An International Journal for Biomedical Sciences - Biomedicine

TRUTH CURES CLING TO TRUTH 

An International Journal for Biomedical Sciences 

Published by: 

Indian Association of Biomedical Scientists ( IABMS ) 

www.biomedicineonline.org 

(Volume 31, Number 1 (January - March) 2011; Website: www.biomedicineonline.org) 

www.biomedicineonline.org Biomedicine - Vol 31; No.1: 2011 

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Editorial Board 

Publication 

Biomedicine is a quarterly Publication of IABMS. It is indexed in Excerpta Medica, CAB International and Ind.Med. 

Editorial Office 

Dr. Kamakshi Memorial Hospital, 

No.1, Radial Road, Pallikaranai, Chennai - 600100. 

Tel: 044 - 22469200, 044 - 66300300. 

Dr. Ajay Kumar Singh, DRDO,New Delhi. 

Dr. M.A. Hussain, Chennai 

Dr. S. Karthikeyan, Dr. ALMPGIBMS, Chennai 

Dr. R. Sheela Devi, Dr. ALMPGIBMS, Chennai 

Dr. Prathima Chatterjee, Salt lake, Kolkata 

Dr. D. Sakthisekaran, Dr. ALMPGIBMS, Chennai. 

Dr. S. Rajarajan, Presidency College, Chennai 

Dr. S. Venkataraman, Chennai 

Dr. Victor Rajamanickam, Chennai. 

Dr. B.M. Sundaram, Bangaluru 

Dr. V. Madhavachandran, Thiruvananthapuram 

Dr. A. Subramoniam, Thiruvananthapuram 

Dr. Pravati Pal, JIPMER, Puducherry 

Dr. D.C. Mathangi, Chettinad Health City, Chennai 

Dr. T. Devasena, Anna University, Chennai 

International Advisory Council 

Dr. T.G. Govindarajan, 

Dr. Kamakshi Memorial Hospital,Chennai, India. 

Dr. W. Selvamurthy, 

Ministry of Defence, New Delhi, India. 

Dr. A. Krishnamurti, 

Annamalai University, India. 

Dr. S.P. Thyagarajan, 

Ramachandra Medical College & R I, Chennai, India. 

Dr Sanguansak Rerksuppaphol, 

Srinakharinwirot University, Nakhoru Nayok, Thailand 

Dr. M. Ramachandran, 

Atlanta, United States of America. 

Dr. Sashi Bala Singh, 

Ministry of Defence, New Delhi, India. 

Dr. V. Anantharaman, 

Chennai, India. 

Dr. S. Ramakrishnan, 

Chennai, India. 

Dr. Suprita Gupta, 

National Medical College, Birgunj, Nepal. 

Dr. Anita Dushyanth, 

Los Angeles, California, United states of America 

Editor in Chief 

Dr. G. Rajagopal, 

Professor of Biochemistry, 

Dr Kamakshi Memorial Hospital, 

Pallikaranai, Chennai - 600100. 

Email: editorbiomedicine@gmail.com 

Mobile: 919791078027 

Editors 

Dr. (Mrs) S. Pushkala, 

Prof. & HOD, Dept. of Immunology, 

Dr. MGR Medical University, Chennai. 

Dr. T. Tirunalasundari, 

Prof. of Biotechnology, 

Bharatidasan University, 

Tiruchirapalli. 

Dr. Joseph Dominic, 

Medical Oncologist, 

Dr. Kamakshi Memorial Hospital. 

Dr.K. Ramesh Rao, 

Professor & HOD, Dept. of Pathology, 

Chettinad Hospital & Research Institute, Chennai. 

Managing Editor 

Mr. M. Alagusundaram, 

Asst. Prof., Department of Microbiology, 

Asan Memorial College of Arts & Science, Chennai. 

Language Editor 

Mrs. J. Aruna, 

Asst. Prof., Department of English, 

Mohamed Sathak College of Arts & Science, Chennai. 

Advisor, Pathology 

Dr. S. Rajalakshmi, 

Dr. Kamakshi Memorial Hospital, Chennai. 

Disclaimer 

The journal is not responsible for any statements made by authors. 

Copy Right: 

No part of this journal should be reproduced without written 

permission from the Editor in Chief. 

Communication Address 

All correspondence should be addressed to: 

Dr. G. Rajagopal, Editor in Chief, Biomedicine. 


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Biomedicine 

Vol.31; No.1: (January - March) 2011 

Contents 

Topics and Authors Page(s) 

I. Editorial..................................................................................................................................................... 1 

II Former Presidents of IABMS - (1985 - 1987)......................................................................................... 2 

III. Review / Special articles 

1 Health beneficial effects of black tea.............................................................................................3 - 8 

Mahejabeen Fatima and Syed Ibrahim Rizvi 

2 Modification of Lifestyle:Hypertension in Obese........................................................................9-12 

Tandra Majhi, Geeta Jaiswal 

IV. Research Papers 

3 Differential effect of Nardostachys jatamansi rhizome extract on acetylcholinesterase 

in different regions of brain in rats under chronic stress........................................................13-21 

Gloria Karkada, K. B. Shenoy, Harsha Halahalli, K. S. Karanth 

4 Heart rate reserve – A useful tool for evaluation of chest pain in middle aged........................22-26 

Ganashree C P, S M Nataraj, Rajalakshmi R,Vijaya Y Vageesh 

5 Serum α1- antitrypsin level and antioxidant status in smokers with Chronic Obstructive 

Pulmonary disease.......................................................................................................................27-31 

S. Venkata Rao , B.D. Toora, V.S.Ravi Kiran and S. Indira. 

6 Cardiovascular autonomic function tests responses in patients 

with diabetes mellitus and non diabetics..................................................................................31-38 

C.N. Mamatha and Ravipati Sarath. 

7 Association between Serum Lipoprotein(a) Concentrations and Serum 

Triglycerides in Type 2 Diabetes Mellitus..................................................................................39-44 

T. Sharmila krishna, J.N.Naidu, M. Audhisesha Reddy, K.Ramalingam, E. Venkat Rao. 

8 Ameliorative effect of Coccinia grandis in streptozotocin induced diabetic rats......................45-52 

Bhuvaneswari Palanisamy, Krishnakumari Shanmugasundaram and Rajeswari Paramasivam 

9 Testicular toxicity in arsenic exposed albino rats: ameliorative 

effects of ascorbic acid and α-tocopherol...................................................................................53-63 

Avijit Dey, Arindam Bose & Prabir Kr. Mukhopadhyay 

10 Evaluation of antioxidant status in niddm with and without complications.........................64-68 

T. Vivian Samuel, S. Smilee Johncy 

11 Clinico-Biochemical Profile of Hypokalemic Patients.................................................................69-73 

Anil Kumar Pandey, Varsha Vijay Akhade, M Sri Hari Babu,Y Himabindu 

12 Antifungal activity of Bacopa Monniera against Dermatophytic Fungus.................................74-77 

S.R.Ayyappan, R.Srikumar, R.Thangaraj, R.Jegadeesh, L. Hariprasath 

13 Prevalence of Extended Spectrum Beta Lactamase producing 

Enterobacteriaceae in Clinical specimens.................................................................................78-83 

G.S. Ravi, B.V.S.Krishna, Namratha W.Nandihal,Asha B.Patil and M.R. Chandrasekhar. 

14 Anti-inflammatory effects of Allium sativum (Garlic) in experimental Rats............................84-89 

M.K.Jayanthi and Murali Dhar 


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15 Reactive Oxygen Species during hypoxia-reperfusion injury under general anaesthesia..........90-95 

Sanjeev Kumar and Ashok kumar 

16 Assessment of probiotic properties of strains of L.fermentum and L.reuteri isolated 

from human breast milk................................................................................................................96-102 

R. Ilayaraja, Radhamadhavan. 

17 Perinatal androgen levels and Sexual dimorphic digit ratio in Down syndrome Children........103-108 

Suresh Bidarkotimath and S. Viveka 

18 Serum Enzymes, Initial and follow- up Lipid profile in Acute Myocardial Infarction...............109-113 

Suman.S. Dambal, V. Indumati. and P.B. Desai. 

19 Association of serum ferritin levels between rheumatoid arthritic obese and 

Non rheumatoid arthritic obese....................................................................................................114-118 

Tandra Majhi and A.K. Srivastara 

V Case Reports 

20 A case of asymptomatic anomalous pancreatico biliary ductal union 

of pancreatic - biliary type..........................................................................................................119-121 

V.S. Anandarani. 

21 Unusual complication at Feeding Jejunostomy in Boerhaave’s Syndrome.................................122-125 

Vithalkumar. M. Betigeri, Anupama V Betigeri, Nanda Kishore Maroju and 

Kasturi Satya Venkata Kumar Subba Rao 

VI Letters to the Editor 

22 Blood Pressure Changes in menstrual cycle...................................................................................126-127 

L. Rajeshwari and D.H. Rajendra 

23 Measurement of Thyroid Stimulating Hormone in serum as the initial test 

in the assessment of thyroid disorder............................................................................................128-130 

V. S. Ravi Kiran, S.Venkata Rao, K. Ambika Devi 


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Editorial Biomedicine; 2011; 31 ( 1 ) : 1 

A Good Food 

It is a fermented food of India especially southern 

India. Its essential components are parboiled rice 

and dehulled black gram dhal. It is nutritionally good 

and very hygienic. It is named IDLI. Reference to 

Idli is made in Tamil Literature between 100 BC and 

300 AD. 

Preparation 

4 parts of parboiled rice and 1 part of dehulled black 

gram dhal are separately kept soaked in drinking 

water for about one hour. A small quantity of dry 

methi seeds are added to the dhal. The dhal is then 

ground in a wet grinder for 3o minutes with periodical 

addition of traces of water for soft grinding. The 

rice is then ground for 30 minutes similarly. Both 

are mixed and required salt is added. The mixture is 

well mixed and allowed to ferment for about 8-10 

hours or preferably overnight. It is now ready for 

preparing idli by steaming. 

Fermentation 

The fermentation of idli batter is due to the leavening 

action produced exclusively by the activity of a 

lactic acid bacterium, Leuconostoc Mesenteroids. 

It is responsible for the souring as well as gas 

production. In the later stages of fermentation, 

growth of streptococcus faecalis and still later 

pediococcus cerevisiae becomes significant. 

Hygienic Cooking 

Idli is made by steaming in an idli vessel. It is 

therefore a sterile hygienic food. Some may prefer 

cooking in a micro-wave oven. This is not correct. 

Nutritive Value of Idli 

The composition of idli makes it nutritionally good. 

Parboiled rice retains the B vitamins unlike the raw 

rice. It supplements the dietary essential amino 

acid, methionine which is deficient in the black 

gram dhal. Black gram dhal, on the other hand, 

compensates the deficient amino acids, lysine and 

threonine in the rice. Idli therefore contains all the 

eight dietary essential amino acids which the body 

can not synthesize. Besides, while eating idlis, a 

side dish, usually a chilli powder made of dhal, 

and dry chillies is used to which fresh sesame oil 

is added for taste. Sesame oil contains the dietary 

essential fatty acids, which the body needs. The 

calorific value of a 35 gram idli is approximately 70 

kilo calories. The digestibility of idli is very good. 

Cereals contain phytate which interferes with the 

absorption of calcium and iron. But fermentation 

produces the enzyme phytase which reduces the 

phytate content. Fermentation also enhances the 

availability of thiamine and riboflavin. Germination 

and fermentation increase the bioavailability of Zinc 

and Iron from the food grains. 

Antioxidant activities 

The effect of fermentation of idli batter on the 

enhancement of antioxidative activities was reflected 

by a higher total phenol (TPC) content. Fermemtation 

enhanced > 2.5 fold TPC. The fermented batter was 

a better free radical scavenger. 

Food for the sick 

Idli is recommended for patients in hospitals due to 

its hygienic preparation and good digestibility. The 

glycemic response in terms of glycemic index was 

determined in patients with type 2 diabetes mellitus. 

It was observed that consumption of idlis delayed 

the rise in blood glucose level. 

Any time Food 

Idlies can be considered as a fast but ideal and safe 

food. It is an ideal breakfast food. However, it is 

an anytime food. Idlis may be taken for the night. 

Idli may be taken with chilli-dhall mix, sambar or 

coconut chutney. 

G.Rajagopal 

Editor in Chief 


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Former Presidents of IABMS - 5 (1987-1989) Biomedicine; 2011; 31 (1): 2 

Dr.B.S.Gajalakshmi 

M.B.B.S.,M.Sc.,Ph.D.,MNAMS,FIABMS 

Bandi Santhana Krishnan Gajalakshmi(Dr BSG) 

was born in Chennai.She is a founder member of 

IABMS and a life member from 1979.Dr BSG 

had early education in Chennai. She was a student 

of Andhra Medical college, Visakhapatnam and 

obtained MBBS degree in 1957. Her professional 

career began in Stanley Medical College, Chennai 

in 1959 as tutor in Physiology.Under the guidance 

of Dr Sarada Subramaniam, she specialised in 

Physiology with an M.Sc. degree in 1966 and 

later with Ph.D. degree in 1980.She worked in 

several medical colleges of TamilNadu in different 

capacities as Assistant Professor, Associate 

Professor ,and Professor in Physiology.Sh e retired 

as Director and H.O.D. of Physiology from the 

Institute of Physiology and Experimental Medicine, 

Madras Medical College, Chennai. Dr Gajalakshmi 

developed a keen interest in the study of the 

pathophysiological effects of scorpion venom of 

buthus tamulus in different animals.She investigated 

the beneficial effects of Lytic Cocktail therapy in 

mitigating the toxic effects of the venom.These 

studies enabled her to obtain Ph.D. in Physiology 

from the University of Madras. Her contributions 

to the clinico physiological problems of scorpion 

venom received wide recognition especially from 

child specialists.Her other fields of interest are 

Diabetes, Stress and Occupational hazards.She has 

published several research communications and 

scientific papers of topical interest in National and 

International journals. Dr Gajalakshmi is associated 

with the activities of several professional bodies. 

She is a founder member of the Indian Association 

of Medical Scientists (IABMS), Women Doctors’ 

Association,and Madras Institute of Magneto 

Biology. She is a life member of Association of 

Physiologists and Pharmacologists of India(APPI), 

Indian Science Congress Association, Altrusa 

International Inc. and Madras Club Women Service 

Organisation. Dr Gajalakshmi has organised several 

Scientific meetings, conferences and workshops 

under the auspices of IABMS to update information 

especially to young scientists.She was the first person 

to introduce a separate session for medical students 

as well as other faculties in 1982 during the 3rd 

Annual Conference of IABMS at Stanley Medical 

college, Chennai. She was conferred the Fellowship 

title of IABMS, instituted for the first time during 

the Annual Conference held at MMC, Chennai, in 

1989.She delivered the prestigious M.K.Nambiar 

Oration at the 15th Annual Conference of IABMS 

in 1994. Dr Gajalakshmi was elected unanimously 

as President of the 75th platinum jubilee conference 

of the Indian Science Congress held at Pune, in 

1988.In 1991, she gave J.N.Maitra memorial oration 

at the 18th Annual Conference of Physiological 

Society of India.In 1999, at the 86th Indian Science 

Congress she delivered N.M. Basu memorial 

oration. Dr.Gajalakshmi, after retirement from 

Government service, has joined BRS Hospital as 

Director. She is continuing to work part time at this 

hospital. To update knowledge in medical sciences, 

she organises CME programmes and Seminars. She 

became interested in community service after being 

inducted into Altrusa International Inc. Madras. 

She conducted several medical camps covering 

dental,diabetes, speech & learning disability, 

eye,and cancer detection in women. She was the 

course director for Auxilliary Nurse Aid Programme 

of one year duration for Harijan Adidravida girls 

from 1995 till 2000 and 125 girls were trained in 

this programme and absorbed by varioius hospitals 

and clinics. Dr Gajalakshmi has shifted her interest 

now to study Vedanthic literature.She disires to 

be a Vanaprastha. Biomedicine on behalf of all its 

readers sends good wishes to this scientist for a 

happy retired life. 


2

REVIEW ARTICLE Biomedicine; 2011; 31 (1): 3 - 8 

Health beneficial effects of black tea 

Mahejabeen Fatima and Syed Ibrahim Rizvi 

Department of Biochemistry, University of Allahabad, Allahabad 211002, Uttar Pradesh, India. 

(Received 20 th December, 2010; Revised 26 th January, 2011; Accepted 7 th March, 2011) 

Corresponding author: 

Dr. Syed Ibrahim Rizvi 

E-Mail: sirizvi@gmail.com 

Abstract 

Tea is the most common beverage in the world. It is consumed mostly as green tea, oolong, or black tea. 

While there are several informative reviews on the health benefits of green tea, there are few detailed 

articles on black tea. Importantly in India the preferred form of tea is black tea. This review is an attempt to 

bring together relevant findings on black tea including chemistry of processing, metabolism and absorption 

of black tea polyphenols and health benefits. 

Key words: Antioxidant, Black tea, Health, Polyphenols 

Introduction 

Tea is the most popular beverage in the world 

which is manufactured from the plant species 

Camellia sinensis (family Theaceae). It is 

grown around the world specially in China, 

Japan, Nepal, India, and SriLanka. Tea is 

consumed mostly as green tea and black tea, 

approximately 20% of the tea is manufactured 

as green tea while 80% of the approximately 

2.5 million metric tons of manufactured dried 

tea is black tea . A small percentage of tea is 

manufactured as oolong tea which is about 2% 

and usually consumed in Japan and China. The 

effect of tea consumption on human health has 

lately received much attention since tea has a 

high concentration of polyphenols which are 

known to possess antioxidant properties. 

Harvested tea leaves are processed in the factory 

and accordingly there are two types of tea: 

Orthodox, and CTC (Crushing Tearing Curling). 

The Orthodox tea is the whole leaf tea whereas 

CTC tea is widely popular and processed 

through the crush, tear and curl (CTC) method. 

Traditionally, tea is classified on the basis of the 

method of processing tea in to following three 

main types: green tea, oolong tea and black tea. 

After plucking, wilting leaves undergo slight 

enzymatic oxidation by the enzyme polyphenol 

oxidase (PPO), which results in fermentation 

of tea though this is not actual fermentation. 

Green tea is unfermented or nonoxidised 

tea and prepared by destroying the enzymes 

of fresh tea leaves by steaming followed by 

rolling either by hand or rollers, then fired to 

dry. Oolong tea is made by withering and then 

putting through a series of light rollings before 

firing, which arrests oxidation, this process 

is called as semifermentation and results in 

partial oxidation. Black tea is oxidized or fully 

fermented form of tea. During the preparation 

of black tea, leaves are withered to reduce the 


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Beneficial effects of black tea 

moisture and then rolled by CTC or tea roller 

however during the rolling process oxidation 

starts when enzymes come into contact with air, 

this causes the change in color of leaves from 

green to brown. Finally to stop the oxidation 

process tea is passed through hot air dryers. 

Chemical composition of green and black tea 

The chemical composition of tea includes 

polyphenols, amino acids, vitamins, 

proteins, carbohydrates, trace elements. 

In addition, alkaloids such as caffeine 

(1,3,7-trimethylxanthine), theobromine and 

theophylline are also natural compounds 

present in tea. Tea polyphenols are the crucial 

compounds among these that include mainly 

catechins also found in red wines, apples, grapes 

and chocolate. Tea catechins are composed of 

epicatechin (EC), epigallocatechin (EGC) and 

their galloyl esters such as gallocatechin (GC), 

epicatechingallate (ECG), and epigallocatechin 

gallate (EGCG). Among teas, green tea 

contains large amount of catechins and esters 

of gallic acid. Black tea contains several 

polyphenols such as bisflavonols, theaflavins 

(TFs) and thearubigins (TRs), whereas 

oolong tea contains small amount of catechins 

and theaflavins. Theaflavins are mixture of 

theaflavin 3-gallate (TF3G), theaflavin3′-gallate 

(TF3′G), theaflavin3,3′-digallate (TF3,3′DG). 

Black tea extract constitutes approximately 

20-30% polyphenols, among them TRs is most 

abundant comprising about 10±20% with 

relative molecular masses ranging from 700 to 

40,000 Da and TFs with 0.3±2% on a dry weight 

basis . Together they contribute characteristic 

color, strength and body to the tea . TFs give 

reddish orange color whereas TRs brownish 

color to tea. In recent years, TFs have attracted 

considerable interest because of their beneficial 

health properties including anti-inflammatory , 

antimutagenic and anticlastogenic effects (1). 

The chemistry of black tea processing 

Tea catechin flavanols, possess the C6 - C3 - 

C6 skeletal structure, correspond to 2-phenylsubstituted 

benzopyrans and pyrones , and 

precursors incorporated from the common 

shikimic and acetate-malonate biosynthetic 

pathways. 

During the manufacturing of black tea, 

oxidation of catechins takes place, TFs and 

TRs are formed through dimerisation and 

polymerization respectively (Fig: 1). TFs are 

formed through oxidative dimerisation between 

catechin derived quinones and gallocatechin 

with endogenous enzyme polyphenol oxidase 

of tea leaf while TRs are formed through 

the oxidative polymerization of galloyl 

esters of catechins through the formation of 

benzotropolone skeleton (2). 

Metabolism and absorption of black tea 

polyphenols 

These large and highly polar molecules cannot be 

absorbed after oral ingestion but are hydrolyzed 

to their aglycones (nonglycosylated forms) 

by bacterial enzymes in the lower part of the 

intestine (3) and then be incompletely absorbed 

or go through further biotransformation by 

bacteria. Recent studies have investigated that 

after tea consumption catechins are metabolized 

and transformed as sulfated, methylated, or 

glucuronidated derivatives by enzymes such as 

sulfotranferase, catechol-O-methyltransferase 

and glucuronosyltransferase respectively (4) 

and it has been hypothesized that break down 

of flavonoids into smaller phenolic acids 

takes place within the colon from bacterial 

degradation, absorption occurs through the 

small intestine. These phenolic acids can be 

absorbed in the circulatory system (5). 

Health benefits of black tea 

Tea plays a significant role in protecting cell 

membranes from oxidative damage, improving 

intestinal microflora which are beneficial to the 

body and also prevents dental caries (6). Possible 


4


beneficial health effects of black tea polyphenols 

are antioxidative, antithrombogenic, and antiinflammatory 

(1). In vitro studies by using 

animal models suggest that consumption of 

black tea contribute in the prevention of some 

cancers, cardiovascular diseases and to treat 

diabetes in clinical trials (7). Moreover, a 

number of antioxidants present in green and 

black tea, mainly catechins and theaflavins 

have anti-carcinogenic , anti-mutagenic (8) 

and neuroprotective (9) properties. The health 

benefits of black tea are interesting, but more 

research on both animals and humans need to 

be conducted. 

Black tea: positive effects on mood and 

mental performance 

Tea contains a unique amino acid, L-theanine 

(5-N-ethyl-glutamine) which is a glutamic 

acid analogue, a predominant amino acid 

component in tea. It constitutes about 1-2% 

of the dry weight of tea leaves and accounting 

for about 50% of the total free amino acids. 

Several studies prove that L-theanine has an 

effect on brain electrical activity and appears to 

have psychoactive properties and can also act 

as a neurotransmitter. It increases the alphabrain 

waves during resting state (10) that are 

associated with relaxed mental state. By using 

animal models such as rats, it has been found 

that theanine improved memory and learning 

ability by modulating serotonin and dopamine 

levels (11). 

In addition caffeine (1,3,7-trimethylxanthine) 

is associated with stimulatory effects on central 

nervous system and cause increase in basal 

metabolic rate (BMR) by about 3-4% (12). It 

has been shown to stimulate thermogenesis by 

inhibiting the enzyme phosphodiesterase and 

also by the stimulation of substrate cycles such 

as the Cori-cycle and the FFA-triglycreride 

cycle (13). Caffeine and theophylline inhibit the 

enzyme adenosine 3, 5-cyclic monophosphate 

(cAMP) phosphodiesterase due to intracellular 

accumulation of cAMP (14), this is associated 

with freshness in mood which corresponds to 

increased mental alertness (15). It has been 

hypothesized that theophylline might inhibit 

Raf-1-dependent tumor progression in rats, the 

inhibition of theophylline is more than caffeine. 

Recent epidemiological studies have shown a 

reduced risk of Parkinson’s disease associated 

with selective amount of consumption tea (16). 

Black tea: Cancer chemoprevention 

There is evidence that tumor killing is induced 

by black tea polyphenols through apoptosis 

in Swiss mouse bearing Ehrlich’s ascites 

carcinoma (17). Another study reports that 

TFs inhibits the mammalian thioredoxin 

reductase, which is an antioxidant protein, 

regulating cellular functions like cell growth 

and apopotosis (18). It has been shown that 

green and black tea polyphenols inhibit the 

effect of hamster buccal pouch carcinogenesis 

by altering the detoxifying enzymes phase 

I (cytochromeP450s), phase II (glucuronyl 

transferase) and redox status, however black tea 

polyphenols were more effective and evidence 

indicates that it is more potent inhibitor 

for development of cancer (19). Moreover, 

considering the cancer chemoprevention studies 

some researches have shown that TF3,3′DG 

inhibits UVB induced activator protein (AP-1), 

induction and in the down regulation of EGFR 

(epidermal growth factor receptor), which 

regulates cell proliferation differentiation and 

transformation, in different mouse cell lines (JB6 

C141 and A431 ) (20). Consumption of black 

tea increases the plasma antioxidant capacity 

significantly in animal models and in humans 

thus protecting human red blood cells against 

oxidative damage induced by agents such as 

reactive oxygen and reactive nitrogen species 

(21). Black tea polyphenols may suppress the 

free radicals, protect HPF-1 (embryonic human 

lung fibroblasts) cells against H 2 O 2 induced 

damage (22). TFs are capable of anti cancerous 

action by inducing apoptosis as well as efficient 


5


Figure 1. Polyphenol oxidation during black tea processing. 

Figure 2. Metabolism of black tea polyphenols in the human body 


6


inhibitor of antiapoptotic proteins (Bcl-2 

family proteins) (23). Other metabolic effects 

of black tea include reducing the oxidation of 

low density lipoprotein (LDL) that is associated 

with atherosclerosis, improving the plasma 

lipid profiles in animals after high fat diet (24), 

reducing blood glucose and blood triglyceride 

levels in aged rats (25) and antioxidative effect 

leading to alcohol intoxication by normalization 

of cellular metabolism functionings through 

alteration in the antioxidative defence enzymes 

(1). 

Antioxidative properties of black tea 

Antioxidants have the ability to entrap free 

radicals and protect the body from damage 

caused by free radical-induced oxidative stress. 

Tea contains polyphenolic flavonoids which can 

enhance the cellular antioxidant enzyme activity 

or antioxidant defence of the body. In biological 

systems various environmental sources generate 

free radicals and reactive oxygen species which 

may oxidize proteins, nucleic acids and lipids. 

It has been demonstrated through in vitro and 

ex vivo studies that black tea polyphenols 

possess strong antioxidant and metal chelating 

properties, because of the vicinal dihydroxyl 

and trihydroxyl group attached to the flavan- 

3-ol, which prevents the generation of free 

radicals and may protect cells and tissues 

against oxidative damage (26). Researches have 

revealed that TF3 possess higher antioxidative 

activity than EGCG, which is the strongest 

antioxidant among all catechins due to the higher 

number of hydroxyl (OH) groups, which are 

considered to be necessary for exerting radical 

scavenging activity (antioxidative properties), 

than do catechins (27). Investigations show 

that antioxidative properties of theaflavins 

change in the following order: TF3>TF2>TF1 

(28) depending on the position and number of 

hydroxyl group in the molecule (27). Moreover, 

regarding the structural analysis theaflavin 

gallates illustrate stronger antioxidative 

properties compared with free theaflavins due 

to the position residues influences (28). There 

is increasing evidence to show that free radical 

damage contribute to many chronic health 

problems such as cardiovascular diseases 

,diabetes some cancers etc. Many epidemiologic 

and laboratory studies suggest that regular 

consumption of black tea polyphenols 

significantly reduce the risk of many oral cancers 

(29) however more research needs to define the 

real extent of tea consumption associated with 

them in the mammalian system. 

Future prospects 

Several epidemiological studies suggest that 

theaflavins can prevent risk of disease such as 

cardiovascular disease and some cancers. On 

the basis of laboratory studies regular intake 

of black tea can improve the oxidative stress 

biomarkers. From several pharmacological 

observations, worldwide abundance and 

absence of toxicity, theaflavin supplement can 

be used as an important natural therapeutic agent 

in chemoprevention studies and in the field of 

medicine. Further research is needed to explain 

the bioavailability and evaluation corresponding 

to the optimal amount of consumption of black 

tea in mammalian system. 

Acknowledgement 

This work was supported by UGC Major 

Research Project F 37-392/2009 SR sanctioned 

to SIR 

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8

Special article Biomedicine; 2011; 31 (1): 9 - 12 

Modification of Lifestyle:Hypertension in Obese 

*Tandra Majhi, **Geeta Jaiswal 

*Department of Psychiatry,C.S.M.Medical University, Lucnow- 226003, Uttar Pradesh, India. 

**Department of Biochemistry, M.L.N.Medical College, Allahabad -211003, Uttar Pradesh, India. 

(Received 29 th June, 2010; Revised 20 th Decemebr, 2010; Accepted 16 th January, 2011) 

Corresponding Author 

Dr. Tandra Majhi 

Email: tandra.majhi@gmail.com 

rinku.majhi@gmail.com 

Abstract 

Obesity is a complex, multifactorial disease that is associated with essential hypertension in Men & Women. 

In India, hypertension in the general population is largely undetected, and the available data and study 

on the topic is merely a tip of the iceberg. Several large epidemiological studies have documented that 

associated between body weight and blood pressure. The age, physical activity, dietary intake and lifestyle 

definitely influence on body weight associated with blood pressure. When, lifestyle modifications are 

adopted by the obese as a primary prevention strategy in Indian population, it may help to avoid health 

problem due to obesity. 

Key words: Blood Pressure, Serum electrolytes, Life style & Physical activity 

Introduction 

Obesity and hypertension are both public health 

problems in Western society. Results from 

the Framingham study have shown that high 

blood pressure(1) and overweight(2) are both 

independent risk factors for cardiovascular 

disease. Hypertension is one of the most 

common obesity-related complication, and 

about 30% of hypertension individuals can 

be classified as being obese(3). In a group of 

hypertensive women taken from the Nurses 

Health Study(4) , the related risk of fatal and 

nonfatal coronary heart disease increased 

from the lowest to the highest form of obesity. 

Prevalence in India, hypertension in the 

general population is largely undetected, and the 

available data and study on the topic is merely a 

tip of the iceberg. Epidemiological studies show 

that hypertension is present in 25% urban and 

10% rural population in India. There are 31.5 

million hypertension in the rural and 34 million 

in the urban population(5). The estimated total 

number of people with hypertension in India on 

2000 was 60.4 million males and 57.8 million 

females and projected to increase to 107.3 

million and 106.2 million respectively in 2025. 

Obesity and Hypertension 

Hypertension is generally defined as a systolic 

blood pressure of 140 mm Hg and a diastolic 

blood pressure of 90 mm Hg according to the 

sixth report of the Joint National Committee 

on Prevention, Detection Evaluation and 

Treatment of High Blood Pressure(6). Obesity 


9

Modification of life style: Hypertension in Obese 

has shown to be independent risk factor for the 

development of hypertension. Several large 

epidemiological studies have documented the 

association between body weight and blood 

pressure(7,8). The relationship between body 

mass index and blood pressure was studied 

in 25-60 years of age, in a sample from urban 

population. In this study, BMI & WHR were 

significantly associated with systolic and 

diastolic blood pressure and was independent 

of age, sex, lifestyle, dietary intake and sodium 

and potassium excretion. In a recent, updated 

evolution of the Nurses Health Study, a longterm 

follow-up study of more than 80,000 

female nurses, BMIs at 18 years of age and 

middle age were positively associated with the 

occurrence of hypertension(9). Long-term and 

medium term weight loss was associated with 

a reduced risk in the group of women with a 

high baseline BMI. A central fat distribution is 

a better predictor for hypertension than overall 

fat mass. Earlier studies have shown a good 

correlation between the Weight/Height Ratio ( 

WHR)and blood pressure levels(10,11). Later 

studies using computed tomography to measure 

to exact amount of visceral adipose tissue 

showed strong correlation with systolic and 

diastolic blood pressure levels both in European 

and African population (12-15). 

Life style modification and Physical activity 

Importance of lifestyle modification can 

be addressed by the fact that most of the 

community physicians in India who were 

treating hypertension have no time to tell the 

patients regarding lifestyle modifications. 

The low level of physical activity of individuals 

demanded by modern life, therefore, has 

created a condition that has made dietary 

intake a more noticeable component of obesity 

risk than it ever was before. A further lifestyle 

change is the daily stress to which human are 

exposed. There are different physiological 

responses to stress. The so-called “fight or 

flight” response involves stimulation of the 

10 

sympatho-adrenal system, and this response 

has been implicated in hypertension. Increase 

in aerobic physical activity such as brisk 

walking, jogging, swimming or bicycling has 

been shown to lower blood pressure. This 

reduction is independent of weight loss(16). A 

meta-analysis of 54 randomized control trials 

show a net reduction of 3.8 mm Hg in systolic 

and 2.6 mm Hg in diastolic blood pressure 

in individuals performing aerobic exercise. 

Compared to controls, physical activity reduces 

systemic vascular resistance due to decreased 

sympathetic and nervous system activity. This 

is evidence by lower plasma nor-epinephrine 

level in exercising individuals as compared to 

sedentary people(17). 

Dietary salt intake 

Food intake in humans is by nature, very poorly 

controlled. There has never been any real 

evolutionary pressure to suppress the appetite. 

In fact, it is quite the opposite. Because man 

is a hunter-gatherer and because of the annual 

cycles of “feast and famine” the evolutionary 

drive has been to seek food constantly, to gorge 

when it is readily available, and to be able to 

survive periods of famine. 

Our studies have indicated in this respect the 

total caloric (carbohydrates, fats, protein and 

minerals) intake was more than prescribed RDA. 

Raised BMI and WHR of the experimental 

group therefore may be a result of over nutrition 

leading to a highly fed state. The total caloric 

intake was much high in the Indian obese almost 

by 132.21 percent their control counterparts. 

Therefore the of total caloric intake is a major 

and main contributor towards the increase in 

body weight and hypertension in India. In these 

days of increasing economic status it has 

resulted in a higher consumption of sugar, salt, 

meat and saturated fatty acids amongst Indians 

and therefore has increased the possibility 

of obesity with blood pressure. Dietary salt 

intake in associated with blood pressure. 

Epidemiological , clinical , experimental and 

www.biomedicineonline.org Biomedicine - Vol 31; No.1: 2011


randomized controlled trials suggest that health 

intervention, including government policies and 

action to regulate reduction in the salt control of 

processed food are cost-effective ways to limit 

cardiovascular disease(18-21). This could avert 

over 21 million disability –adjusted life-years 

per year worldwide(22). The Dietary Approach 

to Stop Hypertension (DASH) sodium feeding 

study showed that an even lower intake of 

sodium (approximately) by both normotensives 

and hypertensives(23). 

The golden rule is : 

Avoid excessive intake of salt in cooking. 

Increase intake of green vegetables and fruits. 

Avoid foods high in salt. 

Avoid junk foods or fast food which are high in 

salt as well as fat content. 

Obesity and serum electrolytes 

Several large epidemiological studies 

have documented that associated between 

body weight and blood pressure. BMI was 

significantly associated with systolic and 

diastolic blood pressure and was independent 

of age, alcohol intake, smoking habits, sodium 

and potassium excretion. Epidemiological and 

clinical studies have shown that potassium 

intake has an important role in regulating blood 

pressure in both the general population and 

people with high blood pressure(24). From our 

study we found that the mean serum sodium 

levels were significantly higher in Indian obese 

compared to the non-obese group. However 

mean serum potassium levels were significantly 

(47.92%) lower. As observed, raised blood 

pressure and serum sodium may be attributed 

to an increased cardiac output in part due to 

the additional blood flow required for the extra 

adipose tissue. However, blood flow in the heart, 

kidneys, gastro-intestinal tract, and skeletal 

muscle also increased metabolic demands. As 

hypertension is sustained for many months and 

years, total peripheral vascular resistance may 

also be increased. 

Management 

11 

The management of overweight control should 

be influenced by the degree of obesity and over 

risk status of associated diseases. Management 

includes both reducing excess body weight 

and instituting other measures to control 

accompanying risk factors obesity should be 

assessed in terms of BMI, waist circumference 

and the patient’s motivation to lose weight. 

The initial goal of weight loss therapy is to 

reduce body weight to approximately 10% 

from baseline. If one can achieve this goal, 

further weight loss attempted, indicated through 

evolution. Those patients who are unable to 

achieve significant weight loss, they may also 

need to participate in a weight management 

program. 

Weight loss strategies 

Diet therapy 

A diet should create a deficit of 500-1000 kcal/ 

day. Depending on the patient’s risk status, the 

low-caloric diet should be consistent with the 

therapeutic life-style changes defined by the 

National Cholesterol education Program. Apart 

from decreasing saturated fat, total fat should 

be 30% or less of total calories (including low 

carbohydrates & salts), saturated fat should 

not produce weight loss unless the total 

caloric intake is also decreased. Fiber diet and 

nutritional antioxidants (Vit C & E) should be 

encouraged. 

Physical activity 

An increase in physical activity is an important 

component of weight loss therapy although 

physical activity alone will not usually produce 

significant weight loss in obese patients. 

Most weight loss occurs because of decrease 

in caloric intake. Sustained physical activity 

is, however, helpful for maintaining a lower 

weight. Therefore the dietary intake and 

physical activity should be balanced to maintain 

the weight loss. 



Conclusion 

As the prevalence of overweight and obesity 

has increased, so have related health care costsboth 

direct and indirect. It must be noted that 

the studies hav shown a positive relationship 

between obesity and hypertension. It indicated 

that age, physical activity, dietary intake 

(including salt intake) and life-style definitely 

influence body weight and blood pressure levels 

in the obese subjects. If life style modifications 

are adopted by the obese people as a primary 

prevention strategy in Indian population, then 

it may help to avoid health problem specially 

hypertension related with obesity. 

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study of obesity and risk of coronary heart disease in women. 

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2004;18:73-8. 

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RH. Weight and blood pressure: findings in hypertension 

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1610. 

8. Dyer AR, Elliott P on behalf of the INTERSALT to operative 

Research Group. The INTERSALT study: relations 

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1989;3: 299-308. 

9. Huang Z, Willett WC, Manson JE at al Body weight- 

weigth change, and risk for hypertension in women. Ann 

Indian Med. 1998;128:81-88. 

12 

10. Lapidus L, Bengtsson C, Larsson B, Penvert K, Rybo E, 

Sjostrom L. contribution of adipose tissue and risk of cardiovascular 

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the population study of women in Gothenburg. Br Med. J 

1984;289: 1257-1261. 

11. Larsson B, Svardsudd K, Welin L, Wilhelmsen L, Bjorntorp 

P, Tibblin G. Abdominal adipose tissue distribution, 

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1913. Br Med. J 1984;288:1401-1404. 

12. Peirin AN, Sothmann MS, Hoffmann RG et al. Adiposity 

fat distribution and cardiovascular risk. Ann. Intern Med 

1989;110: 867-872. 

13. Kamai M, Tokunaga K, Fujioka S, Yamashila S, Kameda- 

Takemura K, Matsuzawa Y. Decrease in intra abdominal 

visceral fat may reduce blood pressure in obese hypertensive 

women. Hypertension 1996;27: 125-129. 

14. Han TS, Van Leer. EM, Seidell JC, Lean MEJ, Waist circumference 

action levels in the indentification of cardiovascular 

risk factors: prevalence study in a random sample. 

Br Med J 1995; 311: 1401-1405. 

15. Okosum IS, Forrester TE, Rotimi CN, Osotimetion BO, Muna 

WF, Looper RS, Abdominal adiposity in six populations of 

west African descent: Prevalence and population attributable 

fraction of hypertension. Obes Res. 1999;7: 453-462. 

16. He J, Klag MJ, Caballero B et al. Plasma insulin levels 

and incidence of hypertension in African Americans and 

Whites. Arch. Intern Med 1999;159:498-503. 

17. Whetton SP, Chin A, Xin X, He J. Effect of aerobic exercise 

on blood pressure: A meta-analysis of randomized, 

controlled trials. Ann Intern Med, 2002;136:493-503. 

18. Intersalt cooperative research group. INTERSALT- an international 

study of electrolyte excretion and blood pressure: 

results for 24-hour urinary sodium and potassium 

excretion. B, Medical J.1988; 297:319-28. 

19. Forte JG, Miguel JM, Miguel MJ et al. Salt and blood pressure: 

a community trial. J Hum Hypertens. 1989; 3:179-84. 

20. Denton D, Weisinger R, Mundy NI et al. the effect of increased 

salt intake on blood pressure of chimpazees. The 

Natl Med. J 1995;1009-16. 

21. Sacks FM, svet key LP, vollmer WM. Effects on blood 

pressure of reduced dilteway sodium and diatery approaches 

to stop hypertension (DASH) diet. New Eng. J 

Med. 2001; 344:3-10. 

