PLoS ONE
Public Library of Science
image
Microbeam X-ray diffraction study of lipid structure in stratum corneum of human skin
Volume: 15, Issue: 5
DOI 10.1371/journal.pone.0233131
  • PDF   
  • XML   
  •       
Abstract

Human skin, not previously frozen, was studied by small-angle X-ray diffraction. The samples were folded so that a 6μm X-ray beam passed through the top layer of skin, stratum corneum. Diffraction patterns recorded with this method consisted of peaks at about q = 0.5, 1.0 and 1.4 nm-1 in the direction perpendicular to the skin surface more clearly than in previous studies. These peaks are interpreted to arise from lipids between corneocytes. A simple unit of a linear electron density profile with three minima was used to account for the observed intensity profiles. Combinations of calculated diffraction from models with one, two and three units accounted for the major part of the observed diffraction pattern, showing the diversity in the structure of the intercellular lipids.

Yagi, Aoyama, Ohta, and Haverkamp: Microbeam X-ray diffraction study of lipid structure in stratum corneum of human skin

Introduction

The top layer of human skin, stratum corneum (SC), serves as a barrier which protects human body from dehydration and penetration of unwanted substances. Its property is also important in tarnsepidermal drug delivery. SC comprises of inactive cells (corneocytes) in which keratin filaments are densely packed [1] and lipids such as ceramide, cholesterol, fatty acids and glycerides fill the space between the cells. The barrier function is considered to mostly depend on the lipids that fill the gap of less than 50 nm wide. Several lipid molecules, such as ceramides and sphingolipids, are arranged in a lamellar structure with their long axes lying across the gap. Many studies on the intercellular lipid structure have been carried out using electron microscopy, electron diffraction and X-ray diffraction. In most experiments, SC was chemically treated to prepare specimens suitable for each technique. In X-ray diffraction experiments, SC samples were often separated from skin by trypsin digestion [25] and investigated as whole SC. This method allows control of hydration and temperature and helps identify origins of diffraction peaks [2, 4, 5]. Studies on human [3, 6, 7], mouse [4, 5], and pig [2] SCs as well as human skin equivalents [8, 9] showed the presence of common basic lipid structure. X-ray diffraction studies on lamellae of lipid constituents of skin have been also made to investigate lipid phases [1012]. For electron microscopy, it is common to go through the procedures of chemical fixation, dehydration and staining of samples before cutting thin sections [13]. Thus, an observed pattern of density in electron micrographs may not represent electron density in a natural specimen. So far, high-resolution structural studies under a condition that is most similar to a living body were made using cryo-electron microscopy [8, 14]. For this technique, human SC is rapidly frozen by liquid nitrogen and then cryo-sectioned and observed by an electron microscope in a frozen state. Thus, it is possible to obtain more direct information on electron density distribution than conventional electron microscopy. However, to obtain structural information at higher spatial resolution, it is necessary to study SC under physiological conditions with X-ray diffraction. For this purpose, we performed small-angle X-ray diffraction (SAXD) measurements of SC on intact human skin.

Recently, there has been a report of an X-ray diffraction study on intact human skin that was cut into a fine (0.1 mm width) strip [6]. Using an X-ray beam with a diameter of a few micrometers, it is possible to investigate the depth dependence of lipid structures in SC. Our sample is a similar one (from the same supplier) but we employed a technique that does not require cutting a skin sample into a fine strip which might distort the structure. As was in a previous experiment on skin of a transgenic mouse [15], we folded a skin sample and passed an X-ray microbeam at the edge of the sample to obtain diffraction from SC so that we can study skin with X-rays under conditions as physiological as possible. The diffraction patterns thus recorded were found suitable for a detailed structure analysis for which we employed a simple tri-lamellar model.

Materials and methods

X-ray diffraction techniques

The SAXD measurements were performed at BL40XU (High Flux Beamline) in the SPring-8 synchrotron radiation facility (Hyogo, Japan). A quasi-monochromatic high-flux X-ray beam from a helical undulator (λ = 0.083 nm) was focused with two mirrors laid horizontally and vertically [16]. In the experimental hutch, an X-ray beam of about 6 μm in diameter was obtained behind a collimating pinhole (5 μm in diameter) and a guard pinhole (100 μm in diameter) [17]. The X-ray flux was about 1 × 1011 photons/s. The sample-to-detector distance was 1503 mm. The scattering vector q = (4π/λ)sin(2θ/2) was calibrated with powder diffraction from silver behenate, where 2θ is the scattering angle. X-ray diffraction patterns were recorded with an exposure time of 0.1 s using an X-ray image intensifier (V5445P, Hamamatsu Photonics, Hamamatsu, Japan) coupled to a CCD camera (C4880-50-24A, Hamamatsu Photonics) with 1024 x 1024 pixels [18]. The experiment was made at an ambient temperature of about 27 °C.

Human skin samples

Six full thickness human skin samples without adipose tissues were purchased from Biopredic International (France). They were obtained from abdomen of healthy Caucasian females (36–56 years old) in cosmetic surgery eight days before the experiment, punched in a disc with a 10 mm diameter and immersed in a preservation medium. The skin samples were provided without any personal information on the donors except sex, ethnic origin, anatomical site, age, and BMI. As for the ethics concerning the samples and compliance with the laws, the supplier, Biopredic international, provides information (https://www.biopredic.com/rubrique-ethics)). From the nature of the surgery, the range of donors is limited. The samples were transported in a fresh state at 4 °C without freezing. Some of the skins were cleaned with chlorhexidine alcohol or betadine before surgery.

