I'm currently thinking about the measurement of intracellular pH in erythrocytes. Without the equipment for NMR-spec or fluorescence measurement it seems that I would have to go back to the even more old school protocols. Sadly I was unable to find a straight forward description of e.g. the old lysis methods which apparently yielded good results - at least for RBCs.
Could anybody kindly provide a protocol for such an easy/low budget measurement of intracellular pH?
Also besides having the basic lab equipment (pipettes, centrifuge, etc.) I plan on using the commercial blood gas analyser for my pH read out of the lysates. Is this going to be a problem?
I'm aware this sounds quite rustic but I do have quite limited resources.
I would be very grateful for your advice.
Cheers, Chris
You may find the following paper useful
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2275073/
Biochem J. 2007 Nov 1; 407(Pt 3): 343–354.
Published online 2007 Oct 12. Prepublished online 2007 Aug 14.
Evaluation of pH during cytostomal endocytosis and vacuolar catabolism of haemoglobin in Plasmodium falciparum
As well as this:
http://jcs.biologists.org/content/119/6/1016.long
J Cell Sci. 2006 Mar 15;119(Pt 6):1016-25. Epub 2006 Feb 21.
The pH of the digestive vacuole of Plasmodium falciparum is not associated with chloroquine resistance.
More simpler: 2 ml of heparinized blood suffy, follows by 3 minutes centrifugation, taking 100 µl of packed RBCs for pH (7.2), PO2 (>100 mmHg) and PCO2 (~45 mmHg) vs ABG from whole venous blood. There should be a routine analysis, since all blood oxygenation, pH regulation and CO2, HCO3 exchanges are regulated by RBCs.
Plasma have not place in these biochemistry events, and any other tissues are involved, including renal pH regulation controlled by RBCs NH3 exchanges.
In brief, circulating RBCs maintain normal blood and body cell pH within strict limits sustaining the lives ,,,
Split the RBCs sample in two aliquots: one is for the following analyses, the other has to be added to 10-20 volumes (or even more...) of distilled water (possibly at a pH around 7,00), after a brief vortexing, measure the pH with a conventional pH-meter......
Hi Christian
A variation of the method that Argante has suggested is as follows:
Using a blood gas analyser is fine.
For accurate results, reading the pH as quickly as possible is important.
Attached is a useful reference.
Regards, Rosemary
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