Original Paper Skin Pharmacol Physiol 2014;27:181–187 DOI: 10.1159/000353912

Received: January 20, 2013 Accepted after revision: June 19, 2013 Published online: March 6, 2014

Age-Related Changes of Stratum Corneum Functions of Skin on the Trunk and the Limbs N. Sato T. Kitahara T. Fujimura  Biological Science Laboratories, Kao Corporation, Ichikai, Tochigi, Japan

Abstract Background/Aims: Despite our understanding that the care of back and buttock skin is important for elderly nursing patients, the stratum corneum (SC) functions of the skin on the trunk of elderly patients have not been well investigated. Methods: Overall, 41 elderly subjects (average age: 75.9 years, 20 male and 21 female) and 20 middle-aged subjects (average age: 41.3 years, 10 male and 10 female) residing in Tokyo were recruited. Hydration of the SC, transepidermal water loss (TEWL), skin surface pH, total bacteria and inflammatory cytokines in the SC of skin on the buttocks, back, lower leg and inner forearm were measured. Results and Conclusion: The hydration of the SC decreased only on the lower leg with age. TEWL showed no change with age at any site. The pH was significantly higher in elderly skin than in middle-aged skin at all sites. The number of total bacteria on the forearm and back increased with age. The ratio of interleukin1α (IL-1α) and IL-1α receptor antagonist was only higher on the middle-aged forearm compared to the elderly. No remarkable gender difference was found in these parameters

© 2014 S. Karger AG, Basel 1660–5527/14/0274–0181$38.00/0 E-Mail [email protected] www.karger.com/spp

without pH values. We clarified that most of the SC functional parameters change with age on both the trunk and the limbs in a similar manner, suggesting that the limbs are acceptable sites to estimate the SC functions of the trunk of elderly patients. Our study may be useful as basic data for future work to maintain the SC function of elderly patients. © 2014 S. Karger AG, Basel

Introduction

Unlike the skin of young people, the skin of elderly people shows wrinkling, spotting and dryness with associated itching. The values of stratum corneum (SC) capacitance, transepidermal water loss (TEWL) and skin surface pH, all of which are related to skin dryness and itchiness, are key parameters of epidermal SC functions. Age-associated changes in such epidermal SC functions have been reported previously. Wilhelm et al. [1] measured TEWL, SC capacitance, skin surface pH and sebum at 11 body sites of young (mean age: 26.7 years) and elderly (mean age: 70.5 years) healthy subjects in the USA. Their study showed that TEWL and skin surface pH values tended to be higher in the older group, while no such difference was found in SC Tsutomu Fujimura, PhD Biological Science Laboratories Kao Corporation 2606 Akabane, Ichikai, Tochigi 321-3497 (Japan) E-Mail fujimura.tsutomu @ kao.co.jp

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Key Words Stratum corneum · Skin physiology · Trunk of the body · Skin aging

Materials and Methods Ethics The study was performed according to the Declaration of Helsinki. Prior to the start of this study, the protocol was approved by the Ethical Committee of the Kao Corporation (Tokyo, Japan), which consists of 6 members including 4 outside members. All subjects signed an informed consent that acknowledged that participation in the survey was voluntary and that withdrawal of consent would not result in any loss of benefit. Subjects and Measurements A total of 61 healthy men and women residing in and around Tokyo, Japan were recruited in two age groups: elderly (70–89 years) and middle-aged (35–45 years). The elderly group consisted of 20 male subjects (mean age ± SD: 75.9 ± 5.3 years) and 21 female subjects (78.2 ± 5.8 years), while the middle-aged

