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Lipid peroxidation, osmotic fragility and antioxidant status in children with acute post-streptococcal glomerulonephritis
Clinica Chimica Acta 308 Ž2001. 155–161 www.elsevier.comrlocaterclinchim
Lipid peroxidation, osmotic fragility and antioxidant status in children with acute post-streptococcal glomerulonephritis T. Devasena a,) , S. Lalitha b, K. Padma c a
Department of Biochemistry, Center for Micronutrient Research, Faculty of Science, Annamalai UniÕersity, Annamalai Nagar 608 002, Tamil Nadu, India b Department of Obstetrics and Gynaecology, UniÕersity of Michigan, Michigan, MI, USA c Department of Pediatrics, Rajah Muthiah Medical College and Hospital, Annamalai UniÕersity, Annamalai Nagar, Tamil Nadu, India Received 7 July 2000; received in revised form 27 February 2001; accepted 16 March 2001
Abstract Plasma and erythrocyte samples from acute post-streptococcal glomerulonephritis ŽAPSGN. children and control children were enrolled in this study. Lipid peroxidation ŽLPO., measured in terms of thiobarbituric acid-reactive substances ŽTBARS. was found to be significantly increased in plasma and RBCs of APSGN children Ž P - 0.05. than in control children. Osmotic fragility of erythrocytes was examined. RBCs of APSGN patients were found to be osmotically more sensitive towards hypotonic saline Ž50% hemolysis at 7 grl saline. when compared to control RBCs Ž50% hemolysis at 4 grl saline.. The activities of antioxidant enzymes superoxide dismutase ŽSOD., catalase ŽCAT., glutathione peroxidase ŽGPx. and glutathione S-transferase ŽGST. were significantly lowered Ž P - 0.05. in APSGN RBCs when compared to control RBCs. Plasma ascorbic acid, reduced glutathione ŽGSH., RBC ascorbic acid, GSH and RBC total sulphydryl content ŽTSH. were significantly depleted in APSGN children relative to controls. The susceptibility of RBCs of APSGN children to lipid peroxidation was confirmed in this study. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Acute post-streptococcal glomerulonephritis ŽAPSGN.; Lipid peroxidation; Osmotic fragility; Antioxidant status
1. Introduction Free radicals such as superoxide O 2(y, nitric oxide NO ( and hydroxyl radicals OH ( are constantly formed in the human body. Lipid peroxidation ŽLPO. is a highly destructive free radical phenomenon that induces a plethora of alterations of membranes. LPO inactivates number of membrane bound enzymes and
)
Corresponding author. Tel.: q91-4144-38343. E-mail address: [email protected] ŽT. Devasena..
protein receptors, induces alterations of respiratory functions and loss of –SH groups from membranebound proteins, mediate DNA and RNA damage w1x. A variety of antioxidant defenses including the enzymes superoxide dismutase ŽSOD., catalase ŽCAT., glutathione peroxidase ŽGPx., glutathione S-transferase ŽGST., together with the antioxidant nutrients such as ascorbic acid, b-carotene and a-tocopherol w2x, have evolved to protect against these free radicals Interest has grown in the role of oxygen-derived free radicals as mediators of tissue damage in many
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disease processes w3x. Glomerulonephritis is of considerable medical importance as it forms the single most common cause of end stage renal failure w4x. Acute post-streptococcal glomerulonephritis ŽAPSGN. is an inflammatory kidney disease caused by b-hemolytic streptococcal infection of the throat and skin w5x. Epidemics of APSGN have been reported in different parts of the world w6x and this disease was found to be predominant among school children of rural community w7x. Though free radical-mediated LPO have an important pathophysiological role in wide range of human diseases including arthritis w8x aging and cancer w3x and minimal change nephrotic syndrome w9x, there have not been many studies performed on the oxidative changes in APSGN children. Peroxidative processes are facilitated by a high content of unsaturated lipid substrates, aerobic conditions, and the presence of metal ions. In erythrocytes, the content of unsaturated lipid is high, oxygen concentration is increased, and iron, the potent peroxidation catalyst, is present in large quantities w10x. Therefore, erythrocytes have often been considered as a useful model reflecting the susceptibility to LPO w11,12x. Peroxidation taking place in RBCs injures the protein via the depletion of thiol groups and oxidation of amino acids, subsequently leading to changes in the permeability, flexibility, fragility and antigenicity of erythrocytes w13,14x. Therefore, the aim of this study is to detect the occurrence of oxidative stress in RBCs of APSGN children. Erythrocyte LPO, osmotic fragility and antioxidant status Ženzymatic and non-enzymatic. in APSGN children, which could throw light on the exact contribution of LPO to disease pathology, were assessed.
2. Materials and methods 2.1. Chemicals 1,1X ,3, 3X-Tetramethoxy propane,5,5X-dithioŽbis. nitro benzoic acid ŽDTNB., phenazine methosulphate ŽPMS., nitro blue tetrazolium ŽNBT., NADH, reduced glutathione ŽGSH. and thiobarbituric acid ŽTBA. were purchased from Sigma ŽSt. Louis, MO..
