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Developing S yrin OX total antioxidant capacity assay for measuring antioxidants in humans
Author(s) -
Prasetyo Endry N.,
Knes Otto,
Nyanhongo Gibson S.,
Guebitz Georg M.
Publication year - 2013
Publication title -
international journal of experimental pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.671
H-Index - 72
eISSN - 1365-2613
pISSN - 0959-9673
DOI - 10.1111/iep.12001
Subject(s) - antioxidant , abts , chemistry , oxidative stress , radical , antioxidant capacity , peroxidase , chromatography , enzyme , biochemistry , food science , dpph
Summary Accurate monitoring of the antioxidant status or of oxidative stress in patients is still a big challenge in clinical laboratories. This study investigates the possibility of applying a newly developed total antioxidant capacity assay method based on laccase or peroxidase oxidized syringaldazine [Tetramethoxy azobismethylene quinone ( TMAMQ )] which is referred to here as S yrin OX , as a diagnostic tool for monitoring both oxidative stress and antioxidant status in patients. Attempts to adapt the R andox total antioxidant procedure [simultaneous incubation of the radical generating system (metmyoglobin and H 2 O 2 ) and antioxidant sample] for S yrin OX were abandoned after it was discovered that the H 2 O 2 reacted with enzymatically generated TMAMQ and ABTS radicals at a rate of 6.4 × 10 −2 /μM/s and 5.7 × 10 −3 /μM/s respectively. Thus this study for the first time demonstrates the negative effects of H 2 O 2 in the R andox system. This leads to erroneous results because the total antioxidant values obtained are the sum of radicals reduced by antioxidants plus those reacting with the radical generating system. Therefore they should be avoided not only for this particular method but also when using other similar methods. Consequently, S yrin OX is best applied using a three‐step approach involving, production of TMAMQ , recovery and purification (free from enzyme and other impurities) and then using TMAMQ for measuring the total antioxidant capacity of samples. Using this approach, the reaction conditions for application of S yrin OX when measuring the total antioxidant capacity of plasma sample were determined to be 50% (v/v) ethanol/50 mM sodium succinate buffer pH 5.5, between 20 and 25 °C for at least 1 h.