Oxidants and Antioxidants Levels in Pulmonary Tuberculosis ...

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Oxidants and Antioxidants Levels in Pulmonary Tuberculosis ...

Biomedical Research 2012; 23 (3): 385-389 ISSN 0970-938XScientific Publishers of IndiaOxidants and Antioxidants Levels in Pulmonary TuberculosisPatients on Antitubercular TreatmentKanchan Mohod and Satish KumarDepartment of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram (Wardha), Maharashtra, India.AbstractSevere oxidative stress has been reported in tuberculosis patients because of malnutritionand poor immunity. However, our knowledge of the antioxidant profile and its relation tolipid peroxidation in tuberculosis is very limited. We analyzed blood samples of patients withpulmonary tuberculosis and controls for oxidative stress markers i.e.; malondialdehyde(MDA), nitric oxide (NO) and antioxidants i.e.; superoxide dismutase (SOD), reduced glutathione(GSH) and vitamin C. A total of 100 cases of pulmonary tuberculosis confirmed bysputum examination and TB ELISA (20 treated with Antitubercular treatment(ATT), 25 underATT and 55 untreated) and 50 healthy controls were evaluated for above parameters bycalorimetric methods. MDA and NO levels were found to be elevated in all the cases withpulmonary tuberculosis compared to healthy controls. However, a more significant elevationwas observed in untreated tuberculosis cases. Cases treated with ATT also showed elevatedlevels these markers. SOD, GSH and vitamin C levels were found to be decreased in tubercularpatients compared to controls. Our findings show oxidative stress in all the cases of pulmonarytuberculosis irrespective of their ATT status. It could be envisaged that oxidativestress might possibly be associated with the pathology associated. Therefore, suitable antioxidantsupplementations may be helpful to protect the patients from free radical attack.Keywords: Free radicals, Oxidative stress, TuberculosisIntroductionAcceptedTuberculosis (TB) is the most feared and dreadful of allthe many afflictions of the human race and ranks amongthe foremost killers of the 21 st century. World TB Day,24 th March 2007 possesses theme of event “TB any whereis TB everywhere”, reflecting a massage of urgency andshared responsibility. Pulmonary tuberculosis is thecommonest form with onset usually insidious and illnessremains unnoticed for sometime. Recently, free radicals /reactive oxygen species (ROS), the oxidants are paid particularattention as they cause lipid peroxidation; damagingthe compounds of all biochemical classes; includingnucleic acid, lipids, proteins, lipoproteins, carbohydratesand connective tissue micro molecules [1] also play animportant role in the pathogenesis of tissue damage inmany clinical disorders [2]. These may contribute towardsinflammation if not neutralized by antioxidants. Oxidativestress results when oxidants predominate over antioxidants.Oxidative stress plays an important role in thepathogenesis of tuberculosis besides other chronic ailmentsowing to the reasons: result of tissue inflammation,poor dietary intake, free radicals burst from activatedmacrophages, poor immunity etc [3-6]. Mycobacteria, theintracellular pathogens grow and replicate in the hostmacrophages. Mycobacteria can induce ROS and reactivenitrogen intermediate (RNI) by activating phagocytes aspart of their protective immune response [7].Although these are important for host defense againstmycobacterium, yet these promote tissue injury and inflammation.Enhanced ROS production in tuberculosisalso contributes to immunosuppression, particularly inthose with impaired antioxidant capacity. Moreover, themalnutrition that is commonly present in TB patients, canadd to the impaired antioxidant capacity in these patients.The data on oxidants and antioxidants profile in TB patientsseems to be limited. Hence, in the present study weestimated the concentration of these oxidants and antioxidantsin pulmonary tuberculosis and rule out the presenceof oxidative stress and if so, its status in course of antituberculartreatment (ATT).Biomedical Research 2012 Volume 23 Issue 3 385


