ORIGINAL ARTICLE. Janeta Nikolovski 1, Georgios N. Stamatas 2, Nikiforos Kollias 2 and Benjamin C. Wiegand 1

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1 ORIGINAL ARTICLE Barrier Function and Water-Holding and Transport Properties of Stratum Corneum Are Different from and Continue to Develop through the First Year of Life Janeta Nikolovski 1, Georgios N. Stamatas 2, Nikiforos Kollias 2 and Benjamin C. Wiegand 1 Skin water barrier development begins in utero and is believed to be complete by week 34 of gestational age. The goal of this investigation was to assess the dynamic transport and distribution of water of the stratum corneum of infants and compare it to those of adults. The interaction of water with the stratum corneum was assessed by measuring capacitance, transepidermal water loss (TEWL), rates of absorption desorption as well as Raman spectra as a function of depth (a total of 124 infants (3 12 ) and 14 adults (14 73 years)). The results show that capacitance, TEWL, and absorption desorption rates had larger values consistently for infant stratum corneum throughout the first year of life and showed greater variation than those of adults. The Raman spectra analyzed for water and for the components of natural moisturizing factor (NMF) showed the distribution of water to be higher and have a steeper gradient in infants than in adults; the concentration of NMF was significantly lower in infants. The results suggest that although the stratum corneum of infants may appear intact shortly after birth (o1 month), the way it stores and transports water becomes adult-like only after the first year of life. Journal of Investigative Dermatology (28) 128, ; doi:1.138/sj.jid ; published online 17 January 28 INTRODUCTION Human skin barrier development begins in utero during the first trimester with stratification of the epidermis (Holbrook, 1982; Cartlidge, 2). The formation of vernix in the third trimester is thought to contribute to the final steps of barrier maturation (Visscher et al., 25). Epidermal cell maturation occurs continually during this entire process, whereas the stratum corneum (SC) as well as the dermo-epidermal undulations become visible at 34-week gestational age. It is believed that at this point barrier maturation is near complete and the fetal epidermis begins to function as a barrier (Kalia et al., 1998; Segre, 23; Mancini, 24). It is the mature semipermeable SC that provides a protective epidermal barrier, allowing for terrestrial life (Menon, 22). Much clinical evidence exists stressing the importance of the SC and its barrier function for infants and especially for 1 Advanced Technologies, Johnson & Johnson Consumer and Personal Products Worldwide, Skillman, New Jersey, USA and 2 Models and Methods, Johnson & Johnson Consumer and Personal Products Worldwide, Skillman, New Jersey, USA Correspondence: Dr Benjamin C. Wiegand, Johnson & Johnson Consumer and Personal Products Worldwide, 199 Grandview Road, SB226, Skillman, New Jersey 8558, USA. bwiegan@cpcus.jnj.com Abbreviations: NMF, natural moisturizing factor; SC, stratum corneum; SD, standard deviation; TEWL, transepidermal water loss Received 19 April 27; revised 5 September 27; accepted 7 November 27; published online 17 January 28 newborns (Harpin and Rutter, 1983; Evans and Rutter, 1986; Saijo and Tagami, 1991; Hoath, 1997; Kalia et al., 1998; Cartlidge, 2; Yosipovitch et al., 2; Kikuchi et al., 26). s born prematurely (less than 34-week gestational age) exhibit underdeveloped skin barrier function, and much research has focused on determining the effect of gestational age on barrier development (Rutter and Hull, 1979; Wilson and Maibach, 198; Lund et al., 1997; Kalia et al., 1998). Given the dramatic transition from an aqueous to a dry terrestrial environment at birth, studies have dealt with skin barrier adaptation within the first few days or the first month of life (Harpin and Rutter, 1983; Visscher et al., 2; Yosipovitch et al., 2; Hernes et al., 22; Hoeger and Enzmann, 22). There is no clear consensus, however, about the state of the SC barrier in infants after the first month of life (Chiou and Blume-Peytavi, 24). Reports have also varied on the point at which infants acquire an adult-like SC. Based on transepidermal water loss (TEWL) and percutaneous water absorption studies, SC maturity has been reported to occur anywhere from 3 to 37 weeks (Rutter and Hull, 1979; Harpin and Rutter, 1983; Kalia et al., 1998). Others believe that even for term infants the SC water holding and water transport properties are in a state of flux in contrast to adults (Holbrook, 2; Visscher et al., 2). Some investigators have reported TEWL values of term infants to be at adult levels and concluded that term infants are born with a functionally mature SC (Rutter, 1728 Journal of Investigative Dermatology (28), Volume 128 & 28 The Society for Investigative Dermatology

