Analytica MedicaISSN:0974-4142

Volume14 No.1&2 Jan- Dec. 2011

Journal of Scientifi c & Research Society

BLDE UniversityShri B. M. Patil Medical College

Hospital & Research Centre,Bijapur-586 103, Karnataka, India

Analytica MedicaISSN:0974-4142

Volume14 No.1 & 2 Jan -Dec. 2011

Journal of Scientific & Research Society

BLDE UniversityShri B. M. Patil Medical CollegeHospital & Research Centre,Bijapur-586 103, Karnataka, India

ISSN:0974-4142Analytica MedicaVolume.14 No.1 & 2 Jan-Dec. 2011

Analy

tica Me

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Honorary Editor in Chief Dr M. S. Biradar Dean, Faculty of Medicine & PrincipalEditor in Chief Dr S. P. Chowkimath Professor & Head, Dept. of PsychaitryEditors Dr Arun Patil KIMS, Karaad Dr Sharan Badiger Dr Aparna Palit Dr Manpreeth Kour Dr Shailaja PatilAssistant Editors Dr Nilima Dongre Dr Satish PatilEditorial Board Dr. S. D. Desai Dr. Manjunath Aithal HOD, Anatomy HOD, Physiology Dr. D. B. Rathi Dr. B. R. Yelikar HOD, Biochemistry HOD, Pathology Dr. P. K. Parandekar Dr. D. I. Ingale HOD, Microbiology HOD, Forensic Medicine Dr. R. S. Wali Dr. M. M. Angadi HOD, Pharmacology HOD, Community Medicine Dr. N. H. Kulkarni Dr. Vallabha K. HOD, ENT HOD, Ophthalmology Dr. M. S. Mulimani Dr. Tejaswini Vallabha HOD, Medicine HOD, Surgery Dr. P. B. Jaju Dr. S. V. Patil HOD, OBGY HOD, Paediatrics Dr. O. B. Pattanashetty Dr. A. C. Inamdar HOD, Orthopaedics HOD, Dermatology Dr. M. M. Patil Dr. R. S. Babar HOD, Radiology HOD, Pulmonology Dr. S. B. Patil HOD, Urology

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Statementsandopinionsexpressedinthearticlespublishedinthejournalarethoseof authorsandnotnecessarilyoftheEditor.NeithertheEditornorthePublisherguarantees,warrantsorendorsesanyproductorserviceadvertisedinthejournal.

Publishedby:Scientific&ResearchSocietyBLDEU’sShriB.M.PatilMedicalCollege,Hospital&ResearchCentre,Bijapur-586103,Karnataka,INDIAPrintedby:BLDEAOffsetPrinters,Bijapur

CONTENT

S.NO TITLE Page

REVIEW ARTICLES 1 NEWER MODALITIES IN DIAGNOSIS OF TUBERCULOSIS IN CHILDREN. Gobbur.R.H. ............................................................................................01-03 2 NEW RAY OF HOPE IN MANAGEMENT OF BRONCHIAL ASTHAMA : INHIBITORS OF VEGF. C.M.Kulkarni, S.M.Patil, Sumangala Patil, Manjunath Aithala ............04-13

ORIGINAL ARTICLES 3 PROFILE OF MEDICO LEGAL AUTOPSIES IN BIJAPUR Gannur Dayanand, Nuchhi U C, Yoganarasimha K. ..............................31-40 4 STUDY OF LIPID PROFILE IN SUBCLINICAL HYPOTHYROIDISM Anand K Pyati, Debasmita Das, Dileep B Rathi. ..................................41-55 5 EFFECT OF FLUORIDE ON MALONDIALDEHYDE, BONE MINERAL STATUS IN POSTMENOPAUSAL OSTEOPOROSIS Parinita Kataraki, Pragna Rao. ...............................................................56-67

CASE REPORTS 6 A CASE REPORT OF RETINOBLASTOMA PRESENTING WITH ORBITAL CELLULITIS Radhika. Sunil G Biradar, Vallabh K ....................................................68-74 7 EPITHELIOID SARCOMA: A RARE CASE REPORT M.S. Kotennavar, Rohan Khairatkar, Gururaj Padasalagi, Aravind Patil .........................................................75-80

EDITORIAL.....

Sharan Badiger. Department of Medicine.

Review Article

NEWER MODALITIES IN RAPID DIAGNOSIS OF M.TUBERCULOSIS IN CHILDREN

R.H.GobburProfessor, Department of Paediatrics,

BLDEU’s Sri B M Patil Medical College, Bijapur.

ABSTRACTMycobacterium Tuberculosis (MTB) is re emerging as a major killer disease, after

failure of its eradication and with spread of silent epidemic of HIV. Newer modalities like Interferon Gamma assay, BACTEC culture method, and PCR help in diagnosing MTB rapidly and reliably. These diagnostic modalities and DOTS therapy by RNTCP could certainly help in controlling Tuberculosis burden, and to ultimately eradicate MTB from the world.

Key words: MTB rapid Diagnosis, Quantiferron MTB assay, BACTEC, PCR. INTRODUCTION:Tuberculosis is a major disease burden in the world. Every year About 8 million people

are infected by Mycobacterium Tuberculosis (MTB)1. About 2million Indians are affected every year many lakhs of children die of severe form of MTB infection. With the silent pandemic of HIV affecting mainly people of developing countries, and HIV is known to increase the severity and complications of mycobacterium TB, this double curse increase mortality and morbidity due to MTB. Especially number of MDRTB and XDRTB cases increases. XDRTB has mortality rate of almost 50% in persons infected with it.

Rapid and reliable tests to diagnose MTB, to know drug sensitivity/resistance are need of the hour. The rapid diagnostic tests lead to rapid onset of appropriate regime for treating MTB, and also avoid unnecessary exposure to Radiographs, and unnecessary prophylactic Anti-tubercular drug therapy leading to increasing incidence of drug resistance.

The following are the useful newer modalities to diagnose MTB in children:I. QUANTIFERON TB GOLD in tube, Assay: 2 ml of whole blood of infected child

is added to this Tube. The tube which contains synthetic polypeptides ESAT6 (Early Secretory Antigen Target6), CFP 10(Culture Filtrate Protein10) and TB7.7, specific for MTB.(These antigens are absent in BCG)Stimulation of the activated T-Cells of MTB infected child by these specific antigens

report as + /-will be given as computerised report in 12-24 hours.1

Thus quantiferon TB GOLD, is as specific as MTB culture, definitely more sensitive and Specific than Monteux (TST) test. Studies have shown that QTB gold positivity has >90% correlation with that of positive Monteux of >10 mm2.II. LED FLOURESCENT MICROSCOPY: Improves rapidity and sensitivity of smear

positivity, over the conventional microscope. Even 100bacilli/ml sputum are picked as positive, with LED microscope.

III. Newer culture methods, eg; 1) BACTEC Radio metric,2) MGIT, 3) BACT- ALERT 3D Containing Light emitting sensors( LES).

In radio labelled BACTEC assay, radio labelled carbon compound is utilised by the MTB bacilli, leading to release of Radio labelled carbon dioxide. This is quantified by the computer. The time to get positive culture of MTB using BACTEC is about 6 days only

In MB BACT-ALERT 3D, as the M.TB grows, it causes utilization of Carbon, leading to release of CO2, which is absorbed by the light emitting sensors. Because of acidic media produced, the sensor becomes opaque, causing more reflection of fluorescent light, indicating growth of M.TB.

These Liquid cultures show rapid and specific growth, within days, rather than 4-6weeks as noted in classical culture methods like LJ Media. Decontamination of sample is done before culture. Media also contains OADC enrichment & PANTA antibiotic mixture for rapid and specific growth.

Oleic acid acts as metabolic stimulant, Albumin binds to toxic free fatty acid, Dextrose acts as energy source, and Catalase destroys toxic peroxides that may be present in the medium. Polymyxin, Amphotericin-B, Neomycin, Trimethoprim, and Azlocillin antibiotic mixtures (PANTA) prevent growth of contaminants and helps growth of pure culture of MTB. Using these liquid culture media, MTB the isolation rate is about 70 %3.IV. PCR assays (NAAT: Nucleic acid amplification test): Especially Real-Time PCR.

DNA extracts from sample is added to the PCR tube. Thermo stable DNA polymerase (Taq), and specific DNA Primers are used in thermo cycling method of multiplying DNA strands, using, Real-time PCR. This gives quantitative assay, without risk of contamination. The probe has fluorescent D (Dye) and Q (quencher) ends which on separation during synthesis of the complementary strand, release Fluorescence. Each subsequent cycles fluorescence increases in geometric proportion. This florescence recording leads to Quantitative and rapid Assay.

Sensitivity and specificity of PCR assay is over 95%. Rapid Identification of Mycobacterium tuberculosis and Non tuberculous Mycobacteria by Multiplex, Real-Time PCR has sensitivity and specificity of >99%4.

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Advantages: Rapid procedure (3 – 4 hours), High sensitivity (1-10 bacilli / ml sputum) CDC recommends NAAT for all suspected TB cases.

Disadvantage: PCR assay is costly, special equipment and trained personnel are required. There is a low but definite risk of false positivity because of contamination(<5 %). Rarely the specimen contains polymerase inhibitor giving false negativity (in 7% of cases).V. PCR Assay can also be used for rapid diagnosis of antibiotic resistance of M.TB

towards drugs like Rifampicin, INH, SM, Fluroquinolone.CONCLUSION:

Gamma Interferon assays, BAC T-ALET/3D and RT- PCR are the newer modalities for rapid diagnosis of M.TB infection especially in children. If the cost of these is made affordable to the poor, then these investigations will aid tremendously in the control and ultimately eradication of tuberculosis.REFERENCES:1. World Health Organization (WHO) report. Global tuberculosis control.2009.

Available at: http://www.who.int/tb/publications/global_report/2009/key_points/en/

2. Lee, Kim HJ, Lee SW. The clinical utility of tuberculin skin test and interferon-γ release assay in the diagnosis of active tuberculosis among young adults: a prospective observational study. BMC Infect Dis 2011;18:11-96.

3. Claudio Piersimoni, Claudio Scarparo, Anna Paola Callegaro et al. Comparison of MB/BACT ALERT 3D System with Radiometric BACTEC System and Lowenstein-Jensen Medium for Recovery and Identification of Mycobacteria from Clinical Specimens: a Multicentre Study. J Clin Microbiol 2001; 39(2):651–657.