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Skin Pharmacol Physiol 2014;27:181–187 DOI: 10.1159/000353912

group consisted of 10 males (41.3 ± 2.7 years) and 10 females (42.2 ± 3.0 years). The exclusion criteria were as follows: (1) diagnosis of dementia; (2) onset of skin symptoms (itching, yellowing, edema) caused by liver or kidney dysfunction or by diabetes; (3) use of diapers; (4) history of atopic, contact or photosensitivity dermatitis, and (5) menstruation on the day of survey. For the survey, we selected 4 body sites: back, buttock (around the sacrum), inner side of the forearm, and the outer side of the lower leg. All subjects were asked to have either a bath or shower on the day before the survey, but not to apply any type of afterbath moisturizer on the 4 body sites. In addition, taking either a bath or shower on the morning of the survey was prohibited. All measurements and collections were performed in November 2010. Each subject was asked to fill out a questionnaire on activity and health (daily meal frequency, exercise habit, hours of sleep, frequency of bathing or showering), to wear a front-opening hospital gown, to enter a measuring room (22–23 ° C, 50–60% relative humidity) and to lie on their side on a bed. After a 30-min acclimation, skin pH was measured with a pH electrode (Horiba Co. Ltd., Kyoto, Japan) in a drop of ion exchange distilled water, which was applied and collected from a 2 × 2 cm skin surface area on the aforementioned 4 body sites. Subsequently, skin surface microbes were collected by swabbing a 4 × 5 cm area of each region 20 times using a 20-BBL culture swab (Becton Dickinson, Franklin Lakes, N.J., USA) soaked with saline solution. These skin areas were then wiped with Baby Water Wipes (Wakodo, Tokyo, Japan) and left for 15 min to dry. Subsequently, the capacitance of the SC was measured with a Corneometer® CM (Courage & Khazaka Electronic GmbH, Köln, Germany), and the TEWL was then measured with a Tewameter® (Courage & Khazaka), using previously reported precautions [5–7]. Following that, SC samples were collected by sequential tape stripping using 3 pieces of adhesive tape (PPS tape; Nichiban, Tokyo, Japan) and were put in a tube.  

 

Analysis of the Interleukin-1 Receptor Antagonist/Interleukin1α Ratio Phosphate buffered saline at pH 7.5 was added into each tube with the tape strips. Subsequently, each tube was left in an ice bath for 15 min with ultrasound. The supernatant was collected from each tube by centrifugation. Proteins in the supernatant were quantified using a BCA protein assay kit (Santa Cruz Biotechnology, Santa Cruz, Calif., USA). Human interleukin-1α (IL-1α) and IL-1α receptor antagonist (IL-1Ra) were then quantified using a Quantikine® ELISA kit (R&D Systems Inc., Minneapolis, Minn., USA). Finally, the IL-1Ra/IL-1α ratio was calculated. Analysis of Skin Surface Microbes DNA was isolated from microbes collected from swabs by the following procedures: grinding with glass beads, extraction with phenol-chloroform isoamyl alcohol, centrifugation and ethanol precipitation. Bacterial DNA was quantified by real-time PCR using DNA polymerase reagents (TaqMan Universal PCR Master Mix; Applied Biosystems, Foster City, Calif., USA), the universal bacterial primer (Eub-R, 5′-GGACTACCAGGGTATCTAATCCTGTT-3) and the universal bacterial probe (Eub-P, 5′-CGTATTACCGCGGCTGCTGGCAC-3) [8]. Each DNA sample was amplified in 40 cycles of denaturation at 95 ° C for 10 min and annealing at 60 ° C for 60 s.  

 

 

Sato/Kitahara/Fujimura

 

