:310-22.<br />
4. Ganote CE and Humphrey SM. Effects of anoxic or<br />
oxygenated reperfusion in globally ischemic, isovolumic,<br />
perfused rat hearts. Am J Pathol 1985; 120:129-45.<br />
5. Nagaich SS. Studies on the anthelmintic activity of Allium<br />
sativum (Garlic) oil on common poultry worms Ascaridia<br />
galli and Heterakis gallinae. J Parasitol App <strong>An</strong>im Biol<br />
2000; 9:47-52.<br />
6. Durak I, Oztürk HS, Olcay E and Güven C. Effects of garlic<br />
extract supplementation on blood lipid and antioxidant<br />
parameters and atherosclerotic plaque <strong>for</strong>mation process<br />
in cholesterol-fed rabbits. J Herb Pharmacother 2002;<br />
2(2):19–32.<br />
7. Jaiswal SK and Bordia A. Radio-protective effect of garlic<br />
allium sativum linn, in albino rats. Indian J Medical Sci<br />
1996; 50(7):231-233.<br />
8. Vogel HG. <strong>An</strong>algesic, anti-inflammatory and antipyretic<br />
activity in Drug discovery and evaluation pharmacological<br />
assays. 2nd Ed. New York: Springer. 2002:759-767.<br />
9. Winter CA, Risley EA and Nuss GW. Carrageenan - induced<br />
edema in hind paw of rat as an assay <strong>for</strong> anti-inflammatory<br />
drugs. Proc Soc Expt Biol Med 1962; 111:544-547.<br />
10. Vinegar R, Schreiber W and Hugo RJ. Biphasic<br />
development of carrageenan edema in rats. J Pharmacol<br />
Exp Ther 1969; 166: 96-103.<br />
11. Dulin WE. <strong>An</strong>ti-inflammatory activity of delta1-9alphafluorohydrocortisone<br />
acetate. Proc Soc Exp Biol Med<br />
1955; 90(1):115–117.<br />
12. Penn GB and Ash<strong>for</strong>d A. The inflammatory response<br />
to implantation of cotton pellets in the rat .J Pharma<br />
Pharmacol 1963; 15:798-803.<br />
13. Singer FM and Borman A. Cotton pellet inhibtion of<br />
granuloma <strong>for</strong>mation is directly related to anti-inflam- ...<br />
Proc. SOC. Exptl. Biol. Med. 1956; 92: 23.<br />
14. Ali M. Aqueous extracts of garlic (Allium sativum) inhibit<br />
prostaglandin. Prostaglandins Leukotrienes<br />
and Essential Fatty Acids. 1993 ;49( 5) : 855-859.<br />
15. Ali. M. Mechanism by which garlic (allium sativum)<br />
inhibits cyclooxygenase activity. Prostaglandins,<br />
Leukotrienes and Essential Fatty Acids 1995; 53(6): 397-<br />
400.<br />
16. Adetumbi, M. A., and B. H. S. Lau. Allium sativum.<br />
(garlic)-a natural antibiotic. Med. Hypotheses 1983.12:227-<br />
237<br />
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<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 90 - 95<br />
Reactive Oxygen Species during hypoxia-reperfusion injury under general<br />
anaesthesia<br />
*Sanjeev Kumar **Ashok kumar<br />
*Department of <strong>An</strong>aesthesiology and Critical care,Navodaya Medical College,Raichur-<br />
584103,Karnataka, India<br />
**Consultant <strong>An</strong>aesthesiologist,Advanced Medicare Research Institute, Kolkata-700029<br />
(Received 3 rd November, 2010; Revised 5 th January, 2011; Accepted 16 th February, 2011)<br />
Corresponding Author<br />
Dr Sanjeev Kumar,<br />
Email:docsanjeevparmar77@gmail.com<br />
Abstract<br />
Background: Hypoxia – Reperfusion of ischemic tissues results in the <strong>for</strong>mation of toxic ROS (reactive<br />
oxygen species) . The hypoxia – reperfusion event that may occur during intubation apnea and reoxygenation<br />
could be a potent source <strong>for</strong> generation of ROS. The role of calcium is important in maintaining the membrane<br />
ionic equilibrium during hypoxia and reperfusion. This study was designed to find out the effectiveness<br />
of a single dose of calcium channel blocker, nifedipine in attenuating the haemodynamic responses and<br />
generation of ROS.<br />
Methods: Twenty patients undergoing elective surgery under general anaesthesia were randomly assigned<br />
to group IA and IB. In group IA patients were anaesthetized by standard anaesthetic technique while in<br />
group IB sublingual nifedipine (10mg) was given 10 minutes be<strong>for</strong>e induction.Blood (10ml) was collected<br />
at different time intervals to measure peroxidative stress and reactive oxygen scavangers.<br />
Results: In group IA laryngoscopy and intubation caused a significant increase (4.06±0.90 vs 4.63±0.84<br />
nmol/mL, p
ROS during hypoxia-reperfusion injury<br />
acid (HOCL) hydrogen peroxide (H2O2)<br />
and nitric oxide derived peroxynitrate. These<br />
reactive oxygen species (ROS) are produced<br />
in limited amounts under normal conditions<br />
as the intracellular defense system consisting<br />
primarily of enzymatic and nutrient free radical<br />
scavengers are able to inactivate them. Hypoxia<br />
is associated with depletion of tissue content of<br />
antioxidants including superoxide dismutase<br />
(SOD), catalase, glutathione peroxidase and<br />
increases vulnerability to reoxygenation which<br />
causes further reduction of antioxidants(3,4).<br />
Thus the hypoxia – reperfusion event that<br />
may occur during intubation apnoea and<br />
reoxygenation could be a potent source <strong>for</strong><br />
generation of ROS. The role of calcium is<br />
important in maintaining the membrane ionic<br />
equilibrium during hypoxia and reperfusion.<br />
On reperfusion, calcium readily enters into the<br />
cell in exchange <strong>for</strong> sodium. The net increase<br />
in intracellular calcium causes a decrease in<br />
ATP synthesis and subsequent lysis of the cell<br />
(5,6). A further deleterious consequence of<br />
the reperfusion dependent calcium influx is<br />
activation of proteases and phospholipidases;<br />
the latter results in release of free fatty acids and<br />
lysophospholipids (7). These compounds are<br />
toxic in their own right and can also result in the<br />
production of ROS and other cytotoxic products<br />
on reperfusion (8,9). Calcium channel blockers<br />
especially nifedipine has been found to be more<br />
effective in minimizing the haemodynamic<br />
adverse response to laryngoscopy and<br />
intubation and has also been found to act as<br />
an antioxidant when administered be<strong>for</strong>e the<br />
ischemic-reperfusion event (10). There<strong>for</strong>e, this<br />
study was designed to find out a) if intubation<br />
apnoea and reoxygenation generated ROS b)<br />
the effectiveness of a single dose of calcium<br />
channel blocker, nifedipine in attenuating the<br />
haemodynamic responses and generation of<br />
ROS.<br />
Methods<br />
After prior approval from our institutional ethics<br />
91<br />
committee and written in<strong>for</strong>med consent from<br />
patients, twenty patients belonging to ASA I<br />
and II with age between 30-50 years undergoing<br />
elective surgery of an estimated duration of<br />
more than 1 hour were included in the study<br />
and randomly allocated to Group IA and IB.<br />
Patients suffering from any major preexisting<br />
neurological, cardiovascular metabolic<br />
respiratory or renal disease were excluded from<br />
the study. On arrival to the operating room<br />
patients were connected to routine monitoring<br />
devices [HR, NIBP, ECG, SpO2]. Vital<br />
parameters were noted be<strong>for</strong>e during and after<br />
general anaesthesia at regular time intervals.<br />
In Group IA – 10 patients were induced with<br />
injection atropine sulphate 0.6 mg, tramadol<br />
hydrochloride 1 mg/kg, pentothal sodium 5 mg/<br />
kg and succinylcholine 2 mg / kg intravenously<br />
followed by oral endotracheal intubation.<br />
In Group IB- 10 patients were induced with<br />
same technique as above but all these patients<br />
were given sublingual nifedipine 10 mg, 10<br />
minutes be<strong>for</strong>e induction. <strong>An</strong>aesthesia was<br />
maintained with oxygen, nitrous oxide and<br />
intermittent doses of vecuronium bromide<br />
and 0.5%-1% halothane. Following surgery<br />
patients were reversed with neostigmine methyl<br />
sulphate 0.08 mg/kg and atropine sulphate<br />
0.02 mg/kg intravenously and extubated.<br />
Blood (10 ml) was collected in EDTA vials at<br />
different time intervals (pre induction, during<br />
intubation, following 30 min of ventilation and<br />
after 1 hour of ventilation; sample I, II, III and<br />
IV respectively). The following biochemical<br />
investigations were carried out in the blood:<br />
For peroxidative stress – Thiobarbituric acid<br />
reactive substances (TBARS) (11) For enzyme<br />
ROS (Reactive Oxygen Species) scavengers –<br />
Superoxide dismutase (SOD) (12) Glutathione<br />
peroxidase (GPx)(13) Reduced glutathione<br />
(GSH)(14) Catalase (Cat)(15) For nutritional<br />
ROS scavengers(16) - Retinol; ß-Carotene<br />
a-Tocopherol Ascorbic acid These results were<br />
compiled and analyzed statistically by using a<br />
paired t-test. Statistical significance was defined<br />
as P
ROS during hypoxia-reperfusion injury<br />
Table1:Haemodynamic Responses in Group I A and IB<br />
I II III IV<br />
(Group I A ) Heart rate (beats/<br />
min)<br />
(Group I B ) Heart rate<br />
(beats/min)<br />
(Group I A ) Mean BP<br />
(mmHg)<br />
(Group I B ) Mean BP<br />
(mmHg)<br />
(Group I A ) O 2<br />
Saturation(%)<br />
(Group I B ) O 2<br />
Saturation(%)<br />
a=I; b=II; c=III; d=IV * Mean + S.D.I=P
ROS during hypoxia-reperfusion injury<br />
Table 4: Peroxidative Stress and Enzyme antioxidant status in Group I B (n=10)<br />
Parameters I II III IV<br />
TBARS nmol./ml<br />
Catalase nmol./min/mgHb<br />
GSH mg/dl RBC<br />
GP x mg GSH consumed/<br />
min at 37 0 C<br />
4.21±1.38* 5.12 ± 1.27 4.29±1.34 4.19±1.46<br />
b 2<br />
0.69 ± 0.24 0.60 ±0.32 0.59±0.37 0.66±0.32<br />
a 2<br />
42.81±5.69 47.42±5.15 45.91±5.65 47.52±5.83<br />
a 2<br />
3.00 ± 1.11 2.81 ±1.21 3.24±1.05 2.88±1.18<br />
SOD U/mgHb 3.11±0.52 3.04±0.56 3.04±0.56 3.15±0.56<br />
a=I; b=II; c=III; d=IV *Mean + S.D.I=P
ROS during hypoxia-reperfusion injury<br />
minutes of ventilation from 5.12±1.27 nmol/<br />
ml to 4.29±1.34 nmol/ml (II vs III, p
ROS during hypoxia-reperfusion injury<br />
intubation there was evident peroxidative stress<br />
which was accompanied by significant activity<br />
or consumption of the antioxidant defense<br />
mechanism in the body. This correlated with<br />
the haemodynamic responses seen during<br />
this period. Nifedipine when given in a single<br />
dose pre-operatively was found to act as an<br />
antioxidant, while limiting any increase in<br />
peroxidative stress and gearing up antioxidant<br />
mechanism in the body to meet the challenge of<br />
acute oxidant stress.<br />
References<br />
1. Shetty, A.N., Shinde, V.S. and Chaudhari, L.S. A<br />
comparative study of various airway devices as regards<br />
ease of insertion and haemodynamic responses. India J.<br />
<strong>An</strong>aesth2004; 48(2): 134-137.<br />
2. Wilson, I.G., Fell, D., Robinson, S.L. and Smith, G.<br />
Cardiovascular responses to insertion of the laryngeal<br />
mask. <strong>An</strong>aesthesia 1992; 47: 300-302.<br />
3. Guarnieri, C., Flamign, F. and Caldarera, C.M. Role of<br />
oxygen in the cellular damage induced by re-oxygenation<br />
of hypoxic heart. J. Mol. Cell Cardiol 1980; 12:797-808.<br />
4. Kato, R. and Foex, P. Myocardial protection by anesthetic<br />
agents against ischemia – reperfusion injury: an update <strong>for</strong><br />
anesthesiologists. Can J. <strong>An</strong>esth 2002; 49(8): 777-791.<br />
5. Bourdillon, P.D. and Poole Wilson, P.A. The effects of<br />
verapamil, quiescence and cardioplegia in ischemic rat<br />
myocardium. Cir. Res1980; 50: 360-368.<br />
6. Parr, D.R., Wilmhurst, J.M. and Harris, E.F. Calcium<br />
induced damage of rat heart mitochondria. Cardiovasc.<br />
Res1975;. 9: 366-372.<br />
7. Murphy, E., Aitcon, J.F. and Horres, C.R. Calcium<br />
elevation in cultured heart cells, its role in cell injury. Am<br />
J. Physiol 1983; 245: C316-C321.<br />
8. Bersohn, M.M. and Philipson, K.D., YFJ . Sodium calcium<br />
exchange and sarcolemmal enzymes in ischaemic rabbit<br />
heart. Am. J. Physiol 1982; 242: C288-C295.<br />
9. Konotos, H.A., Weiep, P.J.T. and Dietrich, W.D.<br />
Cerebral arteriolar damage by arachidonic acid and PG2.<br />
95<br />
Science1980; 209: 1242-1245.<br />
10. Ohsuzu, F. Effects of calcium antagonists and free radical<br />
scavengers on myocardial ischemia and reperfusion<br />
injury: Evaluation by 31 P-NMR spectroscopy. Japanese<br />
Circulation <strong>Journal</strong> 1989; 53:1138-1143.<br />
11. Ohkawa, H., Ohishi, N. and Yagi, K. Assay <strong>for</strong> lipid<br />
peroxides in animal tissues by thiobarbituric acid reaction.<br />
<strong>An</strong>al Biochem 1979; 95(2): 351-8.<br />
12. Mishra, H.P. and Fridovich, I. The role of superoxide<br />
anion in the autoxidation of epinephrine and a simple assay<br />
<strong>for</strong> SOD. J. Biol. Chem1972; 247: 3170.<br />
13. Pirie. A. Glutathione peroxidase in lens and a source of<br />
hydrogen peroxide in aqueous humour. Biochem1965;<br />
96:243.<br />
14. Beutler, E. and Kelly, B.M. Improved method <strong>for</strong> the<br />
determination of blood glutathione J. Lab. Clin. Med1963;<br />
61: 882.<br />
15. Sinha,A. K.Colorimetric assay of catalase. <strong>An</strong>nal<br />
Biochem1972; 47: 389.<br />
16. Natelson, S Techniques of clinical chemistry. 3rd Ed<br />
Charles. C. Thomas. USA 1971: 162, 288 and 751.<br />
17. Derbyshire, D.R., Chmielewski, A., Fell, D., Vater, M.,<br />
Achola, K. and Smith, G. Plasma Catecholamine responses<br />
to tracheal intubation. Br. J. <strong>An</strong>aesth1983; 55: 855-859.<br />
18. Shribman, A. J., Smith, G. and Achola, K.J. Cardiovascular<br />
and catecholamine responses to laryngoscopy<br />
with and without tracheal intubation. Br. J. <strong>An</strong>aesth1987;<br />
59: 295-299.<br />
19. Kale, S.C., Mahajan, R.P., Jayalakshmi, T.S., Raghavan,<br />
V. and Das, B. Nifedipine prevents the pressor response<br />
to laryngoscopy and tracheal intubation in patients with<br />
coronary artery disease. <strong>An</strong>aesthesia1988; 43:495-497.<br />
20. Puri, G.D. and Batra, Y.K. Effect of nifedipine on cardiovascular<br />
responses to laryngoscopy and intubation. Br. J.<br />
<strong>An</strong>aesth1988; 60:579-581.<br />
21. Mason, R.P. and Trumbore, M.W. Differential membrane<br />
interactions of calcium channel blockers. Implications<br />
<strong>for</strong> antioxidant activity. Biochem. Pharmacol1996; 51(5):<br />
653-60.<br />
22. Silva, |J.M., Filibe, P.M., Fernandes, A.C. and Manso, C.F.<br />
<strong>An</strong>tioxidant effect of drug used in cardiovascular therapy.<br />
Rev. Port. Cardiol1998; 17(6): 495-503.<br />
23. Sugawara, H., Tobisa, K. and Kikuchi, K . <strong>An</strong>tioxidant<br />
effects of calcium antagonists on rat myocardial membrane<br />
lipid peroxidation. Hypertens. Res1996; 19(4): 223-228.<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
96<br />
<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 96 - 102<br />
Assessment of probiotic properties of strains of L.fermentum and L.reuteri<br />
isolated from human breast milk<br />
R. Ilayaraja and Radhamadhavan.<br />
Department of Microbiology, SRM Medical College Hospital & Research Centre,<br />
SRM University, Kattankulathur - 6032023. Tamil Nadu, India.<br />
(Received 27th October, 2010; Revised 10th February, 2011; Accepted 1st March, 2011)<br />
Corresponding Author<br />
R. Ilayaraja<br />
E-Mail: srmmicro@gmail.com.<br />
Abstract<br />
Objectives: To assess the probiotic potential of strains of Lactobacillus fermentum and Lactobacillus<br />
reuteri isolated from human breast milk was investigated.<br />
Materials and Methods: The milk samples were inoculated anaerobically into MRS medium and incubated<br />
<strong>for</strong> 48 hrs at 37 0 C. Probiotic characteristics of the strains were assessed according to the criteria laid down<br />
by WHO namely, tolerance to low pH, bile, NaCl concentration and temperature, Hemolytic activity and<br />
antimicrobial activity.<br />
Results: The cultures were identified based on morphological and biochemical characteristics of Lactobacilli<br />
as per the Bergey’s Manual of Determinative Bacteriology and included growth at 15 0 , 37 0 C and 45 0 C and<br />
fermentation of different carbon sources. Both the strains fulfill the probiotic criteria suggested by WHO.<br />
Interpretation and conclusion: This study revealed that these two strains fulfill characteristics <strong>for</strong> use as<br />
probiotics. L.fermentum had higher probiotic activity compared to L.reuteri.<br />
