Our first step was to assess the potential ability of organic carboxylates to diffuse spontaneously into liposomes without the assistance of a molecular transporter, based on previous reports showing indications that carboxylates might cross the lipid membrane without the assistance of a synthetic anion carrier in lucigenin assays. To gain insight into this, we selected malonic and succinic acids, as well as their deprotonated forms (mono-deprotonated: monosodium malonate, monosodium succinate; di-protonated: malonate, succinate) due to their high solubility in aqueous solutions (Figure ). These compounds also allow us to study how each form affects the emission intensity of lucigenin. Malonate and succinate were added to liposomes loaded with 0.8 mM lucigenin in 225 mM NaNO3, 5 mM HEPES, pH 7. In both cases, when a pulse of the corresponding dicarboxylate (25 mM) was added without a transporter, it did not affect the lucigenin emission intensity over time. However, when Triton X-100 (5% w/w in water) was added, both anions quenched the fluorescence of the lucigenin (Figure ). For monosodium carboxylates, liposomes were loaded with 0.8 mM lucigenin in 225 mM NaNO3, 5 mM MES, pH 5 (MES: 2-(N-morpholino)ethanesulfonic acid). In contrast to the dianions, monosodium malonate and monosodium succinate progressively reduced the lucigenin emission intensity, which supports the observations regarding the spontaneous diffusion of carboxylates through lipid membranes. It should be noted that for these anions, the anion pulse changes the pH value in the liposome solution from 5 to 3.9 and 4.6 for monosodium malonate and monosodium succinate, respectively. These pH values are close to where the maximum fraction for monosodium malonate (96 %) and monosodium succinate (85 %) is expected based on the calculated speciation graphs (Figure ). Finally, malonic and succinic acids were added under the same conditions as monosodium carboxylates. Upon adding malonic or succinic acid to the liposome solution, the pH value dropped from 5 to 2.3 and 3.1, respectively. However, the addition of carboxylic acids and increased acidity did not have a significant effect on the lucigenin emission, even upon lysis. Further experiments using solutions without liposomes showed that neither malonic acid nor succinic acid quenched the fluorescence of lucigenin (Figure ). These results revealed that RCOO -anions, rather than the RCOOH groups, quench the fluorescence of lucigenin. The observed quenching with mono sodium malonate and succinate thus indicates that carboxylates end up inside the liposomes.