Just before the experiment, a skin sample was taken out of the preservation solution and blotted on a filter paper to remove excess solution. Then, it was folded onto a folded aluminum tape so that it formed a pointed edge at the top (S2b Fig). The ends of the sample were fixed together with a paper clip. The skin was mounted on vertical and horizontal translational stages in air and an X-ray microbeam was passed through the SC at the pointed tip. The skin was moved vertically by 5 μm after each exposure to scan the entire thickness of the SC. This vertical scan was performed at five to ten different locations on each skin sample. The total measurement took 15 to 20 minutes. The skin sample was found moist after the experiment and change in the diffraction pattern due to drying was not observed throughout the experiment. A second scan at the same position provided identical results showing absence of a serious radiation damage, but the sample was shifted by about the diameter of the X-ray beam to avoid irradiating the same spot twice.

Data analysis

Lamellar diffraction appeared perpendicular to the skin surface (Fig 1). A detailed consideration on the nature of the diffraction is described in S1 Appendix. A radial distribution profile of diffraction intensity was obtained by summing intensity along the length of an arch at each radial position. For presentation, intensity was multiplied by q for the Lorenz correction. This is necessary to compare with intensity profiles obtained by simulations.

X-ray diffraction patterns from human skin.
Fig 1
(a) A typical X-ray diffraction pattern recorded at the top of the skin where the diffraction peaks were first observed. (b)-(f) Diffraction patterns recorded when the skin was raised with 5 μm steps, moving the X-ray beam towards the bottom of SC. The skin surface is horizontal.X-ray diffraction patterns from human skin.

Results

Experimental

Fig 1 shows a galley of SAXD patterns from human skin. Broad diffraction peaks due to oriented lamellar-like structure are clearly visible. Fig 2 shows intensity profiles along the direction perpendicular to the skin surface (this direction is defined as the meridian) integrated along the arcs. Profiles at six successive positions separated by 5 μm are plotted with displacements. Data from the six samples are shown. The profile at the bottom (black) is where the peaks were first observed and that at the top was recorded after the beam moved by 25 μm into the skin sample. Including the beam size, total of about 30 μm of thickness was investigated. The main features are the three major peaks at about q = 0.5, 1.0 and 1.4 nm-1 along the meridian.

Radial intensity distributions recorded at different depths of the SC.
Fig 2
(a)-(f) were obtained from different skin samples. The sample was raised with 5 μm steps from the black to the brown curve so that the black curve is closest to the skin surface. The profiles are vertically displaced for clarity. A peak at q = 1.85 nm-1 in (e) is due to crystalline cholesterol.Radial intensity distributions recorded at different depths of the SC.

The diffraction profiles in Fig 2a–2e show three major peaks at q = 0.51, 1.03 and 1.4 nm-1, the first two of which may be the first and second orders, but their intensities do not correlate well at different depths: in most of the typical results obtained in this study, the peaks at q = 1.03 and 1.4 nm-1 are much stronger than that at q = 0.51 nm-1 in the region close to the skin surface (black and blue curves), while the 0.51 nm-1 peak becomes stronger in deeper regions (red and brown curves). When measured at the depth where the peaks are most prominent, typically at 10 μm from where the peaks were first observed, the first peak was at q = 0.511 ± 0.005 (mean ± standard deviation, range 0.504 to 0.515) nm-1 and the second was at q = 1.034 ± 0.013 nm-1 (range 1.019 to 1.055). These values are averages over values from five skin samples, each of which was an average of values obtained at 5 to 10 different locations on the skin. The ratio of the two values is close to 2, suggesting that these are the first and second orders of the 12.3 nm periodicity (periodicity d is obtained by d = 2π/q). Additionally, there is a broad peak at around q = 2 nm-1 which may be the 4th order of this periodicity. Fig 2 clearly shows that the peak at q = 1.4 nm-1 , which was observed as a shoulder in a previous report on human skin [6], is indeed a separate peak as observed by Schreiner et al. [7].

When the X-ray beam is close to the skin surface, the peak at q = 0.5 nm-1 is usually weaker than that at q = 1.0 nm-1. In such a case, the 1.0 nm-1 peak tends to be broad and the 1.4 nm-1 peak is weaker (typically, Fig 2a and 2d). The 0.5 nm-1 peak becomes stronger when the beam penetrates 10–15 μm below the surface. Further down, it remains distinct while the peaks at 1.0 and 1.4 nm-1 become weaker and form a single broad peak. When they are merged, a single peak at around q = 1.0 nm-1 with a tail towards higher angles is observed (Fig 2a). Generally, the diffraction peaks from lipids were observed over the depth of 20 μm which is slightly larger than the reported thickness of SC, partly due to the size of the X-ray beam. Thus, the diffraction pattern from deeper regions of SC may include contribution from lipids newly synthesized in stratum granulosum.

