160 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS influence of pH on fluorescence is dismissed by the authors because ANS displacement (as measured by fluorescence intensity) increased from 24% (pH 7 solution) to 34% (pH 9.5 solution), which the authors considered negligible. However, two different values were given in the article for the same test parameter, the measurement of fractional displacement of ANS by water at one minute. Figure 6B shows a value of-0.24 (24%), while Figure 10 shows a value of-0.10 (10%). If the authors had chosen to use 10%, instead of 24%, for comparison to a pH 9.5 solution, the pH effect would be consid- erably increased. Data from our laboratory show that a decrease in pH indeed increases the fluorescence intensity of both Na- and TEA-laurate. Greater solubilization of ANS by TEA-laurate as well as the pH-induced decrease in fluorescence could easily account for any discrepancy between TEA-laurate and SLI in ANS fractional displacement and stratum corneum binding reported in this article. The authors state that the binding strength of ANS to corneum proteins is sufficiently large to overcome the electrostatic effects of a change in the state of ionization of the amino acid residues resulting from a change in pH. It is interesting to note, however, that the authors propose a mechanism for ANS displacement by surfactants by stating that the electrostatic repulsion between the two negatively charged ligands is important. If the electrostatic effects have little or no effect on ANS-binding on corneum proteins, why would these same effects cause ANS to be displaced from stratum corneum by surfactants and why does the electrostatic repulsion affect ANS but not surfactants? Using this technique, the authors contend that the TEA-laurate cleansing bar leaves greater residue on the stratum corneum than the SLI-based cleansing bar. This is simply not the case. Data from our laboratory involving in vivo attenuated total reflectance- Fourier transform infra-red (ATR-FTIR) spectrophotometric analysis of cleansing bar rinsability showed that a SLI-containing synthetic detergent bar left significantly (p 0.01) greater post-rinse residue on the skin than a TEA-soap/glycerin bar following a realistic washing procedure (30-sec wash, 15-sec rinse). Finally, although the words "significant" and "not significant" were used to describe certain results, there was no mention of which type of statistical analysis led to this conclusion, nor was a p value given for any contention of significance. The selective disclosure of data and arbitrary use of biochemical and molecular theory certainly challenge the validity of this paper. Given the current findings involving ANS and TEA-laurate and the cleansing bar FTIR-ATR study, the lack of any statistical analysis, and the experimental inconsistencies/omissions involving test surfactants and pH, it is very difficult to consider any conclusions drawn by this paper to be scientifically valid. REFERENCES (1) J. Slavik, Anilinonaphthalene sulfonate as a probe of membrane composition and function, Blochim. Biophys. Acta, 694, 1-25 (1982). (2) B. Vidal, The use of the fluorescent probe 8-anilinonaphthalene sulfate (ANS) for collagen and elastin histochemistry, J. Histochem. Cytochem. 26, 196-201 (1978). Stephanie K. Ball Yohini Appa Neutrogena Corporation

LETTERS TO THE EDITOR 161 TO THE EDITOR: RESPONSE TO COMMENTS BY S. BALL AND Y. APPA (NEUTROGENA CORPORATION) ON "INTERACTIONS OF CLEANSING BARS WITH STRATUM CORNEUM PROTEINS: AN IN VIT•?O FLUO- RESCENCE SPECTROSCOPIC STUDY" (S. MUKHERJEE, M. MARGOSIAK, K. ANANTHAPADMA- NABHAN, K. Yu, AND M. ARONSON, J. Soc. COSRIET. CHERL, 46, 301-320, 1995). We appreciate the interest the authors have shown in our work on using ANS displace- ment as a tool to measure surfactant-protein interactions. Unfortunately, their com- ments indicate a misinterpretation or misunderstanding of the content of our paper. Ball and Appa claim that it is an "assumption" on our part to suggest that anionic surfactant binding to corneum proteins will displace the ANS bound to the protein. We have clearly shown that corneum-bound ANS quenches tryptophan (protein) emission. The authors may be aware that quenching of fluorescence involves very short range interac- tions, which suggests that the bound ANS is located close to the tryptophan moiety in the protein. Fluorescence lifetime measurements of corncure proteins also agree with this. Treatment with anionic surfactant decreases the ANS emission and--this is im- portant-increases the tryptophan emission. This demonstrates that anionic surfactants displace protein-bound ANS. The conclusion that different anionic surfactants bind to skin differently is also supported by direct binding results with radiolabeled materials. Regarding the authors' contention that the removal of the ANS from the corneum is due to miceliar solubilization, we believe that to be very unlikely. The fluorescence inten- sities of the soak solutions that had been in contact with ANS-treated corneum were negligible compared to the ANS fluorescence intensities from the corneum. For example, in a typical experiment, ANS fluorescence intensities in the soak slurries (0.25% by wt.) of isethionate and TEA soap bars were 0.8 and 0.83, respectively, compared to an ANS intensity in the corneum of around 400. The intensities from the soak solution were comparable to ANS emission in water, i.e., close to zero, suggesting that very little of ANS was being solubilized by the surfactant solutions. We have also compared the affinity of ANS for different surfactant solutions (1.0% by wt.) using fluorescence spectroscopy. The intensity ratios (relative to water) for various solutions are as follows: Em. max. wavelength Sample (nm) Relative intensity Water 500 1.00 TEA (0.04 M pH = 9.6 500 1.07 NaL 495 4.64 TEA/NaL 495 5.26 SLS 495 5.84 SLI 485 8.5O C12(EO) 6 475 124 Octanol 465 438 These results clearly show that the affinity of the ANS probe for various anionic sur- factant systems is quite similar. However, it is significantly higher for a nonionic surfactant. The affinity of ANS for SLI is higher than its affinity for Na/Na-TEA laurate, suggesting that ANS solubilization may play a greater role for isethionate-based sur-