June 2019

Medical Mass Spectrometry Vol. 3 No. 1

Research Paper

Imaging mass spectrometry reveals sodium lauryl sulfate-induced changes
in skin lipoquality, principally affecting sphingomyelin
1,2

1,2

1,2,3

Yusuke Saito , Fumihiro Eto , Shiro Takei

2,4
1,4, Ikuko Yao *, Mitsutoshi Setou

1

Department of Cell Biology and Anatomy, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431‒3192, Japan2
Department of Optical Imaging, Institute for Medical Photonics Research,
Preeminent Medical Photonics Education & Research Center, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431‒3192, Japan
3
Laboratory of Fish Biology, Department of Environmental Biology, College of Bioscience and Biotechnology,
Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487‒8501, Japan
4
International Mass Imaging Center, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431‒3192, Japan
Abstract

Lipids and lipoqualities are important for skin barrier function. In previous reports, repeated irritation by so-dium lauryl sulfate (SLS) was found to reduce epidermal barrier function and epidermal water content. Imaging mass
spectrometry (IMS) can reveal not only the identity and localization of lipids in organs but also the distribution of administered drugs. Here, we attempted to visualize the distribution of SLS and changes in lipid composition in the epidermis of
the skin. Initially, we histologically examined micro damage of the skin using trypan blue in the foot-pad of mice treatedwith SLS. Subsequently, we assessed the invasion of SLS and the effect of SLS on the lipid composition of the skin using
IMS. We found that, relative to the foot-pad of mice treated with saline, the foot-pad of mice treated with SLS exhibited asigniﬁcant change in m/z 817.71 associated with sphingomyelin despite the fact that no noticeable damage was observed in
the layer structure dyed with trypan blue. Furthermore, we proved the penetration of SLS to the epidermis and dermis. The
results suggest that SLS penetrates the skin tissue and alters the lipid composition on the skin surface.
Key words: Skin, Pharmaceuticals, Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS),
Sodium lauryl sulfate, Lipids

Introduction

horny zone. Horny zone can be divided into three layers;

Healthy skin forms a barrier with the epidermis contrib-

horny layer, clear layer and granular layer. Notably, lipids

utes to the defense mechanism against infection from theenvironment, and controls water transpiration. The epider-such as ceramide in the horny zone play an important rolein skin barrier function. In addition, abnormal balance oflipid composition of the horny zone causes various dis-mis is composed of a basal layer, a prickle cell layer, and a

1‒3)

eases

* Corresponding author.

Surfactants ﬁnd widespread use as emulsiﬁers, and as

Ikuko Yao

suspending, solubilizing, and stabilizing agents. Due totheir unique properties, surfactants are used in the produc-

Department of Optical Imaging, Hamamatsu University
School of Medicine, 1‒20‒1 Handayama, Higashi-ku, Hama-tion of many products such as pharmaceuticals, cosmetics,

matsu, Shizuoka 431‒3192, Japan
Tel/Fax: ＋81‒53‒435‒2092

pesticides and detergents. In addition, various detergents

Received January 28, 2019. Accepted February 27, 2019.

are used as skin cleansing products to maintain hygiene.
However, surfactants, in particular the hydrophilic deter-

Epub June 11, 2019.

gent sodium lauryl sulfate (SLS), can change permeability

DOI: 10.24508/mms.2019.06.004

in cell membranes in biological systems .

E-mail: yaoik@hama-med.ac.jp

4)35June 2019

Medical Mass Spectrometry Vol. 3 No. 1

SLS is widely used in industrial cleaners and cosmetics,

dipped in physiological saline for 10 min. Mice were theneuthanized and the foot-pads were excised. Physiologicaland it lowers the surface tension of aqueous solutions and5)

can dissolve lipid membranes . The main function of a skinsaline was applied as a control.

cleanser is to remove dirt and grime from the skin surface.

