Fig 1.
Effect of creatine on AAPH/ H2O2 -induced hemolysis of erythrocytes.
Erythrocytes were treated with the indicated concentrations of H2O2 /AAPH, in presence and absence of Cr. After centrifugation, hemolysis was determined from the absorbance of supernatants as described in Materials and Methods. Results are mean values ± SEM from six independent experiments using blood from different donors. *Significantly different from control (p<0.05).
Fig 2.
Effect of creatine on AAPH/ H2O2-induced lipid peroxidation.
Erythrocytes were treated with the indicated concentrations of H2O2 /AAPH, in presence and absence of Cr. Malondialdehyde levels were determined in hemolysates as an index of lipid peroxidation. Results are mean values ± SEM from six independent experiments using blood from different donors. *Significantly different from control (p<0.05). LPO, lipid peroxidation.
Fig 3.
Effect of creatine on AAPH/ H2O2-induced protein oxidation.
Erythrocytes were treated with the indicated concentrations of H2O2/AAPH, in presence and absence of Cr. Carbonyl content was determined in hemolysates as an index of protein oxidation. Results are mean values ± SEM from six independent experiments using blood from different donors. *Significantly different from control (p<0.05).
Fig 4.
Effect of creatine on AAPH/ H2O2-induced changes in glutathione levels.
Erythrocytes were treated with the indicated concentrations of H2O2/AAPH, in presence and absence of Cr. Reduced glutathione (GSH) levels were determined in hemolysates. Results are mean values ± SEM from six independent experiments using blood from different donors. *Significantly different from control (p<0.05).
Table 1.
Effect of creatine on H2O2-induced changes in methemoglobin levels, activity of erythrocyte membrane bound and antioxidant enzymes.
Fig 5.
Effect of creatine on AAPH/ H2O2-induced changes in ferric reducing ability of cells.
Erythrocytes were treated with the indicated concentrations of H2O2/AAPH, in presence and absence of Cr. FRAP assay was done in hemolysates. Results are mean values ± SEM from six independent experiments using blood from different donors. *Significantly different from control (p<0.05).
Fig 6.
Effect of creatine on AAPH/ H2O2-induced changes in DPPH radical scavenging activity.
Erythrocytes were treated with the indicated concentrations of H2O2/AAPH, in presence and absence of Cr. DPPH assay was done in hemolysates. Results are mean values ± SEM from six independent experiments using blood from different donors. *Significantly different from control (p<0.05).
Fig 7.
SEM images of human erythrocytes.
(A) Untreated control; erythrocytes treated with (B) 20 mM Cr alone (C) 5 mM H2O2 alone and (D) 20 mM Cr + 5 mM H2O2. Treatment conditions are described in Materials and Methods section. Magnification is 1000 fold.
Table 2.
Effect of creatinine on AAPH-induced changes in some erythrocyte parameters.
Table 3.
Effect of creatinine on H2O2-induced changes in some erythrocyte parameters.
Table 4.
Effect of creatine on H2O2-induced changes in some lymphocyte parameters.
Fig 8.
Effect of creatine on H2O2-induced decrease in lymphocyte viability.
Human lymphocytes were treated with H2O2 in presence and absence of Cr and then their viability was determined by the MTT assay. Results are mean values ± SEM from five independent experiments using blood from different donors. *Significantly different from control (p<0.05).
Fig 9.
Analysis of DNA damage by comet assay.
DNA damage in human lymphocytes after incubation with H2O2, in presence and absence of Cr, was studied by the Comet assay as described in Materials and Methods. (A) Tail length (B) Olive tail moment. Results are mean values ± SEM from five independent experiments using blood from different donors. *Significantly different from control (p<0.05).
Fig 10.
Schematic representation of H2O2/AAPH toxicity in human erythrocytes and protective effect of creatine.
AAPH and H2O2 enter the cell and, being strong oxidants, increase the generation of free radicals and reactive oxygen species leading to oxidative stress condition. This converts hemoglobin to methemoglobin and decreases the oxygen carrying capacity of erythrocytes. Glutathione levels are lowered which reduces the antioxidant power of cell and results in protein oxidation, lipid peroxidation and membrane damage. All these factors contribute to cell damage which can reduce the lifespan of erythrocytes in blood (red cell senescence) since damaged erythrocytes are removed from circulation by the spleen. Creatine protects the erythrocytes either by inhibiting the generation of free radicals and reactive oxygen species or by directly quenching them by its antioxidant property. This protects the erythrocytes from oxidative damage and can enhance their lifespan. (GSH, glutathione; MetHb, methemoglobin; ROS, reactive oxygen species; FR, free radicals; PO, protein oxidation; LPO, lipid peroxidation; OS, oxidative stress; RBC, red blood cells).