4. Richardson, D. Samson, N. Banaeil. Rapid Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria by Multiplex, Real-Time PCR_E. T. J Clin Microbiol 2009;47(5) 1497-1502.

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Review Article

NEW RAY OF HOPE IN MANAGEMENT OF BRONCHIAL ASTHMA- ROLE OF INHIBITORS OF VASCULAR

ENDOTHELIAL GROWTH FACTORC.M.Kulkarni1, S.M.Patil2, Sumangala Patil3, Manjunath Aithala3

1Associate Professor, 2Assistant Professor, 3ProfessorDepartment of Physiology, BLDEU’s Shri B.M.Patil Medical College, Bijapur

ABSTRACT:Vascular Endothelial Growth Factor (VEGF) is a mediator of airway inflammation

and remodeling in asthma. Transforming Growth Factor (TGF) – B1 plays pivotal role in tissue remodeling and repair in many chronic lung diseases. Few studies to study interactions between VEGF and TGF B1 have been done in murine models. Ovalbumin inhaled murine model showed typical pathophysiological features of allergic asthma. Administration of VEGF inhibitors showed reduced signs and decreased levels of TGF B1 and peribronchial fibrosis including phosphoinositide 3-kinase (PI3K) activity after ovalalbumin(OVA) inhalation. In addition, the increased TGF – B1 levels and collagen deposition after OVA inhalation were decreased by administration of PI3K inhibitors. These results suggest that inhibition of vascular endothelial growth factor attenuates peribornchial fibrosis, at least when mediated by regulation of transforming growth factor – B1 expression through phosphoinositide 3 –kinase/ Akt pathway in amurine model of allergic airway disease.

Keywords: Airway remodeling, phosphoinositide 3 kinase, subepithelial fibrosis, TGF, VEGF, OVA.

INTRODUCTION:Bronchial asthma is a chronic inflammatory disease characterized by airway wall

remodeling. Airway remodeling is due to an excess of extracellular matrix deposition in the airway wall, which leads to subepithelial collagen deposition1. The histological characteristics of chronic inflammation include angiogenesis, increased connective tissues deposition and cellular proliferation of myofibroblasts. An increase in vessel size, vessel number, vascular surface area and exaggerated expression of vascular endothelial growth factor (VEGF) are documented in the asthmatic airway2. It has been suggested that these vascular alterations contribute to the airway obstruction and airway hyperresponsiveness3.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogenic 4

peptide and plays a key role in vasculogenesis and angiogenesis. VEGF also increases vascular permeability so that plasma proteins, including inflammatory mediators and inflammatory cells, can leak into the extravascular space to allow the migration of inflammatory cells into the airway4. VEGF is a mediator of vascular and extravascular remodeling and inflammation, and thus the inhibition of VEGF may be a good therapeutic strategy. It has been recently shown that VEGF exhibits a critical role in enhancing chronic T-helper type 2 cell (TH2) –mediated inflammation and transforming growth factor (TGF) –B1 production, which in turn may result in airway subepethillial fibrosis5.

TGF-B1 family proteins are influential regulators of tissues remodeling and act as potent inhibitors of proliferation for most cell type6. It has been well established that TGF-B1 plays an important role in the pathogenesis of structural changes, including fibrosis, in a number of chronic lung diseases. Enhanced basal activity of PI3K has been reported in eosiinophils derived from allergic asthmatics. VEGF –induced PI3K activation has been linked to biologically diverse roles of VEGF, such as cell migration, vascular permeability, cell survival and cell proliferation. Moreover, recent studies have revealed that P13K plays a pivotal role in regulation of the TGF –B1 expression6-9.

VEGF InhibitorsAdministration of SU5614, CBO-P11, wortmannin or LY294002 and effects of VEGF

inhibitors on TGF-B1 levels in lung tissues and BAL fluids of OVA sensitized and challenged mice. Western blot analysis revealed that levels of TGF-B1 protein in lung tissues were significantly increased at 7 days after the last inhalation of OVA, compared with the levels in the control mice. The increased TGF-B1 levels after OVA inhalation were decreased significantly by the administration of SU5614 or CBO-P11. Consistent with these results, enzyme immunoassay revealed that the increased TGFB1 levels after the last OVA inhalation were decreased significantly by the administration of SUC5614 or CBO-P11.

Effects of P13K inhibitors of TGF-B1 levels in lung tissues Western blot analysis revealed that levels of TGFB1 protein in lung tissues were

significantly increased 7 days after the last inhalation of OVA, compared with the levels in the control mice. The increased TGF-B1 levels after OVA inhalation were decreased significantly by the administration of LY294002 or wortmannin.

Effects of P13K inhibitors on total lung collagen deposition OVA –sensitized and challenged mice had a significant increase in the levels of lung

collagen deposition compared with control mice, as assessed by determination of total lung collagen content. The increased collagen deposition after OVA inhalation was significantly decreased by the administration of wrotminnin or LY-294002, compared with that of untreated mice.

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Effects of VEGF inhibitors on cellular changes in BAL fluidsThe increased numbers of total cells, lymphocytes, neutrophils and eosinophils were

significantly reduced by the administration of SU5614 or CBO-P11.Effects of VEGF inhibitors on airway hyperresponsiveness These results indicate that administration of SU5614 or CBO-P11 reduces OVA induced

airway hyperresponsiveness.Induction of TGF-B1 production by VEGF and effect of PI3K inhibitor on VEGF

induced TGF-B1 production in A549 lung epethelial cells. Enzyme immunoassays revealed that levels of TGF-B1 protein in A549 cells were increased significantly by treatment with VEGF10.

DISCUSSION: Fibrosis is an important cause of morbidity and mortality in the lung. This is illustrated

in airway disorders such as asthma, which is characterized by chronic inflammation and subepithelial airway fibrosis. TGF – B1 is a multifunctional cytokine that plays pivotal roles in diverse biological processes, including tissue remodelling and repair. In human chronic lung diseases like asthma, TGF- B1 expression has been shown to be increased. Moreover, TGF – B1 administration promotes peribronchial collagen deposition and the increase of TGF – B1 expression in asthma patients seems to correlate with disease severity and the degree of sub epithelial fibrosis. Although some TH2 –inflammatory mediators, such as IL-13 and IL-11, are known to have the ability to enhance TGF-B1 expression, the exact mechanisms for the regulation of TGF- B1 expression remains to be elucidated. A recent study has demonstrated that VEGF enhances TGF- B1 production, which in turn, may result in airway sub epithelial fibrosis). These findings suggest that inhibition of VEGF attenuates peribronchial fibrosis by preventing TGF-B1 production in allergic airway disease5, 11-15.

VEGF can be produced by a wide variety of cells including macrophages, neutrophils eosinophils and lymphocytes. Several studies have also shown that overproduction of VEGF causes airway inflammation and bronchial hyper responsiveness. The present study found that VEGF expression was upregulated and peribronchial fibrosis was also increased in OVA –induced allergic airway disease. Administration of the VEGF inhibitors SU5614 or CBOP-11significantly reduced per bronchial fibrosis induced by OVA inhalation.

Since PI3K was first identified as an activity associated with various oncoprotiens and growth factor receptor, accumulating evidence has indicated that PI3Ks can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, neurite outgrowth, membrane ruffling and super oxide production, as well as actin reorganization and chemotaxis. PI3K activity is also stimulated after antigen

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challenge in a murine model of allergic asthma, and administration of wortmannin or LY-294002, two broad –spectrum inhibitors of PI3Ks, attenuates inflammation and airway hyperresponsiveness. Consistent with these observations the present results have shown that p-Akt protein and PI3K enzyme activity were significantly increased after OVA inhalation and the increased levels of activity were substantially reduced by treatment with VEGF inhibitors SU5614 or CBO-P1116,17.

To summarise, the present data strongly indicates that the inhibition of vascular endothelial growth factor signalling is a potentially powerful therapeutic strategy for sub epithelial fibrosis of allergic airway disease, partly mediated by regulation of transforming growth factor –B1 expression through the phosphoinositide 3-kinase /Akt pathway. Therefore, these findings provide an important mechanism for the use of vascular endothelial growth factor inhibitors to prevent and /or treat sub epithelial fibrosis in allergic airway diseases.

REFERENCES:01. Roche WR, Beasley R, Williams JH, Holgate ST, Subepithelial fibrosis in the

bronchi of asthmatics. Lancet 1989; 1:520-524.02. Lee YC, Lee HK, Vascular endothelial growth factor in patients with acute asthma.

J Allergy Clin Immunol 2001; 107: 1106.03. Kanazawa H, Nomura S, Yoshikawa J. Role of microvascular permeability on

physiologic differences in asthma and eosinophlic bronchitis. Am J Resp Cirt Care Med 2004; 169:1125-1130.

04. Lee YC, Kwak YG, Song Ch. Contribution of vascular endothelial growth factor to airway hyperresponsiveness and inflammation in a murine model of toluene diisocynate –induced asthma .J Immunol 2002;168: 3595-3600.

05. Lee CG, Link H, Baluk P, et al. Vascular endothelial growth factor (VEGF) induces remodelling and enhances TH2-mediated sensitization and inflammation in the lung. Nat Med 2004; 10: 1095-1103

06. Massague J, Blain SW, Lo RS. TGF-β signaling in growth control, cancer, and heritable disorders. Cell 2000; 103: 295-309.

07. Holgate ST, Lackie PM, Howarth PH, et al. Invited lecture: activation of the epithelial measenchymal trophic unit in the pathogenesis of asthma. Int Arch Allergy Immunol 2001; 124: 253-258.

08. Bracke M, van de Graaf E, Lam mers JW, Coffer PJ, Koenderman L. In vivo priming of FcxR functioning on eosinophils of allergic asthmatics. J Leukoc Biol 2000; 68: 655-661.

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09. Thakker GD, Hajjar DP, Muller WA, Rosengart TK. The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. J. Biol Chem 1999; 274: 10002-10007.

10. Lee KS, Min KH, Kim SR, et al. Vascular endothelial growth factor modulates matrix mettaloproteinase-9 expression in asthma. Am J Respir Crit Care Med 2006; 174: 161-170.

