recent advancements regarding vitamin d3 t · sickle cell trait (as) individuals(6). the specific...

Editorial

Recent advancements regarding Vitamin D3

The endocrine system of vitamin D is central to the control of bone andcalcium homeostasis. D2 and D3 are the two major types of Vitamin D.Vitamin D2 is synthesized in plants and fungi, whereas Vitamin D3 or

calcitriol is produced in relatively large amounts in humans and in majorityof vertebrate animals. The active metabolite of Vitamin D, 1α, 25-hydroxyvitamin D3 [1, 25 (OH) 2D3] is involved in calcium and phosphatemetabolism and exert a large no of biological effects. Vitamin D inhibitsparathyroid hormone secretion, adaptive immunity and cell proliferation andat the same time promotes insulin secretion, innate immunity and stimulatescellular differentiation. The role of Vitamin D in immune-regulation has led tothe concept of dual function as both as an important secosteroid hormone forthe regulation of body calcium homeostasis and as essential organiccompound that has been shown to have a crucial effects on the immuneresponses. Altered levels of vitamin D3 have been associated, by recentobservation studies, with a higher susceptibility of immune-mediateddisorders and inflammatory diseases.

Vitamin D3 is photosynthesized in the skin, in which UV light catalyzes the firststep in D3 biosynthesis. The key step of Vitamin D biosynthesis involvesdifferent isoforms of cytochrome P450 family. Following biosynthesis, inactivevitamin D3 or calcidiol is transported to the proximal tubule of kidney, whereit is hydrolyzed to active form.

Significant advancements are made from different studies in defining the roleof Vitamin D in innate immunity. Vitamin D regulates the production of T-cellhelper 1 (Th1) and Th2 cytokines and 1L-17, by which it influences adaptiveimmunity and inflammation. Interestingly, specific T-cell cytokines are able toinfluence the TLR inducedvitamin-D-dependent anti-microbial pathway inhuman monocytes The Th1 cytokine IFN-γ enhances the TLR2/1 inductionof CYP27B1, cathelicidin and DFEB4. Recent studies have shown a markedeffect of Vitamin D3 in activity of cytokine like TNF-α expression ininflammatory disease. The down regulatory effect of D3 is not unique to TNF-α expression and was found to decrease the synthesis of IL-2 in activatedlymphocytes and also inhibited expression of 1L-1, IL-6 and IL-12 in culturedmonocytes and macrophages. The ability of vitamin D3 to suppressmycobacterium TB growth in monocytes has also been linked to theproduction of bactericidal superoxide anions and to the generation ofphagocyte derived reactive oxygen synthesis (NO).

2 Ind. J. Med. Biochem. Vol. 17, No. 2, 2013

Vitamin D acts through its receptor VDR, which upon binding to vitamin D3gets activated and regulate transcription of different classes of genes includingthose involved in immune reaction. Vitamin D receptor or VDR is one of theDNA-binding transcription factor but has an important additional property,which it shares only with some other members of nuclear receptorsuperfamily. VDR can get specifically activated by macromolecularconcentrations of lipophilic molecule, the property which is shared withnuclear receptor nuclear receptor for steroid hormones. Like other membersof the human nuclear receptor superfamily, VDR is characterized by a highlyconserved DNA binding domain (DBD) and a structurally conserved ligand-binding domain (LBD). As observed with other transcription factors, the DBDand of VDR cannot contact more than six nucleotides within the major grooveof the target DNA and the conserved sequence for VDR binding is RGKTSA(R= A or G, K= G or T and S= C or G), which is also referred to as VDREor vitamin D response element. Several recent studies using chromatinimmune-precipitation assay or ChIP technique have shown there are numberof genes that are regulated by VDR. Vitamin D3 stimulation oflymphoblastoma or human monocyte has shown to trigger VDR binding tomore than 1500 sites within the genome. The VDR binding site of this geneis located within the intron some 11okb downstream of TSS.

Number of recent studies suggests that VDR maturation is associated withnumber of diseases mainly due to defects in immunomodulation. VDR issuggested to play important role in miRNA regulation and thus mightindirectly involved in key regulation of other genes. VDR also regulates MCM7gene that encodes MIR 106b and known to regulate expressions of MIR181aand MIR-22 (14,15). Micro RNA regulation is linked to several diseases andtheir presence sometimes used as marker for some diseases.

RFLP studies from different segments of VDR gene demonstratedpolymorphism in the gene and mutations in the VDR gene are associatedwith different disease progression among individuals. These diseases includediabetes, tuberculosis, leprosy, cancer and bone biology which are directly orindirectly associated with vitamin D levels. Analysis of the TaqI polymorphismin the 3’ region of the VDR gene showed that the overall distributions ofgenotypesbetween the groups studied were different. In tuberculoid leprosy,the tt genotype was found at significantly higher frequency than in thecontrols. In contrast, the TT genotype was found at increased frequency in thelepromatous leprosy group compared with the controls. Taken together, allthese information clearly suggesting vitamin D and VDR together plays a verysignificant role in innate and adaptive immunity that controls number ofhuman diseases and new drug regimen must include vitamin D supplementand alternatively VDR therapy might be helpful.

IntroductionSickle cell disease (SCD) is a monogeneticrecessive haemoglobinopathy that affectsmillions throughout the world. It is commonamong people whose ancestors come from Sub-Saharan Africa, South America, Cuba, CentralAmerica, Saudi Arabia, India, andMediterranean countries. SCD is prevalent inIndia especially in states of Chhattisgarh, Odisha,Maharashtra, Telangana and Gujarat amongothers(1). As per ongoing studies, SCD geneaffects about 25 lac people that constitute about10% of the Chhattisgarh state's population(2).Heterozygous state (AS) of this gene referred assickle cell trait, is asymptomatic, but homozygous

state (SS) of this gene causes SCD. This diseaseis a significant cause of morbidity and mortalityin the state. SCD is caused by a point mutation(GAGGTG) in the beta globin gene (11p15.5)leading to replacement of the polar amino acidglutamic acid with a hydrophobic amino acidvaline at the 6th position in beta globin chain ofhaemoglobin molecule(3). These mutant betaglobin chains (βS) in homozygous state lead tothe formation of haemoglobin S (HbS), whichgets polymerized through hydrophobicinteractions under hypoxic conditions, to form arope like fiber composed of seven double strandstwisted helically around a vertical axis(4).Depending on their orientation within the

3

EFFECT OF HYDROXYUREA THERAPY ON ERYTHROCYTE MEMBRANE Na+ K+ ATPASE ACTIVITY, HBF AND

HAEMATOLOGICAL INDICES IN SICKLE CELL DISEASE PATIENTS

H. K. Uraon1a, O. P. Kashyap1a, P. K. Patra1a,2, D. Rath1a,H. Mishra2, S. Phuljhele1b, S. Gupta1a, P. K. Khodiar2

aDepartment of Biochemistry and bPaediatrics1Pt. J. N. M. Medical College, Raipur, India

2Sickle Cell Institute Chhattisgarh, Raipur, India

AbstractSickle cell disease is a monogenetic haemoglobinopathy, which is characterized by polymerization ofabnormal haemoglobin (HbS) to form rope like structures under low oxygen tension, resulting indistortion in the normal shape of RBCs to the sickle shape. Polymerization of HbS is further increasedby dysregulation of cation homeostasis due to activation of some membrane ion channels leading tocellular dehydration. Further, deoxygenation also leads to induction of erythrocyte membrane Na+-K+ATPase activity, which leads to loss of Na+ ions along with water from RBCs and furtheraggravation of cellular dehydration and in turn, HbS polymerization. Foetal haemoglobin (HbF), thelevel of which is increased by Hydroxyurea therapy, is protective in such patients, as it preventspolymerization of HbS molecules in RBCs. In this work we studied the effect of Hydroxyurea therapyon erythrocyte membrane Na+ K+ATPase activity, HbF and haematological indices in sickle celldisease patients. Statistical analyses showed significantly different Na+ K+ATPase activity and HbFlevels between SCD patients on and not on Hydroxyurea therapy. Similarly other parameters were alsoanalyzed and discussed.

erythrocyte, these polymers deform the shape ofthe cell to produce one of several morphologicalforms, mainly sickle shaped, instead of normalbiconcave disc shape. Initially the episodes of sickling are reversible butrepeated episodes of sickling damage theerythrocyte cell membrane and decrease thecell's elasticity(5). Such erythrocytes fail to returnto their normal shape, even under normaloxygen tension. Further, activity of erythrocytemembrane Na+ K+ ATPase is increased in SCDpatients in comparision to normal (AA) andsickle cell trait (AS) individuals(6). The specificratio of Na+ to K+ transportation through Na+K+ATPase on red blood cell membrane is 3:2(7).Thus increased activity of Na+ K+ATPase onRBC membrane leads to disturbed internal ionicmilleu of RBCs, which leads to cellulardehydration and distortion of RBC shape andvolume(8).Till date no pharmaco-therapeutic cure isavailable for this disease. Only disease modifyingtherapies available till date are blood transfusionand Hydroxyurea. Suggested mechanism ofHydroxyurea is elevation of protective HbF levelin RBCs(9). In this study, we have tried to analyzethe effect of Hydroxyurea therapy on HbF level,Na+ K+ATPase activity and otherhaematological parameters and deduce otherpossible mechanisms involved in the therapeuticeffect of Hydroxyurea on SCD patients.

Materials and Methods This study was done in the department ofBiochemistry, Pt. J.N.M. Medical College Raipurafter getting approval by the institutional ethicscommittee.Recruitment of subjectsThe study group comprised of 80 subjects. Thegroup was divided into three subgroups viz.,Normal Controls AA (20 subjects), Sickle Celltrait AS individuals (20 subjects) and SCDpatients (40 subjects). SCD patients’ subgroupcomprised of further two subgroups of 20subjects each, viz., those on Hydroxyurea

therapy (SCD Hyd+) and those not onHydroxyurea therapy (SCD Hyd-). The meanage in the AA , AS , SCD Hyd+ and SCD Hyd-gropus were 17±3.2 , 15.15±1.87, 14.65±2.3and 14.55±2.32 years respectively. Differencesin the male to female ratio in the study groupswere also not statistically significant. Initialidentification of disease status of subjects wasdone using haemoglobin solubility test. Subjectsshowing negative solubility test results, wereidentified as Normal controls. Those showingpositive solubility test results, were subjected tohaemoglobin electrophoresis in alkaline mediumto identify their SCD status. This electrophoresisstep was used to discriminate Sickle Cell traitindividuals and SCD patients. Identified SCDpatients were subjected to High PerformanceLiquid Chromatography (HPLC) to estimate thecontent of Foetal haemoglobin in SCD patients.Patients in Hyd+ group were recruited only if

they were on Hydroxyurea therapy for at leastlast one month. The dose being administered tothem was 10 mg/kg body weight alternate day.Patients in Hyd- group were those, who had nohistory of Hydroxyurea therapy. Patients withany other associated haemoglobinopathy orblood dyscrasias, history of discontinuedHydroxyurea therapy, severe bone morrowdepression, liver & kidney disorders, otherchronic debilitating diseases were excluded fromthis study. Moreover pregnant & lactatingwomen and malnourished children were alsoexcluded(10).

Sample Collection and laboratory analysisAfter getting written informed consent, fivemilliliter of venous blood was collected fromsubjects in vials containing 0.05 ml of EDTA asanticoagulant. Subjects were identified as sicklecell trait (AS) and SCD (SS) throughelectrophoresis. For this purpose, about 300 μlof blood sample was used for alkaline paperelectrophoresis for subjects with positivesolubility test. 5 μl of EDTA added blood samplesof AS and SS subjects were used to measure fetal


haemoglobin level. For this purpose Biorad HbVariants HPLC analyzer was used. Otherhematological parameters viz., haemoglobinlevel, RBC count, total leukocyte count (TLC),mean corpuscular volume (MCV), meancorpuscular haemoglobin (MCH), meancorpuscular haemoglobin concentration(MCHC), were measured by using automatedhematology cell counter analyzer (Mindray BD– 300 plus). Erythrocyte membrane Na+ K+ATPase activity was measured in the form ofreleased of inorganic phosphate (Pi) per mg ofprotein & expressed as (μmol Pi/mg protein/hr)by the action of Phosphatases present in theRBC membrane(7). The phosphate concentrationwas measured by using the standard curve madeusing Fiske & Subbarow principle and themembrane protein was quantified by usingLowry’s principle(11). For measuring totalbilirubin, blood was taken without addinganticoagulant and bilirubin was measured by ILab 650 autoanalyzer. (Fig. 1)

Statistical AnalysisStatistical analysis was done using SPSS version13. Data was presented as mean and standarddeviation (SD). Unpaired Student’s t-test,ANOVA and Post – hoc Bonferroni test wereused to analyze the observed data. A P value<0.05 was considered statistically significant.

ResultsNa+ K+ ATPase activity in RBC membrane:Mean Na+ K+ ATPase activity in terms of μmolPi/mg protein/hr, AA, AS, SCD Hyd- and SCDHyd+ subgroups is depicted in Fig. 2. The Na+-K+ATPase activity in the AA, AS, SCD Hyd- andSCD Hyd+ subgroups was found to be104.52±18.18, 117.93±19.15, 153.60±5.76,135.29±24.30 μmol pi/mg protein/hrrespectively. Applying ANOVA the differencebetween four groups was found to be significant(F=23.629, p<0.0001).The Post – Hoc Bonferroni test was used toestimate the significance of pair-wise difference

of Na+ K+ ATPase activity in terms of μmolPi/mg protein/hr, in AA, AS, SCD Hyd- and SCDHyd+ subgroups. The results obtained aredepicted in Table 1. Difference between AA andAS groups was not found to be statisticallysignificant. While other groups including SCDHyd- vs SCD Hyd+ had statistically significantdifference in Na+ K+ ATPase activity. (Fig. 2)

Foetal Haemoglobin Levels in RBCsThe HbF level in the AA, AS, SCD Hyd- andSCD Hyd+ subgroups is depicted in Fig. 3. HbFlevel in the AA, AS, SCD Hyd- and SCD Hyd+subgroups was found to be 0.58±0.28%,2.55±1.78%, 18.07±3.9% and 22.61±6.22%respectively. Applying ANOVA the differencebetween four groups was found to be significant(F=170.208, p<0.0001). Difference betweenAA and AS groups was not found to bestatistically significant. While other groupsincluding SCD Hyd- vs SCD Hyd+ hadstatistically significant difference in HbF level.(Fig. 3).Results of comparision of Na+ K+ ATPaseactivity and HbF level are provided in Table 1.

Haematological and other parametersDetailed values of haematological and otherparameters are given in Table 2.Applying unpaired student t test the difference ofhaemoglobin concentration between these twogroups was found to be statistically significant(p<0.0001). Applying ANOVA the difference inhaemoglobin concentration between threegroups was found to be significant (F=21.11,p<0.0001). The Post – Hoc Bonferroni testshowed that the SS patients had a significantlylower total RBC count then the AA & ASsubjects. Difference in RBC count between theAA & AS groups was not found to be statisticallysignificant (p value>0.05). Similarly, mean RBCcount SCD Hyd+ group was found to besignificantly higher SCD Hyd- groups (p<0.01).Applying ANOVA the difference in the TLCbetween three groups was found to be

5Hydroxyurea therapy & Biochemical parameters in sickle cell disease 5

statistically significant (F=7.652, p<0.001). ThePost – Hoc Bonferroni test showed that the SCDpatients had a significantly higher TLC then theAA and AS subjects. But the difference betweenthe AA & AS groups was not found to bestatistically significant. There was no significantdifference in TLC count between SCD Hyd+group & SCD Hyd- groups (p=0.086).Applying ANOVA the difference in MCVbetween four groups was found to be significant(F=3.680, p=0.016). The Post– Hoc Bonferronitest showed that the difference in MCV valuesbetween AA–AS, AA–SCD Hy- and AA– SCDHyd+ groups was statistically significant(p<0.01). On the other hand difference in MCVvalues between AS– SCD Hyd-, AS –SCDHyd+ and SCD Hyd- – SCD Hyd+ groups wasnot statistically significant (p>0.05).Applying ANOVA the difference between thevalues of MCH for four groups was found to bestatistically significant (F=3.964, p=0.011). ThePost – Hoc Bonferroni test showed that thedifference in the values of MCH between AA –SCD Hyd- group was statistically significant(p<0.01). But AA – AS , AA – SCD Hyd+, AS -SCD Hyd-, AS - SCD Hyd+ & SCD Hyd- –SCD Hyd+ groups was not statisticallysignificant (p>0.05). Applying ANOVA the difference between thevalues of MCHC for four groups was found tobe significant (F=2.810, p=0.045). The Post –Hoc Bonferroni test shows the differencein thevalues of MCHC between AS – SCD Hyd- wasstatistically significant (p<0.05) but AA – AS ,AA – SCD Hyd-, AA – SCD Hyd+, AS - SCDHyd+ & SCD Hyd- – SCD Hyd+ groups wasnot statistically significant (p>0.05).Another interesting finding was significantdifference in total serum bilirubin level betweenSCD Hyd- and SCD Hyd+ groups (p<0.0001).Applying ANOVA the difference of total serumbilirubin concentration between three groups was

found to be significant (F=21.766, p<0.0001).

