Identification of Phytosphingosine-Based 1-O-Acylceramide in Human Stratum Corneum and Investigation of Its Role in Skin Barrier

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a paper confirming the existence of CER 1-O-ENP in the human stratum corneum and its efficacy on the function of stratum corneum.. This result is very interesting, and the results are very clear. However, there are some points which may have to be revised.

 

Major points

1. There is very little information about human study. The author may need to indicate more clearly and detail.

 

2. The author wrote the function of stratum corneum as barrier function. But the some indicated data is skin moisture content. The barrier function is not equally to moisture content, thus, the author should indicate correctly.

 

3. The author conclude the strength in SC cohesion by analyzing the changes in TEWL following tape stripping. But this effect if not only caused by the strength in SC cohesion but also cutaneous barrier function itself. To clarify the strength in cohesion, the author should measure the amount of removed SC amount by tape stripping.

 

4. The are no discussion about the difference in the result between human test and the stability of micelles (the effect of the concentration of Cer 1-O-ENP).

 

5. There are no discussion about the mechanism of topically applied Cer 1-O-ENP on stratum corneum function. This molecular may be too big to penetrate to stratum corneum of healthy skin.

Author Response

Point-by-point Responses to the Comments

 

Reviewer 1

Comments and Suggestions for Authors

This is a paper confirming the existence of CER 1-O-ENP in the human stratum corneum and its efficacy on the function of stratum corneum.. This result is very interesting, and the results are very clear. However, there are some points which may have to be revised.

Major points

1. There is very little information about human study. The author may need to indicate more clearly and detail.

Response: We describe the scheme of the human study (Fig 2) in the 2.3 Human Study section. We added some more detailed information of the protocols in the section 2.4. Measurement of skin hydration, retention of hydration, and SC cohesion as below;

Line170 ~ Line189

2.4. Measurement of skin hydration, retention of hydration, and SC cohesion

 

2.4.1. The assessment of SC hydration and retention of hydration

The assessment of SC hydration was also performed according to the previously described [32]. To measure hydration, a 2.5 cm2 area of the forearms of all volunteers was treated twice a day with 25 µL (10 µL/cm2) of each test cream. After 28 days of topical applications, skin hydration was evaluated using a Corneometer CM820 device (Courage & Khazaka). Moisture retention was analyzed for three days after stopping the application of respective test creams. Volunteers were asked not to apply any other cosmetic moisturizer during this period. All test sites were acclimated for 30 min in a controlled room (temperature of 24.1 ± 1 °C and relative humidity of 42.5 ± 1%) before measurement. The changed value of arbitrary units was calculated from each baseline and expressed as the mean ± standard error of the mean (SEM).

 

2.4.2. The SC cohesion

         SC cohesion was expressed as D TEWL between baseline and after four weeks of test cream application sites. Volunteers were asked to apply designated test creams to a 2.5 cm2 area of the forearms twice a day. The TEWL was measured to calculate SC cohesion immediately after tape-stripping 15 times with the D-Squame tape in an air-conditioned room at 20.4 ± 0.2°C and 40.5 ± 1.0% relative humidity [36].

 

2. The author wrote the function of stratum corneum as barrier function. But the some indicated data is skin moisture content. The barrier function is not equally to moisture content, thus, the author should indicate correctly.

Response: I appreciate your comment. You made a good point in that the barrier function is not equally correlated to the moisture content. However, the moisture content of the stratum corneum is one of the essential parameters of the skin barrier. As a skin barrier, the stratum corneum protects our body from insults from the environment and prevents the loss of moisture from inside the skin. Several parameters have been used to determine barrier function, such as barrier permeability expressed by TEWL, moisture content measured by Corneometer, and SC cohesion expressed by DTEWL. Skin pH is also an important indicator of a healthy skin barrier. In this study, we tried to show that CER1-ENP enhanced the moisture content by helping strengthen the lipid multilamellar organization

3. The author conclude the strength in SC cohesion by analyzing the changes in TEWL following tape stripping. But this effect if not only caused by the strength in SC cohesion but also cutaneous barrier function itself. To clarify the strength in cohesion, the author should measure the amount of removed SC amount by tape stripping.

