Lactate/hydrogenperoxide measurement - (May/23/2006 )
I would like to measure lactate and hydrogenperoxide levles in my cell culture supernantant - has anyone an easy protocol for me?
These are straightforward spectrometry assays, rather fun to set up and run. The lactate assay may present a few problems ijn the pre-analytical stage in that lactate levels will rise due to glycolysis. To stop this you need to chill your sample or pop in sodium fluoride & potassium oxalate.
As for the assay itself, in the presence of NAD+ and lactate dehydrogenase (pH 9.0), lactate is oxidised to pyruvate, NADH and H+.
Equilibrium actually lies to the left (lactate) but you can add hydrazine which binds to pyruvate preventing the back reaction. Other techniques involve removing pyruvate as it is formed by adding glutamate and alanine transferase.
Detection is by spectrometry at 340nm. Check the linearity you get, I think this method is valid up to 5.6mmol/l (50mg/dL).
As for the H202 assay, you can use the Trinder reaction.
H202 + phenol + 4-aminophenanzone -> quinonamine dye + 2H20
(you need peroxidase as well).
Quinonamine dye is easily measured at 480 to 520nm.
Let me know if you need concentrations and volumes. If you are near a hospital clinical lab you could ask them to run your samples. Certainly, the lactate assay is done in most labs. The H202 is actually coupled to other assays e.g. cholesterol (no-one needs H202 measured). Both methods will be in Methods in Enzymology but do let us know if you need more detail.
Let us know how you get on.
for peroxide detection, you can try ammonium molybdate