22. Murray CSL, Lauer JA, Hutubessy RCW et al. Effectiveness 

and costs of interventions to lower systolic blood pressure 

and cholesterol: a global and regional analysis on reduction 

of cardiovascular- disease risk. Lancet 2003; 361:717-25. 

23. HeJ, Ogden LG, Vupputuri S et al. Dietery sodium intake 

and subsequent risk of cardiovascular disease in over 

weight adults. JAMA 1999; 282:2027-34. 

24. He FJ, Macgregor GA. Potassium intake and blood pressure. 

Am. Hypertens. 1999; 12: 848-51. 


Research Paper Biomedicine; 2011; 31 (1): 13 - 21 

Differential effect of Nardostachys jatamansi rhizome extract on acetylcholinesterase 

in different regions of brain in rats under chronic stress 

*Gloria Karkada **K. B. Shenoy**** Harsha Halahalli ****K. S. Karanth 

*Department of Biotechnology, Nitte Mahalinga Adyanthaya Institute of Technology, 

Nitte - 574110, Karnataka, India. 

**Department of Applied Zoology, Mangalore University, Mangalore-574119 India. 

***Department of Physiology, KS Hegde Medical Academy, Mangalore-575018, India. 

****Department of Pharmacology, KS Hegde Medical Academy, Mangalore-575018. 

(Received 16 th July, 2010; Revised 6 th December, 2010; Accepted 6 th January, 2011) 


Dr K. B. Shenoy 

Email: bshenoyk@gmail.com 

Abstract 

Background and objectives: Nardostachys jatamansi, a popular herb in Indian medicine, has been 

investigated for various effects on central nervous system. However the neurobiological mechanisms 

of its actions are not clear. It was hypothesized that N.jatamansi root ethanolic extract (NJE) modulates 

acetylcholinesterase activity levels in brain regions relevant for learning and memory. 

Methods: A factorial study design was adopted to investigate the effects of two experimental interventions 

- treatment with NJE and chronic restraint stress for 21 days – on AChE activity levels in the hippocampus, 

frontal cortex and striatum. 

Results: Unstressed animals treated with NJE had significantly higher level of AChE activity in the frontal 

cortex (179% higher) and hippocampus( 36% higher) compared to controls. AChE activity was also 

significantly higher (by 54 %) in the frontal cortex of stressed animals and concomitant NJE treatment 

when compared to untreated stressed group. Chronic restraint stress per se had no effect on AChE levels in 

the brain regions studied. 

Interpretation and Conclusions: Enhancement of AChE activity in the frontal cortex following NJE 

administration may be indicative of positive plasticity of cholinergic pathways to the neocortex. The 

implications of this region specific cholinergic modulation for the possible development of N.jatamansi 

based anti-dementia and nootropic interventions remain to be clarified. 

Keywords: Acetylcholinesterase, Cholinergic, Chronic restraint stress, Nardostachys jatamansi, Nootropic 

Introduction 

Nardostachys jatamansi has been valued for 

its therapeutic properties in the Ayurveda and 

Unani traditions of complimentary medicine. 

13 

The rhizomal extracts of N. jatamansi have 

been particularly studied for effects on the 

central nervous system. N. Jatamansi ethanolic 

root extracts (NJE) have been reported to have 

anticonvulsant activity in a rat electroshock 


Effect of Nardostachys jatamansi rhizome in brain 

seizure model (1), anti-parkinsonism activity 

in a rat 6-OHDA model (2), neuroprotective 

effect in a rat cerebral ischemia model (3) and 

antidepressant activity in tail suspension and 

forced swim mouse models of depression (4). 

A few studies have investigated possible 

neurobiological mechanisms of the action of 

N.jatamansi extracts. The anti-parkinsonism 

effect of NJE has been correlated with 

enhanced anti-oxidant defenses, restoration of 

dopamine and its metabolites, enhanced striatal 

dopaminergic D2 receptor binding and tyrosine 

hydroxylase expression (2). The antidepressant 

activity may be related to modulation of levels 

of neurotransmitters such as norepinephrine, 

dopamine, serotonin, 5-hydroxyindoleacetic 

acid and gamma-aminobutyric acid (5). Another 

study in a mouse model of depression has 

suggested altered monoamine oxidase (MAO-A 

& MAO-B) activities and GABAergic function 

(4). Neuroprotection afforded by NJE in a 

model of cerebral ischemia has been attributed 

to its anti-oxidant properties (3). While most 

studies have focused on therapeutic utility of 

NJE in disease models of the CNS, there has 

also been an interest in the possible nootropic 

effects of N. jatamansi. Learning and memory 

as assessed by elevated plus maze and passive 

avoidance task in young mice has been reported 

to be improved by administration of NJE. NJE 

was also shown to reverse age related amnesia 

in older mice. The finding of a reversal of 

scopolamine induced amnesia by NJE has been 

suggested as evidence for possible cholinergic 

modulation by NJE (6). The importance of 

cholinergic function for cognitive functions 

in general is well recognized and insights into 

the pathophysiology of Alzheimer’s disease 

has underlined the role of acetylcholine and 

its metabolism in cognition (7). Estimation of 

acetylcholinesterase (AChE) activity in brain 

regions has been used as a marker of cholinergic 

function. AChE is localized to cholinergic 

synapses and is involved in the hydrolysis of 

acetylcholine (ACh) to choline and acetate 

and thereby terminating the neurotransmitter 

14 

action of ACh. A reduction in AChE activity has 

generally been found in conditions of impaired 

cognition. For instance, AChE activity has been 

found to be reduced in the hippocampus and 

cortex of patients suffering from Alzheimer’s 

disease (8, 9). Evidence from animal models 

of learning and memory impairments has also 

pointed to a correlation between AChE activity 

and cognition. AChE activity levels have been 

reported to be decreased in animals subjected to 

21 days of chronic restraint stress – an animal 

model that has been shown to be consistently 

associated with hippocampus dependent 

learning and memory impairments (10, 11). 

Animal models of stress are increasingly being 

used to understand neurobiological bases of 

neuropsychiatric disorders and therapeutic 

strategies. There is significant evidence to link 

stress to the etiopathology of affective disorders 

and cognitive impairment (12). In this context 

a few studies have evaluated the efficacy of 

NJE in preventing or reversing the deleterious 

effects of stress on the central nervous system. 

For instance, pre-treatment with NJE has been 

reported to reduce markers of stress such as 

elevation of plasma corticosterone, gastric 

ulceration and increase in adrenal and spleen 

weights in rats subjected to an acute stress of 

restraint and cold exposure. These anti-stress 

effects were attributed to anti-oxidant actions 

of NJE (13). N. jatamansi has been suggested 

to have a beneficial impact on learning and 

memory and also to be protective against stress. 

N. jatamansi is therefore a promising candidate 

for a nootropic agent. However, the mechanism 

of action of NJE with respect to its nootropic 

effect is not clear. We hypothesized that NJE 

has a modulatory effect on the cholinergic 

system which may underlie its nootropic 

effect. We aimed to investigate the efficacy 

of NJE in modulating AChE levels in both 

normal control rats as well as rats which were 

subjected to chronic restraint stress which has 

been previously reported to alter AChE levels. 

AChE activity levels were measured in the 

hippocampus and frontal cortex – two brain 



areas traditionally recognized to be important for 

declarative learning and memory as well as the 

striatum which served as a control brain region. 

A factorial study design was adopted in which 

groups of animals differed systematically in 

terms of the interventions – treatment with NJE 

and exposure to chronic restraint stress. This 

allowed us to apply robust statistical methods 

to analyze the effects of NJE treatment on both 

normal control rats as well as stressed rats 

Materials and Methods 

Experimental subjects 

The study was conducted at K S Hegde Medical 

Academy, Mangalore. The Institutional animal 

ethics committee approved the experimental 

protocols. All efforts were made to minimize 

both the suffering and the number of animals 

used. Inbred Wistar strain male rats from the 

institutional animal house, aged between 60-80 

days, weighing 180-200gms served as subjects. 

The rats were reared in a 12 hour light-dark 

environment with ad libitum access to food and 

water except during restraint stress procedure. 

A factorial study design was adopted to delineate 

the effects of two experimental interventions 

treatment with NJE chronic restraint stress – 

on AChE activity levels in three brain regions. 

Thirty two animals were randomized to four 

groups (N = 8 for each group). 

Group 1: Normal control group (NC) 

Group 2: N. jatamansi root extract treated group (JM) 

Group 3: Chronic restrai nt stress group (RS) 

Group 4: Chronic restraint stress + N. jatamansi 

root extract treatment group (RS+JM) 

Preparation and administration of N. 

jatamansi extract 

Briefly, botanically identified roots and 

rhizomes of N. jatamansi were purchased from 

a recognized and licensed supplier (Amsar 

Private Ltd-Indore). An ethanolic extract was 

prepared following standardized methods 

15 

described earlier (14). They were cut into 

small pieces, powdered and refluxed with 95% 

ethanol in a Soxhlet extractor for 6-8 hours. The 

extract was evaporated to dryness under reduced 

pressure and temperature using a rotary vacuum 

evaporator and the dried residue was stored at 

4º C. The yield of dry extract from the crude 

powder was about 10%. Based on the previous 

studies a dose of 200mg/kg body weight was 

decided and the extract was administered orally 

for 21 days. 

Chronic restraint stress 

Chronic restraint stress involved placing the 

rats in a wire mesh rodent restrainer for 6 

hours/day for 21 days. This form of stress has 

been reported to elevate cortciosterone levels, 

increase adrenal and spleen weights and cause 

gastric ulcers (15, 10). 

Corticosterone estimation to establish 

effectiveness of stress 

The effectiveness of the chronic restraint stress 

procedure was established in separate cohort 

of six rats of the same mean age and weight as 

the study groups. Blood samples were obtained 

from each animal on Days 0, 3, 10 and 21 (Day 1 

being the start of the stress procedure). For each 

collection, animals were lightly anesthetized 

with ether and rapidly bled from the retroorbital 

sinus into collection tubes containing 

sodium citrate as anticoagulant. Samples were 

centrifuged at 4º C for 15 min at 3000 rpm. 

Serum aliquots were aspirated and stored in 

sealable polypropylene microcentrifuge tubes 

at −70º C until assayed for serum corticosterone 

concentrations using a Enzyme Immuno Assay 

(EIA) kit obtained from Cayman chemicals Inc 

USA(16). 

Estimation of AChE activity 

The frontal cortex (FC), hippocampus (HP) 

and striatum (ST) were rapidly dissected 



out, weighed and homogenized in 0.1M 

Phosphate buffer (pH 8).The homogenates were 

centrifuged at 10,500rpm for 20 minutes at 4°C 

to obtain post mitochondrial supernatant, which 

was stored at -70°C till further use. AChE 

activity was estimated by Ellman’s method 

(17) with modifications as described elsewhere 

(11). The esterase activity was measured by 

providing the substrate acetylthiocholine iodide 

(ATC). Thiocholine released by the cleavage of 

ATC by AChE is allowed to react with the -SH 

reagent 5,5’-dithiobis-(2-nitrobenzoic acid) 

(DTNB), which is reduced to thionitrobenzoic 

acid, a yellow colored anion with an absorption 

maxima at 412nm. Change in the absorbance 

at 412nm was noted every 2 min for 10 min at 

412 nm using a spectrophotometer. The activity 

was calculated and expressed as micromoles 

hydrolyzed per min per gram of tissue. 

Statistical analysis 

AChE activity levels were entered into a threeway 

ANOVA with Stress (Levels: No Stress, 

Restraint Stress), Treatment (Levels: No 

Treatment, NJE Treatment) and Region (Levels: 

Frontal Cortex, Hippocampus, Striatum) as the 

three factors to assess between-subjects main 

effects and the interaction between the three 

factors. If the main effects were significant 

(p


Table 1: AChE activity in the hippocampus(HP), frontal cortex(FC) and striatum(ST) 

HP FC ST 

NC 2.75+0.68 2.21+0.80 4.55+2.58 

JM 3.74+1.41 6.18+1.17 4.17+1.67 

RS 1.99+0.11 3.43+1.79 4.92+3.87 

RS+JM 3.30+1.10 5.27+2.69 3.48+1.03 

Values represent mean+standard deviation of AChE activity. NC: Normal control group, JM: NJE treated group, RS: 

Chronic restraint stress group, RS+JM: Chronic restraint stress + NJE treatment group. N = 8 for each group 

Figure 1: Serum corticosterone levels in the rats subjected to chronic restraint stress for 21 days. Values represent mean 

± standard error of mean (N = 6). Repeated Measures ANOVA: F(3, 12) = 22.547, p


Figure 2: Profile plots showing the effect of NJE on AChE activity levels (µmol/min/g) in the hippocampus (HP), 

frontal cortex (FC) and striatum (ST). 

(A) Three-way ANOVA analysis with the three factors being Stress (2 levels: No stress, Chronic restraint stress), Treatment (2 levels: 

No treatment, NJE treatment) and Brain Region (3 levels: hippocampus, frontal cortex, striatum) as the three factors. Significant main effects of 

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]. 

(B) Bonferroni adjusted pairwise comparisons of AChE activity in HP, FC and ST between Untreated (Normal Control) and NJE treated 

(unstressed) groups. *p Untreated. 

(C) (C) Bonferroni adjusted pairwise comparisons of AChE activity in HP, FC and ST between Chronic restraint stressed (Untreated) and 

NJE treated (chronic restraint stressed) groups. *p Untreated. 

Values represent mean ± standard error of mean. 

Figure 3: Regional differences in mean AChE activity levels in Untreated and NJE treated animals (Both unstressed 

controls and chronic restraint stressed combined for comparison). HP: hippocampus, FC: frontal cortex, ST: striatum. 

*pHP; #pFC; $pHP; ^pST. Bonferroni adjusted comparisons. Values 

represent mean ± standard error of mean. 


18


HP (p < 0.01) and FC (p < 0.05). There were 

no significant differences between FC and HP. 

However, in animals treated with NJE, AChE 

levels in the FC were significantly higher 

compared to both HP (p < 0.01) and ST (p < 

0.05). There were no significant differences 

between FC and ST in this group. In the animals 

which received NJE treatment, AChE level in 

the FC was significantly greater than in those 

which did not receive such treatment (p < 0.001) 

(Figure 3). No such effect of treatment was seen 

in the other brain regions studied. 

Thus treatment with NJE was associated with 

significantly greater AChE in the frontal cortex 

irrespective of whether the animals were 

subjected to chronic restraint stress or not. 

Discussion 

The current study assessed the effects of two 

interventions – 21 day treatment with NJE 

and 21 days of chronic restraint stress – on 

AChE activity in three brain regions, viz. 

hippocampus, frontal cortex and striatum. A 

factorial analysis revealed that NJE treatment 

had a differential effect on AChE activity on 

the three brain regions studied in both normal 

control animals as well as animals which were 

subjected chronic restraint stress. Stress per se 

had no effect on AChE activity levels. 

To the best of our knowledge, this is the first 

report of the modulatory effects of NJE on 

regional brain AChE activity. NJE has been 

shown to modulate other neurotransmitter 

systems in the brain. For instance, 15 days 

of NJE administration has been shown to 

increase norepinephrine, dopamine, serotonin, 

5-hydroxyindoleacetic acid and gammaaminobutyric 

acid in animal the rat brain (5). 

NJE treatment has also been reported to increase 

activity levels of MAO-A and MAO-B thus 

possibly influencing catecholamine levels in the 

mouse brain (4). However, the effect of NJE on 

AChE levels or other indicators of cholinergic 

function has not been studied so far. A previous 

study reported a reversal of scopolamine 

19 

induced amnesia by NJE administration. This 

is suggestive of a modulatory effect of NJE on 

cholinergic neurotransmission. The finding of 

increased AChE activity levels in the present 

study lends further support to the possibility 

that cholinergic modulation is involved in the 

nootropic effects of N. jatamansi. 

The interpretation of the finding of increased 

AChE activity levels in terms of its implications 

on learning and memory is complex. Generally, 

a decrease in AChE activity has been correlated 

with impairment of cognitive functions, 

particularly of hippocampal and neocortical 

dependent learning and memory. This is 

exemplified by the finding of decreased AChE 

activity levels in the hippocampus and cortex 

of post-mortem brain samples from patients 

with Alzheimer’s disease (9). Decreased AChE 

activity in the hippocampus has also been 

described animal models of impaired learning 

and memory. Chronic restraint stress for 21 

days which causes demonstrable deficits in 

hippocampal dependent spatial and reference 

memory errors has been associated with 

decreases AChE activity (10, 11). Conversely, 

interventions which reverse chronic restraint 

stress induced learning-memory deficits have 

been associated with restoration of AChE 

levels in the hippocampus (11). These findings 

may be interpreted as evidence for a positive 

correlation between AChE activity levels in the 

hippocampus / neocortex and learning-memory 

capabilities of the mammalian brain. AChE 

activity levels in the hippocampus has been 

shown to correspond to neuronal activity levels 

in the septo-hippocampal cholinergic pathway 

(18)and lesions interrupting these projections 

is associated with decreased AChE activity in 

their target regions (18, 19). Thus AChE activity 

levels may reflect structural and functional 

integrity of cholinergic pathways. It may 

therefore be speculated that the enhancement 

of AChE activity in the frontal cortex following 

21 days NJE administration may be indicative 

of positive plasticity of cholinergic pathways 

to the neocortex. However, it must be noted 



that an enhanced AChE activity could result 

in accelerated ACh hydrolysis at cholinergic 

synapses thus compromising cholinergic 

modulation of cognitive functions. Thus the 

true implications of the elevated AChE activity 

levels in the frontal cortex resulting from NJE 

treatment needs to be assessed in conjunction 

with its behavioral effects. AChE activity levels 

were highest in the striatum followed by the 

hippocampus and frontal cortex. This is in 

concordance with the findings of Hammond 

& Brimijoin (9) in human post-mortem brain 

samples. Though the AChE activity levels in 

the chronic restraint stress group did not differ 

significantly from the normal control group, 

mean AChE activity level in the hippocampus 

was lower in the stressed group (Table 1). 

This supports previous findings of reduced 

hippocampal AChE in chronic stress (10, 11). 

In the present study, chronic restraint stress for 

21 days did not have significant effect on AChE 

activity levels in the hippocampus, frontal 

cortex or striatum. This is contrary to some 

studies which have reported reduced AChE 

activity in the hippocampus and frontal cortex 

in similar models of chronic restraint stress 

but is in concordance with studies that have 

reported that chronic stress does not affect the 

basal cholinergic function (20). The absence of 

change in AChE activity levels is unlikely to be 

due to an ineffectiveness of the stress protocol 

as significant elevation of serum corticosterone 

during the early period of the chronic restraint 

stress was observed as has been described in 

earlier studies (15). The interesting finding to 

emerge from this study is the elevation in AChE 

activity levels in the frontal cortex associated 

with NJE treatment in both chronically stressed 

rats as well as normal control unstressed rats. 

This observation suggests the possibility that N. 

jatamansi has a nootropic effect that can enhance 

cognitive function even in normal unperturbed 

animals. However it remains to be seen whether 

the elevated AChE activity translates to an 

enhanced behavioral performance. 

Conclusion 

20 

The novel finding that 21 days of administration 

of NJE preferentially elevates AChE activity 

levels in the frontal cortex suggests a possible 

mechanism for a nootropic effect of N. 

jatamansi. The implications of this region 

specific cholinergic modulation for the possible 

development of N. jatamansi based antidementia 

and nootropic interventions remains 

to be clarified. 

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Alterations in the Levels of Biogenic Amines, Amino 



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Chatterjee S, et al.Stress modulating antioxidant effect of 

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22 

Biomedicine; 2011; 31 (1): 22 - 26 

Heart rate reserve – A useful tool for evaluation of chest pain in middle aged 

*C.P. Ganashree, S M Nataraj, R. Rajalakshmi,Vijaya Y Vageesh 

Department of Physiology, JSS Medical College, Mysore - 570015, Karnataka. 

* PGIBMS awardee at 31st Annual conference of IABMS 

(Received 12th November, 2010; Revised 20th December, 2010; Accepted 10th January, 2011) 

Corresponding author 

Ganashree C P 

E-mail: ganaraghu@yahoo.com 

Abstract 

Background: Chronotropic incompetence (CI) is the inability of heart rate response to meet metabolic 

demand. It is an attenuated heart rate response to exercise and is a predictor of all- cause mortality in healthy 

populations. 

Objectives: To study heart rate reserve and CI in middle aged with non specific chest pain to isotonic 

exercise. 

Methods: This is a retrospective cross sectional study carried out in the department of Medicine, JSS 

hospital. Study group comprised of 43 patients(Males-30 and Females-13) in the age group 40-50 years 

with history of nonspecific chest pain but normotensive, nondiabetic and normal resting ECG. Standard 

Bruce protocol was followed. Two groups were made-Group A(6 patients with typical angina) and Group 

B(37 patients with non typical angina). 

Chronotropic incompetence was concluded if derived value is less than 85% of the patient’s heart rate 

reserve. 

Results: Mean Heart rate reserve was significantly lowered in Group A when compared to Group B (60.4750 

vs78.8978, p value

Heart rate reserve in the evaluation of chest pain 

events in the next five years than was ST 

segment depression(1). Many further studies 

have confirmed that chronotropic incompetence 

is an independent predictor of risk in patients 

with coronary artery disease; although 

inferior to myocardial perfusion scanning in 

the prediction of cardiac death, it does add 

incremental prognostic value to this test(2). 

Chronotropic incompetence is also predictive 

of adverse cardiac events and total mortality 

in apparently healthy individuals even after 

adjustment for factors such as age, ST segment 

shift, physical activity, and traditional coronary 

disease risk factors(3). Further confirmation of 

the prognostic value of the heart rate response 

to exercise has been demonstrated in a study of 

about 6000 healthy male Parisian civil servants 

who underwent exercise testing between 1967 

and 1972(4). A low heart rate response to 

exercise proved to be a powerful predictor of 

both sudden death and total mortality though 

not of death from ‘‘non-sudden myocardial 

infarction’’. This finding was particularly 

striking as the lack of heart rate response in 

these individuals was relative; subjects who 

achieved less than 85% of their predicted 

maximum peak heart rate were excluded from 

the study. Chronotropic incompetence (CI) is 

the inability to proportionally increase heart 

rate (HR) to meet the increase in metabolic 

demand (4). It can refer to the inadequacy of 

the sinus node or of the escape pacemaker 

in the case of heart block. It can also refer to 

the inadequacy of AV nodal regulation of 

the supraventricular pacemaker as in atrial 

fibrillation. The clinical manifestations of CI 

are protean and include the inability to achieve 

maximal HR, a delay in achieving maximal 

HR, inadequate submaximal or recovering HR 

or rate instability with exertion (5,6). It is not 

known whether chronotropic incompetence is 

independently predictive of all-cause mortality 

among patients referred for stress testing after 

23 

accounting for myocardial perfusion defects. 

The purpose of this study was to examine the 

association of chronotropic incompetence with 

myocardial perfusion defects and to determine 

the ability of chronotropic incompetence to 

predict all-cause mortality in a low-risk cohort 

of patients referred for exercise treadmill test. 

Materials and methods: 

This is a retrospective cross sectional study 

conducted in Treadmill Unit, Department of 

Medicine, JSS Hospital, JSS Medical College, 

Mysore. Approval from the Ethical committee 

was obtained prior to the commencement of the 

study. 

Subjects: 

Study group included Forty three patients 

comprising 30 males and 13 females belonging 

to the age group of 40-50 years. They came with 

a history of non-specific chest pain. They were 

normotensive, nondiabetic and had normal 

Electrocardiogram (ECG) at rest. Simple 

random selection was made. Patients with 

evidence of recent myocardial infarction, angina 

pectoris of recent onset or with a pronounced 

change in the severity or frequency of angina 

were excluded from the study. Standard Bruce 

Protocol was followed. Subjects were divided 

into two groups-Group A (6 patients with 

typical angina) and Group B (37patients with 

non typical angina). 

Clinical data 

Before exercise testing, a structured interview 

and chart review yielded data on symptoms, 

medications, coronary risk factors, prior cardiac 

events and a number of cardiac and noncardiac 

diagnosis. Resting hypertension was defined 

as a resting systolic blood pressure ≥140 mm 

Hg, a resting diastolic blood pressure of ≥90 



mm Hg or treatment with antihypertensive 

medications(7). Assessment of diabetes was 

based on questioning and medication use. 

Exercise Testing 

Treadmill testing was carried out according to 

standard protocols, usually Bruce or modified 

Bruce(8,9). To facilitate estimation of exercise 

capacity, leaning on handrails during exercise 

was explicitly not allowed. During each 

stage of exercise, data on symptoms, rhythm, 

heart rate, blood pressure (by indirect armcuff 

sphygmomanometer), estimated work 

load in metabolic equivalents (METs) and ST 

segments were collected and entered on-line. 

Estimated functional capacity in METs was 

estimated from standard published tables(9). 

Based on protocol and total time completed in 

the final stage; a MET is a measure of oxygen 

consumption equal to 3.5 ml/kg/min, which 

represents basal, resting metabolic needs. When 

ST segments were interpretable, an ischemic 

response was considered present if there was 

≥1 mm of horizontal or down sloping STsegment 

depression of 80 ms after the J-point 

or if there was ≥1 mm of additional ST-segment 

elevation in leads without pathologic Q waves. 

If a patient had more than one treadmill exercise 

echocardiogram performed during the study 

period, only the first one was considered for 

analysis. Chronotropic incompetence was 

first assessed as failure to achieve 85% of the 

age-predicted heart rate. This method may be 

confounded by effects of age, physical fitness 

and resting heart rate, so chronotropic response 

was also assessed by calculating the ratio of 

heart rate reserve (HRR) used at peak exercise; 

this chronotropic index has been described in 

detail elsewhere(3,10). In an analogous fashion, 

the percent HRR used is: 

%HRR used = (HR peak -HR rest ) / (220-age- 

HR rest ) x 100. 

24 

In a group of healthy, non-hospitalized 

adults, the ratio of percent HRR used during 

exercise was approximate. Thus, chronotropic 

incompetence can be defined as a percent HRR 

used ratio of


Group Statistics 

Table 1: Heart rate reserve (in mean±SD) in patients with angina and without angina 

Angina No.of patients Mean Std. Deviation T-test P value 

Positive 6 60.4750 19.26660 

Negative 37 78.8978 13.87697 

P value


capacity, cardiovascular risk factors, and ST 

segment depression. Results of this study was 

similar to Jouven et al(11) reported that impaired 

ability to increase HR during exercise stress 

testing is a powerful predictor of the risk of 

cardiovascular mortality in 5713 asymptomatic 

subjects. However, the underlying mechanisms 

are not completely understood. 

Chin et al (14) reported that 72% of the patients 

with chronotropic incompetence had significant 

coronary heart disease. Patterson et al(15). 

showed that achieving

27 


Serum α 1 - antitrypsin level and antioxidant status in smokers with Chronic 

Obstructive Pulmonary disease 

*S. Venkata Rao , **B.D. Toora, ***V.S.Ravi Kiran and ****S. Indira 

*Department of Biochemistry, Katuri Medical College, Chinakondrupadu, Guntur - 522 019, 

Andhra Pradesh, India. 

** Aarupadai Veedu Medical College, Pondicherry ,607402, India. 

***Alluri Sitarama Raju Academy of Medical sciences, Eluru-534005, West Godavari Dist., 

Andhra Pradesh, India. 

**** S.D.M.S.College,Vijayawada - 520010, Andhra Pradesh, India. 

(Received 12 th August, 2010; Revised 28 th February, 2011; Accepted 8 th March, 2011) 


Dr.S.Venkata Rao, 

E-mail: s_vrao11@yahoo.co.in 

Abstract 

Background & objectives: Smoking induces slow respiratory problems including chronic obstructive 

pulmonary disease (COPD). Cigarette smoke contains free radicals and decreases alpha1 antitrypsin levels 

in the body. The aim of the present study was to study the relationship between levels of α 1 antitrypsin and 

antioxidant status in smokers with COPD. 

Methods: A 20 controls without smoking and a total of 80 COPD cases with history of smoking were 

studied by spirometry and classified in to four groups according to GOLD. The α 1 antitrypsin, superoxide 

dismutase, vitamin E and vitamin C were measured in all the subjects respectively. 

Results:Controls had a normal spirometry. Cases showed a decrease in FEV1% with corresponding increase 

in the number of cigarettes/day. The α1 antitrypsin, superoxide dismutase, vitamin E and vitamin C were 

decreased proportionately with increase in the number of cigarettes/day in cases when compared to the 

controls respectively. The difference in the means of each parameter between different case groups and 

controls is statistically highly significant. 

Interpretation & conclusion:Smoking inhibits α 1 antitrypsin. Cigarette smoke contains free radicals. The 

soot attracts neutrophils to the site of which releases more free radicals. Thus there is more elastase and less 

protease inhibitor, leading to lung damage. Our study showed decreased α 1 antitrypsin and antioxidant levels 

in smokers due to the increased inhibitory and consumption of antioxidants in the body due to increased 

production of free radicals respectively. 

Introduction 

Cigarette smoking is the major global health 

hazard and it successfully induces several 

respiratory and the major systemic problems. 

Cigarette smoke contains more than 5000 

different chemicals of which many are oxidants, 

including Hydrogen Peroxide, Oxygen free 

radical ,Hydroxyl free radical and nitric 

oxide(1). Cigarette smoke can be separated into 

a gas and a tar phase and both of these contains 

abundant oxidants. Iron is present in cigarette 

smoke and in the epithelial lining fluid, iron 

catalyze the production of free radicals through 


Serum α 1 - antitrypsin in smokers with COPD 

the Fenton’s and Haber- weiss reactions. Air 

pollutants are another exogenous source of 

reactive oxygen species in the lungs (2). Smoking 

induced chronic obstructive pulmonary disease 

(COPD) is the fourth most common cause of 

death in adults (3).The increased oxidative 

burden occurs in lungs of patients with chronic 

obstructive pulmonary disease and this results 

in an imbalance between oxidants and anti 

oxidants,(4) leads to pathogenesis of COPD(5) 

The other reasons are due to excessive smoking, 

occupational hazards, emphysema and air 

pollution (6). Oxidative stress is also thought to 

play an important role in the diseased aspect of 

the lungs with systemic consequences ,such as 

muscle dysfunction and weight loss(7). 

Global Initiative on Obstructive Lung Disease 

(GOLD) guidelines defined smoking induced 

COPD as “a disease state characterized by air 

flow limitation which is to a greater extent 

irreversible. Air flow limitation is progressive 

and with an abnormal inflammatory response 

of the lungs to noxious particles or gases (8). 

In alveolar capillary units the unopposed 

actions of proteases and oxidants results in 

destruction of the alveoli and Emphysema 

appears. Smoking induced COPD comprising a 

chronic bronchitis, emphysema, smaller air way 

disease. Clinical features are similar exertional 

dyspnoea , cough and sputum production in 

usually a prolonged period. Pathogenesis is 

after cigarette smoke, 90% of all patients with 

COPD are smokers, almost 20% of these 

smokers develop the condition (9). Persistent 

reduction in FEV1 (forced expiratory maneuver) 

is the common finding in COPD. Diagnosis is 

made by measurement of pulmonary function. 

(10). Assessment of FEV1/FVC (FEV1, forced 

expiratory volume in one second, FVC, forced 

vital capacity ) is the indicator for early airway 

obstruction. A decline in the pulmonary function 

is a good index every year (11). A reduced 

FEV1/FVC ratio is characteristic. \ 

In general the smoking, finally produce 

COPD within less than 10 pack /10 years. 1 

pack a year is equal to 20 cigarettes/day/1 year. 

28 

The oxidants are counteracted by enzymatic 

antioxidants in airways are superoxide 

dismutase, glutathione peroxidase, catalase, 

glutathione-S-transferase, xanthin oxidase, etc. 

The non-enzymatic antioxidants are Vitamin C, 

Vitamin E (Alfa Tocoferol), urate, lipoic acid 

and bilirubin(3) RTLF (Respiratory-tract lining 

fluid) that covers the respiratory epithelium 

contains Vitamin C, reduced glutathione (GSH), 

urate, Vitamin E and extracellular superoxide 

dismutase (SOD)(9), pulmonary antioxidant 

system is excellently adaptive.. Hence the 

evaluated parameters in this regard are Alfa 1 

Antitrypsin, SOD, Vitamin E and Vitamin C 

in the prognostic study of COPD patients. 

Methods 

A total of 80 COPD male cases with history of 

smoking and 20 age and sex matched healthy 

controls with no smoking, attending our hospital 

were included in the study. Furthermore 80 

COPD cases with history of smoking were 

classified in to four groups according to GOLD 

and number of cigarettes/day. The study was not 

done in the acute phase but in the stable chronic 

phase. All subjects were studied by spirometry 

and classified into controls and four case groups 

according to the Global Initiative for Chronic 

Obstructive Lung Disease (GOLD)(12) as 

follows : 

Controls : n = 20, no smoking, normal 

spirometry, no risk 

Group I : n = 20, 5 – 10 cigarettes/day, FEV1/ 

FVC ‹ 70%, FEV1 greater than or equal to 80% 

predicted, mild COPD. 

Group II : n = 20, 11 – 15 cigarettes/day, FEV1/ 

FVC ‹ 70%, FEV1 greater than or equal to 50% 

to ‹ 80% predicted, moderate COPD. 

Group III : n = 20, 16 – 20 cigarettes/day, FEV1 

greater than or equal to 30% to ‹ 50% predicted, 

moderate COPD. 

Group IV : n = 20, ≥ 20 cigarettes/day, FEV1/ 

FVC ‹ 70%, FEV1 ‹ 30% predicted or FEV1 ‹ 


Serum α 1 - antitrypsin in smokers with COPD 

50%, severe COPD. 

The Serum α1 antitrypsin was estimated by 

quantitative turbidimetric test kit of Spinreact 

Company. The antioxidant status was assessed 

by estimating superoxide dimustase,vitamin E, 

and vitamin C. 

SOD was assayed by the method of Marklund 

and Marklund (13) modified by Nandi et al . 

(14). This method is based on the ability of 

SOD to inhibit autoxidation of pyrogallol under 

specific conditions. Reading was taken at 420 

nm and expressed as units/ml. 

Vitamin E was estimated in serum by the method 

of Baker and Frank (15), which is based on the 

Table 1. 

29 

reduction of ferric to ferrous ions by tocopherol, 

which then forms a red colored complex with 

2,2′,bipyridyl that is read at 520 nm. The level 

of vitamin E was expressed as mg/l serum. 

Plasma VitaminC was estimated by Aye 

Kyaw’s Method(16) where phosphotungstic 

acid first deprotienize the plasma and then react 

with ascorbic acid to produce blue color. 

Statistical analysis 

Statistical analysis of data of alpha1 antitrypsin and antioxidant parameters 

Statistical analysis was done using SalStat 

statistical software. ANOVA test was used 

to compare the means between controls and 

different case groups at 5% level of significance 

Group Control Group-I Group-II Group-III Group-IV p – value 

No. of cigarettes/day 

Nil 5 – 10 11 – 15 16 – 20 ≥20 

n 20 20 20 20 20 

Forced 

expiratory 

volume 

(FEV1%) 

α1 Antitrypsin 

(mg/dl) 

Superoxide 

dismutase (U/ 

ml) 

Vitamin C 

(Ascorbic 

acid) (mg/dl) 

Vitamin E 

(tocopherol) 

(mg/dl) 

80.20000 

± 

3.33404 

160.15000 

± 

22.96972 

3.32400 

± 

0.25270 

0.40100 

± 

0.04471 

1.52250 

± 

0.27376 

(Values are expressed in mean ± SD) 

65.75000 

± 

7.35473 

85.10000 

± 

3.44735 

3.02000 

± 

0.57546 

0.32500 

± 

0.05296 

1.26450 

± 

0.32806 

63.75000 

± 

10.06231 

72.30000 

± 

3.77108 

2.29300 

± 

0.29491 

0.30200 

± 

0.06387 

1.19600 

± 

0.22526 

38.10000 

± 

3.87842 

36.90000 

± 

5.91964 

2.12850 

± 

0.40081 

0.24200 

± 

0.04396 

0.77700 

± 

0.09200 

22.80000 

± 

2.33057 

45.75000 

± 

8.76521 

1.69500 

± 

0.22130 

0.20800 

± 

0.03270 

0.50100 

± 

0.10642 

0.0000001 

0.0000001 

0.0000001 

0.0000001 

0.0000001 


Discussion 

In table-1, the values are expressed in terms of 

mean ± SD and One-way analysis of variance 

(ANOVA) was used to compare the means 

between controls and different cases groups 

respectively at 5% level of significance. The α1 

Antitrypsin was decreased proportionately with 

increasing number cigarettes per day as shown 

in the table-1. The difference in the means 

between controls and different case groups is 

statistically highly significant as p = 0.0000001 

as shown in table-1. The antioxidant parameters 

like superoxide dismutase, vitamin E and 

vitamin C were also decreased proportionately 

with increasing number cigarettes per day 

respectively as shown in the table-1. The 

difference in the means of each parameter 

between controls and different case groups was 

statistically highly significant as p = 0.0000001 

as shown in the table-1. 

Conclusion 

Our study shows, depending on the severity 

of smoking, α1 Antitrypsin and antioxidant 

parameters like superoxide dismutase, vitamin 

E and vitamin C were found to be decreased 

proportionately . The α1 Antitrypsin as it 

is an acute phase protein, it may increase 

in the early phase but later it decreases 

respectively. However, the α1 Antitrypsin 

level was studied during stable chronic phase 

of COPD. Our findings had a correlations 

with other similar studies like Somayajulu 

G.L: et al., (17) and Sayyad et al(18) 

. 

Smoking oxidizes the Methionine (358-residue) 

of alpha -1 antitrypsin and thus inactivates 

the protein. Hence such alpha-1 antitrypsin 

molecule can’t bind to the protease ‘active 

elastase’ and thus proteolysis of lung and tissue 

damage occurs accelerating the development of 

Emphysema. Smoking inhibits α1 antitrypsin. 

Cigarette smoke contains free radicals. The 

soot attracts neutrophils to the site of which 

30 

releases more free radicals. Thus there is more 

elastase and less protease inhibitor, leading 

to lung damage. Our study showed decreased 

antioxidant levels in smokers due to the 

increased consumption of antioxidants in the 

body which in turn due to increased production 

of free radicals respectively. 

Our study reveals that, along with specific 

treatment, the COPD cases should be treated with 

naturally occurring antioxidants like vitamin E, 

vitamin C and beta carotene respectively. This is 

important because, there is increased oxidative 

stress, decreased antioxidants and decreased 

alpha-1 antirypsin activity in smokers with 

COPD cases. 

Smoking cessation is the single most effective 

and cost-efficient intervention for reducing the 

risk of COPD and stopping its progression, 

according to the guidelines. “Even a brief, threeminute 

period of counseling to urge a smoker to 

quit can be effective, and at the very least this 

should be done for every smoker at every visit,” 

they stress. 

References 

1. Pryor W A .,Stone K.Oxidants in cigarette 

smoke;Radicals,Hydrogen peroxide,Peroxy nitrate, and 

Peroxy nitrite,Ann N Y Acad Science1993.:686;12- 

27;(Medicine). 

2. Macnee W,Donaldson K,.Environmental factors in COPD. 

Voelkel N F ,Mac Nee W.eds.Chronic Obstructive Lung 

Disease. London.B C Deekar.,2002;pp.145-160. 

3. Maria Szilasi, Tamas D, Zoltan N, Janos S. Pathology of 

chronic obstructive pulmonary disease. Pathology Oncology 

Research Vol.12, No.1, 2006. 

4. Halliwell B . Antioxidants in human health and disease. 

Annu Rev Nutr 1996;16:33-50. CrossRef 

5. McNee W. Oxidants/antioxidants and COPD. Chest 

2000; 117 5: Suppl. 1, S303–S317. 

6. Rogerio RJ, R Lapa E S,Cellular and biochemical bases of 

chronic obstructive pulmonary disease. Journ.Bras.Pneumol.2006;32(3):241-8. 

7. Langen R C, Korn S H, Wouters E F.ROS in local and 

systemic pathogenesis of COPD, Free Radi Biol Med 

2003:35:226-235.(Cross ref)(Medicine). 

8. Barnes, PJ, Shapiro, SD, Pauwels, RA Chronic obstruc- 


tive pulmonary disease:molecular and cellular mechanisms. 

Eur Respir J 2003;22,672-688 

9. Frank J.Kelley. Vitamins and respiratory disease: anti 

oxidant micronutrients in pulmonary health and disease. 

Proceedings of the Nutrition Society,2005, 64,510-526. 

10.Kasper Braunwald, Fauci, Harrison Principles 

of Internal Medicine,16 th edi,P.No.1548. 

11.John E Repine A B,Ida Lankhorst.Oxidative stress in 

Chronic Obstructive Pulmonary Disease. Am J Resp Crit 

Care Med Vol.156.pp.341-357,1997. 

10. The GOLD Workshop Panel. Global Strategy for the Diagnosis, 

Management, and Prevention of Chronic Obstructive 

Pulmonary Disease: NHLBI/ WHO Workshop Report. 

