I am trying to measure a snapshot of the hydrogen peroxide levels in plasma using a quick assay that consists of Horse Radish peroxidase (HRP).
Whenever I spike the plasma samples to be tested, with a specific concentration of hydrogen peroxide, the hydrogen peroxide is undetectable when measured 15 minutes later. This suggests that any endogenous hydrogen peroxide in the plasma will decompose just as fast as soon as the plasma samples are brought to room temperature.
Is there a way to stop hydrogen peroxide levels in my plasma samples from decomposing that wouldn't affect HRP activity of the assay?
Hi Andrey,
That was very useful thanks you. My only concern is that my assay uses HRP to detect hydrogen peroxide. Therefore azide would not be suitable. Do you know if 2-vinyl pyridine will affect HRP activity?
As for 3-AT, based on the publication you were referring to-- it inhibits the correct folding of catalase once it has been expressed by the cell. From my understanding this means it will have no effect on fully active catalase in plasma. Correct?
Hi Andrey,
unfortunately, you just can't measure plasma H2O2 in that way for three main reasons:
1. Erythrocytes consume H2O2 in a subsecond time scale (pseudo-first-order rate constant ~6/s, as estimated here Article Hydrogen peroxide metabolism and sensing in human erythrocyt...
). This is much faster than you can inhibit peroxiredoxins, GPx and catalase, not to mention draw blood in the first place.2. Second, endothelial NADPH oxidases in endothelial cells are rapidly activated by blood flow disturbances such as caused by the very action of drawing blood. They release superoxide to the circulation, which is rapidly dismutated to H2O2 + O2 by ecSOD.
3. As discussed in the paper cited above and supported by recent experiments in the zebrafish (Article Image-Based Measurement of H 2 O 2 Reaction-Diffusion in Wou...
), except near inflammation sites plasma H2O2 levels are submicromolar and most likely nM-level.Therefore, in order to determine physiologically meaningful plasma H2O2 concentrations you would need a non-invasive method with at least nM sensitivity. No such method is yet available, to my knowledge.
Dr. Armindo Salvador gave good reasons to suggest that H2O2 measurements will be prone to error, But before doing anything, Noha, ask yourself: OK, I have some number, what does it mean? What does it add to our understanding Oxidative stress? Surprisingly (for you) this information will be useless. To explain why, would take too much time and space in this notice. The main reason, H2O2 is not dangerous, particularly in the blood..
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