12th International Symposium on Bioluminescence & Chemiluminescence

Symposium abstracts:

The ABEL^® peroxynitrite antioxidant test with Pholasin^® measures the antioxidant capacity of plasma to protect against peroxyl radical attack

Knight, Jan¹, Ganderton, Maria¹, Hothersall, John², Nourooz-Zadeh, Jaffar³

1.Knight Scientific Limited, 15 Wolseley Close Business Park, Plymouth, PL2 3BY, UK Email: Knightscientific@cs.com

2.University College London, Institute of Urology& Nephrology, , University College London, Middlesex Hospital Campus, London W1W 7EY UK

Email: J.hothersall@ucl.ac.uk

3. Department of Medicine, University College London, Middlesex Hospital Campus, London W1W 7EY UK

Email: rmhajnz@ucl.ac.uk

Pholasin^®, reacts with peroxynitrite to emit light. In the ABEL^® (Analysis By Emitted Light) antioxidant assay peroxynitrite is formed by the reaction between superoxide and nitric oxide, released simultaneously and continually from SIN-1 (3-morpholino-sydnonimime HCl). When SIN-1 is injected into a microplate well containing Pholasin^®, light of gradually increasing intensity is detected reaching a peak after a few minutes. When antioxidants capable of scavenging peroxynitrite are present, these compete with Pholasin^® for peroxynitrite delaying the time at which the peak occurs. The antioxidant capacity of the sample is equated to VEA, a Vitamin E analogue (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) with a standard curve derived from the times to peak of different concentrations. This assay was used to assess the capacity of samples of plasma to protect against peroxyl radical attack. The peroxyl radical generator, 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH;10mM) was heated at 50^oC with human plasma for up to 300 minutes; water replaced AAHP in controls; samples were taken at recorded times. The rate of consumption of antioxidants increased with increasing time of exposure to AAPH; after 240 minutes 82-87% had been consumed; between 240 and 300 minutes all the antioxidants had been consumed and the oxidized plasma demonstrated pro-oxidant activity. There was no significant loss of antioxidants from the controls.

This is a preprint of an article accepted for publication in Luminescence: Copyright 2001 John
Wiley & Sons, Ltd (Wiley website)