Bethesda, Md: National Heart, Lung, and Blood Institute; 

2001. NIH Publication No. 2701. 

11. Marklund S, Marklund G (1974): Involvement of the superoxide 

anion radical in the autoxidation of pyrogallol 

and a convenient assay for superoxide dismutase, Eur J 

31 

Biochem, 47: 469-74. 

12. Nishal HK, Sharma MP, Goyal RK, Kaushik GG (1998): 

Serum superoxide dismutase levels in diabetes mellitus 

with or without microangiopathic complications, JAPI, 

46(10): 853-5. 

13. Baker H, Frank O. Tocopherol. In: Clinical vitaminology, 

methods and interpretation, New York Inter Science Publisher, 

John Wiley and Sons, Inc. 1968;172–3 

14. Aye Kyaw. A simple colorimetric method for ascorbic 

acid determination in blood plasma.Clin Chim Acta 1978; 

Vol.86: 153-7. 

15. Somayajulu, G.L; Raja Rao D, Reddy P.P. Serum 

α1antitrypsin in smokers and non smokers. Indian Jour of 

Clinical Biochemistry 1996/11 70-72 

16. A.K.Sayyed, K.H.Deshpande, A.N. Suryakar, R.D, Ankush 

and R.V Katkam. Oxidative Stress and serum 

α1antitrypsin in smokers. Indian Jour of Clinical Biochemistry 

2008/23(4) 375-377. 


32 


Cardiovascular autonomic function test responses in patients with diabetes 

mellitus and non diabetics. 

Mamatha C.N. Ravipati Sarath 

Department of physiology.Hassan Intitute of Medical Sceinces, Hassan, Karnataka, India. 

Department of physiology. School of Medical Sciences, 

University Of Hyderabad, Andhra Pradesh, India. 

(Received 1 st November 2010; Revised 18 th January 2011; Accepted 30 th January 2011) 


Dr.Mamatha 

E-Mail: mamathramesh78@gmail.com 

Abstract 

Introduction: The present study is designed with the objective of investigating the relation between cardiovascular 

autonomic function and diabetes mellitus.Diabetic autonomic neuropathy (DAN) is a serious and 

common complication of diabetes and well studied form of DAN. 

Materials and Methods: A total 60 diabetic patients who belong to age group 40 – 60 years attending the 

diabetic clinic and medical out patient department are selected as test group. 30 non – diabetic patients 

matched for age and sex as that of test group, attending medical OPD for other complaints in the J.S.S. 

Hospital, Mysore, are selected as control group. After thorough examination of the subjects as per the 

proforma, the standard autonomic function tests based on cardiovascular reflexes were performed. ECG 

and heart rate were recorded at baseline. Deep breathing test ,heart rate variation to Valsalva manoeuvre, 

heart rate response to standing ,blood pressure response from supine to standing were performed to assess 

parasympathetic functions of the heart. Blood pressure response to sustained handgrip test is performed to 

assess sympathetic function. 

Results and Discussion: The results obtained were treated statistically by appropriate methods and compared 

between the groups.There was a statistically significant reduction in the test group for all the cardiovascular 

autonomic functions. Both parasympathetic and sympathetic cardiovascular responses were 

significantly decreased in diabetes compared to age-matched healthy controls. 

Conclussion: Study has been conluded with the association of Cardiovascular autonomic neuropathy in 

patients with diabetes as the disease progresses affecting both sympathetic and parasympathetic components. 

keywords: Diabetes mellitus; cardiovascular autonomic neuropathy(CAN); cardiovascular reflex tests. 

Introduction: 

Diabetes mellitus (DM) is a metabolic disorder 

characterized by hyperglycemia due to absolute 

or relative deficiency of insulin. The increased 

morbidity and mortality is due to its complications 

like neuropathy, nephropathy, and retinop- 

athy etc, especially seen in 40 to 60 years age 

group. Diabetic autonomic neuropathy (DAN) 

is a serious and common complication of diabetes. 

DAN may affect many system throughout 

the body Eg: CVS, GIT, Genitourinary system. 

Because of its association with a variety of adverse 

outcomes including cardiovascular deaths 


Cardiovascular autonomic function test responses in patients in diabetes mellitus 

due to silent myocardial ischemia, cardiovascular 

autonomic neuropathy (CAN) is the most 

critically important and well studied form of 

DAN. One of the studies showed CAN results 

from damage to the autonomic nerve fiber that 

innervate the heart and blood vessels and results 

in abnormalities that is heart controle and vascular 

dynamics (1). 

Previous studies showed that long term poor 

glycemic controle can only increase the risk of 

developing advanced diabetic neuropathy(2). 

But present studies have confirmed the presence 

of autonomic neuropathy at presentation 

itself (3) . Some manifestations of autonomic 

neuropathy may even precede the diagnosis of 

diabetes by several years (4).So these evidences 

supports that all patients with diabetes regardless 

of metabolic controle are at risk for autonomic 

neuropathy. 

In that CAN,a common form of autonomic 

dysfunction found in patients with diabetes 

mellitus. Cardiovascular disease is the leading 

cause of death among patients with diabetes. 

The presence of cardiovascular autonomic neuropathy 

has been associated with substantially 

increased risk of cardiovascular mortality in diabetes. 

Quantitative cardiovascular autonomic 

function tests are widely used to detect, verify 

and quantify the cardiovascular autonomic dysfunction. 

The determination of the presence of CAN is 

usually based on a battery of autonomic function 

tests rather than just on one test. A number 

of autonomic function tests considered reliable, 

reproducible, simple and quick to carry out and 

all of them are non – invasive. Despite its relationship 

to an increased risk of cardiovascular 

mortality and its association with multiple 

symptoms and impairments, the significance 

of DAN has not been fully appreciated. So the 

present study is undertaken to assess the severity 

of adverse effects of diabetes on autonomic 

functions of CVS which helps in early detection 

of CAN in asymptomatic diabetic and there by 

promotes timely diagnostic and therapeutic intervention. 

Materials and Methods: A total of 

33 

60 diabetic patients who belong to age group 

40 – 60 years attending the diabetic clinic and 

medical out patient department in the J.S.S 

Hospital, Mysore, are selected as test group. 30 

non – diabetic patients matched for age and sex 

as that of test group, attending medical OPD for 

other complaints in the J.S.S. Hospital, Mysore, 

are selected as control group. 

Inclusion criteria for study group Cases of already 

diagnosed diabetes mellitus since 5 

years who are full filling WHO criteria’s are selected 

as test group. 

Cases who are in the age group of 40 – 60 years 

are selected. Exclusion criteria for study group 

Non-diabetic patients are excluded from the 

study 

Patients taking medications other than oral hypoglycemics 

that could influence the autonomic 

functions and drugs those could affect the cardiovascular 

functions are excluded. Disease 

states like alcoholic neuropathy that could affect 

autonomic functions are excluded. Patients 

suffering from cardiac disorders are excluded. 

Inclusion criteria for control groups Subject 

must be non-diabetic Subjects matched for age, 

sex as that of test group are included as controls. 

Subjects attending medicine out patient department 

for various complaints at J.S.S. Hospital 

are included.Exclusion criteria for control 

group Already diagnosed diabetic patients are 

excluded Subjects taking medication that could 

affect cardiovascular functions and those who 

are suffering from cardiac disorders are excluded 

as controle group. 

Methods 

The subjects were instructed not to have coffee, 

tea, cola 12 hours before the tests and were 

asked to have light breakfast two hours before 

the tests. The subject was asked to relax in supine 

position for 30 minutes. The resting heart 

rate was recorded on a standard ECG from lead 

II, at a paper speed of 25 mm/sec. BP was measured 

with sphygmomanometer by the standard 

auscultatory RivaRocci method. The cardiovas- 



cular tests performed are detailed below in the 

order of execution. These tests were demonstrated 

to the subjects. 

Materials: 

Autonomic function tests will be carried out by 

using Electro cardiograph Sphygmomanometer 

Hand grip dynamometer Procedure of autonomic 

evaluation. In the early 1970’s five simple non 

invasive cardiovascular reflex tests were proposed 

that is valsalva manoeuver, HR response 

to deep breathing, HR response to standing up 

, BP response to standing up are performed to 

assess the parasympathetic and BP response to 

sustained handgrip to assess the sympathetic 

function have been widely used in a variety of 

studies(5). 

1.Deep breathing test: 

In the sitting position subject was asked to 

breath quietly and deeply at the rate of 6 breaths 

per minute. A continuous ECG was recorded for 

six cycles with marker to indicate the onset of 

each inspiration and expiration. The maximum 

and minimum R-R intervals were measured 

during each breathing cycles and converted to 

beats per minute. The result was then expressed 

as mean of the difference between maximum 

and minimum heart rate for six measured cycles 

in beats per minute(6).Deep breathing 

difference(DBD)=mean of heart rate differences 

in 6 breath cycles.A normal response is a 

difference of 15 beats/min or more,11-14 beats/ 

min borderline and less than 10 beats/min was 

considered abnormal. 

2.Heart-Rate variation to Valsalva Manoeuver: 

The subject was seated comfortably and was 

asked to blow into a mouthpiece connected to a 

mercury sphygmomanometer and holding it at 

a pressure of 40 mm of mercury for 15 seconds, 

while a continuous ECG was being recorded. 

34 

The ECG was continued to be recorded after release 

of pressure at the end of 15 seconds for 30 

seconds. The heart rate changes induced by the 

valsalva manoeuver was expressed as the ratio 

of the maximal tachycardia during the manoeuver 

to the maximal bradycardia after the manoeuver. 

This ratio was defined as the Valsalva 

ratio and was calculated as the ratio of maximum 

R-R interval after the manoeuver to minimum 

R-R interval during the manoeuvre(7). 

Valsalva ratio(VR)= maximal tachycardia/maximum 

bradycardia= maximum R-R 

interval/minimum R-R interval. 

A value of 1.10 or less is defined as an 

abnormal response, 1.11-1.20 as borderline, and 

1.21 or more as a normal response(8). 

3. Heart rate response to standing - Postural 

Tachycardia Index (PTI) : 

The subjects were asked to lie on the examination 

table quietly while heart rate is being recorded 

on ECG. They were then asked to standup 

unaided and ECG was recorded for 1 minute. 

The shortest R-R interval at or around 15th beat 

and longest R-R interval at or around 30th beat 

was measured. The result was expressed as ratio 

of 30/15. 

PTI= Longest R-R interval at 30th beat / shortest 

R-R at 15th beat. A ratio of 1.00 or less was 

defined as an abnormal response, 1.01-1.03 as 

borderline and 1.04 as normal response(9). 

4. Blood Pressure Response To Standing 

(Orthostatic test): 

The subject was asked to rest in a supine position 

for 5 minutes. The resting BP was recorded. 

The subject was then asked to stand unaided 

and remain standing unsupported for 3 minutes. 

The BP was recorded at 30 seconds and 3 minutes 

after standing up. The difference between 

the resting and standing BP levels was calculated. 

The fall in systolic BP at 30 seconds on 

standing noted. A fall of 30 mm Hg or more was 

defined as abnormal, fall between 11-29 mm Hg 



Table: Cardiovascular autonomic function tests: 

Results 

Heart Rate response tests 

(Parasympathetic function) 

Heart rate response to Valsalva 

Manoeuver (VR) 

Heart rate response during deep 

breathing (DBD) 

Table 1 : Comparison of DBD in test and control group and results of T-test : values mentioned are mean and 

SD in each group. 

Group Mean ± SD T P 

Test 12.36 ± 4.79 

Control 14.96 ± 5.51 

-2.306 0.023 

Heart rate response to deep breathing were analysed between the two groups. DBD was found to be significantly lesser in the test group when 

compared to control group (P < 0.023). 

Table 2 : Comparison of VR in test and control group and results of T-test : values 

mentioned are mean and SD in each group. 

Group Mean ± SD t P 

Test 1.16 ± 0.77 

Control 1.25 ± 0.98 

-4.365 0.00 

Heart rate response to valsalva manoeuver were analysed between the two groups. VR was found to be significantly lesser in the test group when 

compared to control group (P < 0.00). 

Table 3 : Comparison of PTI in test and control group and results of T-test : 

values mentioned are mean and SD in each group. 

Group Mean ± SD t P 

Test 1.02 ± 0.12 

Control 1.08 ± 0.87 

-2.57 0.01 

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 

control group (P< 0.01). 

Normal Borderline Abnormal 

1.21 or more 1.11-1.20 1.10 or less 

15 beats/min 

Or more 

11-14 

beats/min 

10 beats/min 

or less. 

Heart rat response t standing (PTI) 1.04 or more 1.01-1.03 1.00 or less 

Blood Pressure (BP) Reponse 

tests (sympathetic function) 

BP response to standing 

(fall in systolic BP) 

BP response to sustained hand 

grip (rise in diastolic BP) 

10 mm Hg 

Or less 

16 mm Hg 

Or more 

11-29 mm Hg 

11 - 15 mm Hg 

30 mm Hg 

Or more 

10 mm Hg 

Or less 


35


Table 4 : Comparison of fall in SBP (Orthostatic test) in test and control group and results of T-test : values 

mentioned are mean and SD in each group. 


Test 9.20 ± 4.02 

Control 17.73 ± 6.41 

as borderline and fall of 10 mm Hg or less was 

considered norma(10). 

5.Blood Pressure Response To Sustained 

Handgrip: 

In this test, sustained muscle contraction is 

measured by a handgrip dynamometer, causes a 

rise in systolic and diastolic blood pressure and 

heart rate. The dynamometer is first squeezed 

to isometric maximum , then held at 30% maximum 

for 5 min. if possible, although even 3 

minutes may be adequate. Blood pressure was 

recorded in the non exercising arm thrice at 

1-minute interval during the procedure. The 

maximum reading of the diastolic blood pressure 

was taken as the final value. Then the 

rise in diastolic blood pressure was calculated 

by subtracting resting diastolic blood pressure 

from this value. A rise in DBP of less than 10 

mm Hg was defined as abnormal, 11-15 mm Hg 

as borderline and 16 mm Hg or as normal(11). 

Statistical Analysis of Data: 

Following statistical methods were employed 

in the present study: 

1. Contingency coefficient test: The Crosstabs 

procedure forms two-way and multiway 

tables and provides a variety of tests and 

36 

measures of association for two-way tables. 

The structure of the table and whether categories 

are ordered determine what test or 

measure to use. 

2. Univariate ANOVA:The Univariate procedure 

provides analysis of variance for one 

dependent variable by one or more factors 

and/or variables. You can investigate 

interactions between factors as well as the 

effects of individual factors, some of which 

may be random.Independent samples ‘t’ 

test:The Independent-Samples T Test procedure 

compares means for two groups of 

cases. 

Discussion: 

6.65 0.00 

Blood pressure response to standing were evaluated between the two groups. There was a significant decline in orthostatic test in test group (P < 

0.00). 

Table 5 : Comparison of rise in DBP in test and control group and results of T-test: values mentioned are 

mean and SD in each group. 


Test 12.76 ± 4.80 

Control 18.20 ± 3.41 

-5.52 0.00 

Blood pressure (rise in DBP) response to sustained hand grip was evaluated between test group and control group. Blood pressure response to 

sustained hand grip was found to be significantly reduced in test group (P < 0.00). 

Diabetes mellitus is a strong risk factor for 

cardiovascular disease. DAN is a risk factor 

that independently increases cardiovascular 

risk in people with diabetes mellitus. Various 

mechanisms are included in the impairment 

of autonomic function which leads to exercise 

intolerance, orthostatic hypotension, painless 

myocardial ischemia and increased risk of 

mortality. Alternatively, the metabolic and 

vascular changes associated with diabetes eg: 

dysfunction of the vascular endothelium may 

adversely affect cardiovascular autonomic 

function. 



Cardiovascular autonomic function tests are 

widely used to detect, verify and quantify the 

cardiovascular autonomic dysfunction. They 

have been tested for their validity and reliability. 

Against this background, the present study 

was undertaken to evaluate cardiovascular 

autonomic function tests in diabetes and match 

the data so obtained with healthy non diabetic 

individuals as controls. 

Heart rate response to deep breathing:- 

This evaluated in 90 subjects, 60 of them 

diabetics comprising the test group and 30 were 

non diabetic patients as control group. The 

mean and standard deviation of test group and 

control group are shown in Table 1. The mean 

and standard deviation of test group and control 

group were 12.36 ± 4.79 and 14.96 ± 5.51 

respectively. 

DBD was significantly reduced in test group 

when compared to control group (P


Blood Pressure response to sustained 

handgrip 

. The mean and standard deviation of test 

group and control group are shown in Table 

5. The mean and standard deviation of test 

group and control group were 12.76 ± 4.80 

and 18.20 ± 3.41 respectively There was a 

significant decline in sustained handgrip test 

in test group when compared to control. (P < 

0.00). In a study it was found that in patients 

with type 2 diabetes showed a decline in 

isometric exercise induced increase in heart 

rate and blood pressure response in subjects 

with CAN(14). The main finding of this study 

is that, decreased Heart rate variability and 

blunted blood pressure responses, as measured 

by cardiovascular testing, is associated with 

diabetic patients. Hence, it is suggested that 

cardiovascular autonomic functions decline in 

diabetic patients as the disease progresses. 

Conclusion: 

Diabetic autonomic neuropathy is a 

heterogenous condition that comprises a wide 

range of dysfunction and whose development 

might be attributed to diabetes per se or to factors 

associated with the disease. Both sympathetic 

and parasympathetic fibres may be affected 

with parasympathetic dysfunction preceding 

sympathetic dysfunction.The etio pathology 

of Diabetic Autonomic Neuropathy is multifactorial.It 

has been postulated that metabolic 

consequences of hyperglycemia rather than 

the type of diabetes may lead to autonomic 

damage. One factor of particular interest is 

oxidative stress. This occurs when there is 

an increase within cells of certain reactive 

molecules containing oxygen. These can react 

with components of cells, including nerves, 

and cause damage. Alternatively, the metabolic 

and vascular changes associated with diabetes 

38 

may adversely affect almost all the organs of the 

body, particularly the cardiovascular system. 

References: 

1. Schumer MP, Joyner SA, Pfeifer MA. Cardiovascular au- autonomic 

neuropathy testing in patients with diabetes. Diabetes 

spectrum 1998; 11: 227-231. 

2. mustonen J, Vusitipa M, Mantysaari M. Changes on autonomic 

nervous function during the 4-year follow-up in 

middle-aged diabetic and non-diabetic subjects initially 

free of coronary heart disease. J. Intern. Med – 241 1997; 

227-235. 

3. Gries FA, Spuler M, Lessmann F. Diabetic cardiovascular 

autonomic neuropathy multicenter study group, The epidemiology 

of diabetic neuropathy. J. Diabetes complications 

1992; 6: 49-57. 

4. Wcin TH, Albers JW. Diabetic neuropathies. Phys Med 

Rehabil Chin N Am. 2001; 12: 307-320. 

5. Ewing DJ, Martyn CN, Young RJ, Clarje BF. The value of 

cardiovascular autonomic function test, 10 years experience 

in diabetes. Diabetes case 1985; 491-498. 

6. Piha SJ. Cardiovascular autonomic function tests. Responses 

in healthy subjects and determination of age related 

reference value. Rehab research centre 1988;1-148. 

7. Hirsch JA, Bishop B. Respiratory sinus arrhythmia in humans: 

how breathing pattern modulates heart rate. Am J 

Physiol 1981;241:H620-H629. 

8. Levin AB. A Simple test of cardiac function based upon 

the heart changes induced by valsalva manoeuver. Am J 

Cardiol 1966;18:90-99. 

9. Ewing DJ, Hume L, Campbell IW, et al. Autonomic mechanism 

in initial heart rate response to standing. J Appl 

Physiol 1980;49:809-814. 

10. Piha SJ. Cardiovascular responses to various autonomic 

tests in males and females. Clin Auton Res 1993;3(1):15- 

20. 

11. Piha SJ. Cardiovascular autonomic reflex tests: Normal 

responses and age related reference values. Clin Physiol 

1991;11:277-290. 

12. Moran A, Palmas W, Field L. et al. Cardiovascular autonomic 

neuropathy is associated with microalbuminuria in 

older patients with type2 diabetes. The American Diabetes 

Association. Diabetes Care 2004;27:972-977. 

13. Purewal TS, Watkins PJ. Postural hypotension in diabetic 

autonomic neuropathy: a review. Diabet Med 

1995;12:192–200.. 

14. Kahn J, Zola B, Juni J, Vinik AI. Decreased exercise heart 

rate in diabetic subjects with cardiac autonomic neuropathy. 

Diabetes care 1986; 9: 389-394. 


39 


Association between Serum Lipoprotein(a) Concentrations and Serum 

Triglycerides in Type 2 Diabetes Mellitus. 

*T. Sharmila krishna, *J.N.Naidu, *M. Audhisesha Reddy, *K.Ramalingam, 

**E. Venkat Rao. 

*Department of Biochemistry and ** Department of Community Medicine , Narayana Medical 

College , Nellore, Andhra Pradesh, India. 

(Received 5 th October 2010; Revised 7 th December 2010; Accepted 10 th January 2011) 


Dr. T.Sharmila Krishna 

Email: sharmilakrishna01@yahoo.in 

Abstract 

Introduction: Diabetes mellitus is a chronic metabolic disorder that is often associated with dyslipidemia, 

leading to cardiovascular complications. Studies have shown Lipoprotein(a) [Lp (a)] as a new independent 

risk factor for coronary artery disease . Concentrations of Lp(a) among diabetics remains controversial. 

Objectives: This study was aimed to evaluate the Lp(a) levels and its relation with other serum lipids 

among type 2 diabetics, first degree relatives (FDRs) with a family history of type 2 diabetes mellitus and 

controls. 

Materials And Methods: A total of 55 subjects, both males and females were studied (type 2 diabetics n 

= 21, FDRs n=19 and controls n = 15). Type 2 diabetics were further sub-grouped into diabetics with < 5 

years and those with ≥ 5 years duration. FBS, PPBS, total cholesterol, triglycerides, LDL-c , HDL-c were 

estimated by Spectrophotometry. Lipoprotein(a) and HbA1c were estimated by Immnunoturbidimetry. 

Results: The mean Lp(a) levels were lower among type 2 diabetics and FDRs compared to controls, 

though it was statistically not significant. A significant negative correlation (p value < 0.007) was observed 

between Lp(a) and serum triglycerides concentrations among diabetics of ≥ 5 years duration. 

Conclusion: Lp(a) concentrations were negatively correlated with triglyceride levels in type 2 diabetics. 

Therefore, our results suggest that the treatment of diabetic dyslipidemia may indirectly affect Lp(a) concentrations. 

So, care must be taken while treating dyslipidemia in type 2 diabetics. 

Key words : Lipoprotein (a) , Triglycerides , Type 2 diabetes mellitus. 

Introduction: 

Lipoprotein (a) was first identified by Karl Berg 

in 1963 (1). It consists of an LDL-like particle 

and the unique glycoprotein, apolipoprotein (a) 

[apo (a)] that is covalently linked to apo-B 100 

via a disulfide linkage (2). Elevated levels of Lp 

(a) are found to be an independent risk factor for 

Coronary artery disease in NIDDM (3,4,5). The 

apolipoprotein (a) of Lp(a) shares high sequence 

homology (75 – 90% ) with plasminogen, 

suggesting Lp(a) inhibits fibrinolysis and 


Serum LP ‘a’ and TG in Diabetes Mellitus 

contribute to the thrombotic phenomenon. 

Further, LDL-like structure contributes to 

atherogenicity leading to Ischemic heart disease 

(6, 7). Studies regarding levels of Lp(a) in type 

2 diabetics still remains controversial, some 

reporting higher levels ( 8,9,10,11 ), some lower 

(12, 13), while few have shown no significant 

change (14, 15 ,16). Variations in Lp (a) levels 

are mainly under genetic control (17). Various 

other factors like ethnicity, diet, drugs and 

hormones also affect serum Lp (a) levels (18,19, 

20 , 21). There are very few studies showing the 

influence of various serum lipids on Lp (a) levels 

in diabetics. Although an inverse correlation 

between serum Lp (a) and serum triglycerides 

has been reported in non-diabetic subjects, there 

is scanty literature on this relationship among 

the diabetic population. 


This study was conducted among the out patients 

attending the General Medicine department 

at Narayana General Hospital, Nellore over a 

period of 6 months. 

A total of 55 subjects were enrolled for the study 

(type 2 diabetics 

n= 21, non-diabetic first degree relatives 

(FDRs) n=19 and healthy controls n=15). 

Type 2 diabetic subjects were further subdivided 

based on duration of diabetes into those with < 

5 yrs duration and those with ≥5 yrs duration. 

All of them were in the age group of 20-70 

years and both sexes were included. Informed 

oral consent was taken from all subjects. 

Inclusion criteria: 

Subjects should have been diabetic for at least 

one year duration. 

To avoid possible transient increase of Lp (a) 

after starting insulin treatment, we excluded all 

patients in whom insulin was initiated during 

40 

the first two months preceding the study. 

Blood samples were collected after 12 hours 

of overnight fasting. 5 ml of venous blood 

was collected and part of the blood was 

transferred into EDTA anticoagulant containing 

tube and sodium fluoride containing tube for 

estimation of HbA1c and glucose respectively. 

Remaining blood was allowed to clot and the 

serum separated was used to analyze lipids 

immediately or stored at – 20°C and analyzed as 

batch for Lp(a). Glucose and lipid profile were 

estimated by Automated chemistry analyzer 

[ Humastar 300 (Human Gm BH , Germany)] 

using available commercial kits. VLDL was 

calculated as triglycerides divided by 5. 

Lipoprotein (a) (TULIP QUANTIA KIT) and 

HbA1c were estimated by Immunoturbidimetry. 

The reference value for Lp(a) is less than 30 mg/ 

dl. For adequate quality control, both normal 

and abnormal reference control serum solutions 

and calibrators were run before each testing. 

Statistical analysis & Results: 

Data were analyzed using statistical software 

SPSS 12.0 version. Values were expressed 

as mean ± standard error of mean (SEM) for 

continuous variables. Correlations between the 

different lipids and serum Lp (a) were done 

using Spearman σ correlation. A non-parametric 

test, Mann –Whitney test was used to compare 

the means between the groups of subjects owing 

to skewing of the data. A p value of < 0.05 

was considered statistically significant. The 

average age and standard deviation of type 2 

diabetics, non-diabetic FDRs and controls were 

52.7 yrs ± 9.15 , 32.4 yrs ± 9.91 and 31.2 yrs ± 

7.35 respectively. Table 1 shows the values of 

the biochemical parameters in type 2 diabetic 

subjects and controls. Table 2 shows the values 

of the biochemical parameters in type 2 diabetic 

subjects and FDRs. Table 3 shows the values 

of the biochemical parameters in FDRs and 



Table 1: Comparison of various biochemical parameters in subjects with type 2 diabetes mellitus and the 

controls. 

Whole group of Diabetics (n=21) Controls (n = 15) 

p Value 

FBS (mg/dl) 144.6 ± 8.88 81.2 ± 2.84 0.000* 

PPBS(mg/dl) 210.0 ± 14.88 96.2 ± 4.05 0.000* 

HbA 1 C (%) 6.92 ± 0.30 5.06 ± 0.12 0.000* 

T.Cholesterol (mg/dl) 175.7 ± 11.64 146.8 ± 7.79 0.140 

S.Triglycerides (mg/dl) 189.5 ± 26.39 120.7 ± 12.86 0.052 

S.VLDL (mg/dl) 51.71 ± 14.49 24.1 ± 2.57 0.038* 

S.LDL (mg/dl) 96.33 ± 7.42 87.19 ± 5.09 0.511 

S.HDL (mg/dl) 41 ± 2.94 35.5 ± 2.78 0.282 

Lp(a) (mg/dl) 19.19 ± 3.08 29.6 ± 4.81 0.105 

Values are expressed as Mean ± SEM. n=number of subjects , *p < 0 .05 Significant 

Table 2: Comparison of various biochemical parameters in subjects with type 2 diabetes mellitus and the FDRs. 

Whole group of Diabetics 

(n=21) 

FDRs (n = 19) 

p Value 

FBS (mg/dl) 144.6 ± 8.88 86.4 ± 2.0 0.000* 

PPBS (mg/dl) 210.0 ± 14.88 101.7 ± 2.96 0.000* 

HbA 1 C (%) 6.92 ± 0.30 5.25 ± 0.127 0.000* 

T.Cholesterol (mg/dl) 175.7 ± 11.64 181 ± 11.3 0.655 

S.Triglycerides (mg/dl) 189.5 ± 26.39 165 ± 18.5 0.616 

S.VLDL (mg/dl) 51.71 ± 14.49 33.1 ± 3.7 0.473 

S.LDL (mg/dl) 96.33 ± 7.42 103.4 ± 6.8 0.297 

S.HDL (mg/dl) 41 ± 2.94 44.8 ± 2.9 0.238 

Lp(a) (mg/dl) 19.19 ± 3.08 20.7 ± 4.5 0.818 

Values are expressed as Mean ± SEM. n=number of subjects , *p < 0 .05 Significant. 


41


Table 3: Comparison of various biochemical parameters in FDRs and controls. 

FDRs(n=19) Controls(n=15) p Value 

FBS (mg/dl) 86.4 ± 2.0 81.2 ± 2.84 0.138 

PPBS (mg/dl) 101.7 ± 2.96 96.2 ± 4.05 0. 263 

HbA 1 C (%) 5.25 ± 0.127 5.06 ± 0.12 0. 314 

T.Cholesterol (mg/dl) 181 ± 11.3 146.8 ± 7.79 0. 24 

S.Triglycerides (mg/dl) 165 ± 18.5 120.7 ± 12.86 0.071 

S.VLDL (mg/dl) 33.1 ± 3.7 24.1 ± 2.57 0.07 

S.LDL (mg/dl) 103.4 ± 6.8 87.19 ± 5.09 0.077 

S.HDL (mg/dl) 44.8 ± 2.9 35.5 ± 2.78 0.031 

Lp(a) (mg/dl) 20.7 ± 4.5 29.6 ± 4.81 0.191 

Values are expressed as Mean ± SEM. n=number of subjects, *p < 0 .05 Significant. 

Figure 1: Scattered diagram showing Correlation between Serum Lp(a) &Serum Triglycerides in diabetics of ≥ 5 

years duration. 


42


controls. The mean (± SEM) Lp (a) levels were 

lower in type 2 diabetics (19.1± 3.8), compared 

to FDRs (20.7± 4.5) and controls (29.6 ± 

4.8) and also they were lower in FDRs when 

compared to controls, though statistically not 

significant. 

Figure 1 shows a significant negative correlation 

(p value 0.007) between serum Lp(a) and serum 

triglycerides among type 2 diabetic subjects of 

≥ 5 yrs duration . No significant correlation was 

observed between serum Lp (a) and other lipids 

among type 2 diabetics of < 5 yrs duration, nondiabetic 

FDRs and controls. 

Discussion: 

To the best of our knowledge this is the first 

study in India done on the relationship between 

serum Lipoprotein (a) and other serum lipids 

in type 2 diabetes mellitus. Our study revealed 

that Lp (a) concentrations were lower in type 2 

diabetics compared to non-diabetic FDRs and 

controls. Calmarza et al and Ali AlBahrani et 

al (22, 23 ) noticed lower Lp (a) levels among 

type 2 diabetics compared to controls, similar 

to our findings. We also observed that FDRs 

had lower Lp (a) concentrations compared to 

controls. But this is in contrast to the study 

done by Agathoklis Psyrogiannis et al (24) who 

noticed that normoglycemic offspring of type 2 

diabetes mellitus had higher Lp (a) levels than 

those without family history of type 2 diabetes 

mellitus. The present study revealed a significant 

negative correlation ( p value 0.007) between 

serum Lp (a) and serum triglycerides among 

type 2 diabetics of ≥ 5 years duration. Similar 

finding was noted by Hernandez et al (25 ) using 

a different method for Lp (a) measurement. Low 

levels of Lp (a) have been detected in patients 

with lipoprotein lipase deficiency by Sandholzer 

et al (26) and it is tempting to speculate that 

diabetic subjects have some degree of functional 

lipoprotein lipase deficiency that is implicated in 

43 

the pathogenesis of hypertriglyceridemia. The 

inverse relationship between Lp (a) and serum 

triglycerides is due to the fact that apo (a) is also 

present in Triglyceride rich particles (TRPs). 

Apo (a) containing - TRPs, in parallel with 

chylomicron remnants , would be rapidly taken 

up by the liver through the remnant receptor 

pathway, thus the lower levels of lipoprotein (a) 

in patients with elevated triglycerides could be 

the result of rapid catabolism of TRP apo (a) 

compared with the slower apo (a) catabolism 

in the LDL density range (23, 25). Supportive 

evidence to our findings was reported by Ko et 

al in their study that improving insulin resistance 

by rosiglitazone (insulin sensitizing agent ) was 

associated with lowering of triglycerides and 

increase in lipoprotein (a) concentrations (27) . 

In conclusion, we observed a significant inverse 

correlation between serum Lp (a) and serum 

triglycerides in type 2 diabetics of ≥ 5 years 

duration. This study highlights that the serum 

Lp (a) concentrations depend upon other lipid 

parameters. Therefore, Lipoprotein (a) should 

be estimated and its interpretation should be 

cautiously exercised and care must be taken 

while treating dyslipidemia in diabetics. 

More prospective studies should be conducted 

in order to substantiate the findings. 

Limitations of our study : 

Firstly, the present study was performed on a 

population confined to a particular area; the 

results do not necessarily apply to other racial 

groups. Secondly, the small sample size and 

further sub grouping of type 2 diabetic subjects 

combined with sample storage and method 

of estimation might have affected the results. 

Lastly, due to lack of funding from external 

agencies larger studies could not be performed. 



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Ameliorative effect of Coccinia grandis in streptozotocin induced diabetic rats 

Bhuvaneswari Palanisamy, Krishnakumari Shanmugasundaram and Rajeswari 

Paramasivam 

Department of Biochemistry, Kongunadu Arts and Science College, Coimbatore – 641 029, Tamilnadu, INDIA. 

(Received 30 th September 2010; Revised 25 th December 2010; Accepted 10 th January 2011) 


Dr. S. Krishnakumari, 

E-mail: krishnashanmugambc@gmail.com 

Abstract 

Background & Objectives: Diabetes, a life long progressive disease has become one of the most 

challenging health problems of the 21 st century. India faces a grave health care burden due to the high 

prevalence of type-II diabetes and its sequalae. In the ongoing search for more effective and safer plant 

drugs, we have evaluated the hypoglycemic effects of methanolic extract of Coccinia grandis by analyzing 

the key enzymes of glycolysis, gluconeogenesis and Citric acid cycle in liver. 

Methods: Four groups of six wistar albino rats of 150-200 g each were grouped into control, diabetic, 

diabetic treated and plant treated. After treatment for 20 days the animals were sacrificed and the 

biochemical parameters like blood glucose, serum insulin, hemoglobin, glycosylated hemoglobin, liver 

glycogen, hexokinase, glucose-6-phosphatase, fructose-1, 6-diphosphatase, succinate dehydrogenase and 

malate dehydrogenase levels were measured. 

Results: Blood glucose and glycosylated hemoglobin levels were siginificantly increased whereas the 

serum insulin and hemoglobin levels were siginificantly decreased in diabetic rats and reverted to near 

normal in treated groups. Glucose-6-phosphatase and fructose-1, 6-diphosphatase levels were siginificantly 

increased whereas the liver glycogen, hexokinase, succinate dehydrogenase and malate dehydrogenase 

levels were siginificantly decreased in diabetic rats and reverted to near normal in treated groups. 

Interpretation & Conclusion: From the above results it is evident that the methanolic extract of Coccinia 

grandis have a potential effect to control hyperglycemia in streptozotocin-nicotinamide induced diabetic 

rats. 

Key words: enzymes, gluconeogenesis, citric acid cycle, streptozotocin, Coccinia grandis 

Introduction 

Diabetes mellitus in all its heterogeneity has 

taken the center stage as one of the ultimate 

medical challenges. It is a group of metabolic 

diseases characterized by hyperglycemia 

resulting from defects in insulin secretion, 

insulin action or both (American Diabetes 

Association, 2003). The prevalence of diabetes 

45 

in India is very rapidly rising and it is estimated 

that by the year 2010, 20% of all type II patients 

in the world would be contributed from India. 

Indians are also susceptible to the major 

complications related to diabetes like coronary 

artery disease, nephropathy and retinopathy. 

Prevalence of the complications is higher in 

low socio-economic groups due to lack of good 

control of glycaemia and hypertension and 


Ameliorative effect of Coccinia grandis in diabetic rats 

also due to behavioral factors. The direct and 

indirect costs involved in the treatment of the 

chronic disease especially when associated with 

the vascular complications are enormous. There 

is an urgent need to implement preventive 

measures to reduce the high morbidity and 

mortality and also to reduce the cost burden to 

the patients and to the society (Ramachandran 

et al., 2002) Synthetic hypoglycemic agents 

can produce serious side-effects that endanger 

the life of the diabetic patients. This leads to 

increasing demand for natural products with 

antidiabetic activity and less side-effects. 

WHO has suggested the evaluation of plant 

potentials as effective therapeutic agents, 

especially in areas where we lack safe modern 

drugs. In modern medicine no satisfactory 

single effective therapy is still available to cure 

diabetes mellitus. Also, some of the world’s 

richest nations are driving the hardest bargains, 

despite the fact that the benefits of treatment are 

global and by advising that to develop the much 

needed research capacity, developing countries 

should no more rely on the industrialized world, 

but find their own specific solutions (Iaccarino, 

2004). An in-depth research into the plants 

growing around us will reveal the goldmine 

lying unexposed within plants. By undertaking 

this type of investigation, it is possible to provide 

alternate raw materials for the ever growing 

pharmaceutical industry and it play a vital role 

in providing medicines need to be efficacious, 

selective, specific in their action and safe with 

minimal side effects. Increasing evidence from 

both experimental and clinical studies suggests 

that oxidative stress plays a major role in the 

pathogenesis of diabetes mellitus. Free radicals 

are formed disproportionately in diabetes by 

glucose oxidation, non-enzymatic glycation 

of proteins, and the subsequent oxidative 

degradation of glycated proteins (Mehta et al., 

2006). Abnormally high levels of free radicals 

and the simultaneous decline of antioxidant 

defence mechanisms may lead to the damage 

of cellular organelles and enzymes, increased 

lipid peroxidation, and development of insulin 

46 

resistance. These consequences of oxidative 

stress may promote the development of 

complications of diabetes mellitus. 

Diabetes is a state of increased oxidant stress 

and there is evidence that oxidation may play 

a role in the genesis of complications. Chronic 

hyperglycemia is a major initiator of diabetic 

microvascular complications (e.g., retinopathy, 

neuropathy, and nephropathy). Glucose 

processing uses a variety of diverse metabolic 

pathways, hence chronic hyperglycemia can 

induce multiple cellular changes leading to 

complications. It is well established now that 

increased oxidative stress plays a major role 

in the development of diabetic complications 

(Maritim et al., 2003 and Soliman, 2008). 

Further, lipid peroxidation and antioxidant 

enzymes in blood have been cited as markers 

for vascular injury/microangiopathy in diabetes 

mellitus in several studies (King, 2008). 

Material and Methods 

This study was conducted in the Department 

of Biochemistry, Kongunadu Arts and Science 

College, Coimbatore, Tamilnadu. 

Collection of plant material and preparation 

of plant extract: 

The fresh leaves of Coccinia grandis 

(Linn.) Voigt (Family: Cucurbitaceae) 

Syn. Coccina indica (Wight & Arn) were 

collected from Karur district, Tamilnadu, 

India. Taxonomic authentication was done 

by Dr.V.S.Ramachandran, Taxonomist, 

Department of Botany, Bharathiar University, 

Coimbatore, Tamilnadu, India. The leaves were 

washed with water, shade dried and powdered. 

The powdered material (10g) was extracted with 

100ml of methanol using Soxhlet apparatus 

and filtered. The filtrate was concentrated and 

dried under reduced pressure and controlled 

temperature. 

Chemicals: 

The chemicals and solvents used in the study 



were of highest purity and analytical reagents 

grade. They were purchased from SD Fine 

Chem., Himedia and Qualigens, India. 

Selection of animals: 

Female albino rats of Wistar strain weighing 

150 200g were purchased from animal breeding 

centre of Kerala Agricultural University, 

Mannuty, Thrissur, Kerala India. Animals were 

provided with standard pellet diet (AVM feeds, 

Coimbatore) and water was provided ad-libitum 

and maintained under standard laboratory 

conditions. The animals were allowed to get 

acclimatized to the laboratory conditions for 

one week. The experiments were conducted 

according to the ethical norms approved by 

ministry of social justices and empowerment, 

Government of India and Institutional 

Animal Ethics committee Guide (Approval No: 

659/02/a/CPCSEA). 

After one week acclimatization period, the 

animals were divided into four groups with six 

animals in each. 

Group I : Control rats fed with standard 

pellet diet and water. 

Group II : Rats induced with 110 mg/ 

kg b.wt Nicotinamide 

followed by 65 mg/kg 

b.wt Streptozotocin intra 

venous. 

