This observational study was conducted at B.S. Medical College and Hospital, Bankura, West Bengal, India, from June 2004 to April 2007. The study protocol was approved by the Institutional Ethical Committee (vide Memo No 3/BSMC/04dt 14-05-2004). A total of 133 participants (aged 8-62 years) were enrolled for the study.

The study was conducted in two phases at an interval of one-month.

The subjects were divided into three groups:

Group 1: Control subjects (n=30): The healthy relatives of the tubercular patients, without any clinical signs, symptoms or X-ray finding suggestive of tuberculosis or any sort of diseases were recruited. Their sputum was negative for acid fast bacilli.

Group 2: Disease-control subjects (Lung disease control (n=27): Included OPD patients suffering from Respiratory Tract Infection (RTI) or bronchiectasis or bronchial asthma or bronchogenic carcinoma. None of them had any clinical signs or symptoms of tuberculosis. The subjects were under individual treatment procedure (not certainly under A-TB drug regime).

Group 3: Tubercular subjects (n=76): The Tubercular (TB) patients (irrespective of age, sex and socioeconomic status) admitted in the Isolation Ward and patients attending the Outpatient Department of B.S. Medical College and Hospital were taken into account. Based upon the prior diagnosis, the patients were categorised as Subgroup A: Pulmonary (n=42) and Subgroup B: Extra- Pulmonary (n=34). In all these tubercular subjects the anti-TB drug (A-TB) therapy was started between 0-15 days. Multi Drug Resistant (MDR) TB cases were not considered in this study. The tubercular subjects were under Directly Observed Treatment (DOT) programme under Revised National Tuberculosis Control Programme (RNTCP).

Nota Bene (NB): All the subjects under study (Groups 1,2, 3A and 3B) had normal liver and kidney functions and normal serum glucose level.

The detailed purpose of the study was explained to all the participants and informed verbal consent was obtained, before collection of blood samples. About 2 mL of morning blood sample was collected by venipuncture in plain vacutainer. The samples were allowed to clot for ~2 hours and the serum was obtained by centrifugation at 2200 rpm for 15 minutes. After the centrifugation, the serum was transferred to clean and sterile eppendorf tubes and was stored at 2-4°C until further analysis. The serum samples of lung disease control and tubercular subjects were recollected after 30 days of usual treatment. The serum SOD levels were assayed on the same day of collection.

Serum SOD activity was assayed using Ransod Kit {Randox Laboratories Ltd., Cat No. SD125} as per the manufacturer’s instruction. The absorbance was measured at 505 nm using a spectrophotometer (Baush and Lomb Co.). SOD was measured from the degree of inhibition of the reaction and expressed as unit/mL (U/mL). One unit of SOD is equal to the amount of SOD required to cause 50% inhibition of reduction rate (per minute), i,e., the change of absorbance per minute [13].

For determination of the sensitivity of SOD enzyme activity towards NaCN, the sera of tubercular subjects (both pulmonary and extra-pulmonary) were incubated with 40 μm, 400 μm and 4000 μm of NaCN for two hours at 37°C. For control measures, purified M.tuberculosis iron-cofactored SOD enzyme and also human erythrocyte Cu-Zn co-factored SOD were incubated with the same concentration of NaCN. The activity of SOD enzyme for all the samples was measured using Ransod kit (Randox Laboratories Ltd.,).

For determination of percentage of inhibition of SOD activity, SOD levels prior to incubation with NaCN were taken as baseline 100% value and changes in the SOD activity on incubation with varying concentration of NaCN (40,400 and 4000 μm) were assayed.

The results were analysed using Statistical Software for Social Sciences (SPSS, version 21.0). The level of significance was assessed using independent Student t-test. p<0.05 was considered statistically significant.

The serum SOD levels of both pulmonary and extra-pulmonary tubercular subjects (anti-TB drugs started between 0-15 days) were significantly higher than normal control subjects (p<0.01) and disease control subjects (p<0.01). After an additional treatment with A-TB drug therapy for one month, the serum SOD level fell dramatically (0.05>p>0.02) [Table/Fig-1].