The peak at q = 1.0 nm-1 is slightly variable in its position across SC. When it appears together with the peak at q = 0.5 nm-1, it is close to q = 1.0 nm-1, while it is often around q = 1.05 nm-1 when the peak at q = 0.5 nm-1 is not observed in the upper part of the SC (Fig 2b and 2d). However, at the depth where the peak is highest, its position is fixed within 1% as described above.

The sixth skin sample gave an anomalous diffraction pattern. As seen Fig 2f, there is a strong and broad peak around q = 0.4 nm-1 which moves towards higher angles at deeper regions. However, the profiles in Fig 2f are similar to others in the region q > 0.7 nm-1 . Thus, the intercellular lipid structure was the same as in other samples. The origin of the peak is unknown, but this sample may have been obtained from a person who used a particular kind of lipids for skin care which produced an additional large peak in the small angle region. This result shows variability of human skin samples and suggests danger in working only on a very small number of samples. Small variations in relative intensity of the peaks were also found among the other five samples (particularly, Fig 2b).

Structure model

We attempted to explain the experimental results using lipid structure models. In Fig 1b, the peaks at q = 0.5, 1.0 and 1.4 nm-1 appear at the medium depth of SC. The former two peaks may be indexed as first and second orders, but the peak at 1.4 nm-1 cannot be indexed. To account for the observed diffraction profiles, models of electron density distribution were constructed. These are based on the electron micrographs of Swartzendruber et al. [13], but the distance between bands and their heights did not strictly follow the micrographs which were obtained from the sections of chemically fixed, dehydrated, and stained samples. In these electron micrographs, RuO4 was used to stain lipid and thus the density mainly indicates distribution of RuO4. Although RuO4 is assumed to react polar lipids [19], it is uncertain which lipids are preferentially stained. The simplest unit in the model consists of a central density minimum and two minima that were at 5.0 nm on either side of the center with the same depth (Fig 3a). Profiles of all minima were assumed to be Gaussians. Diffraction intensity expected from this unit gives broad peaks at around q = 0.55 and 1.1 nm-1 (Fig 3d). When two such units were superposed with a separation of 13.0 nm (Fig 3b), the resultant electron density profile resembles a model of tri-lamellar structure (the so-called Landmann unit) [13, 20]. X-ray diffraction expected from this model has peaks at q = 0.5, 1.0 and 1.3 nm-1 (Fig 3d). With three units (Fig 3c), there are small additional peaks other than the major three peaks (Fig 3d). When the diffraction intensities from the one-, two- and three-unit models are added with a ratio of 3:3:1, the resultant profile (Fig 3e) resembles the observed profile with peaks at q = 0.56, 1.04, 1.41 nm-1 and a broad baseline between the latter two peaks. This is similar to the experimentally observed profile in Fig 2.

Models based on electron microscopy and calculated diffraction from them.
Fig 3
(a) An electron density model of one lamellar unit. There is an electron sparse region, corresponding hydrocarbon chains of lipids, in the center and there are two such regions at 5 nm on each side. All low electron density regions are simulated by a Gaussian with a full-width at half maximum of 3.5 nm. (b) A model in which the model (a) was duplicated in the right with a shift of 13 nm. (c) A model with three identical units. The third unit is shifted by -13 nm from the first unit. (d) X-ray diffraction expected from the two-unit model (black) and the three-unit model (blue). (e) Mixture of one-, two-, and three-unit models with a ratio of 3:3:1.Models based on electron microscopy and calculated diffraction from them.

Attempts were also made to use density profiles obtained by cryo-electron microscopy. Since the micrographs were obtained from unstained sections, the intensity in them should represent electron density in the specimen. However, the density distribution with five lucent bands presented in Fig 2b by Iwai et al. [8] produces peaks at q = 0.6 and 1.1 nm-1 but only a very weak peak at 1.4 nm-1. It is possible that underfocusing to obtain high contrast (Iwai, personal communication) in the micrographs emphasized the periodicity in electron density.

Discussion

In this study, we used human skin samples that had been kept wet and unfrozen until the experiment. Compared with the results of a study which used a thin section of a skin that had been kept frozen [6], the X-ray diffraction pattern obtained with the present technique gave more distinct peaks (Fig 1). Possibly, freezing may affect the lipid structure, but there may be also damages caused in the cutting procedure. As other factors that may influence the diffraction pattern, the sample setting is also slightly different, and the tension on the skin in the present experiment may have induced orientation. Similarities to the diffraction pattern from a live mouse skin [15], which was obtained with the same sample setting as in the present work, suggest that the current sample handling technique maintained the SC structure well and this lamellar structure is common in mammalian skins. Possible effects of a long period in preservation solution on the gross lamellar structure is not evident in comparison with the results on a live mouse skin.

From the crystallographic consideration described in detail in S1 Appendix, the change in the diffraction pattern observed when the beam moves deeper into the SC mainly represents differences in the lipid structures at different depths. The pattern obtained close to the skin surface represents diffraction purely from the upper part of SC. The patterns at deeper depths are summation of ones coming from different parts along the beam, but the largest contribution comes from the part where the layers of intercellular lipids lie parallel to the beam.