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capacitance. Marrakchi and Maibach [2] measured these parameters at 9 sites (7 facial sites, the neck and the forearm) in young (mean age: 29.8 years) and elderly (mean age: 73.6 years) subjects of European and American populations. Their study showed that on the forearm of the older group compared to the younger group, SC capacitance was higher and TEWL was lower. In addition, pH values at several facial sites and at the forearm were reported to be significantly higher in the older group than in the younger group. In a Chinese population, Man et al. [3] measured the SC capacitance, sebum and pH of skin on the forearm and forehead and analyzed their relationships with age and gender. They reported that the male elderly group (over 70 years old) had a lower forearm SC capacitance and a significantly higher forearm skin pH than the male middle-aged group (aged between 36 and 50 years). Those previous studies were conducted focusing on the SC function of the face and the forearm. Despite our understanding that the care of back and buttock skin is important for elderly nursing patients, and despite the fact that the itchiness and/or dryness of the skin occurs mainly on the trunk skin in the elderly, the SC functions of skin on the trunk have not been well investigated. Nakagami et al. [4] compared the SC functions in perianal sites of elderly patients with or without incontinence, and found that the SC capacitance, skin pH and the amount of SC ceramide were greater in the incontinence group than in the healthy group. However, these results were obtained only from elderly patients, and therefore it is insufficient to understand the SC functions of skin on the trunk at different ages. In the present study, we investigated age-related changes of SC functions of the skin on the trunk and limbs as reference sites.

Table 1. Summary of subjects in this study

Middle-aged

Elderly

Gender

Age, years

daily meal frequency (average/day)

Exercise habit

Hours of sleep (average/day)

Frequency of (bathingb average/day)

men (n = 10)

41.3±2.8

2.7 times

3/7 (yes/no)

average: 6.0 h range: 5.5–7 h

more than once/day: 9 once/2 days: 1

women (n = 10)

42.2±3.0

2.9 times

4/6 (yes/no)

average: 6.3 h range: 5–7 h

more than once/day: 9 once/2 days: 1

men (n = 20)

75.9±5.3

2.9 times

yes: all subjectsa

average: 6.6 h range: 4–9 h

more than once/day: 13 once/2 or more days: 7

women (n = 21)

78.2±5.8

3.0 times

yes: all subjectsa

average: 6.9 h range: 4–9 h

more than once/day: 19 once/2 days: 2

Age is expressed as mean ± SD. a  Includes light exercise such as walking, stretching exercise. b Includes showering bath.

Results

middle-aged group, the capacitance was significantly lower on the leg, and the total bacterial count was significantly greater on the forearm. The pH values of the elderly group tended to be higher than those of the middleaged group, but these differences were not significant.

Discussion

Table  1 summarizes the conditions of the subject’s health. Elderly subjects were in good physical condition as were the middle-aged subjects regarding daily meal frequency, exercise habit, hours of sleep and frequency of bathing or showering. Figure 1 summarizes SC capacitance, TEWL, skin surface pH, total bacterial count and the IL-1Ra/IL-1α ratio in the mixed gender group. The results of comparisons between the age groups were as follows (in the elderly group compared to the middle-aged group): (1) SC capacitance was significantly lower on the leg; (2) TEWL was similar in all 4 body sites; (3) skin pH was significantly higher in each of the 4 body sites; (4) the total bacterial count was significantly greater on the forearm and the back, and (5) the IL-1Ra/α/IL-1α ratio was significantly lower in the forearm. Figures 2 and 3 summarize values of women and men, respectively. In women, the skin pH levels in the 4 body sites were significantly higher in the elderly group than in the middle-aged group. No significant differences were observed in other measurements. In men, when the values of the elderly group were compared to those of the

Hydration State of the SC It is unclear whether the SC capacitance becomes lower in association with age. Firooz et al. [9] reported an age-dependent decrease of capacitance, but other reports are contradictory about that. Marrakchi and Maibach [2] reported an age-dependent increase of SC capacitance while Wilhelm et al. [1] reported that there was no agedependent difference. Interestingly, Man et al. [3] reported that capacitance changes with age only in men but not in women. In contradiction to that, Ohta et al. [10] reported that skin capacitance is higher in postmenopausal women than in pre- or perimenopausal women. This contradiction might be due to regional differences between the arm [3] and the forehead [10]. The present study showed that there was an age-dependent difference or a menopause influence on the SC on the trunk of the body, as reported previously for the face and the forearm. On the other hand, SC capacitance was significantly lower on the lower leg of the elderly male group than in the middle-aged male group. This specificity of male lower leg skin needs to be investigated further.