In the experiment showing the extent of trypan blue (TB)
inﬁltration, the foot-pad was washed for 10 min, immersed

However, removal of useful skin lipids results in transientdermatitis and is often followed by a long-lasting hyporeactivity to irritants. Repeated irritation with SLS reduced epi-

in 5% aqueous TB solution for 60 min, and ﬁnally washedlightly with running water or physiological saline. The tis-dermal barrier function and epidermal water content. In

sues for optical microscopy were sectioned to 30 μm thick-recent years, many studies have investigated skin irritation

ness at −20°
C using a Leica CM1950 cryostat (Leica. However, the speciﬁc molecules involved in theMicrosystems, Wetzlar, Germany). Sections were thenby SLS5‒7)mechanism have not been elucidated.

placed on a glass slide and embedded in ultraviolet curable

Imaging mass spectrometry (IMS) is a powerful tool that

resin. The background color of each image obtained byallows simultaneous mapping of many molecules in a tissue
8)
section by a single measurement . Our group has devel-

optical microscopy (OLYMPUS, cellSense) was corrected

oped original IMS equipment in collaboration with Shimadzu Corporation of Japan. The method extends tech-(Wayne Rasband, National Institutes of Health, USA) wasTMusing Adobe Photoshop

and was then combined. ImageJ

used for image analysis.

niques for molecular proﬁling of different tissue samples,
9)
10)
11)
12)
such as the brain , liver , testis , oral tissue , whole-

Tissue Preparation for IMSof rodents, and colon cancer inand involves sample preparation, and a nanopar-

For IMS, the skin tissues subjected to saline or SLS treatments were directly snap-frozen in liquid nitrogen and sec-

imaging has resulted in many developments for assessing

tioned to 15 μm thickness. Continuous frozen sections were
thaw-mounted on MAS coated glass slides (Matsunami

the localization of molecular species in biological samples.

Glass Industries, Ltd., Osaka, Japan). We used 1.0 mL of

Several applications represent the direct entailment of this

CHCA solution (37.8 mg/mL in 60% acetonitrile containing

technology to basic clinical research. In this study, we used
IMS to clarify the effect of SLS on the lipoquality of skin

0.1% TFA) as the matrix to detect SLS. The matrix solution
was uniformly sprayed on the tissue surface using a 0.2-mm

which is crucial for its barrier function.

nozzle-caliber air brush (Procon Boy FWA Platinum; Mr.

Materials and Methods

Hobby, Tokyo, Japan). We used 1.0 mL of DHB solution(50 mg/mL in 50% methanol) as a matrix for lipid detec-

13)

14)

eye , and retina
15)

humans16)
ticle-based ionization process in IMS . Notably, MALDI

tion, by spraying on sections with an air-brush (Procon Boy

Reagents

α -Cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) were obtained from Bruker Dal-

FWA platinum; Mr. Hobby, Tokyo).

tonics (Leipzig, Germany). SLS (purity＞99.5%) was pur-

MALDI-IMS

IMS experiments were performed in the positive ionization mode and negative ionization mode using MS-IT-TOF

chased from Nacalai Chemicals (Kyoto, Japan) and wasused as received.

(Mass microscope prototype; Shimadzu Corporation,

Animal experiments

Kyoto, Japan) in the linear positive and negative modes.

We used the foot-pad skin of 8-week-old C57BL/6J male

The Nd:YAG laser at 355 nm was used at 25% energy andmice from Japan SLC (Shizuoka, Japan). The care and useof laboratory animals was in accordance with the Animal

1000 Hz repetition rate. The interval between data points
was 10 μm, sufﬁcient to cover the entire section. Mass spec-

Experiment Regulations of the Hamamatsu University

tra were obtained with a scanning mass range of 250 to 300

School of Medicine. Mice were anesthetized with chloral

Da for detecting SLS and with a range of 600 to 910 Da forhydrate (400 mg/kg, i.p. using a 3.5% aqueous solution)

detecting lipids with a mass resolution of 10,000. Images ofand mounted on a stereotaxic apparatus.
The time schedule of the experiment is shown in Fig. 1A.

skin sections were acquired using the Mass microscopeprior to LDI. Data were analyzed using Imaging MS Solu-

The mouse foot-pad was treated with 5% SLS for 1 h andtion software (ver.1.01.02, Shimadzu) and IMAGERE36

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Medical Mass Spectrometry Vol. 3 No. 1

Experimental design and changes in mouse foot-pads after SLS treatment.

Fig. 1.