Response: You made a good point again. It has always been controversial what the primary determinant that enhances SC cohesion is. Corneodesmosome(CD) is a primary factor responsible for SC cohesion. Meanwhile, according to Peter Elias (SKIN BARRIER), the lipid matrix in the stratum corneum's intercellular space could also enhance SC cohesion to some extent. In this study, we tried to measure the SC cohesion as a whole, not to measure the amount of CD. So, we expressed the SC cohesion by calculating the difference in the TEWL (ΔTEWL) value before and after the test cream application by conducting a fixed number of tape strippings. As shown in reference 36, conducting a fixed number tape of strippings (15 times in this study) to measure SC cohesion is believed to be a good alternative protocol.

4. The are no discussion about the difference in the result between human test and the stability of micelles (the effect of the concentration of Cer 1-O-ENP).

Response: I appreciate your suggestion. I added some speculative discussion points that may explain the difference in the results between the human test and the stability of the lipid lamellar LC in the formulation.

Line 389 ~ Line 387

“Data from human studies was not well correlated with that from the experiment for the effect of Maltese cross formation, which exhibited a dose-dependent effect of ENP. With increasing concentration of ENP, the Maltese cross formation was significantly increased with enhanced stability. This difference could be attributed to the difference between in vivo human ceramide complexity and a simple and defined in vitro experimental condition where only limited classes of ceramides were involved. There are more than 20 classes of ceramides with several hundreds of different ceramide species.”

5. There are no discussion about the mechanism of topically applied Cer 1-O-ENP on stratum corneum function. This molecular may be too big to penetrate to stratum corneum of healthy skin

Response: The intercellular space of the human stratum corneum is filled with skin physiological lipids, such as ceramide, cholesterol, and free fatty acid, in the form of the multi-lamellar structure surrounding corneocytes. Ceramide comprises 50% of the total lipid in the stratum corneum. It has been shown that topically applied ceramide quickly permeates into the intercellular space, especially when it is formulated together with cholesterol and free fatty acid to form a liquid crystal, suggesting a lamellar structure. We demonstrated that ceramide could permeate into the deep layers of the skin barrier within 30 min (J Cosmet Dermatol. 2022;21:4931–4941). 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a nice work showing the presence of phytosphingosine-based 1-O-acylceramide in human stratum corneum and its role in skin barrier. Regarding skin barrier function, the stratum corneum is a permeability barrier of the skin. To confirm the role of phytosphingosine-based 1-O-acylceramide in the skin barrier function, please also show the effect of this ceramide on the permeability function (e.g skin permeability assay).

Author Response

Point-by-point Responses to the Comments

Reviewer 2 

Comments and Suggestions for Authors

This is a nice work showing the presence of phytosphingosine-based 1-O-acylceramide in human stratum corneum and its role in skin barrier. Regarding skin barrier function, the stratum corneum is a permeability barrier of the skin. To confirm the role of phytosphingosine-based 1-O-acylceramide in the skin barrier function, please also show the effect of this ceramide on the permeability function (e.g skin permeability assay).

Response: Thank you for your comment. In this study, we focused on the human study. In a separate biophysical characterization study, we have shown that the presence of CER 1-O ENP in a molecular dynamic simulation model retarded the penetration of an ethanol molecule through the SC lipid multilamellar matrix. (Ref. 38: Molecular Dynamics Investigation into CerENP’s Effect on the Lipid Matrix of Stratum Corneum. J. Phys. Chem. B 2024, 128, 5378−5386)

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by King et al., titled "Identification of phytosphingosine-based 1-O-acylceramide in the human stratum corneum and investigation of its role in the skin barrier" by King et al., offers an innovative perspective by identifying the presence of CER 1-O-ENP in human SC and its roles roles in skin barrier functions. The research presents valuable implications for cosmetic science, especially for moisturizers production.

However, there are some improvements that need to be addressed.

1. Abstract: 

I think that the abstract can be improved. I would avoid the repetition of "CER 1-O-ENP" too many times, it can be redundant. 
I would also replace "TEWL" with the full form, in this way it could be of better understanding for a wider audience.

2. Introduction:

Line 40 add "et al." after "Suzuki M."

Lines 50 to 61 "CER 1-O-ENS" is very redundant and makes difficult to read the text. I would, if possible, improve the readability of this section.

3. Materials and. Methods:

Improve quality of Table 1, it seems like a pasted blurred image.

Line 162 there is a typo error, remove one ")".