Stratum Corneum Functions at Various Body Sites

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Statistics After excluding outlier data of each group using the SmirnovGrubbs rejection test, Student’s t test was used to determine statistical significance (p < 0.05). The outliers were excluded from all figures.

40

Inner forearm

Lower leg

Back

Buttock

0

b

**

Lower leg

Back

Buttock

Lower leg

n = 19

4 2

Inner forearm

Lower leg

Back

Buttock

Back

Fig. 1. Summary of comparisons between middle-aged and elderly

n = 40

n = 17

n = 39

n = 38

5

Inner forearm

*

6

0

d

*

Elderly subjects

15 n = 40

Ratio of IL-1Ra/IL-1į

e

8

Middle-aged subjects

*

0

Buttock

Buttock

n = 41 (except number caption). a Hydration (capacitance). b TEWL. c Skin surface pH. d Total number of bacteria. e Ratio of

subjects for several physiological measurement parameters. Middle-aged subjects, n = 20 (except number caption); elderly subjects,

IL-1Ra/IL-1α. * p < 0.05, ** p < 0.01, determined by Student’s t test.

TEWL Value TEWL measurement is a standard method for characterizing skin barrier function. Concerning the age-related changes in TEWL values, there have been several previous reports [1, 2, 9] indicating that the TEWL value declines with age. However, we found no significant difference with age in any of the 4 body sites, either in the individual gender groups or in the mixed gender group. Some reports have shown a racial difference in SC functions. The TEWL value is known to vary among Asians, Africans and Europeans residing in the same region [11], and therefore a racial difference may explain this inconsistency.

Skin Surface pH An acidic skin surface pH is known to play a critical role in antibacterial and barrier functions of the skin, and also in SC desquamation. Previous studies showed that the skin pH value increases with age on the forehead and the forearm [1–3, 12, 13]. In one such study, Zlotogorski [12] reported that the facial skin surface pH of subjects over 80 years of age showed 0.5 units higher pH values than those of the younger group. A similar difference in pH has been reported by Marrakchi and Maibach [2] and also by Man et al. [3]. In this study, skin surface pH increased with age at all body sites, which indicates that previous findings about

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Inner forearm

20

10

Back

n = 40

* n = 40

5

c

Lower leg

n = 39

*

**

Total bacteria (logx)

pH

7

4

Inner forearm

10

8

6

10

n = 38

a

20

n = 40

0

**

30

n = 40

50

TEWL (g·m–2 h–1)

Hydration (AU)

100

40

a

Inner forearm

Lower leg

Back

Buttock

b

*

**

**

Inner forearm

Lower leg

Back

Buttock

Inner forearm

Lower leg

Back

Buttock

8 6 4 2 0

d

Buttock

Elderly women

0

Inner forearm

Lower leg

Back

Buttock

Fig. 2. Summary of comparisons between middle-aged and elderly

women for several physiological measurement parameters. Middle-aged women, n = 10 (except number caption); elderly women,

age-dependent changes on the limbs (forearm and lower leg) also apply to the trunk of the body. However, it is noteworthy that the age-associated increase was less in men compared to women. Such a gender difference in skin surface pH was reported in elderly men over 70 years [3]. These results indicate that skin pH on the trunk of the body increases with age in a similar manner to the limbs. In addition, the increase was greater in women than in men.

n = 21 (except number caption). a Hydration (capacitance). b TEWL. c Skin surface pH. d Total number of bacteria. e Ratio of

IL-1Ra/IL-1α. * p < 0.05, ** p < 0.01, determined by Student’s t test.