11. Elias JA, Zhu Z, Chupp G, Homer RJ. Airway remodelling in asthma. J Clin Invest 1999; 104: 1001-1006

12. Lee CG, Kang HR, Homer RJ, Chupp G, Elias JA. Transgenic modelling of transforming growth factor-B1: role of apoptosis in fibrosis and alveolar remodelling. Proc Am Thorac Soc 2006; 3: 418-423

13. Chu HW, Trudeau JB, Balzar S, Wenzel SE, Peripheral blood and airway tissue expression of transforming growth factor B by neutrophils in asthmatic subjects and normal control subjects. J Allergy Clin Immunol 2000; 106: 1115-1123

14. Minshall EM, Leung DY, Martin RJ, et al. Eosinophil associated TGF-B1 mRNA expression and airway fibrosis in bronchial asthma. Am J Respir Cell Mol Physiol 2004; 286: L539-L545.

15. Li ZD, Bork JP, Krueger B, et al. VEGF induces proliferation, migration, and TGF-B1 expression in mouse glomerular endothelial cells via mitogen activated protein kinase and phosphatidylinositol 3-kinase. Biochem Biophys Res Commun 2005; 334: 1049-1060.

16. Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002; 296: 644-64617. Kilic E, Kilic U, Wang Y, Bassetti CL, Marti HH, Herman DM. The

phosphatidylinositol-3 kinase/Akt pathway mediates VEGF’s neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia. FASEB J 2006; 20: 1185-1187

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Original Article

PROFILE OF MEDICO-LEGAL AUTOPSIES IN BIJAPURGannur Dayanand G1, Nuchhi U C2, Yoganarasimha K3

1Associate Professor, 2Assistant Professor, 3Professor, Department of Forensic Medicine,

BLDEU’s Shri B M Patil Medical College & Research centre Bijapur. Karnataka

ABSTRACT:A 3-year study was carried out on the cases of unnatural deaths subjected to Medico-

legal autopsies from 2006 to 2008. The main objectives of the study were: (a) To ascertain the various aspects of unnatural deaths, (b) To analyze the probable reasons for the same and (c) To find remedial measures to bring down the incidence. The incidence of unnatural deaths was found to be persistently increasing. Maximum number of such deaths 266 (29%) belonged to the age group of 21 - 30 years. Male: female ratio was 1.1: 1. Rural population was more prone to poisoning whereas the urban became victim of road-traffic accidents. Males preferred poisoning and hanging whereas females preferred self-immolation (burns) to end their own lives. Suggestions relating to road safety, decreasing the stress of the modem mechanical life-style, educating the public in general and regarding the availability, use and storage of poisonous substances in particular have been put forward, while highlighting the social evil of dowry system prevailing in India.

Keywords: Unnatural Deaths, Dowry Deaths, Road traffic accidents.INTRODUCTION:Unnatural deaths are known to claim a substantial number of lives world over, with

the vehicular accidents accounting for a lion’s share. The vehicular accident rate per thousand vehicles is greater in developing countries than in the developed. In India, one person dies in less than every five minutes due to vehicular accidents1 and the accident rate i.e. number of accidents per hundred thousand populations is 24.3. The increased pace of mechanization, increasing number of fast moving vehicles, unskilled or semi skilled drivers, drunken drivers and the woefully inadequate road system have ushered in this man made epidemic in India. Ignorance and intentional violation of traffic rules, encroachment of the roads by shopkeepers, hawkers and stray animals play an important role in contributing to the increase of vehicular accidents2. Poisoning is a major problem all over the world, though the type of poison and the associated morbidity and mortality varies from place to place and changes over a period of time. The use of poisons for suicidal and homicidal purposes dates back to the Vedic era in India. The exact incidence of this problem in India remains uncertain, but, it is reported that 1 to 1.5 million cases

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of poisoning occur every year, of which nearly 50,000 die3.The last quarter of the century has seen tremendous advances in the fields of agriculture, industrial technologies and medical pharmacology. These advances have been paralleled with remarkable changes in the trends of acute poisoning in developing countries, including India 4. Fire and its searing / cleansing powers have been held in great reverence and fear in the Indian psyche. This extended to cleansing and blessing of human bonds and relationships over it. Even Shushruta’s ancient medical treatise gave it the final sterilizing / cleaning authority. From this background, setting oneself on fire may have been arrived at, as an Indian means of honorable suicide5. The burn fatalities in India go beyond the meaning implied in the term ‘accident’ and the impact they cause, no longer remains confined to the family but spreads far wide to be aptly termed as a ‘Social Calamity’6. The prevailing system of dowry, which is mainly responsible for all such deaths, is a product of emerging capitalist ethos - the offshoot of an unequal society, a result of rampant consumerism, aided and abetted by the black-market economy. Its increasing incidence is symbolic of continuing erosion and devaluation of women’s status in independent India7.

The other means of unnatural deaths -include hanging, drowning, jumping from height, etc for suicidal purposes. This is so because methods used by individuals bent on self- destruction depend upon the availability of the lethal instruments8-15. Snake bites, electrocution, anaphylactic deaths, etc categorized under “others” constitute a substantial number of unnatural deaths in this part of the world because of the lack of infrastructural facilities for timely management of such patients. Undiagnosed and sudden deaths are registered to be under suspicious circumstances and inquest proceedings initiated by the police, only to find on postmortem examination, that in most of such cases a disease process was responsible for the death. Crime rate in a community is directly linked with the rate of poverty and illiteracy16. India being a poor country with a high unemployment and illiteracy rate, the crime rate though disproportionate, still contributes its bit towards unnatural deaths.

METHODS: The present study was carried out from January 2006 to December 2008 in the

Department of Forensic Medicine & Toxicology at Shri B. M. Patil Medical College and hospital, Bijapur, Karnataka India. The study comprises 266 cases of unnatural deaths subjected to medico legal autopsy. The information regarding age, sex, socioeconomic background, marital status and the circumstances leading to such deaths was obtained from the relatives / friends of the deceased, hospital records and the concerned investigating agencies. The reports of the relevant samples /viscera subjected to chemical analysis on autopsy to establish the poison consumed in suspected cases of poisoning and to establish / rule out any intoxication in other cases were thoroughly scrutinized. The use of kerosene oil was also subject to confirmation from the reports of chemical

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analysis in cases of burns. The reports of relevant samples preserved during autopsy and subjected to histopathological examination to arrive at a conclusion regarding the cause of death due to a disease process, but under suspicious circumstances were also taken into consideration.

RESULTSOut of total number of 266 cases of unnatural deaths/deaths under suspicious

circumstances autopsied during the period study from Jan 2006 to Dec 2008. Table-1: Age wise distribution of cases

Age yrs Number of cases Percentage (%)1-10 08 3.0011-20 49 18.4221-30 79 29.7031-40 59 22.1841-50 48 18.0451-60 10 3.76>60 13 4.88Total 266 100

Table-2: Sex wise distributionSex Number of cases Percentage (%)

Male 156 58.65Female 116 41.35Total 266 100

Table-3: Manner of unnatural deathYear wise 2006 2007 2008 Percentage (%)Suicidal 30 31 35 36.10

Accidental 50 59 59 63.15Homicidal 1 - 01 0.75

Total 81 90 95 100Table-4: Distribution of cases according to the incidence

Incidence Number of cases %Poisoning 124 46.61

Burns 71 26.69Road traffic accidents 60 22.56

Asphyxial deaths 12 4.5Total 266 100

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Table-5: Area wise distributionArea Number of cases %Urban 96 36.09Rural 170 63.90Total 266 100

DISCUSSION:Unnatural death is one of the indicators of the level of social and mental health.

Responsibility for prevention of violence in our society does not rest only on the law- enforcement personnel. Public health and other human service agencies must assist in preventing primary violence as they have done to prevent other major causes of morbidity and mortality. The present study reveals that poisoning was the commonest method employed for suicides and there was an appreciable increase in the percentage of suicide from 24.68% in 1994 to 51.9 % in 2003. Different reports published from time to time have reported a suicide rate in India as 43 per 100,00017, 28.57 per 100,00018, 38 per 100,000 19, 29 per 100,00020 & 22.83 per 100,00021. The age group, 16-30 years, was most prone to suicide, accounting for 73 % suicidal deaths. This is in conformity with the various studies conducted at different places22-25. The high suicide rate among the adolescents and young maybe attributed to various socio-economic factors viz. lack of employment opportunities, urbanization, break-up in the family support system, economic instability etc. Different agrochemicals especially Aluminum Phosphide marketed as tablets, has emerged as a dangerous weapon to human lives on account of its easy availability, non availability of an effective antidote, being cheap, efficacious and easy to use and is now the single most frequent suicidal method in Northern India.

In the present study, the poisoning accounted for 124(46.61%), whereas burns clamied71 (26.69%) lives, road traffic accidents were 60 (22.56%) and mechanical asphyxial death 12(4.5%). The higher incidence of unnatural female deaths due to burns in the age group of 21 - 30 years, helps to emphasize the fact that the burn fatalities in India go beyond the meaning implied in the term ‘accident’ to be aptly termed as a ‘Social Calamity’. These deaths in general and homicidal and suicidal burn deaths in particular have genuinely been termed as ‘Bride Burning’ or ‘Dowry Deaths7. The high incidence of burn deaths, especially among the young females is often attributed to cooking on open unguarded flames. Loose, voluminous, highly inflammable, synthetic garments / saris of the victims are alleged to catch fire suddenly while cooking. Kerosene oil, match sticks, and other cooking material, being easily available in houses, is usually preferred by Indian women to commit suicide, and as for killing, it helps to hide not only the torture and other means of violence but also helps to tamper with or even destroy the circumstantial evidence.

The other means of unnatural deaths include drowning for suicidal purposes and 12

constituted about 35(36.10%) of the total suicidal deaths. This is so because methods used by individuals bent on self destruction depend upon the availability of the lethal instrument that varies from place to place and community to community. Major part of India having rural population with agriculture as the main employment, agrochemical poisoning is more prevalent. The ongoing revolution of evolving faster and better means of transport, the world over, has brought along with it an important and unwelcome guest - road traffic accidents. These have taken an almost epidemic form in the recent past. This is particularly true of our country where one person becomes victim of this man made dragon in less than every five minutes 8. The decrease in the number of road-traffic fatalities, from 14% in 1994 to 8% in 2003, observed in our study is in contrast to the reports from other parts of India that have registered a regular increase27-29. The main reason for this welcome improvement in Bijapur is perhaps the strict implementation of the traffic rules by the authorities, and the citizens abiding-by the rules imposed on them. Wide and well-maintained roads have also played an important role in bringing down the accident rate in the city.

