Optimising hydrogen peroxide measurement in exhaled breath condensate.
BACKGROUND: Exhaled breath condensate (EBC) analysis has been proposed as a non-invasive method of assessing airway pathology. A number of substances, including hydrogen peroxide (H2O2), have been measured in EBC, without adequate published details of validation and optimisation.OBJECTIVES: To explore factors that affect accurate quantitation of H2O2 in EBC.MATERIALS AND METHODS: H2O2 was measured in EBC samples using fluorometry with 4-hydroxyphenylacetic acid. A number of factors that might alter quantitation were studied including pH and buffering conditions, reagent storage, and assay temperature.RESULTS: Standard curve slope was significantly altered by pH, leading to a potential difference in H2O2 quantification of up to 42%. These differences were resolved by increasing the buffering capacity of the reaction mix. H2O2 added to EBC remained stable for 1 h when stored on ice. The assay was unaffected by freezing assay reagents. The limit of detection for H2O2 ranged from 3.4 nM to 8.8 nM depending on the buffer used.CONCLUSIONS: The reagents required for this assay can be stored for several months allowing valuable consistency in longitudinal studies. The quantitation of H2O2 in EBC is pH-dependent but increasing assay buffering reduces this effect. Sensitive reproducible quantitation of H2O2 in EBC requires rigorous optimisation.[1]References
- Optimising hydrogen peroxide measurement in exhaled breath condensate. Brooks, W.M., Lash, H., Kettle, A.J., Epton, M.J. Redox Rep. (2006) [Pubmed]
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