Microbes It is now widely accepted that human skin harbors hundreds of bacterial strains and those microbes contribute to the skin surface functions. Inter- and intraindividual dif-

ferences in the distribution of skin microbes have been reported previously [14–16]. The growth of skin microbes strongly depends on the pH of the skin surface. Lambers et al. [17] reported that an acidic environment between pH 4 and 4.5 is desirable for the growth of microflora. On the other hand, it has been reported that the growth of bacterial species depends on the skin surface pH [18, 19]. In this study, the total number of skin microbes increased with age, and the age-dependent increase was greater in men than in women. This result does not agree with our results that the increase of pH was less in men than in women. In addition, no body regional difference was observed between the limbs and the trunk of the

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5

n = 20

10

n = 18

15

n=8

Ratio of IL-1Ra/IL-1į

Back

Middle-aged women

20

e

Total bacteria (logx)

pH

**

5 4

Lower leg

10

7

c

Inner forearm

n = 20

0

8

6

10

n = 20

0

20

n = 20

50

30

n = 20

TEWL (g·m–2 h–1)

Hydration (AU)

100

40

50

a

*

Inner forearm

Lower leg

Back

Buttock

b

Total bacteria (logx)

pH

6 5

Inner forearm

Lower leg

Back

Buttock

0

e

Lower leg

Back

Back

Buttock

8

Lower leg

Back

Buttock

6 4 2 0

d

*

Inner forearm

Buttock

men for several physiological measurement parameters. Middleaged men, n = 10 (except number caption); elderly men, n = 20

body. We consider that this inconsistency is not due to washing because all samples were collected more than 12 h after a bath or shower. The total number of skin microbes was not a significant parameter that changed with age or with body sites. IL-1Ra/IL-1α Ratio IL-1α and its receptor antagonist IL-1Ra are cytokines involved in the acute immune responses to UV or microbial stimuli [20, 21]. IL-1Ra serves to block IL-1β signaling by binding to IL-1α. The IL-1Ra/IL-1α ratio was reported to determine IL-1α activity [21]. Hirao et al. [22] found that the IL-1Ra/IL-1α ratio was higher in sun-exSkin Pharmacol Physiol 2014;27:181–187 DOI: 10.1159/000353912

n = 18

n=9

n=9

n = 18

n = 19

n=9

Inner forearm

Fig. 3. Summary of comparisons between middle-aged and elderly

186

Lower leg

Elderly men

15

5

Inner forearm

Middle-aged men

n = 19

Ratio of IL-1Ra/IL-1į

20

10

10

10

7

4

20

0

8

c

30

(except number caption). a Hydration (capacitance). b TEWL. c Skin surface pH. d Total number of bacteria. e Ratio of IL-1Ra/

IL-1α. * p < 0.05, determined by Student’s t test.

posed facial skin than in sun-protected inner forearm skin. This ratio decreased with age on the face but did not change on the forearm. They considered that an appropriate balance between IL-1α and IL-1Ra may help to maintain the homeostasis of the skin. In our study, a significant age-related difference in the IL-1Ra/IL-1α ratio was found only on the forearm in the mixed gender group. No significant difference was found on the sun-protected back or buttock skin. We consider that these differences are due to the sun exposure level caused by outdoor activities of the subjects. Our finding indicates that the IL-1Ra/IL-1α ratio is not a parameter that changes with age or with body sites. Sato/Kitahara/Fujimura

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0

TEWL (g·m–2 h–1)

Hydration (AU)

100

Conclusions

No significant differences were detected between the skin on the trunk and the limbs in most of the SC functional parameters. No remarkable gender difference was found in these parameters and sites except for the pH value. We clarified that most of the SC functional parameters change with age on both the trunk and the limbs in a similar manner. In conclusion, these results suggest that the limbs are acceptable sites to estimate the SC functions

of the trunk of elderly patients. Our study may be useful in providing basic data for future work to maintain the SC function of elderly patients.

Acknowledgments Authors pray for the reposes of Dr. Shunichi Akiba, Kao Corporation.

References

Stratum Corneum Functions at Various Body Sites

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Age-related changes of stratum corneum functions of skin on the trunk and the limbs.

Despite our understanding that the care of back and buttock skin is important for elderly nursing patients, the stratum corneum (SC) functions of the ...
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