Key words: MRS medium, Probiotic, antimicrobial therapy, Lactobacillus.<br />
Introduction:<br />
Over the past 15 years, there has been an<br />
increase in research on probiotic bacteria and<br />
a rapidly growing commercial interest in the<br />
use of probiotic bacteria in food, medicine and<br />
as supplements. (17). A variety of probiotic<br />
bacteria have been targeted as potential<br />
therapeutic agents and include Lactic acid<br />
bacteria (LAB), Bifidobacteria, Saccharomyces<br />
and streptococci. Today with waning efficacy<br />
of antibiotics and dramatic resurgence of<br />
infectious disease, there is an intense need of<br />
an alternative to existing antibiotic-dominated<br />
therapies Probiotics are substances secreted by<br />
one microorganism which stimulates the growth<br />
of another. A more <strong>for</strong>mal definition widely<br />
used is “A live microbial food supplement<br />
which beneficially affects the host animal by<br />
improving its intestinal microbial balance”. <strong>An</strong><br />
expert panel commissioned by FAO (Food and<br />
Agriculture Organization) and WHO (World<br />
Health Organization) defined probiotic as “live<br />
microorganism”, which when administrated in<br />
adequate amounts confers a health benefit on<br />
the host. (2). Bacteria most commonly used<br />
in probiotic preparations belong to genera of<br />
lactobacillus, Bifidobacterium, Enterococcus,<br />
Bacillus and Streptococcus. Some fungal<br />
strains belonging to saccharomyces were also<br />
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properties of L.fermentum and L.reuteri<br />
used (3). A number of clinical studies have been<br />
per<strong>for</strong>med on the ability of probiotic bacteria<br />
to prevent or treat gastro intestinal infections.<br />
Bacteria belonging to the two genera namely<br />
Lactobacillus and Bifidobacterium are the most<br />
commonly used probiotic bacteria. Some of the<br />
well planned, double-blind, placebo-controlled<br />
studies suggested that lactobacillus strains are<br />
the most promising strains in the prevention of<br />
diarrhea. Recently, it was shown that in children<br />
admitted to hospital, the risk of acquiring<br />
nosocomial diarrhea was reduced 30% to<br />
7% in the non breast fed children receiving<br />
prophylactic therapy with lactobacillus GG.<br />
(5). According to 1997 issue of the journal of<br />
Pediatrics Gastroenterology and Nutrition,<br />
L.reuteri is an effective treatment <strong>for</strong> Rota viral<br />
diarrhea in children. Currently used in vitro tests<br />
<strong>for</strong> study of probiotic properties of bacterial<br />
strains include tolerance to gastric acidity ,<br />
bile salt , NaCl concentration and temperature,<br />
hemolytic activity and antimicrobial activity,<br />
adherence to mucous and/ or human epithelial<br />
cells and cell lines, antimicrobial activity<br />
against potentially pathogenic bacteria, ability<br />
to reduce adhesion of pathogens to surfaces,<br />
and resistance to spermicides (applicable to<br />
probiotics <strong>for</strong> vaginal use. (4). The aim of this<br />
study was to isolate bacterial strains from human<br />
breast milk and to study antimicrobial activity,<br />
and other characteristics of a good probiotic<br />
Materials and Methods<br />
Isolation and Identification:<br />
The lactobacillus strains were isolated from<br />
human breast milk on solid Man-Rogosa-<br />
Sharpe media (MRS)(g/l, peptone 10.0, meat<br />
extract 8.0, yeast extract 4.0, D(+)glucose 20.0,<br />
dipottassium hydrogen phosphate 2.0, tween<br />
80 - 1.0, di-ammonium hydrogen citrate 2.0,<br />
sodium acetate 5.0, magnesium sulphate 0.2,<br />
manganese sulphate 0.04, supplemented with<br />
14.0 g agar respectively). The bacteria were<br />
grown at 37 0 C in microaerophillic condition<br />
with out shaking. The lactobacillus strains were<br />
97<br />
identified as described by Bergey’s Manual of<br />
Determinative Microbiology (18). Purity of<br />
the strains were verified by three successive<br />
subculture from the single colony. For long<br />
term preservation of cultures, MRS broth was<br />
supplemented with 10% glycerol.<br />
Carbohydrate Fermentations<br />
Isolates were characterized according to their<br />
carbohydrate fermentation profiles by testing<br />
against 14 different carbohydrates. Each sugar<br />
solutions was prepared at a final concentration<br />
of 10% (w/v) with phenolphthalein red as pH<br />
indicator, and the solutions were filter sterilized<br />
with filters (0.22 μm pore diameter). All the<br />
reactions were per<strong>for</strong>med in duplicate including<br />
suitable positive and negative controls After<br />
overnight incubation at 37°C, the turbidity and<br />
the color change from red to yellow indicated<br />
positive fermentation results (1).<br />
Gas Production from Glucose<br />
The production of gas during glucose<br />
fermentation was observed by placing an<br />
inverted Durhams tube in MRS broth and<br />
inoculated with 1% overnight fresh cultures.<br />
Then the test tubes were incubated at 37 °C <strong>for</strong><br />
24 hrs. Accumulation of air bubble in Durham<br />
tubes after 24 hrs indicated CO2 production<br />
from glucose (16).<br />
Growth at Different Temperatures<br />
5ml MRS containing bromecresol purple<br />
indicator, was inoculated with fifty μl of<br />
overnight cultures in triplicate incubated <strong>for</strong> 5<br />
days at 15 °C, 37 0 C and 45 °C. Growth at any<br />
temperatures was indicated by the turbidity and<br />
change of colour from purple to yellow.<br />
Growth at Different NaCl Concentrations<br />
Isolates were tested <strong>for</strong> their tolerance against<br />
different NaCl concentrations. Test media<br />
containing bromecresol purple indicator were<br />
prepared with 3different concentrations NaCl<br />
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properties of L.fermentum and L.reuteri<br />
(1%, 2% and 3%) and transferred into tubes<br />
in 5 ml. These tubes were inoculated with 1%<br />
overnight cultures and then incubated at 37 °C<br />
<strong>for</strong> 5 days. The change of the colour from purple<br />
to yellow was noted.<br />
<strong>An</strong>tibiotic susceptibility profile:<br />
<strong>An</strong>tibiotic susceptibility profile was determined<br />
on MRS agar medium against 13 different<br />
antibiotic discs procured from Hi-media, India.<br />
by disk diffusion method (6).<br />
<strong>An</strong>timicrobial activity of Bacteriocin:<br />
<strong>An</strong>timicrobial action of lactobacillus strains was<br />
determined against indicator bacteria by agar<br />
well diffusion method (7). A total of 11 clinical<br />
isolates were obtained from department of<br />
Microbiology, SRM Medical college hospital &<br />
research centre. Out of these twelve, Four were<br />
Gram positive (Staphylococcus aureus, MRSA<br />
(Methicillin Resistant Staphylococcus Aureus).<br />
Streptococcus pneumoniae and Enterococcus<br />
sps) and seven were Gram negative (Escherichia<br />
coli, Klebsiella pneumoniae, Pseudomonas<br />
aeruginosa, Proteus sps, Salmonella typhi,<br />
Shigella dysentriae, and Vibrio cholera).<br />
Supernatant of lactobacilli sp representing the<br />
Bacteriocin were monitored <strong>for</strong> antibacterial<br />
activity against indicator bacteria inoculated on<br />
Muller Hinton Agar (MHA). A volume of 50<br />
µl of cell free supernatant was filled in 5 mm<br />
diameter sealed wells cut in the MHA agar. The<br />
diameter of the zone of inhibition was measured<br />
with caliper after 24 hr of incubation.<br />
Haemolytic activity<br />
Haemolytic property was evaluated on nutrient<br />
agar plate supplemented with 5% sheep blood<br />
which was incubated at 37 0 C <strong>for</strong> 24 hrs. (15).<br />
Resistance to Low pH<br />
Resistance to pH 3 <strong>for</strong> 3 hours is often used in<br />
vitro assays to determine the resistance to gastric<br />
acid, .considering the duration of stay of food<br />
98<br />
in the stomach (11). For this purpose, 16-18 hr<br />
old cultures were inoculated in 10 ml of 0.05 M<br />
sodium phosphate buffer adjusted to pH 2.0 to<br />
7.0 with 1N HCl and samples were incubated<br />
at 37 0 C <strong>for</strong> 3 hrs. The resultant growth was<br />
serially diluted to 10 fold dilution by phosphate<br />
buffer pH 7.0. Viable microorganisms in<br />
different dilutions were enumerated by pour<br />
plate techniques incubated at 37 °C under<br />
anaerobic conditions <strong>for</strong> 48 h. The survival rate<br />
was calculated as the percentage of colonies<br />
grown on MRS agar compared to the initial cell<br />
concentration. Each experiment was per<strong>for</strong>med<br />
in triplicate.<br />
Bile salt resistance:<br />
The ability to grow in the presence of 0.3%,<br />
0.6%, 0.9% and 1.2% bile salts was determined<br />
in MRS broth. (13). The growth was examined<br />
after 24 hours under anaerobic conditions of<br />
incubation at 37 0 C by plate count method and<br />
compared to those grown in the absence of bile<br />
salt. The experiment was per<strong>for</strong>med in triplicate.<br />
Results and Discussion:<br />
Human breast milk samples were obtained<br />
from lactating women of 20-36 years. of age.<br />
The samples were collected 6 to 32 days after<br />
delivery. Lactobacilli are Gram positive,<br />
microaerophilic, catalase negative, oxidase<br />
negative, non-spore <strong>for</strong>ming and non-capsulated<br />
rods. Microscopically they are short to long<br />
rods that appear as single cells, in pairs and in<br />
short chains. Surface colonies on MRS agar<br />
plate are 0.5 to 2 mm in dm, circular, lenticular,<br />
creamy-white. As per their Carbohydrate<br />
fermentation profile the isolates were identified<br />
as Lactobacillus reuteri, and Lactobacillus<br />
fermentum.<br />
Table.1 shows the result obtained <strong>for</strong> antibiotic<br />
susceptibility of the two species. Both the species<br />
were resistance to Vancomycin and sensitive to<br />
Ampicillin, Chloramphenicol, Clindamycin,<br />
Rifampicin, Amikacin, Gentamycin and<br />
ciprofloxacin. Most spp showed resistance to 3<br />
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properties of L.fermentum and L.reuteri<br />
Table 1. <strong>An</strong>tibiotic resistance profiles of the tested probiotic strains (<strong>An</strong>tibiotic Conc. range, µg/ML):<br />
L.f- Lactobacillus fermentum, L.r- Lactobacillus reuteri, S- sensitive, R- resistant<br />
22<strong>An</strong>tibiotics (µg) L.f 1 L.f 2 L.f 3 L.f 4 L.r 1 L.r 2 L.r3 L.r 4<br />
Ampicillin (10) S S S S S S S S<br />
Amikacin (32) S S S S S S S S<br />
Cefoxitin (30) S S R S S S S S<br />
Clindamycin (2) S S S S S S S S<br />
Ciprofloxacin (30) S S S S S S S S<br />
Cloramphenicol (30) S S S S S S S S<br />
Gentamycin (5) S S S S S S S S<br />
Kanamycin (30) R R R R R S R R<br />
Oxacillin (1) S S S R R S S S<br />
Rifampicin (5) S S S S S S S S<br />
Tetracycline (30) R R R S R S S S<br />
Vancomycin (30) R R R R R R R R<br />
SG- slight growth, G-growth, NG- no growth, NH- non hemolytic<br />
Table. 2. Resistance to temperature, bile and hemolysin production:<br />
Resistance To Temp Resistance Of Nacl In % Type Of Hemolysis<br />
15 0 C 37 0 C 45 0 C 1% 2% 3%<br />
L.fermentum SG G G G G NG NH<br />
L.reuteri SG G G G G NG NH<br />
Figure 1. <strong>An</strong>timicrobial activity of culture supernatant against<br />
pathogens<br />
Zone of Inhibition<br />
14<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
S,aure<br />
us<br />
Entero<br />
coccu<br />
MRSA E.coli K.pne<br />
umoni<br />
L.fermentum 11 10 12 11 10 10 8 11 11 10<br />
L.reuteri 10 8 9 10 10 10 7 9 11 9<br />
Clinical pathogens<br />
P.aeru Proteu<br />
ginosa s spp<br />
S.typh<br />
i<br />
S.dys<br />
entriae<br />
V.chol<br />
era<br />
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99
properties of L.fermentum and L.reuteri<br />
OD Values<br />
Percentage of viable strains (%)<br />
1.8<br />
1.6<br />
1.4<br />
1.2<br />
1<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Fig 2. Resistance to Low pH<br />
pH2 pH3 pH4 pH5 pH6 pH7<br />
Fig.3. Tolerance of 0.3% of Bile salt<br />
L.fermentum<br />
L.reuteri<br />
0 hrs 5 hrs 10 hrs 15 hrs 20 hrs 25 hrs 30 hrs 35 hrs 40 hrs 45 hrs 50 hrs<br />
Control L.fermentum L.reuteri<br />
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100
properties of L.fermentum and L.reuteri<br />
of the 13 antibiotics tested. i.e. to Vancomycin,<br />
tetracycline and kanamycin. Four spp (three<br />
L.fermentum and one L.reuteri) showed multiple<br />
resistances to 3 different antibiotics, namely<br />
cefoxitin, oxacillin and kanamycin. Lactic<br />
acid bacteria (LAB) especially lactobacilli are<br />
normal inhabitants of intestinal tract of humans<br />
and animals and are also found in milk and<br />
milk products. (Mitsuoka 1992).. In our study<br />
all lactobacilli inhibited the growth of S.aureus,<br />
MRSA (Methycillin Resistant Staphylococcus<br />
Aureus), Enterococcus spp, E.coli, Klebsiella<br />
pneumonia. Pseudomonas aeruginosa, Proteus<br />
spp, Salmonella typhi, Shigella spp, Vibrio<br />
chlorae. The strongest antimicrobial effect<br />
was shown by L.fermentum against indicator<br />
bacteria when compared to L.reuteri. (Fig. 1).<br />
The antimicrobial action may be attributed to<br />
lactic acid production, bacteriocins and some<br />
peptide with inhibitory properties (8). <strong>An</strong>other<br />
criterion <strong>for</strong> a good probiotic is the ability to<br />
grow at different temperatures. At the end of<br />
5 days incubation, all isolates could grow at<br />
15, 37 and 45 °C. Growth at 15C was minimal<br />
compared to that in 37 and 45 (Table 2). All of<br />
the isolates were able to grow upto 2% NaCl<br />
concentration. Lactobacillus isolated from<br />
human breast milk were not hemolytic to sheep<br />
blood (Table 2).and is a desirable property <strong>for</strong><br />
an ideal probiotic strain (9). The effect of pH<br />
ranging from 2.0 to 7.0 on the L.fermentum<br />
and L.reuteri was studied. It was found that<br />
they could survive a pH of 2.0, (78%) even<br />
though growth was better in 3.0 to 7.0. (Figure<br />
2). Resistances to low pH are one of the major<br />
selection criteria <strong>for</strong> probiotic strains and reflect<br />
its ability to withstand conditions in stomach and<br />
small intestine (12).Bile tolerance by probiotics<br />
has been shown to be strain- and bile typedependent,<br />
with resistance levels ranging from<br />
bile concentrations of 0.125 to 2.0% (10). Bile<br />
tolerance has been described as an important<br />
factor <strong>for</strong> the survival and growth of LAB in the<br />
intestinal tract. While both strains isolated from<br />
human breast milk could tolerant 0.3% Ox gall<br />
bile, there were some minor differences. (Figure<br />
101<br />
3.)In general, the required concentration of bile<br />
salts considered necessary to screen <strong>for</strong> resistant<br />
strains <strong>for</strong> human use is 0.3%. (14).<br />
Conclusion:<br />
This study was focused on exploring the potential<br />
features of strains isolated from human milk <strong>for</strong><br />
use as probiotics. Lactobacillus strains constitute<br />
the normal bacterial flora of the human breast<br />
milk. L.fermentum and L.reuteri were the most<br />
predominant isolates and exhibited many probiotic<br />
qualities. They were inhibitory to the most<br />
predominant pathogens isolated from clinical<br />
specimen. The ability to survive acidic conditions,<br />
tolerance to heat (45C), 0.3% bile and 2% NaCl,<br />
represent qualities of an ideal probiotic and may<br />
have potential applications to provide balanced<br />
intestinal microbiota in children with diarrhea. This<br />
study also emphasizes the value of breast feeding in<br />
prevention of infective diarrhea in children.<br />
References:<br />
1. Roos, S., Engstrand L., Jonsson H. Lactobacillus gastricus<br />
sp. nov., Lactobacillus antri sp. nov., Lactobacillus<br />
kalixensis sp. nov. and Lactobacillus ultunensis sp. nov.,<br />
isolated from human stomach mucosa. <strong>International</strong> <strong>Journal</strong><br />
of Systematic and Evolutionary Microbiology. 2550;<br />
55:77-82.<br />
2. Food and Agricultural Organization of the United Nations<br />
