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

Xiaoping Liu, Jay L. Zweier

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)894-901
Number of pages8
JournalFree Radical Biology and Medicine
Volume31
Issue number7
DOIs
StatePublished - Oct 1 2001

Keywords

  • Electrochemical sensor
  • Free radicals
  • Hydrogen peroxide
  • Hydrogen peroxide electrode
  • Leukocytes
  • NADPH oxidase

ASJC Scopus subject areas

  • Medicine(all)
  • Toxicology
  • Clinical Biochemistry

Fingerprint Dive into the research topics of 'A real-time electrochemical technique for measurement of cellular hydrogen peroxide generation and consumption: Evaluation in human polymorphonuclear leukocytes'. Together they form a unique fingerprint.

Cite this