Group III : Diabetic rats treated with 

methanolic extract of 

Coccinia grandis 200 

mg/kg b.wt for 20 days 

orally 

Group IV : Rats treated with methanolic 

extract of Coccinia 

grandis 200 mg/kg b.wt 

for 20 days orally 

Sample Collection: 

After the experimental regimen, the animals 

were sacrificed by cervical dislocation under 

47 

mild chloroform anesthesia. Blood was collected 

by an incision made in the jugular veins and 

the serum was separated by centrifugation at 

2000 rpm for 20 minutes. The liver was excised 

immediately and thoroughly washed in ice cold 

physiological saline. A 10% homogenate of the 

washed tissue was prepared in 0.1M Tris HCl 

buffer (pH 7.4) in a potter homogenizer filled 

with a Teflon plunger at 600 rpm for 3 mins. 

Blood glucose was done by the method 

of Beach and Turner(1958), serum insulin by 

the method of Anderson (1993), hemoglobin 

by the method of Drabkin and Austin (1932), 

glycosylated hemoglobin by the method of 

Sudhakar and Pattabiraman (1981), liver 

glycogen by the method of Morales et al (1973), 

hexokinase by the method of Branstrup (1957), 

glucose – 6 – phosphatase by the method 

of Koide and Oda (1959), fructose – 1, 6 – 

diphosphatase by the method of Ganccedo and 

Ganccedo (1971), succinate dehydrogenase by 

the method of Slater and Bonner (1952) and 

malate dehydrogenase by the method of Mehler 

et al (1948) levels were estimated. Statistical 

analysis was performed using SPSS package, 

version 6.0. The values were analyzed by one 

- way analysis of variance (ANOVA) followed 

by Least Significant Difference (LSD). All the 

results were expressed as mean ± SD for six 

rats in each group p


The treatment with the plant extract caused a 

significant decrease in the elevated blood glucose 

and increase in the lowered insulin and glycogen 

levels to near normal glycemic concentration 

in streptozotocin induced diabetic rats, which 

is an essential trigger for the liver to revert 

its normal homeostasis during experimental 

diabetes. Normal animals treated with the 

plant extract at a dosage of 200 mg/kg body 

weight showed a slight reduction in the blood 

glucose which may be due to the hypoglycemic 

property of Coccinia grandis while insulin and 

glycogen levels remained normal. The levels 

of total hemoglobin and HbA 1c in control and 

experimental animals are depicted in Table 2. 

The diabetic rats showed a significant decrease 

in the level of total hemoglobin and increase in 

the level of HbA 1c when compared with normal 

control rats. The level of total hemoglobin 

was significantly increased and HbA 1c was 

significantly decreased by the administration of 

plant extract in diabetic rats. Normal animals 

treated with the plant extract at a dosage of 200 

mg/kg body weight did not show any significant 

change in hemoglobin and HbA 1c levels. 

The activity of the enzymes involved in 

carbohydrate metabolism such as hexokinase, 

glucose – 6 – phosphatase and fructose- 1, 

6- diphosphatase in the liver of normal and 

treated rats were shown in Table 3. A significant 

decrease in hexokinase activity was observed in 

the liver of diabetic rats, whereas the activities of 

hepatic glucose-6-phosphatase and fructose-1, 

6-bisphosphatase were significantly increased 

compared with normal rats. Treatment with the 

plant extract to the diabetic rats resulted in a 

significant increase in hexokinase activity and 

decrease in gluconeogenic enzymes activity in 

the liver of diabetic rats. Oral administration of 

plant extract to normal rats resulted in a nonsignificant 

decrease in hepatic gluconeogenic 

enzymes and the activity of hexokinase was not 

altered significantly compared with normal rats. 

Table 4 depicts the effect of methanolic extract 

of Coccinia grandis on malate dehydrogenase 

and succinate dehydrognase in the liver of 

48 

control and experimental animals. In the 

present study, significant decrease in malate 

dehydrogenase and succinate dehydrogenase 

activities in the liver of diabetic animals was 

observed and the levels increased significantly 

in the liver of treated diabetic animals. There 

was no significant change in the levels of 

SDH and MDH in normal animals which 

were administered with the plant extract. An 

increase in succinate dehydrogenase and malate 

dehydrogenase activities in treated animals 

indicates better utilization of energy yielding 

intermediates by TCA cycle. 

Discussion 

Reduction of glucose transporter expression to 

prevent cytotoxic effect of STZ (Thulesen et 

al., 1997) and decrease in β cell sensitivity to 

glucose due to long term hyperglycemia (Curry, 

1986) is believed to be the possible causes of 

high level of glucose in the blood stream of 

STZ – induced diabetic rats. The increased 

glucose level in the diabetic animals might be 

due to the destruction of the pancreatic cells 

caused by the streptozotocin induction and the 

glucose level was found to be near normal in the 

treated animals which may be due to the insulin 

like activity of Coccinia grandis leaf extract 

by activating the glucose uptake by the cells. 

Lowered blood glucose levels in the diabetic 

rats might be due to increasing glycogenesis, 

inhibiting gluconeogenesis in the liver or 

inhibiting the absorption of glucose from the 

intestine. From the results of clinical studies by 

Knekt et al., 2002, it is evident that the reduction 

of hyperglycaemia is the most important factor 

in the prevention of chronic microvascular 

complications of diabetes mellitus (retinopathy, 

nepropathy, neuropathy and diabetic foot) as 

well as in the prevention of the accelerated 

atherosclerosis-related conditions (myocardial 

infarction, stroke). The mode of action of the 

active compound(s) of the plant material is 

probably mediated through enhanced secretion 

of insulin from the β-cells of Langerhans or 



Results 

Table 1: Effect of methanolic extract Coccinia grandis on blood glucose, serum insulin and liver glycogen in 

control and experimental rats 

Parameters Blood glucose Serum insulin Liver glycogen 

(mg/dl) (IU/ml) (mg/g tissue) 

Group I 105.50±15.37 10.32±0.28 39.56±0.44 

Group II 406.00±15.16a * 4.24±0.31a * 21.70±0.44 a * 

Group III 268.83±36.59b * 6.38±0.20b * 37.13±0.39 b* 

Group IV 83.50±12.88c ns 10.94±0.24 c ns 39.72±0.30c ns 

Values are expressed as mean±SD (n=6)(p


through extrapancreatic mechanism (Akhtar et 

al., 2007). The STZ – induced diabetes causes 

the destruction of β cells of the Islets, which 

leads to a reduction in insulin release and high 

blood glucose level namely hyperglycemia. 

The observed increase in the levels of serum 

insulin indicates that Coccina grandis extract 

stimulates insulin secretion by the closure of 

K + -ATP channels, membrane depolarization 

and stimulation of Ca 2+ influx, an initial key 

step in insulin secretion from the remnet β- 

cells or from regenerated β-cells. Previous data 

shows that, ferulic acid a phenolic compound 

increases insulin release in clonal β- cells RIN- 

5F (Nomura et al., 2003). The treatment with 

the Coccinia grandis extract has enhanced the 

rate of glycogenesis as indicated by higher 

amounts of hepatic glycogen in the diabetic 

treated group. Glycogen content of normal 

animals in fasting stage was only slightly higher 

than the diabetic animals and this may be due to 

the degradation of glycogen to maintain normal 

blood glucose levels, whereas glycogen levels 

in diabetics were found to be very low despite 

high blood glucose levels possibly due to lower 

levels of glycogen synthase activity (Singh, 

2001). Accumulation of glycogen in liver of 

treated animals is somewhat similar to that 

reported during insulin therapy. The glycogen 

content is decreased in liver muscles of diabetic 

rats (Grover et al., 2000). Similar observations, 

i.e. hypoglycaemic activity and improved levels 

of hepatic glycogen, were reported by Kedar 

and Chakrabarti (1982) in diabetic animals 

with the treatment of Momordica charantia 

for 3 weeks. Pectin from ivy gourd (Coccinia 

grandis) has shown significant hypoglycemic 

activity in rats, stimulating glycogen synthetase 

activity and reducing phosphorylase activity 

(Grover et al., 2002). The excess of glucose 

present in the blood during diabetes, which 

react with hemoglobin and form glycosylated 

hemoglobin. The various proteins including 

hemoglobin undergo an enzymatic glycation 

in diabetes. Glycosylated hemoglobin was 

found to be increased in diabetes mellitus and 

50 

the amount of increase is directly proportional 

to that of fasting blood glucose level (Sheela 

& Augusti, 1992). Lowered levels of total 

hemoglobin were observed in diabetic rats 

which might be due to the increased formation 

of HbA 1c . Hyperglycemia is the clinical 

hallmark of poorly controlled diabetes, which 

is known to cause glycation, and also known as 

non-enzymatic glycosylation. HbA 1c was found 

to increase in patients with diabetes mellitus 

and the increase was directly proportional to the 

fasting blood glucose levels (Alberti, 1982). 

In experimental diabetes, enzymes of glucose 

metabolism are markedly altered. One of the 

key enzymes in the catabolism of glucose is 

hexokinase, which phosphorylates glucose and 

converts it into glucose-6-phosphate (Laakso et 

al., 1995). Hexokinase insufficiency in diabetic 

rats can cause decreased glycolysis and decreased 

utilization of glucose for energy production 

(Gancedo & Gancedo, 1971). Hexokinase is 

significantly reduced in diabetic rats; this may 

be the reason for the diminished consumption of 

glucose in the system and increased blood sugar 

levels. The gluconeogenic enzyme, glucose- 

6-phosphatase is a crucial enzyme of glucose 

homeostasis because it catalyses the ultimate 

biochemical reaction of both glycogenolysis 

and gluconeogenesis (Mithievre et al., 1996). 

Increased glucose-6- phosphatase activity in 

diabetic rats provides hydrogen, which binds 

with NADP + in the form of NADPH and enhances 

the synthesis of fats from carbohydrates (i.e. 

lipogenesis) (Bopanna et al., 1997) and finally 

contributes to increased levels of glucose in the 

blood. Increased hepatic glucose production in 

diabetes mellitus is associated with impaired 

suppression of the gluconeogenic enzyme 

fructose-1, 6-bisphosphatase. Activation of 

gluconeogenic enzymes is due to the state 

of insulin deficiency, because under normal 

conditions, insulin functions as a suppressor of 

gluconeogenic enzymes. In diabetic condition, 

hexokinase activity has been decreased, 

which may be due to loss of insulin receptors 

(Garvey et al., 1985). Previous studies showed 



that the caffeic acid seemed to suppress the 

hepatic glucose output by enhancing hepatic 

glucose utilization and inhibiting glucose over 

production in db/db mice (Jung et al., 2006). 

Administration of the plant extract to the 

diabetic rats significantly reduced the levels of 

plasma glucose, which activates hexokinase and 

increase glycolysis and utilization of glucose 

for energy production. Similar effects of 

mangiferin, in streptozotocin induced diabetic 

rats were reported (Sellamuthu et al., 2009). The 

hepatic gluconeogenic enzymes (glucose-6phosphatase 

and fructose-1, 6-bisphosphatase) 

were significantly increased in diabetic state 

(Shulman, 2000). The activities of the two 

enzymes may be due to the increased synthesis 

of enzymes contributing to the increased glucose 

production during diabetes by the liver (Pari et 

al., 2005). In diabetic rats treated with the extract 

modulates and regulates the activities of these 

enzymes, either through regulation by cyclic 

AMP or by metabolic activation or inhibition 

of glycolysis and gluconeogenesis. In previous 

reports, para-methoxycinnamic acid markedly 

reduced hyperglycemia in STZ-induced diabetic 

rats by increasing glycolysis and inhibiting 

gluconeogenesis (Adisakwattana et al., 

2005). When Coccinia indica and Momordica 

charantia extracts were administered to diabetic 

rats, the results indicated that there was lowering 

of blood glucose by depressing its synthesis 

through depression of the key gluconeogenic 

enzymes glucose-6-phosphatase and fructose- 

1,6-biphosphatase and also by enhancing 

glucose oxidation by the shunt pathway through 

activation of its principal enzyme, G6PDH 

(Shibib et al.,1993). 

Malate dehydrogenase plays an 

important role in the citric acid cycle by 

providing oxaloacetate for the formation of 

citrate with acetyl-CoA for generating malate 

which can feed the cytosolic gluconeogenic 

pathway. These enzymes are reported to be 

inhibited in tissues of diabetic animals in several 

studies (Lemieux et al., 1984). From our results, 

we can conclude that the lowered insulin level 

51 

plays an important role in the pathogenesis of 

diabetes and its complication thereby increasing 

glucose level in blood. Since the methonolic 

extract of Coccinia grandis has the capacity to 

activate the glucose uptake into the cells and 

enhanced reduction in gluconeogenesis they 

act as an antihyperglycemic agent and since it 

increases the utilization of the energy yielding 

intermediates by TCA cycle it is insulin mimetic. 

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Biomedicine; 2011; 31 ( 1 ) : 53 - 63 

Testicular toxicity in arsenic exposed albino rats: ameliorative effects of ascorbic 

acid and α-tocopherol 

*Avijit Dey, **Arindam Bose,Prabir Kr. *Mukhopadhyay 

*Department of Physiology, Presidency College, Kolkata-700 073, India 

**Department of Microbiology, Immunology & Molecular Biology, Choitram Hospital & Research 

Center, Manikbagh Road, Indore, Madhya Pradesh - 452014, India. 

(Received 30th August 2010; Revised 25th November 2010; Accepted 30th December 2010) 


Dr. Prabir Kr. Mukhopadhyay 

E-mail: p.mukherjeepresi@gmail.com 

Abstract: 

Backgound and objective: The study was designed to evaluate the protective effect of combined 

administration of ascorbic acid and α-tocopherol on the arsenic induced testicular toxicity. 

Methods: The histoarchitecture of testis along with sperm number, viability and motility were counted to 

study the testicular gametogenic toxicity. The levels of malondialdehyde (MDA) and conjugated dienes in 

testicular tissue were measured for evaluation of oxidative stress and the activities of testicular superoxide 

dismutase (SOD) and catalase were also measured. 

Results: Arsenic trioxide treatment (3 mg/kg body weight/ day) in a single dose for 30 consecutive days 

caused increase in seminiferous tubular luminal space coupled with reduced accumulation of spermatozoa 

and disarray in cellular organization. Other significant changes were decrease in sperm count, viability and 

motility. This treatment was also associated with significantly high levels of MDA and conjugated dienes 

along with significant inhibition of the SOD and catalase activities in the testicular tissue. All these changes 

were reversed with ascorbic acid and α-tocopherol co-administration. Supplementation of ascorbic acid 

(200 mg/kg body weight/day) and α-tocopherol (400 mg/kg body weight/day) along with same arsenic 

exposure caused partial restoration of normalcy. All these sperm physiological changes and altered gonadal 

features, both histomorphometric and histological observations were found significantly ameliorated. The 

other studied changes were significantly reversed. 

Interpretation and conclusion: Results of this study propose that co-treatment of ascorbic acid and 

α-tocopherol to arsenic-exposed rats protects the testicular antioxidant system and causes effective recovery 

from the toxic effects of arsenic on testes. 

Key words: arsenic, testis, oxidative stress, ascorbic acid, α-tocopherol. 

Introduction 

It has become evident that increasing human 

activities have modified the global cycle of 

heavy metals and metalloids, including the 

toxic non-essential elements like arsenic, 

53 

As(1). Among these metals, arsenic exhibits a 

complex metabolism and is possibly the most 

abundant and potential carcinogen (2). Arsenic 

is available to the human population through 

sources that include drinking water, food and 

air. Population exposed to arsenic contaminated 


Effects of ascorbic acid and α-tocopherol in arsenic toxicity 

drinking water includes those in Taiwan, 

Chaina, Europe, United States, Bangladesh and 

India (3, 4). Studies done in September 2006 by 

the School of Environmental Studies, Jadavpur 

university, Kolkata, for the assessment of 

ground water arsenic contamination in 19 

districts of West Bengal, India have revealed 

a gruesome picture which depicts that out of 

80.2 million 4.6 million people in West Bengal 

are at risk of drinking arsenic contaminated 

water containing arsenic > 50 µg/L, where 

the WHO recommended maximum arsenic 

contaminant level (MCL) is 10 µg/L (5). 

Consumption of arsenic containing drinking 

water has been associated with cancers (lung, 

bladder, skin etc.) and other chronic diseases 

such as dermal, cardiovascular, neurologic, 

and diabetes (6). A lot of potential toxic effects 

are also observed on the reproductive system 

also. In female rats, the associated noticeable 

changes are the reductions of ovarian weight 

along with associated decrease of plasma levels 

of LH, FSH and the steroidogenic enzymes 

like ∆ 5 -3β-hydroxysteroid dehydrogenase (∆ 5 

-3β-HSD), 17β- hydroxysteroid dehydrogenase 

(17β-HSD) (7). On male reproduction, the 

studied effects of arsenic toxicity include the 

reduction of the weight of testes and accessory 

sex organs (8), inhibition of androgenic and 

gametogenic potentials of testis (8). Arsenic 

exposure has shown to depress the functions of 

antioxidant defense system leading to oxidative 

damage to cellular macromolecules (9). 

According to the recent report, arsenic can also 

alter the cellular functions (10) and can also 

cause DNA damage (10). Studies suggest that 

arsenic compounds may also exert their toxicity 

through the generation of reactive oxygen 

species (ROS), such as superoxide radical, 

hydroxyl radical, hydrogen peroxide and nitric 

oxide during their metabolism in the cells (9, 11) 

and thus may cause cellular damages (12). ROS 

attacks and causes peroxidation of sperm plasma 

membrane polyunsaturated fatty acids (PUFA) 

resulting in the formation of malondialdehyde 

(MDA) (13), conjugated diene (CD) radicals 

54 

(14). Male infertility is reflected by low sperm 

count, low sperm motility and bad quality of 

sperms (8). Seminiferous tubular diameter is 

reported to show a dose and duration dependent 

reduction (15). Degeneration and loss of germ 

cells, marked atrophy of seminiferous tubules 

and Leydig cell are also reported (15). 

Recent studies have shown that ascorbic acid 

is able to block the arsenic induced impairment 

of male reproductive functions and testicular 

oxidative changes in mice (16) and could also 

protect the liver and kidney of rats from arsenic 

induced oxidative stress (17). Administration 

of ascorbic acid with α-tocopherol exhibits 

profound effects in preventing protein 

oxidation and DNA damage in rats (6). The 

same supplementation could also prevent lipid 

peroxidation and protect the antioxidant system 

in arsenic-intoxicated rats (18). Our present 

study is an attempt to investigate whether the 

combined oral application of ascorbic acid 

and α-tocopherol can ameliorate the arsenic 

induced oxidative stress mediated alterations of 

testicular histoarchitecture, sperm physiology 

and testicular antioxidant defense system by 

their cumulative antioxidant actions. 

Materials and Methods: 

Animal selection and drug treatment: 24 adult 

male albino rats of the Wister stain weighing 

130±10 mg were selected for this experiment. 

The animals were maintained under standard 

laboratory conditions (14 hrs light: 10 hrs dark, 

25±2° C) with free access to food and water. All 

animal experiments were performed according 

to the ethical guidelines suggested by the 

Institutional Animal Ethics Committee (IAEC) 

guided by the Committee for the purpose of 

Control and Supervision of Experiments on 

Animals (CPCSEA), Ministry of Environment 

and Forest, Government of India. For the 

experiments, rats were randomly selected into 

three groups consisting eight rats in each: 

group I, control; group II, arsenic treated; and 

group III, arsenic+ ascorbic acid+ α-tocopherol 



supplemented. The animals of group I and II 

were provided with a control diet composed of 

71% carbohydrate, 18% protein, 7% fat, and 4% 

salt mixture and vitamins (19). For chronic oral 

exposure to arsenic a dose was selected (3 mg / 

kg body wt / day), which is within the range of 

LD 50 of 70 kg body wt. human (1-4 mg /kg) and 

lesser than one thirteenth of LD 50 value of rats 

(40mg/kg) (20). Accordingly, rats of group II 

and III were orally treated with aqueous solution 

of arsenic trioxide, 3mg /kg body wt/ day for 

30 days. The rats of group III, in addition, were 

treated with ascorbic acid (200mg / kg body wt 

/ day, dissolved in water) and α-tocopherol (400 

mg / kg body wt / day, dissolved in olive oil) 

by oral gavage for once a day. To overcome 

the impact of any altered food intake, control 

group (group I) rats were pair-fed with other 

experimental groups, II and III. 

Food and water intake and body wt. of 

the rats were monitored throughout the 30 days 

of the experiment. 

Rats were sacrificed 24 hrs after 

the last treatment by light ether anesthesia. The 

testes were dissected, weighted and one testis 

of each rat was used for histological study and 

the other, for the assessment of levels of free 

radicals and scavenger enzyme activities. 

Study substance: Arsenic trioxide was used for 

this experiment, purchased from Loba Chemical 

Pvt. Ltd., Mumbai, India. α-tocopherol was 

purchased from Himedia, Mumbai, India and 

L-ascorbic acid was purchased from SRL Pvt. 

Ltd., Mumbai, India. 

1. Permanent slide preparation for 

histological study: Bouin’s fixed testes were 

embedded in paraffin wax and blocks were 

prepared. 5μm thin sections were cut from the 

middle portion of the testes with a high precision 

microtome (IEC Microtome, USA) and stained 

with Hematoxylin and Eosin (H/E). 

2. Sperm viability count: Immediately after the 

sacrifice cauda portion of the epididymis was 

cut. The cauda was kept in 1 ml diluent (21). 

55 

This was kept for 5 mins at 37°C. It was then 

taken out, and an incision was given through 

the cauda and sperms were dispersed in the 

fluid. 40 μl of this spermatozoal suspension was 

transferred to an eppendrof. This was stained 

with EosinY and Nigrosin (40 μl each). This 

was mixed gently and 25 μl from the mixture 

was taken on a grease free slide and a smear 

was drawn and dried. The number of viable 

and non-viable sperms was counted under light 

microscope (OLYMPUS, CH 20i TR). 

3. Sperm count: From the dispersed 

spermatozoal suspension, 25 μl was charged 

on a Neubauer Haemocytometer (22) and the 

numbers of sperms were counted and calculated 

using WBC chambers. 

4. Sperm motility count: The cauda epididymis 

from all three groups was obtained as mentioned 

earliar and each cauda was kept in 1 ml PBS 

0.2mol, pH 7.4, at 37°C (23). 25 μl of this was 

taken on a clean slide covered with a cover slip 

and was observed under a microscope. Total 

numbers of motile sperms per 100 sperms 

were counted. The readings were taken at the 

beginning of 1 st hr, 2 nd hr, 3 rd hr of the experiment 

(the temperature of spermatozoal suspension 

was maintained at 37°C throughout the process). 

5. Testicular conjugated dienes measurement: 

Testicular conjugated dienes were measured 

according to the method of Slater (1984) (24). 

Testicular tissue was homogenized in 0.1mol 

phosphate buffer (pH 7.4) containing 0.1mol 

Na 2 HPO 4 and 0.1mol anhydrous NaH 2 PO 4 at 

a tissue concentration of 50 mg/ml. Lipids 

of the homogenizing mixture (0.5 ml) were 

extracted with chloroform-methanol (2:1) 

mixture. It was then centrifuged at 1000g for 5 

minutes at room temperature. Chloroform was 

evapourated and the lipid residue was dissolved 

in 1.5 ml cyclohexane. The absorbance of 

this dissolved conjugated dienes was read 

spectrophotometrically at 233 nm. 

6. Testicular malondialdehyde measurement: 

Malondialdehyde (MDA) levels in the 

testicular tissues from rats of all groups were 

measured biochemically following the method 



of Ohkawa et al. 1979 (25). Tissue samples 

were homogenized in 0.1mol phosphate buffer 

(pH7.4) at tissue concentration of 50 mg/ml. 

The homogenizing mixture (0.5 ml) was mixed 

with 0.5 ml 0.9% saline and 2ml of TBA-TCA 

mixture( 0.392 gm thiobarbituric acid in 75 ml 

of 0.25 N HCl with 15 gm trichloroacetic acid, 

volume upto 100ml by 95% ethanol) and boiled 

for 10 minutes. The mixture was then cooled 

to room temperature and centrifuged at 4,000 

rpm for 10 minutes. The whole supernatant was 

taken in spectrophotometric cuvette and read at 

535 nm. 

7. Testicular catalase activity: The testicular 

catalase activity was measured from all groups 

following the method of Beers and Sizer (1952) 

(26). Testicular tissue was homogenized in ice 

cold medium containing 0.05 mol Tris Hcl 

buffer( pH- 7.0) at a tissue concentration of 20 

mg/ml. The mixure was centrifused at 10000g 

for 20 minutes at 4°C. In a spectrophotometric 

cuvette 0.5 ml hydrogen peroxide solution (100 

μl 30% H 2 O 2 + 99 ml double dist water) and 2.5 

ml double distilled water were mixed well and 

read at 240 nm. Then 40 μl of the supernatant 

was added, mixed well and six readings were 

taken at 30 second intervals. 

8. Testicular superoxide dismutase (SOD) 

activity: Testicular SOD was measured 

according to the method of Martin et al (1987) 

(27). To measure the testicular SOD activity 

the tissue was homogenized in an ice cold 

medium containing 0.1mol phosphate buffer 

(pH 7.2) at a tissue concentration of 50mg/ml. 

The homogenate was centrifuged at 10,000g for 

20 min at 4ºC. In a spectrophotometer cuvette 

3ml of 0.05M phosphate buffer with EDTA 

was added and then 50µl of haematoxylin was 

added and checked OD at 30 second intervals 

with sample and without sample at 560 nm. 

9. Statistical analysis: The data were expressed 

as mean ± SEM (Standard error of mean). 

For statistical analysis, the quantitative data 

of each parameter from the different groups 

were analyzed by Student’s “t” test. The 

mean ± SEM was calculated for each group 

56 

and the corresponding level of significance 

was calculated. Statistical software system 

Minitab version 2009 was employed in the 

entire methodologies. p


Table 1: Effect of ascorbic acid and α-tocopherol co-administration on the arsenic trioxide induced alterations 

in spermatozoal status of mature albino rats. 

Parameter 

Studied 

Control 

Group-I 

(Mean 

±SEM) 

1)Sperm count (10 6 /cauda) 102.07 

±9.71 

2)Sperm viability(%) 28.06 

±0.49 

3)Sperm motility(%) Ist hr. 87.75 

±0.99 

2nd hr. 81.0 

±0.75 

3rd hr. 73.37 

±0.78 

Treated 

Group-II 

(Mean 

± SEM) 

61.76 

±0.45 

14.80 

±0.98 

58.87 

±2.89 

54.87 

±1.12 

41.0 

±1.57 

Percentage 

decreased 

Significance 

level 

I vs II 

39.49 p


Fig. 4: Effect of ascorbic acid & α-tocopherol co-administration on arsenic induced alterations in sperm count (values are expressed in 

mean ±SEM). 

10^6/cauda 

120 

100 

80 

60 

40 

20 

0 

Fig. 5: Effect of ascorbic acid & α-tocopherol co-administration on arsenic induced alterations in sperm viability (values are expressed in 

mean ±SEM). 

percentage 

30 

25 

20 

15 

10 

5 

0 

Group-I Group-II Group-III 


Fig. 6: Effect of combined application of ascorbic acid & α-tocopherol on arsenic induced alterations in sperm motility (values are expressed 

in mean ±SEM). 

Percentage 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 


Ist hr. 

2nd hr. 

3rd hr. 


58


Fig. 7: Effect of ascorbic acid & α-tocopherol co-administration on arsenic induced alterations in testicular conjugated dienes level (values 

are expressed in mean ±SEM). 

Fig. 8: Effect of ascorbic acid & α-tocopherol co-administration on arsenic induced alterations in testicular MDA level (values are expressed 

in mean ±SEM). 

‘ 

nmole/ mg of tissue 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

nmole/mg of tissue 

60 

50 

40 

30 

20 

10 

0 



Fig. 9: Effect of ascorbic acid & α-tocopherol co-administration on arsenic induced alterations in testicular CAT activity (values are 

expressed in mean ±SEM). 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

Fig. 10: Effect of ascorbic acid & α-tocopherol co-administration on alterations in testicular SOD activity induced by arsenic (values are 

expressed in mean ±SEM). 

unit/ mg of tissue 

mmole H2O2 

consumption/mg of 

1.6 

1.4 

1.2 

0.8 

0.6 

0.4 

0.2 

tissue/min 

1 

0 

59 





1a. Section of control testis showing normal features x 10 

1b. Section of one seminiferous tubule of control testis showing normal features x 40 

2a. Section of arsenic treated testis showing increases in luminal areas and reduced spermatozoal mass x 10 

2b. Section of arsenic treated one seminiferous tubule showing disintegrated cell membrane and disorganized cellular orientation x 10 

3a. Section of antioxidant vitamins supplemented testis showing features towards normalcy x 10 

3b. Section of antioxidant vitamins supplemented one seminiferous tubule showing almost normal features x 40 


60


values nearer to that of control (table-1, fig.6). 

The mean ± SEM (Standard error of mean) was 

calculated for each groups and the corresponding 

level of significance was calculated by using the 

previous statistical analysis. 

3. Testicular conjugated dienes: Testicular 

conjugated dienes level was significantly 

increased (p


radicals and thus the tocopheroxyl radical, α TO . 

is formed (36) which is regenerated to α-TOH 

by ascorbate present in the aqueous phase (37). 

This reduction of α-TO . by ascorbate prolongs 

the life span of this very important antioxidant, 

α-tocopherol and the resulting dehydroascorbate 

which is having little antioxidant activity is again 

reduced back to ascorbate by GSH dependent 

reductase. Therefore the group III rats which 

received the combined application of ascorbic 

acid and α-tocopherol with arsenic treatment 

showed minimal level of alterations in the 

studied parameters suggesting the free radical 

mediated toxicity in testicular gametogenic 

activity caused by arsenic.Alterations of 

membrane potentials, loss of cellular functions 

and macromolecular damages caused by arsenic 

toxicity occur before the initiation of cell damage. 

This co-administration of antioxidant vitamins 

possibly prevents the cellular disorganization 

by maintaining the antioxidant defence system 

in the seminiferous tubules (fig.3a & 3b). 

Previous studies on arsenic induced subjects 

with the same antioxidant vitamins have also 

showed that these have the modulatory activity 

on arsenic induced apoptosis by improving 

the cellular antioxidant status and scavenging 

the free radicals (38). Here the co-treatment 

of ascorbic acid and α-tocopherol shows its 

efficacy in the prevention of testicular toxicity 

by restoring the antrioxidant status of the 

testicular tissue. Thus our observations indicate 

that the dietary supplimentation of ascorbic acid 

and α-tocopherol could ameliorate the arsenic 

induced testicular toxicity. 

Conclusion: 

The study reveals that the detrimental effects 

on gametogenic functions of rat testis during 

the chronic arsenic exposure can be reversed 

by simultaneous administration of antioxidant 

vitamins like ascorbic acid and α-tocopherol, 

as the loss of antioxidant defense system is one 

of the major causes behind the arsenic induced 

damages. Clinical trials with these vitamins 

62 

may be undertaken to find an easy and effective 

strategy to combat the detrimental effects on the 

reproductive system of the human population 

exposed to this silent environmental toxicant 

which is gradually increasing day by day and 

imposing a serious threat to people worldwide. 

Acknowledgement: 

The authors gratefully acknowledge the 

financial assistance from the Minor Research 

Project [No.F.PSW-074/09-10 (ERO)] provided 

by the University Grants Commission, New 

Delhi, India. 

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Evaluation of antioxidant status in Type 2 Diabetes Mellitus with and without complications 

*T. Vivian Samuel, **S. Smilee Johncy 

*Department of Biochemistry, **Department of Physiology, J J M Medical College, Davangere, 

Karnataka - 577004, India. 

(Received 21 st June 2010; Revised 11 th December 2010; Accepted 02 th January 2011) 


Dr. T. Vivian Samuel, 

Email : tviviansamuel@yahoo.co.in 

Abstract 

Background & objectives:Oxygen free radicals have been implicated in pancreatic beta cell damage 

and genesis of diabetes as well as in pathogenesis of its complications like nephropathy, retinopathy and 

neuropathy. The present study was aimed to evaluate lipid peroxidation and antioxidant status in Noninsulin 

dependent diabetes mellitus (NIDDM) and to assess the same between NIDDM subjects with and 

without complications. 

Methods: 35 control subjects and 89 NIDDM were studied. Lipid peroxidation in terms of serum 

malondialdehyde (MDA), by thiobarbituric acid method, erythrocyte superoxide dismutase (SOD), reduced 

glutathione (GSH) and serum vitamin C were estimated using spectrophotometer. Fasting blood glucose 

was also measured. 

Results : The mean MDA levels in controls, NIDDM without complications and NIDDM with complication 

were 3.83 ± 0.26 nmol/ml, 4.73 ± 0.51 nmol/ml and 5.64 ± 0.50 

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. 

Lipid peroxidation was significantly raised in NIDDM with complications than in NIDDM without 

complications and in control subjects. The mean SOD, GSH, Vit C levels were significantly lowered in 

NIDDM. 

Interpretion & Conclusion: The results suggest that the antioxidant deficiency and excessive peroxidation 

damage appear very early in NIDDM, well before the development of complications. Therapeutic measures 

to increase antioxidants and control lipid peroxidation are warranted for effective control of its complications. 

Keywords: Ascorbic acid, Malondialdehyde, Reduced glutathione, Superoxide dismutase. 

Introduction: 

In diabetes mellitus there is an imbalance in 

the antioxidant protective mechanism, leading 

to oxygen stress in the cells. Toxic oxygen 

derived products are generated in all aerobic 

cells which include superoxide radical (O – 

2 ), 

hydrogen peroxide (H O ) and hydroxyl radical 

2 2 

(OH – ), the latter being the most lethal1 . All 

64 

biomolecules may be attacked by free radicals, 

but lipids are the more susceptible. The human 

cells are abundant and rich sources of PUFA, 

hence are readily attacked by oxidizing radicals 

by a process known as lipid peroxidation to 

form lipid peroxides. This is a self perpetuating 

chain reaction and highly damaging 2 . One of 

the most important intracellular antioxidant 

substances is an enzyme, superoxide dismutase 


which scavenges free radicals by converting the 

harmful superoxide ion into stable hydrogen 

peroxide. Ascorbic acid’s role as an antioxidant 

is indicated by its known free radical scavenging 

action. As a reducing and antioxidant agent, it 

directly reacts with O – and OH and various lipid 

2 

hydroperoxides. As an antioxidant, Vitamin C 

exerts a sparing effect on the antioxidant actions 

of Vitamin E and selenium. 3 Glutathione in 

erythrocyte exists in reduced form (GSH). GSH 

serves as both a nucleophile and an effective 

reductant by interacting with numerous 

electrophilic and oxidizing compounds such as 

H O , O 2 2 – and OH. Reduced glutathione readily 

2 

interacts with free radicals, notably hydroxyl 

and carbon radicals, by donating a hydrogen 

atom. Such reactions can provide protection by 

neutralizing reactive OH, which is considered 

a major source of free radical damage 4 . Tare 

RS and co-workers illustrated the prevalence 

of oxidative stress in diabetes by a highly 

significant increase in concentration of MDA 

and diminished activity of SOD in comparison 

to the control subjects. MDA exhibited a 

positive correlation with fasting blood glucose 

and glycosylated hemoglobin emphasizing the 

phenomenon of antioxiditive glycosylation 

of proteins 5 . Hyperglycemia and oxidative 

stress has been recognized as cardinal features 

of diabetes. Hyperglycemia causes glucose 

dependent chemical alterations in body proteins 

(non-enzymatic protein glycation) responsible 

for changes in protein structure and function, 

contributing to the pathophysiology of diabetes 

and its complications. The present study aims 

to assess the lipid peroxidation and antioxidant 

status in non-insulin dependent diabetes mellitus 

(NIDDM) and also their correlation to glycemic 

control and development of complications. 

Materials and methods : 

The present study was conducted on NIDDM 

patients and control subjects. 35 control 

subjects who were healthy non-smokers and 

non-alcoholics at the time of study and 89 

65 

NIDDM patients who were on treatment were 

studied. The selected subjects included both 

males and females in the age group of 40-65 

years. Among the NIDDM patients, 49 were 

without any complications and 40 were with 

microvascular complications like Retinopathy, 

Peripheral neuropathy, Nephropathy and both 

Retinopathy and Peripheral neuropathy. NIDDM 

patients with infectious diseases, smokers and 

alcoholics were all excluded from this study. 

Overnight fasting blood sample was collected 

for estimation of fasting blood glucose, serum 

MDA, serum ascorbic acid, erythrocyte SOD 

and reduced glutathione. Fasting blood glucose 

was estimated by O-Toluidine method6 . Serum 

MDA was estimated by Thiobarbituric acid 

(TBA) method, in which one molecule of MDA 

reacts with two molecules of TBA and yields a 

pink crystalline pigment which is measured at 

535 nm7 . Serum ascorbic acid was estimated by 

2,4 – dinitrophenyl hydrazine (DNPH) method 

in which ascorbic acid is oxidized by copper 

to form dehydroascorbic acid, which when 

treated with DNPH and sulfuric acid forms 

orange colour which is measured at 520 nm8 . 

Blood reduced glutathione was estimated by 

5,5 dithiobis – 2 – nitrobenzoic acid (DTNB) 

method. DTNB is readily reduced by 

sulphydryl compounds, forming a highly 

coloured yellow anion. Optical density is 

measured at 412 nm. 9 Superoxide dismutase 

in hemolysate was estimated using Nitroblue 

Tetrazolium (NBT). Illumination of riboflavin 

in the presence of oxygen and electron donors 

like methionine or EDTA generates superoxide 

anion. The reduction of nitroblue tetrazolium 

– by O was followed at 560 nm using a 

2 

spectrophotometer10 . 

Statistical analysis : 

All the values are expressed as their Mean ± S.D. 

Data were subjected for analysis using unpaired 

‘t’ test, for comparison between two groups and 

One way ANOVA (F-test) for multiple group 

comparison. 


Table – 1 : Comparison of FBS, MDA, Vit C, GSH and SOD between controls, NIDDM with complications and 

NIDDM without complications 

Groups No 

FBS 

(mg/dl) 

MDA 

(nmol/ml) 

Vit. C 

(mg/dl) 

GSH 

(mg/dl) 

SOD 

(U/ml) 

Controls (I) 35 97.7 ± 5.7 3.83 ± 0.26 1.58 ± 0.10 54.6 ± 3.25 5.16 ± 1.03 

NIDDM without 

complications(II) 

NIDDM with 

complications(III) 

49 207.0 ± 21.2 4.73 ± 0.51 0.89 ± 0.20 48.6 ± 2.49 3.91 ± 0.49 

40 258.1 ± 20.7 5.64 ± 0.50 0.67 ± 0.07 44.9 ± 1.81 3.48 ± 0.27 

I versus II p < 0.001 p < 0.001 p < 0.001 p < 0.001 p< 0.001 

I versus III p < 0.001 p < 0.001 p< 0.001 p< 0.001 p < 0.001 

II versus III p < 0.001 p < 0.001 p< 0.001 p < 0.001 p< 0.001 

Table – 2 : Comparision of FBS, MDA, Vit. C, GSH and SOD between NIDDM without complications and 

NIDDM with different complications 

Groups No. 

NIDDM without 

complications (II) 

NIDDM with retinopathy 

(III a) 

NIDDM with Neuropathy 

(III b) 

NIDDM with 

Nephropathy(IIIc) 

NIDDM with retinopathy 

Neuropathy (III d) 

FBS 

(mg/dl) 

MDA 

(nmol/ml) 

Vit. C 

(mg/dl) 

GSH 

(mg/dl) 

SOD 

(U/ml) 

49 207.0 ± 21.2 4.73 ± 0.51 0.89 ± 0.20 48.6 ± 2.49 3.91 ± 0.49 

10 255.8 ± 13.7 5.65 ± 0.21 0.71 ± 0.06 45.2 ± 1.59 3.56 ± 0.25 

11 249.6 ± 17.2 5.60 ± 0.19 0.67 ± 0.08 45.8 ± 1.73 3.63 ± 0.22 

10 250.3 ± 12.1 5.73 ± 0.20 0.68 ± 0.07 44.8 ± 1.51 3.49 ± 0.29 

9 279.9 ± 25.1 5.59 ± 1.02 0.61 ± 0.06 43.5 ± 1.89 3.21 ± 0.21 

II versus III a p < 0.001 p < 0.001 p < 0.05 p < 0.001 p < 0.05 

II versus III b p < 0.001 p < 0.001 p < 0.01 p < 0.001 N.S. 