CONCLUSIONl Maximum Deaths were reported in the Age group of 21-30 years followed by 31-

40 years and 41-50 year group.l Sex wise distribution were Maximum males then the females.l Maximum Victims had succumbed to poisoning. While burn and road traffic

accidents were found to be II and III most common cause of death.l Most of the cases belong to rural area and due to low education and awareness,l Manner of Death were Maximum due to an Accidental followed by suicidal and

homicidal is the least.REFERENCES:

1. Banerjee K.K. Study of thoraco-abdominal injuries in fatal road traffic accidents in northeast Delhi. JFMT 1997; 15(1): 40-43.

2. Sharma BR Harish D. Sharma V. et al. Road Traffic Accidents- a demographic and topographic analysis; Med. Sci Law 2001;41(3):266-274. 3. Aggarwal P, Handa R, Wali J.P. Common Poisoning in India. Proceedings of

National Workshop on Practical and Emergency Toxicology. 1998;1:25-31. 4. Singh D, Jit I. Changing Trends of acute Poisoning in Chandigarh Zone. Am J

Forensic Med. Patho. 1999;20(2):203-210. 5. Murty O.P, George Paul. Bride burning and burns - certain differentiating aspects

thereof; JFMT 1995;12(3& 4):13-26. 6. Sharma BR, Naik RS, Anjankar AJ.Epidemiology of burnt females. The Antiseptic 1991; 88(11):570-572.

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7. Sharma BR, Harish D, Sharma V et al. Kitchen Accidents Vis-à-vis Dowry deaths. Burns 2002;28 (3): 250 - 253.

8. Avis SP, Archibald JF. Asphyxial Suicide by Propane inhalation & plastic Bag suffocation. Journal of Forensic Sciences. 1994; 39 (1): 253-256.

9. Harruff RC, Llewellyn AL, Clark MA et al. Firearm Suicides during Confrontation with Police Journal of Forensic Science JFSCA.1994; 39 (2): 402-411.

10. Gupta BD, Jani CB, Patel BJ, Shah PH.Homicide - Suicide Deaths (Dyadic) Two Case reports. JFMT 2000; 17 (1): 31-37.

11. Bullock MJ & Diniz D. Suffocation Using Plastic Bags A Retrospective Study of Suicide in Ontario, Canada, Journal of Forensic Sciences, JFSA 2000; 45 (3): 608-613.

12. Campman SC, Springer FA, Henrikson DM. The chain Saw: An Uncommon means of committing Suicide. J. Forensic Sci. 2000; 45 (2): 471-473.

13. Siciliano C, Costantinides F, Bernasconi P. Suicide using a Hand Grenade J Forensic Sci. 2000: 45 (1): 208-210.

14. Marc B, Bandry F, Douceron H et al. Suicide by Electrocution with Low-Voltage Current. J Forensic Sci. 2000; 45 (1): 216-222.

15. Cooper PN & Milroy CM. Violent Suicide in South Yorkshire, England. Journal of Forensic Sciences, JFSA 1994; 39 (3): 657-667.

16. Aryappan A and Jayadev CJ. Society in India; Social Science Publication; 1985.page No.?

17. Rao Venkoba. Suicide attempters in Madurai, JIMA 1971; 57: 278. 18. Nandi DN, Banerjee G and Boral GC. Suicide in West Bengal, A Century apart.

Indian Journal of Psychiatry. 1978; 28: 59-62. 19. Sharma SK. Current Scenario of poisoning in Indian JFMT 1998; XV (1): 89-94. 20. Shukla GD, Verma BL & Mishra DN. Subde in Jhansi City. Indian Journal of

Psychiatry 1990; 32: 44-51. 21. Sharma PG and Sawang DG. Suicide in rural areas of Warangal district, Indian

Journal of Psychiatry 1993; 3: 79-84. 22. World Health Organization; Suicide and attempted suicide in young people, Geneva:

WHO; 1974. 23. McClure GMG Recent Trends in Suicides amongst the young; British Journal of

Psychiatry 1984; 144: 134-138. 24. Bhullar DS, Oberoi SS, Aggarwal OP et al.Profile of Unnatural deaths (between

18-30 years of age) in GMCH Patiala, (India) JFMT 1996;13 (3): 5-8. 25. Sharma BR. Trends of Poisons and Drugs used in Jammu JFMT 1996;13 (2): 7-9.

14

Original Article

STUDY OF LIPID PROFILE IN SUBCLINICAL HYPOTHYROIDISM

Anand K Pyati, Debasmita Das, Dileep B RathiDepartment of Biochemistry, BLDEU’s Shri B M Patil Medical College, Bijapur, Karnataka.

ABSTRACT: Overt hypothyroidism has been found to be associated with abnormal lipid profile and

cardiovascular disease. Whether subclinical hypothyroidism and thyroid autoimmunity are also risk factors for cardiovascular disease is controversial. Sixty subjects comprising 30 subclinical hypothyroidism patients and 30 age & sex matched healthy controls were included in the study. In all the subjects BMI, lipid profile tests were analyzed by standard methods. We found that, serum mean levels of total cholesterol, triglycerides were significantly higher in SCH patients when compared with controls and the difference was statistically significant (p < 0.05). The percentage of subjects having higher BMI, elevated TC, LDL, TG, and decreased HDL was higher in SCH patients than in controls. Among the SCH patients, TSH levels were positively correlated with that of total cholesterol and triglycerides. In conclusion, patients with subclinical hypothyroidism exhibit increased levels of the atherogenic parameters (mainly TC, TG & LDL), which may predispose to the development of atherosclerotic coronary artery disease (CAD).

Key-words: Subclinical hypothyroidism, Dyslipidemia, Cardiovascular risk, Euthyroid

INTRODUCTION:Subclinical hypothyroidism (SCH) is defined as a serum TSH concentration above the

statistically defined upper limit of the reference range when serum T4 (T4) concentration is within its reference range1. SCH is a common problem, with a prevalence of 3% to 8% in the general population. The prevalence increases with age and is higher in women. After the sixth decade of life, the prevalence in men approaches that of women, with a combined prevalence of 10%2.

Subclinical thyroid disease is mainly a laboratory diagnosis. Patients with subclinical disease have few or no definitive clinical signs or symptoms of thyroid dysfunction1. The most common cause of elevated TSH is autoimmune thyroid disease. Previous radioiodine therapy, thyroid surgery, and external radiation therapy can also result in mild thyroid failure. Transient SCH may occur after episodes of postpartum, silent, and granulomatous thyroiditis3.

15

Despite some conflicting results, many studies found that subjects with subclinical hypothyroidism have higher total cholesterol and low density lipoprotein cholesterol levels than euthyroid subjects. Few studies have showed that subjects with subclinical hypothyroidism have increased C-reactive protein values. Subclinical hypothyroidism has been associated with increased risk for atherosclerosis. However, data on coronary heart disease (CHD) in subjects with subclinical hypothyroidism are conflicting4.

Small percentage of these patients advance to overt hypothyroidism each year, lipid abnormalities are reported to be more common in patients with overt hypothyroidism and are thought to contribute to the disproportionate increase in cardiovascular risk in these persons. Controversy continues over whether elderly individuals should be screened for subclinical hypothyroidism. The decision about whether to screen patients for this disorder is clouded by inconsistent evidence of association of dyslipidemia and other risk factors of cardiovascular disease with SCH and also any benefit from early treatment. A few trials have found that persons with subclinical hypothyroidism who are given L-thyroxine experience some improvements in their energy level and feelings of well-being5.

Cardiovascular diseases (CVDs) are the most common cause of mortality, primarily affecting older adults. Heart disease causes nearly 7,00,000 deaths annually in the United States. Although established risk factors explain most cardiac risks, significant attention has been focused on alternative biochemical markers to assist in identifying those at risk of a clinical cardiac event. Previous studies have suggested that abnormal levels of thyroid stimulating hormone (TSH) may represent a novel cardiac risk factor6.

There are few population-based studies that have compared lipid profile parameters in subclinical hypothyroidism patients with euthyroid persons. So the purpose of this study is to determine whether the known risk factors for the CAD such as lipid abnormalities are more significant in patients with subclinical hypothyroidism when compared with those in euthyroid individuals.

METHODS:A cross sectional study of lipid profile in subclinical hypothyroidism subjects was

conducted from April 2011 to December 2011. We selected 30 subclinical hypothyroidism cases aged ≥ 40 years from among the patients referred by the physicians to the clinical biochemistry department, BLDEA Hospital and Research Centre, Bijapur, Karnataka, (attached to teaching institute, Shri B M Patil Medical College, Bijapur.) and 30 age and sex matched healthy euthyroid controls from the general population according to the inclusion and exclusion criteria mentioned below. This study was approved by the Ethical and Research Committee of BLDEU’s Shri B M Patil Medical College, Bijapur and all the subjects gave an informed consent before undergoing further investigations.

Inclusion criteria: Subclinical hypothyroidism cases having TSH in the range of 4.50 to 14.99 mU/L and a T4 value greater than 4.5 μg/dL. The euthyroid controls having normal TSH values.

16

Exclusion criteria: Obese (BMI ≥ 30 kg/m2) subjects, smokers, those with known hypothyroidism, previous radioactive iodine therapy, thyroidectomy, external radiation, consumption of drugs known to cause SCH, primary or secondary dyslipidemia, diabetes mellitus, renal and hepatic failure, or other systemic diseases were excluded from the study.

The subjects’ BMI was determined from height and weight. BMI (Kg/m2) = Weight/(Height)2. Venous blood samples were collected from all the subjects at 8 AM following a 12 hours fast, in a plain bulb with all the aseptic precautions. Blood samples were centrifuged within 30 minutes at 3000 rpm for 5 min. and serum was separated. Serum samples were stored at -20° C until assayed.

Serum T3, T4 and TSH levels were measured by ELISA method7,8,9 using immunoassay analyzer. Serum total cholesterol (TC) and triglycerides (TG) were determined by enzymatic colorimetric assay10 (CHOD-PAP method using Statfax-2000 semiautoanalyzer). High-density lipoprotein cholesterol (HDL-C) was determined enzymatically in the supernatant after dextran–magnesium-induced precipitation of other lipoproteins. Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula11.

Statistical analysis:Descriptive data is presented as mean ± SD and range values. Statistical analysis was

carried out using unpaired Student’s t-test for all variables. The Pearson’s correlation coefficient is used to assess the degree of association between two variables. For all the tests, p-value of 0.05 or less was considered for statistical significance.

