Level of oxidative stress in the red blood cells of patients with liver cirrhosis.
Methods: Fifty patients admitted with the complication of liver cirrhosis (with bleeding complications, n=30 and without bleeding complications, n=20) were included in the study. Age and sex matched normal healthy volunteers (n=45) served as controls. The levels of oxyhaemoglobin and methaemoglobin were assayed in the red blood cells. Oxidative stress markers such as lipid peroxides, lipid hydroperoxides and nitric oxide were determined along with enzymatic antioxidants. Membrane bound adenosine triphosphatases, cytosolic glucose-6-phosphate dehydrogenase and NADH-methaemoglobin reductase were also measured. The levels of cholesterol and total phospholipids were assessed in red blood cell membrane. The osmotic fragility of red blood cells was monitored using different concentrations of sodium chloride.
Results: The level of methaemoglobin was significantly higher (P < 0.001) in the red blood cells of liver cirrhotic patients with bleeding complication compared to that of non bleeding patients. The activity level of NADH-methaemoglobin reductase was significantly lower (P<0.001) compared to that of normal subjects. Levels of oxidative stress markers including nitric oxide were found to be higher in patients. The levels of enzymatic antioxidants were low except of glutathione peroxidase. The activity levels of adenosine triphosphatases were also found to be significantly lower (P<0.001) in patients compared to normal subjects. A significant alteration (P<0.05) was found in membrane cholesterol/phospholipid ratio of cirrhotic bleeders. Osmotic fragility of red blood cells was also altered in patients.
Interpretation & conclusion: In cirrhotic condition red blood cells are subjected to severe oxidative stress with significant alterations in the membrane properties.
Key words Bleeding--cirrhosis--membrane integrity--methemoglobin--oxidative stress--red blood cells
Cirrhosis is a worldwide health problem affecting 15-20 per cent of the total population. It is the end stage of liver fibrosis characterized by nodule formation (1). Life-threatening complications associated with cirrhosis are portal hypertension, and oesophageal variceal bleeding (2). Some patients also suffer with external bleeding.
Cirrhosis is commonly associated with abnormalities in the systemic circulation and impaired primary haemostasis (3). Gastrointestinal and oesophageal variceal bleeding are the major causes of death in patients with cirrhosis. The red blood cells (RBC) in haemorrhagic blood have been found to be lysed at 20-30 per cent level (4). In general, the oxidative stress and the red cell membrane integrity play important role in cell lysis. Many reports showed the defective function of platelets as the cause of variceal bleeding in cirrhosis (5). But, the information regarding functional status of RBC in cirrhosis is limited. Therefore, the present study was undertaken to demonstrate the level of oxidative stress, enzyme activities and lipids responsible for the membrane integrity of RBC in patients with liver cirrhosis.
RBCs are subjected to oxidative stress during their normal aerobic functions. But, in normal healthy subjects this stress is balanced by a powerful enzymatic and nonenzymatic antioxidant system, failing which may probably disturb capillary perfusion and result in cell lysis (6). So, the level of oxidative stress markers and antioxidants were determined in RBCs of patients with fiver cirrhosis.
Experimental studies have shown that elevated nitric oxide (NO) production plays an important role in platelet abnormalities in liver cirrhotic patients (7). It has been reported that under hypoxic conditions, NO accelerates its own consumption by increasing its entry into RBC where it is metabolized to form peroxynitrite (8). So the levels of NO, nitrosothiol (NOSO), oxyhaemoglobin (oxyHb) and methaemoglobin (met-Hb) were quantified in RBCs along with met-Hb reductase activity.
Material & Methods
Subjects: All consecutive patients registered in the Department of Surgical Gastroenterology and Proctology, Stanley Medical College and Hospital, Chennai, and Department of Digestive Health Diseases, Government Peripheral Hospital, Gandhi Nagar, Chennai, during July 2005-May 2006 were enrolled in this study. The sample consisted of 50 patients in the age group of 30-40 yr of both sex. The sample size was decided by the Ethical Committee based on the statistical data available in the Hospital Registration Unit. The liver cirrhotic patients were divided into 2 groups. Patients with bleeding complication were considered as group 2 (n = 30) and non-bleeders were considered as group 3 (n = 20). Age and sex matched healthy subjects including the relatives of the patients and staff members with normal liver functions confirmed by liver biochemistry were included as control subjects in group 1 (n = 45). Case history collected from the patients furnished the details regarding their habits and symptoms. The bleeding complication of the patients was confirmed by Doppler use, ultrasound and endoscopy. Group 2 patients had variceal bleeding (n = 22). Few patients also had external bleeding in nose (n = 5) and in gums (n = 3). The clinical data such as platelet count, levels of albumin, bilirubin and prothrombin time, etc. were also recorded. All these analytic procedures were done by using standard kits in the laboratory of Biochemistry-UGC Project Wing at Bharathi Women's College, Chennai.