and World Health Organizations. Posting data: Regulatory<br />
and clinical aspects of dairy probiotics. Food and Agricultural<br />
Organization of the United Nations and World<br />
Health Organizations expert consultation report. Food<br />
and Agricultural Organization of the United Nations and<br />
World Health Organizations working group report. 2001;<br />
(Online).<br />
3. Gibson GR, Roberfroid MB. Dietary Modulation of the<br />
human clonic microbiota; Introducing the concept of prebiotics.<br />
J. Nutr. 1995; 125;1401-12.<br />
4. S.K.Dash. PhD, President and Director of Research UAS<br />
Laboratories, 9953 Valley View road, Eden Prairie, Minnesota<br />
55344, USA. Selection criteria <strong>for</strong> probiotics. Paper<br />
presented in XXXVII Dairy industry Conference, Feb 7-9,<br />
2009. Kala Academy, Panjim, Goa.<br />
5. Szajewska H, Kotawska M, Mrukowicz J.Z, Armanska<br />
M, and Mikotajczyk W. Efficacy of Lactobacillus GG in<br />
prevention of nosocomial diarrhea in infants. <strong>Journal</strong> of<br />
pediatrics. 2001; 138, 361-365.<br />
6. NCCLS (National Committee <strong>for</strong> Clinical Labotratory<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
properties of L.fermentum and L.reuteri<br />
Standards). NCCLS document M-100-S9. Per<strong>for</strong>mance<br />
standards <strong>for</strong> antimicrobial susceptibility testing 9 th in<strong>for</strong>mation<br />
Supplement. NCCLS: Weyne Pa. 1999.<br />
7. Flemming H.P., J.L.Etchells and R.L.costilow. Microbial<br />
inhibition by an isolate pedicoccus from cucumber brines.<br />
Appl. Microbiol. 1985; 30: 1040-1042.<br />
8. Strus. M, K.Pakosz, H.Gociniak, A.Przondomordarska,<br />
E.Roy nek.H, Pituch, F, Meisel.Miko Ajczyk and<br />
P.R.Heczko. <strong>An</strong>tagonistic activity of Lactobacillus strains<br />
against anaerobic gasterointestinal tract pathogens. (Helicobacter<br />
pyroli, Campylobacter coli, C.jejuni, Clostridium<br />
difficile). Med. Dosw. Microbiol. 2001; 53: 133-142.<br />
9. De Vuyst, L., Foulquie and H.Reverts. Screening <strong>for</strong> enterocins<br />
and detection of hemolysin and Vancomycin resistance<br />
in Enterococci of different origins. Intl. J. Food<br />
Microbiology., 2003; 84: 299-318.<br />
10. Lian W.C. Hsiao, H.C., and Chou. C.C. Viability of microencapsulated<br />
bifidobacteria in stimulated gastric juice and<br />
bile solution. Inter. J. Food Microbiol., 2003; 82: 293-301.<br />
11. Prasad. J, Gill. H, Smart. J, and Gopal P.K. Selection and<br />
Characterization of Lactobacillus and Bifidobacterium<br />
strains <strong>for</strong> use as probiotic. <strong>International</strong> Dairy <strong>Journal</strong>.<br />
1998; 8:993-1002.<br />
12. Çakir. I. Determination of some probiotic properties on<br />
102<br />
Lactobacilli and Bifidobacteria. <strong>An</strong>kara University thesis<br />
of Ph.D. 2003.<br />
13. Awan, J.A. and S.U. Rahman. Microbiology Mannual.<br />
Unitech Communications, Faisalabad, Pakistan, 2005;<br />
pp:45-51.<br />
14. Gilliland, S. E. Beneficial interrelationships between<br />
certain microorganisms and human: Candidate microorganisms<br />
<strong>for</strong> use as dietary adjuncts. J. Food Prot., 1990;<br />
42:164-167.<br />
15. Lombardi, A. Dal Maistro. L, De Dea.P, Gatti, M. Giraffa,<br />
G. A polyphasic approach to highlight genotypic and<br />
phenotypic diversities of Lactobacillus helverticus strains<br />
isolated from dairy starter cultures and cheeses. <strong>Journal</strong> of<br />
Dairy Research, 2002; 69, 139-149.<br />
16. Davis. D.H.G. The Classification of Lactobacilli from the<br />
human mouth. <strong>Journal</strong> of General Microbiology, 1995; 13,<br />
n.3, p. 481-493,<br />
17. Scarpellini E, Cazzato A, Lauritano C, Gabrielli M, Lupascu<br />
A, Gerardino L. Abenavoli L, Petruzzellis C, Gasbarrini<br />
G, Gasbarrini A.Probiotics: Which and when? Dig Dis<br />
2008; 26:175-182.<br />
18. Michael, T. Family Lactobacillaecea. In: Star, P. (Eds).<br />
The Prokaryotes. New York, Blackwell Press, USA, 1981;<br />
pp:609-619.<br />
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103<br />
<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 103 - 108<br />
Perinatal androgen levels and Sexual dimorphic digit ratio in Down syndrome<br />
Children<br />
Suresh Bidarkotimath and S. Viveka<br />
Department of <strong>An</strong>atomy, A J Institute of medical sciences, Mangalore - 575004. Karnataka, India.<br />
(Received 26 th July, 2010; Revised 23 rd December, 2010; Accepted 11 th March, 2011)<br />
Corresponding Address<br />
Dr. Suresh Bidarkotimath<br />
Email: bidarkotimath@gmail.com<br />
Abstract<br />
Background and Objectives:- Down syndrome being the major common cause of mental retardation<br />
in children is most commonly studied chromosomal anomalies. The sexual abnormalities in it include<br />
undescended testis, late sexual maturity. It has been proven that the androgen levels in children and adults<br />
with this syndrome are low. To establish the androgen levels in early perinatal period the digit ratio can be<br />
used. Digit ratio is the ratio between the length of 2nd finger and 4th finger as measured <strong>for</strong>m the bases of<br />
digit to their tip. Digit ratios are determined by the early perinatal androgen levels and it remains stable after<br />
early life. Digit ratios are shown to be sexually dimorphic.<br />
Methods:- Digit ratios of 23 Down syndrome patients were analyzed which included 13 males and 10<br />
females from Saanidhya Samarth Center, Mangalore, and compared it with the age related control group<br />
of 50 children.<br />
Results :- It was found that the ratios are sexually dimorphic in Down syndrome patients [The mean of<br />
2D:4D ratios of males right hand was 0.961 (SD 0.042) and left hand was 0.977 (SD 0.067). The mean of<br />
2D:4D ratios of females right hand was 0.90 (SD0.053) and left hand was 0.939 (SD 0.075)] and they are<br />
statistically different from the control group.<br />
Interpretation and Conclusion:- It is hypothesized that the ratios are sexually dimorphic as in general<br />
public that there is no difference between the perinatal androgen levels in Down syndrome patients as<br />
compared with normal counterparts.<br />
Key words: Down syndrome, Digit ratio, Perinatal androgen levels.<br />
Introduction<br />
The ratio of index finger (2D) to the ring finger<br />
(4D) length is referred as digit ratio (2D:4D).<br />
The digit ratio and its association to the human<br />
characteristics has been the focus of much<br />
research in recent years. The digit ratios are<br />
determined by taking the lengths of the fingers<br />
are taken from the proximal crease of the<br />
digit to their tip(1) and taking their ratios. It<br />
has been widely accepted that the sex differ-<br />
ence in 2D:4D ratio arises early in development<br />
and the ratios are slightly lower in males than<br />
females, making it – sexually dimorphic. This<br />
dimorphism arises due to the near equal lengths<br />
of 2nd and 4th fingers(2) in males. Once established<br />
in early neonatal life digit ratio assumed<br />
to be stable in later life. The digit ratio has been<br />
reported to be associated with several characteristics<br />
such as fetal growth, congenital adrenal<br />
hyperplasia, developmental psychopathology,<br />
autism and Asperger’s syndrome(3). Little is<br />
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Perinatal androgen levels, digit ratio in Down syndrome<br />
known about 2D:4D ratio variations in Down<br />
syndrome patients. Down syndrome (trisomy<br />
21, Manglolism) is the most common chromosome<br />
disorder and single most common cause<br />
of moderate mental retardation. It is associated<br />
with impairment of cognition and characteristic<br />
facial and other dysmorphic features. It<br />
is also associated with undescended testis and<br />
delayed appearance of secondary sexual features<br />
in male children(4) . Down children attain<br />
a significantly lesser height than their normal<br />
counterparts(5). It has been confirmed that<br />
levels of circulating androgens as evidenced<br />
by urinary testosterone and epiandrosterone<br />
levels are low. Though much is known about<br />
the adult androgen levels and related clinical<br />
problems, there is relative paucity of knowledge<br />
on the perinatal androgen levels in Down<br />
syndrome. The Homeobox genes Hox a and d<br />
control the differentiation of the urinogenital<br />
system, and may there<strong>for</strong>e indirectly influence<br />
the prenatal production of testicular androgen<br />
and the development of the digits (6). Prenatal<br />
testosterone comes from maternal testosterone,<br />
which may pass across the placenta and enter<br />
the fetal bloodstream, and from the fetus itself,<br />
which secretes increasing amounts from about<br />
8 weeks (the time of Leydig cell differentiation)<br />
to mid-gestation (7). From that point levels<br />
slowly decrease until a few months after birth<br />
when it has reached the low level characterizing<br />
childhood (8). The fetal source is dependent<br />
on the differentiation of the testes (9). Perinatal<br />
androgen levels attain maximum level at 3 – 6<br />
months of intrauterine life and there is slight increase<br />
be<strong>for</strong>e delivery. At puberty there is slow<br />
but sustained increase in the testosterone levels<br />
be<strong>for</strong>e attaining the adult levels. There is substantial<br />
evidence that digit ratio is established<br />
early in intrauterine life under the influence<br />
of androgens. Low androgen levels during the<br />
late intrauterine life around the time of delivery<br />
causes relative delay in the descending of testis.<br />
In this study we tried to find out whether there<br />
are any alterations in early perinatal androgen<br />
levels as depicted by digit ratio.<br />
Materials and methods:<br />
104<br />
We have selected 23 Karyotypically diagnosed<br />
cases of Down syndrome (all having 21 trisomies)<br />
children from Saanidhya Center <strong>for</strong> disabled,<br />
Mangalore. All subjects were unrelated<br />
Caucasians. Their age ranged from 6 to 22<br />
years. The digits lengths were determined by<br />
taking photographs of the hands of the selected<br />
children by using Digital camera (Sony India)<br />
with 8MP resolution, auto ISO having light<br />
background. The digit lengths were measured<br />
from the ventral proximal crease to the tip of the<br />
fingers both on ulnar and radial aspect (in order<br />
to account the sloppy proximal crease) by using<br />
‘measure tool’ of Adobe Photoshop CS5(10).<br />
Similar set of data was also taken from age related<br />
normal children. The direct measures of<br />
digits using measuring tool like vernier calipers<br />
and measures from photocopies produce comparable<br />
2D:4D ratios . The data was tabulated<br />
and analyzed using student’s t test.<br />
Results:<br />
Out of 23 Downs cases selected, 13 were males<br />
and 10 were females. Repeatability of 2D:4D<br />
from all 23 hands were as follows: males (n=13)<br />
r1=0.86; females (n=10) r1= 0.92. There was<br />
significantly greater variance between subjects<br />
than within-subject error (repeated measures<br />
ANOVA: males, F=13.70, p=.0001; females,<br />
F= 23.22, p=.0001).We concluded that our measurements<br />
represented real differences between<br />
subjects. The mean of 2D:4D ratios of males<br />
right hand was 0.961 (SD 0.042) and left hand<br />
was 0.977 (SD 0.067).The mean of 2D:4D ratios<br />
of females right hand was 0.90 (SD0.053)<br />
and left hand was 0.939 (SD 0.075) as shown<br />
in Graph:1 .In accord with previous workers<br />
males have statistically significant lower ratios<br />
than the females. The difference between the<br />
Down syndrome male and female patients were<br />
statistically significant (right hand t = 42, p ><br />
0.001, left hand t = 10, p > 0.001). The mean<br />
of 2D:4D ratios of males of age related control<br />
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Perinatal androgen levels, digit ratio in Down syndrome<br />
group was – right hand 0.990 (SD 0.020) and<br />
left hand 0.970 (SD 0.040). The mean of 2D:4D<br />
ratios of females of age related control group<br />
was – right hand 1.050 (SD 0.030) and left hand<br />
1.050 (SD 0.030). The difference between the<br />
males and females of control group were statistically<br />
significant (right hand t = 8.37, p ><br />
0.001 and left hand t = 7.94, p > 0.001). There<br />
is statistical difference between the digit ratios<br />
of Down syndrome and control group in both<br />
males (right hand t= 2.355, p > .001) and females<br />
(right hand t= 8.04, p > .001).<br />
Discussion<br />
Digit ratio:<br />
It has been known <strong>for</strong> more than a century that<br />
males and females tend to differ in the relative<br />
lengths of their index (2D) and ring (4D) fingers.<br />
The concept of digit ratio is popularized by an<br />
evolutionary psychologist, Prof John Manning<br />
and it’s been a decade since he published his<br />
views on it. He describes the digit ratio as a living<br />
fossil and a record of factors the fetus was<br />
exposed to at a critical time <strong>for</strong> the development<br />
of many other things.<br />
The digit ratios are sexually dimorphic and it has<br />
been proved that these ratios retain its dimorphism<br />
in infants, children and adults. A study by<br />
Stephens and Shepard has proved that this ratio<br />
is dimorphic even in fetuses, providing much<br />
needed support <strong>for</strong> the theory of establishment<br />
of the digit ratio in early neonatal peroid (11).<br />
In males, the second digit tends to be shorter<br />
than the fourth, and in females the second tends<br />
to be the same size or slightly longer than the<br />
fourth, making 2D:4D ratio sexually dimorphic<br />
with mean male 2D:4D lower than mean female<br />
2D:4D(12). The dimorphism patterns are well<br />
recognized in laboratory animals as well. Digit<br />
ratio has shown variations with different race<br />
and ethnicity. The differentiation of the digits<br />
is under the control of Homeobox or Hox genes<br />
(the posterior-most Hoxd and Hoxa genes),<br />
which also control the differentiation of the tes-<br />
105<br />
tes and ovaries (13). This common control of<br />
the distal limbs and genital bud may be seen<br />
when progressive removal of posterior Hox<br />
gene function results in loss of digits, genital<br />
bud derivatives, and fertility. It is suggested that<br />
the common control of the differentiation of the<br />
gonads and digits may mean that the functioning<br />
of the <strong>for</strong>mer may be reflected in the <strong>for</strong>mation<br />
of the latter (14). Polymorphisms within<br />
Hoxd and Hoxa may there<strong>for</strong>e result in variation<br />
in gonad and digit <strong>for</strong>m and function. The<br />
<strong>for</strong>mer could then affect Leydig cell differentiation<br />
and there<strong>for</strong>e the production of testosterone<br />
(15). Patterns of 2D:4D ratios may there<strong>for</strong>e<br />
reflect aspects of gonadal function such as the<br />
production of testosterone and estrogen.<br />
Several studies have found a significant positive<br />
relationship between 2D:4D ratio and medical<br />
problems like attension deficit hyperactive<br />
disorder (ADHD)(16) , autism, aggression in<br />
males, sporting ability in females. In contrast to<br />
this, a significant negative relationship has been<br />
found between the 2D:4D ratio and physical<br />
competence.<br />
There is some evidence that 2D:4D ratio may<br />
also be indicative <strong>for</strong> human development and<br />
growth. Ronalds et al. (2002) showed that men<br />
who had an above average placental weight and<br />
a shorter neonatal crown-heel length had higher<br />
2D:4D ratios in adult life(17) . Moreover, studies<br />
about 2D:4D correlations with face shape<br />
suggest that testosterone exposure early in life<br />
may set some constraints <strong>for</strong> subsequent development.<br />
Prenatal sex steroid ratios (in terms<br />
of 2D:4D) and actual chromosomal sex dimorphism<br />
were found to operate differently on<br />
human faces, but affect male and female face<br />
shapes by similar patterns. However, exposure<br />
