REGIONAL VARIATION IN FAAs IN STRATUM CORNEUM 305 RATIONALE AND FAA ANALYSIS Filaggrin deimination and proteolysis produces most of the SC free amino acids (15–17). Further processing converts histidine to urocanic acid (4) and arginine to citrulline (18,19). The presence of high urocanic acid and citrulline concentrations indicates NMF formation from fi laggrin (4). Glutamic acid undergoes further reaction to pyrrolidone carboxylic acid (PCA) (20,21). Lactates, urea, sugars, and ions are also components of NMF. Based on available analytical techniques, we evaluated the free amino acid compo- nent of NMF. The tapes were extracted with 300 μl of 6mM perchloric acid spiked with 10 μl of 2 μmol/ml α-amino-n-butyric acid (AABA) for three hours. The tape was re- served for protein analysis. Samples were analyzed using the AccQ-Tag system (Waters Corp.) on a C-18 reverse-phase column (25-cm length, 4.6-mm internal diameter). The excitation wavelength was 250 nm with emission at 395 nm, and a column temperature of 40°C with a run time of 40 minutes. The HPLC results were standardized to the amount of protein removed by the individual tape using the Pierce BCA protein assay (22). The rate of moisture accumulation (MAT) was measured with a NOVA® Dermal Phase Meter (NOVA® Technology, Portsmouth, NH). The method uses changes in skin capaci- tive resistance (the ratio of charge to potential on an electrically charged isolated conductor) under probe occlusion to determine skin hydration (23). STATISTICS General linear models (GLM) and analysis of variance (ANOVA) methods were used to evaluate the effects of body site on the protein removed, FAA, and hydration (SPSS and SigmaStat, SPSS, Chicago, IL). A value of p 0.05 was considered statistically signifi cant. Amino acid values are given as log10 values (pmoles) normalized to protein (μg), and proteins are given as μg/ml. Values are reported as estimates plus/minus stan- dard error. To account for the high inter-individual variability, the subject was included as a factor/covariate in the statistical model (GLM). Site effects were assessed for tape 1 and for cumulative FAA normalized to cumulative protein for the sums as follows: sum 1+3, sum 1+3+5, sum 1+3+5+10, and sum 1+3+5+10+15. RESULTS AND DISCUSSION STUDY 1 The soluble protein from tape 1 was signifi cantly higher for the calf versus the forearm ( p 0.05). The cumulative protein for tapes 1, 3, 5, 10, and 15 was not different. FAAs were higher for the calf for tape 1 and the cumulative values except for glutamic acid ( p 0.05). MAT was not different for the sites. STUDY 2 Tape 1 protein was signifi cantly lower for the calf versus the torso (ANOVA on ranks, Dunn’s method post hoc versus torso control, p = 0.02). No site differences were noted

JOURNAL OF COSMETIC SCIENCE 306 for the cumulative protein values (Table I). High inter-individual variability was found for protein levels from the torso (Table I). For tape 1, the jaw site had lower citrulline than the back, torso and calf, and lower serine than the calf. For the cumulative levels, the total FAA (log10 pmoles/μg protein) was lower for the jaw than for all other sites as fol- lows: 3.12 ± 0.1, jaw 3.41 ± 0.1 back 3.26 ± 0.1 torso 3.51 ± 0.1 calf (F = 2.9, p = 0.05). Citrulline was signifi cantly lower for the jaw than for all other sites, and arginine was signifi cantly lower for the calf (Figure 2). Cumulative citrulline (normalized to cumula- tive protein) for tape 1, sum 1+3, sum 1+3+5, sum 1+3+5+10, and the sum of all tapes is shown versus the respective cumulative protein amounts in Figure 3. Similar trends were observed for total FAA and individual amino acids. With SC depth, FAA fi rst in- creased and then leveled off for the back and torso. The increase continued throughout the sampled region for the jaw. In contrast, the calf site remained relatively constant. MAT was signifi cantly lower for the jaw than for either the torso or back ( p 0.05, Table I), but the jaw and calf were not different. There were no signifi cant correlations between FAAs and MAT values for either study, as indicated in Figure 3. At the outermost SC, the calf site had greater FAA than the forearm for comparable pro- tein removed. FAAs were lowest for the jaw compared to the torso, back, and calf. The outcomes do not support the study hypothesis, i.e., body site variations in skin hydration (MAT) could not be attributed to differences in FAA levels. One important limitation in this study is that we examined only about 40% of the components of NMF (14). We did Figure 2. Cumulative FAA by cumulative protein is shown as log10 pmoles of amino acid normalized to μg protein. Citrulline was signifi cantly lower for the jaw than for all other sites, and arginine was signifi cantly lower for the calf (*p 0.05). Table I Study 2 Protein Levels and Rate of Moisture Accumulation (MAT) for Back, Torso, Calf, and Jaw Sites Site Rate of moisture accumulation (MAT) (cru/sec) Protein tape 1 (μg/ml) Protein: Cumulative tapes 1,3,5,10,15 (μg/ml) Back 2.5 ± 1.2 8.5 ± 2.3 49.2 ± 22.3 Torso 3.4 ± 2.0 22.3 ± 15.7 81.4 ± 23.0 Calf 1.6 ± 0.9 18.8 ± 3.9** 107.2 ± 22.0 Jaw 0.9 ± 0.7* 14.1 ± 3.1 66.2 ± 21.9 * Signifi cant difference from back and torso (F = 5.06, p = 0.01). ** Calf and torso sites different by ANOVA on ranks, post hoc Dunn’s versus torso contol ( p 0.05).