A: Time schedule of the TB immersion experiment. Mouse hind feet were immersed in 5% SLS aqueous solution for 60 min andthen washed with physiological saline for 10 min. These samples were used for IMS. To evaluate TB penetration, mouse feet subjected to SLS or control treatment were further immersed in 5% TB for 60 min, followed by sectioning. B: Workﬂow of imaging
mass spectrometry. Multiple sections were sliced using a cryostat and were placed on conductive material. The ion image for speciﬁc signals was reconstructed from the obtained spectra. C: Optical images of swollen mouse foot-pads immersed in 5% SLS for
60 min. D: Cross-section optical images of control (left) and SLS-treated (right) mouse foot-pads stained with TB. E: Gray scale in‒y′indicating histological differences in the sites stained with TB. Arrows indicate thetensity of color in the yellow line x–y and x′
position of the skin surface. The vicinity of the boundary between the surface, epidermis, and dermis is indicated by a dotted line.
Scale bar represents 20 μm. F: The degree of staining of the skin cross section was estimated by the integrated value of TB intensity
in Fig. 1E. SLS, sodium lauryl sulfate; TB, trypan blue; ED, epidermis; Der, Dermis.

TM

VEAL

mulating time＝221 ms; repeating number＝1; and 10 μm

MS software. The experimental design is shown

interval between data points. The other settings were thein Fig. 1B.

same as those used for the MS analysis of precursor ions.

MS/MS analysis

Mass spectra were obtained with a scanning mass range

In order to obtain sufﬁcient signals, one skin sample was

between 50 and 300 Da with a mass resolution of 10,000.

used for each measurement. The molecular weight range
for the ion trapping was 1.0 Da around the m/z of each pre-Statistical analysis

A Student s t-test was used for comparison between
groups. All data are presented as mean±SEM of the num-cursor ion. The setting of the IMS analysis was as follows:
laser intensity for fragmentation＝50; gas level＝50; accu37

June 2019

Medical Mass Spectrometry Vol. 3 No. 1

Mass microscope detected SLS signal

ber of biological replicates (noted in the ﬁgure legends).

In a previous report, repeated exposure to surfactants

Results and Discussion

such as SLS was shown to cause skin function to fail . We

Effect of SLS on skin surface histology

assume that SLS inﬁltrates the skin surface. IMS is useful

6)Here, we histologically evaluated the effect of SLS onfor directly detecting a target substance without requiring askin surface. Our results show that SLS caused swelling ofmouse foot-pad, as shown in Fig. 1C‒F, but the structure oflabel. Therefore, we tried to detect SLS on the skin surface

the skin surface was maintained. The images of the foot-Saline and SLS solution were dropped on a mouse tissue

pad of mice treated with SLS or saline are shown in Fig.
1C. Strong swelling was observed in the foot-pad of mice

section, MS spectra are shown in Fig. 2A (saline), and 2B

treated with SLS than in mice treated with saline.

obtained at m/z 265.15. To conﬁrm the signal of m/z 265.15
was derived from SLS, we performed MS/MS measure-using IMS.

(SLS). In the region where SLS was dropped, a peak was

Subsequently, the extent of TB staining and invasion was
histologically measured to observe the extent of damage ofthe swelling in the foot-pad. The experimental procedure isment of the target substance and compared the peaks

shown in Fig. 1A. In appearance, (Fig. 1C below), foot-in Fig. 2C, a strong peak was observed at m/z 96.96, whichpads of mice treated with SLS were less stained than those

was considered to be fragmented and was detected in the
portion indicated by the arrow in the SLS structural for-obtained by fragmentation form standard. From the resultsof mice treated with saline. We obtained an optical image toobserve the microstructure of the foot-pads, and the image

mula shown in Fig. 2C. These results suggested that the m/z

was analyzed to evaluate the extent of TB staining andinvasion. Cross-sectional images were corrected in Photo-265.15 is a peak derived from SLS.

shop (Adobe), converted to grayscale, and a graph was

SLS inﬁltrated into the skin of the mouse foot-pad

obtained by digitizing the gray value in the line connecting

Previous experiments revealed that SLS swells the skinof the mouse foot-pad. The result implied that SLS inﬁl-points x to y, as shown in Fig. 1D, using Image J software,

trated into the skin of the mouse foot-pad. In addition, we

the results are shown to the left of Fig. 1E. Swelling of the
foot-pad of mice treated with SLS was also observed in theproved detection of SLS in IMS. Here, we investigated thespectrum of SLS in mice foot-pad treated with SLS using

optical images. However, noticeable layer structure damage
by invasion of TB was not observed in the foot-pad of mice