DiscussionThis study was undertaken to determine thealteration in erythrocyte membrane Na+-K+ATPase activity, HbF level and haematologicaland other parameters in sickle cell diseasepatients under Hydroxyurea therapy. This studyhas revealed that the increased Na+-K+ ATPaseactivity in SCD patients may be decreased tonormancy by the use of Hydroxyurea therapy.This may be an additional mechanism ofHydroxyurea, in treatment of SCD patients inaddition to increasing HbF levels. ThusHydroxyurea may be helpful in maintaining theinternal ionic milleu of RBCs and thus indirectlythe shape and volume of RBCs. As evident fromseveral studies, this study also has confirmed thatHydroxyurea increases the HbF level insideRBCs in SCD patients(12). As far as use ofHydroxyurea is concerned, this study has shownthat Hydroxyurea improves RBC count in SCDpatients. Total serum bilirubin level also hasshown significant improvement upon use ofHydroxyurea. Other parameters including TLC, MCV, MCH,MCHC have not shown any significantdifference between the groups on therapy andthose not on Hydroxyurea therapy. As the sizeof sample included in this study was small, thefindings may be validated using a larger data set.

ConclusionThis study showed that the increased Na+ K+ATPase activity in SCD patients may bedecreased to normal level by the use ofHydroxyurea therapy. This effect may be anadditional mechanism of Hydroxyurea, intreatment of SCD patients in addition toincreasing HbF levels. Further studies may beundertaken with larger data sets to validate thefindings in this study.



Fig. 1 : Standards and standard curve for phosphate estimation

Fig. 2 : Mean values of Na+ K+ ATPase activity in RBC membrane

Fig. 3 : Mean values of foetal haemoglobin (HbF)


Table 1 : Statistical significance of difference in Na+ K+ ATPase activity and HbF level

Comparison groups p value for difference in p value for differenceNa+ K+ ATPase activity in HbF level

AA – AS > 0.05 > 0.05

AA – SCD Hyd- < 0.01 < 0.01

AA – SCD Hyd+ < 0.01 < 0.01

AS – SCD Hyd- < 0.01 < 0.01

AS – SCD Hyd+ < 0.01 < 0.01

SCD Hyd- – SCD Hyd+ < 0.01 < 0.01

Table 2 : Values of haematological and other parameters

Haematological and other AA AS SCD Hyd+ SCD Hyd-ParametersHaemoglobin (g%) 12.73 ± 2.18 11.8 ± 2.31 10.75 ± 1.70 7.32 ± 1.26

RBC count(X10 12/L) 4.21 ± 0.59 4.11± 0.60 3.75 ± 0.66 2.98 ± 0.75

TLC ( /Cumm) 7.46 ± 2.60 7.16 ± 2.12 9.04 ± 3.67 11.09 ± 3.70

MCV (fl) 87.51 ± 9.70 79.22 ± 10.74 80.42 ± 8.36 78.30 ± 10.11

MCH (hg) 30.28 ± 4.29 27.66 ± 3.88 27.69 ± 2.83 26.31 ± 3.76

MCHC (g/dl) 34.58 ± 1.84 34.98 ± 1.32 34.54 ± 1.33 33.65 ± 1.44

Total Biirubin (mg%) 0.54 ± 0.22 1.01 ± 0.52 1.51 ± 0.88 2.40 ± 1.12

AA= Normal Control AS = Sickle cell traitSCD = Sickle cell discase Hyd+ = hydroxyurea therapy presentHyd- = Hydroxyurea therapy absent

Reference

1. Deshmukh P, Garg BS, Garg N, Prajapati NC,Bharambe MS. Prevalence of Sickle Cell Disorders inRural Wardha. Indian J Community Med 2006; 31(1):1-3.

2. Patra P K, Chauhan V S, Khodiar P K, Dalla AR,Serjeant GR. Screening for the sickle cell gene inChhattisgarh state, India: an approach to a majorpublic health problem. J Community Genet 2011;2(3):147-51.

3. Turnpenny P, Ellard S. Emery's Elements of MedicalGenetics. Philadelphia: Elsevier Churchill Livingstone;2012.

4. Eaton WA, Hofrichter J. Sickle cell hemoglobinpolymerization. Adv Protein Chem 1990; 40:63-279.

5. Turhan A, Weiss LA, Mohandas N, Coller BS, FrenettePS. Primary role for adherent leukocytes in sickle cellvascular occlusion: a new paradigm. PNAS 2002;99(5): 3047-51.

6. Clark MR, Morrison CE, Shohet SB. Monovalentcation transport in irreversibly sickled cells. JCI 1978;62(2): 329-37.

7. Json G, Samson A, Joy Z. Na+/K+ ATPase Activity innormal & sickle cell erythrocytes. Chemistry &

material research. 2012; 2(5). Available from:http://www.iiste.org/Journals/index.php/CMR/article/view/17083/17442

8. Izumo H, Lear S, Williams M, Rosa R, Epstein FH.Sodium-potassium pump, ion fluxes, and cellulardehydration in sickle cell anemia. J Clin Invest 1987;79(6):1621-28.

9. Goldberg MA, Brugnara C, Dover GJ, Schapira L,Lacroix L, Bunn HF. Hydroxyurea and erythropoietintherapy in sickle cell anemia. Semin Oncol 1992; 19(3Suppl 9):74-81.

10. Ballas SK, McCarthy WF, Guo N, DeCastro L,Bellevue R, Barton BA, Waclawiw MA. Exposure tohydroxyurea and pregnancy outcomes in patients withsickle cell anemia. J Natl Med Assoc 2009; 101(10):1046-51.

11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.Protein measurement with the Folin phenol reagent.J Biol Chem 1951; 193(1): 265-75.

12. Kinney TR, Helms RW, O'Branski EE, Ohene-Frempong K, Wang W, Daeschner C, et al. Safety ofhydroxyurea in children with sickle cell anemia: resultsof the HUG-KIDS study, a phase I/II trial. PediatricHydroxyurea Group. Blood 1999; 94(5):1550-54.


IntroductionMycobacterium tuberculosis is a frequent causeof pneumonia in areas with high burden ofpulmonary tuberculosis(1). Clinicians arefrequently faced with the challenge ofdifferentiating between pulmonary tuberculosisand pneumonia due to other bacterialpathogens. The varying clinical and radiographicpresentation and a low sensitivity of AFBmicroscopy make it even more difficult todistinguish between the two(2). Differentialdiagnosis between the two is important asprompt isolation of pulmonary tuberculosispatients and initiation of treatment is necessary.Serum levels of CRP,an acute phase reactantprotein synthesized by the liver followingstimulation by various cytokines including tumor

necrosis factor-α and interleukin-6 (IL-6),markedly increase within hours after infection orinflammation. Since pneumonia is aninflammation of the lung, C-reactive protein(CRP), could be used as a marker ofinflammation to detect the extent of injury(3).Numerous studies have demonstrated increasedlevels of CRP in patients with pulmonarytuberculosis and non-tubercular bacterialpneumonia, but it’s utility to discriminatebetween the two has yet not been fully evaluatedin our part of the world(4,5).Being an inexpensive and readily available test,we explored the usefulness of CRP levels as auseful adjunctive test in the early differentiationof pulmonary tuberculosis from non-tubercularbacterial pneumonia.

10

USEFULNESS OF LEVELS OF C - REACTIVE PROTEIN IN DIFFERENTIATING BETWEEN PULMONARY TUBERCULOSIS

AND NON-TUBERCULAR BACTERIAL PNEUMONIA

Shivani Jaswal, Varinder Sainia, Jasbinder Kaur, Harjeet Kaur, Seema GuptaDepartment of Biochemistry and aPulmonary Medicine,

Government Medical College & Hospital, Chandigarh, India

AbstractDifferential diagnosis between pulmonary tuberculosis (TB) and non-tubercular bacterial pneumoniais important as prompt isolation of pulmonary tuberculosis patients and initiation of treatment isnecessary. The aim of the study was to explore means of discriminating between the two, based onlevels of C-reactive protein (CRP).100 patients presenting with signs and symptoms of pneumoniaformed the study group and were compared with 25 age and sex matched healthy controls. Levels ofCRP were estimated by standardized immunoturbidimetric methods on HITACHI 902.Sensitivity andspecificity of a range of CRP levels was investigated and the cut off values that resulted in bestdiscrimination were calculated using Youden’s index. 54 patients were diagnosed with tubercularpneumonia and 46 had non-tubercular bacterial pneumonia. The levels of CRP were significantlylower (p<0.001) in patients with TB as compared to non-tubercular bacterial pneumonia (medianlevels 4.25 mg/L and 23.9mg/L respectively). The best cutoff was found to be for CRP levels < 5mg/L,where the sensitivity for detecting TB was 78% and specificity was 56%. The positive predictive value(PPV) for CRP levels <5mg/L was calculated to be 66.7% and negative predictive value (NPV)65.7%.The high sensitivity and negative predictive value in differentiating between pulmonarytuberculosis and non-tubercular bacterial pneumonia suggests a supplementary role of CRP for thediagnostic exclusion of pulmonary TB from non-tubercular bacterial pneumonia in areas with highprevalence of the disease

Materials and MethodsThe study was conducted in the Department ofBiochemistry &the Department of PulmonaryMedicine, Government Medical College &Hospital, Chandigarh, India. The study wasplanned as a descriptive cross-sectional andobservational study comprising of 100 patientswith signs and symptoms of pulmonarytuberculosis or bacterial pneumonia, attendingthe OPD of the Department of PulmonaryMedicine.Patients with previously diagnosed oron treatment of pulmonary tuberculosis orbacterial pneumonia and patients with any othersystemic / infective illness were excluded fromthe study.Clearance from the Institutional ethical andresearch committee was obtained and informedconsent was taken from all participants of thestudy. Clinical data collected from the patientsincluded vital signs, symptoms and Chest X Rayfeatures. Lab measurements were conducted onthe day of the enrolment. Levels of CRP wereestimated in blood samples by standardizedimmunoturbiditimetric methods on HITACHI902 Autoanalyzer.The diagnosis of TB and Pneumonia was doneby the clinician.Patients were considered to havepulmonary TB when M. tuberculosis wascultured from the sputum or lavage fluidcombined with a lung parenchymal lesion.Bacterial pneumonia was diagnosed when thepatient had infiltrate on chest X-ray, whichresolved completely with antibiotic treatmentand culture of sputum was negative for M.tuberculosis.The median levels of CRP in the study groupswere statistically compared using Mann Whitneytest. Sensitivity and Specificity of a range of CRPlevels was investigated and the cutoff values thatresulted in best discrimination were calculatedusing Youden’s index. The optimal cut-off levelswere further investigated using receiveroperating characteristic (ROC) analysis.

ResultsThe study group comprised of 100 patientspresenting with the sign and symptoms ofpneumonia. The demographic characteristics ofthe subjects of the study are shown in table 1.54% of the cases were diagnosed withpulmonary tuberculosis and 46% had non-tubercular bacterial pneumonia. The patients inthe two subgroups were age and sex matched.Incidence of cough, sputum and fever werefound to be significantly more (p<0.001) inpatients with non-tubercular pneumonia, whileweight loss was found to be significantly more(p<0.001) in patients with pulmonarytuberculosis.Levels of CRP were found to be significantlymore (p<0.001) in the patients of non-tubercular bacterial pneumonia when comparedto the patients of pulmonary tuberculosis.The best cut-off level for differentiating betweenpulmonary tuberculosis and non-tubercularbacterial pneumonia was found to be a CRPlevel of <5 mg/L with the highest Youden’s Indexof 0.53, and a sensitivity of detecting pulmonarytuberculosis of77% and specificity of 76% asshown in Table 2.CRP levels of <5 mg/L werefound to have a positive predictive value of79.25% (95% CI: 65.89%-89.14%) and anegative predictive value of 74.47% (95% CI:59.65%-86.04%) for diagnosis of pulmonarytuberculosis in the patients of the study group.Fig. 1 shows the Receiver OperatingCharacteristic curve for levels of CRP. Area underROC for levels of CRP <5 mg/L was found to be0.74, suggestive of the usefulness of these levelsin discriminating between pulmonarytuberculosis and non-tubercular bacterialpneumonia

DiscussionIndia has the highest burden of TB in the world,an estimated 2 million cases annually, andaccounting for approximately one fifth of theglobal incidence(6).It is estimated that about 40%of the Indian population is infected with TB

11C - reactive Protein in Tuberculosis 11

bacteria, the vast majority of whom have latentrather than active TB disease. It is also estimatedby the World Health Organization (WHO) that300,000 people die from TB each year inIndia(7).Pulmonary tuberculosis is a granulomatousinfectious disease of lung caused byMycobacterium tuberculosis. There are variedpresentations of pulmonary tuberculosis,pneumonia being the most common(8). Non-tubercular bacterial pneumonia is an acuteinflammation of lung parenchyma mostcommonly caused by Streptococcuspneumonae(9). It is the third leading cause ofdeath worldwide with a high rate ofhospitalization; complication and mortality(10).Almost 20% of the hospitalized patients needintensive care admissionand 10% of progress tonon-resolving pneumonia(8,11).A rapid diagnosis and appropriate antibiotictreatment are therefore, essential to reduce themorbidity and mortality of Pneumonia. In areaslike ours with a high burden of Tuberculosis, thedifferential diagnosis of pulmonary tuberculosisfrom non-tubercular pneumonia becomesdifficult owing to the similar clinicalpresentations. Sputum culture forMycobacterium tuberculosis is the gold standardfor diagnosis and sputum Z-N staining is mostcommonly used. The AFB smear test lacksspecificity(12). These sputum negative patientscan be differentiated from non-tubercularpneumonia by other modality of investigation(sputum culture for Mycobacterium, Bronchiallavage or Histopathological examination) whichare time taking, costly and require specialist.Therefore an adjunct diagnostic test that candifferentiate between pulmonary tuberculosisand non-tubercular bacterial pneumonia at anearly stage of the disease will be of great clinicalimportance in terms of isolating patients withpulmonary tuberculosis and administering

appropriate anti-tubercular medication orantibiotic treatment.CRP is an acute phase reactant, synthesized byhepatocytes under the influence of interleukin-1arising at sites of infection, inflammation andtrauma(3). The ability of serum CRP levels toidentify etiology of pneumonia and predictprognosis has been investigated(4,5). The utility oflevels of CRP as a marker of bacterial infection inthe lower respiratory tract has also been studiedin several populations(13). In a study conductedby Choi M et al, median CRP levels were foundto be lower in patients with tuberculosis ascompared to patients with non-tubercularpneumonia(14).In a similar study conducted byYoung Ae Kang et al, the role of levels of CRP indifferentiating between pulmonary tuberculosisand pneumonia was emphasized(15). The results of our study also suggest theusefulness of serum levels of CRP indistinguishing between pulmonary tuberculosisand non-tubercular bacterial pneumonia. Wefound significant low levels of CRP in patientswith tuberculosis as compared to those with non-tubercular bacterial pneumonia. We report thebest cut-off for distinguishing between the two asserum CRP levels of <5 mg/L suggestive ofpulmonary tuberculosis, with a high sensitivityand negative predictive value.Thus, in conclusion, we suggest that serum levelsof CRP might have an important role in thediagnostic algorithm of pneumonia as rapid,inexpensive and non-invasive test for theexclusion of pulmonary tuberculosis from non-tubercular bacterial pneumonia in areas withhigh prevalence of the disease.

AcknowledgementWe acknowledge the financial support extendedby The Department of Science &Technology,Chandigarh Administration, for carrying out thiswork.


