Enzymatic Oxidative Stress Indicators and Oxidative Stress Index in Patients of Leprosy

Introduction: Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae. Oxidative stress caused by Reactive oxygen species (ROS) plays a crucial role in the pathogenesisis of leprosy. Objective: To measure the enzymatic oxidative stress indicators and oxidative stress index in patients of leprosy and compare them with healthy age & sex matched controls. Materials and Methods: In this prospective study 30 untreated leprosy patients were included in the study and matched with 60 healthy controls. Biochemistry estimation was done with blood samples and MDA (lipid peroxidation), Nitric oxide (NO), (SOD) Superoxide dismutase (antioxidant enzyme), total oxidant status (TOS), total antioxidant status(TAS) and oxidative stress index (OSI) were estimated. Results: Highly significant rise (p<0.0001) in serum MDA, NO, TOS and OSI was seen in leprosy patients when compared with controls with highly significant decline in SOD and TAS. Conclusion: The study confirms the oxidative stress in leprosy and suggests antioxidant therapy may be used as an adjuvant in the treatment of leprosy along with MDT.


Introduction
L eprosy (Hansen's disease) is a chronic, granulomatous, mu la ng, visible, ancient, s gma zing disease caused by Mycobacterium leprae and clinically manifests as nodules, plaques, thickened dermis and peripheral nerves. 1 Oxida ve stress (OS) caused by Reac ve oxygen species (ROS) plays a crucial role in the pathogenesisis of leprosy. 2 OS is defi ned as excessive genera on of ROS. 3 Normally the an oxidant defense system is responsible for balancing the produc on of ROS but in leprosy the produc on of ROS exceeds the normal limits resul ng in OS causing further insult to cells, ssue and biomolecules. 3Experimental studies support the possible rela on between ROS and leprosy. 4e 'an oxidant defense system' comprises of enzyma c scavangers of ROS, like SOD (Superoxide dismutase), catalase and glutathione peroxidise and nonenzyma c an oxidants like vitamin E, C and glutathione. 3This dynamic defense system removes the aggressive oxygen species and keeps the concentra on of ROS at normal levels. 3S have only transient existence and cannot be measured directly in vitro; hence the end products of OS are used as laboratory markers. 5Among the biomarkers of OS, Lipid peroxidation (LPO) and Protein carbonylation are the most frequently used reliable laboratory parameters. 5Measurement of serum antioxidants is another way to investigate OS. 6 SOD catalyzes the dismutation of superoxide ion into oxygen and hydrogen peroxide and is considered most reliable to determine enzymatic OS. 6 The present study was aimed to analyze the relationship between OS indices, and enzymatic OS indicators in leprosy cases and to fi nd out whether these changes have a signifi cant association with bacterial load and type of leprosy.

Recruitment of pa ents and controls:
In this prospec ve study, conducted in a ter ary care centre of North India, newly diagnosed adult leprosy pa ents registered in leprosy clinic from July 2014 to December 2015 were included based on below men oned inclusion and exclusion criterion.The study was approved by the Ins tu onal ethical commi ee and is registered in CTRI (CTRI/2017/08/009369).Newly diagnosed leprosy pa ents before the start of an -leprosy treatment, were selected as cases.The pa ents were categorized according to the WHO classifi ca on based on clinical, histological and bacteriological criteria. 8Clinical details included the number and distribu on of lesions, pa ern of nerve involvement, and complica ons including reac ons, neuri s and deformi es.The pa ents who were in reac ons, taking any form of an oxidant/mul vitamin supplementa on, giving history of any other infec ous diseases or other major systemic illnesses were excluded from the study.The Controls were age and sex matched and were apparently healthy individuals on general physical examina on and without any past history of any chronic disease or leprosy.Above men oned exclusion criteria for cases were also applied for controls.Persons with history of any addic on like smoking or alcoholic habits and other chronic skin or systemic diseases like diabetes mellitus, cancer or hypertension were also excluded from the study.
A er ini al screening according to the inclusion and exclusion criteria of all the pa ents, 60 were deemed fi t for the inclusion in the study.Informed and wri en consent were taken from the pa ents for inclusion in the study.Out of 60, only 30 pa ents gave the consent for inclusion in the study.Blood sample was collected from 60 age and sex matched healthy controls and 30 leprosy Pa ents cons tuted the study groups.