The clinical and diagnostic characteristics of the patients and controls are presented in Table I. Five milliliters of blood was collected from each patient before any prescribed treatment and processed for RBC preparation. The proposed plan of work was approved by the Ethical Committee of Stanley Medical College and Hospital. Written consent was obtained from each subject.
Reagents: Reduced glutathione, Sulphanilamide, N-(1-Napthyl) ethylene diamine dihydrochloride (NEDD), NADH, 2,6-dichlorophenol indophenol (DCPIP) were purchased from Sigma-Aldrich Company, Bangalore, India. All the other reagents and chemicals used were of analytical grade.
Methods: RBC membrane was isolated according to the procedure of Dodge et al (9). The concentration of oxy-and met-Hb were determined spectrophotometrically in the haemolysate by measuring the absorbance at 577 and 630 nm as described by Winterbourn (10) (oxyHb [concentration of heme, [micro]M] = 66 [A.sub.577] - 80 [A.sub.630]; metHb [concentration of heme, [micro]M] = 279 [A.sub.630] - 3 [A.sub.577]). The levels were expressed as percentage of total Hb.
NADH- met-Hb reductase activity was assayed by measuring the rate of decrease in absorbance at 600 nm resulting from the reduction of DCPIP (11). The enzyme activity was expressed as units/100mg Hb.
The method of Draper & Hadley (12) was adopted for the estimation of lipid peroxides (LPO). The colour intensity of the pink coloured chromogen was measured at 533 nm. The lipid peroxide content was expressed as mM malondialdehyde/g Hb. Lipid hydroperoxides (LHP) were detected by their ability to oxidize ferrous ion under acidic condition in the presence of xylenol orange resulting in the formation of a chromophore with absorption maxima at 560 nm (13). The values were expressed as nM/g Hb.
Estimation of NO in terms of total nitrite was based on the original methods of Fiddler (14) using Griess reagent. Hydrogenated cadmium beads were used as the reducing system. The level was expressed as nM nitrite/mg Hb. The total-SH content was determined by the method of Moron et al (15). The addition of dithionitrobenzoic acid (DTNB) to compounds containing sulphydryl groups yield a yellow coloured complex, the intensity of which is directly proportional to the amount of -SH groups and measured at 412 nm. The values were expressed as jag GSH/g Hb.
Estimation of nitrosothiol (NOSO) is based on the method of Cook et al (16). The concentration was expressed as nM nitrite/mg Hb. Glucose-6-phosphate dehydrogenase (G6PD) was estimated by the method of Deutsch (17). The activity was expressed as units/g Hb.
Superoxide dismutase (SOD) activity was assayed by the method of Kakkar et al (18) and was expressed as units/g Hb. The method of Sinha (19) was adopted for the assay of catalase (CAT). The activity was expressed as [micro]M [H.sub.2][O.sub.2] decomposed/min/g Hb. The method of Rotruck et al (20) was adopted for the assay of glutathione peroxidase (GSH-Px). The remaining GSH content was measured by Ellman's reaction. The activity was expressed as nM GSH consumed/min/mg Hb.
[Na.sup.+] [K.sup.+] ATPase activity was measured according to the method of Bonting (21) in the presence of [Na.sup.+] and [K.sup.+] ions. The level of [Mg.sup.2+] dependant ATPase was determined by the method of Ohinishi et al (22) and [Ca.sup.2+] dependant ATPase by the method of Hijerton and Pan (23) using ATP as substrate in the presence of [Mg.sup.2+] and [Ca.sup.2+] ions respectively. The activity was expressed as nM of phosphorus liberated/min/mg protein.
