Study of Oxidative Stress in Different Forms of Leprosy.

BACKGROUND: Leprosy is a chronic infectious disease caused by Mycobacterium leprae. India records the highest number of new leprosy cases in the world. Oxidative stress may play a significant role in leprosy. AIM: The aim of the study was to evaluate oxidative stress in various forms of leprosy and compared to healthy controls.
MATERIALS AND METHODS: Seventy newly diagnosed, untreated leprosy patients were selected as cases and sixty healthy controls. Oxidative stress was evaluated by measuring serum malondialdehyde (MDA) level and superoxide dismutase (SOD) activity. Student's unpaired t-test and Anova (Analysis of Variance) test were used for analysis of data. P < 0.05 was considered as significant.
RESULTS: There was a statistically significant increase in the mean values of serum MDA level, MDA/SOD and a decrease in serum SOD activity in cases when compared to controls and the values were significantly associated with increased duration, bacterial load and multibacillary type in leprosy.
CONCLUSION: Our study suggests that there was oxidative stress in leprosy. This warrants antioxidant supplementation to prevent tissue injury.

What was known?

Oxidative stress plays a key role in the pathogenesis of leprosy

Introduction

Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae. Although the prevalence of leprosy has decreased over past 20 years, the incidence of newly detected cases is still high.[1] India records the highest number of new leprosy cases in the world.[2] Depending on host resistance, leprosy may present as tuberculoid or lepromatous type with a spectrum of intermediate stages appearing between the two.[3]

The pathogenesis of leprosy has been found to be influenced by a number of factors including oxidative stress (OS).[45] The major defense against microbial infection is the macrophage system. The infected macrophages show increased phagocytosis and oxygen consumption known as “respiratory burst,” associated with production of free radicals like reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide and hydroxyl radicals.[6] These free radicals apart from killing the bacteria, also damage host tissues mainly lipids, proteins and nucleic acids.[45]

Cells have the “antioxidant defense” system that comprises enzymatic antioxidants like superoxide dismutase (SOD), catalase, peroxidase and non-enzymatic antioxidants like Vitamins A, E, C and glutathione.[6] Under normal conditions, the production of ROS is presumed to be in balance with antioxidant defenses. OS is a severe disruption of balance in favor of ROS.

In leprosy, this delicately maintained physiological balance is shifted in favor of ROS from phagocytes. The drugs used in multidrug therapy (MDT) also generate ROS and may further increase the damage to host tissues.[57] In addition to increased production of ROS, decreased anti-oxidants contribute to OS in leprosy.[89] Malnutrition co-exists with the depletion of micronutrients and antioxidant vitamins in leprosy and aggravates infection,[10] while infection with intracellular M. leprae adversely affects the nutritional status.[11]

Prime targets of peroxidation by ROS are polyunsaturated fatty acids (PUFA) in membrane lipids, which are degraded to malondialdehyde (MDA).[6] The level of MDA in serum serves as a marker of cellular damage due to free radicals.[12] Another way of studying OS is to investigate the serum antioxidants.[13] SOD is an enzymatic antioxidant that catalyzes the dismutation of superoxide ion into oxygen and hydrogen peroxide. The ratio of MDA/SOD in serum may be considered as an index of OS.[14]

Few studies have been done to report OS in leprosy and its association with disease parameters. Evaluation of OS in leprosy can be of benefit in the prognosis and treatment of the disease.

Aim

The present study was aimed at evaluating oxidative stress in leprosy and to find out whether these changes have a significant association with the duration, bacterial load and type of leprosy.

Materials and Methods

A case-control study was conducted with leprosy patients as cases and healthy volunteers as controls. This study was done during the period from July 2009 to March 2010. Newly diagnosed leprosy patients before the start of MDT, attending the Hansen's clinic of Dermatology Department in Osmania General Hospital, Hyderabad were selected as cases. The patients who were taking any form of antioxidant/multivitamin supplementation or suffering from reactions, ulceration, co-infection and history of smoking, infectious diseases or other major illnesses were excluded from the study. Controls include healthy individuals, without any past history of leprosy disease. Persons with history of smoking or alcoholic habits and other skin diseases and systemic diseases like diabetes mellitus or hypertension were excluded from the study.

This study was approved by institutional ethical committee. After prior consent from the patients, slit skin smear was done to grade Bacteriological Index (B.I) according to Ridley's logarithmic scale.[15] The diagnosis of leprosy is based on clinical grounds into Paucibacillary (P.B) {2 to 5 skin patches} and multibacillary (M.B) {≥6 skin patches} cases as per the WHO Operational classification.[1] Under aseptic precautions, 5 ml of fasting venous blood samples were collected and serum was used for the estimation of MDA by the thiobarbituric acid reactive substances assay method[16] and SOD activity by inhibition of the auto-oxidation of adrenaline method.[17] In the present study, serum total protein values were estimated by Biuret method to express the SOD activity as a fraction of serum total protein. The MDA/SOD ratio was estimated as an index of OS.

The data was analyzed by using SPSS 15.0 version. The results were expressed as Mean ± Standard Error (S.E). Student's unpaired t-test and “ANOVA” (Analysis of Variance) test were used to analyze the data. P < 0.05 was considered as significant.