Although the separations between lucent bands in electron micrographs such as 11, 6.5, 4.3 nm [8, 20] sometimes coincide with the d-spacings of the diffraction peaks, such correspondence may not explain the origin of X-ray diffraction peaks, because the observed diffraction profile is a Fourier transform of the electron density distribution. Generally, distance between density maxima or minima does not directly correspond to an X-ray diffraction peak unless it is repeated regularly. Thus, we constructed structural models based on electron microscopy to account for the major features of the diffraction pattern (Fig 3).

Based on the discussion by Swartzendruber et al. [13], the unit in our model represents corneocyte lipid envelopes and their shared monolayer, while the two-unit model corresponds to the so-called Landmann unit [13, 21] that is made of two closely apposed bilayers. It has been shown that intercellular spaces are mostly filled by either one, two, or three such units. It was indeed found in this study that the X-ray diffraction pattern from SC can be explained by summation of diffraction patterns from different numbers of units.

It has been reported that there are two periodicities in lipids in SC. One is a long periodicity of 11–13 nm that gives rise to an order of diffraction peaks at multiples of q = 0.5 nm-1 and a short periodicity of 6 nm that gives rise to a peak at q = 1.0 nm-1 [3]. Neither of the two periodicities can account for the peak at q = 1.4 nm-1 (d = 4.5 nm). The present simulation shows that such observation may be accounted for by a single phase of lipids made of two and three repeats of the basic unit. When the number of repeats is small, the simulation predicts three peaks (at q = 0.5, 1.0 and 1.4 nm-1). Also, the broad intensity between the peaks at q = 1.0 and 1.4 nm-1 may be explained by diffraction from the one-unit structure. Differences in the relative intensity of peaks observed in different samples may be explained by different ratios of the three structures. The broad intensity peak at q = 1.0 nm-1 and a shift of the 0.5 nm-1 peak towards higher angles at the lower part of SC (Fig 1b) suggest less contribution from the two- and three-unit structures. Thus, the molecular arrangements in intercellular lipids may change across SC.

Although this simple model accounts for major features of X-ray diffraction from SC, it is still imperfect. For example, the diffraction profiles at the top and bottom parts of SC are not satisfactorily explained by different ratios in summation. Lipid molecules are covalently attached to corneocyte envelope [22], so that the electron density distributions with different numbers of the units may not be represented by simple superposition of the same density profile. However, a more detailed analysis was not attempted because there were considerable variations in the experimentally observed X-ray diffraction patterns recorded at different locations on the skin, suggesting there may be other contributions such as the intercellular lipids with a short periodicity [3] or materials other than intercellular lipids.

Conclusions

We established a technique to study structure of SC in intact human skin with X-ray diffraction, which is convenient and can be applied to various skin samples. It can be used to study effects of environmental changes and drugs on lipid structure in skin. We demonstrated that the obtained diffraction pattern can be analyzed with a simple model based on the tri-lamellar structure.

Acknowledgements

We would like to thank Prof. Ichiro Hatta, Dr. Ichiro Iwai (Shiseido Co.), Dr. Takuji Kume (Kao Co.) and Dr. Ryuji Ohgaki (Osaka University) for discussion. An initial trial experiment on mouse skin was conducted with an approval of SPring-8 Program Review Committee (2013A1097).

References

1 

L Norlén. . Stratum corneum keratin structure, function and formation—a comprehensive review. Int J Cosmetic Sci. 2006;28:, pp.397–425.

2 

JA Bouwstra, GS Gooris, W Bras, DT Downing. . Lipid organization in pig stratum corneum. J Lipid Res. 1995;36:, pp.685–95.

3 

JA Bouwstra, GS Gooris, JA van der Spek, W Bras. . Structural investigations of human stratum corneum by small-angle X-ray scattering. J Invest Dermatol. 1991;97:, pp.1005–12. , doi: 10.1111/1523-1747.ep12492217

4 

JA Bouwstra, GS Gooris, JA van der Spek, S Lavrijsen, W Bras. . The lipid and protein structure of mouse stratum corneum: a wide and small angle diffraction study. Biochimica Biophysica Acta. 1994;1212:, pp.183–92.

5 

I Hatta, N Ohta, K Inoue, N Yagi. . Coexistence of two domains in intercellular lipid matrix of stratum corneum. Biochimica Biophysica Acta. 2006;1758:, pp.1830–6.

6 

J Doucet, A Potter, C Baltenneck, YA Domanov. . Micron-scale assessment of molecular lipid organization in human stratum corneum using microprobe X-ray diffraction. J Lipid Res. 2014;55(11):, pp.2380–8. , doi: 10.1194/jlr.M053389

7 

V Schreiner, Gert S Gooris, Stephan Pfeiffer, Ghita Lanzendörfer, Horst Wenck, Walter Diembeck, et al. Barrier characteristics of different human skin types investigated with X-ray diffraction, lipid analysis, and electron microscopy imaging. J Invest Dermatol. 2000;114:, pp.654–60. , doi: 10.1046/j.1523-1747.2000.00941.x

8 

I Iwai, H-M Han, L den Hollander, S Svensson, L-G Öfverstedt, J Anwar, et al. The human skin barrier is organized as stacked bilayers of full extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety. J Invest Dermatol. 2012;132:, pp.2215–25. , doi: 10.1038/jid.2012.43

9 

J Vicanová, ST Boyce, MD Harriger, AM Weerheim, JA Bouwstra, M Ponec. . Stratum corneum lipid composition and structure in cultured skin substitutes is restored to normal after grafting onto athymic mice. Journal of Investigative Dermatology Symposium Proceeding. 1998;3(2):, pp.114–20. , doi: 10.1038/jidsymp.1998.24

10 

JA Bouwstra, GS Gooris, A Weerheim, J Kempenaar, M Ponec. . Characterization of Stratum Corneum Structure in Reconstructed Epidermis by X-ray Diffraction. J Lipid Res. 1995;36(3):, pp.496–504.

