A real-time electrochemical technique for measurement of cellular hydrogen peroxide generation and consumption: Evaluation in human polymorphonuclear leukocytes

There has been a long-standing need for sensitive and specific techniques for hydrogen peroxide (H₂O₂) measurement. We describe the development and application of a highly sensitive electrochemical sensor, utilizing a membrane-coated platinum microelectrode, suitable for real-time measurement of hydrogen peroxide generation and consumption in biochemical or cellular systems. This sensor provides high sensitivity enabling measurement of hydrogen peroxide down to 5-10 nM concentrations. We demonstrate that it can be used to measure the magnitude and time course of H₂O₂ generation from the NADPH oxidase in leukocytes as well as the rate of H₂O₂ degradation. After human polymorphonuclear leukocytes (PMNs) were activated by phorbol 12-myristate acetate, H₂O₂ concentration increased with time and reached a peak concentration, from 5 to 15 μM in PMNs prepared from different individuals, within 3 to 8 min, then decreased slowly. The H₂O₂ concentration in the solution is less than the total H₂O₂ generation from the activated PMNs because a part of H₂O₂ generated is decomposed. H₂O₂ in solution, generated from the PMNs, was rapidly consumed after the activated PMNs were treated with 10 μM diphenylene iodonium (DPI). The rate of H₂O₂ consumption was measured following the addition of exogenous H₂O₂. The total production of H₂O₂ from the activated PMNs was calculated from the measured H₂O₂ concentration and the rate of H₂O₂ consumption. This technique enables sensitive and continuous real-time measurement of H₂O₂ concentration and total H₂O₂ generation in cellular or enzyme systems without addition of any detection reagents.