Materials and Methods
Five ml blood was drawn from median cubital vein of the pa ents into plain tubes.To separate the serum from the plasma sample, it was centrifuged at 5000 × g for 5 min at room temperature.All serum samples were stored at -20°C un l me of processing.
Lipid peroxida on was measured by the quan fi ca on of MDA (Malondialdehyde) in blood samples of pa ents using the thiobarbituric acid-reac ve substances (TBARS) assay. 8NO (Nitric oxide) was evaluated based on the principle that nitrate (NO3-) present in the serum samples is reduced to nitrite using nitrate reductase and the method is known as Griess Method. 9Measurement of total oxidant status (TOS) was based on principle that oxidant present in the sample oxidizes the ferrous ion-o-dianisidine complex to ferric ions.The oxida on reac on is enhanced by glycerol molecules, which are enormously present in the reac on medium.During this oxida on reac on, the ferric ions make a coloured complex with xylenol orange in an acidic medium and the intensity of this colour is measured spectrophotometrically.This is directly related to the total amount of oxidant molecule present in the sample. 10 The oxida ve stress index (OSI) was calculated from a percent ra o of total oxidant level to the total anoxidant (TAS) level as explained elsewhere. 22I=[ TOS (μmol H 2 O 2 equivalent/L) / TAS (μmol Trolox equivalent/L) X100

Sta s cal analysis:
Results were expressed as mean±SD and these were evaluated using student paired and unpaired t test and z test.P value was considered highly signifi cant if <0.0001 and signifi cant if <0.05.

Results
The fi nal study comprised of 30 pa ents and 60 healthy controls.Table 1 lists the general demographic and clinical characteris cs of case group.Controls were age and sex matched and hence not sta s cally diff erent from study pa ents (Table 1).

Status of oxida ve stress indices in cases and controls:
Total oxidant status was 34.90±15.55mmol in controls and increased in cases 150.06±44.43mmol.This diff erence was highly signifi cant, p value was <0.0001.On the contrary, total an oxidant status was 1.09±0.12μmol in controls and got decreased in cases, 0.39±0.26μmol.This diff erence was also highly signifi cant, p value was <0.0001.On calcula ng the oxida ve stress index there was highly signifi cant increase in cases, 73.39±29.98mmol as compared with controls, 3.15±1.37mmol (Table 3).

Baseline comparison of oxida ve stress parameters with bacterial load and type of leprosy.
The total oxidant status (TOS), total an oxidant status (TAS), oxida ve stress index (OSI) and Malondialdehyde (MDA) levels were decreased in Bacteriological index = 0 in comparison to Bacteriological index ≥ 1However, this was not sta cally signifi cant.The Nitric oxide (NO) and Superoxide dismutase (SOD) were increased in Bacteriological index = 0 in comparison with Bacteriological index ≥ 1, And this increase was also not signifi cant.
The total an oxidant status (TAS) and oxida ve stress index (OSI) and Malondialdehyde (MDA) levels were decreased in Paucibacillary in comparison to Mul bacillary with no sta s cal signifi cance.
The total oxidant status (TOS), Nitric oxide (NO) and Superoxide dismutase (SOD) were increased in Paucibacillary in comparison with Mul bacillary with no signifi cant diff erence (Table 4).