All other chemicals and solvents were of analytical grade and obtained from S.D. Fine Chemicals ŽBombay. and Fisher Inorganic and Aromatic ŽChennai.. 2.2. Patients Seventeen children suffering from APSGN aging between 2 and 10 years and 20 control children of both sexes chosen from the Pediatric Ward of Raja Muthiah Medical College Hospital, Annamalai University, Annamalai Nagar, Tamil Nadu, India, were studied for the present investigation. Informed consent was obtained from the parents of all the children. APSGN children follow skin infection in the form of infected scabies. The criteria for inclusion of APSGN patients in our study were macroscopic hematuria, acute onset of edema, oilguria, proteinuria, a recent history skinrthroat infection, hypertension, cough, urinary red cell casts, and decreased serum complement levels. Control children had normal urinary findings, with no skin or pharyngeal infection. 2.3. Methods Venous blood was collected from APSGN and control children into heparinized tubes and centrifuged at 1200 = g for 10 min. Plasma was separated and buffy coat was discarded by aspiration. Erythrocytes were washed thrice with cold physiological saline and stored at y48C until analysis. To determine the degree of free radical-induced LPO, one approach is to measure the concentration of compounds thought to be products of free radical attack upon endogenous molecules. Measurement of malondialdehyde by thiobarbituric acid reaction remains the most widely used method for assessing lipid peroxidation due to its simplicity and sensitivity although it has long been criticized as lacking specificity w15x. This assay was based on the formation of a red adduct Žabsorption maximum: 532 nm. between thiobarbituric acid and malondialdehyde, a colorless end product of LPO w16x. Plasma and RBC LPO was measured according to the method of Okhawa et al. w17x and Donnan w18x, respectively.
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at 48C, the reaction was stopped by the addition of glacial acetic acid. The color formed at the end of the reaction was extracted into butanol layer and measured at 520 nm. One unit of the activity was taken as the enzyme reaction, which gave 50% inhibition of NBT reduction per mg of hemoglobin. Catalase ŽCAT. ŽEC 1.11.1.6. catalyse the decomposition of H 2 O 2 producing water and oxygen. CAT was assayed colorimetrically by the method of Sinha w25x. The hemolysate preparation was allowed to split H 2 O 2 for 30 s in the presence of phosphate buffer—0.01 molrl, pH 7. The reaction was stopped by the addition of dichromateracetic acid mixture. The reaction mixture was heated at 608C for 10 min. In the presence of H 2 O 2 dichromate in the acetic acid was converted to perchromic acid and then to chromic acetate when heated. The chromic acetate formed was measured at 620 nm. Values were expressed as mmolrl of H 2 O 2 consumed per minute per mg of hemoglobin. Glutathione peroxidase ŽGPx. ŽEC 1.11.1.9. was estimated by the method of Rotruck et al. w26x. A determined amount of hemolysate was allowed to react with H 2 O 2 in the presence of reduced glutathione and 0.4 molrl Tris buffer ŽpH 7.0. for 10 min. Then the remaining GSH was allowed to react with DTNB and the yellow color developed was read at 412 nm. 2GSHq H 2 O 2
GPx
GSSGq 2H 2 O
6
Activity was expressed as mg of GSH consumedr minrmg of hemoglobin. Glutathione S-transferase ŽGST. ŽEC 2.5.1.18. activity was assayed by the method of Habig et al. w27x. GST catalyse the reaction of 1-chloro-2,4-dinitrobenzene ŽCDNB. with the thiol group of glutathione in the presence of phosphate buffer Ž0.3 molrl, pH 6.5.. CDNBq GSH
GST
CDNB–S-glutathione
6
Reduced glutathione ŽGSH. protect cells against oxidative stress and harmful by products of aerobic life such as hydrogen peroxide, organic peroxide and singlet oxygen. The concentration of GSH in plasma and RBCs was measured by Ellman’s method w19x, based on the development of yellow color when 5,5X-dithioŽbis. nitro benzoic acid ŽDTNB. was added to the protein-free supernatant of the plasma andror RBCs. The reaction was carried out in the presence of phosphate buffer pH 8.0. Ascorbic acid along with GSH and vitamin E plays a key role in protecting cells against oxidative damage due to its ability to interact with variety of oxygen species w20x. Plasma and RBC ascorbic acid concentrations were determined through the method of Roe and Keuther w21x. Protein was precipitated by 6% trichloroacetate acid. Ascorbic acid present in the protein-free supernatant was converted to dehydro ascorbic acid by mixing with acid washed norit and was then coupled with 2,4-dinitro phenyl hydrazine ŽDNPH.. The coupled DNPH was treated with sulfuric acid and converted into an orange red color compound, which was read colorimetrically at 520 nm. The total sulphydryl content ŽTSH. of the erythrocytes was measured according to the method of Sedlak and Lindsay w22x. This method was based on the development of yellow color when DTNB was added to the compounds containing sulphydryl groups to form 1-nitro,5-mercapto benzoic acid, which was read at 412 nm. To determine the activities of RBC antioxidant enzymes, RBC lysates were prepared by freezing and thawing three times in dry ice. The lysates were diluted to 1:5 with distilled water and frozen at y48C until analysis. The hemoglobin concentration in the lysates was determined by Drabkin and Austin’s method w23x. Activities of all antioxidant enzymes assayed were expressed as units of enzyme per mg of hemoglobin. Superoxide dismutase ŽSOD. ŽEC 1.15.1.1. catalyzes the dismutation of superoxyl radical ŽO 2(y . producing H 2 O 2 and a molecule of oxygen. SOD activity was determined by the method of Kakkar et al. w24x. This method was based on the inhibition of formation of NADH-phenazine methosulphate nitro blue tetrazolium formazan. The reaction was initiated by the addition of NADH. After incubation for 90 s
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The CDNB–glutathione conjugate absorbs light at 340 nm and the activity of the enzyme was estimated by the changes in optical density at this wavelength. Values are expressed as mmolrl of CDNB–GSH conjugate formed per min per mg of hemoglobin.