2 2b). SC hydration measured by skin conductance seems to consistently show reduced levels upon birth and elevated levels thereafter (Visscher et al., 2; Yosipovitch et al., 2; Giusti et al., 21; Hoeger and Enzmann, 22). Despite reports of normal basal barrier function at birth, full-term infant skin is known to exhibit a greater tendency to develop irritant/allergic contact dermatitis, as well as to be prone to higher percutaneous absorption, prompting some to suggest that barrier function is not fully developed at birth (Rutter, 2a; Behne et al., 23). Natural moisturizing factors (NMFs) in the SC are known to be involved in barrier function and serve as efficient humectants. NMF concentration has been shown to decline with age and to be impacted by skin disease and environmental damage (Rawlings and Harding, 24). The goal of this study was to investigate the water storing and water transport properties of the SC of infants in the first year of life with traditional tools, such as TEWL, as well as skin capacitance, absorption desorption, and Raman confocal spectroscopy, and to compare these to the properties of adult SC. In the rest of the document, we will use the expression water-handling properties to signify the water transport and water storing properties of the SC. RESULTS The water-handling properties of infant and adult skin were measured using both a static and a dynamic approach. Static or steady-state measurements relate to the presence and distribution of water within the SC, whereas the dynamic measurements refer to the uptake and release of water from the SC. Note that the data presented are for a population pulled from five independent studies as explained in the Materials and Methods section. A post hoc analysis has shown that in all cases where infant data are compared to adult data, the calculated statistical power exceeds.95, except where noted otherwise. Static (steady-state) measurements Skin conductance is higher in infants. Skin hydration was evaluated using skin conductance measurements (NOVA DPM meter) on the upper ventral (inner) arms and lower dorsal (outer) arms. Skin conductance values as a function of age for the upper ventral arm site are shown in Figure 1a. skin displays higher conductance values than adult skin. Similar results were found for the lower dorsal arm area (data not shown). The data were grouped by age (3 6, 7 12, 13 48, and adults). Conductance values of infant skin (3 12 ) were significantly higher when compared to adult skin (Figure 1b and c). There were no significant differences between infants aged 3 6 and those aged Linear regression analysis of the entire data set showed age to be a significant factor contributing to hydration values and accounting for 44% of the variation. A large intersubject variability was found in the skin conductance measurements of the infant population (Figure 1a). The coefficient of variance for all of the infant groups was 2 5 times higher compared to that for the adult group for the lower dorsal site (Table 1). Similarly, the coefficient of variance for the conductance measurements on the upper ventral arm site was higher for the 3- to 6- and 7- to 12-month-old groups than the older groups. Conductance (NOVA DPM, a.u.) Age () Age (years) Conductance (NOVA DPM, a.u.) Conductance (NOVA DPM, a.u.) Figure 1. Skin conductance is higher in infants. (a) Age distribution of skin conductance measurements (a.u. ¼ arbitrary units) on the ventral side of the upper arm. (b) Data taken from the upper ventral arm were grouped by age: 3 6 (N ¼ 21), 7 12 (N ¼ 46), (N ¼ 17), and adults (N ¼ 71). Skin conductance values of the upper ventral arm decrease after the first 12 of life (*Po.5, 3 12 vs adult). (c) Data taken from the lower dorsal arm were averaged similarly. Skin conductance values of the lower dorsal arm decrease after the first 12 (*Po.5, 3 12 vs adult). Data shown as mean±sd

