Arch Dermatol Res (2004) 296: 163–168
DOI 10.1007/s00403-004-0499-7
O R I GI N A L P A P E R
Meike Bock Æ Nanna Yvonne Schürer
Hans Joachim Schwanitz
Effects of CO2-enriched water on barrier recovery
Received: 19 April 2004 / Revised: 12 July 2004 / Accepted: 14 July 2004 / Published online: 7 August 2004
Springer-Verlag 2004
Abstract The objective of the present study was to
evaluate the impact of CO2-enriched water on barrier
recovery of detergent-damaged skin compared to tap
water employing bioengineering methods and thin-layer
chromatography (TLC) analysis of stratum corneum
(SC) lipids. Irritation of the skin was elicited on the
forearms of 20 volunteers using 1% sodium lauryl sulphate (SLS). The degree of skin irritation was followed
over 10 days in terms of skin colour reflectance
(L*a*b*), transepidermal water loss (TEWL), and skin
capacitance expressed as median values. For TLC
analysis, SC lipids were extracted prior to and during the
observation period. Clinical examination showed the
efficacy of CO2-enriched water on barrier recovery.
Compared to unenriched tap water, CO2-enriched water
produced a significant (P<0.01) increase in total SC lipids and in particular in the ceramide fraction. Furthermore, TEWL was significantly (P<0.01) lower in
skin treated with CO2-enriched water than in skin treated with unenriched water. These findings may indicate
that rinsing with CO2-enriched water enhances (1) clinical regeneration of detergent-damaged skin, (2) epidermal lipid synthesis, and (3) barrier repair after
detergent-induced perturbation.
Keywords Stratum corneum lipids Æ Epidermal
barrier Æ Ceramides Æ Bioengineering methods Æ CO2
Introduction
It is known that the stratum corneum (SC) is of great
importance for proper skin barrier function. The integM. Bock (&) Æ N. Y. Schürer Æ H. J. Schwanitz
Department of Dermatology,
Environmental Medicine and Health Theory,
University of Osnabrück, Sedanstr. 115,
49090 Osnabrück, Germany
E-mail: [email protected]
Tel.: +49-541-4051813
Fax: +49-541-9692445
rity of this barrier is largely dictated by the presence of
extracellular lipids which consist of a mixture of free
fatty acids, cholesterol and ceramides together with
smaller amounts of cholesterol sulphate, glucosyl ceramides and phospholipids [7]. In diseased skin an impaired barrier function can often be ascribed to an
altered lipid composition and organization [9].
Enhancement of barrier repair after perturbation by
stimulation of epidermal lipid synthesis and the relevant
enzyme activities has been the subject of dermatological
research.
Permeability barrier recovery is delayed either when
perturbed skin sites are exposed to neutral pH buffers
[22] or when endogenous acidifying mechanisms are
blocked [10]. Extracellular processing of certain polar
precursors to nonpolar lipids needed for barrier function
and its maintenance depends on an environment with a
low extracellular pH [27]. The low extracellular SC pH
controls the formation and maintenance of the apolar
lamellar body-derived SC lipids. Two enzymes that are
required for lipid processing and require an acidic milieu
for optimal activity, b-glucocerebrosidase [15] and acidic
sphingomyelinase [17], are secreted at the SC granulosum interface. While acidification contributes to normal
barrier function, neutralization alone provokes aberrant
permeability barrier homeostasis and decreased SC
integrity [13, 22].
Little information is available on the effects of CO2
on SC lipid synthesis and barrier repair. Naturally
occurring carbonated water has been used since the
fourth century to treat skin lesions, and throughout
medical history CO2 has been observed to have ‘healing
powers’ even for ‘skin rashes’ and ‘ulcers’ [14]. Pilot
studies have revealed an enhanced clearing of skin lesions after irritation following treatment with CO2-enriched water compared to unenriched tap water [5].
CO2 dissolves slightly in water to form a weak acid
called carbonic acid. Under pressure water will absorb
more CO2 so that the solution contains more carbonic
acid. Within the viable nucleated epidermis carboanhydrases are responsible for the metabolization of CO2
164
to carbonic acid, which may dissociate into bicarbonate
ions and protons [12]. Carboanhydrases are not present
in the SC. Bicarbonate ions, which may not penetrate as
well as CO2 into the viable epidermal layers, cannot be
retrieved from the SC [30, 31]. Therefore, CO2 metabolization to carbonic acid may affect the physiological
pH gradient within the epidermal layers.
The purpose of this study was to analyse the efficacy
of topically applied CO2-enriched water upon (a) barrier
recovery of detergent-damaged skin evaluated by bioengineering methods and (b) lipid synthesis by barrierperturbed epidermis.