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Table 1 Levels of plasma and erythrocyte TBARS of control and APSGN children
Table 2 Plasma and RBC ascorbic acid, plasma and RBC GSH and RBC total thiol content ŽTSH. of control and APSGN children
Parameters
Control Ž ns 20.
APSGN Ž ns17.
Parameters
Control Ž ns 20.
APSGN Ž ns17.
Plasma TBARS Žnmolrml. Erythrocyte TBARS Žpmolrmg Hb.
2.16"0.06 1.50"0.02
7.19"0.08 ) 5.08"0.08 )
Plasma GSH Žmgrdl. Erythrocyte GSH Žmgrdl. Plasma ascorbic acid Žmgrdl. RBC ascorbic acid Žmgrdl. RBC TSH Žmgrml.
35.50"0.20 46.50"0.60 0.75"0.05 0.90"0.10 10.50"0.56
28.3"0.23 ) 38.80"0.15 ) 0.41"0.01) 0.44"0.05 ) 6.50"0.55 )
Values are mean"S.D. Values in parentheses indicate number of samples. ) Values are statistically significant, P - 0.05.
Erythrocyte osmotic fragility was determined through the method of Dacie w28x. Small aliquots of samples were mixed with a large excess of buffered saline solutions of varying concentrations Ž1–9 grl. in separate tubes. Simultaneously, an aliquot of sample was also mixed with distilled water. All the tubes were incubated at room temperature for 30 min and centrifuged at 1200 = g for 10 min. The supernatant from each tube was read colorimetrically at 540 nm using the supernatant of the tube with RBCs and 9 grl saline as blank. A value of 100% lysis was assigned to the supernatant of the tube with RBC and distilled water. The percentage hemolysis at each
Values are mean"S.D. Values in parentheses indicate number of samples. ) Values are statistically significant, P - 0.05.
saline concentration was calculated using the formula % hemolysis O.D. of the tube with RBCsq Saline s O.D. of the tube with RBCsq distilled water = 100 2.4. Statistical analysis Values are expressed as mean " S.D. Statistical analysis was performed using the Mann–Whitney U-test Žnon-parametric.. The null hypothesis was rejected for P - 0.05.
Fig. 1. Osmotic fragility curves of control and APSGN children Ž n s 15. Žsamples of 15 children selected from the two groups were tested twice.. Mean of % hemolysis was determined and plotted against the corresponding saline concentration. 50% hemolysis of control and APSGN RBCs occurred at 7 and 4 grl of saline, respectively Ž P - 0.05.. Control RBCs were capable of resisting the hypotonicity until a concentration of 4grl. However, APSGN RBCs could resist itself until a saline concentration of 7 grl, owing to increased susceptibility to LPO.
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3. Results
4. Discussion
APSGN children produced a three-fold increase in plasma and RBC peroxide formation Žmeasured in terms of TBARS. when compared with control children ŽTable 1.. Mean values of % hemolysis were plotted against saline concentration and the osmotic fragility curves of control and APSGN erythrocytes shown in Fig. 1. Hemolysis in hypotonic saline was highest in erythrocytes from APSGN children. Control erythrocytes showed the lowest percent lysis when compared to APSGN erythrocytes Ž P - 0.05.. The curve of the APSGN erythrocytes was shifted towards slight right. Fifty percent hemolysis of APSGN erythrocytes occurred at a saline concentration of 7 grl. In control RBCs, 50% hemolysis occurred at a saline concentration of 4 grl. Therefore, the control RBCs were less fragile and able to withstand the hypotonicity whereas APSGN erythrocytes were more fragile and less resistant to hypotonicity. Table 2 represents the levels of plasma and erythrocyte ascorbic acid, plasma and erythrocyte GSH and erythrocyte TSH of APSGN and control children. The levels of ascorbic acid, GSH and TSH were found to be significantly decreased in APSGN relative to children in the control group. A significant decrease in the activities of SOD, CAT, GPx and GST were observed in APSGN children when compared to control children. The data are shown in Table 3.