11 

JA Bouwstra, J Thewalt, GS Gooris, N Kitson. . A Model Membrane Approach to the Epidermal Permeability Barrier: An X-ray Diffraction Study. Biochemistry. 1997;36(25):, pp.7717–25. , doi: 10.1021/bi9628127

12 

D Groen, GS Gooris, JA Bouwstra. . New insights into the stratum corneum lipid organization by X-ray diffraction analysis. Biophysical Journal. 2009;97:, pp.2242–9. , doi: 10.1016/j.bpj.2009.07.040

13 

DC Swartzendruber, PW Wertz, DJ Kitko, KC Madison, DT Downing. . Molecular models of the intercellular lipid lamellae in mammalian stratum corneum. J Invest Dermatol. 1989;92:, pp.251–7. , doi: 10.1111/1523-1747.ep12276794

14 

A Al-Amoudi, J Dubochet, L Norlén. . Nanostructure of the Epidermal Extracellular Space as Observed by Cryo-Electron Microscopy of Vitreous Sections of Human Skin. J Invest Dermatol. 2005;124:, pp.764–77. , doi: 10.1111/j.0022-202X.2005.23630.x

15 

N Nakagawa, M Yamamoto, Y Imai, Y Sakaguchi, T Takizawa, N Ohta, et al. Knocking-in the R142C mutation in transglutaminase 1 disrupts the stratum corneum barrier and postnatal survival of mice. J Dermatol Sci. 2012;65(3):, pp.196–206. , doi: 10.1016/j.jdermsci.2011.12.011

16 

K Inoue, T Oka, T Suzuki, N Yagi, K Takeshita, S Goto, et al. Present Status of high flux beamline (BL40XU) at SPring-8. Nuclear Instruments and Methods A. 2001;467–468:, pp.674–7.

17 

N Ohta, T Oka, K Inoue, N Yagi, S Kato, I Hatta. . Structural Analysis of Cell Membrane Complex of a Hair Fibre by Micro-beam X-ray Diffraction. J Appl Crystallogr. 2005;38:, pp.274–9.

18 

Y Amemiya, K Ito, N Yagi, Y Asano, K Wakabayashi, T Ueki, et al. Large-aperture TV detector with a beryllium-windowed image intensifier for X-ray diffraction. Rev Sci Instrum. 1995;66:, pp.2290–4.

19 

KC Madison, DC Swartzendruber, PW Wertz, DT Downing. . Presence of intact intercellular lipid lamellae in the upper layers of stratum corneum. J Invest Dermatol. 1987;88:, pp.714–8. , doi: 10.1111/1523-1747.ep12470386

20 

JR Hill, PW Wertz. . Molecular models of the intercellular lipid lamellae from epidermal stratum corneum. Biochimica Biophysica Acta. 2003;1616:, pp.121–6.

21 

L Landmann. . Epidermal permealibity barrier: transformation of lamellar granule-disks into intercellular sheets by a membrane-fusion process, a freeze-fracture study. J Invest Dermatol. 1986;87:, pp.202–9. , doi: 10.1111/1523-1747.ep12695343

22 

DC Swartzendruber, PW Wertz, KC Madison, DT Downing. . Evidence that the corneocyte has a chemically bound lipid envelope. J Invest Dermatol. 1987;88(6):, pp.709–13. , doi: 10.1111/1523-1747.ep12470383


12 Feb 2020

PONE-D-19-36009

Microbeam X-ray Diffraction Study of Lipid Structure in Stratum Corneum of Human Skin

PLOS ONE

Dear Dr. Yagi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please consider carefully the comments from the reviewers and recommendations for change, and address each of these recommendations. Where you disagree with a particular comment from the reviewers please identify this with a justification for your disagreement. 

We would appreciate receiving your revised manuscript by Mar 28 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

    A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
    A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
    An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Richard G. Haverkamp, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

3. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript uses x-ray diffraction on natural abdominal skin of healthy patients to probe the lipid organization of the SC. It is a nice set of experiments that can be compared to past work on mice and extracted SC lipid studies. Overall, this appears to confirm similar work by Bouwstra and others that there is an existence of a short and long periodicity phase.

However, one aspect that is unclear is how the preserving fluid might influence the structure of the SC lipid phase. Clearly this work does not suffer from hydration issues but could the preserving fluid alter the lipid phase? Some discussion on this should be provided.

Reviewer #2: The authors propose that X-ray diffraction can be used to study the structure of lipids in the stratum corneum of the skin. They present preliminary data in the current study that supports the initial assumption. Fresh skin, not previously frozen, was examined using SAXS in the area of the stratum corneum. Diffraction peaks were observed at the lipid range. The results are at preliminary level, but are quite interesting. There are a few points that have to be addressed:

1. The appendix is an extended discussion of the data findings and should be included in the Discussion section of the paper.

