Intracellular hydrogen ion (H+) buffering power, conventionally defined as the amount of acid or base that would have to be introduced into the cell cytosol to decrease or increase ipH by one pH unit, is generally said to increase as intracellular pH (ipH) decreases. This implies that the cell has a lesser capability to resist acute acid or base perturbations at its steady state ipH than at any lower ipH. We re-examined this notion, reasoning that the logarithmic nature of the pH unit could limit the validity of the conventional expression of buffering power in imparting physiologic insight into the mechanisms of cellular H+ homeostasis. The mathematical derivation of the formula, Δi[NH4+]/ΔipH, conventionally used to estimate buffering power using the NH4Cl technique, revealed that this parameter is, by design, inversely proportional to the exponential of ipH. This a priori dependence on pH dictates an increase in buffering power with decreasing ipH, and thereby interferes with the assessment of the physiologic capability of the intracellular milieu to buffer protons at different ipH levels. To circumvent this problem, buffering power was defined as the amount of hydrogen ions that would have to be added to or removed from the cell to effect a change in the concentration of H+ in the cell cytosol of 1 mM (a term heretofore referred to as the cell H+ buffering coefficient). The mathematical derivation of the formula used to calculate the cell H+ buffering coefficient, Δi[NH4+]/ΔA[H+] i, does not suffer from an a priori dependence on ipH. Using this approach we found that in rat thymocytes suspended in a HCO3/CO2 solution, the total cell buffering coefficient decreased as ipH was lowered from 7.3 to 6.6. The decrease in total cell buffering coefficient was associated with a decrease in the buffering power of buffers other than CO2/HCO3 (that is, the intrinsic buffering coefficient), therefore unravelling a pattern of dependence on ipH that is the opposite of that portrayed by the conventional expression of the buffering power. The component of intracellular buffering contributed solely by the HCO3/CO2 system also decreased with decreasing ipH as expected in an open buffer system. We, thus, conclude that the intrinsic cell H+ buffering power, estimated from the use of the cell buffering coefficient, decreases as ipH is reduced from 7.3 to 6.6 whereas it increases when estimated by the conventional approach. The coefficient of cell H+ buffering, unlike the traditional buffering power, provides insight into the physiologic ipH dependency of the cell's capability to resist acute acid or base perturbations.
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