3 SC water content is higher in infants. Skin hydration was assessed by in vivo Raman confocal spectroscopy. The confocal arrangement allows the acquisition of Raman spectra as a function of depth into the skin with a lateral resolution of 1 mm (x and y directions) and a depth resolution of 5 mm (z direction). The spectra were then analyzed for water content, and water concentration profiles were calculated as a function of depth into the skin. Although water profiles have been reported from adult subjects (Caspers et al., 21; Hellemans et al., 25), this method was applied for the first time on infant skin in this study. Measurements on infant skin revealed differences in water distribution in the SC as compared to adults (Figure 2a). The infant group showed a higher amount of water on the skin surface ( mm depth), as well as within the SC and throughout the first 26 mm from the skin surface. No significant differences in water concentration as a function of depth were found between infants of less than 1 year of age compared to infants older than 1 year of age. s were Table 1. Coefficients of variance for the skin conductance measurements Coefficient of variance Upper ventral Lower dorsal N found to have both a steeper water gradient in the SC (the gradient was calculated as the slope of the linear portion of the concentration profile, 4 14 mm) compared to adults and a higher water content within the first 2 mm from the surface of the SC (Figure 2b and c). Note that in the case of Figure 2b, a post hoc analysis showed that the statistical power for the comparison between infant and adult data is.831. The strength of the conclusion for a difference in the water profiles, however, is supported by the data in Figure 2c, where the statistical power is.988. Dynamic measurements Endogenous water loss through the skin is higher in infants. Measurements of TEWL on the upper ventral arm as a function of age are shown in Figure 3a. Similar results were obtained for the lower dorsal arm area (data not shown). skin appears to lose water at higher rates than adult skin. The average values of measurements of TEWL for all infant groups and for both skin sites investigated were significantly higher than for adults (Figure 3b and c). No significant difference was found between the infant groups of 3 6 and 7 12 for the upper ventral arm site, where a decreasing trend was seen for the lower dorsal arm site vs age. A large intersubject variability in TEWL measurements was found among the infant subjects (Figure 3a). Calculation of coefficients of variance for each age group revealed higher values of variance in the younger group (3 6 old) compared to either the 7- to 12-month-old or the adult group (Table 2). The coefficient of variance of all infant TEWL measurements from the upper ventral arm site was higher than that of adults. Water gradient (slope from 4 14 mm) Water concentration (mass %) , , Depth (μm) AUC ( 2 mm) Figure 2. Water content of the infant SC is higher than that of the adult SC. (a) The average distribution of water on the lower ventral arms of infants (aged 3 33, N ¼ 13) and adults (N ¼ 13) was measured within the top 4 mm of the skin by Raman confocal microspectroscopy. SC contains more water than adult SC (*Po.5 up through the first 26 mm). Data shown as mean±sd. (b) The water distribution within infant SC demonstrates a steeper slope (at depths of 4 14 mm). Data shown as mean±sd, *Po.5. (c) SC contains higher concentration of water within the top 2 mm than adult SC, calculated by the area under the curve (AUC). Data shown as mean±sd, *Po Journal of Investigative Dermatology (28), Volume 128

4 TEWL (g m 2 hour 1 ) TEWL (g m 2 hour 1 ) Age () Age (years) s s TEWL (g m 2 hour 1 ) Figure 3. TEWL values are higher in infants. (a) TEWL from the upper ventral arm for each subject was measured and plotted against age. (b) Data taken from the upper ventral arm were averaged for infants ranging in age from 3 to 6 (N ¼ 19), 7 to 12 (N ¼ 31), and adults (N ¼ 71). TEWL values from the upper ventral arm are higher for the first 12 of life compared to adult (*Po.5 for both age ranges). (c) Data taken from the lower dorsal arm were averaged similarly. TEWL values from the lower dorsal arm are higher in the youngest subjects (*Po.1, 3 6 vs 7 12 ) and higher among all infants tested than that of adults (*Po.5, 3 6, 7 12 vs adult). Data shown as mean±sd. Table 2. Coefficients of variance for the TEWL measurements Coefficient of variance Upper ventral Lower dorsal N TEWL, transepidermal water loss. Exogenous water absorption and desorption were higher in infants. Water absorption and desorption were measured on the lower arm of both infants and adults. The