Hi Stefan,

Good question. Heating samples at 70degC may not be a good idea considering possible adverse effect of elevated temperature on different heat labile substances present in the biological fluids including ATP itself. though ATP soultion is fairly stable in aqueous solution, the situation is different in biological fluids. Ecto-ATPase is present in extracellular fluids and it hydrolyzes extracellular nucleoside tri- and/or diphosphates with great efficiency.

Pharmacological inhibitor of such ecto-ATPase is available commercially (ARL 67156) which could be the best possible measure to prevent ATP hydrolysis in biological fluids such as yours. We always use ARL for measuring extracellular ATP released in different ex vivo experiments with a good luck.

Hope that helps.

-Best

ATP stability at high temperature depends on the time of exposition and on the nature of the fluid (pH, composition .....). Any way ATP is much more stable than is commonly believed. The best you can do is to mimic your experiment adding some ATP and measuring the recovery at the end of the experiment. Good luck

Hi Maria,

Your idea is good. But this will only work if the ATP released in the fluid is high enough (in the uM range) to be compared with the the exogenous added ATP . Loss of ATP (released in true experimental condition) may not be significantly detectable at a very lower concentration with adequate precision and accuracy. We work on ATP release in ocular lens and ATP release measured by exposing the lenses to hyposmotic solution remains in the pmole range (per 100ul sampling) and no way it will be equivalent to several uM concentration. I am not very familiar for the possible ATP release by the cardiac tissue. If the experiment needs longer duration to get a descent detectable amount of ATP release still that may not be the true available concentration at that instance as ectoATPase may have already hydrolyzed a significant amount of ATP.

Stefan should definitely follow your suggestions but incorporating ARL 67156 irrespective of the experimental conditions will allow him to measure ATP with greater accuracy in a valid way.

@Stefan,

You can download the paper Hyposmotic stress causes ATP release and stimulates Na,K-ATPase activity in porcine lens" free through my RG page for further reading.

I would suggest de-proteinating your sample (use TCA for example) to stop all ATP hydrolysis. I have been doing similar studies and have found that the cell lysis buffer that comes with the Perkin-Elmer ATPlite kit is very effective at stopping all ATPase activity in samples. I usually harvest my samples, quench ATPases with lysis buffer, and then assay remaining ATP levels in all samples at the end of the study.

Thanks for all your answers. I included the ARL67156 inhibitor which worked really fine. I also heated the samples to 95deg for 1 minute though... I have tested that now with an ATP solution and found that it didn't affect the luciferase readout. I did that because I am concerned that because ARL67156 does not inhibit 100%, ATP will be degraded significantly during the handling process of all the probes (I had a lot).

Hi Stefan,

Good to know that ARL I suggested worked well for your experiment.

Best-

Dear Stefan,

In the right environment you can even boil ATP (it is an extraction method for ATP in bacteria). I think, however, that you need to inactivate not only ATPases but also other ATP degrading enzymes. Adenylate kinase, at least from bacteria, is e.g. not inactivated by boiling. All ATP degrading enzymes require divalent metal ions like Mg2+ or Ca2+. Consequently they show little activity in the presence of excess EDTA. If you freeze the samples ATP should be stable. Adding a strong detergent as DTAB will inactivate all enzymes completely within a few seconds. This is the safest method (extracts can be stored for several days in the fridge). For a sensitive assay of ATP and several other metabolites you can use the firefly luciferase reaction. I can provide a detailed technical support based on my long experience in the field. Further info on what you want to measure, what type of samples and whaat instrumentation you have access to would be interesting.

Looking forward to hear from you.

Best regards,

Arne

[email protected]