Mohod/KumarThe present study was carried out in the Department ofBiochemistry at Mahatma Gandhi Institute of MedicalSciences, Sevagram. The study was designed as case controlstudy. A total of 100 pulmonary tuberculosis casesattending District Tuberculosis Center, Wardha were includedin this study. According to the AFB grading andATT treatment status, the cases were categorized intofresh untreated TB1 (n=55), under treatment TB2 (n=25)and treated ? (n=20). Age and sex matched healthy individuals(n=50) without past or present history of pulmonaryor extra pulmonary TB or any other aliments wereincluded as Controls. About 5 ml venous blood was collectedform each subject after obtaining inform consentprocured and analyzed for MDA, NO, SOD, GSH andvitamin C.Estimation of malondialdehyde (MDA)MDA was assessed in serum by modified TCA - TBAmethod of Stater. [8]. 0.5 ml of serum sample of the eachsubject was taken in test tubes and 3 ml of 10 % TCA wasadded, mixed well and left to stand for 10 minutes atroom temperature, followed by centrifugation for 15 minutesat 5000 rpm. 2 ml of supernatant fluid from theabove mixture was taken to which 1.5 ml of 0.67 % TBAwas added. A pale pink colour developed, where intensitywas measured at 530 nm.Estimation of nitric oxide (NO)test marked test tube, 1.5 ml plasma followed by additionof 2 ml distilled water, 8 ml of disodium phosphatebuffer, then 1ml of DTNB reagent added to it, and mixedwell. The colour developed rapidly, stable for 10 min. Areagent blank was made using 2 ml of distilled water, 8ml of phosphate buffer and 1 ml of DTNB. Reading wastaken at 412 nm in the spectrometer.Estimation of plasma vitamin CVitamin C was assayed by colorimetric method as describedby Aye Kyaw [12]. 1 ml of fresh plasma wastaken in a centrifuge tube, 1 ml of colour reagent wasadded and mixed thoroughly with a glass rod and allowedto stand for 30 min at room temperature ( reaction iscomplete within 30 min and colour is stable).The mixture was then centrifuged at 3000 rpm for 15 min.The blue colored supernatant was transferred to anothertest tube carefully with the help of pipette without disturbingthe precipitate. Absorbance at 700 nm was readagainst a blank, constituted with distilled water, which issubjected to all treatment simultaneously as test sample.For every set a standard and a blank were run through theprocedure.ResultsNO was estimated in plasma by Griess reagent assay ofLee. [9]. 50 ml of plasma sample was taken in a tube. Toit 450 ml of distilled water and 500 ml of Griess reagentwere added. The reading was taken at 550 nm against theblank containing 500 ml distilled water and 500 ml Griessreagent.Estimation of superoxide dismutase (SOD)SOD was assayed in serum utilizing the principle of inhibitionof auto-oxidation of pyrogallol by SOD enzymeusing the method of Marklund. [10]. The assay mixture ina 3 ml volume consisted of 300 ml of Pyrogallol (0.2mM), 300 ml of EDTA (1 mM), and varying concentrationof standard SOD enzyme or 100 ml of serum in airequilibrated Tris HCI buffer (50 mM, pH - 8.2). Pyrogallolwas added after the addition of all other reagents tostart reaction. Initial 60 sec period was considered as inductionperiod of enzyme. So after 60 sec change in absorbanceat 420 nm at 10 sec interval was recorded to aperiod of 4 mints. Change in absorbance per mint wascalculated.Estimation of reduced glutathione (GSH)GSH was assayed in serum by the method of Beutler.[11]. Two test tubes marked test and blank were taken. InThe MDA (nmol/ml) levels in untreated PTB (TB1)group (8.25+1.21), under ATT (TB2) [6.44+0.62] andtreated (TB3) group (5.39+0.49) were found to be significantlyhigh compared to the levels in healthy controlgroup (4.50+0.43). The NO (µM) levels in TB1(0.54+0.10), TB2 (0.40+0.08) and TB3 (0.30+0.06) werealso found to be significantly high compared to the levelsin healthy control group (0.19+0.05). The levels weredecreased with clinical improvement, but still remainedhigh after a therapy compared to controls, probably due toATT interference (Table 1).The enzyme SOD (unit/ml) activity in TB1 (2.11+0.59),TB2 (2.44+0.34) and TB3 (2.71+0.49) was significantlylow compared to the healthy controls (3.42+0.52). Activitiesof GSH (mg/dl) in TB1 (0.11+0.02), TB2 (0.16+0.04)and TB3 (0.22+0.06) were significantly low compared tonormal healthy controls (0.31+0.07). The vit C (mg/dl)levels in TB1 (0.29+0.05), TB2 (0.43+0.06) and TB3(0.48+0.07 ) were found to be significantly low comparedto the levels in normal healthy controls (0.61+0.1) [Table2]. The levels were increased with clinical improvement,but still remained low after a therapy compared to controls,probably due to ATT interference.Biomedical Research 2012 Volume 23 Issue 3 386


Table 1. Mean (SD) levels of oxidants in different status of pulmonary tuberculosis (PTB) cases and controls.ParametersTB1(n=55)TB2(n=25)TB3(n=20)Controls(n=50)MDA (nmol/ml) 8.25 + 1.21 * 6.44 + 0.62 ** 5.39 + 0.49 ** 4.50 + 0.45NO (µM) 0.54 + 0.10 * 0.40 + 0.08 ** 0.30 + 0.06 ** 0.19 + 0.05Significant (P value


though treated well. It is known that tuberculostatics,anticancer and analgesic drugs generate ROS in thecourse of their metabolisms, which are consequently leadingto oxidative stress beside other toxic effect. Thetreatment with isoniazid (INH) inhibits the biosynthesis ofmycolic acid in the cellular wall of mycobacterium fromone part [23, 24] and the result in ROS generation duringINH metabolism from other [25-27]. Via the ROS production,isoniazid injures the DNA, protein etc. and this isprobably one cause for its adverse side effects, includingits oxidative hepatotoxicity. Walubo [28] further mentionedthat the deficiency of total antioxidant status mightmarkedly increase oxidative stress, possibly affecting theimmune response and predisposing to drug toxicity. Thisis not surprising as regression of TB is a long-lastingprocess, even up to eighteen months after treatment. Plit.[15] demonstrated that even after six months of apparentlysuccessful chemotherapy, tuberculosis was associatedwith increased oxidative stress, which was characterizedby increased levels of circulating lipid peroxides andlow plasma concentration of vitamin E and antioxidant.Wild. [24] found that TB patients showed an increase intotal antioxidant status during therapy but low comparedto controls.Also in extra-pulmonary forms, Sudha. [29] indicated thatthe low blood antioxidant status of tubercular meningitis(TBM) patients compared to controls improved aftertreatment, suggesting the role of radicals in TBM. Similarly,Srinivas [30] reported increased levels of lipid peroxidationproduct MDA and reduced antioxidant defenceslike SOD, GSH, vitamin C and vitamin E in both untreatedand treated urogenital tuberculosis patients comparedto controls.TB patients are unable to produce sufficient amount ofantioxidants to cope up with the increased oxidative stressin them. Antioxidants supplementation along with antituberculartherapy may prove beneficial and may help infast recovery in the management of these cases. Wellcontrolledclinical intervention trails are desirable for futureresearch in this area.AcknowledementsWe thank Mr. D. Mehta, Presidant, Kasturba Health Societyand Dr. Chabra, Dean Mahatma Gandhi Institute ofMedical Sciences for their encouragement. We also thankDr. Kalsait, District Tuberculosis Officer, Wardha fortheir active support.References1. Carrol CE. Oxygen free radicals and human disease. JAnn Int Med 1987; 107: 526-45.Mohod/Kumar2. Sinclair AJ, Barnett AH, Lunec J.. Free radicals andantioxidant system in health and disease. JAMA 1991;7: 409-17.3. Jack Cl, Jackson Ml, Hind CR. Circulating markers offree radical activity in patient with pulmonary tuberculosis.J Tuber Lung Dis 1994; 75: 132-137.4. Grimble RF. Malnutrition and the immune responseImpact of nutrients on cytokine biology in infection. JTrans R Soc Trop Med Hyg 1994; 88: 615-619.5. Nathan CE, Brukner LH, Silverstein SC, Conn ZA.Extra cellular cytolysis by activated macrophages andgranulacytes. Pharmacologic triggering of exfactorcells and the release of hydrogen peroxide. JExp Med 1979; 149: 84-99.6. Favier A, Sappey C, Laclerc P, Faure P, Micound M.Antioxidant status and lipid peroxidation in