IMS (Fig. 2D, E).

treated with SLS as well as treated with saline. The foot-To compare the extent of inﬁltration of SLS, we divided

pad of mice treated with SLS had a lower gray value in the
ED layer than in the foot-pad of mice treated with saline.

the skin into stratum corneum (SC), stratum lucidum (SL),We randomly selected ﬁve points and created a similar

regions. The yellow dotted line shows the position of thegraph. We conﬁrmed that the result is reproduced.

stratum granulosum (SG), stratum basale (SB), and dermis

In addition, this experiment was performed for three skin

skin surface. As shown in Fig. 2A, m/z 265.15 wasobserved at a high intensity on the skin surface of the foot-samples, and the sum of the gray values up to the epidermis

pad of mice treated with SLS as indicated by the dotted line

was averaged and shown in Fig. 1F. The total gray value
was signiﬁcantly lower for the foot-pads of mice treated

and was found to reach the dermis. We evaluated thesewith SLS than that for foot-pads of mice treated with saline.
Normal skin has a moisture content of 20‒30% and its

the skin. The intensities in the range selected from theimaging results using spectral intensities in each layer ofimage processing software were compared and the result isshown in Fig. 2E. In the foot-pad of mice treated with SLS,

lamellar structure is stabilized. However, it is suggested
that the foot-pad of mide treated with SLS swelled more

a strong intensity was obtained in the vicinity of the stratum

than that of mice treated with saline because of SLS inﬁltration, despite no noticeable damage in the layer structure.

corneum, and a stronger intensity was shown closer to thesurface. In the foot-pad of the mice treated with saline, such

Furthermore, the fact implies that SLS inﬁltration in thelocations where it occurred caused an increase in water per-spectra were obtained in neither the epidermis nor the dermis. In addition, the existence of m/z 265.15 was also con-meability and swelling.

ﬁrmed on the slide glass. However, this may be because of
the SLS dissolved with acetonitrile attached to the surface
38June 2019

Fig. 2.

Medical Mass Spectrometry Vol. 3 No. 1

Detection of sodium lauryl sulfate on the surface of mouse food pad by Mass Microscope.
A: Mass spectrum of a region where saline was dropped on a mouse tissue section as a control. B: Mass spectrum of a region where
5% SLS was dropped. A peak derived from SLS was conﬁrmed at m/z 265.15. C: MS/MS result of m/z 265.15 as a precursor ion
after dropping SLS standard along with matrix solution onto the untreated tissue section. The obtained spectrum showed a peak at
m/z 96.96 conﬁrming the presence of SLS. D: Imaging of SLS detected as the ion at m/z 265.15 in the surface of mouse food pad.
The yellow dotted line shows the position of the skin surface. Scale bar represents 100 μm. E: The average signal intensities of theion at m/z 265.15 in the skin treated with or without SLS. SLS, sodium lauryl sulfate; SC‒SL, stratum corneum‒stratum lucidum;
SG‒SB, stratum granulosum‒stratum basale; Der, Dermis.

SLS-induced changes skin lipoquality, principally
affecting sphingomyelinof the glass slide during atomization of the matrix at thetime of sample preparation. As the visualized data show therespective positional information and spectrum intensity, it

Here, we demonstrated the effect of SLS on the distribu-

is possible to conﬁrm the existence of the target substancein each organization and to compare them by using the soft-tion of various kinds of lipid molecular species on skin of
the mouse foot-pad via IMS. We obtained six types ofware by selecting an arbitrary area of the sample. In theimages of locations where lipids were considered to bepresent of the mouse foot-pads treated with SLS and saline

results in Fig. 2B, the signal intensity decreased gradually(Fig. 3). We show that the peaks at m/z 608.48, m/z 769.58,
m/z 796.55, m/z 817.71, m/z 844.55, and m/z 894.62 corre-

from the upper layer to the lower layer of the skin of the
SLS. Therefore, this result shows the degree of inﬁltrationof SLS into the skin of the foot-pad of mice treated withspond to lyso-phosphatidylcholine (LysoPC) (24:0), sphingomyelin (SM) (d18:1/18:0), phosphatidylcholine (PC)SLS.

