Improved Chemiluminescence Assay for Measuring Antioxidant Capacity of Seminal Plasma
An improved enhanced chemiluminescence antioxidant assay utilizes horseradish peroxidase conjugate and luminol to produce a cell-free oxygen radical generating system. We introduce the use of a peroxidase enzyme stabilizer to prolong the production of oxygen radicals at a steady rate. Addition of antioxidants temporarily interrupts oxygen radical generation, resulting in an inhibition curve. A linear relationship exists between the area of the inhibition curve and the molar quantity of added antioxidant used to quantify total nonenzymatic antioxidant capacity (TAC) in biological fluids including seminal plasma. We streamline the existing enhanced chemiluminescence technique by using a microtiter plate luminometer. A plate luminometer is as accurate as a tube luminometer in measuring TAC, using identical reaction volumes. As little as 1–50 μL of sample may be analyzed. A plate luminometer can detect molar Trolox equivalents as low as 12.5 μM, compared to 25 μM in tube luminometer, using identical volumes. The plate luminometer assay is made even more rapid with use of an injector.
Key wordsOxygen radicals Antioxidant Total antioxidant capacity Oxidative stress Luminometer Chemiluminescence Semen Seminal plasma
The authors thank Ashok Agarwal, Ph.D. of the Cleveland Clinic, Cleveland, Ohio, for sending us his TAC protocol, which we modified for this study. We also thank Professor Seymour Klebanoff, M.D. Ph.D., of the Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, for allowing us to use his tube luminometer and for illuminating discussions. The Paul G. Allen Foundation for Medical Research supported this research.