patients infectedwith HIV. J Chem Biol Interact 1994; 91: 165-180.7. Beers RF, Sizer IW. A spectrophotometer method ofmeasuring the breakdown of hydrogen peroxide bycatalase. J Biol Chem 1952; 195: 133-1408. Stater T F and Swayer B C. Colorimetric method fordetermination of lipid peroxidase. J. Biochem 1971;123: 805-814.9. Lee Du, Kang YJ, Park MK, Lee YS. et al. Effects of13-alkyl-substituted berberine alkalaids on the expressionof Cox-ll TNF-a, iNOS and IL-12 production inLPS - stimulated macrophages. J Life science 2003; 73:1401-1412.10. Marklund S, Marklund G. Estimation of superoxidedismutase. J Eur Biochem 1974;16: 47: 469-74.11. Beutler E, Duron O, Kelly B. Improved Method for thedetermination of blood glutathione. Journal of Laboratoryand Clinical Medicine 1963; 61: 882.12. Aye Kyaw. Biochemistry Research Division. Dept of.Medical Research, Clinical Chimica 1978; 86: 153-15713. Reddy YN, Murthy SV.Krishna DR, Prabhakar MC.Role of free radicals and antioxidants in tuberculosispatients. J Ind Tuberc 2004; 51: 213-218.14. Madefo H, Lindtjorn B, Aukrust P, Berge RK. Circulatingantioxidants and lipid peroxidation products inuntreated tuberculosis patients in Ethiopia. J Am ClinNutr 2003; 78: 117-12215. Plit ML, Theron AJ, Fickl H, Van Renseburg CE,Pendel S, Anderson R. Influence of antimicrobial chemotherapyand smoking status on the plasm concentrationof vitamin C, E and Beta carotene, acute phasereactants, iron and lipid peroxides in patient with pulmonarytuberculosis. J Int Tuberc Lung Dis 1998; 2:590-596.16. Rwangabwoba JM, Fischman H, Semba RD. 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Oxidants and Antioxidants in Tuberculosis18. Winkler BS, Orselli SM, Rex TS. The redox couplebetween glutathione and ascorbic acid: a chemical andphysiological perspective. Free Radic Biol Med 1994;17: 333-349.19. Meister A. Glutathione - ascorbic acid antioxident systemin animals. J Biol Chem 1994; 269: 9397-9400.20. Niki E, Noguchi N, Tsuchihashi H, Gotoh N. Interactionamong vitamin C, vitamin E and B caroteine. Am JClin Nutr 1995; 62 (Suppl): 13225-13265.21. Rock CL, Jacob RA, Bowen PE. Update on the biologicalcharacteristics of the antioxidant micronutrients:vitamin C, vitamin E and the carotenoids. J Am DietAssoc, 1996; 96: 693-704.22. Siez H, Stabl W. Vitamin E and C, B carotene andother carotenoids as antioxidants. Am J Clin Nutr 1995;62: 13158-13218.23. Betts J C, Melaren A, Duncan K.Signature gene expressionprofiles discriminate between isoniazidthiolactomycinand trilosan treated Mycobacterium tuberculosis.Antimicrobial agents and chemotherapy2003; 47: 2903-2913.24. George KM, Yuan Y, Sherman DZR, Berry CE. Thebiosynthesis of cycloropanated mycolic acid in Mycobacteriumtuberculosis. J Biol Chem Online, 1995; 270:27292-27298.25. Wang J, Burger Y, Drlica K. Role of superoxide incatalase-peroxidase mediated isoniazid action againstMycobacteria. Antibacterial agents and chemotherapy1998; 42: 709-711.26. Johnson K, King D, Schultz P. Studies on the mechanismof action of isoniazid and ethionamide in the chemotherapyof tuberculosis. J. of American chemotherapysociety 1995; 117: 5009-5010.27. Albano E, Tomasi A. Spin trapping of free radicalsintermediates produced during the metabolism of isoniazidand iproniazid in isolated hepatocytes. Biochemistryand pharmacology 1987; 36: 2913-2920.28. Walubo A, Smith PJ, Folb PI. Oxidative stress duringantituberculosis therapy in young and elederly patients.Biomed Environ Sci 1995; 8: 106-110.29. Sudha K, Rao A, Rao SN. Rao A. Oxidative stress antioxidantsin tubercular meningitis. Indian J of ClinicalBiochemistry 2002; 17: 34-41.30. Srinivas S. Pragasam V.Muthu V.Oxidative stress inurogenital tuberculosis patients:a predisposing factorfor renal stone formation- amelioration by vitamin Esuppl. Clin Chim Acta 2004; 350: 57-63.Correspondence:Kanchan MohodDepartment of BiochemistryMahatma Gandhi Institute of Medical SciencesSevagram (Wardha) 442 102, MaharashtraIndiaPhone: 09370316651Fax: 07152 284038e mail: drkanchanmohod@gmail.comBiomedical Research 2012 Volume 23 Issue 3 387

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