II versus III c p < 0.001 P < 0.001 p < 0.01 p < 0.001 p < 0.05 

II versus III d p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 

Table – 3 : Analysis of MDA, Vit. C, GSH, SOD in NIDDM patients depending on the glycemic control 

Groups No. of cases 

MDA 

(nmol/ml) 

Vit. C (mg/dl) GSH (mg/dl) 

SOD 

(U/ml) 

A 18 4.29 ± 0.33 1.04 ± 0.27 50.7 ± 1.7 4.17 ± 0.41 

B 45 5.18 ± 0.48 0.78 ± 0.12 46.8 ± 2.2 3.71 ± 0.44 

C 26 5.66 ± 0.59 0.65 ± 0.05 44.5 ± 1.6 3.43 ± 0.23 

F – value 41.7 43.3 54.0 19.4 

p – value < 0.001 < 0.001 < 0.001 < 0.001 

Groups A – FBS < 200 mg/dl B – FBS 200 – 250 mg/dl C – FBS > 250 mg/dl 


66

Results 

It is evident from the Table 1, that there is highly 

significant (p < 0.001) increase in FBS and MDA 

levels in NIDDM patients with complications 

than in NIDDM patients without complications 

and in controls. There is highly significant (p < 

0.001) decrease in vitamin C, GSH and SOD 

in NIDDM patients with complications than 

in NIDDM patients without complications 

and in control subjects. It is evident from the 

Table 2, that there is highly significant (p < 

0.001) increase in FBS and MDA in NIDDM 

patients with more than one complications 

when compared with NIDDM patients with 

one complication and NIDDM patients without 

any complications. There is highly significant 

(p < 0.001) decrease in Vitamin C, GSH and 

SOD in NIDDM patients with more than one 

complication when compared with NIDDM 

patients with one complication and NIDDM 

patients without any complications. As shown 

in Table 3, it is evident that in NIDDM patients, 

the mean MDA level increases with poorly 

controlled blood glucose level. On the other 

hand the mean Vitamin C, GSH and SOD levels 

decrease with uncontrolled blood glucose level. 

Discussion : 

Free radical mediated cytotoxic process of 

lipid peroxidation appears to have a role in 

development of the multifactorial disease, 

diabetes mellitus. The role of oxygen free 

radicals in diabetes and its complications is 

being propagated and the weight of supporting 

evidence is steadily increasing. In diabetes 

mellitus there is an increase in the rate of 

production of free radicals and H 2 O 2 due to the 

autoxidation of glucose. It is known that chronic 

hyperglycemia leads to glycosylation of proteins 

like Hb which could also lead to the formation 

of free radicals because of autoxidation of 

the Amadori products. In poorly controlled 

diabetes mellitus, glucose oxidation through 

67 

the pentose phosphate pathway leads to an 

excessive formation of NADPH, which in turn 

can promote lipid peroxidation in the presence 

of cytochrome P-450 system. Oxyhemoglobin 

in erythrocytes could act like cytochrome 

P-450 in the presence of NADPH and this could 

induce increased lipid peroxidation 11 . 

According to Ranjini K. Sundaram et al., 

lipid peroxidation was significantly raised 

within the first 2 years of diagnosis. MDA was 

significantly elevated in NIDDM and appeared 

to be associated with the multiplicity of 

complications 12 . In our study, mean MDA level 

in NIDDM patients increased progressively 

with the increase in blood glucose level. MDA 

levels have a positive correlation with increase 

in blood glucose levels. Similar findings have 

been reported by many workers, Sushil K. Jain 

et al., Gallou G.et al 13 . The mean MDA level was 

elevated in NIDDM patients with complication 

when compared to NIDDM patients without 

any complications and in control subjects. 

MDA concentration was significantly higher in 

NIDDM than in control and also increased in 

patients macroangiopathy than patients with no 

vascular complications 14 .A likely explanation 

for a lower Vitamin C status in diabetics is that 

ascorbic acid is actively transported into the cells 

in its partially oxidized form as dehydroascorbic 

acid, which is promptly converted to ascorbic 

acid within the cell. The carrier of ascorbic 

acid transport serves also to transport glucose 

and is inhibited in transporting ascorbic acid 

by the hyperglycemia of diabetes. The carrier 

number is also subject to increase by insulin and 

is believed to be low in diabetes 15 . Increased 

antioxidative glycosylation of hemoglobin may 

lead to imbalanced generation of free radicals 

like superoxide, thereby causing depletion of 

SOD which quenches it. Diminished activity 

of SOD points out to an exhausted antioxidant 

reserve which further exacerbates the oxidative 

stress. Excessive peroxidation is associated 

with reduced SOD activity in diabetes. Loss 

of SOD activity in the erythrocytes appear 

to be a function of the duration of diabetes. 


SOD, inhibited by glycosylation, is lowered 

in poorly controlled diabetes mellitus. Due to 

the absence of protein synthesizing machinery 

in the erythrocytes, the inactivation SOD by 

glycosylation may be dominant factor in the loss 

of SOD activity observed. SOD deficiency is seen 

within 2 years of the detection of NIDDM and 

further these development of complications 16 . 

In diabetic subjects, the increased sorbitol 

synthesized caused NADPH depletion, which 

when deficient, limits the reduction of GSSG 

to GSH. Therefore, a major decrease in GSH 

may profoundly impair free radicals scavenging 

activity, resulting in exacerbated cell damage 

after exposure to free radical generated by 

glucose autoxidation. The levels of glutathione 

are regulated by glutathione peroxidase and 

glutathione reductase. The decreased activity of 

glutathione reductase in diabetics together with 

the decreased transport rate of GSSG indicates 

that regeneration and transport systems, which 

decrease intracellular GSSG, are impaired in 

diabetics, when erythrocytes are exposed to 

oxidative stress 17 . 

Conclusion : 

Therapeutic measures to increase antioxidants 

and control lipid peroxidation are warranted 

for effective control of complications. From 

the observations reported, it may be concluded 

that antioxidant deficiency and excessive 

peroxidative damage appear very early in 

NIDDM, well before the development of 

complications. 


Authors are grateful to the Head of the 

Department Biochemistry, Principal, J J M 

Medical College,Davengere for their support 

and encouragement. Authors are deeply 

indebted to all volunteers who participated in 

this study. 

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possible pathogenesis of diabetic angiopathies. Life Sci 

1984;34(6):577-84. 

16) Peuchant E, Delmes MCB, Couchouron A, Dubourg L, 

Thomas MJ, Peromat A et al. Short term insulin therapy and 

normoglycemia. Effects on erythrocyte lipid peroxidation 

in NIDDM patients. Diabetes Care 1997;20(2):202-7. 

17) Murakami K, Kondo T, Ohtsuka Y, Fujiwara Y, Shimada 

M, Kawakami Y. Impairment of glutathione metabolism 

in erythrocytes from patients with diabetes mellitus. 

Metabolism 1989;38(8):753-8. 


Clinico-Biochemical Profile of Hypokalemic Patients 

69 


*Anil Kumar Pandey, **Varsha Vijay Akhade, *** M Sri Hari Babu,****Y Himabindu 

*Department of Physiology, GSL Medical College & General hospital, 

Rajahmundry - 533294, Andhra Pradesh, India. 

**Department of Physiology Bidar Institute of Medical Sciences, Bidar, Karnataka 

***Department of General Medicine, GSL Medical College, 


****Department of Obstetrics & Gynaecology, GSL Medical College, 


(Received 21 st September,2010;Revised 20 th December,2010; Accepted 3 rd March,2011) 

Corresponding Author: 

Dr. Anil Pandey, 

E-mail: drpandeyak@yahoo.co.in 

Abstract 

Background and Objectives: Hypokalemia is a commonly encountered electrolyte disturbance with 

diverse range serious manifestations involving cardiovascular & neuromuscular systems. The kidney 

determines potassium homeostasis and excess potassium is excreted in the urine. The objective of the 

study was to find out clinico-biochemical spectrum of hypokalemia with special reference to preventable 

causative factors, so as to help early detection and appropriate management of such cases. 

Materials & Methods: Etiology of hypokalemia was assessed in patients admitted to Govt. Medical 

College & New Civil Hospital, Majura Gate, Surat over a period of 9-12 month. This was correlated with 

the clinical outcome and other clinical variables. Serum potassium was estimated by ion selective electrode 

method. During this period 896 investigations for electrolytes were carried out. 

Results: Out of these 378 investigations showed dyselectrolytemia. Hypokalemia was present in 76 patients. 

In majority of the cases hypokalemia was found to be associated with inadequate and/or inappropriate 

administration of fluid and/or drugs to the patients. However, patients receiving diuretics were at the highest 

risk as 39 out of 87 patients belonged to this category. Hypokalemia was observed in both the young as well 

as elderly patients. 

Conclusion: Hypokalemic patients with ECG changes had significant lower potassium levels than patients 

without ECG changes. 

However, the absolute correlation between the degree of potassium deficiency and adverse side effects 

was not found, possibly because the occurrence of side effects is related to both the degree and duration of 

potassium deficiency and underlying disease process. 

Keywords: Hypokalemia, Electrolyte, Diuretics. 

Introduction 

Hypokalemia is recurrently encountered in 

clinical medicine and has been anticipated to 

occur in approximately 20% of patients admitted 

to general medical and ICU wards(1). It may 

result from inadequate diet, transcellular shift 

(movement of potassium from serum into cells) 


Clinico - Biochemical Profile of Hypokalemic Patients 

and medications (2), Renal & Gastrointestinal 

losses and medications especially diuretics 

(13). Kypokalemia is also seen for 24 hours 

after Insulin injection (3). Patients may be 

asymptomatic, identified only on routine 

electrolyte screening, or may have serious 

manifestations like muscular paralysis, cardiac 

arrhythmias and cardiac arrest (4-6). Untreated 

hypokalemia is associated with morbidity 

and mortality. As timely intervention yields 

encouraging results, early detection with high 

of suspicion is critical to survival. 

Most cases of hypokalemia occur in the setting of 

specific disease states. Patients receiving thizide 

and loop diuretics are at the highest risk, with as 

many as 50% developing serum potassium level 

of less than 3.5 meq/L (7).Thiazide diuretics are 

more likely to cause hypokalemia than loop or 

osmotic diuretics. Individuals with secondary 

hyperaldosteronism, whether due to CCF, 

hepatic insufficiency, or nephritic syndrome may 

also exhibit hypokalemia. Though laboratory 

tests are the gold standard test for diagnosing 

changes in the serum electrolyte concentration, 

there may be delays in obtaining the results. 

The electrocardiogram (ECG) may be a useful 

diagnostic tool, if the clinician is aware of the 

possible changes resulting from abnormalities 

in the serum potassium concentration (10). 

In view of varied manifestations and etiological 

factors, an effort was made to study the clinicobiochemical 

spectrum of hypokalemia with 

special reference to preventable causative 

factors, so as to help early detection and 

appropriate management of such cases. 


A prospective hospital based study was 

conducted at New Civil Hospital and Govt. 

Medical College, Surat during the period from 

April 2003 to August 2004. Laboratory reports 

of in-patients were screened to identify cases of 

hypokalemia. Relevant history including that of 

treatment, mode of presentation, investigation 

results, etiological diagnosis, clinical outcome 

70 

and other clinical variables were recorded. 

Serum potassium and sodium were estimated 

by ion selective electrode method on Instalyte, 

Transasia. The hypokalemia was graded as 

mild (3-3.5), moderate (2-3 meq/L) and severe 

(


deficits like quadriparesis, paraparesis and 

monoparesis being more commonly seen with 

severe hypokalemia. The extent of neuromuscular 

manifestations were significantly 

related to degree of hypokalemia. 

The various etiologies of hypokalemia included 

severe gastrointestinal losses from diarrhea 

and vomiting, renal tubular disorders, diabetic 

ketoacidosis, dialysis or inadequate replacement 

during prolonged parenteral nutrition or due to 

administration of nephrotoxic drugs or laxatives 

( Table I). 

Two cases of post – insulin infusion also 

presented with signs of hypokalemia. One 

patient admitted with loose motions was 

normokalemic on admission but was found 

to be having raised blood sugar. Accordingly, 

insulin was administered. Next day, the serum 

potassium level dropped to 0.9 meq/L. Another 

patient of ALL on chemotherapy developed 

acute pancreatitis. Investigations reveals 

normokalemia but random blood sugar was 92. 

mg/dL, for which insulin was administered. Next 

day the investigations reveledhypokalemia. 

These two patients had normal levels os 

sodium, while other patients of hypokalemia 

also had hyponatremia. 

A patient of aplastic anemia with skin infection 

was being treated with amphotericin B. Initially 

the patient was normokalemic but during the 

course of 12-15 days of treatment , gradually 

developed hypokalemia. This was because of 

toxic effects of drugs on renal tubules. A total 

six patients died during the period of study. 

Discussion 

Hypokalemia has diverse clinical manifestations. 

Mild hypokalemia is usually asymptomatic, 

while moderate hypokalemia results in 

confusion, disorientation, weakness and 

discomfort of muscles. On occasion, moderate 

hypokalemia causes cramps during exercise. 

Another symptom of moderate hypokalemia 

is a discomfort in the legs that is experienced 

while sitting still. The patient may experience 

71 

an annoying feeling that can be relieved by 

shifting the positions of the legs or by stamping 

the feet on the floor. Severe hypokalemia 

results in extreme weakness of the body and 

on occasion, in paralysis. The paralysis that 

occurs is flaccid paralysis or limpness. Paralysis 

of muscles of the lungs results in death. In the 

present study, 5 severely hypokalemic patients 

manifested with severe weakness with inability 

to stand and walk. Out of 5 patients, 4 patients 

developed severe hypokalemia gradually over a 

period of seven days. 

Another dangerous outcome of severe 

hypokalemia is abnormal heart beat (arrhythmia) 

that can lead to death from cardiac arrest. Several 

prospective studies shown that hypokalemia 

predisposes the patients to the development of 

a variety of ventricular arrhythmias, including 

a fatal ventricular fibrillation. However, cardiac 

arrhythmia was not detected in the present. Since 

hypokalemia may cause arrhythmia, the ECG 

is usually carried out in these patients. In our 

study ECG was carried out in 37 out of aq total 

of 76 patients of hypokalemia. The ECG in 14 

patients showed changes due to hypokalemia, 

while the remaining 23 patients did not show 

any ECG changes. This result is in conformity 

with an earlier report (9). The probable reason 

for the inconsistency could be that the ECG 

reflects tissue potassium level rather than the 

serum potassiums. 

Diuretics are used to treat a number of medical 

conditions, including hypertension, congestive 

cardiac failure, liver disease and kidney disease. 

However, diuretic treatment can produce 

hypokalemia as a side effect and is the most 

common cause of hypokalemia in the elderly 

patients. The use of furosemide and thiazide, 

the two commonly used diuretic drugs were 

also the majaor cause of hypokalemia (n = 29) 

(38%) in the present study. 

More than 98% of the total body potassium 

is present in the intracellular compartment, 

predominantly in the skeletal muscle cells, 

enabling small changes in the distribution 

of potassium to alter the extracellular 



concentration markedly. Certain hormones 

such as, insulin activates Na + -K + ATPase, 

which results in active potassium uptake. 

Active insulin administration produces rapid 

potassium shift from from the extracellular to 

intracellular space, resulting in hypokalemia. 

Our study revealed two such cases of post 

insulin hypokalemia. One patient presented 

with loose motion and the other was a case 

of ALL. Both the cases were normokalemic 

on admission. Routine investigations of the 

first patient revealed very high blood sugar 

for which insulin was administered. The 

patient of ALL was put on chemotherapy who 

developed acute pancreatitis during the course 

of his illness and blood sugar was found to be 

raised. Accordingly, insulin was administered. 

Following day of insulin administration, 

the investigation of both patients revealed 

hypokalemia strongly suggesting that the drug 

had caused their hypokalemia. The condition of 

both the patients improved and the serum levels 

changed to normokalemia following potassium 

supplementation subsequently. 

Other common causes of hypokalemia are 

excessive diarrhea or vomiting. Diarrhea results 

in various abnormalities, such as dehydration 

(loss in body water), hyponatremia and 

hypokalemia. Little potassium is excreted in 

the stool under normal conditions because of 

a low stool volume and a low stool potassium 

concentration. However in case of diarrhea, 

substantial amount may be excreted by this 

route. In our study the presenting clinical feature 

of loose motions was present in 9 cases. All the 

patients had concomitant hyponatremia also. 

Vomiting or nasogastric suctioning, can lead to 

hypokalemia due to prolonged loss of gastric 

contents. A small part of this potassium loss 

is direct because these body fluids contain 

5-8 meq/L potassium. More importantly, 

concomitant alkalosis and intravascular volume 

depletion contribute to renal potassium loss. 

Metabolic alkalosis results in bicarbonateuria, 

which increases potassium excretion both 

72 

directly and, as a cation to balance the negative 

charge of bicarbonate ions, and indirectly, 

through stimulation of urinary sodium excretion, 

leading to worsening of intravascular volume 

depletion (13). All the seven hypokalemic 

patients who presented with vomiting in our 

study had concomitant hyponatremia. 

Hypokalemia is known to be associated 

with adverse outcomes and may have fatal 

consequences (10). Six of our patients died 

during the hospital stay mostly due to effect 

of underlying disease. Despite frequent 

measurement of serum potassium levels in the 

hospital patients, hypokalemia is a relatively 

common occurrence. Potassium deficiency 

alters the function of several organs and most 

prominently affects the heart and neuromuscular 

system(12,14). These effects ultimately 

determine the morbidity and mortality related to 

this condition. Although hypokalemic patients 

with ECG changes had significant lower 

potassium levels than patients without ECG 

changes. However, the absolute correlation 

between the degree of potassium deficiency 

and adverse side effects was not found, possibly 

because the occurrence of side effects is related 

to both the degree and duration of potassium 

deficiency and underlying disease process. 

The accurate treatment of hypokalemia requires 

correct identification of the cause. Hypokalemia 

can be associated either with normal or decreased 

total body potassium content. Normal total 

body potassium with hypokalemia is a result of 

potassium redistribution from the extracellular 

to the intracellular space (10). All the 3 patients 

put on the insulin therapy had shown ECG 

changes. The hypokalemia in these patients is 

a result of redistribution of potassium. Total 

body potassium depletion can result from either 

renal or extrarenal potassium losses (13). We 

suggest that the clinician evaluating a patient 

with hypokalemia consider two broad groups 

of etiologies; redistribution and true potassium 

deficit (table I). 

Initial treatment of hypokalemia is often 



inadequate, and sometimes treatment is delayed. 

Hospital-acquired hypokalemia was found to 

result from inadequate and / or inappropriate 

administration of fluids and / or drugs to patients. 

Patients receiving diuretics were at highest risk 

with 29 patients developing serum potassium 

levels of less than 3.5 meq/L. fifty percent of 

such patients have shown ECG changes as 9 

out of 17 patients in which ECG was carried 

out had changes related to hypokalemia (10). 

Hypokalemia was observed in both the young 

and elderly. It was associated with adverse 

outcomes. It may have fatal consequences, but 

timely intervention yields encouraging results 

(11). 

Conclusion 

A total of 976 investigations on electrolytes 

were carried out. In all 421 investigations 

had electrolyte imbalance. Hypokalemia was 

present in 76 patients in whom a total of 287 

investigations were carried out. ECG was done 

in 37 patients, out of which the findings were 

consistent with hypokalemia in only 14 patients. 

Remaining 23 patients did not show ECG 

changes. However, the absolute correlation 

between the degree of potassium deficiency and 

adverse side effects was not found, possibly 

because the occurrence of side effects is related 

to both the degree and duration of potassium 

deficiency and underlying disease process. 

References 

73 

1. Gary GS, and Brenner BM. Fluid and electrolyte disturbance. 

In: Harrison’s Principles of Internal Medicine. Mc- 

Graw-Hill, New York,14 th edi.272-4. 

2. Cohn JN, Kowey PR, Welton PK. New guidelines for potassium 

replacement in clinical practice: a contemporary 

review by the national council on potassium in clinical 

practice. Arch Intern Med 2000; 160 (16); 2429-2436. 

3. Massmi M, Akikatsu N and Yohei T. Electrolyte disorder 

following massive insulin overdose in a patient type II 

Diabetes. Intern Med 2000;39:55-57. 

4. Knochel JP. Neuromuscular manifestation of electrolyte 

disorders.Am J med 1982; 72: 521-35. 

5. Helfant RH. Hypokalemia and arrhythmias. Am J Med 

1986; 80 (suppl 4-A): 13-22. 

6. Cannon P. Recognizing and treating cardiac emergencies 

due to potassium imbalance. J Cardiovascular Med 1983; 

4:467-76. 

7. shilliday IR. Loop diuretics in the management of acute 

renal failure. A prospective , double blind, placebo control, 

randomized study. Nephrol Dial transplant 1997; 12:2592. 

8. Mandal AK. Hypokalemia and hyperkalemia. Med Clin 

North Am 1997;81:611-39. 

9. Singhi S, Marudkar A. Hypokalemia in a pediatric intensive 

care unit. Indian pediatr 1995; 33:9-14. 

10. A Webster, W Brady, F Morris Recognising signs of danger: 

ECG changes resulting from an abnormal serum 

potassium concentration.Emergency Medicine Journal 

2002;19:74-77; doi:10.1136/emj.19.1.74 

11. Kamel KS: Disorder potassium homeostasis: An approach 

based on pathophysiology. Am j Kidney Dis 1994; 24:597,. 

12. Latronico N, Shehu I, Seghelini E. Neuromuscular sequelae 

of critical illness. Curr Opin Crit Care 2005;11(4):381-90. 

13. Assadi F. Diagnosis of hypokalemia: a problem-solving approach 

to clinical cases. Iran J Kidney Dis 2008;2(3):115-22. 

14. Guideline of ECC Committee, Subcommittees and Task 

Forces of the American Heart Association. 2005 American 

Heart Association Guidelines for Cardiopulmonary Resuscitation 

and Emergency Cardiovascular Care. Circulation 

2005;112(24)(Suppl-IV),1-203. 


74 


Antifungal activity of Bacopa monniera against Dermatophytic Fungus 

*S.R.Ayyappan, **R.Srikumar, ***R.Thangaraj, ****R.Jegadeesh, ****L. Hariprasath 

* Department. of Microbiology,Sathyabama University Dental College & Hospital 

Jeppiaar Nagar, Old Mahabalipuram Road,Chennai 600 119, India 

** Department of Microbiology,Bharathidasan University College for Women, 

Orathanadu-614625,Thanjavur, Tamilnadu, India 

*** Ecoscience Research Foundation,East Coast Road, Palavakkam, Chennai 600041 

Tamil Nadu, India. 

**** Center for Advance Studies in Botany, University Of Madras, 

Guindy Campus Chennai- 600 025, Tamil Nadu, India. 

(Received 12 th June, 2010; Revised 10 th December, 2010; Accepted 20 th February, 2011) 


Dr.R.Srikumar, 

E-mail: rsrikumar_2003@yahoo.co.in 

Abstract 

Background & Objectives: Dermatophytes are fungi that can cause infections in keratin tissue constitute 

a serious problem, especially in tropical and subtropical countries. Antimicrobials of plant origin are effective 

in the treatment of infectious diseases while simultaneously mitigating many side effects that are often 

associated with synthetic antimicrobials. The lack of data on antifungal activity of Bacopa monniera makes 

this study unique. 

Methods: In the present study crude aqueous and ethanolic extract of B. monniera were prepared and their 

antifungal effects were assessed against the dermatophytic fungi namely Aspergillus niger, Aspergillus 

flavus, Trichophyton rubrum and Microsporum. 

Results: Result showed ethanolic extract had high inhibitory action against the studied fungus when 

compared to aqueous extract. In both ethanolic and aqueous extract the maximum inhibition activity was 

observed against T. rubrum 80% followed by Microsporum, A. flavus and A. niger. In addition this study 

also showed ethanolic extract is an ideal solvent. 

Conclusion: The present study revealed that B. monniera have antifungal activity. Among the extracts 

evaluated in this study, ethanolic extract showed highest antifungal activity. 

Key words: Bacopa monniera; Dermatophyte; Antifungal activity 

Introduction 

Dermatophytes are fungi that can cause 

infections of the skin, hair, and nails due to their 

ability to utilize keratin. The organisms are 

transmitted by either direct contact with infected 

host by direct or indirect contact with infected 

exfoliated skin or hair. These infections, which 

constitute the most frequent fungal diseases in 

human, are widespread in tropical countries. 

Due to the increasing development of drug 

resistance in human pathogens as well as the 


appearance of undesirable effect of certain 

antimicrobial agents, there is a need to search for 

new agents. Plants are among the most important 

and common sources of potentially valuable 

new drugs. Medicinal plants are widely used for 

treatment of diseases all over the world. Herbal 

medicines have been used in treatment of the 

various diseases and for maintenance health (1). 

In addition antimicrobials of plant origin are 

effective in the treatment of infectious diseases 

while simultaneously mitigating many side 

effects that are often associated with synthetic 

antimicrobials (2). B. monniera belongs to the 

family Scrophulariaceae, commonly called 

as Brahmi, Neer brahmi etc. B. monniera is a 

small creeping herb with numerous branches, 

small oblong leaves and light purple or small 

and white flowers, with four or five petals. B. 

monneri is reported for its tranquilizing (3), 

sedative (4), stabilization of mast cells (5), 

anti-inflammatory activity via inhibition of 

prostaglandin synthesis (6), anticancer effect by 

inhibiting the DNA replication in cancer cells 

(7). The present study describes the antifungal 

potential of aqueous and ethanolic extract of B. 

monniera against the dermatophytic fungi. 


Plant Material 

B. monniera was collected from the Anna 

Sidha College, Chennai, Tamil Nadu, India and 

taxonomically identified by, the Department of 

Botany, University of Madras, Chennai, India, 

and specimens were deposited at an herbarium 

(No: CASBH12). 

Aqueous Extract 

The entire plant of B. monniera was grounded 

into fine powder, maintained at 60 ºC for 3 hr in 

sterile distilled water. The resulting suspensions 

were filtered and evaporated for dryness at 60°C 

in vacuo. 

Ethanolic Extract 

75 

B. monniera (coarse powder) was put in a 

soxhlet extractor containing 70% of ethanol. 

The resulting extract was preserved at 5 o C in an 

airtight bottle until further use. 

Assay of Antifungal Activity 

The fungus A. niger, A. flavus, T. rubrum 

and Microsporum used in this study were 

maintained by culturing on Sabouraud dextrose 

agar (SDA) at 28 °C. The antifungal test was 

performed by employing the method described 

by Yongabi with slight modification (8). In 

briefly 500mg of each extract was diluted with 

the assay media (100 ml). A uniform portion of 

the test fungi was removed using a 5mm steel 

borer and aseptically placed on the assay media. 

All plates were carefully sealed all around with 

a masking tape to avoid any aerial contaminants 

and carefully incubated at 28°C for 7 days. The 

rate of mycelia growth was measured in mm on 

8 th day. Ketoconazole (50mg/ml) was used as an 

experimental positive control and water served 

as the negative control. 

Calculation 

Percentage of Mycillial inhibition = [(dc-dl)/dc] 

x 100 

dc = colony diameter in negative control, d1 

colony diameter in extract treatment 

Results 

The studied aqueous and ethanolic crude 

extracts had antifungal activities against the 

studied fungi, but the activity of inhibition 

varied for the fungi with respect to the type of 


Table. 1 Effect of aqueous and ethanolic extracts of B. monniera on the growth rate of A. niger, A. flavus, T. 

rubrum and Microsporum sp (inhibition expressed in percentage). 

extract (Table. 1). In aqueous extract maximum 

inhibition activity was observed against T. 

rubrum (40%) followed by Microsporum 

(38%), A. niger (31.4%), and A. flavus (31.2%). 

In ethanolic extract the maximum inhibition 

activity was observed against T. rubrum (50%) 

followed by Microsporum (47.6%), A. niger 

(42.8%) and A. flavus (40.6%). Overall result 

showed ethanolic extract had high inhibitory 

action against the studied fungus with respect to 

the aqueous extract. 

Discussion 

Plant Extract A. niger A. flavus T. rubrum Microsporum 

Aqueous extract of B. 

monniera 

Ethanolic extract of B. 

monniera 

The emergence of anti-fungal resistant strain 

of various fungi particularly dermatophyte 

has prompted research into developing new 

strategies for fighting fungal infections which 

may be less toxic to man (9). Both the aqueous 

and ethanolic extracts of B. monniera showed 

inhibitory activity against the studied fungus 

(Table. 1). Ethanolic extract showed high 

inhibitory action against the studied fungus 

when compared to aqueous extract. Most of 

the phytochemcials already identified in herbs 

are reportedly aromatic or saturated organic 

molecules which make ethanol as an ideal 

solvent this might be the reason for a better 

antifungal activity observed in the ethanolic 

extract (10). The inhibitory action of both the 

31.4 31.2 40 38 

42.8 40.6 50 47.6 

Ketoconazole Control 71.4 75 85 76.1 

76 

extracts observed in the present study might be 

due to rupture of the cytoplasmic membrane of 

the fungal cell which may leads to the damage 

of intracellular components (11). 

Conclusion 

This study evidently concludes that B. monniera 

have anti-fungal activity. The exact mechanism 

behind the antifungal activity of B. monniera 

warrants further studies. 

Acknowledgement 

The authors are thankful to Dr.R.Sheela Devi, 

Associate Professor, Department of Physiology, 

University of Madras, Chennai, Tamilnadu, 

India. 

References 

1. Sahito SR, Memon MA, Kazi TG, Kazi GH. Evaluation 

of mineral contents in medicinal plant Azadirachta indica 

(neem). J Chem Soc Pak 2003; 25: 139-143. 

2. Kokosha L, Polesny Z, Rada V, Nepovim A, Vanek 

T. Screening of some Siberian medicinal plants for 

antimicrobial activity. J Ethnopharmacol 2002; 82: 51-53. 

3. Aithal HN and Sirsi M. Pharmacological investigation of 

Herpestis monneri. Indian J Pharmacol 1961; 23: 2-5. 

4. Malhotra CL and Das PK. Pharmacological studies of 

Herpestis monneri. Indian Journal of Medical Research 

1959, 47: 244-305. 

5. Samiulla DS, Prashanth D, Amit A. Mast cell stabilizing 

activity of Bacopa monnieri. Fitoterapia 2001; 72: 284-285. 


6. Jain P, Khanna NK, Trehan T, PendseVK and Godhwani 

JL. Antiinflammatory effects of an Ayurvedic preparation, 

Brahmi Rasayan, in rodents. Indian J Exp Biol 1994; 32: 

633-636. 

7. Elangovan V, Govindasamy S, Ramamoorthy N, 

Balasubramaanian K. In vitro studies on the anticancer 

activity of Bacopa monnieri. Fitoterapia 1995; 66: 211-215. 

8. Yongabi KA, Dukku UH, Agho MO, Chindo IY. Studies 

on the antifungal properties of Urtica dioica, Urticaceae. J 

Phytomedicine and Therapeutic 2000; 5: 39-43. 

77 

9. Patterson TF, Revankar SG, Kirkpatrick WR, Dib O, 

Fothergill AW, Redding SW, Sutton DA, Rinaldi MG. Simple 

method for detecting fluconazole-resistant yeasts with 

chromogenic agar. J Clin Microbiol 1996; 34: 1794-1797. 

10. Cowan MM. Phytochemicals: health protective effects. 

Can J Diet Pract Res 1999; 60: 78-84. 

11. Chuang P, Lee C, Chou J, Murugan M, Shieh B, Chen H. Antifungal 

activity of crude extracts and essential oil of Moringa 

oleifera Lam. Bioresour Technol 2007; 98: 232–236. 


78 


Prevalence of Extended Spectrum Beta Lactamase producing Enterobacteriaceae 

in Clinical specimens 

*G.S. Ravi, **B.V.S.Krishna, ***Namratha W.Nandihal,***Asha B.Patil and 

****M.R. Chandrasekhar. 

* Department of Microbiology, PES Institute of Medical Sciences and Research, 

Kuppam,Chittoor - 517425, Andhra Pradesh, India. 

**Department of Microbiology & Infection Control,University Hospital of North Staffordshire 

NHS Trust,Central Pathology Laboratories,Hartshill Road, Stoke on Trent ST4 7PX, 

United Kingdom. 

***Department of Microbiology,Karnataka Institute of Medical Sciences, 

Hubli - 580022, Karnataka, India. 

*** Department of Microbiology, Karnataka Institute of Medical Sciences, 


****Department of Microbiology,Belgaum Institute of Medical Sciences, 

Belgaum - 590001, Karnataka, India. 

(Received 1 st November, 2010; Revised 2 nd January, 2011; Accepted 20 th January, 2011) 


Dr. Ravi. G. S 

Email: ravinavilehal@yahoo.com 

Abstract 

Background and objectives: Extended spectrum beta lactamases (ESBLs) are broad spectrum β-lactamase 

enzymes found in variety of Enterobactriaceae. Most strains producing these enzymes become highly 

effective at inactivating various β- lactam antibiotics and are frequently resistant to many other classes 

of antibiotics. The incidence of infection due to ESBL producing Enterobacteriaceae has markedly 

increased in recent years. The present work was undertaken to study the prevalence of ESBL producing 

Enterobactriaceae in clinical specimens at a tertiary care hospital. 

Methods: A total of 313 culture isolates of Enterobactriaceae obtained from different clinical specimens 

were included. All the isolates were subjected to screening tests for ESBL production & later for confirmation 

by phenotypic confirmatory combination disc diffusion test. 

Results: Out of 313 strains tested, 248 were short listed as potential ESBL producer by screening test and 

200(63.89%) were confirmed to be ESBL producers. ESBL rates were high among Klebsiella oxytoca 

(89.47%), Klebsiella pneumoniae (71.87%), and Escherichia coli (62.19%). They were commonly isolated 

from blood (92.30%), CSF (78.57%) and Urine (65.21%). The multidrug resistance was significantly higher 

among ESBL producers than in non-ESBL producers (p value of

Enterobacteriaceae in Clinical specimens 

Introduction 

Extended spectrum b-lactamases (ESBLs) 

are enzymes that confer resistance to and 

hydrolyze expanded spectrum cephalosporins 

like ceftazidime, cefotoxime, monobactam 

-azteronam and related oxyimino b-lactams as 

well as to older penicillins and cephalosporins 

(1,2). They arise from mutations in the genes 

for common plasmid mediated b-lactamases 

especially TEM and SHV enzymes, that alter the 

configuration of the enzyme near its active site 

to increase the affinity and hydrolytic ability of 

the b-lactamase for oxyimino compounds while 

simultaneously weakening the overall enzyme 

efficiency. Wide spread use of third generation 

cephalosporins and aztreonam is believed to 

be the major cause of the mutations leading to 

emergence of ESBLs (3). 

The first bacterial isolate capable of hydrolyzing 

extended spectrum cephalosporins producing 

an extended spectrum b-lactamase, SHV-2 was 

identified in Klebsiella ozaenae in Greece in 

1983 (4). There after, within one year there was 

wide spread dissemination of ESBL producing 

organisms in hospitals of Europe (5). The 

prevalence of ESBLs among clinical isolates 

varies from country to country and from 

institution to institutions. The first reported 

outbreak of ESBL producing organisms 

occurred in France in 1985(1, 6). More recently, 

outbreaks have occurred worldwide, including 

several cities in the United Sates (4, 7, 8, 9 ). 

ESBLs were initially associated with nosocomial 

outbreaks caused by a single enzyme producing 

strains. Recently more complex situations with 

a significant increase in community isolates 

producing ESBLs have been reported. It is 

necessary to identify the prevalence of these 

ESBL strains in hospitals and to characterize 

their epidemiology to control the spread of 

these strains (10). This upward trend in the 

prevalence of pathogens producing ESBLs is 

of increasing clinical concern. Infections with 

79 

these ESBL producing organisms continue to 

be associated with higher rates of mortality, 

morbidity and health care costs (11). Also 

ESBL mediated resistance poses problems for 

invitro susceptibility testing and reporting (2). 

Prevalence of ESBLs in India varies from 6 

to 87% (12). With this background, the present 

study was undertaken to know the prevalence 

of ESBL producers among Enterobacteriaceae 

isolates and to compare antimicrobial 

susceptibility pattern among ESBL producers & 

Non ESBL producers, and to suggest appropriate 

therapeutic options. 


The study was conducted at Microbiology 

laboratory, KIMS Hospital Hubli, Karnataka. 

Clinical Isolates: A total of 313 consecutive 

non repeat culture isolates Enterobacteriaceae 

were obtained from 280 different specimens 

from different specialty wards of Surgery, 

Orthopaedics, Otolaryngology and Neonatal 

intensive care unit (NICU) over a period of one 

year. The isolates were identified on the basis of 

conventional microbiological procedures. 

Antimicrobial susceptibility test 

Antimicrobial susceptibility testing was 

determined by Kirby-Bauer disc diffusion 

method as per NCCLS recommendations 

(13). Antimicrobial disc used were 

Ampicillin(10µg), Co-trimoxazole(1.25/23.7 

5µg),Amoxycillin-clavulanicacid(20/10µg),T 

etracycline(30µg), Gentamicin(10µg),Amika 

cin(30µg),Netilmicin(30µg),Ciprofloxacin(5 

µg),Cephalexin(30µg),Cefuroxime(30µg),C 

ephotaxime(30µg),Ceftriaxone(30µg),Cefop 

erazone(30µg),Celftazidime (30µg), Cefixime( 

30µg),Cefpodoxime(30µg), Imipenem (10µg). 

Antimicrobial susceptibility patterns of ESBL 

producers and non producers were compared. 



Screening test for ESBLS 

As per the NCCLS guidelines, isolates 

showing inhibition zone size of ≤22mm 

with Ceftazidime(30µg), ≤25 mm 

with Ceftriaxone(30µg) and ≤27 mm 

Cefotaxime(30µg) were identified as potential 

ESBL producers and short listed for confirmation 

of ESBL production. NCCLS Phenotypic 

confirmatory combination disc diffusion test 

for detection of ESBL was performed (13, 14). 

This test requires the use of third generation 

cephalosporin antibiotic disc alone and in 

combination with clavulanic acid. In this study, 

a disc of Ceftazidime (30 µg) alone and a disc 

of Ceftazidime +clavulanic acid (30µg/10µg) 

were used. Both the discs were placed at a 

distance of 25 mm apart, centre to centre on 

Mueller Hinton Agar (MHA) plate inoculated 

with a bacterial suspension of 0.5 McFarland 

turbidity standards and incubated overnight at 

37 O C. Difference in zone diameters with and 

without clavulanic acid were measured. 

Interpretation 

An increase in inhibition zone diameter of 

5mm for a combination disc of ceftazidime 

+ clavulanic acid disc versus the inhibition zone 

diameter around the ceftazidime disc alone, 

confirms ESBL production. 

Quality control 

Escherichia coli ATCC 25922. A 2mm increase 

in zone diameter for ceftazidime clavulanic acid 

disc compared with ceftazidime disc as negative 

control. Klebsiella pneumoniae ATCC 700603. 

A 5mm increase in ceftazidime clavulanic acid 

disc zone diameter as positive control. 

The plates for the susceptibility tests were 

prepared in the laboratory. Mueller – Hinton 

Agar (Dehydrated, Hi-Media) was used for 

80 

all antimicrobial susceptibility tests. All the 

antibiotic discs were obtained from Hi-media 

laboratories Pvt. Limited, Mumbai, India. 

Statistical Analysis 

Chi-square test was used with appropriate 

correction for the observation. Where the cell 

frequency was less than five, Fisher exact test 

was applied to see the significance of difference 

between the resistance levels of various drugs in 

ESBL producer strains and non-ESBL producer 

strains using EPI 6 software. 

Results 

A total of 313, non-duplicate isolates belonging 

to the family Enterobacteriaceae recovered 

from 280 different clinical samples of 279 

patients (183Male/96 Female) submitted for 

microbiological analysis from different clinical 

specialties, were studied. The major source of 

bacterial isolates were pus(215), blood(39), 

urine(23) and CSF(14), followed by Ear swab(8), 

Sputum(5), Ascitic fluid(3), Pleural fluid(3), 

Bile(2) and Throat swab(1) (Table 1). Genus 

Klesbsiella was the most common pathogen 

isolated from the tested samples, constituting 

115/313(36.74%) of the total isolates, Klebsiella 

pneumoniae represented 96/313(30.67%) and 

Klebsiella oxytoca19/313(6.07%). Followed 

by Escherichia coli 82/313(26.17%). Of the 

313 isolates, subjected for screening test, 248 

were short listed as potential ESBL producers 

with initial NCCLS screening method. NCCLS 

Phenotypic confirmatory identified 200 out of 

248 as ESBL producers, indicating a prevalence 

of 63.89% of ESBL production amongst all 

Enterobacteriaceae. 

Distribution of ESBL producers varied among 

different species of Enterobacteriaceae. Rates 

were high among Klebsiellae oxytoca(89.47%), 

Klebsiellae pneuminae(71.87%), Escherichia 



coli(62.19%), Citrobacter freundii(62.50%), 

Proteus mirabilis(61.11%), Proteus 

vulagaris(50.00%) and Providencia 

species(44.00%) which were the major isolates 

in the group. Even among the organisms detected 

in less numbers, ESBLs were detected in 30% of 

Citrobacter koseri and in 100% of Enterobacter 

species and Morganella morgagni (Table 2). 