13C - reactive Protein in Tuberculosis 13

Table 1 : Demographic profile of subjects of the study group

Types of Pneumonia Non-tubercular Pulmonary P valuebacterial pneumonia tuberculosis

Age 40.57±16.35 years 38.40±16.32 years >0.5

Sex Males 66.6% 60.3% >0.5

Females 33.4% 39.7% >0.5

Incidence Sputum 93% 76% <0.001

of symptoms Cough 95.6% 71.7% <0.001

Fever 91.10% 65.2% <0.001

Weight loss 27.10% 53.50% <0.001

Median Levels 23.90 4.25 <0.001of CRP (mg/L)

Table 2 : Number of patients according to level of CRP, using a range of cutoffs,sensitivities and specificities

CRP No. of patientslevels Non- Sensitivity Specificity Youden’s 1-specificity

Tubercular tubercular IndexPneumonia Pneumonia

< 2 mg/L 18 7 0.33 0.84 0.17 0.16

<5 mg/L 42 11 0.77 0.76 0.53 0.24

<10 mg/L 48 19 0.88 0.58 0.46 0.42

<15 mg/L 52 31 0.96 0.32 0.28 0.68

Reference

1. Westgard JO, Barry PL. Beyond quality assurance:committing to quality improvement. Lab Med1989;20:241–7.

2. Nevalainen D, Berte L, Kraft C, Leigh E, Picaso L,Morgan T. Evaluating laboratory performance onquality indicators with six sigma scale. Arch Pathol LabMed 2000;124(4):516–9.

3. Coskun A. Six sigma and calculated laboratory tests.Clin Chem 2006;52:770–1.

4. Roger G. Schroeder a,*, Kevin Linderman a,1,Charles Liedtke b,2, Adrian S. Choo Six Sigma:Definition and underlying theory Journal ofOperations Management 2008; 26:536–554.

5. Westgard JO: 2006, Quality control. How labs canapply six sigma principles to quality control planning.Clin Lab News 32:10–12.

6. Revere L, Black K. Integrating six sigma with totalquality management: a case example for measuringmedication errors. J Healthc Manag 2003;48:377–91.

7. Tetrault G. Evaluating laboratory performance withthe six sigma scale. Arch Pathol Lab Med2000;124(12):1748–9.

8. Westgard JO, Westgard SA. The quality of laboratorytesting today an assessment of r metrics for analyticquality using performance data from proficiencytesting surveys and the CLIA Criteria for Acceptable

Performance. J Clin Pathol 2006;125: 343–54.

9. Westgard JO. Quality control. How labs can apply sixsigma principles to quality control planning. Clin LabNews 2006;32:10–2.

10. Willard MD, Tvedten H: 2004, Small animal clinicaldiagnosis by laboratory methods, 4th ed. WBSaunders, St. Louis, MO.

11. Westgard JO. Internal quality control: planning andimplementation strategies. Ann Clin Biochem2003;40:593–611.

12. Westgard JO, Groth T, Aronsson T, Falk H, de VerdierCH. Performance characteristics of rules for internalquality control: probabilities for false rejection anderror detection. Clin Chem. 1977;23:1857–67.

13. Vander Meer V, Neven AK, van der Brock PJ,Assadelft WJ. Diagnostic value of C-reactive proteinin infection of the lower respiratory tract. Systematicreview. BMJ 2005; 331:26.

14. Choi CM, Kang CI, Jeung WK, Kim DH, Lee CH, YimJJ. Role of C-reactive protein for the diagnosis of TBamong military personel in South Korea. TheInternational Journal of Tuberculosis and LungDisease. 2007;11(2):233-236.

15. Kang YA, Kwon SY, Yoon HIL, Lee CT. role of CRPand Procalcitonin in differentiation of Tuberculosisfrom bacterial community acquired pneumonia.Korean J Intern Med 2009;24(4):337-342.


Fig. 1 : Receiver Operating Characteristic curve for levels of CRP in patients of the study group

IntroductionVitamin-D is predominantly known for itsimportance in calcium metabolism andhomeostasis. Emphasis has now been shiftingfrom the involvement of vitamin-D in bonehealth to its non-skeletal effects. The expressionof Vitamin D receptors (VDR) is noted in mosttissues and cells of the human body includingliver, pancreas, and several immune cellsincluding monocytes, macrophages, Tlymphocytes, B lymphocytes, natural killer (NK)cells, and dendritic cells (DC), with expressionmost abundant on the epithelial cells of thegastrointestinal tract. The pleotropic functions ofvitamin D including anti inflammatory, antiapoptotic, anti fibrotic roles has been gaining

influence in recent years(1). The production ofvitamin-D on exposure to sunlight depends onincident angle of the sun and latitude, season,time of sun exposure and duration, skinpigmentation, aging and sunscreen usage.Recent studies have shown increased incidenceof vitamin-D deficiency throughout the worldand its association with various co-morbidconditions(2,3).Contradictory to the belief that vitamin-Ddeficiency must be a rare occurrence in tropicalcountry like India receiving sunlight throughoutthe year, studies have shown the prevalence ofvitamin-D deficiency in our country upto anextent of 90%(4).However, data to reflect on the magnitude of

15

HYPOVITAMINOSIS D AND ASSOCIATED CO-MORBIDITIES IN A TERTIARY CARE HOSPITAL SETTING

B. Gayathri, R Sujatha, Ghalib NehaDepartment of Biochemistry

PSG Institute of Medical Sciences and Research, Coimbatore, Tamilnadu-641004

AbstractVitamin-D is conventionally known for its importance in calcium metabolism and homeostasis.Emphasis has now been shifting from the involvement of vitamin-D in bone health to its extra-skeletalconcern. Data to reflect on the magnitude of vitamin-D deficiency and to create an insight into theprevalence of vitamin-D deficiency in hospitalised patients and its association with various co-morbidconditions is very scarce. So this study is carried out with the objective of assessing vitamin-D levelsin a hospital patient population and its association with co-morbid illnesses. Estimated Vitamin-Dlevels were obtained from the medical records who were admitted to a tertiary care hospital during theperiod of August 2012-March 2014. Details of the patients including age, gender, diagnosis andassociated co-morbidities were taken from the medical records. The study included 450 in-patients outof which 231 were males and 219 females. Our study showed that the prevalence of vitamin-Ddeficiency was 83% which was nearly equal in males and females. Further analysis showed that themost common morbidities associated with vitamin-D deficiency was type 2 diabetes mellitus andsystemic hypertension. Nearly 34.6% of vitamin-D deficient subjects had chronic liver disease andneurological disturbances each. The other common co-morbidities associated were cancer, thyroiddisorders, tuberculosis and autoimmune diseases.Epidemic of vitamin-D deficiency in India is likely to significantly contribute to the enormous burdenon the healthcare system. Adequate supplementation of vitamin-D to the population could have amajor impact in the prevention of vitamin-D deficiency and thereby reducing the trouble caused bythe most common co-morbidities associated with its deficiency.

vitamin-D deficiency and to create an insight intothe prevalence of vitamin-D deficiency inhospitalised patients and its association withvarious co-morbid conditions in our country isvery scarce. So this study is carried out with theobjective of assessing vitamin-D levels in atertiary care hospital and associated co-morbidconditions.

Materials and MethodsEstimated Vitamin-D levels were obtained fromthe medical records who were admitted to atertiary care hospital during the period of August2012-March 2014, after the clearance fromInstitutional Human Ethics Committee. Detailsof the patients including age, gender, diagnosisand associated co-morbidities were taken fromthe medical records. Serum 25-hydroxy vitamin-D was measured in Roche e411 auto-analyzerusing dedicated kits by Electro-chemiluminescence method. Normal serumreference interval was 30-70ng/mL. Vitamin-Dlevels of 20-<30ng/mL was classified as vitamin-D insufficiency and levels <20ng/mL wereclassified as vitamin-D deficiency(VDD). VDDwas further classified based on Lips classificationas mild (10-<20ng/mL), moderate(5<10ng/mL) and severe (<5ng/mL)(5,6).

ResultsA Vitamin-D levels of 450 patients attending to atertiary care hospital were included and analysedin the study after obtaining clearance fromInstitutional Human Ethics Committee. Meanage of the study population was 51.50±17.48years. The study group included 231 males and219 females. The age-wise distribution of thestudy population is depicted in Fig 1. Theprevalence of vitamin-D deficiency in males andfemales are illustrated in the graph in Fig 2. Outof the 450, 389 (86.4%) had low vitamin-Dlevels.

DiscussionIn the present study, it was revealed that out of

450 in-patients in whom vitamin-D levels wereestimated, 389 patients (86.4%) had lowvitamin-D levels (<30ng/mL). Further theprevalence of low vitamin-D levels were nearlyequal in both males (84%) and females (89%).The most common age group in which vitamin-D levels were insufficient was 41-60 years whichis consistent with previous studies whichreported older adults are at increased risk ofdeveloping low vitamin-D levels (<30ng/mL).This is partly because as age advances, skincannot synthesize vitamin-D efficiently. Also theyare likely to spend more time indoors and haveinadequate intake of the vitamin(7). The most common co-morbid conditionassociated with vitamin-D deficiency in our studywas type 2 diabetes mellitus. In our study, it wasfound that 47% of patients with severe vitamin-D deficiency had diabetes mellitus. Lee et al.found that 89% of their study population ofdiabetic patients suffered from vitamin-Ddeficiency and just 9 out of 300 persons hadsufficient vitamin-D concentration(8). This mightbe due to the direct role of vitamin-D in theactivation of pancreatic beta-cell or indirect roleby regulation of calcium homeostasis. The next common illness associated withvitamin-D deficiency was found to be arterialhypertension. In our study about 30% of thepatients with decreased vitamin-D levels hadassociated systemic hypertension. This supportsthe antihypertensive and vascular protectiveeffects of vitamin-D, such as suppression of therenin-angiotensin-aldosterone system and anti-atherosclerotic properties includingimprovements of endothelial function(9).The co-morbid condition associated withvitamin-D deficiency in about 34.6% of subjectswith low vitamin-D levels had chronic liverdisease. Impaired conversion of vitamin-D to its25-hydroxylated form in the liver is the majormechanism for the resulting vitamin-Dinsufficiency in these patients. Photo conversionin skin is normal in these patients(10). Our findingswere consistent with the findings of Fisher et al


which reported the prevalence of vitamin-Ddeficiency high in liver disease patients(86.3%)(11).The other common morbidities associated withvitamin-D inadequacy in decreasing order offrequency were neurological disturbances,thyroid dysfunction, malignancy, respiratorydiseases and autoimmune diseases. Nearly anequal number of patients had neurologicaldisturbances as liver disease. Vitamin-D has beenfound to function as a modulator in braindevelopment and neuro-protectant. It alsoexhibits an association with the regulation ofnerve growth factor synthesis which isresponsible for the growth and survival ofneurons(12).About 21.3% of vitamin-D deficient individualshad thyroid dysfunction. Extensive studies in theassociation of vitamin-D inadequacy and thyroiddysfunction are scarce. Amal Mackawy and hisco-workers(13) studied the effect of vitamin-Ddeficiency on thyroid gland in experimentalstudy and reported that a lack of vitamin-Dcontributed to the possibility of low thyroidhormones. In our study, 13 out of 291 vitamin -D deficientindividuals had tuberculosis. This was consistentwith recent meta-analysis which showed thatVitamin-D deficiency was associated with higherrisk of active tuberculosis(14). Vitamin-D wasestablished to have a protective role in thetreatment of tuberculosis about 6 decades

ago(15), but the idea was unobserved.Vitamin-D deficiency has been found to developas a pandemic in recent years. It has also beenassociated with several co-morbid conditionswhich contribute the huge economic burden onour country. Vitamin-D estimation should beincluded in routine laboratory analysis andvitamin-D supplements provided to reduce therisks associated with vitamin-D deficiency and itsassociated co-morbidities.

ConclusionVitamin-D deficiency is likely to play animportant role in the very high prevalence ofrickets, osteoporosis, cardiovascular diseases,diabetes, cancer and infections such astuberculosis in India. Owing to its multifariousimplications on health, the epidemic of vitamin-D deficiency in India is likely to significantlycontribute to the enormous burden on thehealthcare system of India. Most of the widelyconsumed food items such as dairy products arerarely fortified with vitamin-D. Fortification ofstaple foods with vitamin-D is the most viablepopulation based strategy to achieve vitamin-Dsufficiency. Vitamin-D supplements are availableat affordable prices, so that propersupplementation of the vitamin to the populationcould have a major impact in the prevention ofvitamin-D deficiency and thereby reducing thetrouble caused by the most common co-morbidities associated with its deficiency.

17Hypovitaminosis D and Associated Co-morbidities 17

Reference

1. Holick MF. Sunlight and vitamin-D for bone healthand prevention of autoimmune diseases, cancers, andcardiovascular disease. Am J Clin Nutr2004;80(6,suppl): 1678S-1688S.

2. Hodgkin P, Kay GH, Hine PM, et al. Vitamin-Ddeficiency in Asians at home and in Britain. Lancet1973;167-171.

3. Harinarayan CV, Joshi SR. Vitamin-D status in India-Its implications and Remedial Measures. J AssocPhysicians India 2009;57:40-48.

4. Goswami R, Gupta N, Goswami D, Marwaha RK,Tandon N, Kochupillai N. Prevalence and significanceof low 25-hydroxyvitamin-D concentrations in healthysubjects in Delhi.Am J Clin Nutr. 2000; 72 : 472-5.

5. Holick, M F Vitamin-D deficiency. N Engl J Med 2007,357 : 266–281.

6. Lips P. Vitamin-D deficiency and secondaryhyperparathyroidism in the elderly:consequences forbone loss and fractures and therapeutic implications.Endocr Rev 2001;22:477-501.

7. Institute of Medicine, Food and Nutrition Board.Dietary Reference Intakes for Calcium and Vitamin-D.Washington, DC: National Academy Press, 2010

8. Lee JI, Oh SJ, Ha WC, et al. Serum 25-hydroxyvitamin-D concentration and arterial stiffness

among type 2 diabetes. Diabetes Res Clin Pract 2012;95: 42-7.

9. Li YC. Molecular mechanism of vitamin-D in thecardiovascular system. J Investig Med 2011; 59 : 868-71.

10. Jung RT, Davie M, Siklos P, et al. Vitamin-Dmetabolism in acute and chronic cholestasis. Gut1979;20(10):840–7.

11. Fisher L, Fisher A. Vitamin-D and parathyroidhormone in outpatients with noncholestatic chronicliver disease. Clin Gastroenterol Hepatol2007;5(4):513–20.

12. Garcion, E., Wion-barbot, N., Montero-menei, C. N.,Berger, F., & Wion, D. (2002). New clues aboutvitamin D functions in the nervous system. TrendsEndocrinol Metab 2002 13(3):100-105.

13. Amal Mavkawy, Bushra Al-ayed, Bashayer Al-rashidi.Vitamin D deficiency and its association with thyroiddisease. Int J Health Sci 2013;7(3):267-275.

14. Liu PT, Modlin RL. Human macrophage host defenseagainst Mycobacterium tuberculosis. Curr OpinImmunol 2008; 20:371–6.

15. Adams JS, Chen H, Chun R et al. Substrate andenzyme trafficking as a means of regulating 1,25-dihydroxyvitamin-D synthesis and action: the humaninnate immune response. J Bone Miner Res 2007; 22(Suppl. 2):V20–4.

19Hypovitaminosis D and Associated Co-morbidities 19

Table 1: Vitamin D deficiency associated co-morbidities in percentage

Disease Severe Moderate Mild deficiency Vitamin-D associated deficiency deficiency insufficiency

Males Females Males Females Males Females Males FemalesTotal Total Total Total Total Total Total Total35 37 46 49 77 85 36 24

Liver diseases 37 8 30 4 26 7 25 17Diabetes mellitus 49 41 33 10 23 21 44 25Hypertension 23 41 33 10 26 21 36 46Cancer 9 5 7 2 1 4 11 0Respiratory diseases 17 5 7 0 0 0 8 0Autoimmunediseases 11 5 0 4 0 1 0 17Neurology 14 27 13 31 21 21 42 17Thyroid 0 22 4 8 1 11 8 29

IntroductionNon- alcoholic fatty liver disease (NAFLD) is themost common cause of liver dysfunctionworldwide. NAFLD may progress to non-alcoholic steatohepatitis (NASH) and in turnCirrhosis(1). All stages are associated withaccumulation of fat in the liver cells(2). NAFLDwas initially believed to be a benign condition.Recent studies have shown it to be associatedwith obesity, type II diabetes mellitus,dyslipidemia and hypertension with insulinresistance being a common factor. Theseconditions cluster to form the metabolicsyndrome, which causes high risk forcardiovascular disease(3).The diagnosis of NAFLD requires a combination

of invasive and non-invasive tests. Mild tomoderately elevated serum levels of aspartateamino transferase (AST) and alanineaminotransferase (ALT) or both are the mostcommon findings(4). However some studiessuggested that use of liver enzymes as a markerof NAFLD underestimate its prevalence(5).Ultrasound has a sensitivity of 89% and aspecificity of 93% in detecting steatosis,sensitivity and specificity of 77% and 89% indetecting increased fibrosis(6). This study was conducted with the aim ofdetermining the prevalence of NAFLD in theapparently healthy population and establishinga relationship between NAFLD and metabolicsyndrome.