2. In the appendix at the bottom of the second page, the authors are attempting to explain the difference in the diffraction patterns between different spots in the tissue. It is not very clear and it is rather confusing. They use wording such “wavier” that does not really apply. The fact that the difference between having defined “lines” or diffuse “arcs” in a diffraction pattern indicates that there are highly ordered structures aligned towards one direction or different directions, respectively was not mentioned. This section has to be rewritten and corrected.

3. In the results section at line 174 the authors highlight the risk of working with a small amount of samples. However, only 6 samples were used in the current study. The authors should consider including more samples in the presented manuscript in order to back up their data.

4. In the same section as above, the authors mention that the last sample was different to the rest ones and it is assumed that the donor of this particular sample might have had a particular condition. In addition to more samples, the study should consider the age and condition of donors and create different groups for comparison.

5. The data presented in the current study is interesting. However, it should be considered as preliminary data and more effort should be invested in order to make it more complete. As it was mentioned already more samples will be necessary in order to prove that the results are consistent and not circumstantial. Electron microscopy technique should be employed at the same samples in order to back up the X-ray diffraction results, especially to show the different directions the lipids can be arranged within the layer. Microscopy is referred in the manuscript, but it was not performed in the samples that were used in the current study.

Reviewer #3: I read the manuscript Microbeam X-ray Diffraction Study of Lipid Structure in Strstum corneum of Human Skin with great interest. The manuscript reports on the diffraction patterns as a function of depth in ststum corneum of fresh skin.

Although the manuscript reports on the X-ray diffraction curves as a function of depth in stratum corneum and reveals different profiles at different depths, which is of interest, this reviewer has major comments on this manuscript. These are listed below:

Major comments

Introduction: the introduction is not very well written and does not explain what is known in the field of stratum corneum lipids.

For example, there is one publication in which isolated fresh skin has been measured and compared with isolated stratum corneum from the same donor using X-ray diffraction and showed that the diffraction profiles are very similar. (Schreiner et al, Journal Invest. Dermatology, 2000) Therefore, it is absolutely not done to suggest that trypsin digestion changes the properties of the stratum corneum. If you suggest this, please show the data, that means measure using the same donors isolated stratum corneum and fresh skin and compare the curves.

The authors cannot simply argue that hydration has been carried out under unnatural conditions. What do the authors mean by this? Specify the papers describing this, but do not make a general comment. Most studies have been done by hydrating the stratum corneum at room temperature at a fixed relative humidity. In the studies described here, probably the stratum corneum was fully hydrated (Transport in medium), which is also not natural. Especially having the skin for a longer time period at high humidity may have an effect.

Many more studies on stratum corneum have been performed (mouse skin, pig skin, human skin equivalents) sometimes also as function of temperature, in several cases showing that the peaks disappear at the same temperature, indicating that these are attributed to the same lamellar phase. Nothing is mentioned about this. Also studies using isolated pig or human ceramides are relevant as these provide also useful information.

Methods:

Although it is excellent to see the curves as function of depth, these curves have already been changed by subtracting the curve obtained at perpendicular orientation in which a minimum diffraction of the lipids has been detected. However, I would like to see the original curves for at least two reasons:

a. The lipid peaks are very broad, which makes subtraction a difficult procedure.

b. The scattering at low angle is very steep, which make subtraction also a difficult procedure.

I cannot rule out that peak positions are sensitive to this procedure.

Results

Model calculations.

The authors use the RuO4 profiles of Swarzendruber to calculate the intensities of the peaks. However, these profiles are in fact a print of the real structure as it visualizes the position of RuO4. It is even not clear to which parts of the lipids RuO4 is fixed. So no information can be drawn about the underlaying structure, only that there is a certain repeat in the structure. This should be very clearly stated.

Minor comments

Page 3: electron diffraction cannot detect the lamellar structures

If the curvature may effect the diffraction profile, why not measuring with a straight oriented sample.

If the beam location gradually changes with 5 micrometer steps, then the total length over which has been measured is 30 micrometer (including the size of the beam).

Line 136 sentence is a repeat of the previous sentence. Second part of thst sentence is not clear.

Line 146/147: The accuracy of the q values is not realistic. Later on it is explained there are differences between donors. This is probably standard deviation of the mean? Not taking into account different donors? See remark line 166

Line 221: freezing of skin samples can induce holes in the lipid structure and therefore the repeating pattern is interrupted.

Line 249: In many other publications Bouwstra always attributed the 1.4 nm-1 peak to the 12-13 nm lamellar phase. So this remark is quite biased and should be changed.

Line 261: there is no plasma membrane in strstum corneum

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.


9 Apr 2020

I would like to thank the three reviewers for valuable comments to improve the manuscript. It has been revised to accommodate the comments. For each comment, a response is provided below.

In the revised manuscript, a few errors and typos were corrected. Particularly, the number of scans on each sample was in fact between five and ten. Previously it was described as 10 to 20, but this includes scans that were made to confirm radiation damage or test scanning software.

All the changed parts are in red.

The line numbers refer to those in the revised manuscript.

