Paital Biswaranjan
Biochemistry Unit, Department of Zoology and Biotechnology, Utkal University, Bhubaneswar 751004, India ; Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Biomed Res Int. 2014;2014:342958. doi: 10.1155/2014/342958. Epub 2014 May 19.
Hydrogen peroxide (H2O2) level in biological samples is used as an important index in various studies. Quantification of H2O2 level in tissue fractions in presence of H2O2 metabolizing enzymes may always provide an incorrect result. A modification is proposed for the spectrofluorimetric determination of H2O2 in homovanillic acid (HVA) oxidation method. The modification was included to precipitate biological samples with cold trichloroacetic acid (TCA, 5% w/v) followed by its neutralization with K2HPO4 before the fluorimetric estimation of H2O2 is performed. TCA was used to precipitate the protein portions contained in the tissue fractions. After employing the above modification, it was observed that H2O2 content in tissue samples was ≥ 2 fold higher than the content observed in unmodified method. Minimum 2 h incubation of samples in reaction mixture was required for completion of the reaction. The stability of the HVA dimer as reaction product was found to be > 12 h. The method was validated by using known concentrations of H2O2 and catalase enzyme that quenches H2O2 as substrate. This method can be used efficiently to determine more accurate tissue H2O2 level without using internal standard and multiple samples can be processed at a time with additional low cost reagents such as TCA and K2HPO4.
生物样品中的过氧化氢（H₂O₂）水平在各种研究中被用作一个重要指标。在存在H₂O₂代谢酶的情况下对组织组分中的H₂O₂水平进行定量，可能总是会得出错误的结果。本文提出了一种对高香草酸（HVA）氧化法中H₂O₂进行荧光分光光度测定的改进方法。该改进包括先用冷的三氯乙酸（TCA，5% w/v）沉淀生物样品，然后在用K₂HPO₄中和后再进行H₂O₂的荧光测定。TCA用于沉淀组织组分中所含的蛋白质部分。采用上述改进方法后，观察到组织样品中的H₂O₂含量比未改进方法中观察到的含量高≥2倍。反应混合物中的样品需要至少孵育2小时才能完成反应。发现作为反应产物的HVA二聚体的稳定性>12小时。该方法通过使用已知浓度的H₂O₂和作为底物淬灭H₂O₂的过氧化氢酶进行了验证。该方法可以有效地用于更准确地测定组织H₂O₂水平，无需使用内标，并且可以使用TCA和K₂HPO₄等额外的低成本试剂一次处理多个样品。