to very high levels of testosterone and/or estrogen<br />
in the womb may have also negative effects.<br />
Fink et al. (2004) found that men with low<br />
2D:4D ratios (indicating high testosterone) and<br />
women with high 2D:4D ratios (indicating high<br />
estrogen) express lower levels of facial symmetry(18).<br />
There is some evidence that testosterone<br />
facilitates the differentiation of the brain<br />
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Perinatal androgen levels, digit ratio in Down syndrome<br />
both prenatally and postnatally. There have<br />
been many extensions of this, such as the Geschwind–Galaburda<br />
hypothesis, that immune<br />
disease and autism(19) are related to prenatal<br />
testosterone, this also explaining why more<br />
men are left-handed, autistic, etc. than women.<br />
Prenatal exposure to testosterone is thought to<br />
promote the development of the right-hemisphere<br />
and increase the incidence of sinistrality.<br />
As such low 2D:4D was found to be associated<br />
with improved left-hand per<strong>for</strong>mance. It has<br />
been showed that the 2D: 4D ratio in patients<br />
suffering from polycystic ovarian disease are<br />
not substantially decreased and in turn the perinatal<br />
androgen levels are not greatly elevated in<br />
them(20).<br />
Down syndrome hand characteristics:<br />
Among many phenotypic findings of Down<br />
syndrome the most important ones are the<br />
hand characteristics, physical traits (ear length<br />
and internipple distance) and clinical findings<br />
(Brushfield spots, wide-spaced first toe, and<br />
excess back neck skin)(21) . Down syndrome<br />
is associated with many hand characteristics,<br />
which can be classified into three main categories<br />
namely hand lines, dermatoglyphics and<br />
hand morphology. Simian crease (single palmar<br />
distal transverse crease) extended proximal<br />
palmar transverse crease , single interdigital<br />
crease on 5th (and sometimes on 4th) finger are<br />
the more common hand crease characteristics.<br />
Simian crease is found in more than a quarter<br />
of Down syndrome patients according to studies<br />
by C Plato(22). Other studies have indicated<br />
that the simian is also very significant when<br />
present only in the right hand; when present<br />
only in the left hand it is especially significant<br />
when combined with a Sydney line in the right<br />
hand Simian crease incidence is increased in<br />
fetuses with Down syndrome Simian crease is<br />
reported in more than third of the cases of the<br />
cases from India by Maina and colleagues(23)<br />
. Extended proximal transverse palmar crease<br />
(Sydney line) is again a significant sign of Down<br />
106<br />
syndrome according to Purvis- Smith (28). Its<br />
incidence is more than 15% (33). Deckers and<br />
Oorthuys have indicated that the extended distal<br />
transverse crease is significant when observed<br />
in the left hand; in the right hand it is significant<br />
when combined with a simian crease in the left<br />
hand . There are reports of single flexion crease<br />
on 4th and 5th fingers. Ulnar loops (especially<br />
the index finger), radial loops on the ring and<br />
index, loop between the base of the index and<br />
middle finger and/or the middle and ring fingers,<br />
hypothenar ulnar loops, whorls, or carpal<br />
loops and distally located axial triradii are the<br />
more common dermatoglyphic characteristics<br />
of Down syndrome. The ulnar loops in Down<br />
syndrome tend to be vertically oriented and Lshaped<br />
- with the ‘open’ side directed to the side<br />
of the little finger. <strong>An</strong>other common variant is:<br />
9 (or 8) ulnar loops combined with a radial loop<br />
on the ring finger or little finger. Radial loop is<br />
most frequently seen on the ring finger (the radial<br />
loops are usually vertically oriented and L<br />
shaped - with the ‘open’ side directed to the side<br />
of the thumb. A radial loop on the thumb, index<br />
finger or middle finger may not point towards a<br />
Down syndrome characteristic hand. The ‘palmar<br />
ridge line’ starts in the triradius below the<br />
index finger. In Down syndrome the path of<br />
the ‘palmar ridge line’ ends often just above or<br />
close to the point where the ‘upper transverse<br />
crease’ exits the palm. In the normal population<br />
the path of the ‘palmar ridge line’ is usually<br />
found completely below the ‘upper transverse<br />
crease’. In Down syndrome the alignment of<br />
ridges over the distal palmar area is - partly due<br />
to the short, broad hand shape - nearly always<br />
rather ‘transverse’. This is usually indicated by<br />
the combination of a palmar ridge line which exits<br />
the palm above (or just below) the heart line,<br />
combined with another palmar ridge line which<br />
exits the palm between the pointer finger and<br />
the middle finger. Characteristic palmar axial<br />
triradius is also advocated as one of the features<br />
of this syndrome(24). In Down syndrome quite<br />
often 3 or more triradii can be observed on the<br />
hypothenar due to the presence of various types<br />
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Perinatal androgen levels, digit ratio in Down syndrome<br />
of large loops, multiple loops, or whorls(25).<br />
The morphological changes in the hand include<br />
short thumb, short and incurved little finger,<br />
brachydactyly (short fingers), broad – short<br />
palm, hyperextensible joints..A low 2D:4D has<br />
been reported to be correlated with an increased<br />
preference to use the left hand (20). Low 2D:4D<br />
in children is associated with an increased risk<br />
of autism. This may be because 2D:4D ratio is<br />
itself related to high prenatal testosterone(26).<br />
2D:4D is associated with measures of size at<br />
birth in males, sperm counts , family size , age<br />
at breast cancer presentation and age at myocardial<br />
infarction(27). Our study of 2D:4D ratios<br />
showed that males have slightly but statistically<br />
significant lower ratios than the females indicating<br />
the sexual dimorphism. The difference<br />
between the males and females digit ratio were<br />
substantial than compared with the difference<br />
of the same in the control group. 2D: 4D ratios<br />
in the Down syndrome group on an average are<br />
smaller in value (e.g. right hand ratios, males-<br />
0.961 and females – 0.90) than the age related<br />
control group (right hand ratios, males – 0.99<br />
and females 1.050) accounted by short fingers<br />
of Down syndrome patients and this difference<br />
is statistically significant. Sexual dimorphism is<br />
established in spite of brachydactyly in Down<br />
syndrome patients. As the digit ratios of both<br />
males and females of Down syndrome patients<br />
varied significantly from the control group, it<br />
may be said that yet another factor determines<br />
the relative lengths of the digits in Down syndrome<br />
apart from factors which decreases the<br />
lengths of all the fingers (resulting in brachydactyly).<br />
Comparing the ratios with the age related<br />
normal children is expected to neutralize<br />
any subtle changes in the digit ratios as a part of<br />
growth during childhood. Our study definitely<br />
fall short in generalizing the statement that digit<br />
ratio across all patients with Down syndrome<br />
is sexually dimorphic as the sample size of the<br />
study is too small. In our study, we did not find<br />
a significant relationship between 2D:4D ratio<br />
and the presence or absence of minor or major<br />
congenital abnormalities.<br />
Conclusions:<br />
107<br />
We conclude that the digit ratio in Down syndrome<br />
is sexually dimorphic and thus it may be<br />
hypothesized that the early perinatal androgen<br />
levels are same as normal intrauterine development.<br />
The sexual alterations seen in Down<br />
syndrome are due to alterations during the descent<br />
of the testis. This view is supported by the<br />
normal or near normal development of the other<br />
genital parts. There is yet another biological<br />
factor responsible <strong>for</strong> the hand morphological<br />
manifestations in Down syndrome – as the digit<br />
ratios are statistically different from the age<br />
related counterparts. It may be viewed that the<br />
factors responsible <strong>for</strong> the generalized shortening<br />
of the fingers are also affecting the relative<br />
lengths of the digits resulting in this kind of difference<br />
in digit ratio.<br />
Acknowledgements:<br />
We thank Mr. Vasanth Kumar Shetty, Principal,<br />
Saanidhya Samarth Center, Mangalore <strong>for</strong> allowing<br />
us to conduct the study. We extend our<br />
thanks to Dr. Ramesh Pai, Dean, A J Institute<br />
of Medical <strong>Sciences</strong> <strong>for</strong> allowing us to proceed<br />
with the study. Many thanks to all the children<br />
of Saanidhya Samarth Center.<br />
References<br />
1. Manning, John T. Digit Ratio: A Pointer to Fertility,<br />
Behavior and Health. New Jersey : Rutgers University<br />
Press, 2002. pp. 24-40.<br />
2. Frietson Galis, Clara M. A., Ten Broek, Stefan Van<br />
Dongen, Liliane C. D. Wijnaendts. Sexual Dimorphism in<br />
the Prenatal Digit Ratio (2D:4D). Arch Sex Behav. 2010,<br />
39, pp. 57-62.<br />
3. J.Manning, A.Stewart, P.Bundred, R.Trivers. Sex and<br />
ethnic differences in 2nd to 4th digit ratio of children.<br />
Early Human Development. November 2004, Vol. 80, 2,<br />
pp. 161-168.<br />
4. Culley, Alvin Petersa and William. Urinary levels of<br />
testosterone and epitestosterone in down’s syndrome<br />
(mongolism). Clinica Chimica Acta. August 1969, Vol. 25,<br />
2, pp. 24-40.<br />
5. Siegfried M. Pueschel. Adolescent Development in Males<br />
With Down Syndrome. Am J Dis Child. 139, 1985, 3, pp.<br />
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Perinatal androgen levels, digit ratio in Down syndrome<br />
236-238.<br />
6. Kondo T, Zakany J, Innis J, Duboule D. Of fingers, toes<br />
and penises. Nature. 1997, Vol. 390, 29, p. 10.<br />
7. JM, Tanner. Foetus into man: Physical growth from<br />
conception into maturity. Cambridge : Harvard Press, 1990.<br />
8. Lording DW, & Dekretser DM. Comparative ultrastructural<br />
and histochemical studies of the interstitial cells of the<br />
rat testis during fetal and postnatal development. <strong>Journal</strong><br />
Reproduction and fertility. Vol. 29, pp. 261-269.<br />
9. Christoph J. Kemper, <strong>An</strong>dreas Schwerdtfeger. Comparing<br />
indirect methods of digit ratio (2D:4D) measurement.<br />
American <strong>Journal</strong> of Human Biology. November 2008,<br />
Vol. 21, 2, pp. 188-191.<br />
10. 10. Trent D. Stephens, Thomas H. Shepard. The Down<br />
syndrome in the fetus. Teratology. August 1980, Vol. 22,<br />
1, pp. 37 - 41.<br />
11. Baker, F. <strong>An</strong>thropological notes on the human hand. Am<br />
<strong>An</strong>thropol. 1, 1888, pp. 51–76.<br />
12. Peichel, C. L., Prabhakaran, B., & Vogt, T. F. The<br />
mouse Ulnaless mutation deregulates posterior Hoxd.<br />
Development. 24, 1997, pp. 3481-3492.<br />
13. Manning J T, Scutt D, Wilson J Lewis-Jones D I. The ratio<br />
of 2nd to 4th digit length: a predictor of sperm numbers and<br />
levels of testosterone, LH and oestrogen. Hum Reprod. 13,<br />
1998, pp. 3000-3004.<br />
14. Manning JT, Trivers RL, Thornhill R, Singh D. The 2nd:4th<br />
digit ratio and asymmetry of hand per<strong>for</strong>mance in Jamaican<br />
children. Laterality. 2000, Vol. 5, 2, pp. 121- 132 .<br />
15. McFadden D, Westhafer JG, Pasanen EG, Carlson<br />
CL and Tucker DM. Physiological evidence of<br />
hypermasculinization in boys with the inattentive subtype<br />
of attention-deficit/hyperactivity disorder (ADHD).<br />
Clinical Neuroscience Research. 2005, pp. 233–245.<br />
16. G. Ronaldsa, D.I.W. Phillips, K.M. Godfreya, J.T. Manning.<br />
The ratio of second to fourth digit lengths: a marker of<br />
impaired fetal growth? Early human Development. June<br />
2002, Vol. 68, 1, pp. 21-26.<br />
17. Fink, Bernhard, . Second to fourth digit ratio and facial<br />
108<br />
asymmetry. Evolution and Human Behavior. march 2004,<br />
Vol. 24, 2, pp. 125-32.<br />
18. Baron-Cohen S and Wheelwright S. The empathy quotient:<br />
an investigation of adults with Asperger syndrome or high<br />
functioning autism, and normal sex differences. <strong>Journal</strong> of<br />
Autism and Developmental Disorders. april 2004, Vol. 34,<br />
2, pp. 163-175.<br />
19. Chizen Marla E. Lujan, Terri G. Bloski, Donna R., Denis<br />
C. Lehotay and Roger A. Pierson. Digit ratios do not serve<br />
as anatomical evidence of prenatal androgen exposure in<br />
clinical phenotypes of polycystic ovary syndrome. Human<br />
Reproduction. 2010, Vol. 25, 1, pp. 204-211.<br />
20. A. Rex, M. Preus. A diagnostic index <strong>for</strong> Down syndrome.<br />
The <strong>Journal</strong> of Pediatrics. June 1982, Vol. 100, 6, pp. 903-<br />
906.<br />
21. Plato, Chris C., Cereghino, James J. and Steinberg,<br />
Florence S. Palmar Dermatoglyphics of Down’s Syndrome:<br />
Revisited. Pediatric Research. march 1973, Vol. 7, 3, pp.<br />
111-118.<br />
22. Maina P Kava, Milind S Tullu, Mamta N Muranjan, K.M<br />
Girisha. Down syndrome: Clinical profile from India.<br />
Archives of Medical Research. January 2004, Vol. 35, 1,<br />
pp. 31-35.<br />
23. Borgaonkar DS, Davis M, Bolling DR, Herr HM.<br />
Evaluation of dermal patterns in Down’s syndrome by<br />
predictive discrimination. I. Preliminary analysis based on<br />
frequencies of patterns. Johns Hopkins Med J. 1971, Vol.<br />
128, 3, pp. 141-152.<br />
24. Blanka A. Schaumann, Milton Alter. Dermatoglyphics in<br />
medical disorders. 1978. pp. 82-83. 15.<br />
25. JT Manning, S Baron-Cohen, G Sanders. The 2nd to 4th<br />
Digit Ratio and Autism. Developmental Medicine and<br />
Child Neurology. 2001, Vol. 43, pp. 160-164.<br />
26. Manning JT, Bundred PE. The 2nd to 4th digit ratio and<br />
age at first myocardial infarction in men: evidence <strong>for</strong> a<br />
link with prenatal testosterone? Br J Cardiol. Vol. 8, pp.<br />
720-723.<br />
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<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 109 - 113<br />
Serum Enzymes, Initial and follow- up Lipid profile in Acute Myocardial Infarction<br />
*Suman.S. Dambal, **V. Indumati. and ***P.B. Desai<br />
*Department of Biochemistry, Karnataka Institute of Medical <strong>Sciences</strong>,<br />
Hubli - 580029, Karnataka, India.<br />
**Department of Biochemistry, Vijayanagar Institute of Medical <strong>Sciences</strong>,<br />
Bellary - 583104, Karnataka, India<br />
***Department of Biochemistry, Jawaharlal Nehru Medical College,<br />
Belgaum - 590010, Karnataka, India.<br />
(Received 8 th October, 2010; Revised 12 th February, 2011; Accepted 28 th February, 2011)<br />
Corresponding author<br />
Dr. Suman.S.Dambal<br />
E-mail: sumandambal25@gmail.com<br />
Abstract<br />
Background & Objectives: Despite impressive studies in the diagnosis and management of Acute<br />
Myocardial Infarction (AMI) over the last three decades it continues to be a major public health problem in<br />
the world. There is paucity of literature on the enzyme levels in Stable <strong>An</strong>gina Patients. Hence the present<br />
study was taken up to study the diagnostic values of serum enzymes in AMI and as well as in Stable <strong>An</strong>gina.<br />
We also studied the Lipid Profile four weeks after AMI with initial levels measured within 24 hours of onset<br />
of symptoms.<br />
Methods: 30 cases of AMI, 30 cases of Stable <strong>An</strong>gina and 30 age and sex matched controls were studied.<br />
The sample was analysed <strong>for</strong> Serum Total CK, CK-MB, AST, LDH, Total cholesterol, HDL-C, LDLC,<br />
VLDL, Non-HDLC and triglycerides.<br />
Results: There was a significant increase in Total CK, CK-MB, AST and LDH levels in AMI patients when<br />
compared to both normal controls and Stable <strong>An</strong>gina patients(P
Bio Chemical profile in Acute MI<br />
Introduction<br />
A great deal of evidence is now available to<br />
show that the incidence of Myocardial Infarction<br />
varies from place to place due to changes in food<br />
habits, climate, type of work and ethnic origin.<br />
Indian immigrants to the west have higher<br />
