316 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS DISCUSSION Fluorescence spectroscopic techniques have been used extensively in the biophysical literature to study protein-ligand interactions and are well suited for probing surfac- tant-corneum protein interactions. Studies with radiolabeled material, although useful for measuring deposition, do not provide any information on the location or molecular interactions. Infrared (IR) spectroscopy has been used to monitor deposition and ad- sorption of fatty acid soap residues on human stratum corneum (20). IR spectroscopy has the advantage of being non-perturbing. However, it is often not sufficiently sensitive to study interactions of a small amount of deposited material with the components of corneum. The ANS displacement method used here is similar to a probe extraction technique recently used by Paye eta/. (9), where the irritation potential of surfactants and cleansing products was correlated with their ability to extract the probe dansyl chloride from stratum corneum. The present method is also somewhat similar to the dye deposition technique developed by Imokawa eta/. (2). In that study, the decrease in the deposition of an acidic dye, indigo carmine, onto a surfactant-treated skin was used as the measure for in vivo deposition to a number of pure anionic surfactants. Presumably, the indigo carmine, like ANS, binds to the same sites in the corneum proteins as do anionic surfactants. However, for complex formulated products such as cleansing bars, tech- niques involving probe extraction might be more reliable, as these products often leave deposits on skin that might interfere with the staining of the dye. The advantages of using ANS are its well-characterized spectral properties and an extensive literature on its binding properties with proteins and lipids (12). Additionally, ANS does not impart any coloration to skin under visible light, making it potentially more suitable for in vivo studies over the other two dyes. Although steady-state fluorescence measurements are relatively easy to carry out, the proper interpretation of the results requires a knowledge of the location of the probe and how the changes in the probe environment may be reflected in different measurable spectroscopic properties such as quantum yield and shape of the emission spectrum. To gain such information, it is essential to use probes with well-understood binding and spectral properties. Without a proper understanding of the location of the probe, and its response to such parameters as pH and specific counter ion effects, conclusions drawn from changes in its spectral properties might be erroneous. This is particularly true for complex cleansing products such as soaps or detergents. The ANS displacement results as well as the direct surfactant binding results presented earlier clearly showed that the TEA-laurate-based soap left much more residue on the corneum than did the syndet bar based on SLI. Importantly, this ranking of residual surfactant bound to the corneum as measured by ANS probe displacement does not agree with the conclusions reported by Wortzman eta/. (8). In that study the amount of the fluorescent dye, fluorescein, that was deposited onto skin from surfactant solutions spiked with this dye was assumed to give a relative measure of the amount of surfactant bound to the skin. Since a TEA-based soap composition similar to bar B gave a lower level of bound fluorescence relative to an isethionate composition similar to Bar A, as measured by their procedure, Wortzman eta/. concluded that the TEA soap composition gave the least residue. This conclusion is opposite to what has been found in the present study and prompted us to consider the Wortzman eta/. study in some detail.

SURFACTANT-SKIN INTERACTIONS 317 The Wortzman study (8) involved spiking a bar slurry with a known amount of fluo- rescein, rubbing the skin with the fluorescein-spiked slurry, gently washing the skin with a limited amount of ambient-temperature water, extracting the residual amount of fluorescein deposited on skin using a methanol:water (80:20) solvent system, and quan- tifying the amount of fluorescein spectrophotometrically using its absorbance at 280 nm. Their results showed that the amount of fluorescein left behind on skin from a TEA-based bar was lower than the amount left from a pure soap and an isethionate-based bar. The authors interpreted these results to mean that the TEA-laurate bar left the least amount of residue from the bars on skin. The inherent assumption in this argument, as stated earlier, is that the amount of fluorescein left on skin is proportional to the amount of surfactant left on skin from the bar. They justified the latter assumption by deter- mining the amount of fluorescein and soap left behind on a grooved glass slide by washing it gently with ambient-temperature water. We believe, for a number of reasons to be discussed below, that the fluorescein deposition does not reflect the inherent tendency of the surfactant to interact with skin and cause damage. In general, two types of probes are used in the study of adsorption of surfactants to surfaces. The first kind is a hydrophobic coadsorbing or tracking probe that will par- tition into surfactant aggregates in solutions as well as at interfaces (21,22). In this case, the surfactant solution is usually spiked with the probe and exposed to the test substrate. The result is an increase in the probe binding with an increase in the adsorption/binding of surfactants to the substrate. The second kind of probe is a competitive binding probe that will compete with the surfactant for adsorption sites. Such probes are generally amphiphilic such as ANS. Experiments with the competitive probe are usually done by preadsorbing the probe and monitoring its displacement by the surfactant. Thus an increase in surfactant binding will be accompanied by an increase in probe displacement. If, on the other hand, the competitive probe is added along with the surfactant, the result will be essentially a lower binding of the probe to the test substrate, with higher levels of surfactant binding. Clearly, in contrast to the tracking probe, the binding of the competitive probe will decrease with an increase in surfactant binding. In this regard, ANS, the anionic probe, is a competitive probe that will compete with anionic surfactants for cationic binding sites present on the substrate. Interestingly, fluorescein, being a weakly ionizable carboxylic acid (Figure lB pK a = 6.5), can behave as either a nonionic or an anionic dye, depending on the solution pH employed. Importantly, fluorescein is in its anionic form below the neutral or alkaline pHs encountered in the bar slurry considered here. This will make fluorescein a competitive probe like ANS rather than a coadsorbing probe as assumed by Wortzman et al. (8). In fact, it is shown in a separate communication (23) that fluorescein does not partition into sodium lauroyl isethionate, sodium dodecyl sulfate, or sodium laurate-type anionic surfactant aggre- gates, indicating that fluorescein binding cannot reflect anionic surfactant binding. It is also shown elsewhere (23) that the residual amount of fluorescein left on skin from cleansing slurries is primarily related to the pH and the type of counter ions in the aqueous phase and their effects on the solubility of fluorescein, rather than to intrinsic interactions of surfactants with skin. As mentioned earlier, fluorescein behaves as a very weak competitive probe rather than as a tracking probe (23). To illustrate the behavior of a competitive probe in a study similar to that by Wortzman eta/., we have essentially repeated their study, using ANS as the competitive probe.