Weak-acid, 31P-nuclear magnetic resonance (NMR) and microelectrode techniques for measuring intracellular pH (pHi) are compared by demonstration of their use in rat liver. The ultimate test of suitability of these methods is the confidence with which they can be used to clarify aspects of metabolic regulation, translocation of substances across biological membranes, and the control of cell pH itself. Though resting pHi in perfused liver is fairly similar with all three techniques, substantial quantitative differences between values obtained with 31P NMR and microelectrodes are revealed after addition of fructose to the perfused liver preparation. The use and limitations of weak-acid methods in determining the mechanism of inhibition of gluconeogenesis from lactate by acidosis and in determining the pH responsiveness of the lactate transporter in the hepatocyte plasma membrane are demonstrated. Microelectrode-derived values of pHi are probably referable to the bulk phase of the cytosol, whereas values from the other two methods are more complex in their interpretation. Microelectrode and NMR methods have the great advantage of being non-destructive, and continuous records may be obtained.
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