20

CLINICOPATHOLOGICAL PROFILE OF NONALCOHOLIC FATTYLIVER DISEASE (NAFLD) AND ITS ASSOCIATION WITH

METABOLIC SYNDROME

M. M. Chatterjee, Quazi. S. Haque, F. Jamal1

Department of Biochemistry, Hind Institute of Medical Sciences, Safedabad Barabanki U.P (India) and

1Dr. Ram Manohar Lohia University, Faizabad, U. P. (India)

AbstractAll patients attending the health checkup had their blood pressure, height and weight, waistcircumference measurements, blood sugars, lipid levels and ultrasound abdomen done. Theprevalence of NAFLD among these subjects was determined and the presence of risk factors formetabolic syndrome in each individual was analyzed. A relationship between NAFLD and metabolicsyndrome was then established. Result of the 1003 people 225 (22.6%) had NAFLD with higherprevalence among males 164/565 (29%) than among females 61/438 (13.9%). In the NAFLD groupnormal body mass index (BMI) was present in only 49/225 (20%) of the subjects while 119/225(52.8%) were overweight and 56/225 (24.8%) were obese. Though liver enzymes were normal themean AST among cases was 37.41 + 14.50 and 33.93 + 14.15 among controls and the mean ALTwas 38.74 + 17.96 among cases and 31.62 + 13.49 among controls. Prevalence of metabolicsyndrome was 106/225 (47%) among cases and 179/778 (23%) among controls. A diagnosis of fattyliver on ultrasound in an asymptomatic person should be taken as an indicator of metabolic syndromeand its progression to cardiovascular disease.

Materials and MethodsAll the patients of age group 20-60 yearsattending OPD for routine health checkupformed the control group. They were subjectedto their height, weight, waist circumferencemeasurements, blood pressure, BMI, Liverfunction test, lipid profile, fasting blood glucose(FBS), post prandial blood sugar (PPBS) andultrasound examination of abdomen. NAFLDwas defined as fatty liver not resulting fromexcessive alcohol consumption on(>20grams/day) drugs, toxins infection diseasesor any other identifiable exogenous cases(7).This was an ultrasound based study. USGfindings revealed that fatty liver was diagnosedas diffuse increase in parenchymal ecogenicitywith progressive loss of clarity of portal veins andincreased attenuation of sound by the liver(8).Those with USG evidence of fatty liver alongwith levels of AST> 55u/dl and ALT> 70 u/dlhad their blood further investigated for thyroidstimulating hormones, hepatitis B virus, hepatitisC virus, serum ceruloplasmin, alpha-1 antitrypsislevels and serum transferring levels to rule outother causes of liver disease. The diagnosis ofNAFLD was made only after excluding them.Normal weight has been defined as BMI from20-25, over weight from 25-30 and obesity from30-35.Metabolic syndrome was identified by findingany three of the five risk factor(9).Those with normal ultrasound formed thecontrol group.All the observation and data were analyzed inthe statistical package social sciences (SPSS).The level of significance was set as P<0.05.

ResultsThe health checkup was attended by 1026people of which 23 had to be excluded due tosignificant alcohol consumption. Hence ourstudy group comprises of 1003 people whichhad 438 females and 565 males.Of the 1003 people 225 people had USGevidence of fatty liver. Thus the prevalence of

NAFLD was 225/1003 (22.6%). It was presentamong 61/438 (13.9) females and 164/565(29%) males. Of the 225 people with NAFLDonly 1 person (0.4%) was underweight with aBMI less than 20.Normal BMI was observed in normal and overweight respectively 49(21.7%) 119(52.8%),while 56(24.8%) of them had frank obesity.Among those with sonographic evidence of fattyliver, only 12 subjects had elevatedtransaminases yet one had values correspond tothe definition of NASH which meant elevatedliver enzymes by 1.5 to 5 times. Although most of the subjects in present study(Table 1) had liver enzymes with in the normalrange yet the mean values were higher with ASTbeing 37.41+14.50 in cases and 33.93+17.96 and 31.62+13.49 among cases andcontrols respectively. Hypertriglyceridemia waspresent in the NAFLD group with mean of170.02+88.90 mg% and among controls it was132.56+69.77 mg%. Levels of low densitylipoproteins (LDL) were 109.18+29.72 mg%and 107.08+32.18 mg% respectively for casesand controls.The total cholesterol levels were 187.92+36.72 mg% for cases and controls. The totalcholesterol levels were 187.92+36.32 mg% forcases and 181.81+37.33 mg% for controls. Thelow HDL level was low for cases with meanbeing 46.61+9.48 mg% and 49.60+12.74 mg% for controls.The FBS was elevated with cases having124.62+45.83 mg% and controls with 109.89+37.80 mg%.Post prandial sugars were higher in cases156.93+78.33 mg% and among controls it was129.59+63.70 mg%. The BMI was higher inNAFLD group, mean of 28.58+4.25 and amongcontrols it was 25.67+5.05.Metabolic syndrome (Table 2) was present in106/225(47%) of cases and 179/778(23%) ofcontrols. Impaired blood glucose levels wereseen in 163/225(72.4%) of cases and 438/778(56.3%) of controls. Hypertension was prevalent

21Clinicopathological Profile of Nonalcoholic Fatty Liver Disease


in 64/225(28.4%) of NAFLD group as against147/778(18.9%) of controls. Elevated triglycerides were present in98/225(43.6%) of cases and 213/778(27.4%) ofcontrols and low HDL levels were seen in66/225(29.3%) of cases and 247/778(31.7%) ofcontrols. Abdominal obesity was present in106/225(47.1%) of cases and 303/778(38.9%)of controls. Other than HDL, all had significantP value at P<0.05.Not even a single risk factor of metabolicsyndrome (Table 3) was present in 10/225(0.4%) of cases and 124/778(15.9%) of controls,while one risk factor was prevalent in 37/225(16.4%) of cases and 258/778(33.2%) ofcontrols. The presence of two risk factors weresimilar in both groups at 63/225(28%) amongcases and 206/778(26.5%) among controls.Three risk factor were present in 66/225(29.3%)of NAFLD group and 129/778(16.6%) ofcontrols.Four risk factors were noted in 37/225(16.4%)and 46/778(5.9%) of cases and controlsrespectively. All the component of metabolicsyndrome were present in 9/225(4%) of casesand 6/778(0.8%) of controls.

DiscussionNAFLD has been reported in 10% to 24% of thegeneral population in various countries(10). AnUSG based study conducted in India has showna prevalence of 24.5%(11). The present studyshowed a prevalence of 22.6% with a higherprevalence among male than among females.The results of the present study were almostsimilar to that study.Marchesani et al. showed that 80% of patientswith NAFLD were Obese(12). Our study had only21% of the population with normal BMI whilethe remaining 79% were either overweight orobese. Although majority of the cases hadnormal liver enzymes the mean values of theenzymes were higher in the NAFLD group ascompared to the controls.

Goland et al. observed that patients with NAFLDhad a significantly higher BMI of 31.14 vs 26.4,higher blood glucose level of 100.6 vs 83.3 andtriglyceride values of 200 vs 126 respectively(13).Lee et al. showed higher anthropometrics valueamong controls. The ALT and AST levels werehigher with an increase in total cholesterol,triglycerides, atherogenic index, FBS, systolicand diastolic blood pressure and a decrease inHDL cholesterol in the USG diagnosed patientsas against controls. There was no significantdifference in the LDL cholesterol values amongboth the groups(14).There is a strong association between NAFLDand metabolic syndrome with its prevalencebeing double in the NAFLD group as comparedto the controls. The liver histology is closelyassociated with the number of risk factors(15).Our study showed the increased presence ofnumber of risk factors of metabolic syndrome inthose with ultrasonic evidence of fatty liver ascompared to the controls NAFLD can rightly becalled the hepatic component of metabolicsyndrome. This was a fact finding study.Currently, this disease is a public health problemworld wide, due to the high prevalence, highlevel of disability associated and high cost for thehealth system(16,17).

ConclusionWe thus concluded that evidence of fatty livershould be taken seriously as a predictor ofmetabolic syndrome. It is atherogenic andpredisposes to diabetes, hypertension,dyslipidimia and has a strong potential forcoronary vascular disease. Prevention is betterthan cure. Hence a diagnosis of NAFLD onultrasound in an asymptomatic patient shouldalert as of the preventable metabolic syndromeand its progression to coronary vascular diseasemaking it necessary to take the same precautionswe would take for any other predictors ofmetabolic syndrome.

Clinicopathological Profile of Nonalcoholic Fatty Liver Disease 23

Table 1 : Comparison of lipid profile, glucose level and anthropometric values betweenthe two groups

Mean + SDParameters NAFLD Group Control Group P Value

(N=225) (N=225)(N=778) <0.05

HDL (mg%) 46.61 + 9.48 49.60 + 12.74 -

LDL (mg%) 109.18 + 29.72 107.08 + 32.18 <0.05

FBS (mg%) 124.62 + 45.83 109.89 +37.80 <0.05

PPBS (mg%) 156.93 + 78.83 126.59 + 63.70 -

Total Cholesterol (mg%) 187.92 + 36.32 181.81 + 37.33 <0.05

TGL (mg%) 170.02 + 88.90 132.56 + 69.77 <0.05

SGOT (μ/dl) 37.41 + 14.50 33.93 + 14.15 <0.05

SGPT (μ/dl) 38.74 + 17.96 31.62 + 13.49 <0.05

BMI (cm) 28.58 + 4.25 25.67 + 5.05 <0.05

Waist circumference (cm) 99.96 + 10.01 93.22 + 11.04 <0.05

Table 2 : Prevalence of different parameters between two study groups

NAFLD Control P ValueTotal No. of Patients enrolled (N=225) (N=778)

Prevalence of metabolic syndrome 106(47.1%) 179(23.0%) <0.05

Prevalence of impaired blood glucose 163(72.4%) 438(56.3%) <0.05(FBS>100mg %)

Prevalence of hypertension (B.P.>130/85) 64(28.4%) 147(18.9%) <0.05

Prevalence of elevated triglycerides 98(43.6%) 213(27.4%) <0.05(>150mg/dl)

Prevalence of low HDL (<40mg% in males 66(29.3%) 247(31.7%)& <50 mg% in females)

Prevalence of increased waist circumference 106(47.1%) 303(38.9%) <0.05(>88cm. in females & >102cm. in males)

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15. Dietel M, Arps H, Klapdor R, Muller- Hagen S, Sieck


Table 3 : Distribution of risk factors among study groups

NAFLD Group Control GroupRisk Factor (N=225) (N=778) P Value

No. % No. %

No Risk Factor 10 0.04% 124 15.9% <0.05

1 Risk Factor 37 16.4% 258 33.2% <0.05

2 Risk Factor 63 28.0% 206 26.5% -

3 Risk Factor 66 29.3% 129 16.6% <0.05

4 Risk Factor 37 16.4% 46 05.9% <0.05

5 Risk Factor 09 04.0% 06 00.8% <0.05

M and Hoffman L. Antigen detection by monoclonalantibodies CA125 in normal and tumor tissue andpatients sera. J of cancer research and clinicaloncology 1996;111:257-265.

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20. Gargano G et al. The role of tumor markers in ovariancancer. Clan Exp Obsetet Gynecol 1990;17:23-29.

21. Ni G, Bai XF, Mao YL, Shao YF, Wu JX, Shan Y et al.The clinical value of serum CEA, CA19-9 and CA 242in diagnosis and prognosis of pancreatic cancer. Eur JSurg Oncol 2004;31:164-169.

22. Abad A, Cazorla E, Ruiz F, Aznar I, Asins E, LlixionaJ. Meig’s syndrome with elevated CA125:case reportand review of the literature. Europian J of Obstetricsand Gynecology and Reproductive Biology 1999;82:97-99.

23. Margel D, Tal R, Neuman A, Konichezky M, Sella A,Baniel J. Serum tumor markers predict extravesicaldisease in clinical stage T2 bladder cancer, J Urol2006;175:1253-57.

24. Oliva E, Amin MB, Jlemez R, Young RH. Clear cellcarcinoma of urinary bladder:A report andcomparison of four tumors of mullerian origin nineprobable urothelial origin with discussion ofhistogenesis and diagnostic problems. The AmericanJ of Surgical Pathology 2002;26:190-97.

25. Steinberg WM, Gelfand R, Anderson KK, et al.Comparison of the sensitivity and specificity of the CA19-9 and carcinoembryonic antigen assays indetecting cancer of the pancreas. Gastroenterology.1986;90:343-9

26. Saraswati A, Malati T. Superiority of CA 125 over CA19-9 and CEA for epithelial ovarian malignanciesIndian J Clin Biochem. 1995;10:23-8.

27. Pall M, Iqbal J, Singh SK, Rana SV. CA19-9 as aserum marker in urothelial carcinoma. Urol Ann.2012;4(2):98-101.

28. Vestergaard EM, Wolf H, and Orntoff TF. Increasedconcentration of genotype interpreted CA19-9 in urineof bladder cancer patients mark diffuse atypia of theurothelium. Clinical Chemistry 1998;44(2):197-204.

29. Cook AM, Huddart RA, NormanA, Dearnaley DP,Horwich A. The utility of tumor markers in assessingthe response to chemotherapy in advance bladdercancer. British J Cancer 2000;82(12):1952-57.

30. Kau SY, Shyr YM, Su CH, Wu CW, Lui WY.Diagnostic and prognostic values of CA19-9 and CEAin periampullary cancers. J Am Coll Surg1999;188:415-20.

31. Bender DP, Sorosky JI, Buller RE, Sood AK. SerumCA125 is an independent prognostic factor in cervicaladenocarcinoma Am J Obstet Gynecol2003;189(1):113-17.

32. Naik DS, Sharma S, Ray A, Hedau. Epidermal growthfactor receptor expression in urinary bladder cancer.Indian J Urol 2011;27:208-14.

33. Parag G et al . Impact of age and gender on theclinicopathological characteristics of bladder cancer.Indian J Urol 2009;25(2) :207-210.

34. Ploeg M, Aben KK, Kiemeney LA. The present andfuture burden of urinary bladder cancer in the world.World J Urol 2009;27:289-93.

35. Parkin DM. The global burden of urinary bladder.Scand J Urol Nephrol Suppl 2008;218:12-20.

Clinicopathological Profile of Nonalcoholic Fatty Liver Disease 25

Introduction Polycystic Ovarian Syndrome (PCOS) issometimes referred to as Sclerocystic OvarianDisease, Stein-Leventhal Syndrome andPolycystic Ovarian Disease (PCOD). PCOS is acomplex, heterogeneous, polygenic endocrinedisorder in women of reproductive age and isconsidered as a multifactorial reproductive,cosmetic and metabolic problem. The etiologyof PCOS is not well understood and itspathophysiological and molecular basis is still apuzzle. PCOS is likely to be the result of anumber of both genetic and environmentalfactors. Some of the contributing factors toPCOS also include a low level of chronicinflammation in the body and fetal exposure tomale hormones. However, androgen excess andinsulin resistance leading to hyper insulinemia

are considered to be the basic defects in PCOSthat was described way back in 1921 by Archard& Theirs as "diabetes of bearded women"(1).The world wide prevalence of PCOS syndromeis 6-10% and in its "classical" form may affect 5-7% of women(2). PCOS is quite common inAsian population. A high prevalence of PCOSup to 35% is reported for the Indian womenwhere the incidence and prevalence of PCOS inoverweight and obese women is greater than20%(3). Women with PCOS are at a higher riskfor a number of illnesses, including high bloodpressure, diabetes, heart disease and othercardiovascular problems and cancer of theuterus, ovary and breast(4).PCOS also presents with a variety of biochemicalabnormalities(5). The most consistentabnormality is hypersecretion of androgens.