(C16:0/C18:2), SM (d18:0/24:0), PC (C16:0/C22:6), andglucosylcermide (GlcCer) (d18:1/C26:0h) (t18:1/C26:1).
Notably, SM (d18:1/18:0), PC (C16:0/C18:2), PC (C16:0/
39

June 2019

Fig. 3.

Medical Mass Spectrometry Vol. 3 No. 1

Speciﬁc lipid reduction in the mouse foot-pad skin after SLS application.

A: Optical cross-sectional image of mouse foot-pad skin. The yellow dotted line shows the skin surface. B‒G: Mass images of ionsat indicated values which correspond to lipids. Histograms show the distribution of their intensity. Scale bar represents 100 μm.

40June 2019

Fig. 4.

Medical Mass Spectrometry Vol. 3 No. 1

Disappearance of SM (d18:0/24:0) signal at m/z 817.17 from the mouse foot-pad skin surface after SLS application.
A: Averaged mass spectrum in the ROI of the Control or SLS applied skin. B: Comparison of the intensities of respective peaks.
White bars indicate Control; black bars represent SLS applied skin.

C22:6), and GlcCer (d18:1/C26:0h) (t18:1/C26:1) havetive SLS treatment. In this study, we suggest the possibility
that a change in SM (d18:0/24:0) on the surface is import-

been previously reported to be expressed on the skin of the17)mouse foot-pad . These four lipid molecular species wereant for skin barrier function.

located deep in the skin and did not change in the mousefoot-pad treated with SLS compared to the those treated

Conclusion

In this study, using IMS, we demonstrated that SLS pene-with saline. Hence, this results suggested that single treatment of SLS did not affect lipids in the deep areas of the

trates the skin and alters the lipid composition of the skinskin.

surface. We found that the lipid composition in the deep

In contrast, as shown in Fig. 3E, SM (d18:0/24:0) was
located on the surface of the skin of mouse foot-pads

parts of the skin does not change. A single treatment of SLS

treated and decreased in the mouse foot-pads treated withskin. Therefore, it is suggested that skin dysfunction due to

SLS compared to those treated with saline. Fig. 4 shows therepeated administration of SLS involves a change in skinhistogram of the abundance ratio of these substances in thelipid composition. Furthermore, various surfactants as well
as SLS can cause removal of lipids. Our study proposes rea-

mainly changes the lipid composition on the surface of theepidermal surrounding area. SM (d18:0/24:0) had a lower
signal intensity in the foot-pads of mice treated with SLS

sons to pay attention to the handling of daily surfactants.

compared to treated with saline. Furthermore, statisticalAcknowledgements

analysis of these data showed that the intensity of SM(d18:0/24:0) was signiﬁcantly reduced in the mice foot-pad
treated with SLS compared with those of mice treated withThis study was ﬁnancially supported by JSPS KAKENHI
Grant-in-Aid for Scientiﬁc Research Grant Numbers

saline.

26460388, 16KT0134 to I.Y, JP15H05898B1 to M. S., by

These results suggest the effect of SLS on lipids, espe-

Imaging Platform supported by MEXT, Japan, and bycially the effect of SM (d18:0/24:0) extraction. SM(d18:0/24:0) exists in the cell membrane at high concentra-

AMED Grant Number JP18gm0910004. The authors also

tions. However, the role of SM (d18:0/24:0) in maintaining

Center and the Advanced Research Facilities and Services,

the barrier function of the skin has not yet been reported.

Preeminent Medical Photonics Education and Research

SLS changed the lipid composition in the epidermis ofmouse foot-pad, although the foot-pads were only treatedCenter (Hamamatsu University School of Medicine, Japan)

thank the staff members of International Mass Imaging

for providing advices and technical assistance.

once. Therefore, it is suggested that the removal effect of
SLS on lipids may promote skin dysfunction under repeti41

June 2019

Medical Mass Spectrometry Vol. 3 No. 1

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