Materials and methods
Subjects
The study group comprised 20 healthy non-preselected
volunteers (15 female, 5 male; aged between 18 and
55 years, average 29,45 years) without any skin diseases.
The study was approved by the local ethics committee
which gave it unanimous approval. Informed consent
was obtained from all participants prior to their inclusion in the study. Subjects were instructed to avoid direct
application of any topical agent to their forearms (test
area) for 5 days prior to and during the investigation.
Procedure
On each forearm, three test areas of diameter 1.5 cm
were marked on clinically normal skin. To induce skin
irritation, 60 ll of a 1% solution of sodium lauryl sulphate (SLS analytical grade, purity >99%, Sigma
Germany) in distilled water was applied to each of the
three test areas under occlusion using large Finn
chambers (12 mm diameter; Scanpor chambers; Hermal,
Reinbeck, Germany) for 2·24 h on two consecutive days
according to the method described by Fartasch [8].
Over a period of 2 weeks the irritated areas on one
arm were flushed with CO2-enriched tap water for 1 min
once daily. Tap water (7–14 scale of hardness, pH
7.9±0.1) at 37C was enriched with CO2 to achieve a pH
of 5.4 at 37C (Carbomed, Ratzeburg). For intraindividual controls the irritated areas of the contralateral
arm were flushed with fresh CO2-unenriched tap water
only.
Bioengineering methods
The following parameters were recorded after a minimum of 30 min of quiet sitting in a controlled environment (temperature 20±0.5C, relative humidity
45±2%) with the forearms lying horizontally on a table.
Transepidermal water loss (TEWL), an indicator of
the integrity of SC barrier function and a sensitive
indicator of surfactant-induced irritation [1, 29], was
measured using a Tewameter 210 (Courage & Khazaka
Electronic, Cologne, Germany). Measurements were
taken in accordance with the guidelines for TEWL
measurements established by the ESCD [24]. Electrical
capacitance, an indicator of skin hydration (RHF), was
measured using a Corneometer CM820 (Courage &
Khazaka) [2]. Skin surface colour was quantified using a
standard tristimulus system as suggested by the Commission Internationale de l’Eclairage (CIE) using a
Chromameter CR-300 (Minolta, Ahrensburg, Germany). The colour is expressed using a three-dimensional coordinate system (L*a*b*). The redness of the
skin is measured on the a* colour coordinate, which is
an indicator of the presence of haemoglobin reflecting
the inflammation level of the skin [11]. All bioengineering measurements were performed daily before treatment.
Thin-layer chromatography
Epidermal lipids were extracted using ethanol/diethylether (3/1, v/v) prior to SLS irritation, immediately after
SLS irritation and 1 and 2 weeks after the rinses with the
CO2-enriched and CO2-unenriched water. For that
purpose, a glass cylinder (2.5 cm diameter) was gently
pressed onto the skin area to be delipidated. Each irritated area was only delipidated once; therefore three
areas of irritation were sufficient to perform the study.
The areas for delipidation were rotated to avoid an
anatomic selection bias.
For the separation of lipids precoated silica gel 60
thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) plates,
20·20 cm and 10·20 cm (Merck, Darmstadt, Germany)
were used. Glacial acetic acid, acetone, diethylether,
chloroform, n-hexane, methanol, chromatography water, petroleum benzene (boiling range 40–60C), sulphuric acid and phosphoric acid (85%) (all from Merck)
were laboratory grade. Pure lipid standards were purchased from Sigma (Munich, Germany) and solubilized
in chloroform/methanol (2/1, v/v). TLC and HPTLC
plates were precleaned overnight in methanol/chloroform/water (20/95/1, v/v/v) using a paper-lined developing chamber (Desaga, Heidelberg, Germany). Plates
were then dried for 10 min by hot air.
Concentrated epidermal lipids were rediluted in
100 ll chloroform/methanol (2/1, v/v) and 20 ll aliquots
were spotted parallel to the bottom edge of a precleaned
TLC plate at a constant distance of 1 cm using a Desaga
TLC application device AS 30. All separations were
carried out in Desaga developing chambers presaturated
with the appropriate solvent system at room temperature. For separation of all major lipid classes, the consecutive solvent system as described by Melnik et al. [23]
was employed. To visualize the chromatographed lipids,
TLC and HPTLC plates were sprayed with an aqueous
solution of 10% CuSO4/8% H3PO4 (Sigma) and charred
at 180C for 30 min on a hot plate [4]. The chromato-
165
grams were evaluated in the remission mode using a
Desaga densitometer CD 60, which is supplied with the
matching densitometric software. Scanning parameters
employed included wavelength of 546 nm, automated
integration of each peak as well as automatic background correction. Identification of each peak was performed
via
direct
comparison
with
the
cochromatographed lipid standard.