26

GENETIC POLYMORPHISM IN CYP17A GENE IN PATIENTS WITH POLYCYSTIC OVARIAN SYNDROME (PCOS) IN

A SOUTH-INDIAN POPULATION

Arindam Basu1, Arghya Sur2, Hemontika Chakraborty1, Prabha Adhikari3

1Department of Biochemistry. Kerala Medical College and Hospital, Mangode, Cherpulassery, Palakkad Dist, Kerala-679503, India

2Department of Physiology, Kerala Medical College and Hospital,Mangode, Cherpulassery, Palakkad Dist, Kerala-679503, India

3Department of Medicine, Kasturba Medical College Hospital, Attavar,Mangalore, Karnataka- 575001, India

AbstractOur study was likely to explore the association with single nucleotide polymorphism in cyp 17a genein patient with PCOS. The study was conducted in Kasturba Medical College Hospital , Mangalore &Kerala Medical College & Hospital, Mangode, Cherpulassery, Palakkad Dist, Kerala. A total of 103female patients with a clinical diagnosis of PCOS attending KMC, hospital, Mangalore, Kerala MedicalCollege & Hospital, Mangode, Cherpulassery, Palakkad Dist, Kerala,. medicine outpatient departmentwere included in the study after obtaining informed consent. Biochemical assays of hormones, lipidprofile, GTT as well as study of genotype and allelic frequencies were done in the hospital centralbiochemistry laboratory. In the radiology department, USG of ovary for diagnosis of PCOS was done.Other clinical diagnosis of PCOS were done in Medicine department. In our study cyp17a gene is notfound to be associated with PCOS in South Indian population.

Because of the high degree of heterogeneity ofPCOS, it is suggested best to consider PCOS asincreased androgens clinically (acne, excessivehair on face, abdomen, or thinning of scalp hair)or in the blood (total or free testosterone,DHEAS), with oligo-ovulation (cycles greater theevery 35 days, low mid-luteal progesterone,monophasic basal body temperature.Studies on etiology of PCOS has yielded somepositive results but the controversy on the modeof inheritance (eg. autosomal dominance,modified autosomal dominance, X-linked,multifactorial) still persists. Thus, there is a greatneed to identify the potential candidate genesthat may have a modest effect individually andin groups in PCOS. Three general geneticmodels have been proposed namely, Singlegene Mendelian model which predicts that thereis single gene defect inherited in a recessive ordominant pattern and that woman who inheritthis defect develop clinically evident PCOS.Multifactorial model where PCOS is consideredas a multifactorial genetic disorder and womencarrying this defect through inheritance orenvironmental factors will have increased risk ofclinical PCOS.Variable expression single genemodel where there is a combination of the abovetwo models. It follows that a single gene defect ispresent but its expression is modified byenvironmental factors. Therefore, a woman whois genetically predisposed but not exposed toenvironmental factors may develop onlysubclinical forms of PCOS and not the fulldisorder. This theory explains the heterogeneousnature of the disorder.Therefore, it can be said that what started initiallyas a gynaecological curiosity, over the years hasbecome a subject of multisystem endocrinopathyknown as PCOS. Considering certain lacunae inthe area of genetics of PCOS as pointed outearlier, we propose to study the family history ofPCOS subjects, not only for PCOS but also for itsrelated conditions. This would help us todetermine the pattern of inheritance of PCOSand related clinical presentations.

AimsTo identify the frequency of the geneticpolymorphisms in cyp17a gene in patients withPCOS.

Materials and MethodsStudy setting, design, sampling and periodof the studyOut-patients and in-patients of Kasturba MedicalCollege Hospital, Attavar, Mangalore, as well asOut-patients and in-patients of Kerala MedicalCollege & Hospital, Mangode, Cherpulassery,Palakkad Dist Kerala diagnosed with polycysticovarian syndrome were included into the study.The study was a descriptive, cross sectional type.Convenient sampling of subjects was done forthe study. The project began in December, 2008and ended in December, 2011 at KasturbaMedical College Hospital, Attavar, Mangalore.Further it has been repeated at Kerala MedicalCollege & Hospital, Cherpulassery, Palakkad,Kerala, from December, 2013 to September,2014. Almost same results were obtained.

Ethical approval Institute's Research ethical committee approvalwas obtained for the study. After obtaininginformed consent from each participant,hundred and three (103) patients with a clinicaldiagnosis of PCOS (where large family trees wasknown) were included in the study. All patientsreceived a long, careful and simple explanationof the purposes of the study and itspathophysiological basis.Criteria for the definition of PCOS :The diagnosis of PCOS was made according tothe ESHRE/ASRM criteria for the PCOSdiagnosis (Rotterdam ESHRE/ASRM-SponsoredPCOS Consensus Workshop Group, 2004)(6)

based on the presence of two of the threefollowing criteria: oligo- and/or anovulation(menstrual dysfunction), clinical and/orbiochemical signs of hyperandrogenism andpolycystic ovaries (PCO) at ultrasonogram(7).Menstrual dysfunction was considered when the

Genetic Polymorphism in CYP17A gene in patients 27

women had oligomenorrhea, defined by six orfewer cycles per year, each cycle with a length ofmore than 35 days, and/or when the patient hadnot had any menstrual bleeding for 3consecutive months during the last year. Clinicalhyperandrogenism was defined by the presenceof hirsutism, represented by a hirsutism score of8 or more. Hyperandrogenism could be clinical(hirsutism, alopecia and/or acne) or subclinical,with only an increase in serum testosteroneand/or dehydroepiandrosterone sulfate(8).Polycystic ovaries were diagnosed by pelvicsonography according to the Rotterdamconference criteria

Patients and Participants103 female patients with a clinical diagnosis ofPCOS and their 291 first and second-degreerelatives were included in the study afterobtaining informed consent.

Inclusion criteria for patients1. Post pubertal females aged up to 35 years.2. Irregular periods (Must have six or fewer

menses /year)3. Have clinical or laboratory evidence of

hyperandrogenism (hirsutism or elevatedtestosterone) and PCO on ultrasound, morethan 9 follicles; [Rotterdam criteria].

4. Who have signed informed consent

Exclusion criteria for patients1. Age > 35 years.2. Diabetes Mellitus > 5 years3. Confirmed malignancy.

Total sample size: (103+291) = 394103 female patients with a clinical diagnosis ofPCOS by Rotterdam criteria attended KMC,hospital, Attavar; Mangalore as well as KeralaMedical College & Hospital, Mangode,Cherpulassery, Palakkad Dist medicineoutpatient department or obstetrics andgynecology department and their first and

second-degree relatives; were included in thestudy after obtaining informed consent.All the subjects including patients and theirfamily members were interviewed in detail andexamined for anthropometry such as BMI,Hirsutism /excess hair, Acne, Baldism,Acanthosis nigricans, Skin tags, Buffalo humps,Moonface, Double chin.1. Biochemical assay such as; Serum fasting

insulin, Cortisol, Testosterone, Dehydroxye-piandostenedione, LH, FSH, TSH weredone in all cases, along with fasting lipidprofile.

2. Blood pressure was measured for all, an oral2 hr GTT was performed after 75 gm ofglucose for all patients.

Genetic analysisStudy of genotype and allelic frequencies weredone by means of PCR- RFLP. DNA wasextracted from heparinised or EDTA blood. Ethical clearance was obtained from ManipalUniversity institutional Ethical Committee as wellas KMCH, Mangode, Cherpulassery institutionalEthical Committee after which the studies wereperformed.

Collection of blood samplesThe blood samples were collected from thepatients with PCOS from coastal districts ofKarnataka state and Kerala state. Patient’shistory was collected and pedigree was drawn.Clinical documentation was undertaken with thehelp of a physician. DNA was extracted fromboth test and control samples following thestandard phenol-chloroform method. PCOSpatients were considered as test and normalindividuals of same family were used as control.Though we recruited 50 female individualdevoid of all phenotypical feature of PCOS andhaving family history of diabetes.

ResultsGenetic Polymorphism: When we screened 103PCOS patients in CYP17A gene for -34T>C.


The allele of gene CYP17A that, contain Cinstead of T is designated as A2 allele. Theunmutated allele with T was designated as A1allele of CYP17A gene. When we screened 103patients with PCOS and their 291 first, andsecond degree relatives with 50 controls for thepresence of polymorphic allele, No patients(0.0%) were heterozygous carrier ofpolymorphic A2 allele (genotype A1A2).Among50 patients 0(0%) carried the (A2) allele in theheterozygous state (A1A2). We found noassociation between - 34T>C polymorphismand PCOS patients (Table 1).

DiscussionFamilial clustering of PCOS has beenconsistently reported, suggesting that geneticfactors play a role in the development of thesyndrome. Sisters, brothers, fathers, mothers,daughters and now even sons of women withPCOS have been found to have a higher risk forexhibiting either hyperandrogenic or metabolic(hyperinsulinemic) traits of the disorder and thusPCOS has become a 'family affair'(10).In the present study we examined the prevalenceof a polymorphism of gene CYP17 promoterand found no association as A1A2=0.0 andA2A2=0.0 genotype frequencies.Echiburu et al (2008)(11) reported a frequency of37% in PCOS subjects with A2 allele in Chileanpopulation, when they examined 159 womenwith clinical and hormonal evidences of PCOSand they concluded the possibility of this genedefect in PCOS to be associated with increase inbody weight, abdominal adiposity and metaboliccomponents.Diamati-Kandarakis et al (2009)(12) describe thesame gene cyp17 polymorphism in Greekpopulation with 50 PCOS subjects and reported58% were heterozygous carriers of thepolymorphic allele and 8% carried A2 allele inhomozygosity, they concluded although this basepair substitution is not a primary genetic defect inPCOS , it may aggravate in clinical picture ofhyerandogenemia, particularly when

homozygosity exist.Gharani et al (1997)(13) also reported a nonsignificant association with cyp17 gene andPCOS, when they examined 96 PCOS womenin white population.In another study Marszalek et al (2001)(14)

illustrate a similar result in Poland population,while they genotyped 56 PCOS women andconcluded the T>C polymorphism of cyp17gene is not associated with steroid hormonesynthesis in PCOS and it is not a primary geneticdefect in this disease.The high incidence of PCOS in first degreerelatives of the affected members, in previousstudies(15,16), suggests a dominant pattern ofinheritance. This is based on the assumption thatat least 50% of the siblings of the PCOSprobands are affected with the disorder(15). Twinstudies on PCOS revealed an incidence of 50%has suggested a complex pattern of polygenicinheritance(17). Other studies have reported that50% hirsutism cases among the affected sistersof PCOS(18). They have shown that somecharacteristics of PCOS inherited differed inproportion; e.g. PCO 73%, hyperandrogenemia87% and hyperinsulinemia 66%. Another reporthas shown 22% of PCOS in affected sisters ofthe proband(16).However when we analyzed clinical conditionsassociated with PCOS, we found a very highassociation suggesting autosomal dominanttransmission. Break up data of the family historydata showed nearly 20% of the fathers of pcoprobands having diabetes mellitus, hypertensionobesity/dyslipidemia each. Also a similarpercentage of mothers of the pco probands hadthe above metabolic syndrome characters.Among the siblings of pco probands, nearly 10%of them had diabetes mellitus, hypertension,obesity/dyslipidemia. However among theuncles, aunts and grandparents of our pcoprobands, the percentage of diabetes mellitus,hypertension, obesity/dyslipidemia was less than15%. When any one of the metabolic syndromecharacter such as diabetes mellitus, hypertension



or dyslipidemia was considered, we foundprevalence of 50% and 54% among the firstdegree and second degree relatives of our PCOSsubjects respectively. Hence this can beconcluded that Cyp17a gene is not associatedwith PCOS in South Indian population.

Reference

1. Archard C, Theirs J. Le virilisme pilaire et son asson a1'insuffisance glycolitique. Bull Acad Natl Med (Paris)1921; 86:51-64.

2. Fauser B, Tarlatzis B, Chang J, et al. 2003ASRM/ESHRE consensus document. Fertil Steril2004; 18: 19-25.

3. Alvarez-Blasco F, Botella-Carretero JI, san Millan JL,Escobar-Morreale HF. Prevalence and characteristicsof the polycystic ovary syndrome in overweight andobese women. Arch Intrn Med 2006; 166: 2081-2086.

4. Daniilidis A, Dinas K. Long term health consequencesof polycystic ovarian syndrome: a review analysis.Hippokratia 2009; 13: 90-92.

5. Kashar-Miller M, Aziz R. Heritability and the risk ofdeveloping androgen excess. J Steroid Biochem MolBiol 1999; 69: 261-268.

6. Rotterdam ESHRE/ASRM-Sponsored PCOSConsensus Workshop Group. Revised 2003consensus on diagnostic criteria and long-term healthrisks related to polycystic ovary syndrome. HumReprod 2004; 19: 41-47.

7. Zawadzki JK, Dunaif A. Diagnostic criteria for

polycystic ovary syndrome: towards a rationalapproach. In: Dunaif A, Givens JR, Haseltine F,Merriam GR, eds. Polycystic ovary syndrome. Boston:Blackwell; 1992: 377-384.

8. Ferriman D, Gallwey JD. Clinical assessment of bodyhair growth in women. J Clin Endocrinol Metab.1961; 21:1440-1447.

9. Carmina E. Prevalence of idiopathic hirsutism. Euro Jof Endocrinol. 1998; 139: 421-423.

10. Xita N, Georgiou I, Tsatsoulis A. The genetic basis ofpolycystic ovary syndrome. Eur J Endocrinol 2002;147: 717-725.

11. Echiburu B, Perez-Bravo F, Maliqueo M, Sanchez F,Crisosto N, Sir-Petermann T., Polymorphism T --> C(-34 base pairs) of gene CYP17 promoter in womenwith polycystic ovary syndrome is associated withincreased body weight and insulin resistance: apreliminary study. Metabolism, 2008; 57: 1765-1771.

12. Diamanti-Kandarakis E, Bourguignon JP, Giudice LC,Hauser R, Prins GS, Soto AM, Zoeller RT, Gore AC.Endocrine-disrupting chemicals: an Endocrine Societyscientific statement. Endocr Rev. 2009; 30: 293-342.

13. Gharani N, Waterworth DM, Batty S, et al.Association of the steroid synthesis gene CYP11a with

Table 1: Genotype frequencies in CYP17a1 genes in patients with PCOS, relatives and controls

Genotype Genotype Genotype GenotypeGene Polymorphism frequency frequency frequency (X2) OR

PCOS Relatives Controls P value

CYP17A1 TT-.1 TT-.1 TT-.1

(RS-743572) - 34T>C TC-.00 TC-.00 TC-.00 - -

CC-.00 CC-.00 CC-.00

AcknowledgementWe acknowledge to all the physicians and nursesof OBG and General medicines departments ofKMCH, Manipal as well as Manipal Universityfor financial support and allowing us to useinstruments for this study. We acknowledgeRoyal Medical Trust to pursue this research workin Kerala Medical College & Hospital, Mangode,Cherpulassery, Kerala.


polycystic ovary syndrome and hyperandrogenism.,Hum Mol Genet. 1997; 6: 397-402.

14. Marszalek B, Laciński M, Babych N, et al.Investigations on the genetic polymorphism in theregion of CYP17 gene encoding 5'-UTR in patientswith polycystic ovarian syndrome., GynecolEndocrinol. 2001; 15:123-128.

15. Govind A, Obhrai MS, Clayton R. Polycystic ovariesare inherited as an autosomal dominant trait: Analysisof 29 polycystic ovary syndrome and 10 control

families. J Clin Endocrinol Metab 1999; 84: 38-43.

16. Legro RS. Polycystic vary syndrome, phenotype andgenotype. Endocrinol and Metab Clin North Am1999; 28: 379-396.

17. Jahanfar S, Eden JA, Warren P et al. A twin study ofpolycystic ovary syndrome. Fertil Steril 1995; 63: 478-486.

18. Norman RJ, Masters S, Hague W. Hyperinsulinemia iscommon in family members of women with polycysticovary syndrome. Fertil Steril 1996; 66: 942-947.

Introduction The incidence and prevalence of type 2 diabetesmellitus(T2DM) is increasing in developingcountries(1). With rapid development of therapy,the mortality from acute complications ofdiabetes has decreased, but mortality fromchronic complications like diabetic nephropathyhas increased(2). A currently favoured hypothesisis that oxidative stress, through a single unifyingmechanism of superoxide production, is thecommon pathogenic factor leading to insulinresistance, β-cell dysfunction, impaired glucosetolerance (IGT) and ultimately to T2DM and itsassociated micro as well as macro vascularcomplications(3,4). Hyperglycaemia and oxidativestress have been implicated in the acceleratedvascular damage associated with diabetes, whicheventually manifests microvascular

complications such as retinopathy, neuropathy,nephropathy and macrovascular disease e.g.peripheral arteriosclerosis, coronary disease,myocardial infarction and stroke. The viciouscycle mechanism between glycation andoxidation (‘glyco-oxidation’) has been proposedas one of the most important pathways. Thisleads to multiple biochemical sequels, whichdisplay a disturbance of oxidative–antioxidativebalance, and creates glyco-oxidative moleculardamage(5,6). The majority of the glyco-oxidationproducts brings about tissue degeneration,particularly in areas of blood vessels and thustake part in the origin of the diabetic vascular latecomplications e.g. nephropathy(7,8). Several studies suggested that, diabetes isassociated with increased modification ofproteins.