RESULTS:This study includes 30 subclinical hypothyroidism patients and 30 age and sex matched

healthy controls.Table 1: Demographics of the sample population

Characteristic Subclinical hypothyroidism (SCH)(n = 30)

Euthyroid controls (EC) (n = 30)

Sex Male Female

0624 06

24 Age 40 – 45 yrs 46 – 50 yrs 51 – 55 yrs 56 – 60 yrs

581007

681204

Table 1 shows age and sex wise distribution of cases and controls studied. The study 17

included 30 cases of subclinical hypothyroidism with a mean age of 51.3 ± 5.6 years, and 30 healthy controls with a mean age of 50.4 ± 5.3 years. Subclinical hypothyroidism and euthyroid control subjects were well matched with respect to age and sex. It is evident from the table that the subclinical hypothyroidism is more common in women among the age group of 51 – 55 years.

Table 2: Comparison of clinical and biochemical parameters between subclinical hypothyoidism subjects and healthy euthyroid controls

Variable SCH Patients(Mean±SD)

Euthyroid cont-rols

(Mean±SD)P value Statistical

significanceAge (yrs) 51.3 ± 5.6 50.4 ± 5.3 - -

BMI (kg/m2) 27.6 ± 4.8 25.9 ± 5.0 0.18 NSTSH (mIU/L) 8.42 ± 2.1 3.37 ± 0.61 0.000 HS

T3 (ng/dL) 146.6 ± 20.0 145.2 ± 18.7 0.78 NST4 (µg/dL) 7.64 ± 1.45 7.97 ± 1.94 0.45 STC (mg/dL) 213 ± 53.7 184.5 ± 43.3 0.025 HSTG (mg/dL) 115.5 ± 40.7 95.3 ± 23.9 0.02 S

LDL (mg/dL) 122.5 ± 34.5 112.3 ± 22.4 0.17 NSHDL (mg/dL) 38.3 ± 8.4 41.5 ± 6.8 0.11 NSP value ≤ 0.05 is considered as statistically significant; BMI = Body mass index; TSH

= Thyroid stimulating hormone; T3 = Tri-iodothyronine; T4 = Tetra-iodothyronine; TC = Total cholesterol; TG = Triglycerides; LDL-C = Low density lipoprotein; HDL-C = High density lipoprotein; S = Statistically significant; HS = Highly significant.

Table 2 shows clinical and biochemical characteristics of the study subjects. Serum mean levels of total cholesterol (213 ± 53.7), triglycerides (115.5 ± 40.7) were significantly higher in SCH patients than in controls (79.4 ± 9.8, 184.5 ± 43.3, 95.3 ± 23.9 respectively) and were statistically significant (p < 0.05). Serum mean levels of BMI (27.6 ± 4.8), fasting glucose (96.3 ± 21.2), HDL – C (38.3 ± 8.4), LDL – C (122.5 ± 34.5) were not significantly different from the values in controls (25.9 ± 5.0, 125.6 ± 16.3, 94.6 ± 15.9, 41.5 ± 6.8, 112.3 ± 22.4 respectively).

Table 3: Risk factors for cardiovascular disease in SCH patientsVariable SCH Patients (%) Euthyroid controls (%)

BMI (> 27.5 kg/ m2) 46.7 33.4TC (> 200 mg/dL) 43.4 16.7TG (> 150 mg/dL) 16.7 3.4

LDL (> 130 mg/dL) 30 16.7HDL (< 30 mg/dL) 20 0

The percentages of patients and controls with increased BMI, abnormal lipid profiles 18

are given in table 3. The percentage of subjects having higher BMI (>27.5 kg/m2), elevated TC (>200 mg/dL), LDL (130> mg/dL), TG (150> mg/dL), and decreased HDL (<30 mg/dL) was higher in SCH patients than in controls.

Among the SCH patients, TSH levels were positively correlated with that of total cholesterol (r = 0.3267) and triglycerides (r = 0.5464). (r = Spearman rank correlation coefficient).

DISCUSSION: The nature and degree of abnormal lipid profiles in overt hypothyroidism has been

demonstrated convincingly in many studies and there is no doubt about the beneficial effects of thyroid hormone replacement therapy on serum lipids and on the risk for cardiovascular disease12. However, the possible effects of subtle alterations of thyroid function as in SCH on lipid profile and atherogenesis remain unclear13. There is, in fact, doubt as to whether SCH should be treated because the evidence in terms of dyslipidemia, hypertension or glucose intolerance provided by different authors is controversial. However, there is increasing evidence that SCH is associated with dyslipidemia and hypertension in the elderly which can be a potential risk factor for the development of CVD in the near future14.

In this study we found that, SCH subjects have statistically significant higher mean levels of serum total cholesterol, TG and thus representing a more atherogenic lipid profile when compared with the age and sex matched euthyroid controls. In a substantial number of studies, TC and LDLc are seem to be elevated in SCH compared to controls. However there are studies which do not confirm these findings.

Previous study results revealed conflicting data on serum lipid concentrations in subclinical hypothyroidism. Total cholesterol and HDL-C were elevated in several studies, but were not different from those in controls in most other studies. Lower serum HDL-C levels were reported in few studies and were not different from euthyroid controls in most other studies15. In our study we found that the percentage of patients with atherogenic lipid profiles (elevated total cholesterol/HDL-C and LDL-C/HDL-C) was higher in SCH patients than in controls.

There is a growing body of evidence indicating that elevated triglyceride levels are an independent risk factor for cardiovascular disease16. Hypertriglyceridemic patients often develop a lipoprotein profile characterized by elevated triglycerides and LDL-C and low HDL-C17. It is estimated that the aggregated risk associated with triglycerides greater than 220 mg/dL and a total cholesterol/HDL-C ratio greater than 5.0 to be 25% of the cardiovascular events18.

A meta-analysis of 17 population-based studies of triglyceride levels and cardiovascular

19

disease identified a 76% increase in cardiovascular disease risk with a 1 mmol/L (89 mg/dL) increase in triglyceride levels19. Our findings are in agreement with previous studies demonstrating that approximately 16.7% of patients with subclinical hypothyroidism had hypertriglyceridemia when compared to 3.4% in control subjects.

The findings of this study must be interpreted within the limitations of the study design. Our assumption that the subclinical hypothyroid group is homogeneous might ignore the possibility that a subgroup of these persons might be at greater risk for hyperlipidemia. In addition, because of the cross-sectional nature of this analysis, it is difficult to ascribe causality to any associations we have found. Because we do not know whether thyroid test abnormalities preceded elevations in triglyceride levels, it cannot be definitely stated that one leads to the other. Further evaluation of this relationship with longitudinal data would be necessary to support a causal link.

CONCLUSION: Patients with subclinical hypothyroidism exhibit increased levels of the atherogenic

parameters (mainly TC, TG & LDL), which may predispose to the development of premature atherosclerotic coronary artery disease (CAD) and seem to weigh in favour of treatment of patients with subclinical hypothyroidism.

REFERENCES:1. Surks MI, Ortiz E, Daniels GH, Sawin CT, Col NF, Cobin RH, et al. Subclinical

Thyroid Disease Scientific Review and Guidelines for Diagnosis and Management. JAMA 2004; 291(2): 228-238.

2. Hollowell JG, Staehling NW, Flanders WD, et al. Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2002;87(2):489-499.

3. Cooper DS. Subclinical hypothyroidism. N Engl J Med 2001;345(4): 260-265.4. Rodondi N, Newman AB, Vittinghoff E et al. Subclinical Hypothyroidism and the

Risk of Heart Failure, Other Cardiovascular Events, and Death. Arch Intern Med. 2005;165:2460-2466.

5. Hueston WJ and Pearson WS. Subclinical Hypothyroidism and the Risk of Hypercholesterolemia. Ann Fam Med 2004;2:351-355.

6. Cappola AR, Fried L P, Arnold AM, Danese MD, Kuller LH, Burke GL, et al. Thyroid Status, Cardiovascular Risk and Mortality in Older Adults. JAMA 2006;295:1033-1041.

7. Soos M, Siddle K. Characterization of monoclonal antibodies directed against 20

human thyroid stimulating hormone. J Immunol methods 1982;51(1);57-68.8. Walker W.H.C. Introduction: An Approach to Immunoassay. Clin Chem 1977; 23:

384. 9. Schuurs AH, Van Weeman BK. Review. Enzyme-immunoassay. Clin Chem Acta

1977;81:1.10. Nader R, Warnick GR. Lipids, lipoproteins, apolipoproteins and other cardiovascular

risk factors. In : Burtis CA, Ashwood ER and Bruns DA, eds. Tietz Text book of clinical chemistry and molecular diagnostics, 4th edn. New Delhi : Elsevier Co., 2006; pp: 916-952.

11. Rifai N, Iannotti E, DeAngelis K, Law T. Analytical and clinical performance of a homogeneous enzymatic LDL – cholesterol assay compared with the ultracentrifugation-dextran sulfate-Mg2+ method. Clin Chem 1998; 44(6): 1242-1250.

12. Tunbridge WMG, Evered DC, Hall R, Appleton D, Brewis M, Clark F et al. Lipid profiles and cardiovascular disease in the Wickham areas with particular reference to thyroid failure. Clin Endocrin 1977; 7: 495–508.

13. Kahaly GJ. Cardiovascular and atherogenic aspects of subclinical hypothyroidism. Thyroid. 2000;10:665–679.

14. Cooper DS. Subclinical thyroid disease: a clinician’s perspective. Annals of Internal Medicine 1998:129;135–138.

15. Kung AWC, Pang RWC, Janus ED. Elevated serum lipoprotein (a) in subclinical hypothyroidism. Clin Endocrinol 1995;63:445–449.

16. Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA 2001;285: 2486–2497.

17. Brewer HB. Hypertriglyceridemia: Changes in the plasma lipoproteins associated with an increased risk of cardiovascular disease. Am J Cardiol 1999;83:3F-12F.

18. Assmann G, Schutt H, Von Eckardstein A. Hypertriglyceridemia and elevated lipoprotein (a) are risk factors for major coronary events in middle aged men. Am J Cardiol 1999;77:1179–1184.