Reviewer #1: This manuscript uses x-ray diffraction on natural abdominal skin of healthy patients to probe the lipid organization of the SC. It is a nice set of experiments that can be compared to past work on mice and extracted SC lipid studies. Overall, this appears to confirm similar work by Bouwstra and others that there is an existence of a short and long periodicity phase.

However, one aspect that is unclear is how the preserving fluid might influence the structure of the SC lipid phase. Clearly this work does not suffer from hydration issues but could the preserving fluid alter the lipid phase? Some discussion on this should be provided.

The samples were investigated eight days after surgery [line 88]. Storage in a preservation solution might alter the lipid lamellar structure, but there is no way to study the effect. One point we would like to make is that the X-ray diffraction profile obtained in the present study is similar to that obtained in a previous study on skin of live wildtype mouse (Nakagawa et al., 2012) [lines 233-235].

Reviewer #2: The authors propose that X-ray diffraction can be used to study the structure of lipids in the stratum corneum of the skin. They present preliminary data in the current study that supports the initial assumption. Fresh skin, not previously frozen, was examined using SAXS in the area of the stratum corneum. Diffraction peaks were observed at the lipid range. The results are at preliminary level, but are quite interesting. There are a few points that have to be addressed:

1. The appendix is an extended discussion of the data findings and should be included in the Discussion section of the paper.

The discussion was summarized in Results section in the previous manuscript. As it is hard to move the entire discussion in the appendix to the manuscript, this summary is now moved to Discussion [lines 236-242]. A brief mention to Appendix is added to Data analysis section [line 107].

2. In the appendix at the bottom of the second page, the authors are attempting to explain the difference in the diffraction patterns between different spots in the tissue. It is not very clear and it is rather confusing. They use wording such “wavier” that does not really apply. The fact that the difference between having defined “lines” or diffuse “arcs” in a diffraction pattern indicates that there are highly ordered structures aligned towards one direction or different directions, respectively was not mentioned. This section has to be rewritten and corrected.

Thank you for the suggestion. Difference between lines and arcs is now explained and this section was rewritten.

3. In the results section at line 174 the authors highlight the risk of working with a small amount of samples. However, only 6 samples were used in the current study. The authors should consider including more samples in the presented manuscript in order to back up their data.

I understand your concern, but the samples used in this study were obtained in cosmetic surgery to remove fat in obese women. Thus, they are all from Caucasian women in their 30s to 50s. It is hard to expand this study because skin samples from other parts of a body or other sex, age or ethnic groups of donors are hard to obtain. This situation is now explained at line 90. Getting skin samples from volunteer donors involves many ethical regulations which are very tight these days, although the situation may be different in each country. The present study was made on limited samples, but it still provides a general view on lipid structure in human skin.

4. In the same section as above, the authors mention that the last sample was different to the rest ones and it is assumed that the donor of this particular sample might have had a particular condition. In addition to more samples, the study should consider the age and condition of donors and create different groups for comparison.

As explained above, it is difficult to obtain samples from different age groups. However, we do not think the intercellular lipid structure in the last sample was different from others. The previous manuscript only described the difference. An additional description on similarity is now added at lines 172-173.

5. The data presented in the current study is interesting. However, it should be considered as preliminary data and more effort should be invested in order to make it more complete. As it was mentioned already more samples will be necessary in order to prove that the results are consistent and not circumstantial. Electron microscopy technique should be employed at the same samples in order to back up the X-ray diffraction results, especially to show the different directions the lipids can be arranged within the layer. Microscopy is referred in the manuscript, but it was not performed in the samples that were used in the current study.

The lipid lamellar structure discussed in this paper is a very basic one and generally considered invariable among humans, even among mammalians to some extent [lines 43-44]. There are small differences among different people as can be noticed in Fig 2, but the basic lipid structure shown in Fig 3 can be used to explain diffraction from humans as well as mammalians. Other techniques also support this view. For example, Swartzendruber et al. found common lamellar structures in mouse, pig, and human skins and proposed a molecular model that was used to construct the model in the present study [line 187]. Detailed structures may be different because lipid compositions are variable among different mammalian species. This needs further studies.

Reviewer #3: I read the manuscript Microbeam X-ray Diffraction Study of Lipid Structure in Strstum corneum of Human Skin with great interest. The manuscript reports on the diffraction patterns as a function of depth in ststum corneum of fresh skin.

Although the manuscript reports on the X-ray diffraction curves as a function of depth in stratum corneum and reveals different profiles at different depths, which is of interest, this reviewer has major comments on this manuscript. These are listed below: Major comments

Introduction: the introduction is not very well written and does not explain what is known in the field of stratum corneum lipids.

For example, there is one publication in which isolated fresh skin has been measured and compared with isolated stratum corneum from the same donor using X-ray diffraction and showed that the diffraction profiles are very similar. (Schreiner et al, Journal Invest. Dermatology, 2000) Therefore, it is absolutely not done to suggest that trypsin digestion changes the properties of the stratum corneum. If you suggest this, please show the data, that means measure using the same donors isolated stratum corneum and fresh skin and compare the curves.