ESBL producers were more commonly isolated 

from blood 36/39(92.30%) followed by CSF 

11/14 (78.57%), urine 15/23(65.21) and pus 

131/215(60.93%) (Table1). The multi drug 

resistance was significantly higher among ESBL 

producers than in non-ESBL producers with 

a p value of


the usage of carbapenem antibiotics as the 

therapeutic alternative to β lactam antibiotics 

as indicated in other studies (22). This is 

especially important when treating serious 

infections. Misuse and overuse of carbapenems 

can lead to emergence of metallobetalactamases 

production among Enterobacteriaceae, which 

can still worsen the therapeutic options, as 

carbapenemases already exist and we have been 

contributing the growing types of them. 

Conclusion 

There is high prevalence of ESBL producers 

among Enetrobacteriaceae isolates, more so 

in Klebsiella species & Escherichia coli. Not 

just the blood & urine samples are the major 

source; even the other clinical samples should 

also be actively screened for ESBL producers 

to address the high prevalence rate of ESBL 

isolates. ESBL producing organisms often 

show co resistance to non β lactam antibiotics, 

resulting in limitation of therapeutic options. 

Use of a proper screening & confirmatory 

test for detection of ESBL producers, along 

with proper monitoring and judicious usage 

of extended spectrum cephalosporins, periodic 

surveillance of antibiotic resistance patterns, 

existence & effective functioning of Infection 

Control committee of doctors and nurses 

(microbiologists should lead the role of Infection 

control), Education of all grades of doctors 

about the dangers of antibiotic use, and having 

effective control over antibiotic prescription in 

the community and in hospitals, would prevent 

the increase occurrence of ESBL producers. 

References 

1. Coudron PE, Molland ES, Sanders CC. Occurrence 

and Detection of Extended Spectrum β-Lactamases in 

members of he family Enterobacteriaceae at A Veterans 

Medical Centre: Seek and You May Find. J Clin Microbial 

1997; 35(10):2593-2597. 

2. Emery CL. Weymouth LA. Detection and Clinical 

82 

Significance of Extended Spectrum b-Lactamases in a 

Tertiary care Medical Center. J Clin microbial 1997; Aug: 

2061-2067. 

3. Chaudhary U, Aggarwal R, Extended Spectrum 

b-Lactamases-An Emerging threat to Clinical therapeutics. 

Ind J Med Microbial 2004; 22 (7): 75-80. 

4. Bradford PA. Extended Spectrum b Lactamases in the 21 st 

Century: Characterization, Epidemiology and Detection 

of This Important Resistance Threat. Clin Microbial Rev 

2001;14(4): 933-951. 

5. Nathisuwan S, Burgess DS, Lewis JS. Extended Spectrum 

b-lactamaes: Epidemiology, Detection and Treatment. 

Pharmacotherapy 2001; 21(8): 920-928. 

6. Sirot DJ, Sirot R, Labia A, Morand P, Courvalin A, 

Darfeuille–Michael R, Perroux, Clunzel R. Transferable 

resistance to third generation cephalosporins in clinical 

isolates of K. pneumoniae, identification of CTX-1, a novel 

b-lactamase. J Antimicrob Chemother 1987; 20: 323-334. 

7. Sirot D. Extended spectrum plasmid–mediated 

b-lactamases. J Antimicrob Chemother 1995; 36: Suppl.A 

:19-34. 

8. Vercauteren E, Deschecmaeker P, Ieven M, Sanders CC, 

Goossens H. Comparison of Screening Methods for 

Detection of Extended Spectrum b-Lactamases and their 

Prevalence among Blood Isolates of E.coli and Klebsiella 

spp. In a Belgian Teaching Hospital. J Clin microbial 1997; 

35 (9): 2191-2192. 

9. Tenover FC, Ranxey PM, Williams PP, Rasheed JK, 

Biddle JW, Oliver A et al. Evaluation of the NCCLS ESBL 

confirmation methods for E.coli with isolates collected 

during project ICARE. J Clin Microbiol 2003; 41(7):3142- 

3146. 

10. Ananthakrishnan AN, Kanungo R, Kumar A, Badrinath S. 

Detection of Extended Spectrum b-Lactamase producers 

among Surgical wound infections and Burns patients in 

JIPMER. Ind J Med Microbiol 2000; 18 (4):160-165. 

11. Manour Al- Zarouni, Abiola Senok, Fatima Rashid, Shaika 

Mohammed Al-Jesmi and Debadatta Panigrahi. Prevalence 

and Antimicrobial susceptibility pattern of Extended 

Spectrum b-Lactamase Producing Enterobacteriaceae in 

United Arab Emirates. Med Princ Pract 2008; 17:32-36. 

12. Parul Aggarwal, Ghosh AN, Satish Kumar, Basu B, Kapila 

K. Prevalence of extended spectrum b-Lactamases among 

Escherichia coli and Klebsiella pneumoniae isolates in a 

tertiary care hospital. Ind J Pathol Microbial 2008; 51 (1): 

139-142. 

13. National Committee for Clinical Laboratory Standards. 

Performance standards for Antimicrobial disk susceptibility 

testing; 12 th Informational Spplement. Wayne, PA, USA: 

NCCLS 2002; M 100-S12 

14. Livermore DM. b Lactamases in laboratory and clinical 

resistance. Clin Microbiol Rew 1995; 8(4): 557-584. 

15. Spanu T, Luzzaro F, Perilli M, Amicosante G, Toniolo 

A, Fadda G et. al. Occurrence of Extended-Spectrum 

b-Lactamases in member of family Enterobacteriaceae 

in Italy. Implications for resistance to b-lactams and other 

antimicrobial drugs. Antimicrobial Agents chemother 

2002; 46 (1):196-202. 

16. Jabeen K, Zafar A, Hasan R. Comparison of double disc 



and combined disc method for detection of Extended 

Spectrum b Lactamases in Enterobacteriaceae. J Pak Med 

Assoc 2003; 53(1):534-536. 

17. Venezia SN, Munz OH, Scwartz D, Turner D, Kuzmenko 

B, Carmeli Y. Occurrence and Phenotypic characteristics 

of Extended Spectrum b Lactamases among the members 

of Enterobacteriaceae, Evaluation of Diagnostic tests. J 

Clin Microbiol 2003; 41(1):155-158. 

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Extended Spectrum b Lactamase producing GNB in a 

83 

tertiary care hospital. Indian J Med Res 2000; 15:153-157. 

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Govind R, Rattan A. Evaluation of methods for Amp C b 

lactamases in Gram Negative clinical isolates from Tertiary 

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Prevalence of Extended Spectrum b Lactamases producing 

Gram negative bacteria in septicemic neonates in a tertiary 

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84 


Anti-inflammatory effects of Allium sativum (Garlic) in experimental Rats 

*M.K.Jayanthi and **Murali Dhar 

*Department of Pharmacology and ** Department of Community Medicine, JSS Medical College, 

(A constituent college of JSS University), Mysore-570015, Karnataka, India. 

(Received 21 st September, 2010; Revised 15 th December, 2010; Accepted 10 th January, 2011) 


Dr Jayanthi M.K. 

Email: dr_jmgatti@yahoo.com 

Abstract 

Background and Objectives:Non-steroid anti-inflammatory drugs (NSAIDs) are known to cause 

adverse effects. On the other hand, garlic is believed to have hypolipidaemic, antibacterial, anti-rheumatic 

and anti-inflammatory properties. Hence present study was undertaken to know the Anti-inflammatory 

effect of allium sativum in Albino Rats. 

Materials and Methods: A total of 36 rats were randomized into two groups of 18 each to be utilized 

by acute (Carrageenin induced paw edema) and chronic (Cotton pellet induced granuloma) inflammatory 

models. Further within each group the animals were randomly allocated to the control, standard (Piroxicam) 

and test (Garlic) drug groups. The rats were fed respective drugs orally 1 hour prior to experimentation. 

Reduction in paw edema size was recorded at four hours and in dry granuloma weight on 8 th day after 

sacrificing the animal. To compare the reductions among three groups, one-way ANOVA was performed. 

Results: The effect of the test drug in terms of percent inhibition was found to be about 50% in acute and 

21% in chronic models. Also, the acute effect was similar to standard drug. Conclusion: In view of the side 

effects of NSAIDs and corticosteroids, it may be envisaged that garlic can be used as an adjuvant in the 

inflammatory disorders. 

Key words: Allium sativum, Anti-inflammatory, Carrageenin, Edema, Granuloma. 

Introduction 

Inflammation is the characteristic response 

of mammalian tissue to injury. Whenever 

tissue is injured, there follows at the site of 

injury a series of events that tend to destroy 

or limit the spread of the injurious agent(1). 

The introduction of acetyl salicylic acid, 

cortisone, gold salts and phenylbutazone for 

the treatment of inflammatory disorders is an 

important milestone in the development of 

clinically useful anti-inflammatory agents (2). 

Currently available anti-inflammatory agents 

are associated with their own side effects. It 

has been estimated that about 34 to 46 percent 

of the users of NSAIDs will sustain some 

gastrointestinal damage due to the inhibition 

of the protective cyclooxygenase enzyme in 

gastric mucosa (3). Therefore, researchers 

have aimed at identifying and validating 


Anti-inflammatory effects of Allium sativum 

plant derived substances for the treatment of 

various diseases. The added advantages of 

indigenous medicinal treatment would include 

its complementary nature to the conventional 

treatment making latter safer, well tolerated 

and economical remedy for inflammatory 

conditions. Allium sativum (Garlic) is a hardy 

perennial bulbous scapigerous herb, with a flat 

stem. The lower portion of the plant forms a 

bulb, which consists of several smaller buds 

called cloves, surrounded by a thin white or 

pinkish sheath. The leaves are flat, narrow 

green; the heads bear small white flowers and 

bulbils. Since ancient days, it has been cultivated 

throughout India, Pakistan, Bangladesh, and 

most tropical countries (4). Garlic has shown 

various activities like hypoglycemic, antiinflammatory, 

anti-cancerous, anti-rheumatic, 

anti-platelet, anti-helmenthic, hypolipidaemic 

and radio protective effects on various animal 

models (5-9). Therefore, present study was 

conducted with the aim of investigating antiinflammatory 

activity of garlic powder. Specific 

objectives of the study were, a) to estimate 

the anti-inflammatory effects of garlic powder 

in acute and chronic inflammatory animal 

models, and b) to compare the effects with that 

of established anti-inflammatory drug, namely, 

Piroxicam. 

Material and Methods 

Preparation of powder: 

Garlic powder was made from chopped 

garlic cloves that were oven-dried and then 

pulverized. The fine powder thus obtained 

was kept in the air tight containers. The yield 

of garlic powder from the garlic bulbs after 

the processing was found to be 250 gm per kg 

of garlic bulbs . A pilot study was done with 

different doses (5 mg/kg, 50 mg/kg, 100 mg/ 

kg and 200 mg/kg) to estimate the dose for the 

study. The ant-inflammatory activity was found 

to be significant at the dose of 100 mg per kg of 

body weight and hence the same dose was used 

in the study. 

85 

Chemicals: Piroxicam, Carrageenin (Sigma) 

and all other chemicals were of analytical grade. 

Animals: Animals used were albino rats of 

either sex, weighing between 120-160 Grams. 

The animals were procured from animal research 

laboratory, National Institute of Mental Health 

and Neuro Sciences (NIMHANS), Bangalore 

and housed in the animal house of the institute 

in groups of 3, at an ambient temperature of 

25±1°C with ad libitum access to food and 

water. The study protocol was approved by 

Institutional Animal Ethics Committee. 

Methods 

To study the anti-inflammatory effect of the 

drugs, two models, namely, Carrageenin 

induced rat paw edema Animal Model and 

Cotton pellet induced Granuloma Animal 

Model were utilized. 

A total of 36 rats were divided into two groups 

of 18 each to be utilized for the two models. 

Further, within each model, animals were 

randomly allocated to the 3 groups of 6 rats 

each; I group: Control (1 ml of Vehicle, 2% 

Gum acacia suspension); II group: Standard 

drug (Piroxicam 100 mg/kg); III group: Test 

drug (Garlic 100 mg/kg). 

Carrageenan induced rat paw edema Animal 

Model 

This is an established animal model to screen 

the acute anti-inflammatory activity of the drugs 

(10, 11). The animals were pretreated with 

drugs orally one hour before the experiment. 

Carrageenan (0.05 ml, 1%) was injected 

aseptically into the subplantar surface of right 

hind paw of each rat. Paw edema was measured 

by Mercury Plethysmograph (UGO Basile, 

Italy) immediately (i.e., at ‘0’ hour) and at the 

end of ‘4’ hours. The difference between the 

measurements at 0 and 4 hours was taken as the 

actual inhibition of edema. 



Cotton pellet induced Granuloma Animal 

Model 

This is an established animal model to screen the 

chronic anti-inflammatory activity of the drugs 

(12). Four sterile cotton pellets weighing 10 mg 

each were implanted subcutaneously in both the 

axilla and groin of each rat. Rats were fed with 

the respective drug daily for 7 days along with 

free access to water and food ad libitum. Later 

the animals were sacrificed on the 8 th day and 

the cotton pellets with granulation tissue were 

removed, cleaned of the extraneous tissue and 

dried in a hot air oven to a constant weight and 

the dry granuloma weight was determined. The 

dry weight of the granuloma (i.e. the amount of 

actual granulation tissue formed) was calculated 

by noting the difference in the dry weight of 

the cotton pellets recorded before and after 

implantation. 

Statistical Analysis: 

The effects of standard and test drugs were 

estimated by working out mean and standard 

deviation of paw edema size (in cm) and dry 

granuloma weight (in mg), followed by the 

estimation of percent inhibition applying the 

formula given below. Percentage inhibition 

by the drug = (W d -W c ) / W c X 100, where 

W d is the effect in intervention group and W c 

is the effect in the control group. One-way 

analysis of variance (ANOVA) was performed 

to test the significance of differences in mean 

paw edema and mean dry granuloma weight 

among the three groups followed by Scheffe’s 

post-hoc test. All the tests of significance 

were interpreted at 5%, 1% or 0.1% level of 

significance. In order to quantify the effects of 

the drugs, we calculated the inflammation in 

the drug groups relative to the control group 

and presented the same using bar diagram. Two 

relative measures were calculated as following. 

Relative effect of a drug = (W d /W c ) X 100 

Results 

86 

Acute anti-inflammatory effect The acute 

anti-inflammatory effect of garlic powder 

and standard drug was studied by looking at 

the changes in Carrageenin-induced Rat Paw 

Edema. This model suggested a statistically 

significant anti-inflammatory effect of both 

the garlic powder and the Piroxicam drug. The 

induced rat paw edema was found to be about 

half in the two drug groups compared to the 

control group. Chronic anti-inflammatory effect 

The chronic anti-inflammatory effect of 

garlic powder and standard drug was studied 

by looking at the changes in Cotton pellet 

induced dry granuloma weight. This model 

also suggested a statistically significant antiinflammatory 

effect of both the garlic powder 

and the Piroxicam drug. The induced granuloma 

in standard and test drug groups was found to 

be respectively 65 and 80 percent of the control 

group. Test drug versus standard drug As for as 

the comparison of the test drug with the standard 

drug is concerned; the two showed a similar 

effect according to paw edema model. However, 

there was stastically significant difference in the 

effect of two drugs according to chronic model. 

In this model, the inhibition was about 21% 

due to the test drug compared to 35% due to 

the standard drug. Thus, although the test drug 

had a significant effect, quantitatively however, 

it was not as much as the standard drug had. 

Discussion 

The introduction of drugs like Aspirin, Cortisone, 

Indomethacin and others has revolutionized the 

treatment of rheumatic and musculoskeletal 

disorders. The amazing efficacy of cortisone 

and phenylbutazone in these inflammatory 

disorders has paved the way for the introduction 

and use of newer anti-inflammatory agents. 

However, the safety factor in respect of both the 

steroidal and nonsteroidal anti-inflammatory 

drugs has been rather intriguing and hence a 

definite need is visualized for the introduction 



Table 1: Mean and standard deviation (SD) of paw edema (in cm) in the three groups under study along with 

the results of analysis of variance (ANOVA) and post-hoc tests 

Group 

No. of 

subjects 

Paw edema (Cms) 

Mean SD 

87 

Inhibition (%) 

Control 6 3.85 0.166 0.0 

Standard drug* 6 1.92 0.128 50.2 

Test drug* 6 2.00 0.139 48.1 

Note: ANOVA significant (p


of safer anti-inflammatory drugs having no 

troublesome adverse effects. There are different 

kinds of garlic oils, garlic powder capsules, 

garlic pills with enteric coatings, garlic pills 

with other herbs added, like cayenne and 

aged garlic extract. Garlic powder used in the 

above study, as the powder form gets absorbed 

readily in the stomach and duodenum. It also 

contains the sulfur containing compounds alliin, 

ajoene, diallylsulfide, dithiin, S-allylcysteine, 

and enzymes, vitamin B, proteins, minerals, 

saponins, flavonoids. 

One of the most biologically active compounds, 

allicin (diallyl thiousulfinate or diallyl disulfide) 

does not exist in garlic until it is crushed or cut; 

injury to the garlic bulb activates the enzyme 

allinase, which metabolizes alliin to allicin. 

In addition allicin is further metabolized to 

vinyldithiines. Ajoene, is another chemical 

constituent thought to be most important to 

health . 

Carrageenin induced rat-paw edema model has 

gained greater importance and support over the 

years, because edema induced by carrageenin 

is reported to have been inhibited by majority 

of the steroidal and the non-steroidal antiinflammatory 

drugs. Moreover, the lesions 

induced by Carrageenin are said to resemble 

histologically those of rheumatoid arthritis 

in human being atleast to a certain extent . 

These observations have justified the use of 

Carrageenin as the prime oedemogen. It has a 

biphasic effect. The first phase is due to release 

of histamine and serotonin (5-HT) (0-2 hours), 

plateau phase is maintained by a kinin like 

substance (3 hours) and second accelerating 

phase of swelling is attributed to PG release (>4 

hours) 

In our study Garlic powder 100 mg/kg, per orally 

significantly reduced edema induced by the 

carrageenin. The results obtained in this model/ 

method suggest that the anti-inflammatory 

activity of garlic powder is almost equal to that 

of the standard drug, namely, piroxicam. 

88 

Cotton pellet induced granuloma is a method 

for testing the proliferative phase i.e. granuloma 

formation, provoked by the subcutaneous 

implantation of compressed cotton pellets. 

After few days histologically giant cells and 

undifferentiated connective tissue can be 

observed along with fluid infiltration. The 

amount of newly formed connective tissue 

can be measured after removal and weighing 

the dried pellets. This model serves as an 

example for chronic inflammatory models. 

Here, the underlying principle of assaying 

the anti-inflammatory activity is based on the 

formation of foreign body granuloma, following 

subcutaneous implantation of cotton-pellets. 

Several authors have used this model for noting 

the anti-granuloma activity of newer agents and 

interestingly, the time-intervals, at which the 

implanted cotton-pellets (with the granuloma) 

have been removed from the sacrificed animals, 

have varied considerably, from a few days to a 

few weeks (13). 

In the present study the implanted cottonpellets 

(with the granuloma) were removed on 

the eighth day after the experimental procedure 

and the dry weight of granuloma served as 

the criterion for assaying anti-inflammatory 

(Anti-granuloma) activity. The results observed 

are suggestive of modest and very low antigranuloma 

activity in respect of piroxicam and 

garlic powder, respectively. 

In an earlier study, aqueous extracts of fresh 

garlic (5, 12.5, 25 and 50 mg/ml) were shown 

to inhibit the synthesis of the prostanoids in 

a dose dependent manner (14). In another 

research, the effects of aqueous extract of raw 

garlic and boiled garlic on cyclo-oxygenase 

activity in rabbit tissues were studied (15). 

Raw garlic inhibited cyclooxygenase activity 

non-competitively and irreversibly. Therefore 

the probable mechanism of action of garlic is 

by inhibiting cyclooxygenase activity. Allium 

sativum yields allicin, a powerful antibiotic. It 

has been claimed that it can be used as a home 

remedy to help speed recovery from strep throat 

or other minor ailments because of its antibiotic 



properties. Recent publications indicate that 

garlic extract has broad-spectrum antimicrobial 

activity against many genera of bacteria and 

fungi. The active component (allicin) has been 

isolated and characterized (16). 

The standardization of the extracts, 

identification and isolation of active principles 

and pharmacological studies of these principles 

may be considered for further detail studies. 

Conclusion 

In general, the medicinal importance of garlic 

preparations has already been substantiated 

by the very availability of “garlic pearls” for 

human use. Present study has shown that Garlic 

has promising anti-inflammatory activity that is 

comparable with that of piroxicam, especially 

in the carrageenin induced paw-edema 

animal model. These findings are valuable 

for identifying lead compounds for antiinflammatory 

drugs, keeping in mind the side 

effects of NSAIDs and corticosteroids. In the 

light of observations made it may be envisaged 

that garlic can be used as an adjuvant in the 

inflammatory disorders. 


Authors are thankful to Dr. Vijayaraghavan, 

Former Professor, Department of Pharmacology, 

Govt. Medical College, Mysore for the guidance 

and support and also to Mr. M. Ganapathy, 

Spicex Limited, Mysore for the processing of 

Garlic powder for this study. 

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12. Penn GB and Ashford A. The inflammatory response 

to implantation of cotton pellets in the rat .J Pharma 

Pharmacol 1963; 15:798-803. 

13. Singer FM and Borman A. Cotton pellet inhibtion of 

granuloma formation is directly related to anti-inflam- ... 

Proc. SOC. Exptl. Biol. Med. 1956; 92: 23. 

14. Ali M. Aqueous extracts of garlic (Allium sativum) inhibit 

prostaglandin. Prostaglandins Leukotrienes 

and Essential Fatty Acids. 1993 ;49( 5) : 855-859. 

15. Ali. M. Mechanism by which garlic (allium sativum) 

inhibits cyclooxygenase activity. Prostaglandins, 

Leukotrienes and Essential Fatty Acids 1995; 53(6): 397- 

400. 

16. Adetumbi, M. A., and B. H. S. Lau. Allium sativum. 

(garlic)-a natural antibiotic. Med. Hypotheses 1983.12:227- 

237 


90 


Reactive Oxygen Species during hypoxia-reperfusion injury under general 

anaesthesia 

*Sanjeev Kumar **Ashok kumar 

*Department of Anaesthesiology and Critical care,Navodaya Medical College,Raichur- 

584103,Karnataka, India 

**Consultant Anaesthesiologist,Advanced Medicare Research Institute, Kolkata-700029 

(Received 3 rd November, 2010; Revised 5 th January, 2011; Accepted 16 th February, 2011) 


Dr Sanjeev Kumar, 

Email:docsanjeevparmar77@gmail.com 

Abstract 

Background: Hypoxia – Reperfusion of ischemic tissues results in the formation of toxic ROS (reactive 

oxygen species) . The hypoxia – reperfusion event that may occur during intubation apnea and reoxygenation 

could be a potent source for generation of ROS. The role of calcium is important in maintaining the membrane 

ionic equilibrium during hypoxia and reperfusion. This study was designed to find out the effectiveness 

of a single dose of calcium channel blocker, nifedipine in attenuating the haemodynamic responses and 

generation of ROS. 

Methods: Twenty patients undergoing elective surgery under general anaesthesia were randomly assigned 

to group IA and IB. In group IA patients were anaesthetized by standard anaesthetic technique while in 

group IB sublingual nifedipine (10mg) was given 10 minutes before induction.Blood (10ml) was collected 

at different time intervals to measure peroxidative stress and reactive oxygen scavangers. 

Results: In group IA laryngoscopy and intubation caused a significant increase (4.06±0.90 vs 4.63±0.84 

nmol/mL, p

ROS during hypoxia-reperfusion injury 

acid (HOCL) hydrogen peroxide (H2O2) 

and nitric oxide derived peroxynitrate. These 

reactive oxygen species (ROS) are produced 

in limited amounts under normal conditions 

as the intracellular defense system consisting 

primarily of enzymatic and nutrient free radical 

scavengers are able to inactivate them. Hypoxia 

is associated with depletion of tissue content of 

antioxidants including superoxide dismutase 

(SOD), catalase, glutathione peroxidase and 

increases vulnerability to reoxygenation which 

causes further reduction of antioxidants(3,4). 

Thus the hypoxia – reperfusion event that 

may occur during intubation apnoea and 

reoxygenation could be a potent source for 

generation of ROS. The role of calcium is 

important in maintaining the membrane ionic 

equilibrium during hypoxia and reperfusion. 

On reperfusion, calcium readily enters into the 

cell in exchange for sodium. The net increase 

in intracellular calcium causes a decrease in 

ATP synthesis and subsequent lysis of the cell 

(5,6). A further deleterious consequence of 

the reperfusion dependent calcium influx is 

activation of proteases and phospholipidases; 

the latter results in release of free fatty acids and 

lysophospholipids (7). These compounds are 

toxic in their own right and can also result in the 

production of ROS and other cytotoxic products 

on reperfusion (8,9). Calcium channel blockers 

especially nifedipine has been found to be more 

effective in minimizing the haemodynamic 

adverse response to laryngoscopy and 

intubation and has also been found to act as 

an antioxidant when administered before the 

ischemic-reperfusion event (10). Therefore, this 

study was designed to find out a) if intubation 

apnoea and reoxygenation generated ROS b) 

the effectiveness of a single dose of calcium 

channel blocker, nifedipine in attenuating the 

haemodynamic responses and generation of 

ROS. 

Methods 

After prior approval from our institutional ethics 

91 

committee and written informed consent from 

patients, twenty patients belonging to ASA I 

and II with age between 30-50 years undergoing 

elective surgery of an estimated duration of 

more than 1 hour were included in the study 

and randomly allocated to Group IA and IB. 

Patients suffering from any major preexisting 

neurological, cardiovascular metabolic 

respiratory or renal disease were excluded from 

the study. On arrival to the operating room 

patients were connected to routine monitoring 

devices [HR, NIBP, ECG, SpO2]. Vital 

parameters were noted before during and after 

general anaesthesia at regular time intervals. 

In Group IA – 10 patients were induced with 

injection atropine sulphate 0.6 mg, tramadol 

hydrochloride 1 mg/kg, pentothal sodium 5 mg/ 

kg and succinylcholine 2 mg / kg intravenously 

followed by oral endotracheal intubation. 

In Group IB- 10 patients were induced with 

same technique as above but all these patients 

were given sublingual nifedipine 10 mg, 10 

minutes before induction. Anaesthesia was 

maintained with oxygen, nitrous oxide and 

intermittent doses of vecuronium bromide 

and 0.5%-1% halothane. Following surgery 

patients were reversed with neostigmine methyl 

sulphate 0.08 mg/kg and atropine sulphate 

0.02 mg/kg intravenously and extubated. 

Blood (10 ml) was collected in EDTA vials at 

different time intervals (pre induction, during 

intubation, following 30 min of ventilation and 

after 1 hour of ventilation; sample I, II, III and 

IV respectively). The following biochemical 

investigations were carried out in the blood: 

For peroxidative stress – Thiobarbituric acid 

reactive substances (TBARS) (11) For enzyme 

ROS (Reactive Oxygen Species) scavengers – 

Superoxide dismutase (SOD) (12) Glutathione 

peroxidase (GPx)(13) Reduced glutathione 

(GSH)(14) Catalase (Cat)(15) For nutritional 

ROS scavengers(16) - Retinol; ß-Carotene 

a-Tocopherol Ascorbic acid These results were 

compiled and analyzed statistically by using a 

paired t-test. Statistical significance was defined 

as P


Table1:Haemodynamic Responses in Group I A and IB 

I II III IV 

(Group I A ) Heart rate (beats/ 

min) 

(Group I B ) Heart rate 

(beats/min) 

(Group I A ) Mean BP 

(mmHg) 

(Group I B ) Mean BP 

(mmHg) 

(Group I A ) O 2 

Saturation(%) 

(Group I B ) O 2 

Saturation(%) 

a=I; b=II; c=III; d=IV * Mean + S.D.I=P


Table 4: Peroxidative Stress and Enzyme antioxidant status in Group I B (n=10) 

Parameters I II III IV 

TBARS nmol./ml 

Catalase nmol./min/mgHb 

GSH mg/dl RBC 

GP x mg GSH consumed/ 

min at 37 0 C 

4.21±1.38* 5.12 ± 1.27 4.29±1.34 4.19±1.46 

b 2 

0.69 ± 0.24 0.60 ±0.32 0.59±0.37 0.66±0.32 

a 2 

42.81±5.69 47.42±5.15 45.91±5.65 47.52±5.83 

a 2 

3.00 ± 1.11 2.81 ±1.21 3.24±1.05 2.88±1.18 

SOD U/mgHb 3.11±0.52 3.04±0.56 3.04±0.56 3.15±0.56 

a=I; b=II; c=III; d=IV *Mean + S.D.I=P


minutes of ventilation from 5.12±1.27 nmol/ 

ml to 4.29±1.34 nmol/ml (II vs III, p


intubation there was evident peroxidative stress 

which was accompanied by significant activity 

or consumption of the antioxidant defense 

mechanism in the body. This correlated with 

the haemodynamic responses seen during 

this period. Nifedipine when given in a single 

dose pre-operatively was found to act as an 

antioxidant, while limiting any increase in 

peroxidative stress and gearing up antioxidant 

mechanism in the body to meet the challenge of 

acute oxidant stress. 

References 

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comparative study of various airway devices as regards 

ease of insertion and haemodynamic responses. India J. 

Anaesth2004; 48(2): 134-137. 

2. Wilson, I.G., Fell, D., Robinson, S.L. and Smith, G. 

Cardiovascular responses to insertion of the laryngeal 

mask. Anaesthesia 1992; 47: 300-302. 

3. Guarnieri, C., Flamign, F. and Caldarera, C.M. Role of 

oxygen in the cellular damage induced by re-oxygenation 

of hypoxic heart. J. Mol. Cell Cardiol 1980; 12:797-808. 

4. Kato, R. and Foex, P. Myocardial protection by anesthetic 

agents against ischemia – reperfusion injury: an update for 

anesthesiologists. Can J. Anesth 2002; 49(8): 777-791. 

5. Bourdillon, P.D. and Poole Wilson, P.A. The effects of 

verapamil, quiescence and cardioplegia in ischemic rat 

myocardium. Cir. Res1980; 50: 360-368. 

6. Parr, D.R., Wilmhurst, J.M. and Harris, E.F. Calcium 

induced damage of rat heart mitochondria. Cardiovasc. 

Res1975;. 9: 366-372. 

7. Murphy, E., Aitcon, J.F. and Horres, C.R. Calcium 

elevation in cultured heart cells, its role in cell injury. Am 

J. Physiol 1983; 245: C316-C321. 

8. Bersohn, M.M. and Philipson, K.D., YFJ . Sodium calcium 

exchange and sarcolemmal enzymes in ischaemic rabbit 

heart. Am. J. Physiol 1982; 242: C288-C295. 

9. Konotos, H.A., Weiep, P.J.T. and Dietrich, W.D. 

Cerebral arteriolar damage by arachidonic acid and PG2. 

95 

Science1980; 209: 1242-1245. 

10. Ohsuzu, F. Effects of calcium antagonists and free radical 

scavengers on myocardial ischemia and reperfusion 

injury: Evaluation by 31 P-NMR spectroscopy. Japanese 

Circulation Journal 1989; 53:1138-1143. 

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

peroxides in animal tissues by thiobarbituric acid reaction. 

Anal Biochem 1979; 95(2): 351-8. 

12. Mishra, H.P. and Fridovich, I. The role of superoxide 

anion in the autoxidation of epinephrine and a simple assay 

for SOD. J. Biol. Chem1972; 247: 3170. 

13. Pirie. A. Glutathione peroxidase in lens and a source of 

hydrogen peroxide in aqueous humour. Biochem1965; 

96:243. 

14. Beutler, E. and Kelly, B.M. Improved method for the 

determination of blood glutathione J. Lab. Clin. Med1963; 

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15. Sinha,A. K.Colorimetric assay of catalase. Annal 

Biochem1972; 47: 389. 

16. Natelson, S Techniques of clinical chemistry. 3rd Ed 

Charles. C. Thomas. USA 1971: 162, 288 and 751. 

17. Derbyshire, D.R., Chmielewski, A., Fell, D., Vater, M., 

Achola, K. and Smith, G. Plasma Catecholamine responses 

to tracheal intubation. Br. J. Anaesth1983; 55: 855-859. 

18. Shribman, A. J., Smith, G. and Achola, K.J. Cardiovascular 

and catecholamine responses to laryngoscopy 

with and without tracheal intubation. Br. J. Anaesth1987; 

59: 295-299. 

19. Kale, S.C., Mahajan, R.P., Jayalakshmi, T.S., Raghavan, 

V. and Das, B. Nifedipine prevents the pressor response 

to laryngoscopy and tracheal intubation in patients with 

coronary artery disease. Anaesthesia1988; 43:495-497. 

20. Puri, G.D. and Batra, Y.K. Effect of nifedipine on cardiovascular 

responses to laryngoscopy and intubation. Br. J. 

Anaesth1988; 60:579-581. 

21. Mason, R.P. and Trumbore, M.W. Differential membrane 

interactions of calcium channel blockers. Implications 

for antioxidant activity. Biochem. Pharmacol1996; 51(5): 

653-60. 

22. Silva, |J.M., Filibe, P.M., Fernandes, A.C. and Manso, C.F. 

Antioxidant effect of drug used in cardiovascular therapy. 

Rev. Port. Cardiol1998; 17(6): 495-503. 

23. Sugawara, H., Tobisa, K. and Kikuchi, K . Antioxidant 

effects of calcium antagonists on rat myocardial membrane 

lipid peroxidation. Hypertens. Res1996; 19(4): 223-228. 


96 


Assessment of probiotic properties of strains of L.fermentum and L.reuteri 

isolated from human breast milk 

R. Ilayaraja and Radhamadhavan. 

Department of Microbiology, SRM Medical College Hospital & Research Centre, 

SRM University, Kattankulathur - 6032023. Tamil Nadu, India. 

(Received 27th October, 2010; Revised 10th February, 2011; Accepted 1st March, 2011) 


R. Ilayaraja 

E-Mail: srmmicro@gmail.com. 

Abstract 

Objectives: To assess the probiotic potential of strains of Lactobacillus fermentum and Lactobacillus 

reuteri isolated from human breast milk was investigated. 

Materials and Methods: The milk samples were inoculated anaerobically into MRS medium and incubated 

for 48 hrs at 37 0 C. Probiotic characteristics of the strains were assessed according to the criteria laid down 

by WHO namely, tolerance to low pH, bile, NaCl concentration and temperature, Hemolytic activity and 

antimicrobial activity. 

Results: The cultures were identified based on morphological and biochemical characteristics of Lactobacilli 

as per the Bergey’s Manual of Determinative Bacteriology and included growth at 15 0 , 37 0 C and 45 0 C and 

fermentation of different carbon sources. Both the strains fulfill the probiotic criteria suggested by WHO. 

Interpretation and conclusion: This study revealed that these two strains fulfill characteristics for use as 

probiotics. L.fermentum had higher probiotic activity compared to L.reuteri. 

Key words: MRS medium, Probiotic, antimicrobial therapy, Lactobacillus. 

Introduction: 

Over the past 15 years, there has been an 

increase in research on probiotic bacteria and 

a rapidly growing commercial interest in the 

use of probiotic bacteria in food, medicine and 

as supplements. (17). A variety of probiotic 

bacteria have been targeted as potential 

therapeutic agents and include Lactic acid 

bacteria (LAB), Bifidobacteria, Saccharomyces 

and streptococci. Today with waning efficacy 

of antibiotics and dramatic resurgence of 

infectious disease, there is an intense need of 

an alternative to existing antibiotic-dominated 

therapies Probiotics are substances secreted by 

one microorganism which stimulates the growth 

of another. A more formal definition widely 

used is “A live microbial food supplement 

which beneficially affects the host animal by 

improving its intestinal microbial balance”. An 

expert panel commissioned by FAO (Food and 

Agriculture Organization) and WHO (World 

Health Organization) defined probiotic as “live 

microorganism”, which when administrated in 

adequate amounts confers a health benefit on 

the host. (2). Bacteria most commonly used 

in probiotic preparations belong to genera of 

lactobacillus, Bifidobacterium, Enterococcus, 

Bacillus and Streptococcus. Some fungal 

strains belonging to saccharomyces were also 


properties of L.fermentum and L.reuteri 

used (3). A number of clinical studies have been 

performed on the ability of probiotic bacteria 

to prevent or treat gastro intestinal infections. 

Bacteria belonging to the two genera namely 

Lactobacillus and Bifidobacterium are the most 

commonly used probiotic bacteria. Some of the 

well planned, double-blind, placebo-controlled 

studies suggested that lactobacillus strains are 

the most promising strains in the prevention of 

diarrhea. Recently, it was shown that in children 

admitted to hospital, the risk of acquiring 

nosocomial diarrhea was reduced 30% to 

7% in the non breast fed children receiving 

prophylactic therapy with lactobacillus GG. 

(5). According to 1997 issue of the journal of 

Pediatrics Gastroenterology and Nutrition, 

L.reuteri is an effective treatment for Rota viral 

diarrhea in children. Currently used in vitro tests 

for study of probiotic properties of bacterial 

strains include tolerance to gastric acidity , 

bile salt , NaCl concentration and temperature, 

hemolytic activity and antimicrobial activity, 

adherence to mucous and/ or human epithelial 

cells and cell lines, antimicrobial activity 

against potentially pathogenic bacteria, ability 

to reduce adhesion of pathogens to surfaces, 

and resistance to spermicides (applicable to 

probiotics for vaginal use. (4). The aim of this 

study was to isolate bacterial strains from human 

breast milk and to study antimicrobial activity, 

and other characteristics of a good probiotic 


Isolation and Identification: 

The lactobacillus strains were isolated from 

human breast milk on solid Man-Rogosa- 

Sharpe media (MRS)(g/l, peptone 10.0, meat 

extract 8.0, yeast extract 4.0, D(+)glucose 20.0, 

dipottassium hydrogen phosphate 2.0, tween 

80 - 1.0, di-ammonium hydrogen citrate 2.0, 

sodium acetate 5.0, magnesium sulphate 0.2, 

manganese sulphate 0.04, supplemented with 

14.0 g agar respectively). The bacteria were 

grown at 37 0 C in microaerophillic condition 

with out shaking. The lactobacillus strains were 

97 

identified as described by Bergey’s Manual of 

Determinative Microbiology (18). Purity of 

the strains were verified by three successive 

subculture from the single colony. For long 

term preservation of cultures, MRS broth was 

supplemented with 10% glycerol. 

Carbohydrate Fermentations 

Isolates were characterized according to their 

carbohydrate fermentation profiles by testing 

against 14 different carbohydrates. Each sugar 

solutions was prepared at a final concentration 

of 10% (w/v) with phenolphthalein red as pH 

indicator, and the solutions were filter sterilized 

with filters (0.22 μm pore diameter). All the 

reactions were performed in duplicate including 

suitable positive and negative controls After 

overnight incubation at 37°C, the turbidity and 

the color change from red to yellow indicated 

positive fermentation results (1). 

Gas Production from Glucose 

The production of gas during glucose 

fermentation was observed by placing an 

inverted Durhams tube in MRS broth and 

inoculated with 1% overnight fresh cultures. 

Then the test tubes were incubated at 37 °C for 

24 hrs. Accumulation of air bubble in Durham 

tubes after 24 hrs indicated CO2 production 

from glucose (16). 

Growth at Different Temperatures 

5ml MRS containing bromecresol purple 

indicator, was inoculated with fifty μl of 

overnight cultures in triplicate incubated for 5 

days at 15 °C, 37 0 C and 45 °C. Growth at any 

temperatures was indicated by the turbidity and 

change of colour from purple to yellow. 

Growth at Different NaCl Concentrations 

Isolates were tested for their tolerance against 

different NaCl concentrations. Test media 

containing bromecresol purple indicator were 

prepared with 3different concentrations NaCl 



(1%, 2% and 3%) and transferred into tubes 

in 5 ml. These tubes were inoculated with 1% 

overnight cultures and then incubated at 37 °C 

for 5 days. The change of the colour from purple 

to yellow was noted. 

Antibiotic susceptibility profile: 

Antibiotic susceptibility profile was determined 

on MRS agar medium against 13 different 

antibiotic discs procured from Hi-media, India. 

by disk diffusion method (6). 

Antimicrobial activity of Bacteriocin: 

Antimicrobial action of lactobacillus strains was 

determined against indicator bacteria by agar 

well diffusion method (7). A total of 11 clinical 

isolates were obtained from department of 

Microbiology, SRM Medical college hospital & 

research centre. Out of these twelve, Four were 

Gram positive (Staphylococcus aureus, MRSA 

(Methicillin Resistant Staphylococcus Aureus). 

Streptococcus pneumoniae and Enterococcus 

sps) and seven were Gram negative (Escherichia 

coli, Klebsiella pneumoniae, Pseudomonas 

aeruginosa, Proteus sps, Salmonella typhi, 

Shigella dysentriae, and Vibrio cholera). 

Supernatant of lactobacilli sp representing the 

Bacteriocin were monitored for antibacterial 

activity against indicator bacteria inoculated on 

Muller Hinton Agar (MHA). A volume of 50 

µl of cell free supernatant was filled in 5 mm 

diameter sealed wells cut in the MHA agar. The 

diameter of the zone of inhibition was measured 

with caliper after 24 hr of incubation. 