Upon incubation with NaCN, no significant inhibition was observed in the serum SOD activity in the test sera of tubercular subjects. Very low degree of inhibition in the activity of SOD was observed in M.tuberculosis Fe-co-factored SOD and human erythrocyte showed significant inhibition of Cu-Zn SOD activity when incubated with NaCN. Significant inhibition was also observed in serum SOD activity of normal control and lung disease control subjects [Table/Fig-2]. This suggests that the SOD activity in tubercular subjects was of Mycobacterial origin rather than the human (host) origin.

Superoxide dismutase is a ubiquitous metalloenzyme that maintains an optimal redox environment in the bacterial cytoplasm and prevents DNA cross-linking, lipid peroxidation and cysteine-cysteine bonding of essential enzymes [14]. M. tuberculosis secretes large amount of leader peptide-independent iron-co-factored SOD (FeSOD or SOD-A) in the extracellular medium during initial stages of growth [7,15,16]. Fe-SOD scavenges ROIs and contributes to the pathogenicity by inhibiting NF- κB activation and mononuclear cell apoptosis [17,18].

In the first phase of the present study, the serum SOD activity was measured in tubercular subjects on A-TB drugs started between 0-15 days and then after one-month of A-TB treatment. Significantly elevated serum SOD levels were observed in both pulmonary and extra-pulmonary tubercular subjects, which can be used as diagnostic marker for early detection of tuberculosis infection. The serum SOD levels fell by an average of 31% in pulmonary TB subjects and 29% in patients with extra-pulmonary TB after one month of A-TB drug treatment. These findings were similar to Nag D et al, study where SOD levels of tubercular subjects decreased after one-month of A-TB treatment and zinc supplementation [19]. The supplemented zinc binds to thiol group of the free iron necessary for bacterial growth and thus reduces the level of SOD [19-21]. These variations in enzymatic activity and the degree of abundance can be used as a framework for the development of a simple assay to diagnose tuberculosis.

In the second phase of the study, an increase in the degree of inhibition of serum SOD activity was observed in normal and lung disease control subjects on incubation with increasing concentration of NaCN. Cyanide and its derivatives are metabolic inhibitors that cause oxidative stress by inhibition of antioxidant enzymes. Mammalian SOD enzymes (SOD-A and SOD-C) are co-factored with Copper (Cu) and Zinc (Zn) whereas M. tuberculosis SOD enzyme (SOD-A) is complexed to iron (Fe) [9,22]. Human erythrocyte Cu-Zn co-factored SOD showed enhanced inhibition towards NaCN, whereas Fe-co-factored SOD was found to be relatively resistant to NaCN. These varied responses of host and bacterial SOD activity towards NaCN, adds up to the specificity of the assay. These findings also suggest the presence of Fe co-factored bacterial SOD in the serum and thus justifies the elevated SOD levels in tubercular subjects.

In some patients, the SOD levels remained constant at the beginning of the A-TB drug treatment which indicated the primary drug resistance, whilst some patients initially responsive to treatment developed secondary drug resistance and their SOD levels rebounded. Therefore, the patients on A-TB drug treatment should be regularly screened to determine the effectiveness of the ongoing treatment.

The sample size of the study was small, further studies on larger sample size are required. The study was very fundamental, there is scope to include E.coli Fe-co-factored SOD in determining the degree of inhibition by NaCN and the exact degree of resistance of Fe-co-factored M.tuberculosis SOD towards NaCN can also be estimated.

The SOD assay serves as an effective technique for early diagnosis of tuberculosis to prevent the transmission by timely institution of suitable treatment (A-TB drugs). The assay also helps in monitoring course of treatment, detection of primary and secondary drug resistance and diagnosis of both pulmonary and extra-pulmonary, as well as human and bovine tuberculosis cases. Incubation with NaCN, increases the sensitivity of the assay and help in differentiation of human and myco-bacterial origin SOD. Minimal serum requirement and rapid processing time, the assay readily may be used in developing countries with limited resources.