Thank you for pointing out this important paper. I agree there is no evidence that trypsin treatment changes the lipid structure. Thus, the statement is retracted. The paper is now cited as a previous study on human skin [line 43, line 153]

The authors cannot simply argue that hydration has been carried out under unnatural conditions. What do the authors mean by this? Specify the papers describing this, but do not make a general comment. Most studies have been done by hydrating the stratum corneum at room temperature at a fixed relative humidity. In the studies described here, probably the stratum corneum was fully hydrated (Transport in medium), which is also not natural. Especially having the skin for a longer time period at high humidity may have an effect.

Introduction was revised according to the comments [lines 42-45]. A possible problem related to transport in preservation solution is mentioned and discussed [lines 233-235].

Many more studies on stratum corneum have been performed (mouse skin, pig skin, human skin equivalents) sometimes also as function of temperature, in several cases showing that the peaks disappear at the same temperature, indicating that these are attributed to the same lamellar phase. Nothing is mentioned about this. Also studies using isolated pig or human ceramides are relevant as these provide also useful information.

These points are mentioned in Introduction [lines 40-46].

Methods:

Although it is excellent to see the curves as function of depth, these curves have already been changed by subtracting the curve obtained at perpendicular orientation in which a minimum diffraction of the lipids has been detected. However, I would like to see the original curves for at least two reasons:

a. The lipid peaks are very broad, which makes subtraction a difficult procedure.

b. The scattering at low angle is very steep, which make subtraction also a difficult procedure.

I cannot rule out that peak positions are sensitive to this procedure.

In the data analysis presented in this paper, background subtraction was not performed. This is corrected now. The diffraction profiles presented in this paper may look rather flat. This is because the profiles were obtained by circular summation (not average) and because a Lorenz factor was applied. This is the reason the higher angle region tends to be enhanced. This procedure is explained [lines 108-112].

Results

Model calculations.

The authors use the RuO4 profiles of Swarzendruber to calculate the intensities of the peaks. However, these profiles are in fact a print of the real structure as it visualizes the position of RuO4. It is even not clear to which parts of the lipids RuO4 is fixed. So no information can be drawn about the underlaying structure, only that there is a certain repeat in the structure. This should be very clearly stated.

This point is now explained [lines 189-192].

Minor comments

Page 3: electron diffraction cannot detect the lamellar structures

This is corrected [line 38].

If the curvature may effect the diffraction profile, why not measuring with a straight oriented sample.

It is hard to obtain a flat, oriented skin, as explained in Appendix page 2-3.

If the beam location gradually changes with 5 micrometer steps, then the total length over which has been measured is 30 micrometer (including the size of the beam).

This is now written in experimental [lines 122-123].

Line 136 sentence is a repeat of the previous sentence. Second part of thst sentence is not clear.

This is a repetition. It is removed.

Line 146/147: The accuracy of the q values is not realistic. Later on it is explained there are differences between donors. This is probably standard deviation of the mean? Not taking into account different donors? See remark line 166

These are in fact standard deviations. It is rather surprising the SD is as small as 1%. The range of values is now given (0.504-0.515 for q=0.511, and 1.055-1.019 for q=1.034). The variation in peak position described at lines 165-167 took place when different depth of stratum corneum was interrogated. At the depth where the peaks are strongest, their positions were not variable [lines 168-169].

Line 221: freezing of skin samples can induce holes in the lipid structure and therefore the repeating pattern is interrupted.

This possibility is pointed out [line 226].

Line 249: In many other publications Bouwstra always attributed the 1.4 nm-1 peak to the 12-13 nm lamellar phase. So this remark is quite biased and should be changed.

This statement was removed.

Line 261: there is no plasma membrane in strstum corneum

This is corrected [line 275].

Submitted filename: rebuttal2.docx

29 Apr 2020

Microbeam X-ray Diffraction Study of Lipid Structure in Stratum Corneum of Human Skin

PONE-D-19-36009R1

Dear Dr. Yagi,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Richard G. Haverkamp, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:


1 May 2020

PONE-D-19-36009R1

Microbeam X-ray Diffraction Study of Lipid Structure in Stratum Corneum of Human Skin

Dear Dr. Yagi:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Richard G. Haverkamp

Academic Editor

PLOS ONE

https://www.researchpad.co/tools/openurl?pubtype=article&doi=10.1371/journal.pone.0233131&title=Microbeam X-ray diffraction study of lipid structure in stratum corneum of human skin&author=Naoto Yagi,Koki Aoyama,Noboru Ohta,Richard G. Haverkamp,Richard G. Haverkamp,Richard G. Haverkamp,Richard G. Haverkamp,&keyword=&subject=Research Article,Research and Analysis Methods,Structural Characterization,X-Ray Diffraction,Biology and Life Sciences,Biochemistry,Lipids,Physical Sciences,Physics,Waves,Diffraction,Biology and Life Sciences,Anatomy,Integumentary System,Skin,Medicine and Health Sciences,Anatomy,Integumentary System,Skin,Biology and Life Sciences,Biochemistry,Lipids,Lipid Structure,Physical Sciences,Physics,Condensed Matter Physics,Electron Density,Research and Analysis Methods,Spectrum Analysis Techniques,Electron Diffraction,Biology and Life Sciences,Anatomy,Integumentary System,Skin,Skin Physiology,Medicine and Health Sciences,Anatomy,Integumentary System,Skin,Skin Physiology,