Lipids were extracted from erythrocyte membrane by the method of Folch et al (24). The lipid extract was redissolved in chloroform-methanol mixture and aliquots of the final lipid extract were used for the estimation of cholesterol and phospholipids. Total phospholipids were estimated in terms of inorganic phosphorus by the method of Fiske and Subbarow (25) after perchloric acid digestion. Cholesterol was estimated by using ferric acetate-uranyl acetate reagent (26). The lipid levels were expressed as [micro]g/ mg protein. The osmotic fragility of erythrocytes was measured in heparinized blood by suspending the red cells at different concentrations of sodium chloride ranging from 0.8 to 0.1 per cent. The percentage haemolysis was measured at 560 nm in the supernatant (27). Heinz bodies were counted as violet coloured objects after staining the blood smear with 0.1 per cent crystal violet solution. The count was expressed as number of stained cells/100 total RBCs.
Statistical analysis: Data were analyzed using SPSS for Windows V.7.5. One way analysis of variance (ANOVA) was performed to find out the significance of variations between three groups followed by Student's t-test and Spearman's correlation test was conducted for correlation analyses.
A significantly low level of oxyHb and high level of metHb were observed in cirrhotic patients with bleeding complication. The activity level of NADH-met-Hb reductase was also found to be significantly low (P<0.001) (Table II). The levels of LPO, LHP and NO in the RBCs of liver cirrhotic patients were significantly high in bleeders (P<0.001) and in nonbleeders (P<0.05) compared to controls.
In the RBCs of patients with liver cirrhosis significantly low level of thiol content was observed when compared to that of normal subjects. The cirrhotic patients showed a significant elevation (P<0.001) in the level of NOSO (Table III). The activity level of GSHPx in both the groups of cirrhotic patients was found to be significantly high (P<0.05, <0.001) when compared with normal controls. Low activity levels of superoxide dismutase and catalase were seen in cirrhotic cases (Table III).
The activity levels of adenosine triphosphatases in the RBC membrane were found to be low in cirrhotic patients. Statistically, significant difference were also seen in the activity levels of [Na.sup.+], [K.sup.+]-(P<0.001) and [Mg.sup.2+]-dependant ATPases (P<0.01) among the bleeders and non-bleeders, whereas [Ca.sup.2+]-dependant ATPase did not show any significant difference (Table IV). The membrane cholesterol-phospholipid (C/PL) ratio was also significantly higher (P<0.01) in patients with both bleeding and non-bleeding complications compared to that of normal subjects. Level of osmotic fragility of RBCs was determined at different concentrations of sodium chloride. Lysis of cirrhotic RBCs was observed at less hypotonic sodium chloride concentration when compared to that of normal RBCs (Fig.)
A significant negative correlation between LPO-antioxidants and metHb-NADH reductase (-0.4812, P<0.05) and a significant positive correlation between LPO-NO, LPO-metHb and NO-metHb (Table V), were observed.
The results of our study showed high level of met-Hb and low level of oxyhaemoglobin in the RBCs of liver cirrhotic patients. The high level of met-Hb in cirrhotic RBC might be partly due to low level of met-Hb reduction.
Hypoxia has been reported in experimentally induced cirrhosis by Corpechot et al (28). Hypoxemia has also been shown in hepatic cirrhosis (29). The present results showed high level of met-Hb in RBCs of patients with liver cirrhosis. The oxygen carrying capacity of met-Hb is significantly less which might cause hypoxia in liver cirrhotic patients.
NO level measured in terms of total nitrite content was found to be high in the RBCs of liver cirrhotic patients. There exists a crucial relationship between NO, oxygen, Hb and RBC for appropriate dilation of blood vessels and delivery of oxygen to tissues. It has been shown that RBC membrane has little pumps for NO (30) and most of the Hb binds NO to one of the four iron atoms, the same place it binds oxygen and renders NO to a sulphur atom at another specific site creating NOSO.
Free radical mediated oxidative stress was evidenced by the elevated levels of LPO and LHP in liver cirrhotic patients. Imbalance between the pro-oxidants and/ or free radicals on one hand and antioxidising systems on the other may be responsible for oxidative stress. The targeted cells are represented by the cell membrane which is particularly rich in unsaturated fatty acids, sensitive to oxidation reactions. The accumulation of lipid peroxides introduce hydrophilic moieties into the membrane hydrophobic phase and thus alters membrane permeability and cell functions (31).