There is yet no evidence to suggest that APSGN is partially caused by an increase in LPO and deficiency of antioxidant enzymes. On the other hand an increase in reactive oxygen species could result from neutrophils, which has been observed to infiltrate the renal glomeruli in APSGN w29x. This could be the reason for the observed increase in plasma and RBC TBARS in APSGN when compared to control children. The osmotic fragility test of red cells is often performed to evaluate the sensitivity of erythrocytes towards hypotonic saline, i.e., this test reflects the ability of RBCs to take up a certain amount of water before lysing. The ability of normal red cell to withstand its hypotonicity results from its biconcave shape, which allows the cell to increase its volume by about 70% before the surface membrane is stretched. Once this limit is reached, lysis occurs w30x. Therefore, measurement of osmotic fragility provides a useful indicator as to whether a patient’s red cells are normal. Our results shows that in RBCs of APSGN children LPO occur fast enough to injure erythrocytes and invoke hemolysis ŽTable 1.. The end products of LPO like malondialdehyde could induce modification in structure, fluidity and permeability of erythrocyte membrane leading to membrane damage, decreased stability and increased sensitivity of the cells towards hypotonic saline w30x. The RBC GSH level of APSGN children was found to be lower than control children. According to Levander and Welsu w31x, RBCs with decreased GSH content show a shortened survival time and increased susceptibility to hemolysis. This could also lead to increase erythrocytic osmotic fragility. Our earlier studies on RBCs from minimal change nephrotic syndrome have shown that an increase in LPO and a decrease in GSH led to an increase in RBC osmotic fragility w9x. Therefore, it could be inferred that increased LPO and decreased GSH levels plays a causal role in increasing the osmotic fragility of the erythrocytes of APSGN children. APSGN patients showed a significant decrease in the plasma erythrocyte GSH concentration than control children. Reduction in GSH content in kidney disease has also been reported by Milner et al. w32x. RBCs have several lines of defense against oxidative
Table 3 Activity of RBC superoxide dismutase ŽSOD., catalase ŽCAT., glutathione peroxidase ŽGPx. and glutathione S-transferase ŽGST. of control and APSGN children Parameters
Control Ž ns 20.
APSGN Ž ns17.
SOD ŽUrmg Hb. CAT Žmmol H 2 O 2 rsrmg Hb. GPx Žmg GSHrminrmg Hb. GST Žmg GSH–CDNB conjugaterminrmg Hb.
4.45"0.18 26.30"0.20 8.56"0.18 1.40"0.22
2.49"0.19 ) 14.50"0.20 ) 4.55"0.14 ) 0.225"0.01)
Values are mean"S.D. Values in parentheses indicate number of samples. ) Values are statistically significant, P - 0.05.
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stress. GSH together with its related enzymes ŽGPx and GST. mainly interact with hydrogen peroxides and lipid peroxides and functions as the major scavenger of ROS in RBCs w33x. Thus, the detoxification role of GSH could attribute to the decreased levels of GSH in plasma and RBCs of APSGN children. The plasma and erythrocyte ascorbic acid level was significantly lower in APSGN patients than in control children ŽTable 2.. This may have been due to its utilization to protect cells against oxidative damage via its rapid interaction with reactive oxygen species generated by cells infiltrating the glomeruli w20,34x. APSGN is an inflammatory disease w5x, while SOD has been implied as an anti-inflammatory compound protective against superoxide produced by glomerular-infiltrating polymorphonuclear cells w35x. A decrease in RBC SOD activity in APSGN children was found to be due to its synergistic action with GSH, in which GSH scavenges a wide range of free radicals generated during LPO of renal glomeruli and RBCs, while SOD removes the superoxide produced in this process. The observed decrease in CAT activity in APSGN children could be due to its protective antioxidant effect against LPO w36x. CAT is protective against renal mesangial cell killing induced by oxidants, which are generated by infiltrating leucocytes w37x. A significant decrease in the activity of the two GSH-dependent enzymes, GPx and GST were noticed in APSGN children. GSH is the natural substrate for both these enzymes w10x. In the presence of GSH, GPx and GST were utilized as a defense against cytotoxic oxygen species ŽO 2(y, OH ( .-induced LPO. GST acts as a detoxifying enzyme by promoting the conjugation of toxic electrophiles to GSH w38,39x. It could be emphasized that glutathione-related enzymes are primarily committed to protect against erythrocyte oxidation in APSGN children.
5. Conclusion From these preliminary studies on LPO, osmotic fragility and antioxidant status in RBCs of APSGN children, we emphasize three major findings. First, an increase in the oxidative stress-induced LPO, and
an increase in osmotic fragility. Second, a decrease in the level of reduced glutathione, ascorbic acid and total thiol content. Third, a decrease in the activities of the major antioxidant enzymes ŽGPx, GST, SOD and CAT. has been observed. Based upon these findings, we can clearly state that in APSGN children, blood loses its antioxidant defense system and the children might suffer from prolonged oxidative stress with an increased susceptibility to LPO and osmotic lysis. Therefore, if these APSGN children are supplemented with antioxidants Že.g., vitamin E, Curcumin, etc.. along with antibiotics, it could prove beneficial in quick amelioration of the disease. Investigations on the effectiveness of antioxidant supplementation with antibiotic to APSGN children are underway in our laboratory.