Data analysis
Statistical analysis was conducted with SPSS (version
11.0). Bioengineering data are presented as delta values
(baseline after irritation adjusted). Differences between
sites treated with CO2-enriched water and unenriched
water were tested for statistical significance using the
Mann-Whitney U-test for non-normally distributed
non-paired data. The Wilcoxon signed rank test was
used to compare the results during the course of each
treatment. The chosen level of significance was P £ 0.05.
Results
Cumulative irritation with 1% SLS over 2·24 h led to
eczematous skin reactions, clearly visible and objectively
detectable by the bioengineering techniques used. Visual
examination showed that CO2-enriched water accelerated clearing of skin irritation compared to contralateral
sides rinsed with unenriched water. Figure 1 shows both
arms of a representative volunteer after 1 week of daily
rinses with unenriched water (Fig. 1a) or CO2-enriched
water (Fig. 1b). The side treated with unenriched water
showed erythema, papules and infiltration (Fig. 1a),
whereas the side treated with CO2-enriched water
(Fig. 1b) showed only a discrete postinflammatory hyperpigmentation and lichenification. Figure 2 shows
comparable results from another volunteer after a 5-day
treatment regimen. The side treated with unenriched
water (Fig. 2a) still showed a marked eczematous reaction with infiltration, papules and serocrusts, whereas
Fig. 1a, b Volunteer no. 19 (a)
after 1 week of daily rinses with
CO2-unenriched tap water, and
(b) after 1 week of daily rinses
with CO2-enriched tap water
the side treated with CO2-enriched water (Fig. 2b)
showed only discrete erythema, postinflammatory hyperpigmentation and small crusts.
In both test areas SLS irritation led to a significant
increase in TEWL (P<0.01), whereas skin capacitance
decreased significantly (P<0.01). The skin reaction was
also characterized by erythema resulting in a significant
(P<0.01) increase in redness (a*). There were no significant differences among the postirritation values of
the skin physiological parameters (TEWL, RHF,
L*a*b*; data not shown), suggesting that the baseline
values did not influence the relative values (postirritation
values adjusted).
Treatment with CO2-enriched water leads to an
improvement of barrier function measurable
by alterations in skin physiological parameters
TEWL was considerably lower on the side treated with
CO2-enriched water than on the side the side treated
with unenriched water. The time course of TEWL values
is shown in Fig. 3. The values of a* were significantly
lower after 2, 4, 5, 6, 7, 8, and 9 days of treatment in the
areas treated with CO2-enriched water than in the areas
treated with unenriched water (Fig. 4). From day 2 to
day 10 the areas treated with CO2-enriched water
showed higher median RHF values, although the differences in RHF values were statistically significant only
on days 3 (P<0.01) and 8 (P<0.001).
TLC of epidermal lipids showed increased total SC
lipids in irritated skin treated with CO2-enriched water
as compared to the side treated with unenriched water.
While the contralateral side, treated with unenriched
water, reflected the time course of lipid synthesis after
irritation, the side treated with CO2-enriched water
showed an increase in total epidermal lipid content with
a significant increase in SC ceramides. A significant increase in epidermal ceramide content was seen in both
test areas 1 week after perturbing the barrier with 1%
SDS. The median total ceramide peak areas before and
after irritation with 1% SDS as well as after 1 and
2 weeks of treatment with CO2-enriched water or
166
Fig. 2a, b Volunteer no. 7 (a)
after 1 week of daily rinses with
CO2-unenriched tap water, and
(b) after 1 week of daily rinses
with CO2-enriched tap water
Fig. 3 Median values of TEWL
after irritation with SDS 1% for
2·24 h under occlusion (n=20).
The TEWL values were
normalized to 100% at time 0,
and the results presented
represent percentage recovery
over a 9-day follow-up after
barrier perturbation. *P<0.05,
**P<0.01, ***P<0.001 (ns not
significant)
Fig. 4 Median values of a*
after irritation with SDS 1% for
2·24 h under occlusion (n=20).
The a* values were normalized
to 100% at time 0 and the
results presented represent as
percentage recovery over a
9 day follow-up after barrier
perturbation. *P<0.05,
**P<0.01 (ns not significant)
unenriched water are shown in Fig. 5. While the difference in total ceramide content between the two sides was
not significant at time zero, the total ceramide content in
the side treated with CO2-enriched water was significantly higher than in the side treated with unenriched
water after 1 week (P<0.05) and after 2 weeks
(P<0.01) of treatment.
