1. Introduction
Vitamin C (VC) encompasses several vitamers that differ in their protonation and oxidation states and include ascorbic acid (AscH). Vitamin C deficiency results in scurvy [
1]. Membrane proteins like the sodium-ascorbate cotransporters SVCT1 and SVCT2 transport reduced ascorbate, thereby contributing to VC homeostasis in the human body [
2]. However, SVCT1 knockout only marginally affected intestinal VC adsorption in mice [
3], suggesting a role for redundant glucose transporters (GLUT transporters) or significant spontaneous membrane permeability
P of AscH that may ensure sufficient intestinal VC uptake. A more quantitative investigation of SVCT’s biological significance requires knowledge of AscH’s passive membrane permeability. Yet, surprisingly little is known. Since AscH is a weak acid with one of its pK values being equal to 4.17 [
4], it may be anticipated to passively permeate biological membranes—similarly to other weak acids such as salicylic acid [
5] or acetic acid [
6,
7]. The second proton release reaction with a pK value of 11.57 [
8] yields a bivalent anion that due to (i) its extremely low concentration at physiological pH values and (ii) the increased desolvation (Born) energy for bivalent ions cannot make a significant contribution to passive transmembrane VC flux.
With
[
9] the only available literature value differs by up to 10 orders of magnitude from values that maybe derived from (i) the membrane permeability
to the ascorbate anion (
) [
10] or (ii) the oil water partition coefficient of AscH [
11]. The goal of the present work was the characterization of AscH passive membrane transport processes. We did so by exploiting scanning electrochemical microscopy in the vicinity of planar lipid bilayers, as this method previously yielded robust values for various acids and bases [
5,
6,
12,
13].
Membrane conductivity measurements rendered an upper estimate for , which excludes from making a significant contribution to the passive membrane permeation of VC.
3. Results
An AscH transmembrane concentration gradient gives rise to an acid flux. In turn, AscH dissociation in the receiving USL gives rise to a pH shift (
Figure 2). We optimized the pH profile size by using (i) low buffer concentrations in both compartments, (ii) large Na-
l-ascorbate concentrations in the
cis compartment, and (iii) a transmembrane pH gradient. As described in Materials and Methods, we first found
(Equation (10)) and subsequently fitted the set of differential equations (Equations (2)–(9)) to the pH profiles to obtain
P. Both
P and
δ are listed in
Table 2. The USL width
reduces with an increasing AscH gradient. This effect can be attributed to an osmotic water flux [
27] that builds up due to the asymmetric addition of AscH. The resulting convection is not taken into account in the numerical calculation, but acknowledging the different
for the different gradients yields little variance in
P (
).
The pH profiles are not susceptible to the application of a transmembrane potential (
Figure 3). This observation suggests that the ionic form does not permeate the membrane on a large scale. Otherwise, the
anions that were driven by external voltage across the membrane would have affected the deprotonation of the also permeating AscH, that is, the equilibrium AscH concentration adjacent to the receiving interface would have increased simply because the concentration of
increased. In turn, the transmembrane AscH concentration gradient would have diminished, resulting in a smaller flux and smaller pH profiles.
Membrane conductivity measurements (
Figure 4) confirmed the assumption that the
flux is much lower than the AscH flux. Even though the
concentration was raised to 1 M and pH was decreased to match acid’s pK, we did not observe an increment in current >
at
of DC voltage. This translates into a specific membrane conductivity
g <
for a membrane that is
in diameter. Such
g value is on the lower end of values reported for freestanding lipid bilayers (
, [
14,
28]). Attributing
g in its entirety to the permeation of
yields an upper limit for
of
(Equation (1)). Thus,
is at least four orders of magnitude smaller than
P.
4. Discussion
With
P of about
AscH permeates fluid membranes much slower than other weak acids of comparable size—like salicylic acid [
5,
29] or acetic acid [
6]—but is comparable to the membrane permeability of other neutral substances, for example, sorbitol [
30]. The transport rate of the deprotonated species
is at least four orders of magnitude lower, being characterized by
. It is thus even smaller than to the slow transport rate of the smaller chloride ions, for which
were reported [
31].