A cell can tolerate a mild oxidative stress but a higher disturbance between the production of free radicals and the antioxidant action results in membrane damage and corresponding pathological consequences. The direct evidence for catalase as the predominant hydrogen peroxide removing enzyme in human erythrocytes has been given by Mueller et al (32). Superoxide dismutase protect RBCs to a certain degree from the harmful effects of superoxide radicals. Glutathione peroxidase catalyzes the breakdown of inorganic and organic peroxides and prevents lipid peroxidation and protects the cell membrane from oxidative damage (33). Reduced glutathione functions in several ways as a substrate for GSH-Px and glutathione-S-transferase, as well as a direct antioxidant, independent of the enzymes protecting the cell membrane from oxidative damage (15). It is believed that the two enzymes, catalase and GSH-Px, protect the RBCs against peroxides that are generated intracellularly or exogenously. The high activity level of GSH-Px may be due to the counter-regulation with oxidative stress. The oxidative stress was comparatively more in bleeders when compared to that of non-bleeders. Nalini et al (34) have shown a significant positive correlation between gamma glutamyl transferase and lipid peroxides in alcoholic cirrhosis.
RBCs deficient in G6PD are susceptible to oxidation and haemolysis (35). The present study showed decreased level of G6PD which might have contributed for less resistance to hypotonic lysis. This is also supported by the presence of Heinz bodies in the blood smear which are the denatured fragments of Hb due to oxidative stress.
The levels of cholesterol and total phospholipids in RBC membrane were found to be altered in liver cirrhosis patients. It has been shown by Grattaglianoa et al (36) that erythrocyte membrane modification is supposed to reflect those of hepatocytosis. The fluidity of RBC membrane which is also maintained by C/PL ratio was found to be altered in patients with liver cirrhosis.
The activities of membrane ATPases which contain essential--SH groups were found to be significantly low in liver cirrhotic patients. Nicotera et al (37) have associated the loss of critical protein sulphydryl groups with inactivation of [Ca.sup.++]-ATPase. [Na.sup.+]-[K.sup.+]-ATPase, involved in the active transport, is altered inversely with membrane lipid ratio (38). The loss of total--SH content might be responsible for the low level of enzyme activities. Kakimoto et al (39) have shown an inverse relationship between ATPases activity and C/PL ratio of erythrocytes in hepatic cirrhosis. We found that the membrane alterations were comparatively greater in bleeders when compared to those of non-bleeders.
The study on osmotic fragility showed that haemolysis began in less hypotonic solution in the RBCs of liver cirrhotic patients which shows the highly fragile nature of RBCs. This effect may partly be due to the membrane alterations observed in terms of C/PL ratio and ATPase activities. The results of the present study showed that the oxidative stress was high in RBCs of liver cirrhotic patients which was reflected by the altered membrane properties. The level of alterations observed in the biochemical parameters of patients without bleeding complication was comparatively less than that of bleeders.
The authors acknowledge University Grants Commission, New Delhi for the financial support.
Received July 11, 2006