32

ELEVATED ADVANCED OXIDATION PROTEIN PRODUCTS (AOPP)CORRELATES WITH HBA1C IN TYPE 2 DIABETES MELLITUS

Kaushik Kar1, Satwika Sinha2

Department of Biochemistry, 1NRS Medical College, Kolkata, West Bengal, India

2Calcutta National Medical College, Kolkata, West Bengal, India

AbstractIncidence & prevalence of type 2 DM is increasing in developing countries like India as also its chroniccomplications. Oxidative stress is believed to be a common pathogenic factor for these graveconsequences. Advanced oxidation protein product (AOPP) is a marker of protein damage whereastotal oxidant status (TOS) reflects the severity of oxidative stress. Different results were found bydifferent authors about correlations between these markers and glycemic control. In this cross sectionalstudy, 50 patients of clinically diagnosed type 2 DM & 28 age and sex matched controls were selected,according to inclusion and exclusion criteria. Disease population was further divided in two subgroupsA & B, based on a median HbA1C level of 7.5%. (HbA1c was estimated by boronate affinitychromatography).We analysed the AOPP and TOS values in diabetes patients and compared themwith controls. (They were estimated spectrophotometrically). We also tried to find out any correlationbetween the subgroups and oxidative parameters (AOPP and TOS). Significant increase in meanHbA1C, AOPP(p<0.0001) and increase in mean TOS level (p<0.086) were found in diabetes patientsin comparision to controls. Significant correlations between HbA1C & AOPP was observed whenHbA1C>7.5% (r=0.59p<0.001y=74.71+9.26x). Hence AOPP estimation is very much beneficialin type 2 diabetes patients when HbA1C is >7.5% for prediction of chronic and grave complicationsof type 2 diabetes.

In addition to the formation and accumulationof advanced glycation end products(AGEs), afamily of oxidized protein compounds termedadvanced oxidation protein products(AOPP),has emerged as a novel class of inflammatorymediators. AOPPs are the dityrosine containing and cross linked protein products(9,10), firstdescribed by Witko-Sarasat et al(1996)(11). These compounds are recognized as markers ofoxidative damage to proteins, the intensity of OSand Inflammation)(12). Studies have alreadysuggested that AOPP plays a major role in the development of diabetic nephropathy, 8,10,13 aswell as diabetic retinopathy(14).This structural and functional changes in diabeticnephropathy take place in the kidney during theearly phases of diabetes, prior tomicroalbuminuria(15,16,17).Human bodies are constantly protected againstexcessive oxidative stress by a complex set ofenzymatic and non-enzymatic antioxidantsystems(18). Low levels of ROS are indispensablein many biochemical processes(19); however,overproduction and/or inadequate removal ofROS can result in oxidative stress. Studies havealso suggested that oxidative stress, in terms ofTotal Oxidant Status(TOS) is increased inpatients with diabetic nephropathy as well as indiabetics without nephropathy and increase isrelated to the severity of diabetes(20).Many authors submitted different opinions aboutaltered values of AOPP and TOS indiabetes(6,13,20,21,22). However there is a fewevidence of such studies at our region.Considering these facts we have tried to evaluatethe values of those parameters in diabetes and tofind out if any apparent correlations of them withglycemic control. Moreover, we tried to find outincreased AOPP and TOS values can furtherworsen the hyperglycemia. This review will focuson identification of any oxidative stress factor torecognise and treat this devastating disease earlyin its progression and to postpone or evenprevent the serious complications associatedwith it.

Materials and MethodsStudy design This hospital based, cross sectional, noninterventional study was conducted during theperiod of 2010-12 in the Biochemistrydepartment of NRS Medical College andHospital, Kolkata, West Bengal, India.

Selection of the case groupThe case group (Group-II) primarily included 50(fifty) patients of clinically diagnosed T2DM(diagnosed from history and relevantbiochemical tests), attending the outpatientdepartment (OPD) and admitted in Medicineindoor ward of the institution on conveniencebasis.The patients were selected with the agegroup of 40-60 years following the inclusion andexclusion criteria.American Diabetic Association(ADA) targetedreasonable A1c goal as <7%, and less stringentA1c goal between 7-8%(23). Hence we haveselected a median HbA1C level (7.5%), tosubdivide the case group (n=50) into twogroups(A and B), and to find out if anycorrelation persists between the subgroups andoxidative parameters. Group A comprises of 26patients (HbA1C<7.5%), Group B comprises of24 patients(HbA1C>7.5%).

Exclusion criteria for the case groupHypertensive patients, T2DM patients with acuteand chronic complications, severely ill,unconscious and disabled patients were notincluded in the study along with abnormal liverfunction tests. Patients with recent history ofstroke, myocardial infarction or any diseasewhich can cause oxidative stress were alsoexcluded from the study.

Selection of the control group: 28 age andsex matched healthy control subjects (Group I)were also selected for the study. All controlsubjects were considered from more or lesssimilar geographical area with similarsocioeconomic status with no significant

33Advanced Oxidation Protein Products in T2 DM

difference in their food habit and drinking waterquality.

Exclusion criteria for control subjectsPersons with chronic smoking habits, alcoholaddiction or any drug addiction were excludedfrom the study.

Ethical considerationsWritten consents (informed consents) wereobtained from the participants (disease andcontrol groups). The study protocol wasapproved by the ethics committee of N.R.S.Medical College.

Sample CollectionThe amount of blood collected in absolutefasting condition with all aseptic precautions in2 parts was 5 ml. The first part collected inethylene diamine tetraacetic acid(EDTA) vial forestimation of glycated hemoglobin(HbA1C).Thesecond part collected was allowed to clot andserum was separated for estimation of AOPPand TOS. Measurement of biochemical analytesHbA1C, the index of long term glycemiccontrol,was determined with Micromat II(Biorad) instrument based on boronate affinitychromatography(24).

AOPP AssayDetermination of AOPP (i.e. some oxidationproducts with characteristic absorbance) wasbased on spectrophotometric detectionaccording to Witko-Sarsat et al. (1996) in ourmodification(25). Concentration of AOPP isexpressed in chloramine units (μmol/l).

TOS Assay : Serum TOS was measured usingErel's TOS method(26), which is based on theoxidation of ferrous ion to ferric ion in thepresence of various oxidative species in acidicmedium and the measurement of the ferric ionby xylenol orange.The results were expressed inμmol H2O2 equivalent/l (μmol H2O2 equiv./l).

Data collection and processing forstatistical analysis Statistical analysis was aimed

i) To asses the significance of differencebetween the mean values of serum AOPPand TOS between the Group I (controls)and Group II (cases).

ii) To asses the correlations between HbA1cand oxidative stress parameters (AOPP andTOS) in Group I (controls), Group A(HbA1c<7.5%) and Group B(HbA1c>7.5%).

Statistical methods usedStatistical analysis was done by one way andpost hoc analysis of variance (ANOVA) withBonferroni correction; P value was consideredsignificant at the confidence level of 0.05. Allstatistical analysis were performed using SPSSsoftware version 16.0 for windows.

ResultsTable 1 shows that there is a significant increasein mean HbA1c and AOPP levels in diabetes incomparison to controls. The mean HbA1c levelwas found 8.3% in diabetics in comparison to5.4% in controls (p<0.0001).The mean AOPP level was also observed as139.6 μmol/L in T2DM when compared tocontrols as 84 μmol/L (p<0.0001). The TOSlevels were increased in diabetes but notsignificantly.The mean TOS value was 10.4μmol H2O2 equivalent/l in control subjects incomparison to 11.9 in diabetics.(p<0.086)Furthermore table 2 shows, significantcorrelation between HbA1c and AOPP whenHbA1c level > 7.5%. (r=0.59p<0.001y=74.71+9.26x).

DiscussionThe results of our study have shown that there isa significant increase in mean HbA1C and AOPPlevels in diabetes in comparison to controls.Furthermore, significant positive correlation


between HbA1C and AOPP has also found in ourstudy when HbA1C level >7.5%. The TOS levelswere increased in diabetes but not significantly.The findings in our study keep in tract with theobservations found by Plwowar A(13) whodescribed that AOPP formation is induced byintensified glyco-oxidation process, oxidantantioxidant imbalance and coexistinginflammation. Similar findings were alsoobserved by Pan et al(2010)(27) and Fathy et al(2009)(28). Gil del valy(29) and Fathy et al(28),found correlations between AOPP and HbA1C.Furthermore Fathy et al(28) found correlations ofAOPP with microvascular complications ofT2DM. Kostolonska J(6) observed the significantcorrelations between HbA1C and AOPP in DM1patients of poor glycemic controls.Kalousovaalso suggested that the increase of AOPP wasmore pronounced in T2DM than in T1DM.(P<0.001 in T2DM vs healthy controls, p<0.05in T1DM vs healthy controls) and AOPP is abetter parameter than AGE. In this context, wefound significant correlations between HbA1Cand AOPP at our region.(when HbA1C>7.5%)Previous studies also observed that AOPP is agood marker for progression of diabeticNephropathy(8). Sharada HM(30) observed thatAOPP increased progressively and significantlywith the growth of albuminuria(p<0.01). Asignificant positive correlation was also found byhim between plasma level of AOPP and both of(S) creatinine and Albumin creatinine ratio(ACR).Zi Ziang Ng et al(14) found higher AOPP levels indiabetic retinopathy patients.From our review wecan assume that at our region progress todiabetic nephropathy as well as retinopathy mayappear earlier if HbA1C>7.5%.In our study TOS level was not increasedsignificantly in diabetes. Recently, numerousstable end products of oxidative stress have beenidentified and these include the AOPP(14). Fromour result we can assume that AOPP is a bettermarker than TOS in diabetes.AOPPs challengealso impaired insulin signalling. Oxidative stress

has been proposed already as a causative factorof T2DM and responsible for itscomplications(3,4). Those observations maycontribute the correlation of HbA1C>7.5% andAOPP. Accumulation of AOPP can cause furtherdeterioration of hyperglycemia. Estimation of AOPP as well as HbA1C may beuseful to predict the risk of development ofdiabetic complications(6). AOPP can be a usefulmarker to estimate the degree of proteindamage in diabetic patients(32).Furthermore AOPP is not age and diseaseduration dependent unlike HbA1C

(30). Some havehypothesised that there was a greater frequencyand magnitude of glycaemic excursions in theconventionally treated patients compared withpatients in the intensive treatment group, andthat this increased glycaemic variabilitygenerated more ROS, leading to vasculardamage(4).Based on the results of our study andconsidering all the above facts we can considerAOPP estimation is very much beneficial inT2DM particularly when HbA1C is >7.5% at ourregion for prediction of diabeticcomplications.AOPP have their own particularbiological proprieties, similar to those of AGEs,and also bind to the same receptor, i.e. RAGE(12). There is substantial evidence to support that thebinding of AGE to its receptor (RAGE) isinvolved in the development of microvascularcomplications(33). According to Kalousova et al,AOPP shares common biological effect exertedby AGE, including interaction with RAGE whichultimately leads to neo-vascularisation that couldresult in DR(5). Zi Ziang ng, stated that, thesoluble RAGE (sRAGE), a RAGE isoform lackingthe transmembrane domain, is a recentlydiscovered naturally occurring inhibitor of AGE-RAGE mediated pathological effects. Accordingto him, the increased plasma sRAGE level indiabetic non retinopathy(DNR) and DR patientscould be viewed as a protective reaction tocounterbalance, at least partly, the elevatedplasma pentosidine(AGE) level.

Advanced Oxidation Protein Products in T2 DM 35

AOPP accumulation may be a therapeutic targetat least for the prevention and delaying theprogression of renal complications indiabetics(12). Furthermore, medications targetedto increase the sRAGE level in plasma can alsoprevent the binding the AOPP with RAGE , toreduce the microvacular complications ofT2DM.14

ConclusionOxidative stress has been implicated in theprogression of long-term diabetes complications,including microvascular and macrovasculardysfunction.We have measured AOPP and TOS in type 2diabetes and also tried to asses the correlationbetween AOPP and TOS with HbA1C.Weobserved that AOPP has increased significantly

in type 2 diabetes but increase of TOS was notsignificant. Increase in AOPP also positivelycorrelated with HbA1C when HbA1C is above7.5% in our study. AOPP accumulation canfurther deteriorate hyperglycemia. So werecommend to measure the AOPP particularly ifHbA1C is >7.5%. In view of the socio-economical status of ourcountry, measurement of AOPP is beneficial as itis simple and not too costly and can be used astherapeutic target of the physicians to challengethe chronic complications of diabetes.

AcknowledgementsThe authors acknowledge Professor MohanMondal (Professor and HOD of Biochemistry,NRS Medical College) for his assistance andguidance in this research.


Table 1 : HbA1c,AOPP and TOS levels in control and type 2 diabetes patients

Study Statistics HbA1C% AOPP(μmol/l) TOS(μmolPopulation H2O2 equiv./l)

Group I Mean 5.45 84.23 10.44

(Control) SD 0.2755 12.39 3.4

(n = 28) SEM 0.052 2.3415 0.3232

Group II Mean 8.296 139.6 11.94

(T2DM) SD 1.3019 28.85 3.8

(n = 50) SEM 0.18 4.08 0.321

p-value Significance <0.0001 <0.0001 0.086


Fig. 1 : Correlation between HbA1C & AOPP in Sub-group B(HbA1C > 7.5%) Type 2Diabetes Mellitus patients

Table 2 : Correlation between HbA1C and AOPP levels in type 2 diabetes

Parameter Control (n=28) Sub-Group-A(n=26) Sub-Group-B(n=24)

Correlation

coefficient (r): 0.3469 0.49984 0.5976

HbA1C & AOPP

p-value <0.0001 <0.0001 0.086

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6. Kostolanská J, Jakus V, Barák L. HbA1c and serumlevels of advanced glycation and oxidation proteinproducts in poorly and well controlled children andadolescents with type 1 diabetes mellitus.J PediatrEndocrinol Metab 2009; 22 (5): 433- 42.

7. Bonnefont-Rousselot D. Glucose and reactive oxygenspecies. Curr Opin Clin Nutr Metab Care 2002;5: 561-68.

8. Piwowar A, Knapik-Kordecka M, Szczecinska J,Warwas M. Plasma glycooxidation protein products intype 2 diabetic patients with nephropathy. DiabetesMetab Res Rev 2008; 24: 549-53.

9. Wei XF,Zhou QG,Hou FF,Liu Bei, Liang M.Advancedoxidation protein products induce mesengial cellperturbation through PKC dependent activation ofNADPH oxidase.American J of Physiol 2009; 296(2):427-37.

10. Shi XY,Hou FF,Niu HX et al. Advanced oxidationprotein products promote inflammation in diabetickidney through activation of renal nicotinamideadenine dinucleotide phosphate oxidase.Endocrinology 2008;149 (4):1829-39.

11. Witko-Sarsat V, Friedlander M, Capeillere-Blandin C.Advanced oxidation protein products as a novelmarker of oxidative stress in uraemia. Kidney Int1996; 49: 1304-13.

12. Piwowar A. Advanced oxidation protein products.Part1.Mechanism of formation,characteristics andproperty. Pol Merkur Lekarski 2010; 28(164): 166-9.

13. Piwowar A. Advanced oxidation protein products.PartII.The significance of oxidation protein products in the

pathomechanism of diabetes and its complications.Pol merkur Lekarski 2010;28(165):227-30.

14. Ng ZX,Chua KH,Iqbal T,Kuppusamy UR. Solublereceptor for Advanced Glycation EndProducts(sRAGE)/pentosidine ratio:A potential riskfactor determinant for type 2 diabetic retinopathy. Intj Mol Sci 2013;14(4):7480-91.

15. O’Connon AS, Schelling JR. Diabetes and thekidney.Am.J. Kidney Dis 2005;46:766-73.

16. Newman DD, Mattock MB, Dawnay AB et al.Systematic review on urine Albumin testing for earlydetection of diabetic complications. Health TechnolAssess 2005; 9: xiii- x163.