Lines 173 and 179 instead of the symbols I would use subheadings (e.g., "2.4.1. Assessment of SC hydration").

4. Results:

Improve quality of figures: 3C, 4B and Table 2.

Line 276 there is an error, it should be 3.4.

Line 311 there is an error, it should be 3.5.

Author Response

Point-by-point Responses to the Comments

Reviewer 3

Comments and Suggestions for Authors

The manuscript by King et al., titled "Identification of phytosphingosine-based 1-O-acylceramide in the human stratum corneum and investigation of its role in the skin barrier" by King et al., offers an innovative perspective by identifying the presence of CER 1-O-ENP in human SC and its roles roles in skin barrier functions. The research presents valuable implications for cosmetic science, especially for moisturizers production.

However, there are some improvements that need to be addressed.

1. Abstract: 

I think that the abstract can be improved. I would avoid the repetition of "CER 1-O-ENP" too many times, it can be redundant. 
I would also replace "TEWL" with the full form, in this way it could be of better understanding for a wider audience.

Response: Thank you for your comments. I replaced the CER 1-O-ENP with ENP in short. TEWL is written in the full name as Trans-Epidermal Water Loss

2. Introduction:

Line 40 add "et al." after "Suzuki M."

Lines 50 to 61 "CER 1-O-ENS" is very redundant and makes difficult to read the text. I would, if possible, improve the readability of this section.

Response: I appreciate your elaborate comments. Corrections have been made following your directions. CER 1-O-ENS to ENS, CER 1-O-ENP to ENP, and other 1-O-Acylceramides are also written in abbreviation.

3. Materials and. Methods:

Improve quality of Table 1, it seems like a pasted blurred image.

Line 162 there is a typo error, remove one ")".

Lines 173 and 179 instead of the symbols I would use subheadings (e.g., "2.4.1. Assessment of SC hydration").

Response: Thank you for your kind suggestions. Necessary corrections were made.

Tables and figures will be revised to improve their qualities by requesting an ART WORK SERVICE to the Editor of COSMETICS.

Line 171 and 183 Subheading are inserted

4. Results:

Improve quality of figures: 3C, 4B and Table 2.

Line 276 there is an error, it should be 3.4.

Line 311 there is an error, it should be 3.5.

Response: Thank you for your kind and elaborate suggestions. Necessary corrections were made.

Line 280: 3.4. The influence of CER 1-O-ENP on the skin hydration

Line 315: 3.5. CER 1-O-ENP fostered the SC cohesion

Figures 3C and 4B have been revised, yet we may still need help from the Editor of COSMETICS for ART WORK SERVICE for tables and other figures.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This is a paper confirming the existence of CER 1-O-ENP in the human stratum corneum and its efficacy on the function of stratum corneum. This result is very interesting, and the results are very clear. The revision has some revised parts in the manuscript according to the comment. However, some answers were done only in the response letter (and the answers are not accepted well) and were not added any sentences reflects to the comment in the manuscript. Additionally there are some questions in the added paragraph. Thus, I judge this manuscript is not novelty for acceptance and need some major revision.

 Major points

1. According to the comment that “There is very little information about human study. The author may need to indicate more clearly and detail.”, the detail information was described. But from the paragraphs, there are wonders. The materials were removed by cleansing prior to skin analysis or not, and how long did it passed on measuring following last application. If the materials remains on the skin surface, it may affect the skin moisture content directly and also attach to the skin on tape stripping. Usually the skin measurement is done 24 hours after last application and having skin cleansing prior to that, However, in this manuscript, there are week 4 point and 24 hours after.

 2. According to the comment that “The author wrote the function of stratum corneum as barrier function. But the some indicated data is skin moisture content. The barrier function is not equally to moisture content, thus, the author should indicate correctly.”, authors made rely only in the response letter. Author should describe this in the manuscript. And there are many skin with low TEWL and low water contents, or high TEWL and high water contents. Thus author should divide barrier function and water holding capacity.

 3. According to the comment that “The author conclude the strength in SC cohesion by analyzing the changes in TEWL following tape stripping. But this effect if not only caused by the strength in SC cohesion but also cutaneous barrier function itself. To clarify the strength in cohesion, the author should measure the amount of removed SC amount by tape stripping.”, authors made rely only in the response letter. Author should describe this in the manuscript same as #2. Author referred Peter Elias (SKIN BARRIER) as the lipid matrix in the stratum corneum's intercellular space could also enhance SC cohesion to some extent. But this is one of the result, thus author could not affirm that the SC cohesion was strengthened. Author should use “may”.