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Reprint requests: Dr. A. Geetha, Reader in Biochemistry, Bharathi Women's College (Autonomous), North Chennai Chennai 600108, India email: firstname.lastname@example.org, email@example.com
A. Geetha, M.D. Lakshmi Priya, S. Annie Jeyachristy & R. Surendran *
P.G. Department of Biochemistry, Bharathi Women's College (Autonomous) & * Stanley Medical College & Hospital, Department of Surgical Gastroenterology & Proctology, Chennai, India
Table I. Characteristics of patients and control subjects Characteristics No. of cases Cirrhotic patients Normal Controls Total number 50 45 Male/female ratio 35/15 35/10 Age, yr (range) 30-40 32-45 Transaminases SGOT(< 20 IU/1) 5 45 (> 20 IU/1) 45 - SGPT(< 20 IU/1) 4 45 (> 20IU/1) 41 - Grade (according to Child - Pugh class) A 8 - B 12 - C 30 - Platelet count, cells/[micro]l < 1x[10.sup.5] 35 - > 1x[10.sup.5] 15 - > 2.5x[10.sup.5] - 45 Albumin level, g/dl > 3.5 6 45 2.8 to 3.5 12 - < 2.8 32 - Prothrombin time, sec < 4 7 45 4 to 6 19 - 6 24 - Ascites 30 - SGOT, serum glutamate oxaloacetate transaminase SGPT, serum glutamate pyruvate transaminase Table II. Levels of oxyhaemoglobin, methaemoglobin, NADH-methaemoglobin reductase and oxidative stress markers in the RBC of liver cirrhotic patients and normal controls Normal controls Parameters (n=45) Oxyhaemoglobin 90.2 [+ or -] 18.04 (% of total Hb) Methaemoglobin 19.73 [+ or -] 3.60 (% of total Hb) NADH-methaemoglobin 5.18 [+ or -] 0.7 reductase (units/ nitrite/mg Hb) Lipid peroxides (mM 22.5 [+ or -] 3.3 malondialdehyde/g Hb) Lipid hydroperoxides 39.1 [+ or -] 4.9 (nM/g Hb) Nitric oxide (nM of 2.6 [+ or -] 0.51 nitrite/mg Hb) Cirrhotic patients Parameters Bleeders (n=30) Oxyhaemoglobin 59.48 [+ or -] 1.03 (++) (% of total Hb) Methaemoglobin 29.13 [+ or -] 5.82 (++) (% of total Hb) NADH-methaemoglobin 3.96 [+ or -] 0.4 (++) reductase (units/ nitrite/mg Hb) Lipid peroxides (mM 36.9 [+ or -] 5.1 (++) malondialdehyde/g Hb) Lipid hydroperoxides 49.1 [+ or -] 6.0 (++) (nM/g Hb) Nitric oxide (nM of 3.6 [+ or -] 0.81 (++) nitrite/mg Hb) Cirrhotic patients Non-bleeders Parameters (n=20) Oxyhaemoglobin 86.10 [+ or -] 17.22 (+) ** (% of total Hb) Methaemoglobin 24.35 [+ or -] 4.38 (+) * (% of total Hb) NADH-methaemoglobin 4.09 [+ or -] 0.4 (+) * reductase (units/ nitrite/mg Hb) Lipid peroxides (mM 30.6 [+ or -] 4.2 (+) * malondialdehyde/g Hb) Lipid hydroperoxides 43.5 [+ or -] 5.5 (+) * (nM/g Hb) Nitric oxide (nM of 3.0 [+ or -] 0.72 (+) * nitrite/mg Hb) P (+) < 0.05 (++) < 0.001 compared to normal controls P * <0.05 ** < 0.001 compared to bleeders Table III. Levels of total sulphydryl content, nitrosothiol, glucose 6-phosphate dehydrogenase, glutathione peroxidase, superoxide dismutase and catalase in the RBC of liver cirrhotic patients and normal controls Parameters Normal controls (n=45) Total -SH content ([micro]g 21.6 [+ or -] 3.1 GSH/g Hb) Nitrosothiol (nM nitrite/mg Hb) 1.1 [+ or -] 0.20 Glucose 6- phosphate 0.936 [+ or -] 0.18 dehydrogenase (Unit/g Hb) Heinz bodies (No. of stained 3 [+ or -] 1 cells/100 red blood cells) Glutathione peroxidase (nM 20.70 [+ or -] 8.3 GSH consumed / min/mg Hb) Superoxide dismutase 60.37 [+ or -] 6.38 (units/g Hb) Catalase ([micro]M 322.18 [+ or -] 60 [H.sub.2][O.sub.2] decomposed/min/g Hb) Cirrhotic patients Parameters Bleeders (n=30) Total -SH content ([micro]g 15.6 [+ or -] 1.7 (++) GSH/g Hb) Nitrosothiol (nM nitrite/mg Hb) 1.96 [+ or -] 0.26 (++) Glucose 6- phosphate 0.677 [+ or -] 0.13 (++) dehydrogenase (Unit/g