Vitamin C (ascorbic acid, AscH) is an essential nutrient for human health. Published values for the passive membrane permeability of AscH and ascorbate range over several orders of magnitude and hence appear to be contradictory. In order to characterize AscH-carriers we determined a robust value for its background membrane permeability. Since AscH is a weak acid one can exploit the technique of scanning electrochemical microscopy on a free standing planar lipid bilayer separating two compartments [1]. Addition of AscH to the donating compartment results in a flux across the membrane of the protonated form of the acid only. In the receiving compartment, the acid establishes its protonation equilibrium according to the Henderson-Hasselbalch equation which leads to an increase in proton concentration in the vicinity of the membrane. This locally reduced pH gradually ascends towards the bulk pH within the so-called unstirred water layer. With the aid of custom built pH-sensitive micro-electrodes mounted to a micromanipulator the proton distribution perpendicular to the membrane can be determined with micrometer precision. Finally, the distribution is evaluated with a numerical model [2] taking into account the protonation reactions with buffering agents and water resulting in a permeability of PAscH=10-8 cm/s. This value is validated by kinetic scattering measurements of liposome suspensions on a spectrophotometer. An outward facing gradient of AscH leads to leakage of the acid out of the vesicles which is accompanied by osmotic shrinkage of these. Consequently the reduced scattering ability of the liposome suspension serves as read-out of the process.
[1] Saparov et al. Biophysical journal 90.11 (2006): L86-L88.
[2] Antonenko et al. Biophysical journal 64.6 (1993): 1701.
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