j. Soc. Cosmet. Chem., 48, 159-163 (May/June 1997) Letters to the Editor TO THE EDITOR: The authors of the article "Interactions of cleansing bars with stratum corneum proteins: An in vitro fluorescence spectroscopic study" (Mukherjee et at., J. Soc. Cosmet. Chem., 46, 301-320, 1995) claim that sodium lauroyl isethionate (SLI) surfactant and a cleansing bar containing SLI deposit less surfactant residue on stratum corneum than sodium laurate, sodium lauryl sulfate (SLS), triethanolamine (TEA)-laurate, and a cleansing bar containing TEA soap. The results are based on the assumption that when binding to stratum corneum proteins, surfactants will displace a previously bound fluorescent marker, -12-(9-anthroyloxy)-stearic acid (ANS), and will subsequently cause a decrease in fluorescence of ANS. Unfortunately, there are many omissions and inconsistencies throughout this article that cause the proported results and conclusions to be highly controversial. We will confine our comments to a few main points. First, the authors assert that anionic surfactants do not appreciably solubilize ANS because ANS exhibits "low" fluorescence in solutions of SLS and SLI, compared to a solution of a non-ionic surfactant. Remarkably, ANS solubility interactions with Na- laurate and TEA-laurate, two surfactants mentioned prominently in the article, were not disclosed by investigators. Data from our laboratory, however, show that ANS has a 2 x greater fluorescence intensity in a solution of TEA-laurate than with SLS and SLI. Because TEA-laurate is more likely to solubilize ANS, less ANS will adhere to the skin, more of the ANS will be rinsed from the stratum corneum, and a weaker fluorescence will occur than with SLI, a phenomenon reported in the article. The authors should be aware that if ANS is to be used as a fluorescent marker in surfactant studies, it should have equal and very little, if any, interaction with all test surfactants this was obviously not the case in the Mukherjee et at. study. The authors claim to validate their ANS displacement results by correlating ANS displacement by surfactants with radiolabeled surfactants binding to the stratum corneum. This correlation offers no conclusive evi- dence because results for radiolabeled TEA-laurate binding were not disclosed at either the l-rain or 1-hr time points. In addition, results of radiolabeled Na-laurate binding were only shown at the 1-hr time point, yet the authors conclude that the two events (ANS displacement and binding of radiolabeled surfactant) are linked. Another major defect in this paper is the dismissal by the authors of the contribution of pH to the fluorometric methodology. Previous literature (1,2) reports that the pH of the test solution (e.g., surfactant) greatly contributes to ANS fluorescence, with a higher pH causing a decrease in fluorescence and a lower pH greatly enhancing fluorescence. The 159

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