Quantification of eight tissue kallikreins in the stratum corneum and sweat.

TO THE EDITOR Human tissue kallikreins are a family of 15 trypsin or chymotrypsin-like secreted serine proteases (kallikrein protein (hK1–hK15) found in a variety of tissues (Yousef and Diamandis, 2001). In the stratum corneum (SC), we previously quantified hK5, hK6, hK7, hK8, hK10, hK11, hK13, and hK14 as candidates of desquamation-related proteases and compared the hK levels among various age groups (Komatsu et al., 2005a). For sweat, hK1, hK2, and hK7 levels were quantified previously (Mann et al., 1980; Mayfield et al., 1989; Hibino et al., 1994; Kishi et al., 2004). In this study, we aimed (1) to quantitatively measure multiple hKs in the SC and sweat from normal individuals and from various body regions, (2) to determine the overall enzymatic activities of these samples, (3) to compare the results among body regions and genders, (4) to compare hK levels between the SC and sweat samples, and (5) to examine a possible correlation between the hK amounts and enzymatic activities. The concentrations of hK5, hK6, hK8, hK10, hK11, hK13 and hK14 (trypsin-like hKs), and hK7 (chymotrypsinlike hK; Yousef and Diamandis, 2001) in normal SC and sweat of various body regions were determined (ng/mg total protein) by immunofluorometric ELISA (Tables 1a and b). Informed consent was obtained from all participants and our study was conducted according to the Declaration of Helsinki Principles. The medical ethical committee of Graduate School of Medical Science, School of Medicine, Kanazawa University approved all described studies. In the SC, neither the mean concentration of each hK nor the total concentrations of trypsin-like hKs differed significantly between body regions (Table 1a). hK6, hK10, and hK13 in the SC were present at significantly lower concentrations in females than in males in the SC (Table 1a). In sweat, each hK mean concentration and the total concentration of trypsin-like hKs differed widely among body regions and gender groups with significant differences (Table 1b). hK6, hK10, and hK14 were present at significantly lower concentrations in females than in males. In females, hK concentrations were consistently lowest in the axilla. For hK6, hK8, hK10, hK13, and hK14, sweat from the face contained the highest amounts of hKs (more details are given in Table 1b). hK6 and hK8 are positively stained in both normal human axillary eccrine and apocrine sweat glands; however, hK13 was detected only in the eccrine sweat glands regardless of gender (more details are in Figure S1). The ‘‘trypsin-like enzymatic activity’’ towards Boc-Phe-Ser-Arg-7-amino4-methyl courmarin synthetic substrate (Suzuki et al., 1996) was measured in the SC (Figure 1a) and sweat samples (Figure 1b) from each individual. The SC samples displayed no significant differences at any time point among body region and gender groups (Figure 1a). According to a standard trypsin curve (data not shown), the total trypsin-like activity per milligram of SC was approximately comparable to 675–875 pg of trypsin. In contrast, sweat trypsin-like activities differed significantly among groups (Figure 1b). The trypsin-like activity per gram of sweat was approximately comparable to that of 250–1375 pg of trypsin. The ‘‘chymotrypsin-like enzymatic activity’’ towards MeO-Suc-Arg-ProTyr-para-nitroanilide-HCl synthetic substrate (Suzuki et al., 1996) was also measured in the SC (Figure 1c). There were no significant differences among groups in the SC samples. (Figure 1c). The para-nitroanilide release was insufficient to generate standard chymotrypsin curves. Sweat samples were also used for chymotrypsin-like activity measurements. However, after the samples rapidly released 3–11 nmol of paranitroanilide/g sweat following 1 minute incubation at 371C, the activity was abolished for an unclear reason (data not shown). In the SC, not only the concentration of each hK but also the overall SC enzymatic activities were highly consistent among body regions, suggesting that the hK expression and the SC enzymatic activities may be regulated similarly throughout the SC, at least in the body regions studied. In the SC, hK6 has been detected at higher amounts in males regardless of age (Komatsu et al., 2005a). Higher levels of hK6 in males are also observed across body regions for both the SC and sweat, suggesting that hK6 expression might be sex hormone dependent, as suggested previously (Yousef and Diamandis, 2001; Komatsu et al., 2005a). In sweat, the total concentration of trypsin-like hKs and the trypsin-like enzymatic activity appear to be highly correlated with each other regardless of body region or gender, which might imply that hKs may contribute significantly to the total trypsin-like activity. However, further studies are necessary to elucidate the fraction of hKs that are enzymatically active in sweat.