19. Austin MA. Epidemiology of hypertriglyceridemia and cardiovascular disease. Am J Cardiol 1999;83:13F–16F.

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Original article

EFFECT OF FLUORIDE ON MALONDIALDEHYDE, BONE MINERAL STATUS IN POSTMENOPAUSAL OSTEOPOROSIS

Parinita Kataraki 1, Pragna Rao21 Department of Biochemistry, BLDEU’S Shri B. M. Patil Medical College, Bijapur-586103 Karnataka

2 Professor and Head, Department of Biochemistry, Kasturba Medical College, Manipal, Karnataka.

ABSTRACTOsteoporosis is a major health problem of old age. This silently progressing metabolic

bone disease is widely prevalent in India, the prevalence of osteoporosis increases with age especially involving long bones. It has been observed that females suffer from osteoporosis, after the menopause, accelerated process of osteoporosis occur and it is observed in India that about 50% suffer from osteoporosis and estimated number is 30 million. Fluorosis is a serious public health problem in many parts of the world where drinking water contains more than 1 ppm of fluoride. Higher intake of fluoride will result in dental and skeletal fluorosis and affect collagen synthesis and bone mineralization. Approximately 99% of the body burden of fluoride is associated with calcified tissues. The fluoride concentration in bone is not uniform. Fluoride concentration in bone is not uniform and higher fluoride levels in the body are associated with calcified tissues. The study was conducted on 100 subjects with their consent which consisted of control group of 50 non-pregnant women in their reproductive age group. Study group consisted of 50 women who had attained menopause either naturally or surgically, all residing in the endemic fluorotic area of Nalgonda district, Andhra Pradesh, India. 5 ml of venous blood was collected from both the groups. Serum fluoride levels (0.68_0.39; p<0.005) was statically significant when compared to the controls (0.45 _ 0.28). The biochemical parameters of bone mineral status, serum calcium (9.19 _ 0.620) p= 0.488 and serum phosphorus (4.27 _ 0.71; p = 0.799) was statistically insignificant. Malondialdehyde was measured as an indicator of oxidative stress which was statistically significant (287.86 _ 49.79; p<0.001). Serum malondialdehyde, a lipid peroxidation product, is used as an indicator of oxidative stress which is induced by fluorosis.

Key-words: Fluorosis, post menopausal women, malondialdehyde, calcium, phosphorus, osteoporosis.

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INTRODUCTION:Fluoride is a trace element that is ubiquitously distributed throughout the environment

in a wide range of concentrations especially in soil and water resources1. Fluoride is a potent anion and the most electronegative element as well as being a cumulative toxin2. The recommended concentration range in the drinking water is 0.7 – 1.2 ppm, depending on the average regional temperature. Lower levels are recommended for warmer regions where water intake tends to be higher3.

Fluoride metabolism is pH dependent and that the transmembrane migration of the ion occurs in the form of hydrogen fluoride in response to differences in the pH of adjacent body fluid compartments. About 80 – 90% of the ingested amount is absorbed from the gastrointestinal tract in presence of sufficient concentrations of cations such as calcium and aluminium form insoluble fluoride compounds which decrease its absorption. The half time for absorption is about 30 minutes3.

Being highly soluble in water, environmental fluoride is absorbed easily in the stomach and gut. Although high amount of fluoride in plasma is in bound form, a small fraction of it is in ionic form. Fluoride passes easily through cell membranes in its ionic form. Therefore primary involvement of bone in chronic fluorosis is attributed to the ability of these tissues to accept fluoride ion in exchange for other ions2.

After about 50% of an ingested fluoride dose has been absorbed, plasma concentrations decline rapidly. This is due to renal excretion and uptake by calcified tissues. Fluoride is freely filtered through the glomerular capillaries and then undergoes a variable degree of tubular reabsorption. Among the halogens, the renal clearance of fluoride is unusually high, about 35 ml/min.

Approximately 99% of fluoride in the body fluids is associated with calcified tissues. The fluoride concentration in bone is not uniform. In long bones, the concentrations are highest in the periosteal region. They decline sharply within a few millimetres of the periosteal surface and increase slightly as the endosteal region is approached. Cancellous bone has higher fluoride concentrations than compact bone.

The clearance of fluoride from plasma by the skeleton proceeds at a high rate that exceeds even for calcium. Approximately 50% of the fluoride absorbed each day by healthy young or middle aged adults becomes associated with calcified tissues within 24 hours, while virtually all of the remainder is excreted in the urine.

Uptake of fluoride by bone occurs in stages. The first stage involves fluoride migration into the hydration shells of bone crystallites. These ion-rich aqueous shells are continuous with, or at least available to, the extracellular fluids. Presumably, fluoride in this pool is rapidly exchangeable so that it can undergo net migration in either direction, depending on

23

the relative concentrations in the extracellular fluid and the hydration shells. Later stages involve fluoride association with or incorporation into precursors of hydroxyfluoroapatite and finally into the apatitic lattice it self. Apatitic fluoride re-enters the circulating body fluids as a result of the long term process of bone resorption. 3 Fluorosis is a slow and progressive process causing symptoms related to several systems particularly related to musculo-skeletal system2.

Fluoride causes lipid peroxidation, DNA damage and apoptosis, and there is a positive relationship among these changes. Fluoride induces apoptosis by oxidative stress induced lipid peroxidation, and there by releasing cytochrome C into the cytosol and further triggering caspase cascade leading to apoptotic cell death4.

Hence this study was planned to assess the importance of biochemical parameters in the diagnosis of osteoporosis, bone mineral status and relation between fluoride and oxidative stress in the pathogenicity of postmenopausal osteoporosis in postmenopausal women residing in endemic fluorotic area.

MATERIALS AND METHODS:The study was conducted on 100 subjects with their consent which consisted of control

group of 50 non-pregnant women in their reproductive age group. Case group consisted of 50 women who had attained menopause either naturally or surgically, all residing in the endemic fluorotic area of Nalgonda district, Andhra Pradesh, India.

Women with liver disorders, alcoholism hyper/hypothyroidism and women on medication with vitamin D, calcium and hormone replacement therapy were excluded from the study. 5 ml of random venous blood was drawn from the out patients, with a sterile disposable syringe which was transferred into centrifuge tubes. The sample was centrifuged at 3000 rotations per minute for 10 minutes and the serum was collected from the centrifuge tubes.

Serum calcium was estimated by Q- Cresolpthalein complex kit method Calcium in an alkaline medium combined with o-cresolpthalein complex to form a purple complex. Intensity of colour formed is directly proportional to the amount and calcium present in the sample5,6.

Serum phosphorus was measured by Molybdate UV method, here phosphate ions in an acidic medium react with ammonium molybdate to form a phosphomolybdate complex. This complex has an absorbance in the ultraviolet region and is measured at 340 nm. Intensity of complex formed is directly proportional to amount of inorganic phosphorus present in the sample7,8.

Serum and water fluoride estimation was done using Ion selective electrode (Eutech fluoride electrode). Serum malondialdehyde, an indicator of oxidative stress was estimated

24

by Thiobarbituric acid reactive assay method. One molecule of malondialdehyde in the serum reacts with two molecules of thiobarbituric acid in acidic medium and gives rise to color complex (pink) which is measured at 532 nm against distilled water in a spectrophotometer9.

RESULTS:The present study was undertaken in the Department of Biochemistry, Kamineni

Institute of Medical Sciences, Narketpally, Andra Pradesh, India. The study group included the postmenopausal women aged between 50 to 70 years. The control group women were in their reproductive age group, non-pregnant, healthy individuals aged between 20 to 40 years. The mean age for the patient and control group is shown in the fi gure 1.

Figure 1: Age distribution among patients and controls:

Table: Mean and SD values of serum F, Ca, P and MDA in patients and controls

Biochemical parameters Controls(n=50)

Patients (n=50) p value

Serum fl uoride(ppm) 0.45 ± 0.28 0.68 ± 0.39 <0.005

Serum calcium( mg/dl) 9.26 ± 0.46 9.19 ± 0.62 NS

Serum phosphorus(mg/dl) 4.32 ± 0.86 4.27 ± 0.71 NS

Serum malondialdehyde(nmol/dL) 127.36 ± 47.01 287.86 ± 49.79 < 0.001

The biochemical parameters, serum calcium and phosphorus were measured to know the bone mineral status in patients and controls. The mean values of these parameters in

25

both the groups are shown in the table which do not show a significant difference between the two groups. The p value for serum calcium (p= 0.488) and serum phosphorus ( p = 0.799) are not statistically significant.

Serum malondialdehyde levels were estimated by method of TBARS as an indicator of oxidative stress in both the groups. The mean values for both the groups are shown in table 4, which show the increased values in the patients when compared to that of control group. The mean values of serum malondialdehyde between patient and control groups were statistically significant. (p < 0.001)

Water fluoride from the surrounding villages of Nalgonda district ( n=10 ) was estimated using Ion selective electrode, which showed the mean of 4.35 _ 2.28 ppm. The subjects included in the study were the local residents of Nalgonda district who were exposed to the fluoride consumption for >10 years.

Serum fluoride levels were measured to know the fluorotic state in both case and control groups. The mean values for both the groups are shown which shows the increased levels in the patient group. Statistical analysis shows (p < 0.005) significant difference in patient and control group. This indicates that although both patients and controls are residing in the area of endemic fluorosis, patients had higher levels of fluoride. This could be due to difference in age.

DISCUSSION:Fluoride is toxic to osteoblasts (bone forming cells). A first response to cell injury

is to initiate repair and when that fails the cell dies. In fluoride intoxication the repair mechanism fails, and the result is an initial increase in both bone formation and resorption. This response to cell injury by fluoride leads to pathological bone formation, and such increased formation or decreased resorption of bone results in increased bone mass. When injured osteoclasts die, new osteoclasts are formed from monocytes. The secondary injury of osteoclasts does not result in a paucity of osteoclasts on the surface of fluorotic bone. Fluoride injures cells involved in bone formation, and a poorer quality bone accumulates in patients with increased intake of fluoride10.

The serum fluoride levels of both case and control groups are low compared to that of the water fluoride levels estimated. This is because, the clearance of fluoride from plasma by the skeleton proceeds at a high rate that exceeds even for the calcium. Approximately 50% of the fluoride absorbed each day becomes associated with calcified tissues within 24 hours, while virtually all of the remainder is excreted in the urine3.

Arjun L Khandare et al10 and M Yildiz et al11 showed that there is significant increase in the serum fluoride level in the patients with high fluoride intake. Our study shows siginificant increase in the serum fluoride in the patient group.

In this study, which includes 50 patients and 50 controls, serum calcium, phosphorus 26

are studied as the indicators of bone mineral status. Serum malondialdehyde was assayed as the indicator of oxidative stress. A study conducted by Boink AB et al12 showed that fluoride induces hypocalcemia. The mechanism is based on the formation of fluorapatite which is responsible for the hypocalcemia.

In our study, both patients and controls had normal serum calcium levels. The normal serum calcium levels can be due to the various dietary sources containing calcium which contribute to the distribution of calcium in the body.

