Initial effect of sodium bicarbonate on intracellular pH depends on the extracellular nonbicarbonate buffering capacity

Objective: The effect of sodium bicarbonate on intracellular pH under condi tions close to those in vivo, with both bicarbonate and nonbicarbonate buff ering systems, is unknown. We postulated that this effect depends on the no nbicarbonate buffering capacity because the alkali-induced back-titration o f these buffers results in a concentration-dependent release of CO2 in the extracellular space, leading to a decrease in intracellular pH. Design: The study was conducted in two stages. First, human hepatocytes wer e perfused with pH 7 bicarbonate-buffered medium (5 mM HCO3-, 20 torr Pco(2 )) containing no nonbicarbonate buffer or small amounts (5 mM 4-[2-hydroxye thyl]-1-piperazineethanesulfonic acid [HEPES]) or large amounts 120 mM HEPE S) of nonbicarbonate buffer. Second, the changes in intracellular pH of hep atocytes placed in acidotic human blood (pH 7, 5 mM HCO3-, 20 torr Pco(2) a t three hematocrits (40%, 20%, and 5%) were measured. Setting: Research laboratory at a medical university. Subjects: Cryopreserved human hepatocytes thawed the day before the experim ents. Interventions: Sodium bicarbonate was infused for 10 mins to increase the H CO3- concentration from 5 to 30 mM. In the second part, 20 mM sodium bicarb onate was added directly to the blood bathing the cells. Measurements and Main Results. The intracellular pH was measured with the p H-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein in its est erified form, acetoxy-methyl ester, by using a single-cell imaging techniqu e. Gas analyses were performed before and during the sodium bicarbonate loa d. Sodium bicarbonate caused a decrease in intracellular pH with all media except the artificial medium containing no HEPES. This decrease was small i n media with low nonbicarbonate buffering capacity (5 mM HEPES and 5% hemat ocrit blood) and large in media with high nonbicarbonate buffering capacity (20 mM HEPES and 40% hematocrit blood). The change in intracellular pH was linked closely to the change in Pco(2) caused by the sodium bicarbonate. Conclusions: The effect of sodium bicarbonate on intracellular pH depends o n changes in Pco(2) in the medium bathing the cells. The increase in Pco(2) is correlated with the extracellular nonbicarbonate buffering capacity bec ause of the release of H+ ions coming from the back-titration of these buff ers. We conclude that sodium bicarbonate may exacerbate cell acidosis under buffering conditions close to those in vivo and that the initial changes i n cell pH caused by sodium bicarbonate depend on the extracellular nonbicar bonate buffering capacity.