Discussion
In these studies, distinct differences in the clearing of SLSirritated skin were observed between skin treated with
CO2-enriched water and skin treated with unenriched
water only. Furthermore, total SC lipid and in particular
167
Fig. 5 Median ceramide peak areas before and directly after
irritation, and after 1 and 2 weeks of daily rinses with CO2enriched or CO2-unenriched tap water (n=20). *P<0.05,
**P<0.01, Mann-Whitney U-test (n.s. not significant)
the ceramide content of SLS-irritated skin was enhanced
after treatment with CO2-enriched water. The increase in
SC lipid and ceramide content was accompanied by an
improved SC barrier function, as reflected by a lower
TEWL. Therefore, CO2-enriched water may stimulate
epidermal ceramide biosynthesis which results in a superior lipid barrier. Rawlings et al. [26] have reported similar
effects following L-lactic acid treatment. Twice-daily
application of 4% formulations of L-lactic acid (pH 3.7–
4.0) led to a significantly improved barrier function as
reflected by a reduction in TEWL and an increase in total
SC ceramide content.
As reported previously, ceramides play a primary role
in barrier function homeostasis and consequently in the
control of water evaporation [20]. The fact that ceramides
showed the described alterations in our experiments may
suggest that topical application of CO2 can effect terminal
keratinocyte lipogenesis resulting in quantitative and/or
qualitative changes of barrier repair after perturbation
with 1% SDS. In vitro, CO2 impairs the expression of
several markers of terminal differentiation such as involucrin, transglutaminase-1, filaggrin and loricrin (personal communication, Grether-Beck, Heinrich Heine
Universität Düsseldorf). The reduction in the expression
of these proteins is considered to be the cause of a reduced
and less-adhesive horny layer [6]. The synthesis of interlammellar lipids, which play an important role in lamellar
cohesion and adhesion in the SC layers, is a part of
keratinocyte differentiation [25]. CO2-enriched water may
affect keratinocyte lipid metabolism. The low SC pH is
affected by catabolic processes within the SC including the
production of urocanic acid by the breakdown of filaggrin
[18], the hydrolysis of phospholipids to yield free fatty
acids [10], and the generation of H+ from NHE1 [3].
Recently, the central role of an acidic milieu has
become increasingly understood as a regulating factor
in SC homeostasis with relevance to the integrity of
the barrier function. Mauro et al. [22] analysed the
effects of pH on barrier recovery following acute
barrier perturbation in hairless mice and found that
barrier recovery proceeds normally at an acidic pH. In
contrast, barrier recovery is slowed when treated skin
is exposed to neutral or alkaline pH. The importance
of an acidic pH of the SC for barrier homeostasis is
suggested by the impairment of barrier function associated with alkalization of the skin [28], and the
exacerbation of experimentally induced contact dermatitis at alkaline pH [32]. Furthermore, SC pH may
be important in skin disorders that are accompanied or
caused by pH changes, such as acute eczemas.
CO2 may penetrate the disturbed barrier of SLS-irritated skin lesions more freely than undisturbed epidermal
barrier. The influence of the extracellular acidity at the
boundary between the SC and the stratum granulosum on
lipid processing enzymes such as phospholipases,
b-glucosylceramidase, b-glucocerebrosidase and acid
sphingomyelinase has been discussed as a possible
mechanism of maturation [16]. Whereas the pH optimum
of the SC phospholipase A2 isoform is not known, epidermal b-glucocerebrosidase and sphingomyelinase,
which generate a family of ceramides from glucosylceramide and sphingomyelin precursors, respectively exhibit a
distinct acidic pH optimum [15, 16]. In particular, b-glucocerebrosidase, with an enzyme optimum of 5.6, is involved in the synthesis of the ceramides which are the most
important barrier lipids. Introducing CO2-enriched water
(pH 5.4 at 37C) into topical therapy may support enhanced restoration of a physiological skin pH, resulting in
an increased SC ceramide content.
Barrier recovery is inhibited by high extracellular
Ca2+ and K+ levels and is accelerated by low extracellular concentrations of these ions. These ions control
barrier homeostasis through their effect on lamellar body
secretion [19]. Hypothetically, treatment with a CO2
solution could initiate the formation of KHCO3 and
Ca(HCO3)2 which would result in lower free Ca2+ and
K+ extracellular concentrations. Nevertheless, more
obvious are the findings that the major effect of extracellular pH—in this case the lowering of pH following
treatment with a CO2 solution—is on extracellular processing of lipids independent of ionic effects [22].
In conclusion, topical treatment of eczematous skin
lesions with carbonated water resulted in accelerated
clearing time, higher SC lipid and ceramides contents
and restoration of barrier function as reflected by lower
TEWL values. Further studies are underway to clarify
the specificity and clinical relevance of these findings.
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