 4. According to the comment that “The are no discussion about the difference in the result between human test and the stability of micelles (the effect of the concentration of Cer 1-O-ENP).”, author added well paragraph

 5. According to the comment that “There are no discussion about the mechanism of topically applied Cer 1-O-ENP on stratum corneum function. This molecular may be too big to penetrate to stratum corneum of healthy skin.”, authors made rely only in the response letter. Author should describe this in the manuscript. Especially author referred one manuscript but in their manuscript. Additionally there are sulci cutis on the skin surface and also one tape stripping cannot remove one corneocyte layer equally. Thus some tapes strip has made, some materials which remains on the skin surface may be analyzed (even the materials was an earth pigment.)

 6. Figure 6. A and B indicate same data. Thus, one of the should be removed.

 7. line 43. The authors name is not Dr. Mann but Dr. Man

Author Response

Point-by-point Responses to the Comments

Reviewer

 

Report 2:

This is a paper confirming the existence of CER 1-O-ENP in the human stratum corneum and its efficacy on the function of stratum corneum. This result is very interesting, and the results are very clear. The revision has some revised parts in the manuscript according to the comment. However, some answers were done only in the response letter (and the answers are not accepted well) and were not added any sentences reflects to the comment in the manuscript. Additionally there are some questions in the added paragraph. Thus, I judge this manuscript is not novelty for acceptance and need some major revision.

 Major points

1. According to the comment that “There is very little information about human study. The author may need to indicate more clearly and detail.”, the detail information was described. But from the paragraphs, there are wonders. The materials were removed by cleansing prior to skin analysis or not, and how long did it passed on measuring following last application. If the materials remains on the skin surface, it may affect the skin moisture content directly and also attach to the skin on tape stripping. Usually the skin measurement is done 24 hours after last application and having skin cleansing prior to that, However, in this manuscript, there are week 4 point and 24 hours after.

Response: Thank you for your comments. The human study aimed to measure the cumulative effect of long-term application of the designated moisturizers, which was four weeks. The volunteers were asked to apply test creams twice daily, morning and evening, and not use any other cosmetics on the test sites. They were also allowed to take showers every day.  Therefore, it was very unlikely any test cream materials remained on the skin surface. Wiping out the test sites before corneometry is a routine procedure. Measurements of skin hydration were performed at week 2 and week 4. Concerning the measurement of moisture retention, volunteers were asked not to apply the test creams and to visit the lab to measure Corneometry every day for 3 days. We have added some of the detailed protocols following your comments below;

Line 171~182

  The assessment of SC hydration was also performed according to the previously described [32]. To measure hydration, a 2.5 cm2 area of the forearms of all volunteers was treated twice a day, morning and evening, with 25 µL (10 µL/cm2) of each test cream for four weeks. Skin hydration measurements were conducted in weeks 2 and 4 using a Corneometer CM820 device (Courage & Khazaka). Moisture retention was analyzed for three days after stopping the application of respective test creams. Volunteers were asked not to apply any other cosmetic moisturizer during this period. All test sites were acclimated for 30 minutes in a controlled environment with a temperature of 24.1 ± 1 °C and a relative humidity of 42.5 ± 1%. Before measurements were taken, any materials on the skin sites were gently wiped off using soft tissue paper. The changed value of arbitrary units was calculated from each baseline and expressed as the mean ± standard error of the mean (SEM).

2. According to the comment that “The author wrote the function of stratum corneum as barrier function. But the some indicated data is skin moisture content. The barrier function is not equally to moisture content, thus, the author should indicate correctly.”, authors made rely only in the response letter. Author should describe this in the manuscript. And there are many skin with low TEWL and low water contents, or high TEWL and high water contents. Thus author should divide barrier function and water holding capacity.