17. Hakim, FA. Pflueger A. Role of oxidative stress indiabetic kidney disease. Med Sci Monit Feb 2010; 16(2): RA 37-48.

18. Mates JM, Perez-Gomez C, Nunez de Castro I.Antioxidant enzymes and human diseases. Clin.Biochem 1999; 32(8): 595–603.

19. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M,Telser J. Free radicals and antioxidants in normalphysiological functions and human disease. Int JBiochem Cell Biol 2007; 39(1):44–84.

20. Aslam M, Sabuncu T,Koeyiquit A, Celik H,Selek S.Relationship between total oxidant status and severityof diabetic nephropathy in type 2 diabetic patients.Nutr Metab Cardiovasc Dis 2007; 17(10): 734-40.

21. Kalousova M,Skrha J,Zima T. Advanced glycation endproducts and Advanced oxidation protein products inpatients with diabetes mellitus. Physiol Res2002;51:597-604.

22. Saygili EI,Aksoy SN, Gurler B, Aksoy A, ErelO,Ozaslan M. Oxidant/Antioxidant status of patientswith diabetic and senile cataract. Biotechnol andBiotechnol Eq 2010;24(1):1648-52.

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Introduction Malaria along with six other diseases viz.diarrhea, pneumonia, tuberculosis, measles,hepatitis B and HIV/AIDS account for 85% ofGlobal infectious disease burden1. Thirty-sixpercent of global population i.e. 2020 million in107 countries and territories situated in thetropical and subtropical regions are at the risk ofmalaria. Of the 2.5 million reported cases ofmalaria in the South East Asia, India alonecontributes about 70% of the total cases(1).Plasmodium falciparum is the most deadlyamong the five Plasmodium species that causehuman malaria and it is the world’s second

biggest killer after tuberculosis. It is estimated that3,000 children under the age of five years fallvictim to malaria each day. Around 40% of theworlds population are at risk(2). Severe malariakills over a million people every year. The annualdeath toll can be as high as one in a 100 childrenunder the age of five(3,4).Plasmodium falciparum infection is frequentlycomplicated with multi-organ failure along withor without metabolic dearrangement(5). Cerebralmalaria, un-arousable coma, not attributable toany other cause, is a specific type of severemalaria(4) that even with correct treatment canhave a mortality rate approaching 20%(6).

40

A HOSPITAL BASED RETROSPECTIVE CLINICAL EVALUATIONOF COMBINED ANTI-MALARIAL THERAPY ON THE OUTCOMEOF PLASMODIUM FALCIPARUM MALARIA CASES IN YOUNG

HOSPITALIZED TRIBAL CHILDREN OF TRIPURA :A THREE YEARS STUDY

Sanjib Kumar Debbarma1, Debasis Ray2, Sujit Kumar Chakraborty3, Shishir Kumar3

Departments of 1Paediatrics, 2Pharmacology and 3Community MedicineAgartala Government Medical College. P. O. Kunjaban, Agartala, Tripura

AbstractThis story is aimed at the comparative evaluation of drug in practice in terms of therapeutic use ofantimalarial drugs against plasmodium falciparum (p.f.) positive malaria cases and to compare theefficacy of artesunate and quinine in the treatment of p.f. positive malaria in hospitalized children. Allthe p.f. positive malaria cases who were admitted from May 2008 to Oct 2011 were included in thisstudy. The prescribing pattern of anti-malarial in these cases and its clinical outcome like mortalityand morbidity patterns includes the fever remission time and total hospital stay was assessed from thecase sheets. Out of the total 264 clinically suspected malaria cases, 121(46%) cases were p.f. positiveand among them 31(25.6%) cases received quinine based combination therapy and rest 90(74.4%)received artesunate based combination therapy. Among the 121 p f positive cases 69(57%) wereadmitted with pronounced symptoms of malaria (severe malaria). Severe anaemia and cerebralmalaria were the two commonest presentation of severe falciparum malaria. There was no statisticallysignificant difference of clinical outcome between the both clinical groups in the form of mortality,time for remission of fever (fever clearance time) or total hospital stay. Randomized clinical trial witha large sample size is necessary to compare the efficacy of both the quinine based and the artesunatebased combination therapy for plasmodium falciparum Malaria.

Over the years quinine has been the drug ofchoice for treating severe malaria in children, butdespite its high efficacy the case fatality rate forsevere falciparum malaria in children can be ashigh as 40%. Additionally, in recent years therehave been concerns that the efficacy of quinineis declining in some parts of South East Asia andquinine resistance cases has been documentedin Africa. This led to the launch of a series oftrials to find newer effective drugs that aresuitable alternatives to quinine in terms ofefficacy(7).In anti-malarial combination therapy, thecombination is often more effective and delaythe emergence of resistance. The eachcomponent of the combination therapy mustindependently be sufficiently efficacious intreating malaria(8). Currently two classes of drugs are available forthe parenteral treatment of severe malaria: thecinchona alkaloids (quinine and quinidine) andthe artemisinin derivatives (artesunate,artemether and artemotil)(8).The clinical resistance to quinine therapy hasbeen noticed sporadically in Southeast Asia andWestern Oceania. Therefore, since the last twodecades this drug has been used in combinationwith tetracycline or doxycycline to enhance itseffectiveness. In India resistance has emergedagainst quinine in northeastern states of Indiaand Kolar district in Karnataka(9). Intravenous artesunate is the drug of choice foradults with severe falciparum malaria,particularly if acquired in Asia. But literature, onearlier studies did not identify sufficient data tomake firm conclusions about the treatment ofchildren or the effectiveness of intramuscularartesunate administration. There is an urgentneed to compare the effects of artesunate withquinine in children with severe falciparummalaria(10). In India, malaria is reported from many states,but several deaths, amounting to epidemicproportion, reported every year from differentparts of north-east India. Chloroquine-resistant

malaria and decreased sensitivity to other anti-malarial drugs has been documented(11). The North eastern States of India are highlyendemic area for malaria in terms incidence andtransmission, and the control of P. falciparummalaria poses a great challenge to the societyhampering manpower loss which is reflected bylower scale of development of this part of thecountry in comparison to the rest of thecountry(12).The state of Tripura in the North Eastern Regionof India is surrounded by Bangladesh except inthe north. The areas adjacent to the Indo-Bangladesh border are at risk of malariaoutbreak due to inadequate health infrastructureand lack of vector control operations(13).To the best of our knowledge, there is nopublished data reported from Tripura about thecomparison of artesunate and quinine in thetreatment of severe falciparum malaria inchildren. So there is need to compare theefficacy of artesunate with quinine in children ofTripura especially tribal children from rural areasuffering from severe falciparum malaria so thata cheap drug like quinine is not abandonedcompletly. Agartala Govt. Medical College and GBPHospital is the only tertiary health care centre inGovt. sector with paediatric intensive care unit,where a good number of severe malaria patientsare referred from various parts of the state andtreated every year. We carried out thisretrospective study from the hospital record andthis may be a useful guide to design therandomized control trial (RCT) in future tocompare the efficacy of Quinine with Artesunatebased combination therapy in children especiallyin this part of the country.

Materials and MethodsIt was about three and half years retrospectivestudy and all the tribal children suffering from p.f.Malaria and admitted in paediatric ward fromMay 2008 to Oct 2011 were included for thisstudy. Outcome of two treatment modalities, one

Clinical Evaluation of Combined Anti-malarial Therapy 41

group which received quinine with clindamycin(when age is less than 8 yrs) or with tetracycline(when age is ≥8 yrs) was compared with theother group which received artesunate withclindamycin (when age is less than 8 yrs) or withtetracycline (when age is ≥8 yrs). The currentpractice of treating the young children sufferingfrom severe malaria and its clinical outcome wasincluded in this study and assessed from the casesheets which were collected from the MedicalRecord section of the Agartala Govt. MedicalCollege and GBP Hospital. The cases wereenrolled in this study either as severe or as nonsevere was as per the entry in the bed head ticketdiagnosed by the clinicians. Data collected andrecorded on a pre-tested format that containedinformation on the demography of the patients,type of anti-malarial drugs used, mortality, feverclearance time (fever recovery time), totalhospital stay, adverse effects, neurological sequeland any adjuvant therapy administered.Complete free from fever were considered asrecovery.The collected data were analyzed for statisticalsignificance to compare the clinical outcome ofp.f. malaria cases that were either treated byintravenous quinine or by intravenous artisunatebased combination therapy (ACT). The data were expressed as mean±standarderror of mean (SEM) and in percentage ofoutcome between the groups. Statisticalcomparison between the different treatmentgroups were done using either student’s t test orchi-square test whichever applicable and P<0.05 was considered significant

Results and observationsOut of the total 264 clinically suspected malariacases, 121(46%) cases were p.f. positive and wereincluded for this study. Out of the total 121 cases31(25.6%) cases received quinine basedcombination therapy and rest 90(74.4%) receivedartesunate based combination therapy. Amongthe 121 p.f. positive cases, 69(57%) cases weresuffering from severe malaria and rest 52(43%)

were non severe p. f. positive cases. Out of total69 severe malaria cases, 16(23.2%) were treatedwith intravenous quinine based combinationtherapy and rest 53(76.8%) were treated withatresunate based combination therapy. Thedemographical distributions of severe falciparummalaria cases were shown in Table1. There wasno significant demographical variation betweenthe two groups. Maximum of the p. f. positivepatients (99%) were from rural area.Table 2 shows the age, district wise and differentnative ethnic group distribution of the casesreceived two modalities of treatment. There wasno significant difference between the twotreatment groups in different age group, indifferent native ethnic group and in patients fromdifferent districts.Different diagnostic methods used for diagnosisof 121 p.f. malaria under this study populationwere peripheral smear slide method (60 cases49.6%), QBC (Fluorescence microscopy) (10cases 8.2%) and Rapid diagnostic tests (51 cases42.1%). There was no significant difference inthe choice of methods, for malaria diagnosis.In table 3 the different clinical presentations ofp.f. positive severe malaria cases were shown.Out of the total 69 severe malaria cases, 24(34.8%) and 42 (60.9%) presented with cerebralmalaria and severe anaemia respectively. 16(23.2%) of total severe malaria cases receivedquinine based combination therapy and 53(76.8%) of total severe malaria cases receivedartisunate based combination therapy.Table 4 shows the clinical outcome profile inpatients treated with two modalities of treatment.There were no significant differences in feverrecovery time and total duration of hospital staybetween the two treated groups. Complete freefrom fever were considered as recovery. 41.3%and 17.4% of the total p.f. positive casesreceived blood transfusion and anticonvulsanttherapy respectively. There was no significantvariation regarding receiving blood transfusionand anticonvulsants between the two treatmentgroups.


DiscussionMalaria is one of the commonest potentially fatalinfections in the world with high incidence inSouth East Asia region(14). In India malaria isreported from many states, but deaths are mostlyreported every year from different parts of north-east India(11).In recent years there have been serious concernsover the declining efficacy of quinine in someparts of South East Asia and quinine resistancealso has been documented in Africa. This led tothe launch of a series of trials to find out thedrugs that are suitable alternatives to quinine(7).There is an urgent need to compare the efficacyof artesunate with quinine in children with severefalciparum malaria(10). Since till date there is no published data / recordsfrom Tripura about the comparative efficacy ofartesunate with quinine for the treatment ofsevere falciparum malaria in children areavailable, so need of such study was ademanding one. Randomized Control Trials(RCT) can be designed if any clue can beidentified from such type of retrospective study.The malaria cases are detected throughout the

year in the state but higher incidence is reportedduring May to October(11). Keeping conformitywith higher incidence of malaria cases during thisperiod, for this retrospective study period wasselected from May, 2008 to Oct, 2011.The baseline characteristics of the two druggroups were comparable. The mean age ofchildren suffering from malaria in two treatmentgroup was around 5.2 years to 5.6 years and iscomparable with the observations of Huda SNet al 2003 14.Malaria was diagnosed by standard methods ofdiagnosis like peripheral smear examinationboth thick and thin, QBC (Fluorescencemicroscopy) and Rapid diagnostic test. Therewas no significant difference regarding the choiceof diagnostic methods. Malaria cases were found in all ethnic group ofnative community of Tripura and there was nosignificant difference in the incidence of malaria

cases. Though it was observed that the numberof malaria cases from North Tripura and Dhalaidistrict, treated in AGMC were less but it may notreflect the true incidence as both the districts arefar away from AGMC and might been treated insome other well equipped institutions.In the present study it was observed thatintravenous artesunate based combinationtherapy was preferred (74.4% ) over quininebased combination therapy (23.6%) fortreatment of p.f. positive cases by the cliniciansand this may probably be due to some reportedconcern on the declining efficacy of quinine,both in our country and the South East Asianand African countries7. But there was nosignificant difference in recovery time (feverremission time) and total hospital stay in boththe treatment group and is comparable to thefindings of other studies(7,10,11).Mortality rate in the patients receiving artesunatebased combination therapy was marginally less(13.2% vs 18.8%) than the patients receivedquinine based combination therapy but thisdifference is statistically insignificant. Thisobservation is in consistent with the earlierstudies from other part of the world(7,10,11). This study revealed that the commonestpresentation of severe falciparum malaria waswith severe anaemia followed by impairedconsciousness/coma then by repeatedconvulsion. This is in contrary to theobservations of other studies where cerebralmalaria is the commonest presentation(7,11) ofsevere falciparum malaria and this probably maybe due to the fact that the 99% of this studypopulation are of rural native origin who aremostly from lower socio economic family andsuffering from nutritional anaemia anddeveloped pronounced complication withmalaria. There was no report of adverse drugreactions among the both treated group patients. Due to insufficient case record data, parasiteclearance time and coma recovery time andsevere falciparum malaria presented withhypoglycaemia, hyperpyrexia, metabolic


acidosis could not be assessed. Though theclinicians of this institution preferred artisunatebased combination therapy over quinine basedcombination therapy for treatment of p.f.positive malaria, but this retrospective study doesnot indicate any significant advantage ofatesunate based combination therapy overquinine based combination therapy. Hence a larger randomized control trial and firmclinical evidence is necessary to make a firmconclusion about the comparative efficacy of

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artesunate based combination therapy withquinine based combination therapy among thechildren of this malaria endemic zone of thecountry so that a cheap and useful drug likeQuinine is not rejected by the clinicians.

AcknowledgementAuthors are grateful to the Principal and MedicalRecord section of Agartala Govt. MedicalCollege, Agartala, Tripura India, for providingnecessary facility for this retrospective study.