mortality compared to the other ethnic groups<br />
of the adopting country. In the Asian Indian<br />
study on Coronary artery Disease (CAD), the<br />
prevalence of CAD was found to be 10.2%<br />
compared with 2.5% in whites of the same age<br />
group. Moreover CAD in Asian Indians occur<br />
prematurely, that is atleast a decade or two<br />
earlier than that seen in Europeans (1, 2).<br />
In Indian context, a survey of the incidence of<br />
Acute Myocardial Infarction (AMI) showed<br />
that the disease was seven times more common<br />
among south Indians (3, 4). Coronary Risk<br />
factor evaluation will identify the major risk<br />
factors and their importance in Indian context<br />
(1, 5). Dyslipidemia has been clearly established<br />
as a major risk factor <strong>for</strong> the development<br />
of CAD by a variety of epidemiological,<br />
pathological, genetic and clinical studies.<br />
It is one of the important modifiable risk<br />
factors of CAD. It initiates atherosclerotic<br />
plague <strong>for</strong>mation. Hypercholesterolemia<br />
(increase in LDL-Cholesterol), combined<br />
hyperlipidemia(Increase in LDL-Cholesterol<br />
and Triglycerides) and hypertriglyceridemia<br />
are three important risk factors quoted by the<br />
European Atherosclerotic Society(6, 7).<br />
Despite impressive studies in the diagnosis and<br />
management of AMI over the last three decades<br />
it continues to be a major public health problem<br />
in the world. AMI warrants a time frame in<br />
which prompt and appropriate use of life<br />
saving treatment is of paramount importance<br />
emphasizing, the need <strong>for</strong> early diagnosis.<br />
In view of the enormity of the problem, the<br />
emphasis has now shifted from treatment to<br />
prevention of Myocardial Infarction.<br />
110<br />
Hence the present study was taken up to study<br />
the diagnostic values of serum enzymes in AMI<br />
and as well as in Stable <strong>An</strong>gina. We also studied<br />
the Lipid Profile four weeks after AMI with<br />
initial levels measured within 24 hours of onset<br />
of symptoms.<br />
Materials and Methods<br />
The present study comprises of three groups,<br />
30 patients (Group-I) with AMI admitted to<br />
ICCU, 30 patients (Group-II) with Stable<br />
<strong>An</strong>gina attending outpatient department of KLE<br />
Soceity’s hospital and Medical research Centre,<br />
Belgaum and 30 healthy individuals (Group-<br />
III) from the staff of Jawaharlal Nehru Medical<br />
college, Belgaum. The age group was in the<br />
range of 40-70 yrs.<br />
Inclusion criteria:<br />
Patients with documented evidence of AMI as<br />
per WHO criteria 1985, history of ischaemic<br />
chest discom<strong>for</strong>t lasting more than or equal to 30<br />
minutes associated with characteristic evolution<br />
of ECG changes like ST segment elevation, Q<br />
wave and T wave inversion were included in<br />
study Group-I.<br />
Patients with chest discom<strong>for</strong>t, typically caused<br />
by exertion or emotion lasting <strong>for</strong> 1-5 minutes<br />
and relieved by rest or sub-lingual nitroglycerine<br />
were included in study Group-II.<br />
Exclusion criteria:<br />
Patients with unstable <strong>An</strong>gina and resting ECG<br />
changes, Liver diseases, muscular dystrophy,<br />
history of intramuscular injection prior to or<br />
after the onset of chest pain and subjects having<br />
diseases which are known to influence the blood<br />
lipids or on Lipid lowering drugs were excluded<br />
from the study.<br />
All the three groups were age and sex matched.<br />
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Bio Chemical profile in Acute MI<br />
In<strong>for</strong>med consent was taken from patients and<br />
healthy individuals. The study was approved by<br />
the Ethical & Research Committee of JNMC,<br />
Belgaum on the use of human subjects in the<br />
Research. 2 ml venous blood was collected in a<br />
plain bulb under aseptic precautions from both<br />
patients with AMI and Stable <strong>An</strong>gina <strong>for</strong> enzyme<br />
assay. The next day 2ml of fasting venous blood<br />
was collected between 8 a.m-10 a.m without<br />
any anti-coagulant <strong>for</strong> assay of Lipid profile<br />
from AMI patients. These AMI patients were<br />
asked to come <strong>for</strong> a follow-up after one month<br />
and 2 ml of fasting venous blood was collected<br />
to study the follow-up Lipid profile levels.<br />
Serum was separated after half an hour and<br />
enzyme assay was carried out immediately and<br />
Lipid assay within 12 hours. The sample was<br />
analysed <strong>for</strong> Serum Total Creatine Kinase(CPK),<br />
CK-MB, Aspartate transaminase (AST), Lactate<br />
dehydrogenase(LDH). Different lipid fractions<br />
estimated included Total Cholesterol, high<br />
density lipoprotein-cholesterol (HDL-C), low<br />
density lipoprotein-cholesterol (LDLC), very<br />
low density cholesterol (VLDLC), Non- high<br />
density lipoprotein-cholesterol (Non-HDLC)<br />
and triglycerides[8-11]. One way ANOVA<br />
followed by Bonferroni multiple comparison<br />
test, paired and unpaired students’s ‘t’ was<br />
employed <strong>for</strong> the statistical analysis of the data<br />
to compare the groups.<br />
Results<br />
The comparable study of Enzyme levels in<br />
healthy controls, AMI and Stable <strong>An</strong>gina<br />
patients is shown in Table 1. There was a<br />
significant increase in Total CK, CK-MB,<br />
AST and LDH levels in AMI patients when<br />
compared to both normal controls and Stable<br />
<strong>An</strong>gina patients(P
Bio Chemical profile in Acute MI<br />
Table 1. Serum Enzyme levels in healthy controls, AMI and Stable <strong>An</strong>gina patients<br />
Enzymes Healthy Controls AMI Stable <strong>An</strong>gina<br />
Total CPK 120.0 ± 25.39 282.9 ± 30.13 * 131.07 ± 24.76<br />
CK-MB 14.87 ± 4.92 165.36 ± 22.50 * 15.57 ± 4.18<br />
AST 25.13 ± 7.69 139.6 ± 21.93 * 25.87 ± 8.53<br />
LDH 151.1 ± 43.04 317.73 ± 25.41 * 170.6 ± 33.50<br />
Values are expressed in as IU/L (Mean ± SD). (*p < 0.0001) in AMI patients when compared to both normal controls and Stable <strong>An</strong>gina patients.<br />
There was no significant change in enzyme levels in Stable <strong>An</strong>gina patients when compared to Controls.<br />
Table 2. The Initial and Follow-up Lipid profile in AMI patients<br />
Lipid Profile Healthy Controls AMI MI at Follow-Up<br />
Total Cholesterol 194.23 ± 19.13 223.23 ± 14.98 *** 222.35 ± 15.72<br />
LDL C 124.77 ± 19.41 158.63 ± 18.26 *** 156.0 ± 20.73<br />
TG 105.30 ± 15.43 164.03 ± 20.22 **** 164.40 ± 20.70<br />
VLDL C 21.67 ± 3.19 32.80 ± 4.10 **** 32.89 ± 4.13<br />
HDL C<br />
Non-HDL C<br />
48.20 ± 6.17<br />
146.03 ± 19.08<br />
31.80 ± 4.47 ****<br />
191.53 ± 17.78 ****<br />
33.35 ± 5.32<br />
189.5 ± 19.08<br />
Values are expressed in as IU/L (Mean ± SD). (****p < 0.0001, ***p
Bio Chemical profile in Acute MI<br />
VLDLC, Non-HDLC and significant decrease<br />
in HDLC in AMI patients (which was measured<br />
within 24 hrs of occurrence of MI) as compared<br />
to healthy controls. More recent data suggests<br />
that measurement of Non -HDL Cholesterol<br />
level (Calculated as Total Cholesterol minus<br />
HDL Cholesterol) could be more representative<br />
of all atherogenic, apolipoprotein(apo) B<br />
containing lipoproteins –LDL,VLDL,IDL<br />
and Lipoprotein(a). Although apolipoprotein<br />
B can be assessed directly, measurement<br />
of Non-HDL Cholesterol is more practical,<br />
reliable, inexpensive and can be considered<br />
as a surrogate marker <strong>for</strong> apolipoprotein B in<br />
routine clinical practice (15, 16). There was no<br />
significant difference between the initial and<br />
follow-up Lipid profile (after one Month) in the<br />
AMI group. Our findings are consistent with<br />
other research workers (17, 18, 19).<br />
From the literature it is found that Total<br />
cholesterol and Triglycerides are affected by<br />
factors like caloric intake, exercise etc., and<br />
there<strong>for</strong>e the increase in their values found<br />
in the present study cannot serve as a reliable<br />
prognostic indicator in AMI patients. On the<br />
other hand HDLC is not acutely affected by the<br />
said factors. HDLC could there<strong>for</strong>e serve as a<br />
reliable prognostic indicator <strong>for</strong> patients with<br />
AMI. Although there may be several phasic<br />
changes in serum Lipids during the course of<br />
AMI, Lipid measurements made within 24 hrs<br />
of AMI are still useful guide in order to identify<br />
patients requiring follow-up. Furthermore,<br />
patients are most impressionable <strong>for</strong> dietary<br />
or therapeutic advice in the immediate post<br />
infarction period.<br />
References<br />
1. Rajmohan L,Deepa R and Mhan V. Risk factors <strong>for</strong> CAD<br />
113<br />
in Indians; Emerging trends. I.H.J.2000;52:221-225<br />
2. Krishnamoorthy K.M. Diet and CAD.<br />
I.H.J.1999;51:268-274<br />
3. Malhotra S.L. Geographical aspects of AMI in India with<br />
special reference to patterns of diet and eating. B.H.J<br />
1967;29:337-343<br />
4. Padmini S.F and and Motlag D.B. Lipoprotein Profile<br />
changes among chronic smokers and smokers with M.I.<br />
I.H.J.1987;39:38<br />
5. Bhatia ML. Prevalence of coronary heart disease in India:<br />
a contemporary view. Indian Heart <strong>Journal</strong>. 1995 Jul-Aug;<br />
47(4): 339-42<br />
6. Gore JM, Goldberg RJ, Matsumoto AS, et al. Validity of<br />
serum total cholesterol<br />
7. levels obtained within 24 hours of acute myocardial infarction.<br />
Am J Cardiol 1984;54:722–725.<br />
8. Mahajan A.S., Reddy K.S., Sachdeva U. Lipid profile of<br />
coronary risk subjects following yogic lifestyle intervention.<br />
Ind. Heart J 1999 Jan-Feb, 51(1): 37-40<br />
9. Allain CC. Enzymatic colorimetric method <strong>for</strong> estimation<br />
of serum cholesterol . Clin Chemistry 1974;20: 470.<br />
10. Lopes-Vireflla MF. Enzymatic colorimetric method <strong>for</strong> estimation<br />
of HDL-cholesterol. Clin Chem 1977; 23: 288.<br />
11. Varley H, Gowenlock AH, Bell M. Lipids and Lipoproteins<br />
In: Proteins clinical biochemistry 5th ed, William<br />
Heinemall Medical books Ltd London, 1980; 1.<br />
12. Bucolo D, David H. Quantitative determination of serum<br />
triglycerides by use of enzymes. Clin Chem 1973; 19: 476.<br />
13. Roberts R, Gowda KS, Ludbrook PA, Sobel BE. Specificity<br />
of elevated serum MB creatine phosphokinase activity<br />
in the diagnosis of acute myocardial infarction. Am J Cardiol.<br />
1975 Oct 6; 36(4):433–437.<br />
14. Kibe olaf and Nils J.N. Observations on the diagnostic &<br />
prognostic values of some enzyme tests in MI. Acta Medica<br />
Scandinavica.1967;182(5): 597-610<br />
15. Smith AF, Rad<strong>for</strong>d D, Wong CP, Oliver MF. Creatine kinase<br />
MB isoenzyme studies in diagnosis of myocardial infarction.<br />
Br Heart J. 1976 Mar; 38(3):225–232.<br />
16. <strong>An</strong>ne L.Peters. Clinical relevance of Non-HDL cholesterol<br />
in patients with diabetes. Clinical Diabetes.2008;26(1):3-7<br />
17. Grundy SM. Low-density lipoprotein, non-high-density lipoprotein<br />
and apolipoprotein B as targets of lipid-lowering<br />
therapy. Circulation.2002; 106:2526-2529.<br />
18. Vincelj J, Sucić M, Bergovec M, Sokol I, Mirat J, Romić<br />
Z, Lajtman Z, Bergman-Marković B, Bozikov V. Serum<br />
total, LDL, HDL Cholesterol and Triglyceride related to<br />
age, gender & cigarette smoking in patient with first acute<br />
myocardial infarction. Coll <strong>An</strong>tropol 1997;21(2):517-524<br />
19. Ryder, RE; Hayes, TM; Mulligan, IP; Kingswood, JC; Williams,<br />
S; Owens, D R. How soon after MI should plasma<br />
lipid values be assessed. BMJ 1984;289:1651-1653<br />
20. Sewdarsen M, Vythilingum S, Jialal I, et al. Plasma lipids<br />
can be reliably assessed within 24 hrs after AMI. Postgraduate<br />
Med.J 1998;64:352-356<br />
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<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 114 - 118<br />
Association of serum ferritin levels between rheumatoid arthritic obese and<br />
Non rheumatoid arthritic obese<br />
*Tandra Majhi and **A.K. Srivastava<br />
*Department of Psychiatry, C.S.M.Medical University, Lucknow UP India.226003<br />
**Department of Zoology, C.M.P. Degree College, Allahabad UP India.211002<br />
(Received 29 th June, 2010; Revised 20 th November, 2010; Accepted 10 th January, 2011)<br />
Corresponding author:<br />
Dr. Tandra Majhi,<br />
E-mail: tandra.majhi@gmail.com<br />
Abstract<br />
Background & Objectives: In Iron deficiency anemia basic of ferritin is appreciably reduced. This variable<br />
was determined in 16 obese rheumatoid arthritic patients, aged 30-60 years with matched 28 obese non<br />
rheumatoid arthritic subjects. The BMI cutoff value <strong>for</strong> the both groups were less than 34. It is measured<br />
by the <strong>for</strong>mula.<br />
Methods: Hb% was measured by the cyanide method while serum iron and TIBC were measured by the<br />
kits which were available by commercially. Serum ferritin was measured by the commercially available<br />
ferritin kit (Lilyat Medicine Company USA) Fisher, ‘t’ test and correlation Coefficiency were applied <strong>for</strong><br />
the statistical analysis.<br />
Results: The prevalence of iron deficiency was found 6.49% in males and 14.29% in females with obese<br />
RA. The indirect relationship was found between BMI and Hb%, whereas direct association between age.<br />
A directly proportional relationship found to be BMI age and serum TIBC (r=0.09, P = 0.73; r = 0.04, P =<br />
0.87). While the serum iron (BMI : r = - 0.12, P = 0.69; Age: r = -0.41, P = 0.12) and ferritin (BMI : r =<br />
0.09, P = 0.71; age : r = -0.17, P = 0.52) levels shows significantly negative correlative with BMI and age<br />
in obese rheumatoid arthritic subjects.<br />
Conclusion: These results suggested that the iron deficiency anaemia is associated with obese rheumatoid<br />
arthritic patients.<br />
Keywords: Rheumatoid arthritis, Obesity, Rheumatoid arthritic obese, Iron deficiency, Serum ferritin<br />
Introduction:<br />
Rheumatoid arthritis (RA) is an autoimmune<br />
disorder of unknown etiology characterized<br />
by symmetric, erosive synovitis and, in same<br />
eases, extra articular involvement (1) . <strong>An</strong>aemia<br />
is a frequent finding in patients with rheumatoid<br />
arthritis and its severity roughly parallels the<br />
activity of the disease (2,3). Serum ferritin<br />
concentration has been shown to give an accurate<br />
indication of the amount of storage iron in normal<br />
subjects and in patients with iron deficiency and<br />
overload (4,5). Excess body fat is a prominent<br />
health hazard (6) significantly contributing<br />
to the development of cardiovascular disease<br />
(CVD)(7) About two thirds of patients who<br />
have had a myocardial infarction (MI) exhibit<br />
increased body weight(8). Obesity increases the<br />
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serum ferritin in rheumatoid obese<br />
risk of coronary heart disease (CHD) through<br />
a number of different pathophysiological<br />
pathways, including insulin resistance, type 2<br />
diabetes, hypertension and dyslipdaemia(9,10).<br />
The purpose of this study was to establish<br />
correlation between serum ferritin levels in<br />
rheumatoid arthritic obese patients and nonrheumatoid<br />
arthritic obese controls.<br />
Materials and Methods<br />
Subjects:<br />
The present observational study was conducted<br />
in the Department of Biochemistry M.L.N.<br />
Medical College Allahabad U.P. The patients<br />
clinically diagnosed from OPD who had not<br />
undergone any previous treatment or medication<br />
<strong>for</strong> their arthritis, were chosen <strong>for</strong> the study. The<br />
study was categorized into two groups.<br />
(i) Control Group : Obese subjects were taken<br />
in this group. Those patients were included<br />
in this group who had negative RA Factor<br />
value and who were not suffering from any<br />
pathological and physiological illness.<br />
(ii) Study Group : Under this group, people<br />
suffering from rheumatoid arthritis with obesity<br />
were taken. In this group the inclusion criteria<br />
was positive RA factor value. According to<br />