Response: I appreciate your elaborate comments on the difference between the barrier function and water-holding capacity. The aged skin tends to have low TEWL, yet very dry skin condition probably due to less NMF and lowered ceramide content. We intended to define the skin barrier’s function more broadly. I modified the manuscript describing the relationship between the two concepts, skin barrier function and skin hydration below based on your comments;

Line 376-383

A healthy skin barrier functions effectively due to its composition of ceramides and maintains proper moisture balance, primarily because of a high level of natural moisturizing factors (NMF) found within the corneocytes. It is understood that the long-lasting moisture content in the skin barrier largely depends on the NMF levels in the corneocytes. Thus, the enhanced skin hydration observed with the application of ENP and its long-lasting moisturizing effect can be attributed to the increased NMF, which enhances the water-holding capacity of the corneocytes. Further study will be required to reveal if the NMF level is enhanced by ENP treatment.  

3. According to the comment that “The author conclude the strength in SC cohesion by analyzing the changes in TEWL following tape stripping. But this effect if not only caused by the strength in SC cohesion but also cutaneous barrier function itself. To clarify the strength in cohesion, the author should measure the amount of removed SC amount by tape stripping.”, authors made rely only in the response letter. Author should describe this in the manuscript same as #2. Author referred Peter Elias (SKIN BARRIER) as the lipid matrix in the stratum corneum's intercellular space could also enhance SC cohesion to some extent. But this is one of the result, thus author could not affirm that the SC cohesion was strengthened. Author should use “may”.

Response: Thank you again for your keen comments. An additional description was added to the manuscript Material & Methods; 2.4.2 The SC Cohesion and Discussion as below following your comments;

Line 188~193

D500-D-Squame Pressure Instrument was used for tape stripping in order to keep experi-mental consistency. The pressure applied was 225g/cm2 and the tape stripping was con-ducted in an air-conditioned room at 20.4 ± 0.2°C and 40.5 ± 1.0% relative humidity [36]. D-Squame tape and the D500-D-Squame Pressure Instrument was purchased from CuDerm Corporation, Dallas, USA).

Line 388~391

Corneodesmosome (CD) is a primary factor responsible for SC cohesion. However, the lipid multilamellar organization interdigitated with cornified lipid envelopes (CLE) may also contribute to SC cohesion as a minor determinant. In this study, we tried to measure the SC cohesion as a whole, not to measure the amount of CD. 

4. According to the comment that “The are no discussion about the difference in the result between human test and the stability of micelles (the effect of the concentration of Cer 1-O-ENP).”, author added well paragraph

Response: I appreciate that you accepted our revised description on the difference in the result between human test and the stability of micelles.

5. According to the comment that “There are no discussion about the mechanism of topically applied Cer 1-O-ENP on stratum corneum function. This molecular may be too big to penetrate to stratum corneum of healthy skin.”, authors made rely only in the response letter. Author should describe this in the manuscript. Especially author referred one manuscript but in their manuscript. Additionally there are sulci cutis on the skin surface and also one tape stripping cannot remove one corneocyte layer equally. Thus some tapes strip has made, some materials which remains on the skin surface may be analyzed (even the materials was an earth pigment.)

Response:  Thank you for your comments. It is generally accepted that topically applied ceramide could easily penetrate into the intercellular space of stratum corneum. The MW of Cer 1-O-ENP [1-O-Acyl(18:0)-N(18:10)P(18:0)] is 848.8 Da and the width of the intercellular space of stratum corneum is over 100nm. The space is filled with several layers of lipid lamellar with 12~13nm (LPP) or 5~6nm (SPP) consisted of ceramide, cholesterol and free fatty acid (see the attachment).

Concerning the potential mechanism of ENP, an additional discussion paragraph is inserted as below;

Line 413~419

The boosting effect of ENP can be explained by findings from previous molecular dynam-ics simulations [38]. These simulations indicated that the splayed ENP induces notable alterations in the organization of the lipid matrix. Such changes include modifications in surface morphology and lateral packing density, which contribute to a more compact li-pid matrix within the stratum corneum (SC) lipid lamellar structure, thereby strengthen-ing the permeability barrier.

6. Figure 6. A and B indicate same data. Thus, one of the should be removed.

Response: Thank you for your comments. Yes. You made a good point. We tried to elaborate importance of the effect of ENP on moisture retention and help readers more easily understand. We would like to keep this Figure 6B. I appreciate your kind understanding.

7. line 43. The author's name is not Dr. Mann but Dr. Man

Response: I am very grateful for your precious time reading this manuscript. The correction has been made (Line 43 and reference 18). 

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is well revised.

Author Response

Thank you!