changes in skin hydration due to the addition of water and its subsequent evaporation/dissipation were monitored with measurements of skin conductance. Conductance measurements at each time point before and after the addition of a drop of water onto SC of the lower dorsal arm were taken and then averaged among infants (ages 3 12 ) and then adults. The measured values of conductance were normalized to the initial baseline readings (Figure 4a). The skin of infants was found to absorb water at a faster rate compared to adults. Similarly, the initial water desorption rate was higher for the infant population (Figure 4b), and it approximated the rate of adults for times longer than 45 seconds. The shape of the desorption curves implies exponential kinetics. The adult data can be fit with a single exponential, but for the infant data the initial rapid decline imposes a better fit with a double exponential function. skin appears to absorb a greater amount of water than adult skin and it also gives it up within the first 45 seconds after blotting; thereafter, it behaves similarly to adult skin. The time constant for the water desorption in adults was t ¼ 3 seconds. For the infants, the two time constants were t 1 ¼ 12 seconds and t 2 ¼ 3 seconds corresponding to an initial rapid decay and a secondary slower phase. Water absorption into the SC was also investigated with in vivo Raman confocal microspectroscopy. Measurements of the water concentration throughout the epidermis were taken before and after a 1-second application of water to the SC (Figure 5). This maneuver resulted in significantly higher amounts of water in the top layers of the SC of infants aged 3 12 and had no effect on the water profiles measured in adult skin (Figure 5a and b). The changes in water concentration following this maneuver in infant skin were found to be significant within the first 8 mm of the SC (Po.5 before vs after). NMF concentration is lower in infants Water in the SC may be taken up by corneocytes and/or byproducts of protein degradation called natural moisturizing factors. In vivo Raman confocal microspectroscopy was used

5 to measure NMF concentration profiles through the skin as a function of depth following the method of Caspers et al. (21) (Hellemans et al., 25). Profiles from infants (3 12 Change in conductance (a.u.) Rate of change of conductance Time (seconds) Absorption ( 3 seconds) 9 12 Desorption (3 6 seconds) Figure 4. Exogenous water absorption and desorption rates are higher in infants. The absorption and subsequent desorption of exogenously applied water was monitored by measuring skin conductance before and 1 seconds after water application to the skin of the lower dorsal arm. (a) skin (N ¼ 88) shows a higher change in conductance values from baseline than adult skin (N ¼ 97; *Po.1 at 3 seconds). (b) Rates of change were calculated and averaged from initial to maximum value (rate of absorption) and from maximum to the value at 6 seconds (rate of desorption). skin demonstrates a higher rate of water absorption (*Po.2) and desorption (*Po.1) than adult skin. Data shown as mean±sd. ) show significantly lower amounts of NMF in the first 12 mm of the SC surface than that from adults (Figure 6). DISCUSSION The goal of this study was to investigate the water-handling properties of the SC of infants in the first year of life with traditional tools, such as TEWL, as well as skin capacitance, absorption desorption, and Raman confocal spectroscopy, and to compare these to the properties of adult SC. This information on water handling in the SC might be of value in enhancing our understanding of the barrier function of the SC and its maturation postpartum. SC hydration has been reported to be low at birth in term neonates and to increase with increasing post-natal age (Visscher et al., 2; Yosipovitch et al., 2; Giusti et al., 21; Hoeger and Enzmann, 22; Behne et al., 23). The duration of this increase in SC hydration ranges from 2 weeks after birth (Visscher et al., 2) to 3 9 days after birth (Hoeger and Enzmann, 22). The results of this investigation indicate that the SC of infants (3 12 of age) is significantly more hydrated than adult SC. This conclusion is supported by both measurements of skin conductance and water distribution using Raman confocal microspectroscopy. It should be noted that hydration measurements using skin conductance might be confounded when comparing skin sites of unequal thicknesses. The SC of infants has been reported to be thinner