Acknowledgements We acknowledge Prof. Sathyamoorthy and Prof. Sivasamy, Department of Statistics, Annamalai University, for their assistance in statistical analysis.
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Lipid peroxidation, osmotic fragility and antioxidant status in children with acute post-streptococcal glomerulonephritis T. Devasena a,) , S. Lalitha b, K. Padma c a
Department of Biochemistry, Center for Micronutrient Research, Faculty of Science, Annamalai UniÕersity, Annamalai Nagar 608 002, Tamil Nadu, India b Department of Obstetrics and Gynaecology, UniÕersity of Michigan, Michigan, MI, USA c Department of Pediatrics, Rajah Muthiah Medical College and Hospital, Annamalai UniÕersity, Annamalai Nagar, Tamil Nadu, India Received 7 July 2000; received in revised form 27 February 2001; accepted 16 March 2001
Abstract Plasma and erythrocyte samples from acute post-streptococcal glomerulonephritis ŽAPSGN. children and control children were enrolled in this study. Lipid peroxidation ŽLPO., measured in terms of thiobarbituric acid-reactive substances ŽTBARS. was found to be significantly increased in plasma and RBCs of APSGN children Ž P - 0.05. than in control children. Osmotic fragility of erythrocytes was examined. RBCs of APSGN patients were found to be osmotically more sensitive towards hypotonic saline Ž50% hemolysis at 7 grl saline. when compared to control RBCs Ž50% hemolysis at 4 grl saline.. The activities of antioxidant enzymes superoxide dismutase ŽSOD., catalase ŽCAT., glutathione peroxidase ŽGPx. and glutathione S-transferase ŽGST. were significantly lowered Ž P - 0.05. in APSGN RBCs when compared to control RBCs. Plasma ascorbic acid, reduced glutathione ŽGSH., RBC ascorbic acid, GSH and RBC total sulphydryl content ŽTSH. were significantly depleted in APSGN children relative to controls. The susceptibility of RBCs of APSGN children to lipid peroxidation was confirmed in this study. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Acute post-streptococcal glomerulonephritis ŽAPSGN.; Lipid peroxidation; Osmotic fragility; Antioxidant status
1. Introduction Free radicals such as superoxide O 2(y, nitric oxide NO ( and hydroxyl radicals OH ( are constantly formed in the human body. Lipid peroxidation ŽLPO. is a highly destructive free radical phenomenon that induces a plethora of alterations of membranes. LPO inactivates number of membrane bound enzymes and
)
Corresponding author. Tel.: q91-4144-38343. E-mail address: [email protected] ŽT. Devasena..
protein receptors, induces alterations of respiratory functions and loss of –SH groups from membranebound proteins, mediate DNA and RNA damage w1x. A variety of antioxidant defenses including the enzymes superoxide dismutase ŽSOD., catalase ŽCAT., glutathione peroxidase ŽGPx., glutathione S-transferase ŽGST., together with the antioxidant nutrients such as ascorbic acid, b-carotene and a-tocopherol w2x, have evolved to protect against these free radicals Interest has grown in the role of oxygen-derived free radicals as mediators of tissue damage in many
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disease processes w3x. Glomerulonephritis is of considerable medical importance as it forms the single most common cause of end stage renal failure w4x. Acute post-streptococcal glomerulonephritis ŽAPSGN. is an inflammatory kidney disease caused by b-hemolytic streptococcal infection of the throat and skin w5x. Epidemics of APSGN have been reported in different parts of the world w6x and this disease was found to be predominant among school children of rural community w7x. Though free radical-mediated LPO have an important pathophysiological role in wide range of human diseases including arthritis w8x aging and cancer w3x and minimal change nephrotic syndrome w9x, there have not been many studies performed on the oxidative changes in APSGN children. Peroxidative processes are facilitated by a high content of unsaturated lipid substrates, aerobic conditions, and the presence of metal ions. In erythrocytes, the content of unsaturated lipid is high, oxygen concentration is increased, and iron, the potent peroxidation catalyst, is present in large quantities w10x. Therefore, erythrocytes have often been considered as a useful model reflecting the susceptibility to LPO w11,12x. Peroxidation taking place in RBCs injures the protein via the depletion of thiol groups and oxidation of amino acids, subsequently leading to changes in the permeability, flexibility, fragility and antigenicity of erythrocytes w13,14x. Therefore, the aim of this study is to detect the occurrence of oxidative stress in RBCs of APSGN children. Erythrocyte LPO, osmotic fragility and antioxidant status Ženzymatic and non-enzymatic. in APSGN children, which could throw light on the exact contribution of LPO to disease pathology, were assessed.
2. Materials and methods 2.1. Chemicals 1,1X ,3, 3X-Tetramethoxy propane,5,5X-dithioŽbis. nitro benzoic acid ŽDTNB., phenazine methosulphate ŽPMS., nitro blue tetrazolium ŽNBT., NADH, reduced glutathione ŽGSH. and thiobarbituric acid ŽTBA. were purchased from Sigma ŽSt. Louis, MO..