Discussion
The present study is a prospec ve study which includes 60 cases of healthy controls and 30 cases of leprosy pa ents.In modern era, interest has grown in inves ga ng the role played by reac ve oxygen species or oxida ve stress in leprosy leprosy by inves ga ng one or more of the oxidant markers like Malondialdehyde (MDA) and an oxidant markers, including SOD, catalase, vitamin E, vitamin C. [11][12][13][14] Lipid peroxida on is involved in pathogenesis of many disease processes when the imbalance occurs between produc on of reac ve oxygen species and protec ve an oxidant system.Malondialdehyde (MDA) is a marker of lipid peroxida on. 15In the present study, MDA was signifi cantly high in serum of leprosy pa ents (p <0.0001) in comparison to healthy controls.Agnihotri N, et al (1995) no ced increased levels of lipid peroxida on levels in mice infected with M. leprae. 4Bhadwat VR et al in their study showed increased Malondialdehyde levels in leprosy pa ents and more so in lepromatous leprosy. 16 the present study, the ac vity of serum enzyma c an oxidant superoxide dismutase (SOD) was highly signifi cantly low in leprosy cases as compared to control group (p <0.0001).Previous studies have also reported signifi cant reduc on of ac vity of serum SOD in untreated leprosy pa ents. 19,20Regarding nitric oxide produc on, we observed that their levels were signifi cantly higher in leprosy pa ents as compared to healthy controls (p<0.0001).The reason behind it is that Microbial killing of lepra bacilli results in produc on of free radicals like nitric oxide. 21These fi ndings are similar to the study done by Schalcher TR et al who also reported increased produc on of nitric oxide levels in leprosy pa ents as compared to healthy controls and no change in nitric oxide levels were seen a er treatment with an leprosy drugs 11,14 It seems that the disease process itself appears to be responsible for the detected nitric oxide increase in the body irrespec ve of the an leprosytreatment. 14udies have tried to quan fy oxida ve stress as per evalua ng levels of various enzymes and carbonyl proteins.Some of the research analyst reported changes of these markers like Superoxide Dismutase (SOD), Glutathione peroxidase (GPx), Malondialdehyde (MDA), Nitric Oxide (NO), and Catalase. 1,12But these studies didn't measure the total oxida ve stress unlike our present study.Oxida ve stress can be measured by calcula ng the ra o of total oxidant status (TOS) and total an oxidant status (TAS). 10,22In the present study there was a highly signifi cant increase in the total oxidant status in leprosy pa ents in comparison to healthy controls (P<0.0001)and there was a highly signifi cant decrease in mean total an oxidant status in leprosy in comparison to healthy controls (P<0.0001).This supports the theory that genera on of free radicals are involved in the pathogenesis of leprosy. 1,13en we measured oxida ve stress index(OSI) by ra o of TOS/TAS, Highly Signifi cant increase in the OSI was observed in pa ents of leprosy as compared to healthy controls (p <0.0001).Bhadwat VR et al reported increased oxida ve stress by quan fying ra o of (MDA/SOD) in LL leprosy in comparison to TT leprosy. 16Abdel-Hafez HZ et al also reported similar fi ndings. 18Jyothi P et al reported signifi cant eleva on in oxida ve stress by measuring the ra o of (MDA/ SOD) in MB leprosy. 17Hence this study confi rms the presence of oxida ve stress in leprosy and interven on with an oxidant supplementa on may improve the total oxidant capacity of the pa ent by reducing the produc on of reac ve oxygen species(ROS) which in turn improves the oxida ve stress in leprosy.

Conclusion
Our study confi rmed the oxida ve stress in leprosy with highly signifi cant increase in oxida ve stress index in leprosy pa ents.The enzyma c an oxidant superoxide dismutase (SOD) was reduced in leprosy pa ents indica ng enzyma c stress in leprosy.The drawback in our study was less number of pa ents and that we could not measure the nonenzyma c an oxidants in our pa ents.Further studies need to be done on a larger scale with es ma on of enzyma c, non-enzyma c an oxidants and total oxida ve stress in leprosy pa ents.These fi ndings support a hypothesis that any enzyma c supplementa on might have an a rac ve approach in comba ng oxida ve stress mediated insult during the chronic course of the disease process of leprosy.
Financial disclosure: None.Confl icts of interest to disclosure: None declared.
Superoxide Dismutase (SOD) was measured with help of Caymans superoxide dismutase assay kit, (item number 706002 from Caymen chemical company• 1180 East Ellsworth Road• Ann Arbor, Michigan 48108• USA).Total An oxidant status (TAS) was measured with help of Caymans an oxidant assay kit, (item number 709001 from Caymen chemical company • 1180 East Ellsworth Road• Ann Arbor, Michigan 48108• USA).

Table 1 :
Demographic and clinical characteris cs of case group.

Table 2 :
Status of enzyma c oxida ve stress indicators between cases and controls (Z test).

Table 4 :
Comparison of oxida ve stress parameters with bacterial load and type of leprosy (One way ANOVA test).