than that of adults and this was also confirmed in vivo in our experiments (data not shown). However, measurements of water content with increasing depth into the skin using Raman confocal microspectroscopy confirm that the epidermis of an infant contains significantly more water on the surface, as well as throughout the first 26 mm, than adult epidermis. The greatest difference between the infant and adult epidermal water concentration was seen between 1 and 14 mm from the surface (Figure 2). SC exhibited a significantly steeper water gradient and higher total water amount than adult SC. This may be attributed in part to a thinner SC in infants. These findings are particularly noteworthy considering the biological significance and implication of this water gradient on SC maturation and on cellular differentiation in infants. Skin Water concentration (mass %) s 1 2 Depth (μm) Before After 3 4 Water concentration (mass %) s Before After Depth (μm) Figure 5. Water absorption profiles. The absorption of exogenously applied water was monitored by in vivo Raman confocal microspectroscopy 1 seconds after water application to the skin of the lower ventral arm. Data were averaged before and after water application. (a) A significant amount of water absorption was found within the SC of infants less than 12 old (N ¼ 5; *Po.5 before vs after for first 8 mm). (b) In contrast, no significant water absorption was found in adult skin (N ¼ 7) after water application. Data shown as mean±sd Journal of Investigative Dermatology (28), Volume 128

6 NMF concentration (mass %) TEWL (g m 2 hour 1 ) Depth (μm) 2 4 Conductance (NOVA DPM, a.u.) 6 Figure 6. NMF concentration is lower in infants. The average distribution of NMF on the lower ventral arms of infants (aged 3 12, N ¼ 8) and adults (N ¼ 15) was measured within the top 28 mm of the skin. s have less NMF in the SC and upper epidermis of their arms as compared to adults (*Po.5 up through the first 12 mm). Data shown as mean±sd. Figure 7. A combination graph (TEWL vs conductance) demonstrates the differences in water-handling properties of infant SC vs adult SC. TEWL and conductance values from the upper ventral arm for each subject were measured and plotted against each other. The graph demonstrates the high variability of the state of the infant SC (being in a state of flux ) compared to the well-established water-handling control mechanisms of adult SC. surface morphology, the desquamation process, and epidermal expression of keratins and other proteins are influenced by the amount and distribution of water in the SC (Rawlings et al., 1994; Pierard et al., 2; Sato et al., 2; Bouwstra et al., 23; Fluhr et al., 24; Rawlings and Matts, 25). Measurements of TEWL have been used extensively as an indicator of barrier function. Although it is generally appreciated that gestational age has a profound effect on TEWL, the values reported for infants vary (Harpin and Rutter, 1983; Saijo and Tagami, 1991; Lund et al., 1997; Kalia et al., 1998; Yosipovitch et al., 2; Visscher et al., 22; Agren and Sedin, 26). These inconsistencies may be attributed to differences in environmental acclimation times, number and age range of subjects, body site studied, or the type of instrument used to measure TEWL (that is, closed vs open chamber techniques). Studies in which open chamber instruments were used to assess TEWL for term infant skin have reported TEWL values that were lower than 1 g m 2 hour 1, which are lower than or equal to those reported from adults (Harpin and Rutter, 1983; Saijo and Tagami, 1991; Kalia et al., 1998). On the basis of those data, it was concluded that term infants are born with a functionally mature SC. Higher TEWL values have also been reported from term infants (Lund et al., 1997; Yosipovitch et al., 2). TEWL measurements performed in this study show significant differences between the values obtained from infants less than 1 year of age and those obtained from adults. The average TEWL values reported in this study for infants range from 15 to 3 g m 2 hour 1, consistent with some published reports (Lund et al., 1997; Yosipovitch et al., 2). These data may support the notion that infant skin barrier is not the same as adults and continues to develop during the first year of life. Measurements of TEWL can often be confounded by factors such as environment (that is, ambient humidity), sweat gland activity, and subject stress (Wilson and Maibach, 1982; Chilcott et al., 22). These factors were controlled