All other chemicals and solvents were of analytical grade and obtained from S.D. Fine Chemicals ŽBombay. and Fisher Inorganic and Aromatic ŽChennai.. 2.2. Patients Seventeen children suffering from APSGN aging between 2 and 10 years and 20 control children of both sexes chosen from the Pediatric Ward of Raja Muthiah Medical College Hospital, Annamalai University, Annamalai Nagar, Tamil Nadu, India, were studied for the present investigation. Informed consent was obtained from the parents of all the children. APSGN children follow skin infection in the form of infected scabies. The criteria for inclusion of APSGN patients in our study were macroscopic hematuria, acute onset of edema, oilguria, proteinuria, a recent history skinrthroat infection, hypertension, cough, urinary red cell casts, and decreased serum complement levels. Control children had normal urinary findings, with no skin or pharyngeal infection. 2.3. Methods Venous blood was collected from APSGN and control children into heparinized tubes and centrifuged at 1200 = g for 10 min. Plasma was separated and buffy coat was discarded by aspiration. Erythrocytes were washed thrice with cold physiological saline and stored at y48C until analysis. To determine the degree of free radical-induced LPO, one approach is to measure the concentration of compounds thought to be products of free radical attack upon endogenous molecules. Measurement of malondialdehyde by thiobarbituric acid reaction remains the most widely used method for assessing lipid peroxidation due to its simplicity and sensitivity although it has long been criticized as lacking specificity w15x. This assay was based on the formation of a red adduct Žabsorption maximum: 532 nm. between thiobarbituric acid and malondialdehyde, a colorless end product of LPO w16x. Plasma and RBC LPO was measured according to the method of Okhawa et al. w17x and Donnan w18x, respectively.
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at 48C, the reaction was stopped by the addition of glacial acetic acid. The color formed at the end of the reaction was extracted into butanol layer and measured at 520 nm. One unit of the activity was taken as the enzyme reaction, which gave 50% inhibition of NBT reduction per mg of hemoglobin. Catalase ŽCAT. ŽEC 1.11.1.6. catalyse the decomposition of H 2 O 2 producing water and oxygen. CAT was assayed colorimetrically by the method of Sinha w25x. The hemolysate preparation was allowed to split H 2 O 2 for 30 s in the presence of phosphate buffer—0.01 molrl, pH 7. The reaction was stopped by the addition of dichromateracetic acid mixture. The reaction mixture was heated at 608C for 10 min. In the presence of H 2 O 2 dichromate in the acetic acid was converted to perchromic acid and then to chromic acetate when heated. The chromic acetate formed was measured at 620 nm. Values were expressed as mmolrl of H 2 O 2 consumed per minute per mg of hemoglobin. Glutathione peroxidase ŽGPx. ŽEC 1.11.1.9. was estimated by the method of Rotruck et al. w26x. A determined amount of hemolysate was allowed to react with H 2 O 2 in the presence of reduced glutathione and 0.4 molrl Tris buffer ŽpH 7.0. for 10 min. Then the remaining GSH was allowed to react with DTNB and the yellow color developed was read at 412 nm. 2GSHq H 2 O 2
GPx
GSSGq 2H 2 O
6
Activity was expressed as mg of GSH consumedr minrmg of hemoglobin. Glutathione S-transferase ŽGST. ŽEC 2.5.1.18. activity was assayed by the method of Habig et al. w27x. GST catalyse the reaction of 1-chloro-2,4-dinitrobenzene ŽCDNB. with the thiol group of glutathione in the presence of phosphate buffer Ž0.3 molrl, pH 6.5.. CDNBq GSH
GST
CDNB–S-glutathione
6
Reduced glutathione ŽGSH. protect cells against oxidative stress and harmful by products of aerobic life such as hydrogen peroxide, organic peroxide and singlet oxygen. The concentration of GSH in plasma and RBCs was measured by Ellman’s method w19x, based on the development of yellow color when 5,5X-dithioŽbis. nitro benzoic acid ŽDTNB. was added to the protein-free supernatant of the plasma andror RBCs. The reaction was carried out in the presence of phosphate buffer pH 8.0. Ascorbic acid along with GSH and vitamin E plays a key role in protecting cells against oxidative damage due to its ability to interact with variety of oxygen species w20x. Plasma and RBC ascorbic acid concentrations were determined through the method of Roe and Keuther w21x. Protein was precipitated by 6% trichloroacetate acid. Ascorbic acid present in the protein-free supernatant was converted to dehydro ascorbic acid by mixing with acid washed norit and was then coupled with 2,4-dinitro phenyl hydrazine ŽDNPH.. The coupled DNPH was treated with sulfuric acid and converted into an orange red color compound, which was read colorimetrically at 520 nm. The total sulphydryl content ŽTSH. of the erythrocytes was measured according to the method of Sedlak and Lindsay w22x. This method was based on the development of yellow color when DTNB was added to the compounds containing sulphydryl groups to form 1-nitro,5-mercapto benzoic acid, which was read at 412 nm. To determine the activities of RBC antioxidant enzymes, RBC lysates were prepared by freezing and thawing three times in dry ice. The lysates were diluted to 1:5 with distilled water and frozen at y48C until analysis. The hemoglobin concentration in the lysates was determined by Drabkin and Austin’s method w23x. Activities of all antioxidant enzymes assayed were expressed as units of enzyme per mg of hemoglobin. Superoxide dismutase ŽSOD. ŽEC 1.15.1.1. catalyzes the dismutation of superoxyl radical ŽO 2(y . producing H 2 O 2 and a molecule of oxygen. SOD activity was determined by the method of Kakkar et al. w24x. This method was based on the inhibition of formation of NADH-phenazine methosulphate nitro blue tetrazolium formazan. The reaction was initiated by the addition of NADH. After incubation for 90 s
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The CDNB–glutathione conjugate absorbs light at 340 nm and the activity of the enzyme was estimated by the changes in optical density at this wavelength. Values are expressed as mmolrl of CDNB–GSH conjugate formed per min per mg of hemoglobin.
