Thulfeqar Hamza1,2, Mahmoud Hussein Hadwan2. 1. Chemistry Dept., College of Science, University of Babylon, Iraq. 2. Pathological Analysis Department, Al-Mustaqbal University College, Hilla City, Babylon Governorate, Iraq.
Abstract
BACKGROUND: Accurate estimation of Prx activity poses many complications and interferences. The present protocol is free of interference and provides an effective alternative for the assessment of peroxide with high sensitivity. The assay can be used in clinical pathology laboratories since it is simple, rapid, and inexpensive. The systematic reagent consisted of AFS/ASA which acted as a sensitive probe for peroxide. METHODS: Prx activity was estimated by incubating samples in suitable concentrations of 1,4-dithio-DL-threitol (DTT) and hydrogen peroxide (H2O2) or t-Butyl hydroperoxide (t-BOOH), as the substrates. The enzymatic reaction was inhibited after incubation with a working reagent containing ammonium ferrous sulfate (AFS) and aminosalicylic acid (ASA). RESULTS: Residual peroxide reacted with the working solution to form a brown-colored ferriaminosalicylate (FAS) complex with a maximum absorbance (λmax) of 425 nm. This protocol used sodium azide (NaN3) to eliminate catalase interference and avoided using high concentrations of strong acid to inhibit the Prx reaction. CONCLUSION: We concluded that the new protocol produced the same efficacy as the reference method since a strong correlation coefficient of comparison (r> 0.99) was found between both the FAS and ferrithiocyanate method.
BACKGROUND: Accurate estimation of Prx activity poses many complications and interferences. The present protocol is free of interference and provides an effective alternative for the assessment of peroxide with high sensitivity. The assay can be used in clinical pathology laboratories since it is simple, rapid, and inexpensive. The systematic reagent consisted of AFS/ASA which acted as a sensitive probe for peroxide. METHODS: Prx activity was estimated by incubating samples in suitable concentrations of 1,4-dithio-DL-threitol (DTT) and hydrogen peroxide (H2O2) or t-Butyl hydroperoxide (t-BOOH), as the substrates. The enzymatic reaction was inhibited after incubation with a working reagent containing ammonium ferrous sulfate (AFS) and aminosalicylic acid (ASA). RESULTS: Residual peroxide reacted with the working solution to form a brown-colored ferriaminosalicylate (FAS) complex with a maximum absorbance (λmax) of 425 nm. This protocol used sodium azide (NaN3) to eliminate catalase interference and avoided using high concentrations of strong acid to inhibit the Prx reaction. CONCLUSION: We concluded that the new protocol produced the same efficacy as the reference method since a strong correlation coefficient of comparison (r> 0.99) was found between both the FAS and ferrithiocyanate method.
Authors: Soo Han Bae; Su Haeng Sung; Hye Eun Lee; Ha Tan Kang; Se Kyoung Lee; Sue Young Oh; Hyun Ae Woo; In Sup Kil; Sue Goo Rhee Journal: Antioxid Redox Signal Date: 2012-05-31 Impact factor: 8.401