in this study by performing measurements in a temperature- and humidity-controlled room, allowing an acclimation period to stabilize subjects before measurements were performed, and refraining from taking data from visibly distressed or crying infants. Despite these efforts, a relatively high amount of variability and high TEWL values were seen within data sets (Tables 1 and 2). We believe that the high TEWL values shown for some subjects may be indicative of a mildly stressed subject rather than suggestive of a sustained high water flux. However, the data presented here highlight a very interesting and consistently apparent observation that infant skin does not seem to exist at a constant, steady state regarding its water holding properties. The TEWL data were further plotted vs the conductance values measured at the corresponding sites (Figure 7). This figure demonstrates both the intersubject variability in infants as well as the tight clustering of adult data (lower left part of the figure corresponding to lower values of TEWL and skin conductance). The conclusions drawn based on the measurements of TEWL were confirmed by the water absorption desorption studies. Water absorption and desorption studies have been performed to monitor the rate at which water is taken up by the superficial layers of the SC and then released (Tagami et al., 1982; Segre, 23; Fluhr et al., 24; Elias, 25). skin was found to absorb significantly more water than adult skin, and the initial rate of desorption was higher for infants than for adults; at later time points, the two responses were identical. Interestingly, water desorption was found to follow a different process in infant skin as demonstrated by two distinct rate constants. This implies that a different mechanism of water desorption may exist in infant skin that is absent or present to a lesser degree later in life. Whereas in adult skin water desorption seems to follow a single process, possibly a relatively slower diffusion through the tissue, water desorption from infant skin seems to occur first via a distinct

7 rapid process followed by the slower diffusive process similar to that seen in adults. These conclusions depend strongly on the conductance values corresponding to the maximum amount of water absorbed. The difference between adult and infant values at that time point is statistically significant at confidence levels of Po.1. It can be speculated that water desorption through the superficial layers of the SC in infants is giving rise to this rapid process, indicating once more the different nature of the infant SC water holding properties. The phenomenon of water absorption was also investigated with Raman confocal microspectroscopy. Spectra were taken from specific skin sites before and after a 1-second application of water onto the skin. A significant increase in water content in the first 8 mm of the SC was found in infants after application of water, whereas no change was measured in adult skin during the same experiment. No differences were seen in water absorption using this method with infants older than 1 year of age (data not shown), although further experiments need to be performed with more subjects to better elucidate the transition point in age. The water barrier function of skin is localized in the SC (Norlen, 21; Bouwstra et al., 23). Water has been shown to be inhomogeneously distributed within adult hydrated SC, where it concentrates within corneocytes and lacunae. The data presented in this study suggest the possibility of distinctly different SC barrier and water-handling properties in the skin of infants less than 1-year old compared to adult skin. Natural moisturizing compounds, found in high concentrations in the SC and within corneocytes, are hygroscopic and act as very efficient humectants (Rawlings and Harding, 24). Distribution profiles of NMF as a function of skin depth showed that infant SC contains significantly smaller amounts of NMF than adult SC. The lower amounts of NMF in infant skin could impact the SC s water-handling properties and is one likely mechanism contributing to the present results demonstrating faster water desorption from infant skin. However, less NMF does not sufficiently explain the higher capacity for water uptake seen in infant skin, suggesting a more hygroscopic environment. Skin barrier function and water transport through the skin involve a complex interplay of multiple factors such as corneocyte maturity/hydrophilicity, lipid amount and