excluding criteria, the patients had not been<br />
suffering from any other pathological and<br />
physical illness. The subjects were in the age<br />
of 30-60 years and the BMI cutoff value <strong>for</strong><br />
the both groups were less than 34. In<strong>for</strong>mation<br />
regarding socio-demographic variables such as<br />
age, sex, martial status religions, education and<br />
income were obtained. Body Mass Index was<br />
calculated by the <strong>for</strong>mula, BMI = weight (kg)/<br />
Height 2 (m 2 ). The total participated subjects<br />
were 77 in the study group but only 16 subjects<br />
were chosen <strong>for</strong> the study, which fulfilled the<br />
study criteria. Fifty two subjects were enrolled<br />
in the control group. Whereas 28 subjects<br />
were selected <strong>for</strong> the control group which had<br />
fulfilled the study criteria.<br />
Assessment :<br />
115<br />
Peripheral blood (5ml) was collected in<br />
vacutainer containing EDTA anticoagulant or<br />
no anticoagulant. Hb% was measured by adding<br />
blood or haemolysate to ferricyanide-cyanide<br />
reagent to convert the hemoglobin pigment to<br />
cyanomethamoglobin by method of cyamide.<br />
(11) Serum iron and TIBC were measured by<br />
Kits based on ferrozine method. Serum ferritin<br />
was measured by commercially available<br />
ferritin kit. (Lilyat Medicine Company USA)<br />
Statistical <strong>An</strong>alysis :<br />
Fisher test and’t’ test were used <strong>for</strong> the<br />
comparison of the means ± of the sample <strong>for</strong><br />
these groups. Correlation between BMI, age<br />
and blood variables in patients and control were<br />
calculated by using Correlation coefficient.<br />
Results<br />
The prevalence of iron deficiency was 6.49%<br />
in men and 14.29% in women with obese<br />
rheumatoid arthritis. Only three men, but<br />
none of the women subjects, were diagnosed<br />
normal without iron deficiency anemia. Present<br />
study was done on 16 obese rheumatoid<br />
arthritis patients i.e. 11 females and 5 males<br />
and compared with 28 obese non rheumatoid<br />
arthritic patients i.e. 18 females & 10 males.<br />
There were no significant differences between<br />
the sex, age and BMI in these two groups. Hb%,<br />
Serum iron, TIBC and serum ferritin levels were<br />
significantly low in the study group compared<br />
to the control group. The inverse relationship<br />
was found between BMI and Hb% (r = - 0.168,<br />
P = 0.53), whereas positive association between<br />
was found age and Hb% (r = 0.12, P = 0.65). A<br />
directly proportional relationship was found to<br />
be BMI, age and serum TIBC (BMI: r = 0.09,<br />
P = 0.73; Age: r = 0.04, P = 0.87). The serum<br />
iron correlated significantly with decrease<br />
activity in both BMI (r = -0.12, P = 0.69) and<br />
the age (r = - 0.41, P = 0.12). Percentage of<br />
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serum ferritin in rheumatoid obese<br />
Table 1: Demographic Characteristics of the two groups<br />
Variables ControlGroup N = 28 Study Group N = 16<br />
Sex M : F 10 : 18 5:11<br />
Age (in year) 47.81±11.56 46.94±10.56*<br />
BMI 31.32 ± 1.27 32.23 ± 2.19*<br />
* Non-Significant, Mean ± SD<br />
Table 2: Comparative study of Hb%, Serum iron, TIBC and ferritin levels in obese and obese rheumatoid<br />
arthritic patients<br />
Variables<br />
Control Group<br />
(N=28)<br />
Study Group<br />
(N = 16)<br />
Hb% (gm%) 13.20 ± 1.27 10.34 ± 2.18 *<br />
Serum Iron (μg/dl) 100.34 ± 25.74 45.62 ± 28.15 *<br />
SerumTIBC (μg/dl) 293.94 ± 47.72 183.59 ± 60.59 *<br />
Serum Ferritin (ng/dl) 169.04 ± 57.72 105.36 ± 39.50 **<br />
Value expressed mean ± SD, * P < 0.001 Highly significant and ** P
serum ferritin in rheumatoid obese<br />
Hb was significantly decreased with age while<br />
there was no significant changes with the body<br />
mass index in study group. While serum ferriten<br />
shows negative correlation with BMI (r = -0.09,<br />
P=0.71) and with age (r = -0.17, P = 0.52) in<br />
the study group. Serum iron and ferritin levels<br />
both were significantly decreased with BMI and<br />
age in the obese rheumatoid arthritis patients.<br />
These results suggested that the iron deficiency<br />
anemia was associated with obese rheumatoid<br />
arthritic patients.<br />
Discussion<br />
The result of the study showed that Hb%, serum<br />
ferritin serum iron and TIBC concentration in<br />
the study group were lower than control group.<br />
There was a significant positive correlation<br />
between BMI and age with serum TIBC in<br />
the study group. By contrast ferritin and iron<br />
concentration were lower in obese rheumatoid<br />
arthritis and were positively correlated with BMI,<br />
age findings consistent with the observation that<br />
obesity in an inflammatory state, which increase<br />
acute-phase reactants. Correlation regression<br />
analysis showed that while the best model to<br />
predict serum iron included ferritin and total iron<br />
binding capacity, most of the predicted variance<br />
was accounted <strong>for</strong> by transferring receptor<br />
alone, although inflammatory indices were also<br />
independent predictors. There<strong>for</strong>e it is possible<br />
that lower levels ferritin may increased the risk<br />
of iron deficiency in the rheumatoid arthritis<br />
with obesity. Using serum transferrin receptor<br />
to predict the presence of iron deficiency<br />
by LB Yanoff et al(12). They found higher<br />
odds of iron deficiency in obese vs non-obese<br />
subjects. They showed the ferritin levels tend<br />
to be elevated in obesity-related inflammatory<br />
state, they did not show a difference between<br />
obese and non-obese subjects is the prevalence<br />
of iron deficiency. They explained the<br />
hypoferrimia of obesity appears by true iron<br />
deficiency hypoferrimia and by inflammatorymediated<br />
functional iron deficiency. Elevated<br />
transferring receptor levels correlate well with a<br />
117<br />
lack of stainable iron in bone marrow in normal<br />
subjects as well as in patients with rheumatoid<br />
arthritis, and transferrin receptor reportedly has<br />
a higher sensitivity than ferritin to diagnose<br />
iron deficiency in patients with ferritin elevated<br />
from acute-phase reaction (13,14,15) Similar to<br />
other inflammatory conditions, obesity appears<br />
to be a state in which transferrin receptor is a<br />
useful adjunct to ferritin in the diagnosis of iron<br />
deficiency.<br />
Davidson et al(16) showed that iron deficiency<br />
anaemia in patients with rheumatoid arthritis may<br />
be difficult to distinguish from the microcytic<br />
anemia found in chronic inflammatory disease. In<br />
uncomplicated iron deficiency reduced marrow<br />
iron is a reliable index <strong>for</strong> this deficiency. This<br />
reduction in stainable marrow iron correlation<br />
with both mirocysosis and lower plasma ferritin<br />
concentration (17,18).<br />
On the other hands the etiology of aging in<br />
important to understand, but it is equally<br />
important to differentiate the normal<br />
physiological changes from those associated<br />
with diseases. It may be generalized atrophy<br />
of all muscle accompanied by a replacement of<br />
same muscle tissue by fat deposit. This results<br />
in some loss of muscle tone and strength. Some<br />
specific implications of this are reduced ability<br />
to breath deeply and reduced gastrointestinal<br />
activity which can lead to constipation or<br />
bladder incontinence, particularly in women.<br />
The joints also undergo changes. Arthritis, the<br />
degenerative inflammation of joints, is the most<br />
common chronic condition is the elderly obese<br />
persons with iron deficiency.<br />
In conclusion, the study indicates that decreased<br />
ferritin concentration is associated with obese<br />
rheumatoid arthritis among, middle aged and<br />
elderly. It is possible that lower levels of ferritin<br />
may be increased in the risk of iron deficiency<br />
anemia in the rheumatoid arthritis with obesity.<br />
The etiology appears to be multifactorial and<br />
may include inadequate bioavailable iron<br />
relative to body weight, as well as diminished<br />
intestinal absorption and decreased iron<br />
bioavailability included by inflammatory with<br />
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serum ferritin in rheumatoid obese<br />
excessive adiposity. The precise mechanisms of<br />
the obesity-related, inflammation induced effect<br />
in serum iron remain to be elucidated. Further<br />
researches are needed to confirm and extend<br />
these findings on obesity with rheumatoid<br />
arthritis in large number of subjects.<br />
Reference<br />
1. Harrcis ED Jr. Rheumatoid arthritis : pathophysiology<br />
and implications <strong>for</strong> therapy. N Engl J Med., 1990; 322<br />
: 1277-89.<br />
2. Nilsson F. <strong>An</strong>emia problems in rheumatoid arthritis. Acta,<br />
Med. Scand. 130 suppl. 1948; 210, 72.<br />
3. Jeffrey M.R. Some observations on anemia in rheumatoid<br />
arthritis, Blood. 1953; 8; 502-518.<br />
4. Jacobs A, Mitter F, WorWood M, Beamish MR Wordrop<br />
CA., Ferritin in the serum of normal subjects and patients<br />
with iron deficiency and iron overload. Brit. Med. J.,1972<br />
4, 206-208.<br />
5. Walters GO, Miller FM Worwood M., Serum ferritin concentration<br />
and iron stores in normal subjects. J. Clin.Path.<br />
1973, 26, 770-772.<br />
6. Van Pelt RE, Jones PP, Davy KP, Desouza CA, Tanaka H,<br />
Davy BM et al. Regular exercise and the age related decline<br />
in resting metabolic rate in women. J. Clin. Endocrinol<br />
Metab.1997, 82 : 3208-12.<br />
7. Ross R. Atherosclerosis- an inflammatory diseases N. Eng<br />
1.1 Med. 1999,40: 115-26.<br />
8. Romero-Corral A, Montori VM, Somers VK, Korinek J.<br />
Thomas RJ, Allison TG et. al. Association of body weight<br />
with total mortality and with cardiovascular events in coro-<br />
118<br />
nary artery disease : a systematic review of cohort studies.<br />
Lancet 2006, 368: 666-78.<br />
9. Pi-Sunyer FX. The obesity epidemic: pathophysiology and<br />
consequences of obesity. Obesity Res. 2002 ,10: 975-104.<br />
10. Krauss RM, Winston M, Fletecher BJ, Grundy SM. Obesity<br />
: impact on cardiovascular disease. Circulation,1998,<br />
98; 1472-6.<br />
11. The Haemoglobin Cyanide method. Clin. Chem. Actr.<br />
1961, 6: 538-545.<br />
12. Yanoff LB, Menzie CM, Denkinger B, Sebring NG,<br />
McHugh T, Remaley AT, Yanovksi JA. Inglammation and<br />
iron deficiency in the hypofessemia of obesity. Int. J. Obes.<br />
Sept. 31 (a): 2007,1412-1419<br />
13. Punnonen K, Irjala K, Rajamaki A.,Serum Transferrin receptor<br />
and its ratio to serum feritin in the diagnosis of iron<br />
deficiency, Blood 1997, 89; 1052-1057.<br />
14. Means RT, Jr. Allen J, Seas DA, Schuster SJ.,Serum Soluble<br />
transferrin receptor and the prediction of marrow aspirate<br />
iron results in a heterogeneous group of patients, Clin,<br />
Lab. Haematol. 1999, 21: 161-167.<br />
15. Most AE, Blinder MA, Gronowski AM, Chumley C, Scott<br />
MG.,Clinical utility of the soluble transferrin receptor and<br />
comparison with serum ferritin in several populations Clin.<br />
Chem. 1998, 44: 45-51.<br />
16. Davidson A, Weyden MBVD, Fong H, Breidahl MJ, Rayan<br />
PFRed cell ferritn content: a re-evaluation of indices <strong>for</strong><br />
iron deficiency in the anemia of shumatoid arthritis. Brit.<br />
Med. J. Vol.1984. 289; 648-650.<br />
17. Brink S, Van Schalkwyk DJ. Serum ferritis and mean corpuscular<br />
volume as predictions of bone narrow iron stores,<br />
S. Afr. Med. J. 1982, 61: 432-434.<br />
18. Krause JR, Stok V. Serum ferritin and bone marrow iron<br />
stores. Correlation with absence of iron in biopsay specimens<br />
Am. J. Clin, Pathol.,1979, 72: 817-20.<br />
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Case Reports <strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 119 - 121<br />
A case of asymptomatic anomalous pancreatico biliary ductal union of<br />
pancreatic - biliary type<br />
V.S. <strong>An</strong>andarani.<br />
Departmentof <strong>An</strong>atomy, Sri Ramachandra Medical College, Porur,Chennai - 600116, India.<br />
(Received 9th December 2010; Revised 10th February 2011 Accepted 23rd February 2011)<br />
Corresponding Author<br />
Dr.V.S. <strong>An</strong>andarani.<br />
E-mail: ranironeena@yahoo.co.in<br />
Abstract<br />
<strong>An</strong>omalous pancreatico biliary junction is an unusual variant of pancreatico biliary anatomy of clinical<br />
importance. It is a congenital anomaly where the common duct <strong>for</strong>med by the union of main pancreatic<br />
duct and bile duct is more than 15 mm in length. According to the mode of termination two different types<br />
are described .Each type is associated with specific clinical conditions.<br />
Key words - pancreatic duct, common duct, pancreatico biliary tree.<br />
Introduction<br />
<strong>An</strong>omalous pancreatico biliary ductal union<br />
[APBDU] is a congenital anomaly. The<br />
incidence is not clear because the identification<br />
of this condition is done only during the<br />
investigations <strong>for</strong> the diseases related. It is<br />
a complex anatomical and functional entity<br />
.The main pancreatic duct and bile duct are<br />
joined well outside the duodenal wall to <strong>for</strong>m a<br />
common duct. The common duct of more than<br />
15 mm length is noted as an anomalous ductal<br />
union[1]. It is reported that 16.7% of patients<br />
with carcinoma gall bladder shows associated<br />
APBUD [2]. This congenital anomaly can<br />
influence the degrees of pancreatic fluid<br />
regurgitation which results in an increased<br />
incidence of biliary tract malignancy[3].<br />
Case report<br />
Adult duodeno pancreas specimens were used<br />
<strong>for</strong> a study of the ductal pattern of pancreas. The<br />
specimens were collected from the cadvers of<br />
the dissection hall and 100 specimens including<br />
119<br />
64 males and 36 females were used. The<br />
pancreas was removed along with duodenum<br />
and the bileduct was incised just above the first<br />
part of duodenum. On the posterior aspect of<br />
the specimen the bile duct was traced down to<br />
the junction with the pancreatic duct . From this<br />
point pancreatic ductal system was traced out.<br />
In a specimen collected from a male cadaver of<br />
55 years(died of road traffic accident) pancratco<br />
billiary ductal union was well outside the<br />
duodenal wall. A clean slit was made on the bile<br />
duct away from the union with pancreatic duct<br />
and the incision was extended down and the duct<br />
was opened upto the major duodenal papillae.<br />
This was to confirm whether the common<br />
channel was a true one or if there is any septum<br />
separating biliary and pancreatic passages.<br />
The length of the true common channel was<br />
measured . The true common channel was of<br />
16mm in length. It was <strong>for</strong>med well out side the<br />
duodenal wall (fig-1).There was no apparent<br />
change in the size of common bile duct or the<br />
main pancreatic duct as they approached the<br />
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asymptomatic pancreatico biliary ductal union<br />
Fig-1 specimen showing APBDU of P-B type. A-main pancreatic duct B-common duct of 16mm in length. C.-bile duct.D- accessory<br />
pancreatic duct<br />
ductal junction.<br />
Discussion<br />
In majority the main pancreatic duct and the<br />
common bile duct open into the second part of<br />
the duodenum after the <strong>for</strong>mation of a common<br />
channel . The terminal part of the common<br />
duct shows a dialatation called ampulla. Around<br />
the ampullary region a sphincter is described.<br />
The sphincter choledochus and the sphincter<br />
pancreaticus surrounds the periampullary<br />
parts of the bileduct and main pancreatic duct<br />
respectively. The sphincter of oddi (proper) is<br />
described around the ampulla. These three sets<br />
of sphincters together described as sphincter of<br />
oddi [ 4]. <strong>An</strong>omalous pancreaticobiliary ductal<br />
junction is an abnormal union of the pancreatic<br />
and biliary ducts that is located outside the<br />
duodenal wall and the length of the common<br />
