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 conditions close to those in vivo, with both bicarbonate and nonbicarbonate buffering systems, is unknown. We postulated that this effect depends on the nonbicarbonate buffering capacity because the alkali-induced back-titration of these buffers results in a concentration-dependent release of CO2 in the extracellular space, leading to a decrease in intracellular pH.


METHODS
The study was conducted in two stages. First, human hepatocytes were perfused with pH 7 bicarbonate-buffered medium (5 mM HCO3-, 20 torr Pco2) containing no nonbicarbonate buffer or small amounts (5 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES]) or large amounts (20 mM HEPES) of nonbicarbonate buffer. Second, the changes in intracellular pH of hepatocytes placed in acidotic human blood (pH 7, 5 mM HCO3-, 20 torr Pco2) at three hematocrits (40%, 20%, and 5%) were measured.


METHODS
Research laboratory at a medical university.


METHODS
Cryopreserved human hepatocytes thawed the day before the experiments.


METHODS
Sodium bicarbonate was infused for 10 mins to increase the HCO3- concentration from 5 to 30 mM. In the second part, 20 mM sodium bicarbonate was added directly to the blood bathing the cells.


RESULTS
The intracellular pH was measured with the pH-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein in its esterified form, acetoxy-methyl ester, by using a single-cell imaging technique. Gas analyses were performed before and during the sodium bicarbonate load. Sodium bicarbonate caused a decrease in intracellular pH with all media except the artificial medium containing no HEPES. This decrease was small in media with low nonbicarbonate buffering capacity (5 mM HEPES and 5% hematocrit 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 Pco2 caused by the sodium bicarbonate.


CONCLUSIONS
The effect of sodium bicarbonate on intracellular pH depends on changes in Pco2 in the medium bathing the cells. The increase in Pco2 is correlated with the extracellular nonbicarbonate buffering capacity because of the release of H+ ions coming from the back-titration of these buffers. We conclude that sodium bicarbonate may exacerbate cell acidosis under buffering conditions close to those in vivo and that the initial changes in cell pH caused by sodium bicarbonate depend on the extracellular nonbicarbonate buffering capacity.
Levraut
J
J
Centre Hospitalier Universitaire de Nice-Sophia Antipolis, Département d'Anesthésie-Réanimation, Hôpital Saint-Roch, Nice, France.
Giunti
C
C
Ciebiera
J P
JP
de Sousa
G
G
Ramhani
R
R
Payan
P
P
Grimaud
D
D
D001769:Blood / Q000187:drug effects*; D002021:Buffers; D002478:Cells, Cultured; D006531:HEPES; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000187:drug effects; D008099:Liver / Q000187:drug effects*; D017693:Sodium Bicarbonate / Q000494:pharmacology*
D002021:Buffers; D017693:Sodium Bicarbonate; D006531:HEPES