The mean values of phosphorus in the two groups did not reveal any statistically significant difference. This finding is in accordance with study of S. G. Srikantia et al13 who suggested that serum phosphorus levels were within nomal limits in subjects suffering from various degrees of skeletal fluorosis.

Lipid peroxidation product, serum malondialdehyde was estimated in both the case and control group indicating the oxidative stress in group II. Aysel Guven et al14 conducted a study which included the assessment of the effect of chronic fluoride intoxication on lipid peroxidation by estimating serum malondialdehyde. They found that malondialdehyde levels were increased which could be associated with peroxidation of membrane phospholipids and the accumulation of malondialdehyde. Our study showed a statistically significant difference in malondialdehyde levels in both case and control group.

Oxidative stress is an independent risk factor for osteoporosis15. The role of reactive oxygen species in bone metabolism is unique and dual considering their effect under physiological and pathological conditions. Under physiological conditions, the production of reactive oxygen species by osteoclasts assists in accelerating destruction of calcified tissue and hence assists in bone remodelling16. Oxidative stress is able to inhibit bone cell differentiation of a preosteoblastic cell line and a marrow stromal cell line that undergoes osteoblastic differentiation17. Enhanced osteoclastic and depressed osteoblastic activity is attributable to primary estrogen deficiency characteristic of osteoporosis16. Oxidative stress may contribute to the development or progress of osteoporosis may indicate an underlying, or accompanying state of inflammation18.

A study by Hui Xu, Chun-hong Wang et al., has shown that, both low and high doses of fluoride caused active state of oxidative stress in osteoblasts, suggesting that oxidative stress may be excited by the active osteoblasts viability induced by low dose of fluoride19. Our study showed the normal bone mineral status and increased serum malondialdehyde levels in case group when compared to control group, indicating oxidative stress.

CONCLUSION:Biochemical markers are non-invasive, inexpensive and can be repeated often. Bone

mineral status measured using serum total calcium and phosphorus have less significant role in fluorotic patients. Flurosis induces oxidative stress where serum malondialdehyde, a lipid peroxidation product is used as an indicator of oxidative stress.

27

REFERENCES:1. MaryFran Sowers, Gary M.Whitford, M.Kathleen Clark and Mary L.Jannausch.

Elevated serum fluoride concentrations in women are not related to fractures and bone mineral density. The journal of nutrition, February, 2005; 2247 – 2252.

2. M.Oncu, K.Gulle, E.Karaoz, F.Gultekin, S.Karaoz, I.Karakoyun et al. Biochemical and histopathological effects of chronic fluorosis on lung tissues of first generation rats. Biological Trace Element Research. 2008; 128:109 – 115.

3. G. M. Whitford. Intake and metabolism of fluoride. Adv Dent Res 1994;8:5 – 14.4. Ling-Fei-He, Jian-Gang Chen. DNA damage, apoptosis and cell cycle changes

induced by fluoride in rat oral mucosal cells and hepatocytes. World J Gastroenterol, 2006; 112(4): 1144 – 1148.

5. R.O.Mendez, MA Gomez. Effect of calcium and phosphorus intake and excretion in bone density in postmenopausal women. Annal of Nutrition and metabolism, 2002; 46:249 – 253.

6. Clifford J. Rosen. Biochemical markers of bone turnover a look at laboratory that reflect bone status. Clin. Chem. Acta. 1973; 46: 361 – 367.

7. Clinical utility of biochemical markers of bone remodelling. Clinical Chemistry. 1999; 458: 1359 – 1368.

8. Goodwin J.F. Diagnostic and therapeutic principles. Clin.Chem. 16, 1970; 16: 198 – 204.

9. Nike E. Yamamoto Y. Membrane damage due to lipid peroxidation. American Journal of Clinical Nutrition, 1991; 53: 522 – 524.

10. Arjun L.Kandare, Rao GS, N Balakrishna. Dual x-ray abosrptiometry (DXA) study of endemic skeletal fluorosis in a village of Nalgonda district, Andhra Pradesh. Fluoride, 2007; 40(3): 190 – 197.

11. M.Yildiz, M.Akdogan, N.Tamer, B.Oral. Bone mineral density of the spine and femur in early postmenopausal Turkish women with endemic skeletal fluorosis. Calcif Tissue Int, 2003; 72: 689 – 693.

12. Boink AB, Wemer J, Meulenbelt J. The mechanism of fluoride induced hypocalcemia. Hum Exp Toxicol. 1994; 13(3): 145 – 55.

13. S.G.Srikantia, A.H.Siddiqui. Metabolic studies in skeletal flurosis. Clin. Sci. 1965; 28: 477 – 485.

14. Aysel Guven, Necati Kaya. Effect of fluoride intoxication on lipid peroxidation and reduced glutathione in Tuj sheep. Fluoride 2005; 38(2): 139 – 142.

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15. Meagher EA, Fitzgerald GA. Indices of lipid peroxidation in vivo: strengths and limitations. Free Radical Biol Med 2000; 28: 1745 – 1755.

16. Gulden Baskol, Huseyin Demir, Banu Cavdaroglu, Mevlut Baskol, Derya Kocer. Assesment of paroxinase 1 activity and malondialdehyde levels in patients with osteoporosis. Erciyes Medical Journal. 2007; 29: 268 – 273.

17. Viereck V., Grundker C., Blaschke S., Siggelkow H., Emon G., Hofbauer LC. et al. Oxidative stress modulates osteoblastic differentiation of vascular an bone cells. Free Radical Biol Med. 2001; 31: 509 – 516.

18. M.Akdogan, G.Eraslan, F.Gultekin, F.Sahindokuyucu, D.Essiz. Effects of fluoride on lipid peroxidation in rabbits. Fluoride.2001; 37(3): 185 – 189.

19. Hui Xu, Chun-hong Wang et al, Lipid peroxidation and fluorosis. Fluoride 2003; 36: 165 – 170.

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Case Report

A CASE REPORT OF RETINOBLASTOMA PRESENTING WITH ORBITAL CELLULITIS

Radhika, Sunil G Biradar, Vallabh KProfessor, Department of Opthalmology, BLDEU’s Shri B.M.Patil Medical College, Bijapur.

ABSTRACT:Retinoblastoma is a highly malignant tumor of the eye that manifests most often in the

first 2 years of life. Retinoblastoma is the most common malignant intraocular tumour in childhood; it is aggressive, involving the globe, with posterior extension to the cranium and central nervous system via optic nerve, orbit metastatis spread to whole body. If left untreated mortality rate is approximately 60% in developing countries. Following treatment recurrence rate is low. A four year old girl being HBsAg positive presented with painful swelling in right eye since 6 months with rapid progression from 7 days. On ocular examination, features of orbital cellulitis like periorbital edema and erythema with proptosis and limited ocular movements with intraocular tumour extending into anterior chamber were noted. Enucleation was done. Histopathology showed Retinoblastoma of right eye with infiltration of surrounding soft tisssues. Here we report a case of retinoblastoma in advanced stage presenting with orbital cellulitis.

Key-words: Retinoblastoma, Orbital cellulitis, Enucleation.INTRODUCTION:Retinoblastoma is caused by a mutation in a gene controlling cell division, causing

cells to grow out of control and become cancerous1. It occurs in increased frequency in children born with deletion of long arm of chromosome2.The incidence in various well- studied population groups around the world varies from 1 in 3,300 to 1 in 20,000 live births3-5.The highest incidence was reported by Albert and Colleagues6,who found an incidence of 1:3300 in Haiti. In United States, the annual incidence is 3.5 cases per million children younger than 15 years and account for approximately 2.5% to 4% of all cancers diagnosed in children younger than 15 years7.

The incidence of retinoblastoma decreases with age. Most tumors occur before age 2 years and are diagnosed before age 5 years7. The median age at diagnosis in the absence of a family history is approximately 24 months for unilateral case and at younger age in patients with positive family history. Abramson and associates8 studied familial retinoblastoma and patients with a previously normal eye examination. 62 % of the first eyes were diagnosed

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by 6 months and 90% by 12 months. Retinoblastoma has also been encountered at birth, although it is rare. There has been some suggestion of a slight preponderance in boys, but most series find no sex predilection and no racial predilection. The most frequent clinical findings are leukocoria, strabismus, uveitis, glaucoma, hyphaema, red eye, visual obscuration, orbital cellulitis, endophthalmitis, panophthalmitis.

Thus, the differential diagnosis are coat’s disease, persistent hyperplastic primary vitreous, ocular toxocariasis, familial exudative vitreoretinopathy, cicatricial retinopathy of prematurity, congenital cataract, microbial endophthalmitis9. The approach to treatment has undergone radical change over past few years like intravenous chemotherapy, enucleation, radiation therapy, laser therapy, cryotherapy. The survival rate for the affected children in developed countries is currently about 90-95%9.

CASE:A four year old girl presented to BLDEU’s out patient department of ophthalmology

with complaints of painful swelling in the right eye since six months with rapid progression from seven days and fever of moderate degree, not associated with chills or rigor, more during the night since five days. She also gives history of five such similar episodes in the past five months, history of deviation in the right eye since birth with white reflex in pupillary area noticed at seven months of age. History of nasal squinting of right eye noted at eight months of age. No history of similar complaints in any family members. Features of orbital cellulitis like periorbital oedema, erythema and Proptosis by about 1.5cm were noted in right eye. Ocular movements were limited laterally. Conjunctiva chemosed with cicrumciliary congestion, hazy cornea, pseudo hypopyon and hemorrhage noted in anterior chamber and rest of the details were not made out. Visual acuity was no PL. Left eye was normal. Pallor was noted on general physical examination and systemic examination was within normal limits. Complete blood test showed elevated WBC counts and Hemoglobin of 9.8 gm/dl, urine and stool examination was normal. USG abdomen and X-ray chest were normal.

CT orbit reported mass with features suggestive of Retinoblastoma of right orbit extending retro-orbitally into the soft tissue and involving optic nerve. Enucleation was done, the eyeball with optic nerve was sent for histopathological examination which was reported as Retinoblastoma of right eye with tumour infiltration into anterior and posterior chamber and involving optic nerve and surrounding soft tissue.

