Restricted diffusion of an 19F-labelled organic acid in human erythrocytes analyzed by 19F pulsed field gradient NMR.

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The diffusion coefficient of an 19F-labelled organic acid (3-trifluoromethylhippurate: TFMH) was measured in intact human red blood cells (RBCs) and sealed right-side-out ghosts at 22.5 +/- 0.05 degrees C. Diffusion coefficients were measured using stimulated echo and spin-echo pulsed field gradient sequences. The apparent diffusion coefficient (Da) of TFMH in the intracellular space was much smaller than in the extracellular medium. This was due to restricted diffusion of intracellular TFMH since i) the intracellular Da decreased when the diffusion time was increased, and ii) the smaller Da component disappeared when the membrane was permeabilized with saponin. From the intracellular Da values obtained over a range of diffusion times, from 5 ms to 1 s, the apparent radius of the diffusion barrier was estimated to be 3 +/- 1.1 micron assuming spherical geometry. Despite the high protein concentration in the intracellular space of the intact RBC, the intracellular values of Da were similar to the values obtained in the ghosts at the same diffusion times. We therefore conclude that the small diffusion coefficient of intracellular TFMH is mainly explained by the restricted diffusion due to the cell boundary.