Results

Seventy newly diagnosed leprosy cases and 60 age and sex-matched controls were enrolled after considering the inclusion and exclusion criteria.

The mean values of serum MDA, MDA/SOD were significantly increased and SOD was significantly decreased in cases when compared to controls [Table 1].

Table 1

Serum MDA and SOD values in controls and leprosy patients

The cases (n = 70) were divided into sub-groups according to the duration of the disease into Group 1 with duration <1 yr (n = 41) and Group 2 with duration >1 yr (n = 29). The study parameters were compared between Group 1, Group 2 of leprosy cases and controls by ANOVA test [Table 2]. The mean values of serum MDA, MDA/SOD were significantly increased and SOD was significantly decreased in Group 2 cases in comparison to Group 1 and controls.

Table 2

Comparison of study parameters in Group 1, Group 2 cases and controls

The cases (n = 70) were further categorized on the basis of B.I into Group 3 consisting of B.I negative (n = 43) and Group 4 with B.I positive (n = 27). ANOVA test was used to analyze the study parameters between Group 3, Group 4 cases and controls [Table 3]. The mean values of serum MDA, MDA/SOD were significantly increased and SOD was significantly decreased in Group 4 cases in comparison to Group 3 and controls.

Table 3

Comparison of study parameters in Group 3, Group 4 of cases and controls

The study parameters were compared between M.B (n = 40), P.B (n = 30) leprosy cases as per WHO operational classification and controls by ANOVA test [Table 4]. The mean values of serum MDA, MDA/SOD were significantly increased and SOD was significantly decreased in M.B cases in comparison to P.B and controls.

Table 4

Comparison of study parameters in M.B, P.B cases and controls using ANOVA test

Discussion

Oxidative stress was suggested to play a key role in the pathogenesis of leprosy by earlier workers.[45] The probable mechanism for increased serum MDA in leprosy, as observed in the present study, is lipid peroxidation by free radicals.[4] The possible reason for the decreased antioxidant status in leprosy cases, as observed in the present study by decreased SOD activity in serum, may be increased utilization to combat increased production of ROS,[18] deranged liver function and the free radical producing ability of drugs used in MDT of leprosy.[519] As leprosy is associated with malnutrition and poor socio-economic conditions,[10] depletion of micronutrients and antioxidant vitamins may lead to decreased antioxidant defense. Vitamins A, C, E[818] and antioxidant minerals like magnesium, zinc[20] have been reported to decrease in leprosy. It was proposed that some component of M. leprae might be down regulating the SOD gene in the host macrophages and other tissues.[21]

Thus, the present study supports the existence of OS in leprosy from increased MDA and decreased SOD activity in leprosy patients. Similar findings were observed by other studies.[1418] The OS observed in leprosy patients could mediate inflammatory episodes, organ damage, depressed cell-mediated immune response and degeneration of nerves in leprosy patients.[9]

In the present study, OS was more pronounced with M.B type of leprosy. It was proposed that in MB cases, there is defective monocyte–macrophage function.[2223] The macrophage shows normal phagocytosis, but they are unable to kill the M. leprae due to inadequate superoxide production.[24] In these cases, the source of ROS could be some other subpopulation of phagocytes in which normal respiratory burst occurs like immunologically activated macrophages, neutrophils and some other sources.[21] Further decreased antioxidant status, as observed in the present study by decreased SOD activity, may also contribute to increased OS in MB leprosy.[9] Similar findings were observed by other studies.[18]

In the present study, OS in leprosy was associated with higher bacterial load. This may be due to utilization of biometals like zinc, iron, calcium, etc. from host tissue for the survival of M. leprae, resulting in their decreased bio-availability which could affect the activity of metalloenzymes like SOD.[11] This may lead to decreased anti-oxidant elements in serum and dominance of ROS leading to OS. This finding was in agreement with other studies.[25] In the present study, the OS increased with duration of the disease. Leprosy is a chronic disease. The bacteria multiply with in host cells and this may lead to constant production of ROS. This may be responsible for cumulative damage in patients with increased duration of the disease.

OS may play a role in immune response in leprosy. The rapidly dividing immune cells are extremely sensitive to OS. Cell membrane peroxidation may cause immunosuppression.[8] ROS induces a selective loss of T-lymphocyte signaling molecules and induces T-lymphocyte hypo responsiveness.[26] The present study supports this view as OS observed in leprosy was more in MB, high bacillary load and increased duration. However, the exact role of OS in pathogenesis of leprosy needs to be established from further experimental studies by correlating OS with immunological markers.

Conclusion

The present study suggests the existence of oxidative stress in leprosy and it was more associated with increased duration of the disease, higher bacterial load and MB type. Thus, administration of nutritional anti-oxidants such as beta carotene, lycopene, flavonoids, selenium, vitamins C and E along with MDT may benefit in the treatment and prognosis of the disease, which can be established by follow-up studies.

What is new?

Oxidative stress in leprosy is associated with duration, bacterial load and Multibacillary type of leprosy