channel is more than 15mm[1]. A normally<br />
functioning spincter of oddi and an intramural<br />
ductal junction may prevent the duodenal reflux<br />
into pancreatico biliary ductal tree as well as the<br />
reflux of pancreatic juice into bile duct and vice<br />
verse. A long common channel with an extra<br />
mural ductal junction indicates a dysfuncting<br />
sphincter of oddi [5]. Thus in a case of APBDU<br />
two ducts are always communicating and two<br />
120<br />
way regurgitation ie, pancreatico biliary reflux<br />
and bilio pancreatic reflux might occur[6].<br />
<strong>An</strong>omalous pancreatico biliary ductal union is<br />
significant because of its clinical association.<br />
Two types of APBDU were noted according<br />
to the morphological changes occurring in the<br />
ducts be<strong>for</strong>e<br />
union [1,7].<br />
1. pancreatic - biliary type -- there is no<br />
notable change in ducts [P-B type].<br />
2. biliary – pancreatic type-- there is a<br />
small dilatation in the common duct just proximal<br />
to the ductal union.[B-P type].<br />
In the present case there was no notable<br />
changes in the ductal morphology towards the<br />
ductal union. So the case was included under<br />
P-B type of APBDU. Known associations<br />
of APBDU include bileduct carcinoma, gall<br />
bladder carcinoma and cystic disease of gall<br />
bladder[1,7].The specific association were<br />
noted <strong>for</strong> B-P type with choledochal cyst<br />
and P-B type with gall bladder carcinoma<br />
and biliary pancreatitis[7]. APBDU can be<br />
associated with other congenital anomalies like<br />
divisum of pancreas[8] and multi septate gall<br />
bladder[9]. Among the patients with diseases<br />
of pancreatico biliary tree 8.7% with clearly<br />
visualized pancratico biliary radiograms had<br />
APBDU[6]. The junction of common bile duct<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
asymptomatic pancreatico biliary ductal union<br />
and main pancreatic duct, when extraduodenal<br />
and is unprotected by sphincter of oddi the<br />
reflux of pancreatic exocrine secretions into<br />
the unprotected biliary tree occurs.This results<br />
in inflammation which causes the epithelial<br />
distruction and dilatation of the bileduct[10].<br />
Mizuno M etal [11] suggested that the reflux<br />
of the activated pancreatic juice into the biliary<br />
tract is an impotant factor in the development of<br />
biliary tract carcinoma. The optimal approach<br />
is the prevention of reciprocal reflux of bile and<br />
pancreatic juice . Surgical treatment includes<br />
complete biliary diversion procedures [12]. In<br />
the present study APBDU was asymptomatic<br />
Summary<br />
The most outstanding feature of the normal<br />
anatomy of the extrahepatic biliary system<br />
is its high degree of variability. <strong>An</strong>omalous<br />
pancreatico biliary junction is an unusual<br />
variant of pancraticobiliary anatomy of<br />
clinical importance because it is associated<br />
with increased risk of pancreatitis, diseases of<br />
biliary tree, cystic changes of gall bladder and<br />
also malignant changes of the biliary tree. In<br />
the present study the specimen showed a P-B<br />
type of APBDU without any recorded clinical<br />
complaints related to this congenital anomaly or<br />
morphological changes of associated structures.<br />
Such cases go undetected because as a rouine<br />
these congenital anomalies are reported during<br />
the investigative radiological procedures.<br />
According to the study result the prevalence<br />
of APBDU in a randomized south Indian<br />
population is 1%.But data needs confirmation<br />
from more studies especially by radiological<br />
screening trials as well dissection studies with<br />
more number of specimens.<br />
References<br />
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7. Wang H-P, Wu M-S,LinC-C, Chang L-Y,KaoA-W,Wang<br />
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8. Colm J McMahon, Charles M Vollmer; Jeffrey Goldsmith;<br />
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9. Takafumi Yamamoto,Jun Matsumoto,Shinya Hashiguchi<br />
,Atsumasa Yamaguchi,Koro Sakoda, Chiaki Taki.<br />
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biliary ductal union :A case report World Jornal of<br />
Gastroenterol 2005;11(38);6066-6068.<br />
10. Babbit DD (1969) Congenital choledochal cyst. New<br />
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the commonbile duct and pancreatic bulb. <strong>An</strong>n Radiol<br />
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11. Mizuno M, Kato T, Koyama K (1996) <strong>An</strong> analysis<br />
of mutagens in the contents of the biliary tract in<br />
pancreaticobiliary maljunction.Surg Today 26:597–602<br />
12. Todani T, Watanabe Y, Narusue M, Tabuchi K, Okajima K<br />
(1977) Congenital bile duct cyst; classification, operative<br />
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122<br />
<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 122 - 125<br />
Unusual complication at Feeding Jejunostomy in Boerhaave’s Syndrome<br />
*Vithalkumar. M. Betigeri, **<strong>An</strong>upama V Betigeri, ***Nanda Kishore Maroju and *Kasturi<br />
Satya Venkata Kumar Subba Rao<br />
*Department of Cardiothoracic and Vascular surgery, VMMC and Safdarjung hospital,<br />
New Delhi, India. 110029<br />
**Department of Physiology, ***Department of General Surgery, Jawaharlal Institute of Postgraduate<br />
Medical Education & Research, Puducherry-605006,India.<br />
(Received 17 th November, 2010; Revised 25 th January, 2011; Accepted 16 th February, 2011)<br />
Corresponding author<br />
Dr Vithalkumar. M. Betigeri.<br />
e-mail: vithalkumarmb@gmail.com<br />
Abstract<br />
Spontaneous rupture of esophagus is the most lethal per<strong>for</strong>ation of gastrointestinal tract. Even with typical<br />
presentation and high index of suspicion, this rare cause of per<strong>for</strong>ation is lethal with mortality of 10-50%,<br />
as result of subsequent post operative complications. We report such a case of 24 year old male patient<br />
managed initially by primary repair but later succumbed to rare postoperative complication of jejunojejunal<br />
intussusception at feeding jejunostomy site.<br />
Key Words: Esophageal per<strong>for</strong>ation, Intussusception, Surgery complications<br />
Introduction:<br />
Inspite first description in 1724 by Herman<br />
Boerhaave, spontaneous transmural<br />
esophageal rupture is most lethal per<strong>for</strong>ation<br />
of gastrointestinal tract with mortality of 10-<br />
50%, delayed diagnosis and comorbidities are<br />
historically most often associated factor <strong>for</strong> it(1).<br />
Jejunojejunal intussusception, the telescoping<br />
or an inavagination of a segment of jejunum in<br />
to an adjacent one, with tip of feeding tube as<br />
lead point is rare cause of adult small bowel<br />
obstruction following tube jejunostomy(2).<br />
Case Report:<br />
Twenty hours after initial conservative<br />
treatment elsewhere <strong>for</strong> abdominal pain and<br />
breathlessness after repeated bouts of vomiting<br />
following an alcohol binge, a 24 year old male<br />
was referred in febrile, tachycardia, tachypneic,<br />
condition with blood pressure of 100/80mmHg.<br />
Chest X-ray showed pneumomediastinum.<br />
Contrast barium swallow suspected leak<br />
from the esophagus. Esophagoscopy (Fig.1)<br />
confirmed 5X3 cm ragged edged per<strong>for</strong>ation in<br />
left lateral wall, 4cm above the gastroesophageal<br />
junction. After initial resuscitation with fluids<br />
and antibiotics, patient underwent exploratory<br />
left posterolateral thoracotomy. Linear<br />
esophageal tear (Fig.2) of 8cm length, from 2<br />
cm above the diaphragm to the level of left lung<br />
hilum with ragged and necrotic mucosal edges<br />
was found. Patient underwent debridement,<br />
closure of the esophageal defect in two layers<br />
across Ryle’s tube with left pericardial pad of<br />
fat buttressing, left pleural drainage, Stamm’s<br />
draining gastrostomy and Witzel’s feeding<br />
jejunostomy. Post operative course was<br />
stormy with intermittent episodes of fever,<br />
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Unusual complication at Feeding Jejunostomy<br />
Figure 1-<strong>Biomedicine</strong><br />
Figure 2 -<strong>Biomedicine</strong><br />
Figure 3- <strong>Biomedicine</strong><br />
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123
Unusual complication at Feeding Jejunostomy<br />
staphylococcal growth in the pleural fluid, toxic<br />
granules with left shift, anemia, hypokalemia,<br />
hypocalcemia, hypoalbuminemia. <strong>An</strong>tibiotics<br />
according to culture sensitivity were given.<br />
As barium swallow done on fourteenth post<br />
operative day revealed anastomosis leak and<br />
general condition of the patient was poor,<br />
nutritional status managed with enteral feeding<br />
which was tolerated well.<br />
In subsequent postoperative recovery period<br />
patient had complained of recurrent pain<br />
abdomen without clinical and radiological<br />
evidence of obstruction. Small bowel series<br />
revealed delayed passage of contrast with<br />
retention in stomach, duodenum and proximal<br />
jejunum and feeding tube contrast study<br />
showed normal caliber of the distal bowel.<br />
When initial conservative management to treat<br />
the obstruction was failed, patient underwent<br />
reexploratory laparotomy where we found 20cm<br />
long antegrade jejunojejunal intussusception<br />
(Fig.3), 10 cm distal to the jejunostomy tube<br />
entry site, with normal position of the tube and<br />
two fixation sites of it to the peritoneum and<br />
the tip of tube projecting beyond apex of the<br />
intussusception. Jejunal resection <strong>for</strong> impending<br />
gangrene features and end to end anastomosis<br />
with resiting of feeding jejunostomy was done.<br />
After post operative recovery, jejunostomy<br />
feedings restarted. Subsequent postoperative<br />
period was overwhelmed by sepsis with<br />
poor nutritional recovery and patient died on<br />
eightyfourth postoperative day.<br />
Discussion:<br />
Because of its rarity and atypical clinical<br />
findings(3,4),intussusception poses diagnostic<br />
challenge in adults who uncommonly have<br />
acute intestinal obstruction as a presenting<br />
feature unlike in children, often have symptoms<br />
of partial intestinal obstruction like pain and<br />
vomiting. Adult require resection almost always<br />
as demonstrable pathology is seen in 90%<br />
cases, eventhough the extent of resection and<br />
124<br />
whether or not to reduce it be<strong>for</strong>e resection is<br />
controversial(3,4).<br />
Feeding jejunostomy by various techniques<br />
is well established method to provide enteral<br />
nutrition and medications in patients expected to<br />
require prolonged nutritional support. Although<br />
technically simple, complications are various<br />
(5) with incidence of 8-20% and mortality of<br />
2-10 %( 6).<br />
Our case highlights rare, serious but potentially<br />
reversible jejunojejunal intussusception as a<br />
complication of jejunostomy tube(2) which was<br />
missed preoperatively because, tube feeding<br />
was not interfered inspite intussusception,<br />
clinical and radiological findings did not<br />
differentiated small bowel obstruction due<br />
to intussusception from adhesions or suture<br />
line. Although radiographic findings of<br />
intussusception are incidental and transient<br />
<strong>for</strong> tube jejunostomy patient(7), unresolving<br />
nature of long segment intussusception<br />
resulting in pregangrenous changes, delayed<br />
diagnosis due to atypical presentation made<br />
resection rather reduction a treatment option in<br />
our patient. After successful surgical resection<br />
of jejunojejunal intussusception, we were able<br />
to restart neojejunostomy tube feeding, which<br />
is utmost important in Boerhaave syndrome in<br />
septic profile. Our case highlights importance<br />
of post operative complications that occurred<br />
inspite aggressive primary repair of esophagus<br />
and failure of primary repair possibly due to<br />
distal bowel obstruction which requires high<br />
index of suspicion, as intussusception in adult<br />
is rare and most intussusception are seen on<br />
operating table (3,4), which was managed<br />
conservatively early in course of the disease<br />
that later culminated in to jejunojejunal<br />
intussusception requiring resection and resiting<br />
of jejunostomy tube, further hampering the<br />
nutritional recovery which is utmost important<br />
in patients of Boerhaave’s syndrome with failed<br />
primary repair.<br />
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References:<br />
1. Teh E, Edwards J, Duffy J, Beggs D. Boerhaave’s<br />
syndrome: A review of management and outcome. Interact<br />
CardioVasc Thorac Surg 2007; 6:640-3.<br />
2. Wu TH, Lin CW, Yin WY. Jejunojejunal intussusception<br />
following Jejunostomy. J Formos Med Assoc 2006;<br />
105(4):355-8.<br />
3. Azar T, Berger DL. Adult intussusception. <strong>An</strong>n Surg 1997;<br />
226:134-8.<br />
4. Begos DG, Sandor A, Modlin IM. The diagnosis and<br />
management of adult intussusception. Am J Surg 1997;<br />
125<br />
173:88-94.<br />
5. Pearce CB, Duncan HD. Enteral feeding. Nasogastric,<br />
nasojejunal, percutaneous endoscopic gastrostomy or<br />
jejunostomy: its indications and limitations.Postgrad Med<br />
J; 2002; 78:198-204.<br />
6. Date RS, Clements WD, Gilliland R. Feeding jejunostomy:<br />
is there enough evidence to justify its routine use? Dig<br />
Surg 2004; 21:142-5[Pubmed].<br />
7. Carucci LR, Levine MS, Rubesin SE, Laufer I, Asad S,<br />
Herlinger H.Evaluation of patients with jejunostomy<br />
tubes: imaging findings. Radiology 2002; 223:241-7.<br />
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Letters to the Editor <strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 126 - 127<br />
Blood Pressure Changes in menstrual cycle<br />
The menstrual cycle is much more than a<br />
cycle of periods. Menstruation is only one<br />
manifestation of the ovarian cycle which is itself<br />
is associated with many physical, psychological<br />
and behavioral changes. Numerous clinical<br />
disorders also appear to be modulated by cyclic<br />
ovarian activity. Certain autonomic changes like<br />
blood pressure have also been reported during<br />
different phases of menstrual cycle but are not<br />
well documented and the results are conflicting.<br />
The literature reveals many types of behavioral<br />
and other changes in women especially during<br />
the premenstrual phase. Hence a study was<br />
taken up to know the effect of blood pressure<br />
changes during different phases of menstrual<br />
cycle. 30 lady medical students aged between<br />
18-20 years of J.S.S. Medical College &<br />
Hospital and Dental College & Hospital Mysore<br />
were selected <strong>for</strong> the study. In<strong>for</strong>med consent<br />
was obtained from each subject. The students<br />
having regular menstrual cycle lasting 26-34<br />
days and normal range of hematological and<br />
biochemical parameters within normal limits<br />
and normotensive (< 140/90 mmHg) were<br />
taken as inclusive criteria. The subjects with<br />
irregular menstrual cycle of 35 days history of<br />
diabetes mellitus, hypertension, other endocrine<br />
disorders, history of postural symptoms or<br />
syncopal attacks and history medication during<br />
the study were excluded. Experimentations<br />
were per<strong>for</strong>med in the department of Physiology<br />
J.S.S.Medical College and Hospital, S.S.Nagar,<br />
Mysore in accordance with the ethical standards<br />
of the committee on human experimentation of<br />
the institution according to Helsinki Declaration<br />
of 1975.Subjects were divided into three groups<br />
based on the phase of menstrual cycle (Group-I<br />
–menstrual phase, Group-II –follicular phase<br />
126<br />
and Group-III –luteal phase). Subjects were<br />
monitored <strong>for</strong> Resting blood pressure, Supine<br />
to standing BP, blood pressure changes during<br />
Isometric Handgrip dynamometer test and cold<br />
pressor test. It was observed that in all the tests<br />
per<strong>for</strong>med, systolic blood pressure difference<br />