Table 2 : Age, district wise and ethnic distribution of the p. f. positive malaria cases thatwere treated either with Quinine or ACT in AGMC from May 2008 to Oct 2011

Variables Quinine based combination Artisunate based combination therapy group therapy group

Age 0 to <1 yrs 5 (4.1%) 3 (2.5%)

1-3 yrs 7 (5.8%) 24 (19.8%)

>3 to 6 yrs 9 (7.4%) 26 (21.5%)

More than 6 yrs 10 (8.3%) 37 (30.6%)

District Wise Dhalai 3(2.5%) 8 (6.6%)

Distribution North Tripura 0 1 (0.8%)

South Tripura 5 (4%) 22 (18.2%)

West Trpura 23 (19%) 59 (48.8%)

Different ethnic Tripuri 26 (21.5%) 63 (52.1%)

Tribal groups Halam 2 (1.7%) 9 (7.4%)

Reang 2 (1.7%) 13 (10.7%)

Chakma 1 (0.8%) 4 (3.3%)

Mog 0 1 (0.8%)

Table 1 : Comparative demographic profile of the p. f. positive malaria cases that weretreated either with Quinine or ACT in AGMC from May 2008 to Oct 2011

Variables Quinine based combination Artisunate based combination therapy group therapy group

Total p.f. positive Cases 31(25.6%) 90(74.4%)(N = 121)

Severe malaria out of p.f. 16(23.2% total severe cases) 53 (76.8% total severe cases)Positive Cases (N = 69)

Non-Severe p.f. Positive Cases 15 (28.8%) 37 (71.2%)(N = 52)

Age in yrs. (Mean ± SEM) 5.2 ± 0.7 5.6 ± 0.3

Weight in kg (Mean ± SEM) 14.4 ± 1.3 15.8 ± 0.63

Sex (M:F) 15 : 16 46: 44

Address (Urban : Rural) 0 : 32 1 : 88


Table 3 : Different presentations of the severe p. f. malaria cases that were treated eitherwith Quinine based combination or Atisunate based combination therapy in AGMC fromMay 2008 to Oct 2011

Types Quinine based Artisunate basedcombination therapy combination therapy

p.f. positive severe malaria cases (N = 69) 16 (23.2%) 53 (76.8%)

Impaired consciousness/coma 7 (10.2%) 17 (24.6%)N = 24 (34.8%)

Repeated generalized convulsions 4 (5.8%) 17 (24.6%)N =21(30.4%)

Severe anaemia (Hb <5mg/dl) 9 (13%) 41 (59.4%)N = 50 (41.3%)

Algid Malaria = 1 (1.4%) 0 1 (1.4%)

Anaemia with Renal Failure N= 2 (2.9%) 1(1.4%) 1(1.4%)

Jaundice (Serum Bilirubin >3 mg/dl) 0 1(1.4%)N=1 (1.4%)

Abnormal bleeding and 0 0Disseminated intravascular coagulation

Pulmonary oedema / acute respiratory 0 1(1.4%)distress syndrome N=1 (1.4%)


Table 4 : Comparative clinical outcome profile of the p.f. malaria cases that were treatedeither with Quinine or ACT in AGMC from May 2008 to Oct 2011

Types Quinine based Artisunate basedcombination therapy combination therapy

(No of cases) (No of cases)

Total p.f. positive cases 31(25.6%) 90(74.4%)(N = 121)

p.f. positive severe malaria cases 16(23.2% total 53 (76.8% totalN = 69 (57% of total p.f. positive cases) severe cases) severe cases)

Mortality N= 10 (8.3%) 3 (9.7% within the group) 7 (7.8% within the group)

Mean time of death since admission (hrs) 9.2 ±7.5 23.6 ± 13.7(Mean ± SEM)

Recovery time (Fever clearance Time) 2.5 ± 0.3 3.2 ± 0.2i n days (Mean ± SEM)

Total duration of hospital Stay in days 6 ± 0.7 6.5 ± 0.3(Mean ± SEM)

Blood transfusion Received 9 (29% within the 41 (45.5% within the N =50 (41.3% of total pf positive cases) group) group)

Any anticonvulsant therapy N=21 4(12.9% within the 17 (18.9% within the (17.4% of total pf positive cases) group) group)

Any bleeding episodes 0 0

Any suspected adverse drug reactions 0 0

Corrigendum

The name of authors & affiliation for the paper titled with SERUM URIC ACID LEVEL AS APREDICTOR OF FETAL OUTCOME IN PREGNANCY INDUCED HYPERTENSION,published in IJMB, Volume 16, No. 2 : 2012 is as follows:

Nikunj Modi1, Nalini Kumar2, Dharmesh Gamit3, Tejas Shah4

1Department of Biochemistry, M P Shah Medical College, Jamnagar, Gujarat, India.2Department of Biochemistry, Sir T General Hospital, Bhavnagar, Gujarat, India

3Department of Obs. & Gynecology, M P Shah Medical College, Jamnagar, Gujarat, India4Department of Biochemistry, GMERS Medical College, Valsad, Gujarat, India

But it should be

Nikunj Modi1, Hariom Sharma2, Nalini Kumar3, Dharmesh Gamit4, Tejas Shah5

1Department of Biochemistry, M P Shah Medical College, Jamnagar, Gujarat, India2Department of Biochemistry, Sir T General Hospital, Bhavnagar, Gujarat, India

3Department of Obs. & Gynecology, M P Shah Medical College, Jamnagar, Gujarat, India4Department of Biochemistry, GMERS Medical College, Valsad, Gujarat, India

5Department of Biochemistry, M P Shah Medical College, Jamnagar, Gujarat, India

We are sorry for the mistake.

Editorial Board


IntroductionAlcohol is consumed by a large percentage ofworld’s population and a moderate proportionof them develop clinically significant liverdisease. Alcohol is the common cause of liverdamage worldwide. There is a significant role ofoxidative stress and lipid peroxidation in thepathogenesis of ethanol-induced liver injury.Ethanol consumption results in depletion ofendogenous antioxidant capabilities. Depletionof mitochondrial GSH (Reduced Glutathione)precedes and promotes the progression ofalcoholic liver injury. Heavy drinkers aredeficient in Selenium which is required for theactivity of Glutathione Peroxidase andantioxidant vitamins A, C and E(1).This study aims at assessing the antioxidant

status of the patients with alcoholic cirrhosis byanalyzing selenium and vitamin E levels and toassess its correlation with severity of liverdamage.

Materials and Methods This is an age and sex matched case-controlstudy conducted during the period of March2013 to September 2013. Prior approval wasobtained from the institutional ethicalcommittee. The study population includes twogroups; 50 healthy volunteers who served ascontrols and 50 cases of alcoholic cirrhosis. Thecases were recruited from MedicalGasteroenterology department, Stanley MedicalCollege and Hospital.

49

SELENIUM AND VITAMIN E STATUS IN PATIENTSWITH ALCOHOLIC CIRRHOSIS

U. N. Priyadharshini1, R. Lalitha2

Department of Biochemistry1Government Dharampuri Medical College, Dharmapuri, Tamil Nadu and

2Kilpauk Medical College, Chennai

AbstractThe purpose of the study is to show that oxidative stress and lipid peroxidation plays a significant rolein the pathogenesis of ethanol-induced liver injury. Heavy drinkers are deficient in Selenium, whichis required for the activity of Glutathione Peroxidase and antioxidant vitamins A, C and E. This studyaims at assessing the antioxidant status of the patients with alcoholic cirrhosis and its correlation withseverity of liver damage. The study population includes 50 healthy volunteers who served as controland 50 cases of alcoholic cirrhosis. The blood samples were analysed for selenium, vitamin E and LiverFunction Tests viz. bilirubin, aspartate transaminase, alanine transaminase and albumin. The meanand standard deviation estimated for cases vs controls were Selenium ( 0.68 ± 0.05 vs 1.03 ± 0.04)and vitamin E (19 ± 0.7 vs 29.7 ± 1.2). Selenium and Vitamin E were significantly lower in caseswhen compared to controls.

Inclusion criteriaPatients with ethanol related decompensatedliver disease with portal hypertension wereincluded in the study. All patients had varyinggrades of oesophageal varices as identified by GIendoscopy.

Exclusion criteria1. Non alcoholic liver disease2. Patients on vitamins and minerals

supplementation during the past one year.3. Patients with positive serology for Hepatitis B

and C.4. Patients with other coexisting medical or

surgical illness like CAD, stroke, diabetes etc.

Sample collectionRandom venous blood sample of 5 ml wascollected. The serum was seperated andanalysed for Selenium, Vitamin E, Bilirubin,Aspartate transaminase, Alanine transaminaseand Albumin.

Determination of SeleniumThe sample was acid digested before analysis.The sample of 500μl was taken in a porcelaindish and 1.5 ml of nitric acid : perchloric acidmixture (ratio 5:1) was added and heated on asand bath until complete ashing. The ashedsample was reconstituted with 2 ml of 0.2% nitricacid. The reconstituted sample was centrifugedfor 10 minutes at 2500 rpm and the clearsupernatant was transferred into another glasstube. The digested sample was submitted foranalysis. Quantitative estimation of selenium was done byGraphite Furnace- Atomic AbsorptionSpectrophotometer with Zeeman backgroundcorrection (Perkin Elmer Analyst 700) in theDepartment of Biochemistry, ShankaraNethralaya . Selenium in vapourized sample absorbs energyat wavelength of 196.3 nm. Absorbance at thiswavelength is specific for selenium and isproportional to its concentration(2).

Reference interval: Adult - 0.8 – 2 μmol/l

Estimation of Vitamin ESerum vitamin E was estimated byspectrophotometric method usingbathophenanthroline and is based on thereducing property of vitamin E(3).AlphaTocopherol being a reducing agent can convertferric ion into ferrous ion which can thencombine with the chelating agent,Bathophenanthroline (BA) to produce pinkcoloured Ferrous- BA complex. The absorbanceof this complex is measured at 536 nm which isproportional to the concentration of αTocopherol in the reaction. Orthophosphoricacid is used to inhibit the interference caused byother reducing agents such as β carotene andglutathione and hence increases the specificity ofthe assay.Reference range : Adults : 12 - 42 μmol/LEstimation of Total Bilirubin was done by DiazoMethod of Pearlman & Lee. Aspartatetransaminase (AST) by IFCC Method(4) Alaninetransaminase by modified IFCC method andAlbumin by Bromocresol green dye bindingmethod.

Result and Statistical AnalysisThe statistical analysis was done using GraphPad Prism 6 statistical software. The distributionof age among the control group and cases wereas shown in table no.1. Mean and standarddeviation were estimated for each group (casesand controls). Mean values were compared usingstudent independent ‘t’ test (Table no.2).Pearson’s correlation analysis was done to findout the relationship between Selenium andVitamin E (Table no.3). The relationship isgraphically represented by scatter diagram.Correlation analysis was also done for serumBilirubin with selenium and vitamin E (Table no. 4)

DiscussionThe present study demonstrates the defects inantioxidant status of patients with alcoholic


cirrhosis. A decrease in plasma Selenium hasbeen found previously by Johansson et al(5). Itmay be due to low intake, absorption ormetabolism in alcoholics. Plasma selenium wasfound to be decreased in alcoholic cirrhosispatients but not among alcoholics in abstinence.This shows that selenium deficient state inchronic alcoholics promotes the progression ofliver cirrhosis. Sakena H Rashed et al has foundthe Vitamin E levels to be significantly reduced inpatients with cirrhotic liver(6). In the present studyserum Selenium and Vitamin E levels werefound to be significantly lower in alcoholiccirrhosis patients compared to the control group(Table no 2).The Karl Pearson’s correlationcoefficient between serum selenium and vitaminE showed a significant and positive correlation(Table no 3). Serum Bilirubin shows a significantand negative correlation with selenium andvitamin E levels in cases. P Clot et al has foundthe markers of oxidative damage such as plasmalipid hydroperoxide and erythrocytemalondialdehyde to be 4-5 fold higher in alcoholintake of more than 100g/day(7).This shows an altered redox status in theseindividuals with several derangements in thenatural antioxidant defence mechanisms.

Further validation studies are needed to evaluatethe effects of selenium and vitamin Esupplementation in patients with alcoholic liverdisease in attenuating the progression ofhepatitis to cirrhosis. The huge disease burdennecessitates the urgent need for initiation ofnutritional intervention trials in alcoholichepatitis patients to find out the benefits in termsof reduction of morbidity and mortality.Although corner stone of therapy in alcoholichepatitis is abstinence, there is increased risk ofrecidivism in patients who attempt to cut backbut not stop drinking altogether(8). And even ifthe patient becomes abstinent, the risk ofdeveloping cirrhosis is very high(9). So nutritionalintervention if proven beneficial, it may have asignificant role in treatment of alcoholic hepatitisin future.

ConclusionThe present study shows decreased levels ofantioxidant nutrients in patients with alcoholiccirrhosis. Selenium and vitamin Esupplementation at an early stage of alcoholicliver disease may improve the outcome and longterm prognosis in these patients.

Selenium and Vitamin E Status in Patients With Alcoholic Cirrhosis 51

Table 1: Age distribution among the cases and controls

Group N Mean age (yrs) SD Student ‘t’ test

Case 50 43.14 1.4 P=0.653

Control 50 42.18 1.6 Not significant


Table 2 : Mean, Standard deviation and Test of significance of mean values between casesand controls

Mean ± Standard deviationVariable Cases Controls P value

Bilirubin <0.001(μmol/L) 128.2±22 10.4±0.51 Significant

AST <0.001(μkat/L) 1.6±0.25 0.37±0.01 Significant

ALT <0.001(μkat /L) 0.4±0.04 0.2±0.01 Significant

Albumin <0.001(g/L) 28±3 52±0.6 Significant

Selenium <0.001(μmol/L) 0.68±0.05 1.03±0.04 Significant

Vitamin E <0.001(μmol/L) 19±0.7 29.7±1.2 Significant

Table 3 : Correlation of serum Selenium and Vitamin E in Alcoholic Cirrhosis patients

Variables Pearson’s correlation Significance Interpretationcoefficient(r) (p)

Selenium Significant and positive vs 0.785 <0.0001 correlation

Vitamin E

Table 4 : Correlation of Bilirubin with Selenium and Vitamin E in alcoholic cirrhosispatients

Variable Pearson’s correlation Significance Interpretationcoefficient ( r) (p)

Vitamin Evs -0.529 <0.0001 Significant andBilirubin Negative correlation

Seleniumvs -0.425 0.002 Significant andBilirubin Negative correlation


Reference

1. Thomas D Boyer, Michael P Manns, Arun J Sanyal.Zakim & Boyer’s Hepatology. A textbook of LiverDisease. 6th edition, Elsevier Saunders, 2012; Pg 493-505.

2. Carl A Burtis, Edward R Ashwood , David E Bruns.Tietz Textbook of Clinical Chemistry and MolecularDiagnostics. 4th edition. Elsevier Saunders, 2006; pg73-75, 2253-2302

3. Nassema Mehbooali and Mohammad Perwaiz Iqbal.A simple micro method for determination of plasmalevels of alpha tocopherol in Pakistani normal adults.Pak J Pharm Sci 2008; 21: 361-365.

4. Schumann G, Bonora R, Ceriotti F. IFCC PrimaryReference Procedures for the Measurement ofCatalytic Activity Concentrations of Enzymes at 37°C.Part 5. Reference Procedure for the Measurement ofCatalytic Concentration of AspartateAminotransferase. Clin Chem Lab Med, 2002;40:725–733.

5. U. Johansson, F. Johnsson, B. Joelsson, M. Berglund,B. Akesson. Selenium status in patients with livercirrhosis and alcoholism. British Journal of Nutrition1986; 55: 227-233.

6. Sakena H. Rashed, Fatima A.Mohammed and MafazK.Saeid. Antioxidant status and some biochemicalparameters in cirrhotic liver patients. National Journalof Chemistry 2010; 40: 742-751.

7. P Clot , M Tabone, S Arico, E Albano. Monitoringoxidative damage in patients with liver cirrhosis anddifferent daily alcohol intake. Gut 1994; 35: 1637-1643

8. Pendery ML, Maltzman IM, West LJ. Controlleddrinking by alcoholics? New findings and areevaluation of a major affirmative study.Science1982; 217:169-175.

9. Shumaker JB, Resnick RH and Galambos JT.Alcoholic hepatitis: its therapy and prognosis. ProgLiver Dis 1972; 4:567-588.

Test Aims to Identify Heart Attacks Before They Occur

A point-of-care assay to detect a heart attack about to happen, which would give clinicians time tointervene before myocardial damage occurs, is being developed by genomics researchers. They found11 genes that were either significantly up regulated or significantly down regulated in patients with AMI,compared with control subjects. Their panel had a perfect discriminative ability, as indicated by the1.0 area under the curve (AUC) of receiver operating characteristics (ROC).

"We thought that was pretty awesome, but seemingly unbelievable, so we went back and recruitedanother 25 healthy volunteers, in addition to the 23 crashing patients with acute myocardial infarction.Using this 11-gene model, we were able to see an AUC of ROC analysis of 0.99, which we felt wasquite fantastic," The researchers reported.

Many of the genes included in the panel have already been recognized as being involved ininflammatory signaling and vesicular trafficking, such as endothelin-1, an endothelium-specific proteinimplicated in the risk for cardiac disease.

The team also tested the concept on whole blood samples from AMI patients and healthy controlsubjects, using quantitative real-time polymerase chain reaction (PCR) on a test panel of seven of thegenes, and found an AUC of 0.998.

The seven-gene test was a little less robust when they tested it on a third cohort of patients who hadstable coronary disease. Nearly two-thirds of that control set had previously undergone coronaryangioplasty with stenting or coronary artery bypass graft, and one-quarter had atrial fibrillation. Forthis test run, the AUC was 0.85 — not perfect, but still quite impressive.

"We moved from having to use immunomagnetic separation techniques and microarrays of thousandsof genes to a real-time PCR of just seven genes from whole blood, something that could potentiallybe moved into a real clinical setting for relevance," the researchers explained.

Obesity gastric bands may cause more complications than weight loss

Almost half of patients undergoing gastric banding for obesity need to have the device removed,according to a study published in the Archives of Surgery.

About 60 percent of the 82 patients with the device, Allergan Inc.’s Lap–Band, followed over 12 yearsor more needed additional operations. The minimally invasive surgery led to weight loss in roughly18 percent of the patients. About 40% of the 82 patients had major complications. Another 22 percentexperienced relatively minor complications, and almost 50 percent had to have the bands entirelyremoved. In all but a few cases, inadequate weight loss or device breakdown was the reason for bandremoval. The mean reduction in body mass index was 7.8 points.

Selenium and Vitamin E Status in Patients With Alcoholic Cirrhosis 54

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