Our study is not in line with data reported by a nuclear magnetic resonance (NMR) study, where
and
have been deduced for
and AscH effluxes from fluid dipalmitoyl-lecithin (DPPC) vesicles at 52 °C [
9]. The small difference between
P and
in the NMR study seems to violate membrane electrostatics: the latter imposes a penalty for placing a monovalent ion with a gyration radius
r =
into the bilayer [
32] of about 16.5 kcal/mol [
33]. Substracting (i) 5.4 kcal/mol with which the dipole potential favors anion permeation and (ii) 2.5 kcal/mol for the image energy [
34], we find an additional penalty of 8.6 kcal/mol for the anion, which translates into a drop of roughly six orders of magnitude in permeabilities as compared to the neutral species. In contrast, the NMR data favor the permeation of the neutral species by only a factor of 100.
A comparison with our data suggests that
P has been underestimated and
overestimated in the NMR study: the NMR-based AscH rate transforms into
P for a fluid bilayer at room temperature, which amounts to only 1/10 of the permeability of our planar bilayers. The calculation assumes that the activation energy
EA scales with
P [
35], that is, amounts to
EA ≈ 20 kcal/mol for AscH—a value similar to that of tetraphenylborate [
34] that translocates at
[
36]. The
rate of the NMR study is equivalent to
for a fluid bilayer at room temperature, that is, it matches the value of the upper permeability limit of planar bilayers. The calculation assumes
EA 30 kcal/mol [
34]—as has been reported for Cl
- that permeates at 30 kcal/mol [
34]—as has been reported for Cl
- that permeates at
[
34].
Attempts to derive
P and
from the reaction of
with paramagnetic spin probes that were intercalated in oriented lipid multilayers [
10] resulted in a large overestimation of the transport rate
. It was estimated to be
. If the calculation was correct,
would appear inside lipid vesicles of diameter
d = 100 nm after time [
37]
had elapsed subsequent to
addition to the outer solution. Yet, both a recent time resolved electron paramagnetic resonance(EPR) immersion depth study [
38] and the original EPR study [
10] indicate that it takes
20–30 min to penetrate to a probe that is buried at a depth of 20 Å of a fluid lipid bilayer. The
of
would completely rule out the possibility of using ascorbate to monitor lipid flip-flop [
39], the more so, the neutral species AscH would permeate 10
6 times faster (see Equation (11)).
According to Overton’s rule, both
P and
correlate well with the partition coefficients
Koct/w and
Koil/w between water and octanol or olive oil, respectively [
40]:
Inserting published
Koct/w and
Koil/w into the empirical relations Equations (12) and (13) yields overestimated
P values (
Table 3). The neglect of the acid base equilibrium is a major reason for the failure. For example, Oldendorf [
11] obtains
Koil/w for carbon-radiolabeled AscH in a biphasic system of Ringer’s solution buffered to pH 7.55–7.58 and refined olive oil. Dissecting the contributions of AscH and
by using acid’s pK and the pH in the aqueous phase may be misleading:
P of AscH appears in the cm/s range (see
Table 3). As pointed out before, such high permeability can be excluded. The measurements leading to
= −2.67 by HPLC with a stationary hydrocarbon phase and a mobile solvent phase [
41] may have encountered the same problem: pH is not indicated. Moreover, the range of
of the calibration substances was limited to 0.9–6.5 [
41]. Since it does not embrace
for AscH, we doubt the accuracy of the reported value.
Unfortunately, computations relying on structural similarity (structure-activity-relation, SAR [
42,
43]) or other descriptors (Linear Solvation Energy Relationship, LSER [
44,
45]) include substances with erroneous experimental partition coefficients, which may bias the prediction. Nevertheless, if the scatter of the empirical relations (Equations (12) and (13)) is taken into account, the two computed
values based on SAR [
42,
43] and the LSER value of about 0.15 [
45] yield a
P prediction that is reasonably close to our experimentally determined value.
Our
P value allows a very rough estimation of the AscH transport capacity of the intestinal tract. The flux (
Φ) through the intestinal barrier:
depends on its area
Aint = 32 m
2 [
46] and AscH’s concentration difference Δ
clb between lumen and blood. Assuming that the total luminal concentration (AscH + Asc
−) may be represented by the gastric juice concentration of 90 µmol/L and a plasma concentration of 30 µmol/L [
47] yields Δ
clb = 60 µmol/L × 10
4.17–7.4 = 35 nmol/L. The
P value of 10
−8 cm/s that was obtained at room temperature corresponds to
P ≈ 5 × 10
−8 cm/s at 37 °C—considering an activation energy of 20 kcal/mol. The resulting
Φ = 6·10
−13 mol/s is likely to be an overestimation since the number neglects the significant jejunal fluid secretion. Such
Φ is clearly insufficient as it corresponds to only about 1 µg AscH per day. The required uptake of 30–180 mg [
48] per day requires facilitated transport.