between menstrual and follicular phase and<br />
follicular and luteal phase was statistically<br />
significant (P0.05) in all the<br />
tests except <strong>for</strong> handgrip dynamometer.<br />
The results were consistent with each<br />
of the earlier studies of Hassan, Mehta and<br />
Greenberg which showed an increase in systolic<br />
blood pressure during the luteal phase. They<br />
explained the increased fluid and salt retention<br />
induced by ovarian steroids as the basis <strong>for</strong><br />
the above changes. Estrogen can cause both<br />
a rise in blood pressure and cardiovascular<br />
damage unrelated to hypertension. The most<br />
important estrogen effect is an increase in the<br />
hepatic synthesis of renin substrate, which leads<br />
to an increase in plasma angiotensin-II level<br />
and aldosterone mediated fluid retention. In<br />
addition, arterial walls have estrogen receptors<br />
that may modulate smooth muscle tone and<br />
estrogen increases the vascular sensitivity to<br />
catecholamines. Higher sympathetic activity<br />
was observed due to increased levels of estrogen<br />
and progesterone in the luteal phase. Also the use<br />
of exogenous progesterone is known to increase<br />
arterial blood pressure. Thus it is possible that<br />
elevated progesterone may be responsible <strong>for</strong><br />
the increase in systolic blood pressure in luteal<br />
phase of the menstrual cycle. Physiological and<br />
psychological stress contributes to the blood<br />
pressure rise at premenstrual and menstrual<br />
phase. Fundamentally the mechanism of higher<br />
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systolic pressure in the luteal phase still is<br />
largely unknown.From the present study, it<br />
may be concluded that there was significant<br />
difference in systolic blood pressure during<br />
different phases of menstrual cycle in young<br />
healthy volunteers. Inspite of the limitations in<br />
*L. Rajeshwari and **D.H.Rajendra<br />
127<br />
being noninvasive technique, the present study<br />
provides data that should be clinically useful<br />
to the gynecologists in making a diagnosis of<br />
hypertension, orthostatic changes in blood<br />
pressure.<br />
*Dept. of Physiology,J.S.S.Medical College & Hospital , J.S.S.University<br />
S.S.Nagar, Mysore-570 015, Karnataka, India<br />
** Dept. of Physiology,Mandya Institute of Medical <strong>Sciences</strong>,District Hospital Campus,<br />
Mandya-571 401, Karnataka, India<br />
Corresponding Author<br />
Dr. Rajeshwari L<br />
email: raj0522004@gmail.com<br />
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128<br />
<strong>Biomedicine</strong>; 2011; 31 ( 1 ) : 128 - 130<br />
Measurement of Thyroid Stimulating Hormone in serum as the initial test in<br />
the assessment of thyroid disorder<br />
Thyroxine (T4) and tri-iodothyronine (T3) are<br />
together known as the ‘thyroid hormones’.<br />
They are synthesized in the thyroid gland<br />
by iodination and coupling of two tyrosine<br />
molecules whilst attached to a complex protein<br />
called thyroglobulin. Thyroxine synthesis<br />
and release are stimulated by pituitary thyroid<br />
stimulating hormone (TSH). The secretion of<br />
TSH is controlled by negative feedback by the<br />
thyroid hormones which modulates the response<br />
of the pituitary to the hypothalamic hormone,<br />
thyrotropin-releasing hormone (TRH). Thyroid<br />
hormones are essential <strong>for</strong> the normal maturation<br />
and metabolism of all the tissues in the body.<br />
Laboratory tests of thyroid function are required<br />
to assist in the diagnosis and monitoring of<br />
thyroid disease. Most laboratories offer a<br />
standard ‘profile’ of thyroid function tests (often<br />
TSH and free T4), and per<strong>for</strong>m additional tests<br />
only if these results are equivocal or the clinical<br />
circumstances require it.<br />
Since the release of TSH from the pituitary is<br />
controlled through negative feedback by thyroid<br />
hormones, measurements of TSH can be used as<br />
an index of thyroid function.<br />
If primary thyroid disease is suspected and plasma<br />
TSH concentration is normal, it can be safely<br />
inferred that the patient is euthyroid. In overt<br />
primary hypothyroidism, TSH concentrations<br />
are greatly increased, often to ten or more times<br />
the upper limit of normal. Smaller increases<br />
are seen in borderline cases or sub-clinical<br />
hypothyroidism and TSH measurement is more<br />
sensitive than T4 under these circumstances.<br />
Plasma TSH concentrations are suppressed to<br />
very low values in hyperthyroidism. It has been<br />
suggested that assays measuring ‘free T4 could<br />
be used as first-line tests of thyroid dysfunction.<br />
However, serum TSH measurement provides<br />
a better test because pituitary TSH secretion<br />
is very sensitive to changes in free T4<br />
concentration. Free T4 analyses are invaluable<br />
in diagnoses where binding proteins are altered,<br />
e.g., pregnancy, contraceptive pill, congenital<br />
TBG deficiency and nephrotic syndrome.<br />
There<strong>for</strong>e, TSH can be used as a first line test of<br />
thyroid dysfunction.<br />
A total of 50 sub-clinical primary thyroid<br />
cases, in the both sexes, attending the hospital<br />
and central lab <strong>for</strong> TFT test in ASRAM superspecialty<br />
teaching hospital were included in the<br />
study after obtaining the consent of the patients.<br />
Out of which, 30 were sub-clinical hypothyroid<br />
and 20 were sub-clinical hyperthyroid<br />
respectively. 20, age and sex matched normal<br />
healthy controls (18 – 60 yrs) were included.<br />
Serum TSH, T4 and T3 hormones were estimated<br />
on Human Automated ELISA analyzer, using<br />
Human ELISA kits. TSH was estimated by<br />
sandwich EIA method (normal range = 0.3 – 6.2<br />
mIU/L). T4 was estimated by Competitive EIA<br />
method (normal range = 4.4 – 11.6 µg/dl) and<br />
T3 was estimated by Competitive EIA method<br />
(normal range = 69 – 202 ng/dl) respectively.<br />
A serum TSH value of < 0.3 mIU/L and serum<br />
TSH value > 6.2 – 20 mIU/L was taken as subclinical<br />
level. Statistical analysis was done, using<br />
STAT TM Ver1.1 and SSP statistical software’s<br />
respectively. The Student unpaired (two sample)<br />
t-test was done at 5% level of significance. The<br />
difference in the means of TSH between subclinical<br />
hypothyroid (mean ± SD = 11.336 ±<br />
4.295) and control (mean ± SD = 2.825 ± 0.983)<br />
was statistically highly significant as p< 0.0001.<br />
The difference in the means of T4 between the<br />
sub-clinical hypothyroid (mean ± SD = 6.843 ±<br />
1.807) and control (mean ± SD = 7.220 ± 1.225)<br />
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was statistically insignificant as p< 0.4190. The<br />
difference in the means of T3 between subclinical<br />
hypothyroid cases (mean ± SD = 111.407<br />
± 24.884) and control (mean ± SD = 106.303)<br />
was statistically insignificant as p< 0.4568.<br />
Both T4 and T3 were well in the normal range<br />
in the sub-clinical hypothyroid and values are<br />
not indicating any primary thyroid dysfunction.<br />
The difference in the means of TSH between<br />
sub-clinical hyperthyroid cases (mean ± SD =<br />
0.048 ± 0.074) and control (mean ± SD = 2.825<br />
± 0.983) was statistically highly significant as<br />
p< 0.0001. The difference in the means of T4<br />
between sub-clinical hyperthyroid cases (mean<br />
± SD = 8.805 ± 1.578) and control (mean ±<br />
SD = 7.220 ± 1.225) was statistically highly<br />
significant as p< 0.0011. But, the difference<br />
in the means of T3 between sub-clinical<br />
hyperthyroid cases (mean ± SD = 116.720 ±<br />
38.174) and control (mean ± SD = 106.303<br />
± 21.408) was statistically insignificant as p<<br />
0.2939. Here also, the values of both T4 and T3<br />
were well in the normal range in the sub-clinical<br />
hyperthyroidism and values are not indicating<br />
any of thyroid dysfunction. There<strong>for</strong>e, the only<br />
TFT parameter that is increased or decreased<br />
in the sub-clinical hypo or hyperthyroidism was<br />
the TSH and clearly indicating about primary<br />
thyroid dysfunction. Where as, there is no<br />
change in the concentrations of T4 and T3 and<br />
they were well in the normal range respectively.<br />
From this study, it is very clear that, the TSH<br />
is a better marker <strong>for</strong> the primary thyroid<br />
dysfunction than the other TFT parameters<br />
i.e., either T4 or T3 (fT4 or fT3) itself or as the<br />
initial thyroid function test. From this study, it<br />
is shown that, any primary thyroid disease,<br />
even at sub-clinical level, is first reflected in<br />
the changes in the concentration of TSH in the<br />
blood. In both sub-clinical hypo or hyperthyroid<br />
disease, the only the TFT parameter showing<br />
the thyroid dysfunction is TSH, where as both<br />
the T4 and T3 are well in the normal range<br />
129<br />
respectively. They are not reflecting any of<br />
the thyroid dysfunction and they are not much<br />
useful in the early diagnose of primary thyroid<br />
dysfunction.<br />
If primary thyroid disease is suspected and plasma<br />
TSH concentration is normal, it can be safely<br />
inferred that the patient is euthyroid. In overt<br />
primary hypothyroidism, TSH concentrations<br />
are greatly increased, often to ten or more times<br />
the upper limit of normal. Minimal increases<br />
are seen in borderline cases or sub-clinical<br />
hypothyroidism and TSH measurement is more<br />
sensitive than T4 under these circumstances.<br />
Plasma TSH concentrations are suppressed to<br />
very low values in hyperthyroidism. It has been<br />
suggested that assays measuring ‘free T4 could<br />
be used as first-line tests of thyroid dysfunction.<br />
However, serum TSH measurement provides a<br />
better test because pituitary TSH secretion is very<br />
sensitive to changes in free T4 concentration.<br />
Free T4 analyses are invaluable in diagnoses<br />
where binding proteins are altered, e.g.,<br />
pregnancy, contraceptive pill, congenital TBG<br />
deficiency and nephrotic syndrome. There<strong>for</strong>e,<br />
TSH can be used as a first line test of thyroid<br />
dysfunction. Physiologically, the pituitary gland<br />
serves as a biosensor and regulator of thyroid<br />
hormone activity. When the biologic activity<br />
of thyroxine (T4) falls below the physiologic<br />
set point, the anterior pituitary responds by<br />
increasing the concentration the TSH. When the<br />
biologic activity of T4 exceeds the set point, TSH<br />
production is reduced. There <strong>for</strong>e, in patients<br />
with a normally functioning pituitary gland,<br />
basal TSH can serve as a monitor of the adequacy<br />
of the thyroid function. The TSH concentrations<br />
changes inversely with the logarithm of the free<br />
T4 (FT4) concentrations so that a doubling or<br />
halving of the T4 concentrations corresponds to<br />
equals approximately a 50-fold change in TSH<br />
concentrations. Our study reveals that, TSH is a<br />
simple, better, reliable and sensitive marker <strong>for</strong><br />
the primary thyroid dysfunction. The incidence<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
of thyroid disease is more in females at the age<br />
group 30 – 50 yrs. than males (M: F = 1:6).<br />
The thyroid problem can be easily diagnosed<br />
by estimating the TSH concentration in the<br />
130<br />
blood. The TSH estimation may be used <strong>for</strong><br />
the diagnosis of suspected thyroid dysfunction,<br />
routine screening purpose, as well as <strong>for</strong> the<br />
newborn screening <strong>for</strong> the thyroid disease.<br />
*V. S. Ravi Kiran, **S.Venkata Rao, ***K. Ambika Devi<br />
*Department of Biochemistry , Alluri Sitarama Raju Academy of Medical sciences,<br />
Eluru - 534004, <strong>An</strong>dhra Pradesh, India.<br />
*Department of Biochemistry, Alluri Sitarama Raju Academy of Medical sciences, Eluru-534004,<br />
<strong>An</strong>dhra Pradesh,India.<br />
**Department of Biochemistry,Katuri Medical College,Chinakondrupadu,Guntur<br />
Corresponding Author<br />
V . S . Ravi Kiran<br />
E-mail: ravikiran_vs2001@yahoo.co.in<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
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131<br />
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Dr G.Rajagopal,<br />
Professor of Biochemistry,<br />
Editor in chief, <strong>Biomedicine</strong>,<br />
Dr Kamakshi Memorial hospital,<br />
1, Radial road, Pallikaranai,<br />
Chennai-600100<br />
Tel: 044-66300300; Mobile : 9197078027<br />
Website: www.biomedicineonline.org<br />
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Each submitted paper is reviewed by peer<br />
reviewers after being screened by the editor in<br />
chief. Eachcommunication is given a number<br />
and it is communicated to the author. Normally<br />
an accepted communication is published within<br />
six months from the date of receipt of the<br />
commmunication.<br />
Publication Charges<br />
The publication charges <strong>for</strong> a communication<br />
is INR 400/- per printed page in the journal<br />
<strong>for</strong> all communications from India.For<br />
communications from outside India,the charges<br />
are 12 US dollers per printed page plus another<br />
10 US dollers towards postage <strong>for</strong> despatch of<br />
the hard copy of the journal. This charge is to<br />
be sent by DD in favour of Dr G.Rajagopal,<br />
Editor in Chief, <strong>Biomedicine</strong> payable at<br />
Chennai <strong>for</strong> all authors in India. The <strong>for</strong>erign<br />
authors can send the charges through bank after<br />
getting the in<strong>for</strong>mation from the Editor in Chief.<br />
Each corresponding author is provided with a<br />
hard copy of the journal in which the paper is<br />
published along with two reprints.<br />
Copyright<br />
132<br />
Communications published in <strong>Biomedicine</strong><br />
become the property of the journal.<br />
Authors declaration and copyright <strong>for</strong>m<br />
The manuscript entitled, ..................................<br />
...........................................................................<br />
..........<br />
has not been sent to any other journal <strong>for</strong><br />
publication.We, the undersigned authors declare<br />
that this communication is our original work<br />
done by us.We declare that the article does not<br />
contain any unlawful statements and does not<br />
infringe on the right of others.<br />
Name, address and signature of all authors are<br />
to be given below.<br />
Note: this <strong>for</strong>m should be filled in , signed by<br />
all authors and sent to the Editor in chief of<br />
<strong>Biomedicine</strong> along with the covering letter and<br />
CD of the paper.<br />
www.biomedicineonline.org <strong>Biomedicine</strong> - Vol 31; No.1: 2011
Indian Association of <strong>Biomedical</strong> Scientists Office Bearers, 2010 & 2011<br />
President:.........................................Dr. Ajay Kumar Singh, Delhi.<br />
Immediate Past President:.............Dr. D. Sakthisekaran, Chennai.<br />
General Secretary:..........................Dr. M.A.Hussain, Chennai.<br />
Treasurer:........................................Dr. S. Karthikeyan, Chennai.<br />
Vice Presidents:...............................Dr. R. Sheela Devi, Chennai.<br />
Dr. G.Victor Rajamanickam, Chennai.<br />
Dr. Pratima Chatterjee, Kolkata.<br />
Dr. T. Thirunalasundari, Tiruchirapalli.<br />
Editor –in-Chief:.............................Dr. G. Rajagopal, Chennai.<br />
Joint Secretaries:.............................Dr. U. Subashini, Tiruchengode.<br />
Dr. V. Madhavachandran,<br />
Thiruvananthapuram.<br />
Members of Executive Committee<br />
Dr. T.N.Umamaheswari, Chennai.<br />
Dr.S.Sriramachandramurthy,Visakapatnam.<br />
Dr. .P.Chaurasia, Leh, Ladakh.<br />
Dr. Subrata Ghosh, Kolkata.<br />
Dr. R.Rajendra Prabhu, Chennai.<br />
Dr. Nidhi Sandal, Delhi.<br />
Dr. L.Veerakumari, Chennai.<br />
Dr.Sucheta Kumari, Deralakatte, Mangalore.<br />
Dr. Baljindar Singh, Chandigarh.<br />
Dr. M.P.Narmadha, Tirucehngode.<br />
Dr. K.Sugendran, Bengaluru.<br />
Miss D.Ramya, Chennai.<br />
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