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Table 1 Levels of plasma and erythrocyte TBARS of control and APSGN children
Table 2 Plasma and RBC ascorbic acid, plasma and RBC GSH and RBC total thiol content ŽTSH. of control and APSGN children
Parameters
Control Ž ns 20.
APSGN Ž ns17.
Parameters
Control Ž ns 20.
APSGN Ž ns17.
Plasma TBARS Žnmolrml. Erythrocyte TBARS Žpmolrmg Hb.
2.16"0.06 1.50"0.02
7.19"0.08 ) 5.08"0.08 )
Plasma GSH Žmgrdl. Erythrocyte GSH Žmgrdl. Plasma ascorbic acid Žmgrdl. RBC ascorbic acid Žmgrdl. RBC TSH Žmgrml.
35.50"0.20 46.50"0.60 0.75"0.05 0.90"0.10 10.50"0.56
28.3"0.23 ) 38.80"0.15 ) 0.41"0.01) 0.44"0.05 ) 6.50"0.55 )
Values are mean"S.D. Values in parentheses indicate number of samples. ) Values are statistically significant, P - 0.05.
Erythrocyte osmotic fragility was determined through the method of Dacie w28x. Small aliquots of samples were mixed with a large excess of buffered saline solutions of varying concentrations Ž1–9 grl. in separate tubes. Simultaneously, an aliquot of sample was also mixed with distilled water. All the tubes were incubated at room temperature for 30 min and centrifuged at 1200 = g for 10 min. The supernatant from each tube was read colorimetrically at 540 nm using the supernatant of the tube with RBCs and 9 grl saline as blank. A value of 100% lysis was assigned to the supernatant of the tube with RBC and distilled water. The percentage hemolysis at each
Values are mean"S.D. Values in parentheses indicate number of samples. ) Values are statistically significant, P - 0.05.
saline concentration was calculated using the formula % hemolysis O.D. of the tube with RBCsq Saline s O.D. of the tube with RBCsq distilled water = 100 2.4. Statistical analysis Values are expressed as mean " S.D. Statistical analysis was performed using the Mann–Whitney U-test Žnon-parametric.. The null hypothesis was rejected for P - 0.05.
Fig. 1. Osmotic fragility curves of control and APSGN children Ž n s 15. Žsamples of 15 children selected from the two groups were tested twice.. Mean of % hemolysis was determined and plotted against the corresponding saline concentration. 50% hemolysis of control and APSGN RBCs occurred at 7 and 4 grl of saline, respectively Ž P - 0.05.. Control RBCs were capable of resisting the hypotonicity until a concentration of 4grl. However, APSGN RBCs could resist itself until a saline concentration of 7 grl, owing to increased susceptibility to LPO.
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3. Results
4. Discussion
APSGN children produced a three-fold increase in plasma and RBC peroxide formation Žmeasured in terms of TBARS. when compared with control children ŽTable 1.. Mean values of % hemolysis were plotted against saline concentration and the osmotic fragility curves of control and APSGN erythrocytes shown in Fig. 1. Hemolysis in hypotonic saline was highest in erythrocytes from APSGN children. Control erythrocytes showed the lowest percent lysis when compared to APSGN erythrocytes Ž P - 0.05.. The curve of the APSGN erythrocytes was shifted towards slight right. Fifty percent hemolysis of APSGN erythrocytes occurred at a saline concentration of 7 grl. In control RBCs, 50% hemolysis occurred at a saline concentration of 4 grl. Therefore, the control RBCs were less fragile and able to withstand the hypotonicity whereas APSGN erythrocytes were more fragile and less resistant to hypotonicity. Table 2 represents the levels of plasma and erythrocyte ascorbic acid, plasma and erythrocyte GSH and erythrocyte TSH of APSGN and control children. The levels of ascorbic acid, GSH and TSH were found to be significantly decreased in APSGN relative to children in the control group. A significant decrease in the activities of SOD, CAT, GPx and GST were observed in APSGN children when compared to control children. The data are shown in Table 3.
