Regulation of intracellular pH by human peripheral blood lymphocytes as measured by 19F NMR.

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Abstract
The intracellular pH of human peripheral blood lymphocytes was measured by means of high-resolution 19F NMR spectroscopy using D,L-2-amino-3,3-difluoro-2-methylpropanoic acid (F2MeAla) as a probe. Lymphocytes readily took up the methyl ester of F2MeAla, and endogenous esterase hydrolyzed the ester to the free amino acid inside the cell. This .alpha.-methyl amino acid is not metabolized by the cell, and its 19F NMR spectrum exhibits large pH-dependent shifts as the .alpha.-amino group is protonated. The size of the 19F shifts, the high sensitivity of 19F NMR and the favorable pKa of the .alpha.-amino group of F2MeAla (pKa = 7.3) allowed intracellular pH of lymphocytes to be measured at 25.degree.-30.degree. C with .apprx. 5-min acquisition times. Measurements at various external pH values demonstrated that human peripheral blood lymphocytes regulate their internal pH, a process requiring expenditure of metabolic energy. In the pH range between 6.8 and 7.4, lymphocytes maintain a constant internal pH of 7.7 .+-. 0.06 pH unit. Outside this range, intracellular pH changes with extracellular pH. The accuracy of this 19F pH probe was confirmed by independent measurements of intracellular pH using equilibrium distributions of 5,5-dimethyloxazolidine-2,4-dione.