phase, density of appendages, surface microrelief, as well as diffusion path length, which could all play a role in the differences between infant and adult skin properties. Skin barrier function is undoubtedly influenced by the environment and the dramatic changes that occur with the shift from an aqueous and constant temperature environment in utero to the arid, cooler, and variable conditions of the extra-uterine world. It has been suggested that interaction with the external environment fosters barrier stabilization (Visscher et al., 2). The adaptation period of adult skin barrier function to environmental changes has been shown to be in the order of weeks for mice (Denda et al., 1998; Elias, 25) and weeks to for humans (Chou et al., 25). It has been suggested that post-natal adaptation occurs within the first month in newborn infants (Visscher et al., 2). On the basis of the results of this study, the stabilization or adaptation/maturation period may extend beyond the first year of life. In contrast to the relative steadystate nature of adult skin, infant skin barrier should not be thought of as deficient but may exist in a state of optimization, balancing growth, thermoregulation, water barrier, and protective functions. This adaptive flexibility can be attributed to the higher rate of skin growth in infants and is evidenced further by the age-related decline in the ability to restore barrier during adulthood. On the basis of these results, we conclude that the properties that make infant skin unique continue to persist at least through the first 12 of life. barrier function and water-handling properties of SC continue to be in a state of flux until more stable regulation mechanisms are developed later in life. MATERIALS AND METHODS Clinical protocol Five independent studies were performed on healthy Caucasian male and female infants and female adults without any history of skin disorders. Only healthy infants were recruited. In terms of atopic dermatitis, this corresponds to no eruptions, Hanifin scale of (Hanifin et al., 21). The adult population was with Fitzpatrick skin types I III and the infant population was fair complexioned and equally distributed between males and females. The studies were performed with an independent Institutional Review Board approval and following the Declaration of Helsinki Principles. Subjects and/or parents or legal guardians signed a written informed consent before the start of each study. The studies were performed in New Jersey, and variations due to the time of year were found to be comparatively small in magnitude and did not significantly contribute to the results. A total of 124 infants (3 to 4 years of age) and 14 adults (14 73 years of age) were studied. For all studies, subjects were instructed to avoid use of skin care products on their arms for at least 24 hours before the study. Measurements on the arms were taken after subjects acclimated to an environmentally controlled room (2 25 1C and 4% relative humidity) for a minimum of 15 minutes (except for Raman studies outlined below). Measurements were not taken from visibly distressed or crying infants. Instrumentation Skin conductance was measured to evaluate the hydration state of the SC using the NOVA DPM (NOVA, Portsmouth, NH), which measures an electrical parameter directly relevant to hydration of the upper SC layers (Berardesca, 1997). This instrument will typically report a reading between 9 and 999 DPM units, according to the manufacturer s instruction. TEWL measurements were made using the closed chamber technique with the Delfin VapoMeter (Delfin, Kuopio, Finland). Measurements were standardized following the European Group on Efficacy Measurement of Cosmetics and Other Topical Products (EEMCO) guidelines (Rogiers, 21). Measurements were performed on the lower dorsal and upper ventral sites of randomly chosen arms of each subject, except where noted. We chose the lower dorsal and upper ventral arm sites to examine possible early effects of sun exposure (with the upper inner site being more protected than the lower dorsal site). TEWL values were 1734 Journal of Investigative Dermatology (28), Volume 128