There is yet no evidence to suggest that APSGN is partially caused by an increase in LPO and deficiency of antioxidant enzymes. On the other hand an increase in reactive oxygen species could result from neutrophils, which has been observed to infiltrate the renal glomeruli in APSGN w29x. This could be the reason for the observed increase in plasma and RBC TBARS in APSGN when compared to control children. The osmotic fragility test of red cells is often performed to evaluate the sensitivity of erythrocytes towards hypotonic saline, i.e., this test reflects the ability of RBCs to take up a certain amount of water before lysing. The ability of normal red cell to withstand its hypotonicity results from its biconcave shape, which allows the cell to increase its volume by about 70% before the surface membrane is stretched. Once this limit is reached, lysis occurs w30x. Therefore, measurement of osmotic fragility provides a useful indicator as to whether a patient’s red cells are normal. Our results shows that in RBCs of APSGN children LPO occur fast enough to injure erythrocytes and invoke hemolysis ŽTable 1.. The end products of LPO like malondialdehyde could induce modification in structure, fluidity and permeability of erythrocyte membrane leading to membrane damage, decreased stability and increased sensitivity of the cells towards hypotonic saline w30x. The RBC GSH level of APSGN children was found to be lower than control children. According to Levander and Welsu w31x, RBCs with decreased GSH content show a shortened survival time and increased susceptibility to hemolysis. This could also lead to increase erythrocytic osmotic fragility. Our earlier studies on RBCs from minimal change nephrotic syndrome have shown that an increase in LPO and a decrease in GSH led to an increase in RBC osmotic fragility w9x. Therefore, it could be inferred that increased LPO and decreased GSH levels plays a causal role in increasing the osmotic fragility of the erythrocytes of APSGN children. APSGN patients showed a significant decrease in the plasma erythrocyte GSH concentration than control children. Reduction in GSH content in kidney disease has also been reported by Milner et al. w32x. RBCs have several lines of defense against oxidative
Table 3 Activity of RBC superoxide dismutase ŽSOD., catalase ŽCAT., glutathione peroxidase ŽGPx. and glutathione S-transferase ŽGST. of control and APSGN children Parameters
Control Ž ns 20.
APSGN Ž ns17.
SOD ŽUrmg Hb. CAT Žmmol H 2 O 2 rsrmg Hb. GPx Žmg GSHrminrmg Hb. GST Žmg GSH–CDNB conjugaterminrmg Hb.
4.45"0.18 26.30"0.20 8.56"0.18 1.40"0.22
2.49"0.19 ) 14.50"0.20 ) 4.55"0.14 ) 0.225"0.01)
Values are mean"S.D. Values in parentheses indicate number of samples. ) Values are statistically significant, P - 0.05.
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stress. GSH together with its related enzymes ŽGPx and GST. mainly interact with hydrogen peroxides and lipid peroxides and functions as the major scavenger of ROS in RBCs w33x. Thus, the detoxification role of GSH could attribute to the decreased levels of GSH in plasma and RBCs of APSGN children. The plasma and erythrocyte ascorbic acid level was significantly lower in APSGN patients than in control children ŽTable 2.. This may have been due to its utilization to protect cells against oxidative damage via its rapid interaction with reactive oxygen species generated by cells infiltrating the glomeruli w20,34x. APSGN is an inflammatory disease w5x, while SOD has been implied as an anti-inflammatory compound protective against superoxide produced by glomerular-infiltrating polymorphonuclear cells w35x. A decrease in RBC SOD activity in APSGN children was found to be due to its synergistic action with GSH, in which GSH scavenges a wide range of free radicals generated during LPO of renal glomeruli and RBCs, while SOD removes the superoxide produced in this process. The observed decrease in CAT activity in APSGN children could be due to its protective antioxidant effect against LPO w36x. CAT is protective against renal mesangial cell killing induced by oxidants, which are generated by infiltrating leucocytes w37x. A significant decrease in the activity of the two GSH-dependent enzymes, GPx and GST were noticed in APSGN children. GSH is the natural substrate for both these enzymes w10x. In the presence of GSH, GPx and GST were utilized as a defense against cytotoxic oxygen species ŽO 2(y, OH ( .-induced LPO. GST acts as a detoxifying enzyme by promoting the conjugation of toxic electrophiles to GSH w38,39x. It could be emphasized that glutathione-related enzymes are primarily committed to protect against erythrocyte oxidation in APSGN children.
5. Conclusion From these preliminary studies on LPO, osmotic fragility and antioxidant status in RBCs of APSGN children, we emphasize three major findings. First, an increase in the oxidative stress-induced LPO, and
an increase in osmotic fragility. Second, a decrease in the level of reduced glutathione, ascorbic acid and total thiol content. Third, a decrease in the activities of the major antioxidant enzymes ŽGPx, GST, SOD and CAT. has been observed. Based upon these findings, we can clearly state that in APSGN children, blood loses its antioxidant defense system and the children might suffer from prolonged oxidative stress with an increased susceptibility to LPO and osmotic lysis. Therefore, if these APSGN children are supplemented with antioxidants Že.g., vitamin E, Curcumin, etc.. along with antibiotics, it could prove beneficial in quick amelioration of the disease. Investigations on the effectiveness of antioxidant supplementation with antibiotic to APSGN children are underway in our laboratory.
Acknowledgements We acknowledge Prof. Sathyamoorthy and Prof. Sivasamy, Department of Statistics, Annamalai University, for their assistance in statistical analysis.
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