Hb) Heinz bodies (No. of stained 14 [+ or -] 2 (++) cells/100 red blood cells) Glutathione peroxidase (nM 38.68 [+ or -] 7.7 (++) GSH consumed / min/mg Hb) Superoxide dismutase 52.70 [+ or -] 4.7 (++) (units/g Hb) Catalase ([micro]M 211.78 [+ or -] 50 (++) [H.sub.2][O.sub.2] decomposed/min/g Hb) Cirrhotic patients Parameters Non-bleeders (n=20) Total -SH content 16.2 [+ or -] 1.8 (+) ([micro]g GSH/g Hb) Nitrosothiol (nM nitrite/mg Hb) 1.50 [+ or -] 0.26 (+) ** Glucose 6- phosphate 0.815 [+ or -] 0.17 ** dehydrogenase (Unit/g Hb) Heinz bodies (No. of stained 11 [+ or -] 2 (+) * cells/100 red blood cells) Glutathione peroxidase (nM 32.05 [+ or -] 6.4 (+) GSH consumed / min/mg Hb) Superoxide dismutase 53.88 [+ or -] 4.4 (+) (units/g Hb) Catalase ([micro]M 276.75 [+ or -] 55 (+) * [H.sub.2][O.sub.2] decomposed/min/g Hb) P (+) < 0.05 (++) <0.001 compared to normal controls P * < 0.05 ** <0.001 compared to bleeders Table IV. Activity levels of adenosine triphosphatases and the levels of cholesterol and total phospholipids in the RBC membrane isolated from normal healthy volunteers and from liver cirrhotic patients Normal controls Parameters (n=45) Na+,K+-ATPase (nM of 0.52 [+ or -] 0.10 Pi liberated/min/mg protein) Mg+ -ATPase (nM of Pi 0.15 [+ or -] 0.03 liberated/min/mg protein) Ca2+ -ATPase (nM of Pi 1.33 [+ or -] 0.27 liberated/min/mg protein) Cholesterol [C] ([micro]g/ 9.6 [+ or -] 1.8 mg protein) Phospholipid [PL] 11.6 [+ or -] 2.5 ([micro]g/mg protein) C/PL ratio 0.862 [+ or -] 1.0 Cirrhotic patients Parameter Bleeders (n=30) Na+,K+-ATPase (nM of 0.28 [+ or -] 0.05 (++) Pi liberated/min/mg protein) Mg+ -ATPase (nM of Pi 0.10 [+ or -] 0.03 (++) liberated/min/mg protein) Ca2+ -ATPase (nM of Pi 0.79 [+ or -] 0.1 (++) liberated/min/mg protein) Cholesterol [C] ([micro]g/ 9.8 [+ or -] 2.0 (@) mg protein) Phospholipid [PL] 9.0 [+ or -] 1.8 (+) ([micro]g/mg protein) C/PL ratio 1.1 [+ or -] 0.15 (+) Cirrhotic patients Parameter Non-bleeders (n=20) Na+,K+-ATPase (nM of 0.42 [+ or -] 0.09 ([DELTA)] ** Pi liberated/min/mg protein) Mg+ -ATPase (nM of Pi 0.13 [+ or -] 0.03 (#) ** liberated/min/mg protein) Ca2+ -ATPase (nM of Pi 0.89 [+ or -] 0.17 ([DELTA][DELTA])(@) liberated/min/mg protein) Cholesterol [C] ([micro]g/ 9.7 [+ or -] 1.8 (@) mg protein) Phospholipid [PL] 9.9 [+ or -] 1.8 ([DELTA]) ** ([micro]g/mg protein) C/PL ratio 0.96 [+ or -] 0.09 (#) * Values are mean [+ or -] SD (++) P < 0.001, (+) P < 0.01, (@)--not significant, compared to normal subjects ([DELTA][DELTA]) P < 0.001, ([DELTA]) P < 0.01, (#) P < 0.05, (@)--not significant, compared to normal subjects *** P < 0.001, ** P < 0.01, * P < 0.05, (@)--not significant, compared to bleeders Table V. Rank correlation between oxidative stress markers and antioxidants in patients with liver cirrhosis rs oxyHb-metHb -0.4786 metHb-NADH-metHb reductase -0.4812 NO-LPO 0.4959 LPO-metHb 0.5247 NO-metHb 0.5100 LPO-GSH-Px 0.5028 LPO-CAT -0.4943 LPO-SOD -0.5265 G6PD-GSH-Px -0.5020 All correlation coefficient (Spearman's rho-[r.sub.s) values were significant at P < 0.005 LPO, lipid peroxides; NO, nitric oxide; CAT, catalase; G6PD, glucose-6-phosphate dehydrogenase; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase
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|Author:||Geetha, A.; Priya, Lakshmi; Jeyachristy, S. Annie; Surendran, R.|
|Publication:||Indian Journal of Medical Research|
|Article Type:||Clinical report|
|Date:||Sep 1, 2007|
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