GROSS PHOTOGRAPH OF RIGHT EYE Fig. 1 shows retinoblastoma of right eye with features of orbital cellulitis

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CUT-SECTION OF THE EYEBALL AFTER ENUCLEATION Fig. 2 : shows the tumour infiltration into both anterior and posterior chambers

involving optic nerve

DISCUSSIONRetinoblastoma is a primary malignant intraocular neoplasm that arises from immature

retinoblasts within developing retina. It is the most common primary intraocular malignancy of childhood. Retinoblastoma can be heritable (40%) or non-heritable (60%). During the normal maturation process, human retina is not completely developed at birth. The superior and nasal retina complete this process before the inferior and temporal retina10.If the human retina differentiates in a similar fashion, this may explain why new foci of retinoblastoma tend to appear predominantly in inferior and temporal periphery. Abramson and colleagues observed that the younger the age at diagnosis, the more likely it is for tumor to be seen in the posterior pole. Most common presentation of retinoblastoma is with leukocoria, strabismus, uveitis. Preseptal and orbital cellulitis are rare presenting features. Studies have shown co-relation of orbital cellulitis with advanced intraocular retinoblastoma11.

Histologically, a typical retinoblastoma is composed of small, polygonal cells with scanty cytoplasm and a large, chromatin-rich nucleus that stains deeply with hematoxylin. Retinoblastomas exhibit a unique form of differentiation to produce elements similar to those seen in photoreceptor cells. Three cytologic features of rosette formation may be seen: fleurettes, Homer-Wright rosettes, and Flexner-Wintersteiner rosettes. Fleurettes show differentiation toward photoreceptors and may be seen in other tumors. Homer-Wright rosettes are tumor cells that send neural processes toward a central zone and have no central lumen. Flexner-Wintersteiner rosettes, or true rosettes, are nearly pathognomonic for retinoblastoma. They are characterized by differentiated tumor cells arranged around a central space. Some data suggest that retinoblastoma may be derived from a primitive stem cell of neuroectodermal tumor with the capacity for differentiation in both neuronal and neuroglial directions12.

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Currently employed treatment options for Retinoblastoma are intravenous chemotherapy, enucleation, external beam radiation therapy, plaque radiotherapy, photocoagulation, transpupillary thermotherapy, cryotherapy9. When a child with retinoblastoma is seen for first time, examination should be conducted under anesthesia with complete mydriasis. The size and location and extent of all tumors should be carefully assessed. If a tumor is missed and then subsequently seen in follow-up, it must be assumed that a new tumor has arisen at that site, and further treatment must be undertaken. In unilateral disease, it is usually massive and there is often no expectation that useful vision can be preserved, surgery (enucleation) is usually undertaken and radiation therapy is not given to the tumor bed.

However, patients with unilateral disease have been treated with chemotherapy in an attempt to preserve vision in the affected eye13,14. One study revealed that children with retinoblastoma who present with obvious external findings of leukocoria, strabismus, or red eye detectable by their family or pediatrician most often require enucleation. Children who manifest no obvious external findings can often avoid enucleation15. Systemic therapy should be chosen in more extensive disease. The survival rate for affected children who have retinoblastoma in developed countries is currently about 90-95%9.

REFERENCES:1. Dome JS, Rodriguez-Galindo C, Spunt SL, Santana VM. Pediatric sold tumors.

In: Abeloff MD, Armitage JO, Niederhuber JE, Kastan MB, McKenna WG, eds. Abeloff’s Clinical Oncology. 4th ed. Philadelphia, Pa: Elsevier Churchill Livingstone; 2008:chap 99.

2. Freedman J,Goldberg L. Incidence of retinoblastoma in the Bantu of South Africa.Br J Ophthalmol 1976;60:655-656.

3. Koch E, Naeses P:Retinoblastoma in Sweden 1958-1971. A clnical and histopathological study. Acta Ophthalmol 1979;57:344-350.

4. Suckling RD,Fitzgerald PH, Stewart J, Wells E. The incidence and epidemiology of retinoblastoma in Newzealand: A 30 year survey. Br J Cancer 1982;46:729-736.

5. Paticia Ward,Seymour Packman, William Loughman. Location of the retinoblastoma susceptibility gene(s)and the human esterase D locus. J Medical Genetics 1984;21:92-95

6. Albert DM, Lahav M, Lesser R, Craft J. Recent observations regarding retinoblastoma: Ultrastructure, tissue culture growth, incidence, and animal models. Trans Ophthalmol Soc UK 1974;94:909–928.

7. Pendergrass TW, Davis S. Incidence of retinoblastoma in the United States. Arch Ophthalmol 1980;98:1204–1210.

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8. Abramson DH, Mendelsohn ME, Servodidio CA et al. Familial retinoblastoma: Where and when? Acta Ophthalmol Scand 1998;76:334–338.

9. Augsburger JJ, Bornfeld N, Giblin M. Retinoblastoma. In Yanoff M, Duker JS.Ophthalmology 3rd ed. China: Mosby Elsevier; 2008:887-893.

10. Provis JM, Leech J, Diaz CM et al. Development of the human retinal vasculature: Cellular relations and VEGF expression. Exp Eye Res 1997;65:555–568.

11. Mulcaney PB, Karcioglu ZA, Huaman AM, Al-Mesfer S. Retinoblastoma associated orbital cellultis.Br J Ophthalmology 1998;82:517-521.

12. Shuangsgoti Sj, Chaiwum B, Kasantikul V: A study of 39 retinoblastomas with particular reference to morphology, cellular differentiation and tumour origin. Histopathology 1989;15:113–124.

13. Shields CL, Shields JA: Editorial: chemotherapy for retinoblastoma. Med Pediatr Oncol 2002;38 (6): 377-378.

14. Schouten-Van Meeteren AY, Moll AC, Imhof SM, et al. Overview: chemotherapy for retinoblastoma: an expanding area of clinical research. Med Pediatr Oncol 2002;38 (6): 428-438.

15. Shields CL, Gorry T, Shields JA. Outcome of eyes with unilateral sporadic retinoblastoma based on the initial external findings by the family and the pediatrician. J Pediatr Ophthalmol Strabismus 2004;41 (3): 143-149.

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Case Report

EPITHELIOID SARCOMA: A RARE CASEM.S. Kotennavar1, Rohan Khairatkar2, Gururaj Padasalagi3, Aravind Patil4

1Associate Professor, 2Post graduate, 3Assistant Professor, 4Professor,Department of Surgery, BLDE University’s Shri B.M.Patil Medical College, Bijapur.

ABSTRACT: Epithelioid sarcoma is a rare tumour accounting for less than 1% of soft tissue

sarcomas. It is a distinctive sarcoma of unknown lineage with predominantly epithelioid cytomorphology. It affects mainly adolescents and young adults with median age of 22 yrs. Herewith we report a rare case of epithelioid sarcoma in left axilla involving Pectoralis major and minor muscles.

CASEA 30 year old male patient presented to us with a painless swelling in left axilla since

4 months.The swelling was initially 2cm x1cm in size which increased to a size of 18cm x 15cm over a period of 4 months. It involved whole of left axilla and part of left anterior chest wall. Nipple and areola were not involved. It had bosselated surface and few nodules on surface, variegated in consistency, nontender, fixed to Pectoralis major muscle and overlying skin, left arm movements were restricted due to swelling.

Fig 1: Photograph showing left axillary swelling with nodules and bosselated surface and incisional biopsy wound

Computerised tomography scan was suggestive of malignant necrotic mass lesion in anterior part of axilla with involvement of Pectoralis major and minor muscles with axillary lymphadenopathy. Incision biopsy was suggestive of ‘embryonal rhabdomyosarcoma’.

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Fig 2: CT scan film showing left axillary swelling with necrosis involving Pectoralis major and minor muscles with axillary lymph node

The patient underwent wide local excision with adequate surgical margin and axillary dissection with removal of part of Pectoralis major and minor muscles with primary closure of wound site. Patient underwent a cycle of radiotherapy and is under follow up.

a)Wide local excision of tumour with defect b) Excised tumour mass

c) Defect closed primarily with suction drain

Fig 3. a,b and c: Intraoperative photographs Pathologic examination of excised tumour showed cells arranged in nodular pattern

with central areas of necrosis. Tumour cells were mixture of spindle and epithelioid cells. Mitotic figures were scant. Lymphoplasmocytic infiltration was seen at few foci. Large areas of necrosis present at centre of tumour. Hemorrhagic areas were also seen. Axillary lymph nodes were negative for malignant cells. On Immunohistochemistry, Cytokeratin and Epithelial membrane antigen were positive3. Features were consistent with ‘Epithelioid sarcoma’.

Fig 4: Histopathology slide with classical features of Epithelioid sarcoma.

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DISCUSSION: Epithelioid sarcoma is a distinctive sarcoma of unknown lineage with predominantly

epithelioid cytomorphology. Epithelioid sarcoma is one of the few sarcomas in which lymph node metastases are fairly common, occuring in 20% of patients. It tends to propagate along fascial planes, tendon sheaths, and nerve sheaths and thus requires extensive wide margin excision for complete tumour removal.

Size and location at presentation are predictors of outcome. In a series of 16 patients from Memorial Sloan Kettering Cancer Centre half of the tumours appeared on the trunk and half in the extremities, with 44% of patients having lymph node metastasis and 44% having lung metastasis; the five year survival rate was 66%.

There is a variant of epithelioid sarcoma, ‘proximal type’ seen predominently in pelvis, perineum and genital tract. Most are deep seated tumours and tend to occur in older patients. The proximal variant is associated with a more aggressive clinical course, resistance to radiation and chemotherapy and worse disease specific survival than the conventional variant.

Surgery and radiotherapy remain the mainstay for local control of soft tissue sarcoma. Wide en bloc resection with 1-2 cm margin of uninvolved tissue with adjuvant radiotherapy is most commonly used. Opinion varies regarding the use of postoperative chemotherapy. The most recent study on adjuvant chemotherapy showed no statistical difference between the study and control group4.

REFERENCES1. Armah and Parwani. Epithelioid Sarcoma. Arch Pathol Lab Med. 2009

May;133(5);814-9.2. Enzinger FM. Epithlioid sarcoma: a sarcoma simulating a granuloma or a carcinoma.

Cancer. 1970;26:1029–41.3. Hiroshi Kato, Masahito Hatori, Shoichi Kokubun, et al. CA125 Expression in

Epithelioid Sarcoma. Jpn J Clin Oncol.2004;34(3)149–154.4. DeVita, Hellman and Rosenberg’s Cancer, Principles & Practice Of Oncology. 9th

Ed, Lippincot Williams & Wilkins. Pp-1546.

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B.L.D.E. UNIVERSITY

Smt. Bangaramma Sajjan Campus, Sholapur Road, Bijapur – 586103, Karnataka, India.University: Phone: +918352-262770, Fax: +918352-263303 ,

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