Haemolytic activity 

Haemolytic property was evaluated on nutrient 

agar plate supplemented with 5% sheep blood 

which was incubated at 37 0 C for 24 hrs. (15). 

Resistance to Low pH 

Resistance to pH 3 for 3 hours is often used in 

vitro assays to determine the resistance to gastric 

acid, .considering the duration of stay of food 

98 

in the stomach (11). For this purpose, 16-18 hr 

old cultures were inoculated in 10 ml of 0.05 M 

sodium phosphate buffer adjusted to pH 2.0 to 

7.0 with 1N HCl and samples were incubated 

at 37 0 C for 3 hrs. The resultant growth was 

serially diluted to 10 fold dilution by phosphate 

buffer pH 7.0. Viable microorganisms in 

different dilutions were enumerated by pour 

plate techniques incubated at 37 °C under 

anaerobic conditions for 48 h. The survival rate 

was calculated as the percentage of colonies 

grown on MRS agar compared to the initial cell 

concentration. Each experiment was performed 

in triplicate. 

Bile salt resistance: 

The ability to grow in the presence of 0.3%, 

0.6%, 0.9% and 1.2% bile salts was determined 

in MRS broth. (13). The growth was examined 

after 24 hours under anaerobic conditions of 

incubation at 37 0 C by plate count method and 

compared to those grown in the absence of bile 

salt. The experiment was performed in triplicate. 

Results and Discussion: 

Human breast milk samples were obtained 

from lactating women of 20-36 years. of age. 

The samples were collected 6 to 32 days after 

delivery. Lactobacilli are Gram positive, 

microaerophilic, catalase negative, oxidase 

negative, non-spore forming and non-capsulated 

rods. Microscopically they are short to long 

rods that appear as single cells, in pairs and in 

short chains. Surface colonies on MRS agar 

plate are 0.5 to 2 mm in dm, circular, lenticular, 

creamy-white. As per their Carbohydrate 

fermentation profile the isolates were identified 

as Lactobacillus reuteri, and Lactobacillus 

fermentum. 

Table.1 shows the result obtained for antibiotic 

susceptibility of the two species. Both the species 

were resistance to Vancomycin and sensitive to 

Ampicillin, Chloramphenicol, Clindamycin, 

Rifampicin, Amikacin, Gentamycin and 

ciprofloxacin. Most spp showed resistance to 3 



Table 1. Antibiotic resistance profiles of the tested probiotic strains (Antibiotic Conc. range, µg/ML): 

L.f- Lactobacillus fermentum, L.r- Lactobacillus reuteri, S- sensitive, R- resistant 

22Antibiotics (µg) L.f 1 L.f 2 L.f 3 L.f 4 L.r 1 L.r 2 L.r3 L.r 4 

Ampicillin (10) S S S S S S S S 

Amikacin (32) S S S S S S S S 

Cefoxitin (30) S S R S S S S S 

Clindamycin (2) S S S S S S S S 

Ciprofloxacin (30) S S S S S S S S 

Cloramphenicol (30) S S S S S S S S 

Gentamycin (5) S S S S S S S S 

Kanamycin (30) R R R R R S R R 

Oxacillin (1) S S S R R S S S 

Rifampicin (5) S S S S S S S S 

Tetracycline (30) R R R S R S S S 

Vancomycin (30) R R R R R R R R 

SG- slight growth, G-growth, NG- no growth, NH- non hemolytic 

Table. 2. Resistance to temperature, bile and hemolysin production: 

Resistance To Temp Resistance Of Nacl In % Type Of Hemolysis 

15 0 C 37 0 C 45 0 C 1% 2% 3% 

L.fermentum SG G G G G NG NH 

L.reuteri SG G G G G NG NH 

Figure 1. Antimicrobial activity of culture supernatant against 

pathogens 

Zone of Inhibition 

14 

12 

10 

8 

6 

4 

2 

0 

S,aure 

us 

Entero 

coccu 

MRSA E.coli K.pne 

umoni 

L.fermentum 11 10 12 11 10 10 8 11 11 10 

L.reuteri 10 8 9 10 10 10 7 9 11 9 

Clinical pathogens 

P.aeru Proteu 

ginosa s spp 

S.typh 

i 

S.dys 

entriae 

V.chol 

era 


99


OD Values 

Percentage of viable strains (%) 

1.8 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

120 

100 

80 

60 

40 

20 

0 

Fig 2. Resistance to Low pH 

pH2 pH3 pH4 pH5 pH6 pH7 

Fig.3. Tolerance of 0.3% of Bile salt 

L.fermentum 

L.reuteri 

0 hrs 5 hrs 10 hrs 15 hrs 20 hrs 25 hrs 30 hrs 35 hrs 40 hrs 45 hrs 50 hrs 

Control L.fermentum L.reuteri 


100


of the 13 antibiotics tested. i.e. to Vancomycin, 

tetracycline and kanamycin. Four spp (three 

L.fermentum and one L.reuteri) showed multiple 

resistances to 3 different antibiotics, namely 

cefoxitin, oxacillin and kanamycin. Lactic 

acid bacteria (LAB) especially lactobacilli are 

normal inhabitants of intestinal tract of humans 

and animals and are also found in milk and 

milk products. (Mitsuoka 1992).. In our study 

all lactobacilli inhibited the growth of S.aureus, 

MRSA (Methycillin Resistant Staphylococcus 

Aureus), Enterococcus spp, E.coli, Klebsiella 

pneumonia. Pseudomonas aeruginosa, Proteus 

spp, Salmonella typhi, Shigella spp, Vibrio 

chlorae. The strongest antimicrobial effect 

was shown by L.fermentum against indicator 

bacteria when compared to L.reuteri. (Fig. 1). 

The antimicrobial action may be attributed to 

lactic acid production, bacteriocins and some 

peptide with inhibitory properties (8). Another 

criterion for a good probiotic is the ability to 

grow at different temperatures. At the end of 

5 days incubation, all isolates could grow at 

15, 37 and 45 °C. Growth at 15C was minimal 

compared to that in 37 and 45 (Table 2). All of 

the isolates were able to grow upto 2% NaCl 

concentration. Lactobacillus isolated from 

human breast milk were not hemolytic to sheep 

blood (Table 2).and is a desirable property for 

an ideal probiotic strain (9). The effect of pH 

ranging from 2.0 to 7.0 on the L.fermentum 

and L.reuteri was studied. It was found that 

they could survive a pH of 2.0, (78%) even 

though growth was better in 3.0 to 7.0. (Figure 

2). Resistances to low pH are one of the major 

selection criteria for probiotic strains and reflect 

its ability to withstand conditions in stomach and 

small intestine (12).Bile tolerance by probiotics 

has been shown to be strain- and bile typedependent, 

with resistance levels ranging from 

bile concentrations of 0.125 to 2.0% (10). Bile 

tolerance has been described as an important 

factor for the survival and growth of LAB in the 

intestinal tract. While both strains isolated from 

human breast milk could tolerant 0.3% Ox gall 

bile, there were some minor differences. (Figure 

101 

3.)In general, the required concentration of bile 

salts considered necessary to screen for resistant 

strains for human use is 0.3%. (14). 

Conclusion: 

This study was focused on exploring the potential 

features of strains isolated from human milk for 

use as probiotics. Lactobacillus strains constitute 

the normal bacterial flora of the human breast 

milk. L.fermentum and L.reuteri were the most 

predominant isolates and exhibited many probiotic 

qualities. They were inhibitory to the most 

predominant pathogens isolated from clinical 

specimen. The ability to survive acidic conditions, 

tolerance to heat (45C), 0.3% bile and 2% NaCl, 

represent qualities of an ideal probiotic and may 

have potential applications to provide balanced 

intestinal microbiota in children with diarrhea. This 

study also emphasizes the value of breast feeding in 

prevention of infective diarrhea in children. 

References: 

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sp. nov., Lactobacillus antri sp. nov., Lactobacillus 

kalixensis sp. nov. and Lactobacillus ultunensis sp. nov., 

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of Systematic and Evolutionary Microbiology. 2550; 

55:77-82. 

2. Food and Agricultural Organization of the United Nations 

and World Health Organizations. Posting data: Regulatory 

and clinical aspects of dairy probiotics. Food and Agricultural 

Organization of the United Nations and World 

Health Organizations expert consultation report. Food 

and Agricultural Organization of the United Nations and 

World Health Organizations working group report. 2001; 

(Online). 

3. Gibson GR, Roberfroid MB. Dietary Modulation of the 

human clonic microbiota; Introducing the concept of prebiotics. 

J. Nutr. 1995; 125;1401-12. 

4. S.K.Dash. PhD, President and Director of Research UAS 

Laboratories, 9953 Valley View road, Eden Prairie, Minnesota 

55344, USA. Selection criteria for probiotics. Paper 

presented in XXXVII Dairy industry Conference, Feb 7-9, 

2009. Kala Academy, Panjim, Goa. 

5. Szajewska H, Kotawska M, Mrukowicz J.Z, Armanska 

M, and Mikotajczyk W. Efficacy of Lactobacillus GG in 

prevention of nosocomial diarrhea in infants. Journal of 

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103 


Perinatal androgen levels and Sexual dimorphic digit ratio in Down syndrome 

Children 

Suresh Bidarkotimath and S. Viveka 

Department of Anatomy, A J Institute of medical sciences, Mangalore - 575004. Karnataka, India. 

(Received 26 th July, 2010; Revised 23 rd December, 2010; Accepted 11 th March, 2011) 

Corresponding Address 

Dr. Suresh Bidarkotimath 

Email: bidarkotimath@gmail.com 

Abstract 

Background and Objectives:- Down syndrome being the major common cause of mental retardation 

in children is most commonly studied chromosomal anomalies. The sexual abnormalities in it include 

undescended testis, late sexual maturity. It has been proven that the androgen levels in children and adults 

with this syndrome are low. To establish the androgen levels in early perinatal period the digit ratio can be 

used. Digit ratio is the ratio between the length of 2nd finger and 4th finger as measured form the bases of 

digit to their tip. Digit ratios are determined by the early perinatal androgen levels and it remains stable after 

early life. Digit ratios are shown to be sexually dimorphic. 

Methods:- Digit ratios of 23 Down syndrome patients were analyzed which included 13 males and 10 

females from Saanidhya Samarth Center, Mangalore, and compared it with the age related control group 

of 50 children. 

Results :- It was found that the ratios are sexually dimorphic in Down syndrome patients [The mean of 

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 

2D:4D ratios of females right hand was 0.90 (SD0.053) and left hand was 0.939 (SD 0.075)] and they are 

statistically different from the control group. 

Interpretation and Conclusion:- It is hypothesized that the ratios are sexually dimorphic as in general 

public that there is no difference between the perinatal androgen levels in Down syndrome patients as 

compared with normal counterparts. 

Key words: Down syndrome, Digit ratio, Perinatal androgen levels. 

Introduction 

The ratio of index finger (2D) to the ring finger 

(4D) length is referred as digit ratio (2D:4D). 

The digit ratio and its association to the human 

characteristics has been the focus of much 

research in recent years. The digit ratios are 

determined by taking the lengths of the fingers 

are taken from the proximal crease of the 

digit to their tip(1) and taking their ratios. It 

has been widely accepted that the sex differ- 

ence in 2D:4D ratio arises early in development 

and the ratios are slightly lower in males than 

females, making it – sexually dimorphic. This 

dimorphism arises due to the near equal lengths 

of 2nd and 4th fingers(2) in males. Once established 

in early neonatal life digit ratio assumed 

to be stable in later life. The digit ratio has been 

reported to be associated with several characteristics 

such as fetal growth, congenital adrenal 

hyperplasia, developmental psychopathology, 

autism and Asperger’s syndrome(3). Little is 


Perinatal androgen levels, digit ratio in Down syndrome 

known about 2D:4D ratio variations in Down 

syndrome patients. Down syndrome (trisomy 

21, Manglolism) is the most common chromosome 

disorder and single most common cause 

of moderate mental retardation. It is associated 

with impairment of cognition and characteristic 

facial and other dysmorphic features. It 

is also associated with undescended testis and 

delayed appearance of secondary sexual features 

in male children(4) . Down children attain 

a significantly lesser height than their normal 

counterparts(5). It has been confirmed that 

levels of circulating androgens as evidenced 

by urinary testosterone and epiandrosterone 

levels are low. Though much is known about 

the adult androgen levels and related clinical 

problems, there is relative paucity of knowledge 

on the perinatal androgen levels in Down 

syndrome. The Homeobox genes Hox a and d 

control the differentiation of the urinogenital 

system, and may therefore indirectly influence 

the prenatal production of testicular androgen 

and the development of the digits (6). Prenatal 

testosterone comes from maternal testosterone, 

which may pass across the placenta and enter 

the fetal bloodstream, and from the fetus itself, 

which secretes increasing amounts from about 

8 weeks (the time of Leydig cell differentiation) 

to mid-gestation (7). From that point levels 

slowly decrease until a few months after birth 

when it has reached the low level characterizing 

childhood (8). The fetal source is dependent 

on the differentiation of the testes (9). Perinatal 

androgen levels attain maximum level at 3 – 6 

months of intrauterine life and there is slight increase 

before delivery. At puberty there is slow 

but sustained increase in the testosterone levels 

before attaining the adult levels. There is substantial 

evidence that digit ratio is established 

early in intrauterine life under the influence 

of androgens. Low androgen levels during the 

late intrauterine life around the time of delivery 

causes relative delay in the descending of testis. 

In this study we tried to find out whether there 

are any alterations in early perinatal androgen 

levels as depicted by digit ratio. 


104 

We have selected 23 Karyotypically diagnosed 

cases of Down syndrome (all having 21 trisomies) 

children from Saanidhya Center for disabled, 

Mangalore. All subjects were unrelated 

Caucasians. Their age ranged from 6 to 22 

years. The digits lengths were determined by 

taking photographs of the hands of the selected 

children by using Digital camera (Sony India) 

with 8MP resolution, auto ISO having light 

background. The digit lengths were measured 

from the ventral proximal crease to the tip of the 

fingers both on ulnar and radial aspect (in order 

to account the sloppy proximal crease) by using 

‘measure tool’ of Adobe Photoshop CS5(10). 

Similar set of data was also taken from age related 

normal children. The direct measures of 

digits using measuring tool like vernier calipers 

and measures from photocopies produce comparable 

2D:4D ratios . The data was tabulated 

and analyzed using student’s t test. 

Results: 

Out of 23 Downs cases selected, 13 were males 

and 10 were females. Repeatability of 2D:4D 

from all 23 hands were as follows: males (n=13) 

r1=0.86; females (n=10) r1= 0.92. There was 

significantly greater variance between subjects 

than within-subject error (repeated measures 

ANOVA: males, F=13.70, p=.0001; females, 

F= 23.22, p=.0001).We concluded that our measurements 

represented real differences between 

subjects. The mean of 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 2D:4D ratios 

of females right hand was 0.90 (SD0.053) 

and left hand was 0.939 (SD 0.075) as shown 

in Graph:1 .In accord with previous workers 

males have statistically significant lower ratios 

than the females. The difference between the 

Down syndrome male and female patients were 

statistically significant (right hand t = 42, p > 

0.001, left hand t = 10, p > 0.001). The mean 

of 2D:4D ratios of males of age related control 



group was – right hand 0.990 (SD 0.020) and 

left hand 0.970 (SD 0.040). The mean of 2D:4D 

ratios of females of age related control group 

was – right hand 1.050 (SD 0.030) and left hand 

1.050 (SD 0.030). The difference between the 

males and females of control group were statistically 

significant (right hand t = 8.37, p > 

0.001 and left hand t = 7.94, p > 0.001). There 

is statistical difference between the digit ratios 

of Down syndrome and control group in both 

males (right hand t= 2.355, p > .001) and females 

(right hand t= 8.04, p > .001). 

Discussion 

Digit ratio: 

It has been known for more than a century that 

males and females tend to differ in the relative 

lengths of their index (2D) and ring (4D) fingers. 

The concept of digit ratio is popularized by an 

evolutionary psychologist, Prof John Manning 

and it’s been a decade since he published his 

views on it. He describes the digit ratio as a living 

fossil and a record of factors the fetus was 

exposed to at a critical time for the development 

of many other things. 

The digit ratios are sexually dimorphic and it has 

been proved that these ratios retain its dimorphism 

in infants, children and adults. A study by 

Stephens and Shepard has proved that this ratio 

is dimorphic even in fetuses, providing much 

needed support for the theory of establishment 

of the digit ratio in early neonatal peroid (11). 

In males, the second digit tends to be shorter 

than the fourth, and in females the second tends 

to be the same size or slightly longer than the 

fourth, making 2D:4D ratio sexually dimorphic 

with mean male 2D:4D lower than mean female 

2D:4D(12). The dimorphism patterns are well 

recognized in laboratory animals as well. Digit 

ratio has shown variations with different race 

and ethnicity. The differentiation of the digits 

is under the control of Homeobox or Hox genes 

(the posterior-most Hoxd and Hoxa genes), 

which also control the differentiation of the tes- 

105 

tes and ovaries (13). This common control of 

the distal limbs and genital bud may be seen 

when progressive removal of posterior Hox 

gene function results in loss of digits, genital 

bud derivatives, and fertility. It is suggested that 

the common control of the differentiation of the 

gonads and digits may mean that the functioning 

of the former may be reflected in the formation 

of the latter (14). Polymorphisms within 

Hoxd and Hoxa may therefore result in variation 

in gonad and digit form and function. The 

former could then affect Leydig cell differentiation 

and therefore the production of testosterone 

(15). Patterns of 2D:4D ratios may therefore 

reflect aspects of gonadal function such as the 

production of testosterone and estrogen. 

Several studies have found a significant positive 

relationship between 2D:4D ratio and medical 

problems like attension deficit hyperactive 

disorder (ADHD)(16) , autism, aggression in 

males, sporting ability in females. In contrast to 

this, a significant negative relationship has been 

found between the 2D:4D ratio and physical 

competence. 

There is some evidence that 2D:4D ratio may 

also be indicative for human development and 

growth. Ronalds et al. (2002) showed that men 

who had an above average placental weight and 

a shorter neonatal crown-heel length had higher 

2D:4D ratios in adult life(17) . Moreover, studies 

about 2D:4D correlations with face shape 

suggest that testosterone exposure early in life 

may set some constraints for subsequent development. 

Prenatal sex steroid ratios (in terms 

of 2D:4D) and actual chromosomal sex dimorphism 

were found to operate differently on 

human faces, but affect male and female face 

shapes by similar patterns. However, exposure 

to very high levels of testosterone and/or estrogen 

in the womb may have also negative effects. 

Fink et al. (2004) found that men with low 

2D:4D ratios (indicating high testosterone) and 

women with high 2D:4D ratios (indicating high 

estrogen) express lower levels of facial symmetry(18). 

There is some evidence that testosterone 

facilitates the differentiation of the brain 



both prenatally and postnatally. There have 

been many extensions of this, such as the Geschwind–Galaburda 

hypothesis, that immune 

disease and autism(19) are related to prenatal 

testosterone, this also explaining why more 

men are left-handed, autistic, etc. than women. 

Prenatal exposure to testosterone is thought to 

promote the development of the right-hemisphere 

and increase the incidence of sinistrality. 

As such low 2D:4D was found to be associated 

with improved left-hand performance. It has 

been showed that the 2D: 4D ratio in patients 

suffering from polycystic ovarian disease are 

not substantially decreased and in turn the perinatal 

androgen levels are not greatly elevated in 

them(20). 

Down syndrome hand characteristics: 

Among many phenotypic findings of Down 

syndrome the most important ones are the 

hand characteristics, physical traits (ear length 

and internipple distance) and clinical findings 

(Brushfield spots, wide-spaced first toe, and 

excess back neck skin)(21) . Down syndrome 

is associated with many hand characteristics, 

which can be classified into three main categories 

namely hand lines, dermatoglyphics and 

hand morphology. Simian crease (single palmar 

distal transverse crease) extended proximal 

palmar transverse crease , single interdigital 

crease on 5th (and sometimes on 4th) finger are 

the more common hand crease characteristics. 

Simian crease is found in more than a quarter 

of Down syndrome patients according to studies 

by C Plato(22). Other studies have indicated 

that the simian is also very significant when 

present only in the right hand; when present 

only in the left hand it is especially significant 

when combined with a Sydney line in the right 

hand Simian crease incidence is increased in 

fetuses with Down syndrome Simian crease is 

reported in more than third of the cases of the 

cases from India by Maina and colleagues(23) 

. Extended proximal transverse palmar crease 

(Sydney line) is again a significant sign of Down 

106 

syndrome according to Purvis- Smith (28). Its 

incidence is more than 15% (33). Deckers and 

Oorthuys have indicated that the extended distal 

transverse crease is significant when observed 

in the left hand; in the right hand it is significant 

when combined with a simian crease in the left 

hand . There are reports of single flexion crease 

on 4th and 5th fingers. Ulnar loops (especially 

the index finger), radial loops on the ring and 

index, loop between the base of the index and 

middle finger and/or the middle and ring fingers, 

hypothenar ulnar loops, whorls, or carpal 

loops and distally located axial triradii are the 

more common dermatoglyphic characteristics 

of Down syndrome. The ulnar loops in Down 

syndrome tend to be vertically oriented and Lshaped 

- with the ‘open’ side directed to the side 

of the little finger. Another common variant is: 

9 (or 8) ulnar loops combined with a radial loop 

on the ring finger or little finger. Radial loop is 

most frequently seen on the ring finger (the radial 

loops are usually vertically oriented and L 

shaped - with the ‘open’ side directed to the side 

of the thumb. A radial loop on the thumb, index 

finger or middle finger may not point towards a 

Down syndrome characteristic hand. The ‘palmar 

ridge line’ starts in the triradius below the 

index finger. In Down syndrome the path of 

the ‘palmar ridge line’ ends often just above or 

close to the point where the ‘upper transverse 

crease’ exits the palm. In the normal population 

the path of the ‘palmar ridge line’ is usually 

found completely below the ‘upper transverse 

crease’. In Down syndrome the alignment of 

ridges over the distal palmar area is - partly due 

to the short, broad hand shape - nearly always 

rather ‘transverse’. This is usually indicated by 

the combination of a palmar ridge line which exits 

the palm above (or just below) the heart line, 

combined with another palmar ridge line which 

exits the palm between the pointer finger and 

the middle finger. Characteristic palmar axial 

triradius is also advocated as one of the features 

of this syndrome(24). In Down syndrome quite 

often 3 or more triradii can be observed on the 

hypothenar due to the presence of various types 



of large loops, multiple loops, or whorls(25). 

The morphological changes in the hand include 

short thumb, short and incurved little finger, 

brachydactyly (short fingers), broad – short 

palm, hyperextensible joints..A low 2D:4D has 

been reported to be correlated with an increased 

preference to use the left hand (20). Low 2D:4D 

in children is associated with an increased risk 

of autism. This may be because 2D:4D ratio is 

itself related to high prenatal testosterone(26). 

2D:4D is associated with measures of size at 

birth in males, sperm counts , family size , age 

at breast cancer presentation and age at myocardial 

infarction(27). Our study of 2D:4D ratios 

showed that males have slightly but statistically 

significant lower ratios than the females indicating 

the sexual dimorphism. The difference 

between the males and females digit ratio were 

substantial than compared with the difference 

of the same in the control group. 2D: 4D ratios 

in the Down syndrome group on an average are 

smaller in value (e.g. right hand ratios, males- 

0.961 and females – 0.90) than the age related 

control group (right hand ratios, males – 0.99 

and females 1.050) accounted by short fingers 

of Down syndrome patients and this difference 

is statistically significant. Sexual dimorphism is 

established in spite of brachydactyly in Down 

syndrome patients. As the digit ratios of both 

males and females of Down syndrome patients 

varied significantly from the control group, it 

may be said that yet another factor determines 

the relative lengths of the digits in Down syndrome 

apart from factors which decreases the 

lengths of all the fingers (resulting in brachydactyly). 

Comparing the ratios with the age related 

normal children is expected to neutralize 

any subtle changes in the digit ratios as a part of 

growth during childhood. Our study definitely 

fall short in generalizing the statement that digit 

ratio across all patients with Down syndrome 

is sexually dimorphic as the sample size of the 

study is too small. In our study, we did not find 

a significant relationship between 2D:4D ratio 

and the presence or absence of minor or major 

congenital abnormalities. 

Conclusions: 

107 

We conclude that the digit ratio in Down syndrome 

is sexually dimorphic and thus it may be 

hypothesized that the early perinatal androgen 

levels are same as normal intrauterine development. 

The sexual alterations seen in Down 

syndrome are due to alterations during the descent 

of the testis. This view is supported by the 

normal or near normal development of the other 

genital parts. There is yet another biological 

factor responsible for the hand morphological 

manifestations in Down syndrome – as the digit 

ratios are statistically different from the age 

related counterparts. It may be viewed that the 

factors responsible for the generalized shortening 

of the fingers are also affecting the relative 

lengths of the digits resulting in this kind of difference 

in digit ratio. 

Acknowledgements: 

We thank Mr. Vasanth Kumar Shetty, Principal, 

Saanidhya Samarth Center, Mangalore for allowing 

us to conduct the study. We extend our 

thanks to Dr. Ramesh Pai, Dean, A J Institute 

of Medical Sciences for allowing us to proceed 

with the study. Many thanks to all the children 

of Saanidhya Samarth Center. 

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Dongen, Liliane C. D. Wijnaendts. Sexual Dimorphism in 

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109 


Serum Enzymes, Initial and follow- up Lipid profile in Acute Myocardial Infarction 

*Suman.S. Dambal, **V. Indumati. and ***P.B. Desai 

*Department of Biochemistry, Karnataka Institute of Medical Sciences, 


**Department of Biochemistry, Vijayanagar Institute of Medical Sciences, 

Bellary - 583104, Karnataka, India 

***Department of Biochemistry, Jawaharlal Nehru Medical College, 

Belgaum - 590010, Karnataka, India. 

(Received 8 th October, 2010; Revised 12 th February, 2011; Accepted 28 th February, 2011) 


Dr. Suman.S.Dambal 

E-mail: sumandambal25@gmail.com 

Abstract 

Background & Objectives: Despite impressive studies in the diagnosis and management of Acute 

Myocardial Infarction (AMI) over the last three decades it continues to be a major public health problem in 

the world. There is paucity of literature on the enzyme levels in Stable Angina Patients. Hence the present 

study was taken up to study the diagnostic values of serum enzymes in AMI and as well as in Stable Angina. 

We also studied the Lipid Profile four weeks after AMI with initial levels measured within 24 hours of onset 

of symptoms. 

Methods: 30 cases of AMI, 30 cases of Stable Angina and 30 age and sex matched controls were studied. 

The sample was analysed for Serum Total CK, CK-MB, AST, LDH, Total cholesterol, HDL-C, LDLC, 

VLDL, Non-HDLC and triglycerides. 

Results: There was a significant increase in Total CK, CK-MB, AST and LDH levels in AMI patients when 

compared to both normal controls and Stable Angina patients(P

Bio Chemical profile in Acute MI 

Introduction 

A great deal of evidence is now available to 

show that the incidence of Myocardial Infarction 

varies from place to place due to changes in food 

habits, climate, type of work and ethnic origin. 

Indian immigrants to the west have higher 

mortality compared to the other ethnic groups 

of the adopting country. In the Asian Indian 

study on Coronary artery Disease (CAD), the 

prevalence of CAD was found to be 10.2% 

compared with 2.5% in whites of the same age 

group. Moreover CAD in Asian Indians occur 

prematurely, that is atleast a decade or two 

earlier than that seen in Europeans (1, 2). 

In Indian context, a survey of the incidence of 

Acute Myocardial Infarction (AMI) showed 

that the disease was seven times more common 

among south Indians (3, 4). Coronary Risk 

factor evaluation will identify the major risk 

factors and their importance in Indian context 

(1, 5). Dyslipidemia has been clearly established 

as a major risk factor for the development 

of CAD by a variety of epidemiological, 

pathological, genetic and clinical studies. 

It is one of the important modifiable risk 

factors of CAD. It initiates atherosclerotic 

plague formation. Hypercholesterolemia 

(increase in LDL-Cholesterol), combined 

hyperlipidemia(Increase in LDL-Cholesterol 

and Triglycerides) and hypertriglyceridemia 

are three important risk factors quoted by the 

European Atherosclerotic Society(6, 7). 

Despite impressive studies in the diagnosis and 

management of AMI over the last three decades 

it continues to be a major public health problem 

in the world. AMI warrants a time frame in 

which prompt and appropriate use of life 

saving treatment is of paramount importance 

emphasizing, the need for early diagnosis. 

In view of the enormity of the problem, the 

emphasis has now shifted from treatment to 

prevention of Myocardial Infarction. 

110 

Hence the present study was taken up to study 

the diagnostic values of serum enzymes in AMI 

and as well as in Stable Angina. We also studied 

the Lipid Profile four weeks after AMI with 

initial levels measured within 24 hours of onset 

of symptoms. 


The present study comprises of three groups, 

30 patients (Group-I) with AMI admitted to 

ICCU, 30 patients (Group-II) with Stable 

Angina attending outpatient department of KLE 

Soceity’s hospital and Medical research Centre, 

Belgaum and 30 healthy individuals (Group- 

III) from the staff of Jawaharlal Nehru Medical 

college, Belgaum. The age group was in the 

range of 40-70 yrs. 

Inclusion criteria: 

Patients with documented evidence of AMI as 

per WHO criteria 1985, history of ischaemic 

chest discomfort lasting more than or equal to 30 

minutes associated with characteristic evolution 

of ECG changes like ST segment elevation, Q 

wave and T wave inversion were included in 

study Group-I. 

Patients with chest discomfort, typically caused 

by exertion or emotion lasting for 1-5 minutes 

and relieved by rest or sub-lingual nitroglycerine 

were included in study Group-II. 

Exclusion criteria: 

Patients with unstable Angina and resting ECG 

changes, Liver diseases, muscular dystrophy, 

history of intramuscular injection prior to or 

after the onset of chest pain and subjects having 

diseases which are known to influence the blood 

lipids or on Lipid lowering drugs were excluded 

from the study. 

All the three groups were age and sex matched. 



Informed consent was taken from patients and 

healthy individuals. The study was approved by 

the Ethical & Research Committee of JNMC, 

Belgaum on the use of human subjects in the 

Research. 2 ml venous blood was collected in a 

plain bulb under aseptic precautions from both 

patients with AMI and Stable Angina for enzyme 

assay. The next day 2ml of fasting venous blood 

was collected between 8 a.m-10 a.m without 

any anti-coagulant for assay of Lipid profile 

from AMI patients. These AMI patients were 

asked to come for a follow-up after one month 

and 2 ml of fasting venous blood was collected 

to study the follow-up Lipid profile levels. 

Serum was separated after half an hour and 

enzyme assay was carried out immediately and 

Lipid assay within 12 hours. The sample was 

analysed for Serum Total Creatine Kinase(CPK), 

CK-MB, Aspartate transaminase (AST), Lactate 

dehydrogenase(LDH). Different lipid fractions 

estimated included Total Cholesterol, high 

density lipoprotein-cholesterol (HDL-C), low 

density lipoprotein-cholesterol (LDLC), very 

low density cholesterol (VLDLC), Non- high 

density lipoprotein-cholesterol (Non-HDLC) 

and triglycerides[8-11]. One way ANOVA 

followed by Bonferroni multiple comparison 

test, paired and unpaired students’s ‘t’ was 

employed for the statistical analysis of the data 

to compare the groups. 

Results 

The comparable study of Enzyme levels in 

healthy controls, AMI and Stable Angina 

patients is shown in Table 1. There was a 

significant increase in Total CK, CK-MB, 

AST and LDH levels in AMI patients when 

compared to both normal controls and Stable 

Angina patients(P


Table 1. Serum Enzyme levels in healthy controls, AMI and Stable Angina patients 

Enzymes Healthy Controls AMI Stable Angina 

Total CPK 120.0 ± 25.39 282.9 ± 30.13 * 131.07 ± 24.76 

CK-MB 14.87 ± 4.92 165.36 ± 22.50 * 15.57 ± 4.18 

AST 25.13 ± 7.69 139.6 ± 21.93 * 25.87 ± 8.53 

LDH 151.1 ± 43.04 317.73 ± 25.41 * 170.6 ± 33.50 

Values are expressed in as IU/L (Mean ± SD). (*p < 0.0001) in AMI patients when compared to both normal controls and Stable Angina patients. 

There was no significant change in enzyme levels in Stable Angina patients when compared to Controls. 

Table 2. The Initial and Follow-up Lipid profile in AMI patients 

Lipid Profile Healthy Controls AMI MI at Follow-Up 

Total Cholesterol 194.23 ± 19.13 223.23 ± 14.98 *** 222.35 ± 15.72 

LDL C 124.77 ± 19.41 158.63 ± 18.26 *** 156.0 ± 20.73 

TG 105.30 ± 15.43 164.03 ± 20.22 **** 164.40 ± 20.70 

VLDL C 21.67 ± 3.19 32.80 ± 4.10 **** 32.89 ± 4.13 

HDL C 

Non-HDL C 

48.20 ± 6.17 

146.03 ± 19.08 

31.80 ± 4.47 **** 

191.53 ± 17.78 **** 

33.35 ± 5.32 

189.5 ± 19.08 

Values are expressed in as IU/L (Mean ± SD). (****p < 0.0001, ***p


VLDLC, Non-HDLC and significant decrease 

in HDLC in AMI patients (which was measured 

within 24 hrs of occurrence of MI) as compared 

to healthy controls. More recent data suggests 

that measurement of Non -HDL Cholesterol 

level (Calculated as Total Cholesterol minus 

HDL Cholesterol) could be more representative 

of all atherogenic, apolipoprotein(apo) B 

containing lipoproteins –LDL,VLDL,IDL 

and Lipoprotein(a). Although apolipoprotein 

B can be assessed directly, measurement 

of Non-HDL Cholesterol is more practical, 

reliable, inexpensive and can be considered 

as a surrogate marker for apolipoprotein B in 

routine clinical practice (15, 16). There was no 

significant difference between the initial and 

follow-up Lipid profile (after one Month) in the 

AMI group. Our findings are consistent with 

other research workers (17, 18, 19). 

From the literature it is found that Total 

cholesterol and Triglycerides are affected by 

factors like caloric intake, exercise etc., and 

therefore the increase in their values found 

in the present study cannot serve as a reliable 

prognostic indicator in AMI patients. On the 

other hand HDLC is not acutely affected by the 

said factors. HDLC could therefore serve as a 

reliable prognostic indicator for patients with 

AMI. Although there may be several phasic 

changes in serum Lipids during the course of 

AMI, Lipid measurements made within 24 hrs 

of AMI are still useful guide in order to identify 

patients requiring follow-up. Furthermore, 

patients are most impressionable for dietary 

or therapeutic advice in the immediate post 

infarction period. 

References 

1. Rajmohan L,Deepa R and Mhan V. Risk factors for CAD 

113 

in Indians; Emerging trends. I.H.J.2000;52:221-225 

2. Krishnamoorthy K.M. Diet and CAD. 

I.H.J.1999;51:268-274 

3. Malhotra S.L. Geographical aspects of AMI in India with 

special reference to patterns of diet and eating. B.H.J 

1967;29:337-343 

4. Padmini S.F and and Motlag D.B. Lipoprotein Profile 

changes among chronic smokers and smokers with M.I. 

I.H.J.1987;39:38 

5. Bhatia ML. Prevalence of coronary heart disease in India: 

a contemporary view. Indian Heart Journal. 1995 Jul-Aug; 

47(4): 339-42 

6. Gore JM, Goldberg RJ, Matsumoto AS, et al. Validity of 

serum total cholesterol 

7. levels obtained within 24 hours of acute myocardial infarction. 

Am J Cardiol 1984;54:722–725. 

8. Mahajan A.S., Reddy K.S., Sachdeva U. Lipid profile of 

coronary risk subjects following yogic lifestyle intervention. 

Ind. Heart J 1999 Jan-Feb, 51(1): 37-40 

9. Allain CC. Enzymatic colorimetric method for estimation 

of serum cholesterol . Clin Chemistry 1974;20: 470. 

10. Lopes-Vireflla MF. Enzymatic colorimetric method for estimation 

of HDL-cholesterol. Clin Chem 1977; 23: 288. 

11. Varley H, Gowenlock AH, Bell M. Lipids and Lipoproteins 

In: Proteins clinical biochemistry 5th ed, William 

Heinemall Medical books Ltd London, 1980; 1. 

12. Bucolo D, David H. Quantitative determination of serum 

triglycerides by use of enzymes. Clin Chem 1973; 19: 476. 

13. Roberts R, Gowda KS, Ludbrook PA, Sobel BE. Specificity 

of elevated serum MB creatine phosphokinase activity 

in the diagnosis of acute myocardial infarction. Am J Cardiol. 

1975 Oct 6; 36(4):433–437. 

14. Kibe olaf and Nils J.N. Observations on the diagnostic & 

prognostic values of some enzyme tests in MI. Acta Medica 

Scandinavica.1967;182(5): 597-610 

15. Smith AF, Radford D, Wong CP, Oliver MF. Creatine kinase 

MB isoenzyme studies in diagnosis of myocardial infarction. 

Br Heart J. 1976 Mar; 38(3):225–232. 

16. Anne L.Peters. Clinical relevance of Non-HDL cholesterol 

in patients with diabetes. Clinical Diabetes.2008;26(1):3-7 

17. Grundy SM. Low-density lipoprotein, non-high-density lipoprotein 

and apolipoprotein B as targets of lipid-lowering 

therapy. Circulation.2002; 106:2526-2529. 

18. Vincelj J, Sucić M, Bergovec M, Sokol I, Mirat J, Romić 

Z, Lajtman Z, Bergman-Marković B, Bozikov V. Serum 

total, LDL, HDL Cholesterol and Triglyceride related to 

age, gender & cigarette smoking in patient with first acute 

myocardial infarction. Coll Antropol 1997;21(2):517-524 

19. Ryder, RE; Hayes, TM; Mulligan, IP; Kingswood, JC; Williams, 

S; Owens, D R. How soon after MI should plasma 

lipid values be assessed. BMJ 1984;289:1651-1653 

20. Sewdarsen M, Vythilingum S, Jialal I, et al. Plasma lipids 

can be reliably assessed within 24 hrs after AMI. Postgraduate 

Med.J 1998;64:352-356 


114 


Association of serum ferritin levels between rheumatoid arthritic obese and 

Non rheumatoid arthritic obese 

*Tandra Majhi and **A.K. Srivastava 

*Department of Psychiatry, C.S.M.Medical University, Lucknow UP India.226003 

**Department of Zoology, C.M.P. Degree College, Allahabad UP India.211002 

(Received 29 th June, 2010; Revised 20 th November, 2010; Accepted 10 th January, 2011) 

Corresponding author: 

Dr. Tandra Majhi, 

E-mail: tandra.majhi@gmail.com 

Abstract 

Background & Objectives: In Iron deficiency anemia basic of ferritin is appreciably reduced. This variable 

was determined in 16 obese rheumatoid arthritic patients, aged 30-60 years with matched 28 obese non 

rheumatoid arthritic subjects. The BMI cutoff value for the both groups were less than 34. It is measured 

by the formula. 

Methods: Hb% was measured by the cyanide method while serum iron and TIBC were measured by the 

kits which were available by commercially. Serum ferritin was measured by the commercially available 

ferritin kit (Lilyat Medicine Company USA) Fisher, ‘t’ test and correlation Coefficiency were applied for 

the statistical analysis. 

Results: The prevalence of iron deficiency was found 6.49% in males and 14.29% in females with obese 

RA. The indirect relationship was found between BMI and Hb%, whereas direct association between age. 

A directly proportional relationship found to be BMI age and serum TIBC (r=0.09, P = 0.73; r = 0.04, P = 

0.87). While the serum iron (BMI : r = - 0.12, P = 0.69; Age: r = -0.41, P = 0.12) and ferritin (BMI : r = 

0.09, P = 0.71; age : r = -0.17, P = 0.52) levels shows significantly negative correlative with BMI and age 

in obese rheumatoid arthritic subjects. 

Conclusion: These results suggested that the iron deficiency anaemia is associated with obese rheumatoid 

arthritic patients. 

Keywords: Rheumatoid arthritis, Obesity, Rheumatoid arthritic obese, Iron deficiency, Serum ferritin 

Introduction: 

Rheumatoid arthritis (RA) is an autoimmune 

disorder of unknown etiology characterized 

by symmetric, erosive synovitis and, in same 

eases, extra articular involvement (1) . Anaemia 

is a frequent finding in patients with rheumatoid 

arthritis and its severity roughly parallels the 

activity of the disease (2,3). Serum ferritin 

concentration has been shown to give an accurate 

indication of the amount of storage iron in normal 

subjects and in patients with iron deficiency and 

overload (4,5). Excess body fat is a prominent 

health hazard (6) significantly contributing 

to the development of cardiovascular disease 

(CVD)(7) About two thirds of patients who 

have had a myocardial infarction (MI) exhibit 

increased body weight(8). Obesity increases the 


serum ferritin in rheumatoid obese 

risk of coronary heart disease (CHD) through 

a number of different pathophysiological 

pathways, including insulin resistance, type 2 

diabetes, hypertension and dyslipdaemia(9,10). 