8 compared from 65 infants (pooled from 2 independent studies) and 71 adults. Baseline skin hydration values were compared from 97 infants (pooled from 2 independent studies) and 71 adults. Dynamic water-handling properties of the SC were measured using an absorption desorption technique on the lower dorsal arm (Tagami et al., 1982). After a baseline reading (using the NOVA DPM meter) was taken, a drop of water was applied to the skin for 1 seconds, followed by blotting with a paper towel. Serial conductance measurements were subsequently taken starting 3 seconds after initial water application up to 18 seconds at 15-second intervals. The rate of change was calculated for both the absorption (uptake) and desorption (loss) of water from the skin. The rate of absorption for each subject was calculated from the initial reading to the maximum reading after 3 seconds. The rate of desorption was calculated similarly from the maximum reading at 3 6 seconds. Water desorption curves (conductance vs time) were also fitted to exponential functions. Rates of water absorption and desorption were compared from 88 infants (pooled from 2 independent studies) and 97 adults. Water and NMF concentrations in the skin were measured using in vivo Raman confocal microspectroscopy (Skin Composition Analyzer 351; River Diagnostics, Rotterdam, The Netherlands). This methodology allows for the direct measurement of water within the skin, in contrast to correlative measurements involving electrical techniques (for example, conductance). Spectra in the high wave number region (2, 4, cm 1 with 72 nm excitation wavelength) were taken in 2 mm increments in the z (depth) direction, starting at just above the surface, up to 4 mm into the ventral forearm skin. Spectra were also taken in the fingerprint region at wave numbers of 4 2, cm 1 from which NMF calculations were based. The top of the skin surface was defined as the flat, raised areas of microrelief. Water concentrations (in mass %) were calculated using the intensity ratio between the Raman bands of water (3,35 3,55 cm 1 ) and protein (2,91 2,965 cm 1 ) as described by Caspers et al. (21, 23). Concentration profiles of NMF were determined using the relative signal contributions of NMF and keratin (Caspers et al., 21, 23). NMF profiles were obtained from the lower ventral arms of infants (aged 3 12, N ¼ 8) and adults (N ¼ 15). Dynamic water-handling properties of the skin were also analyzed using this method. Following baseline measurements, a paper towel soaked in water was placed onto the same skin site (forearm) for 1 seconds. The skin was blotted dry and measurements were immediately taken. Water concentration profiles were compared from 12 infants (3 33 old) and 12 adults (their mothers; years old). Water concentration profiles following water absorption were taken from five infants ( old) and seven adults (25 43 years old). From the water concentration profiles, the SC thickness was calculated as the length defined from the point of the beginning of the keratin signal (automatically defined by the acquisition software by River Diagnostics) to the point of water saturation (plateau) considered to be the beginning of the viable epidermis (Egawa et al., 27). Also, the water gradient in the SC was calculated from the water concentration profiles as the slope of the linear portion of the profile at depths 4 14 mm. All statistical analysis was performed using MINITAB Release 14 (Minitab Inc., State College, PA). Comparison of two data sets was determined using Student s t-test, after confirmation of normal distribution using MINITAB s normality test, and statistical significance was assumed at the level of P ¼.5. The data sets that were not normally distributed (skin conductance and TEWL data) were first transformed to create normally distributed data so that we could perform Student s t-test. The transformation used in this case was 1/x 2. CONFLICT OF INTEREST The authors state no conflict of interest. ACKNOWLEDGMENTS We gratefully acknowledge Diana Friscia and Dr Laura McCulloch for their efforts on study design and execution, Dr Anthony Rawlings for insightful discussions and editing of the manuscript, and River Diagnostics (Rotterdam, The Netherlands) for guidance with the in vivo Raman microspectroscopy measurements. REFERENCES Agren JSG, Sedin G (26) Ambient humidity influences the rate of skin barrier maturation in extremely preterm infants. J Pediatr 148:613 7 Behne MJ, Barry NP, Hanson KM, Aronchik I, Clegg RW, Gratton E et al. (23) Neonatal development of the stratum corneum ph gradient: localization and mechanisms leading to emergence of optimal barrier function. J Invest Dermatol 12: Berardesca E (1997) EEMCO guidance for the assessment of stratum corneum hydration: electrical methods. 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