Effects of alkaline buffers on cytoplasmic pH in lymphocytes

To study experimentally possible adverse effects of bicarbonate on cytoplasmic pH.

Open, randomized control trial of white blood cells from human volunteers.

Experimental laboratory in a large university hospital.

Lymphocytes prepared from human blood.

The fluorescent intracellular probe 2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) was used to study the influence on cytoplasmic pH after the addition of different alkaline buffers to lymphocytes with normal as well as decreased initial intracellular pH.

In normal lymphocytes, sodium bicarbonate caused a marked, dose-dependent acidification of the cytoplasm followed by a slow, often unpredictable increase. Ringer's acetate solution decreased the intracellular pH dose-dependently this acidification effect continued throughout the measurements. In contrast, trometamol (tris) and Carbicarb both caused a pronounced dose-dependent and lasting alkalinization of the cytoplasm. Tris buffer mixture (Tribonate) produced a slight initial dose-dependent acidification, followed by a slow increase in cytoplasmic pH to values above those recorded during control measurements. Lymphocytes that were preincubated in acetate showed similar results after addition of tris buffer mixture or sodium bicarbonate. Lymphocytes with intracellular acidosis due to preincubation in an acid buffer demonstrated a more pronounced and dose-dependent decrease of cytoplasmic pH immediately after addition of the bicarbonate-containing buffers (sodium bicarbonate and tris buffer mixture). The decrease was only partly compensated for over the next 10 mins. Only the buffers that were not producing CO2 could fully compensate for the severe extra- and intracellular acidosis imposed on the lymphocytes by preincubation in an acid medium.

Use of bicarbonate-containing buffers often results in an initial decrease of cytoplasmic pH.