The purpose of this study was to establish 

correlation between serum ferritin levels in 

rheumatoid arthritic obese patients and nonrheumatoid 

arthritic obese controls. 


Subjects: 

The present observational study was conducted 

in the Department of Biochemistry M.L.N. 

Medical College Allahabad U.P. The patients 

clinically diagnosed from OPD who had not 

undergone any previous treatment or medication 

for their arthritis, were chosen for the study. The 

study was categorized into two groups. 

(i) Control Group : Obese subjects were taken 

in this group. Those patients were included 

in this group who had negative RA Factor 

value and who were not suffering from any 

pathological and physiological illness. 

(ii) Study Group : Under this group, people 

suffering from rheumatoid arthritis with obesity 

were taken. In this group the inclusion criteria 

was positive RA factor value. According to 

excluding criteria, the patients had not been 

suffering from any other pathological and 

physical illness. The subjects were in the age 

of 30-60 years and the BMI cutoff value for 

the both groups were less than 34. Information 

regarding socio-demographic variables such as 

age, sex, martial status religions, education and 

income were obtained. Body Mass Index was 

calculated by the formula, BMI = weight (kg)/ 

Height 2 (m 2 ). The total participated subjects 

were 77 in the study group but only 16 subjects 

were chosen for the study, which fulfilled the 

study criteria. Fifty two subjects were enrolled 

in the control group. Whereas 28 subjects 

were selected for the control group which had 

fulfilled the study criteria. 

Assessment : 

115 

Peripheral blood (5ml) was collected in 

vacutainer containing EDTA anticoagulant or 

no anticoagulant. Hb% was measured by adding 

blood or haemolysate to ferricyanide-cyanide 

reagent to convert the hemoglobin pigment to 

cyanomethamoglobin by method of cyamide. 

(11) Serum iron and TIBC were measured by 

Kits based on ferrozine method. Serum ferritin 

was measured by commercially available 

ferritin kit. (Lilyat Medicine Company USA) 

Statistical Analysis : 

Fisher test and’t’ test were used for the 

comparison of the means ± of the sample for 

these groups. Correlation between BMI, age 

and blood variables in patients and control were 

calculated by using Correlation coefficient. 

Results 

The prevalence of iron deficiency was 6.49% 

in men and 14.29% in women with obese 

rheumatoid arthritis. Only three men, but 

none of the women subjects, were diagnosed 

normal without iron deficiency anemia. Present 

study was done on 16 obese rheumatoid 

arthritis patients i.e. 11 females and 5 males 

and compared with 28 obese non rheumatoid 

arthritic patients i.e. 18 females & 10 males. 

There were no significant differences between 

the sex, age and BMI in these two groups. Hb%, 

Serum iron, TIBC and serum ferritin levels were 

significantly low in the study group compared 

to the control group. The inverse relationship 

was found between BMI and Hb% (r = - 0.168, 

P = 0.53), whereas positive association between 

was found age and Hb% (r = 0.12, P = 0.65). A 

directly proportional relationship was found to 

be BMI, age and serum TIBC (BMI: r = 0.09, 

P = 0.73; Age: r = 0.04, P = 0.87). The serum 

iron correlated significantly with decrease 

activity in both BMI (r = -0.12, P = 0.69) and 

the age (r = - 0.41, P = 0.12). Percentage of 



Table 1: Demographic Characteristics of the two groups 

Variables ControlGroup N = 28 Study Group N = 16 

Sex M : F 10 : 18 5:11 

Age (in year) 47.81±11.56 46.94±10.56* 

BMI 31.32 ± 1.27 32.23 ± 2.19* 

* Non-Significant, Mean ± SD 

Table 2: Comparative study of Hb%, Serum iron, TIBC and ferritin levels in obese and obese rheumatoid 

arthritic patients 

Variables 

Control Group 

(N=28) 

Study Group 

(N = 16) 

Hb% (gm%) 13.20 ± 1.27 10.34 ± 2.18 * 

Serum Iron (μg/dl) 100.34 ± 25.74 45.62 ± 28.15 * 

SerumTIBC (μg/dl) 293.94 ± 47.72 183.59 ± 60.59 * 

Serum Ferritin (ng/dl) 169.04 ± 57.72 105.36 ± 39.50 ** 

Value expressed mean ± SD, * P < 0.001 Highly significant and ** P


Hb was significantly decreased with age while 

there was no significant changes with the body 

mass index in study group. While serum ferriten 

shows negative correlation with BMI (r = -0.09, 

P=0.71) and with age (r = -0.17, P = 0.52) in 

the study group. Serum iron and ferritin levels 

both were significantly decreased with BMI and 

age in the obese rheumatoid arthritis patients. 

These results suggested that the iron deficiency 

anemia was associated with obese rheumatoid 

arthritic patients. 

Discussion 

The result of the study showed that Hb%, serum 

ferritin serum iron and TIBC concentration in 

the study group were lower than control group. 

There was a significant positive correlation 

between BMI and age with serum TIBC in 

the study group. By contrast ferritin and iron 

concentration were lower in obese rheumatoid 

arthritis and were positively correlated with BMI, 

age findings consistent with the observation that 

obesity in an inflammatory state, which increase 

acute-phase reactants. Correlation regression 

analysis showed that while the best model to 

predict serum iron included ferritin and total iron 

binding capacity, most of the predicted variance 

was accounted for by transferring receptor 

alone, although inflammatory indices were also 

independent predictors. Therefore it is possible 

that lower levels ferritin may increased the risk 

of iron deficiency in the rheumatoid arthritis 

with obesity. Using serum transferrin receptor 

to predict the presence of iron deficiency 

by LB Yanoff et al(12). They found higher 

odds of iron deficiency in obese vs non-obese 

subjects. They showed the ferritin levels tend 

to be elevated in obesity-related inflammatory 

state, they did not show a difference between 

obese and non-obese subjects is the prevalence 

of iron deficiency. They explained the 

hypoferrimia of obesity appears by true iron 

deficiency hypoferrimia and by inflammatorymediated 

functional iron deficiency. Elevated 

transferring receptor levels correlate well with a 

117 

lack of stainable iron in bone marrow in normal 

subjects as well as in patients with rheumatoid 

arthritis, and transferrin receptor reportedly has 

a higher sensitivity than ferritin to diagnose 

iron deficiency in patients with ferritin elevated 

from acute-phase reaction (13,14,15) Similar to 

other inflammatory conditions, obesity appears 

to be a state in which transferrin receptor is a 

useful adjunct to ferritin in the diagnosis of iron 

deficiency. 

Davidson et al(16) showed that iron deficiency 

anaemia in patients with rheumatoid arthritis may 

be difficult to distinguish from the microcytic 

anemia found in chronic inflammatory disease. In 

uncomplicated iron deficiency reduced marrow 

iron is a reliable index for this deficiency. This 

reduction in stainable marrow iron correlation 

with both mirocysosis and lower plasma ferritin 

concentration (17,18). 

On the other hands the etiology of aging in 

important to understand, but it is equally 

important to differentiate the normal 

physiological changes from those associated 

with diseases. It may be generalized atrophy 

of all muscle accompanied by a replacement of 

same muscle tissue by fat deposit. This results 

in some loss of muscle tone and strength. Some 

specific implications of this are reduced ability 

to breath deeply and reduced gastrointestinal 

activity which can lead to constipation or 

bladder incontinence, particularly in women. 

The joints also undergo changes. Arthritis, the 

degenerative inflammation of joints, is the most 

common chronic condition is the elderly obese 

persons with iron deficiency. 

In conclusion, the study indicates that decreased 

ferritin concentration is associated with obese 

rheumatoid arthritis among, middle aged and 

elderly. It is possible that lower levels of ferritin 

may be increased in the risk of iron deficiency 

anemia in the rheumatoid arthritis with obesity. 

The etiology appears to be multifactorial and 

may include inadequate bioavailable iron 

relative to body weight, as well as diminished 

intestinal absorption and decreased iron 

bioavailability included by inflammatory with 



excessive adiposity. The precise mechanisms of 

the obesity-related, inflammation induced effect 

in serum iron remain to be elucidated. Further 

researches are needed to confirm and extend 

these findings on obesity with rheumatoid 

arthritis in large number of subjects. 

Reference 

1. Harrcis ED Jr. Rheumatoid arthritis : pathophysiology 

and implications for therapy. N Engl J Med., 1990; 322 

: 1277-89. 

2. Nilsson F. Anemia problems in rheumatoid arthritis. Acta, 

Med. Scand. 130 suppl. 1948; 210, 72. 

3. Jeffrey M.R. Some observations on anemia in rheumatoid 

arthritis, Blood. 1953; 8; 502-518. 

4. Jacobs A, Mitter F, WorWood M, Beamish MR Wordrop 

CA., Ferritin in the serum of normal subjects and patients 

with iron deficiency and iron overload. Brit. Med. J.,1972 

4, 206-208. 

5. Walters GO, Miller FM Worwood M., Serum ferritin concentration 

and iron stores in normal subjects. J. Clin.Path. 

1973, 26, 770-772. 

6. Van Pelt RE, Jones PP, Davy KP, Desouza CA, Tanaka H, 

Davy BM et al. Regular exercise and the age related decline 

in resting metabolic rate in women. J. Clin. Endocrinol 

Metab.1997, 82 : 3208-12. 

7. Ross R. Atherosclerosis- an inflammatory diseases N. Eng 

1.1 Med. 1999,40: 115-26. 

8. Romero-Corral A, Montori VM, Somers VK, Korinek J. 

Thomas RJ, Allison TG et. al. Association of body weight 

with total mortality and with cardiovascular events in coro- 

118 

nary artery disease : a systematic review of cohort studies. 

Lancet 2006, 368: 666-78. 

9. Pi-Sunyer FX. The obesity epidemic: pathophysiology and 

consequences of obesity. Obesity Res. 2002 ,10: 975-104. 

10. Krauss RM, Winston M, Fletecher BJ, Grundy SM. Obesity 

: impact on cardiovascular disease. Circulation,1998, 

98; 1472-6. 

11. The Haemoglobin Cyanide method. Clin. Chem. Actr. 

1961, 6: 538-545. 

12. Yanoff LB, Menzie CM, Denkinger B, Sebring NG, 

McHugh T, Remaley AT, Yanovksi JA. Inglammation and 

iron deficiency in the hypofessemia of obesity. Int. J. Obes. 

Sept. 31 (a): 2007,1412-1419 

13. Punnonen K, Irjala K, Rajamaki A.,Serum Transferrin receptor 

and its ratio to serum feritin in the diagnosis of iron 

deficiency, Blood 1997, 89; 1052-1057. 

14. Means RT, Jr. Allen J, Seas DA, Schuster SJ.,Serum Soluble 

transferrin receptor and the prediction of marrow aspirate 

iron results in a heterogeneous group of patients, Clin, 

Lab. Haematol. 1999, 21: 161-167. 

15. Most AE, Blinder MA, Gronowski AM, Chumley C, Scott 

MG.,Clinical utility of the soluble transferrin receptor and 

comparison with serum ferritin in several populations Clin. 

Chem. 1998, 44: 45-51. 

16. Davidson A, Weyden MBVD, Fong H, Breidahl MJ, Rayan 

PFRed cell ferritn content: a re-evaluation of indices for 

iron deficiency in the anemia of shumatoid arthritis. Brit. 

Med. J. Vol.1984. 289; 648-650. 

17. Brink S, Van Schalkwyk DJ. Serum ferritis and mean corpuscular 

volume as predictions of bone narrow iron stores, 

S. Afr. Med. J. 1982, 61: 432-434. 

18. Krause JR, Stok V. Serum ferritin and bone marrow iron 

stores. Correlation with absence of iron in biopsay specimens 

Am. J. Clin, Pathol.,1979, 72: 817-20. 

. 


Case Reports Biomedicine; 2011; 31 ( 1 ) : 119 - 121 

A case of asymptomatic anomalous pancreatico biliary ductal union of 

pancreatic - biliary type 

V.S. Anandarani. 

Departmentof Anatomy, Sri Ramachandra Medical College, Porur,Chennai - 600116, India. 

(Received 9th December 2010; Revised 10th February 2011 Accepted 23rd February 2011) 


Dr.V.S. Anandarani. 

E-mail: ranironeena@yahoo.co.in 

Abstract 

Anomalous pancreatico biliary junction is an unusual variant of pancreatico biliary anatomy of clinical 

importance. It is a congenital anomaly where the common duct formed by the union of main pancreatic 

duct and bile duct is more than 15 mm in length. According to the mode of termination two different types 

are described .Each type is associated with specific clinical conditions. 

Key words - pancreatic duct, common duct, pancreatico biliary tree. 

Introduction 

Anomalous pancreatico biliary ductal union 

[APBDU] is a congenital anomaly. The 

incidence is not clear because the identification 

of this condition is done only during the 

investigations for the diseases related. It is 

a complex anatomical and functional entity 

.The main pancreatic duct and bile duct are 

joined well outside the duodenal wall to form a 

common duct. The common duct of more than 

15 mm length is noted as an anomalous ductal 

union[1]. It is reported that 16.7% of patients 

with carcinoma gall bladder shows associated 

APBUD [2]. This congenital anomaly can 

influence the degrees of pancreatic fluid 

regurgitation which results in an increased 

incidence of biliary tract malignancy[3]. 

Case report 

Adult duodeno pancreas specimens were used 

for a study of the ductal pattern of pancreas. The 

specimens were collected from the cadvers of 

the dissection hall and 100 specimens including 

119 

64 males and 36 females were used. The 

pancreas was removed along with duodenum 

and the bileduct was incised just above the first 

part of duodenum. On the posterior aspect of 

the specimen the bile duct was traced down to 

the junction with the pancreatic duct . From this 

point pancreatic ductal system was traced out. 

In a specimen collected from a male cadaver of 

55 years(died of road traffic accident) pancratco 

billiary ductal union was well outside the 

duodenal wall. A clean slit was made on the bile 

duct away from the union with pancreatic duct 

and the incision was extended down and the duct 

was opened upto the major duodenal papillae. 

This was to confirm whether the common 

channel was a true one or if there is any septum 

separating biliary and pancreatic passages. 

The length of the true common channel was 

measured . The true common channel was of 

16mm in length. It was formed well out side the 

duodenal wall (fig-1).There was no apparent 

change in the size of common bile duct or the 

main pancreatic duct as they approached the 


asymptomatic pancreatico biliary ductal union 

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 

pancreatic duct 

ductal junction. 

Discussion 

In majority the main pancreatic duct and the 

common bile duct open into the second part of 

the duodenum after the formation of a common 

channel . The terminal part of the common 

duct shows a dialatation called ampulla. Around 

the ampullary region a sphincter is described. 

The sphincter choledochus and the sphincter 

pancreaticus surrounds the periampullary 

parts of the bileduct and main pancreatic duct 

respectively. The sphincter of oddi (proper) is 

described around the ampulla. These three sets 

of sphincters together described as sphincter of 

oddi [ 4]. Anomalous pancreaticobiliary ductal 

junction is an abnormal union of the pancreatic 

and biliary ducts that is located outside the 

duodenal wall and the length of the common 

channel is more than 15mm[1]. A normally 

functioning spincter of oddi and an intramural 

ductal junction may prevent the duodenal reflux 

into pancreatico biliary ductal tree as well as the 

reflux of pancreatic juice into bile duct and vice 

verse. A long common channel with an extra 

mural ductal junction indicates a dysfuncting 

sphincter of oddi [5]. Thus in a case of APBDU 

two ducts are always communicating and two 

120 

way regurgitation ie, pancreatico biliary reflux 

and bilio pancreatic reflux might occur[6]. 

Anomalous pancreatico biliary ductal union is 

significant because of its clinical association. 

Two types of APBDU were noted according 

to the morphological changes occurring in the 

ducts before 

union [1,7]. 

1. pancreatic - biliary type -- there is no 

notable change in ducts [P-B type]. 

2. biliary – pancreatic type-- there is a 

small dilatation in the common duct just proximal 

to the ductal union.[B-P type]. 

In the present case there was no notable 

changes in the ductal morphology towards the 

ductal union. So the case was included under 

P-B type of APBDU. Known associations 

of APBDU include bileduct carcinoma, gall 

bladder carcinoma and cystic disease of gall 

bladder[1,7].The specific association were 

noted for B-P type with choledochal cyst 

and P-B type with gall bladder carcinoma 

and biliary pancreatitis[7]. APBDU can be 

associated with other congenital anomalies like 

divisum of pancreas[8] and multi septate gall 

bladder[9]. Among the patients with diseases 

of pancreatico biliary tree 8.7% with clearly 

visualized pancratico biliary radiograms had 

APBDU[6]. The junction of common bile duct 


asymptomatic pancreatico biliary ductal union 

and main pancreatic duct, when extraduodenal 

and is unprotected by sphincter of oddi the 

reflux of pancreatic exocrine secretions into 

the unprotected biliary tree occurs.This results 

in inflammation which causes the epithelial 

distruction and dilatation of the bileduct[10]. 

Mizuno M etal [11] suggested that the reflux 

of the activated pancreatic juice into the biliary 

tract is an impotant factor in the development of 

biliary tract carcinoma. The optimal approach 

is the prevention of reciprocal reflux of bile and 

pancreatic juice . Surgical treatment includes 

complete biliary diversion procedures [12]. In 

the present study APBDU was asymptomatic 

Summary 

The most outstanding feature of the normal 

anatomy of the extrahepatic biliary system 

is its high degree of variability. Anomalous 

pancreatico biliary junction is an unusual 

variant of pancraticobiliary anatomy of 

clinical importance because it is associated 

with increased risk of pancreatitis, diseases of 

biliary tree, cystic changes of gall bladder and 

also malignant changes of the biliary tree. In 

the present study the specimen showed a P-B 

type of APBDU without any recorded clinical 

complaints related to this congenital anomaly or 

morphological changes of associated structures. 

Such cases go undetected because as a rouine 

these congenital anomalies are reported during 

the investigative radiological procedures. 

According to the study result the prevalence 

of APBDU in a randomized south Indian 

population is 1%.But data needs confirmation 

from more studies especially by radiological 

screening trials as well dissection studies with 

more number of specimens. 

References 

121 

1. Richer.J.P, Faure .J.P, Morichau-Beauchant Dugue.T,M 

aillot.N,Kamina.P,Carretier.M. Anomalous pancreatico 

biliary ductal union with cystic dilatation,.Surgical and 

radiological Anatomy ,March 1998 :20;139-142. 

2. Kimura.K.,Ohto.M.,Saisho.H.,Unozawa.T.,Tsuchiya.Y.,M 

orita.M.,Ebara.M.,Matsutani.S.,Okulda.K.Association of 

gall bladder carcinoma and anomalous pancreatico biliary 

ductal union.;Gateroenterology1985 Dec;89(6);1258-65. 

3. Roukounakis.N.E, Kuhn.J.A, Mc Carty .T.M, Association 

of an abnormal pancreaticobiliary junction with biliary 

tract cancers. Proceedings (Baylor University Medical 

Center)2000:13:11-13. 

4. Boyden EA. The anatomy of the choledocboduodenal 

junction in man. Surg Cynecol Obstef 1957; 104:641-52. 

5. Kamisawa T, Okamoto A: Biliopancreatic and 

pancreatobiliary refluxes in cases with and without 

pancreaticobiliary maljunction: diagnosis and clinical 

implications. Digestion 2006;73:228–236. 

6. Fumino.S,Tokiwa..K.,Katoh .T,Ono.S.,Iwai.N. New 

insight into bile flow dynamics in anomalous arrangement 

of the pancreatico biliary duct British journal of Surgery 

2002:89(7):865-869. 

7. Wang H-P, Wu M-S,LinC-C, Chang L-Y,KaoA-W,Wang 

H-H, LinJ-T . Pancreatico biliary diseases associated 

with anomalous pancreatico biliary ductal union. 

Gastrointestinal endoscopy1998: 48(2):184-189. 

8. Colm J McMahon, Charles M Vollmer; Jeffrey Goldsmith; 

Alphonso Brown; Douglas Pleskow; Ivan Pedrosa 

.Pancreas(Journal of Neuroendocrine tomours and 

pancreatic tomours and pancreatic diseases and sciences) 

Jan 2010;39.; 101-104. 

9. Takafumi Yamamoto,Jun Matsumoto,Shinya Hashiguchi 

,Atsumasa Yamaguchi,Koro Sakoda, Chiaki Taki. 

Multiseptate gall bladder with anomalous pancreatico 

biliary ductal union :A case report World Jornal of 

Gastroenterol 2005;11(38);6066-6068. 

10. Babbit DD (1969) Congenital choledochal cyst. New 

etiological concept based on anomalous relationships of 

the commonbile duct and pancreatic bulb. Ann Radiol 

12:231 

11. Mizuno M, Kato T, Koyama K (1996) An analysis 

of mutagens in the contents of the biliary tract in 

pancreaticobiliary maljunction.Surg Today 26:597–602 

12. Todani T, Watanabe Y, Narusue M, Tabuchi K, Okajima K 

(1977) Congenital bile duct cyst; classification, operative 

procedure and review of 37 cases including cancer arising 

from choledochal cyst. Am J Surg 134:263–296. 


122 


Unusual complication at Feeding Jejunostomy in Boerhaave’s Syndrome 

*Vithalkumar. M. Betigeri, **Anupama V Betigeri, ***Nanda Kishore Maroju and *Kasturi 

Satya Venkata Kumar Subba Rao 

*Department of Cardiothoracic and Vascular surgery, VMMC and Safdarjung hospital, 

New Delhi, India. 110029 

**Department of Physiology, ***Department of General Surgery, Jawaharlal Institute of Postgraduate 

Medical Education & Research, Puducherry-605006,India. 

(Received 17 th November, 2010; Revised 25 th January, 2011; Accepted 16 th February, 2011) 


Dr Vithalkumar. M. Betigeri. 

e-mail: vithalkumarmb@gmail.com 

Abstract 

Spontaneous rupture of esophagus is the most lethal perforation of gastrointestinal tract. Even with typical 

presentation and high index of suspicion, this rare cause of perforation is lethal with mortality of 10-50%, 

as result of subsequent post operative complications. We report such a case of 24 year old male patient 

managed initially by primary repair but later succumbed to rare postoperative complication of jejunojejunal 

intussusception at feeding jejunostomy site. 

Key Words: Esophageal perforation, Intussusception, Surgery complications 

Introduction: 

Inspite first description in 1724 by Herman 

Boerhaave, spontaneous transmural 

esophageal rupture is most lethal perforation 

of gastrointestinal tract with mortality of 10- 

50%, delayed diagnosis and comorbidities are 

historically most often associated factor for it(1). 

Jejunojejunal intussusception, the telescoping 

or an inavagination of a segment of jejunum in 

to an adjacent one, with tip of feeding tube as 

lead point is rare cause of adult small bowel 

obstruction following tube jejunostomy(2). 

Case Report: 

Twenty hours after initial conservative 

treatment elsewhere for abdominal pain and 

breathlessness after repeated bouts of vomiting 

following an alcohol binge, a 24 year old male 

was referred in febrile, tachycardia, tachypneic, 

condition with blood pressure of 100/80mmHg. 

Chest X-ray showed pneumomediastinum. 

Contrast barium swallow suspected leak 

from the esophagus. Esophagoscopy (Fig.1) 

confirmed 5X3 cm ragged edged perforation in 

left lateral wall, 4cm above the gastroesophageal 

junction. After initial resuscitation with fluids 

and antibiotics, patient underwent exploratory 

left posterolateral thoracotomy. Linear 

esophageal tear (Fig.2) of 8cm length, from 2 

cm above the diaphragm to the level of left lung 

hilum with ragged and necrotic mucosal edges 

was found. Patient underwent debridement, 

closure of the esophageal defect in two layers 

across Ryle’s tube with left pericardial pad of 

fat buttressing, left pleural drainage, Stamm’s 

draining gastrostomy and Witzel’s feeding 

jejunostomy. Post operative course was 

stormy with intermittent episodes of fever, 


Unusual complication at Feeding Jejunostomy 

Figure 1-Biomedicine 

Figure 2 -Biomedicine 

Figure 3- Biomedicine 


123

Unusual complication at Feeding Jejunostomy 

staphylococcal growth in the pleural fluid, toxic 

granules with left shift, anemia, hypokalemia, 

hypocalcemia, hypoalbuminemia. Antibiotics 

according to culture sensitivity were given. 

As barium swallow done on fourteenth post 

operative day revealed anastomosis leak and 

general condition of the patient was poor, 

nutritional status managed with enteral feeding 

which was tolerated well. 

In subsequent postoperative recovery period 

patient had complained of recurrent pain 

abdomen without clinical and radiological 

evidence of obstruction. Small bowel series 

revealed delayed passage of contrast with 

retention in stomach, duodenum and proximal 

jejunum and feeding tube contrast study 

showed normal caliber of the distal bowel. 

When initial conservative management to treat 

the obstruction was failed, patient underwent 

reexploratory laparotomy where we found 20cm 

long antegrade jejunojejunal intussusception 

(Fig.3), 10 cm distal to the jejunostomy tube 

entry site, with normal position of the tube and 

two fixation sites of it to the peritoneum and 

the tip of tube projecting beyond apex of the 

intussusception. Jejunal resection for impending 

gangrene features and end to end anastomosis 

with resiting of feeding jejunostomy was done. 

After post operative recovery, jejunostomy 

feedings restarted. Subsequent postoperative 

period was overwhelmed by sepsis with 

poor nutritional recovery and patient died on 

eightyfourth postoperative day. 

Discussion: 

Because of its rarity and atypical clinical 

findings(3,4),intussusception poses diagnostic 

challenge in adults who uncommonly have 

acute intestinal obstruction as a presenting 

feature unlike in children, often have symptoms 

of partial intestinal obstruction like pain and 

vomiting. Adult require resection almost always 

as demonstrable pathology is seen in 90% 

cases, eventhough the extent of resection and 

124 

whether or not to reduce it before resection is 

controversial(3,4). 

Feeding jejunostomy by various techniques 

is well established method to provide enteral 

nutrition and medications in patients expected to 

require prolonged nutritional support. Although 

technically simple, complications are various 

(5) with incidence of 8-20% and mortality of 

2-10 %( 6). 

Our case highlights rare, serious but potentially 

reversible jejunojejunal intussusception as a 

complication of jejunostomy tube(2) which was 

missed preoperatively because, tube feeding 

was not interfered inspite intussusception, 

clinical and radiological findings did not 

differentiated small bowel obstruction due 

to intussusception from adhesions or suture 

line. Although radiographic findings of 

intussusception are incidental and transient 

for tube jejunostomy patient(7), unresolving 

nature of long segment intussusception 

resulting in pregangrenous changes, delayed 

diagnosis due to atypical presentation made 

resection rather reduction a treatment option in 

our patient. After successful surgical resection 

of jejunojejunal intussusception, we were able 

to restart neojejunostomy tube feeding, which 

is utmost important in Boerhaave syndrome in 

septic profile. Our case highlights importance 

of post operative complications that occurred 

inspite aggressive primary repair of esophagus 

and failure of primary repair possibly due to 

distal bowel obstruction which requires high 

index of suspicion, as intussusception in adult 

is rare and most intussusception are seen on 

operating table (3,4), which was managed 

conservatively early in course of the disease 

that later culminated in to jejunojejunal 

intussusception requiring resection and resiting 

of jejunostomy tube, further hampering the 

nutritional recovery which is utmost important 

in patients of Boerhaave’s syndrome with failed 

primary repair. 


References: 

1. Teh E, Edwards J, Duffy J, Beggs D. Boerhaave’s 

syndrome: A review of management and outcome. Interact 

CardioVasc Thorac Surg 2007; 6:640-3. 

2. Wu TH, Lin CW, Yin WY. Jejunojejunal intussusception 

following Jejunostomy. J Formos Med Assoc 2006; 

105(4):355-8. 

3. Azar T, Berger DL. Adult intussusception. Ann Surg 1997; 

226:134-8. 

4. Begos DG, Sandor A, Modlin IM. The diagnosis and 

management of adult intussusception. Am J Surg 1997; 

125 

173:88-94. 

5. Pearce CB, Duncan HD. Enteral feeding. Nasogastric, 

nasojejunal, percutaneous endoscopic gastrostomy or 

jejunostomy: its indications and limitations.Postgrad Med 

J; 2002; 78:198-204. 

6. Date RS, Clements WD, Gilliland R. Feeding jejunostomy: 

is there enough evidence to justify its routine use? Dig 

Surg 2004; 21:142-5[Pubmed]. 

7. Carucci LR, Levine MS, Rubesin SE, Laufer I, Asad S, 

Herlinger H.Evaluation of patients with jejunostomy 

tubes: imaging findings. Radiology 2002; 223:241-7. 


Letters to the Editor Biomedicine; 2011; 31 ( 1 ) : 126 - 127 

Blood Pressure Changes in menstrual cycle 

The menstrual cycle is much more than a 

cycle of periods. Menstruation is only one 

manifestation of the ovarian cycle which is itself 

is associated with many physical, psychological 

and behavioral changes. Numerous clinical 

disorders also appear to be modulated by cyclic 

ovarian activity. Certain autonomic changes like 

blood pressure have also been reported during 

different phases of menstrual cycle but are not 

well documented and the results are conflicting. 

The literature reveals many types of behavioral 

and other changes in women especially during 

the premenstrual phase. Hence a study was 

taken up to know the effect of blood pressure 

changes during different phases of menstrual 

cycle. 30 lady medical students aged between 

18-20 years of J.S.S. Medical College & 

Hospital and Dental College & Hospital Mysore 

were selected for the study. Informed consent 

was obtained from each subject. The students 

having regular menstrual cycle lasting 26-34 

days and normal range of hematological and 

biochemical parameters within normal limits 

and normotensive (< 140/90 mmHg) were 

taken as inclusive criteria. The subjects with 

irregular menstrual cycle of 35 days history of 

diabetes mellitus, hypertension, other endocrine 

disorders, history of postural symptoms or 

syncopal attacks and history medication during 

the study were excluded. Experimentations 

were performed in the department of Physiology 

J.S.S.Medical College and Hospital, S.S.Nagar, 

Mysore in accordance with the ethical standards 

of the committee on human experimentation of 

the institution according to Helsinki Declaration 

of 1975.Subjects were divided into three groups 

based on the phase of menstrual cycle (Group-I 

–menstrual phase, Group-II –follicular phase 

126 

and Group-III –luteal phase). Subjects were 

monitored for Resting blood pressure, Supine 

to standing BP, blood pressure changes during 

Isometric Handgrip dynamometer test and cold 

pressor test. It was observed that in all the tests 

performed, systolic blood pressure difference 

between menstrual and follicular phase and 

follicular and luteal phase was statistically 

significant (P0.05) in all the 

tests except for handgrip dynamometer. 

The results were consistent with each 

of the earlier studies of Hassan, Mehta and 

Greenberg which showed an increase in systolic 

blood pressure during the luteal phase. They 

explained the increased fluid and salt retention 

induced by ovarian steroids as the basis for 

the above changes. Estrogen can cause both 

a rise in blood pressure and cardiovascular 

damage unrelated to hypertension. The most 

important estrogen effect is an increase in the 

hepatic synthesis of renin substrate, which leads 

to an increase in plasma angiotensin-II level 

and aldosterone mediated fluid retention. In 

addition, arterial walls have estrogen receptors 

that may modulate smooth muscle tone and 

estrogen increases the vascular sensitivity to 

catecholamines. Higher sympathetic activity 

was observed due to increased levels of estrogen 

and progesterone in the luteal phase. Also the use 

of exogenous progesterone is known to increase 

arterial blood pressure. Thus it is possible that 

elevated progesterone may be responsible for 

the increase in systolic blood pressure in luteal 

phase of the menstrual cycle. Physiological and 

psychological stress contributes to the blood 

pressure rise at premenstrual and menstrual 

phase. Fundamentally the mechanism of higher 


systolic pressure in the luteal phase still is 

largely unknown.From the present study, it 

may be concluded that there was significant 

difference in systolic blood pressure during 

different phases of menstrual cycle in young 

healthy volunteers. Inspite of the limitations in 

*L. Rajeshwari and **D.H.Rajendra 

127 

being noninvasive technique, the present study 

provides data that should be clinically useful 

to the gynecologists in making a diagnosis of 

hypertension, orthostatic changes in blood 

pressure. 

*Dept. of Physiology,J.S.S.Medical College & Hospital , J.S.S.University 

S.S.Nagar, Mysore-570 015, Karnataka, India 

** Dept. of Physiology,Mandya Institute of Medical Sciences,District Hospital Campus, 

Mandya-571 401, Karnataka, India 


Dr. Rajeshwari L 

email: raj0522004@gmail.com 


128 


Measurement of Thyroid Stimulating Hormone in serum as the initial test in 

the assessment of thyroid disorder 

Thyroxine (T4) and tri-iodothyronine (T3) are 

together known as the ‘thyroid hormones’. 

They are synthesized in the thyroid gland 

by iodination and coupling of two tyrosine 

molecules whilst attached to a complex protein 

called thyroglobulin. Thyroxine synthesis 

and release are stimulated by pituitary thyroid 

stimulating hormone (TSH). The secretion of 

TSH is controlled by negative feedback by the 

thyroid hormones which modulates the response 

of the pituitary to the hypothalamic hormone, 

thyrotropin-releasing hormone (TRH). Thyroid 

hormones are essential for the normal maturation 

and metabolism of all the tissues in the body. 

Laboratory tests of thyroid function are required 

to assist in the diagnosis and monitoring of 

thyroid disease. Most laboratories offer a 

standard ‘profile’ of thyroid function tests (often 

TSH and free T4), and perform additional tests 

only if these results are equivocal or the clinical 

circumstances require it. 

Since the release of TSH from the pituitary is 

controlled through negative feedback by thyroid 

hormones, measurements of TSH can be used as 

an index of thyroid function. 

If primary thyroid disease is suspected and plasma 

TSH concentration is normal, it can be safely 

inferred that the patient is euthyroid. In overt 

primary hypothyroidism, TSH concentrations 

are greatly increased, often to ten or more times 

the upper limit of normal. Smaller increases 

are seen in borderline cases or sub-clinical 

hypothyroidism and TSH measurement is more 

sensitive than T4 under these circumstances. 

Plasma TSH concentrations are suppressed to 

very low values in hyperthyroidism. It has been 

suggested that assays measuring ‘free T4 could 

be used as first-line tests of thyroid dysfunction. 

However, serum TSH measurement provides 

a better test because pituitary TSH secretion 

is very sensitive to changes in free T4 

concentration. Free T4 analyses are invaluable 

in diagnoses where binding proteins are altered, 

e.g., pregnancy, contraceptive pill, congenital 

TBG deficiency and nephrotic syndrome. 

Therefore, TSH can be used as a first line test of 

thyroid dysfunction. 

A total of 50 sub-clinical primary thyroid 

cases, in the both sexes, attending the hospital 

and central lab for TFT test in ASRAM superspecialty 

teaching hospital were included in the 

study after obtaining the consent of the patients. 

Out of which, 30 were sub-clinical hypothyroid 

and 20 were sub-clinical hyperthyroid 

respectively. 20, age and sex matched normal 

healthy controls (18 – 60 yrs) were included. 

Serum TSH, T4 and T3 hormones were estimated 

on Human Automated ELISA analyzer, using 

Human ELISA kits. TSH was estimated by 

sandwich EIA method (normal range = 0.3 – 6.2 

mIU/L). T4 was estimated by Competitive EIA 

method (normal range = 4.4 – 11.6 µg/dl) and 

T3 was estimated by Competitive EIA method 

(normal range = 69 – 202 ng/dl) respectively. 

A serum TSH value of < 0.3 mIU/L and serum 

TSH value > 6.2 – 20 mIU/L was taken as subclinical 

level. Statistical analysis was done, using 

STAT TM Ver1.1 and SSP statistical software’s 

respectively. The Student unpaired (two sample) 

t-test was done at 5% level of significance. The 

difference in the means of TSH between subclinical 

hypothyroid (mean ± SD = 11.336 ± 

4.295) and control (mean ± SD = 2.825 ± 0.983) 

was statistically highly significant as p< 0.0001. 

The difference in the means of T4 between the 

sub-clinical hypothyroid (mean ± SD = 6.843 ± 

1.807) and control (mean ± SD = 7.220 ± 1.225) 


was statistically insignificant as p< 0.4190. The 

difference in the means of T3 between subclinical 

hypothyroid cases (mean ± SD = 111.407 

± 24.884) and control (mean ± SD = 106.303) 

was statistically insignificant as p< 0.4568. 

Both T4 and T3 were well in the normal range 

in the sub-clinical hypothyroid and values are 

not indicating any primary thyroid dysfunction. 

The difference in the means of TSH between 

sub-clinical hyperthyroid cases (mean ± SD = 

0.048 ± 0.074) and control (mean ± SD = 2.825 

± 0.983) was statistically highly significant as 

p< 0.0001. The difference in the means of T4 

between sub-clinical hyperthyroid cases (mean 

± SD = 8.805 ± 1.578) and control (mean ± 

SD = 7.220 ± 1.225) was statistically highly 

significant as p< 0.0011. But, the difference 

in the means of T3 between sub-clinical 

hyperthyroid cases (mean ± SD = 116.720 ± 

38.174) and control (mean ± SD = 106.303 

± 21.408) was statistically insignificant as p< 

0.2939. Here also, the values of both T4 and T3 

were well in the normal range in the sub-clinical 

hyperthyroidism and values are not indicating 

any of thyroid dysfunction. Therefore, the only 

TFT parameter that is increased or decreased 

in the sub-clinical hypo or hyperthyroidism was 

the TSH and clearly indicating about primary 

thyroid dysfunction. Where as, there is no 

change in the concentrations of T4 and T3 and 

they were well in the normal range respectively. 

From this study, it is very clear that, the TSH 

is a better marker for the primary thyroid 

dysfunction than the other TFT parameters 

i.e., either T4 or T3 (fT4 or fT3) itself or as the 

initial thyroid function test. From this study, it 

is shown that, any primary thyroid disease, 

even at sub-clinical level, is first reflected in 

the changes in the concentration of TSH in the 

blood. In both sub-clinical hypo or hyperthyroid 

disease, the only the TFT parameter showing 

the thyroid dysfunction is TSH, where as both 

the T4 and T3 are well in the normal range 

129 

respectively. They are not reflecting any of 

the thyroid dysfunction and they are not much 

useful in the early diagnose of primary thyroid 

dysfunction. 

If primary thyroid disease is suspected and plasma 

TSH concentration is normal, it can be safely 

inferred that the patient is euthyroid. In overt 

primary hypothyroidism, TSH concentrations 

are greatly increased, often to ten or more times 

the upper limit of normal. Minimal increases 

are seen in borderline cases or sub-clinical 

hypothyroidism and TSH measurement is more 

sensitive than T4 under these circumstances. 

Plasma TSH concentrations are suppressed to 

very low values in hyperthyroidism. It has been 

suggested that assays measuring ‘free T4 could 

be used as first-line tests of thyroid dysfunction. 

However, serum TSH measurement provides a 

better test because pituitary TSH secretion is very 

sensitive to changes in free T4 concentration. 

Free T4 analyses are invaluable in diagnoses 

where binding proteins are altered, e.g., 

pregnancy, contraceptive pill, congenital TBG 

deficiency and nephrotic syndrome. Therefore, 

TSH can be used as a first line test of thyroid 

dysfunction. Physiologically, the pituitary gland 

serves as a biosensor and regulator of thyroid 

hormone activity. When the biologic activity 

of thyroxine (T4) falls below the physiologic 

set point, the anterior pituitary responds by 

increasing the concentration the TSH. When the 

biologic activity of T4 exceeds the set point, TSH 

production is reduced. There fore, in patients 

with a normally functioning pituitary gland, 

basal TSH can serve as a monitor of the adequacy 

of the thyroid function. The TSH concentrations 

changes inversely with the logarithm of the free 

T4 (FT4) concentrations so that a doubling or 

halving of the T4 concentrations corresponds to 

equals approximately a 50-fold change in TSH 

concentrations. Our study reveals that, TSH is a 

simple, better, reliable and sensitive marker for 

the primary thyroid dysfunction. The incidence 


of thyroid disease is more in females at the age 

group 30 – 50 yrs. than males (M: F = 1:6). 

The thyroid problem can be easily diagnosed 

by estimating the TSH concentration in the 

130 

blood. The TSH estimation may be used for 

the diagnosis of suspected thyroid dysfunction, 

routine screening purpose, as well as for the 

newborn screening for the thyroid disease. 

*V. S. Ravi Kiran, **S.Venkata Rao, ***K. Ambika Devi 

*Department of Biochemistry , Alluri Sitarama Raju Academy of Medical sciences, 

Eluru - 534004, Andhra Pradesh, India. 

*Department of Biochemistry, Alluri Sitarama Raju Academy of Medical sciences, Eluru-534004, 

Andhra Pradesh,India. 

**Department of Biochemistry,Katuri Medical College,Chinakondrupadu,Guntur 


V . S . Ravi Kiran 

E-mail: ravikiran_vs2001@yahoo.co.in 


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