The Skin Barrier

Skin is considered the largest organ in the human body, and as such, it serves many roles in health and disease. As a primary function, the skin acts as a barrier against the external environment and keeps important molecules like water and electrolytes from escaping. The key player in these functions is the lipid barrier located at the uppermost layer of the skin known as the stratum corneum (SC). The SC is composed of several layers of corneocytes surrounded by lipids and is commonly known as the “brick and mortar” structure, with the corneocytes being bricks and the surrounding lipids being the mortar. Altogether, the SC acts as the first point of entry for foreign substances and cosmetic products (van Smeden & Bouwstra, 2016). The composition of the SC and its associated lipids is essential for maintaining healthy skin; therefore, cosmetic products should be carefully formulated to help maintain and restore a healthy skin barrier.

The 3:1:1:1 Ratio

The 3:1:1:1 ratio refers to a Ceramide-dominant molar mixture of Cholesterol, Essential Fatty acids (EFA) and Non-essential Fatty acids (NEFA). Individually, these lipids are vital for skin health, and disruption in any one of these components can result in structural abnormalities and impaired barrier function. It has been shown that a combination of all three lipids (ceramides, Cholesterol and fatty acids) is necessary for cosmetic formulations and barrier repair. These studies determined that a 3:1:1:1 ratio helped improve and accelerate barrier recovery compared to other ratios (Man et al., 1993; Mao-Qiang et al., 1996; YANG et al., 1995).

Composition of the Barrier Lipid

The lipid matrix of the SC is dominated by three types of lipids, including 25% cholesterol, 15% free fatty acids, and 50% ceramides based on total lipid mass (Feingold, 2007). The details and importance of each lipid component are highlighted below.

• Function of cholesterol in barrier repair. Cholesterol is responsible for the elasticity of the skin. It keeps the skin from breaking during freezing conditions and prevents it from melting in hot conditions. Its precursor, Cholesterol sulfate, plays a vital role in regulating desquamation (Bouwstra & Ponec, 2006). Its ratio with Cholesterol is crucial in balancing epidermal differentiation and permeability barrier homeostasis (Elias & Feingold, 2006). 
• Function of fatty acids in barrier repair. Most common fatty acids in the SC are straight-chained with 22-24 carbon-chain lengths (“Biochemistry of Human Stratum Corneum Lipids,” 2006). Fatty acids have a diverse function in the skin, but their primary function is to help decrease trans-epidermal water loss by forming lamellar layers with Ceramides and Cholesterol. They also serve as antimicrobial molecules on the upper layers of the SC. Interestingly, they can function both in promoting and preventing inflammation. 
• Function of ceramides in barrier repair. Ceramides are responsible for the formation and organization of the lipid barrier. They form the bulk majority of the barrier lipids. Read more about them here.

Current understanding

The topical application of Ceramides, Cholesterol, and Fatty acids was first investigated by Man et al. (1993) to determine the effect of incomplete and complete lipid mixtures on barrier recovery. In this study, lipid mixtures solubilized in propylene glycol were applied to the acetone-disrupted skin of hairless mice. Barrier recovery was assessed following the application of single lipid species, two-component and three-component lipid mixtures. Man et al. (1993) observed that only a complete mixture of Ceramides, Cholesterol, and free fatty acids in an equimolar ratio (1:1:1) facilitated normal barrier recovery. When single lipid species or two-component mixtures were applied, barrier recovery was inhibited. Fluorescence microscopy and ultrastructural analysis revealed that all three lipid components travel through the stratum corneum and are incorporated into the epidermis. However, single and incomplete mixtures resulted in abnormal lamellar bodies and a structurally imbalanced lipid matrix, whereas the complete lipid mixtures produced normal lamellar bodies and lipid bilayers.

An optimal lipid mixture was subsequently developed by Mao-Qing et al. (1995) during an investigation comparing the abilities of complete lipid mixtures and non-physiologic lipids for barrier correction. The lipid mixture was comprised of Cholesterol, Ceramides, Linoleic acid (EFA), and Palmitic acid (NEFA) in a 3:1:1:1 molar ratio. This mixture was observed to notably accelerate barrier repair when applied to the acetone-disrupted skin of hairless mice. Yang et al. (1995) further investigated the optimal lipid mixture’s ability to facilitate barrier repair following moderate or extensive disruption by acetone, tape stripping, petroleum ether solvents, and detergents. In this study, a 1.5% solution containing Cholesterol, Ceramides, Linoleic acid (EFA), and Palmitic acid (NEFA) in a 4.3:3:1.08:1 ratio was solubilized in a Propylene glycol and Ethanol vehicle. The optimal lipid mixture was shown to improve the rate of barrier recovery following an extensive insult by acetone and petroleum ether, as well as moderate and extensive disruption by tape stripping. However, when the barrier sustained injuries from various detergents, the lipid mixture did not appear to accelerate barrier repair in all cases. Specifically, when the barrier was disrupted using sodium dodecyl sulfate (SDS) and ammonium laurel sulphate (ALS), no improvement in barrier function was observed. Yang et al. (1995) hypothesized that certain detergents may cause more extensive damage to the stratum corneum, such as the denaturation of proteins, that cannot be corrected through a topical application of lipids. It is also feasible that detergents cause more than just superficial damage to the stratum corneum, penetrating and disturbing the deeper layers of the epidermis.

With early evidence demonstrating an optimal lipid mixture, Mao-Qing et al. (1996a) explored various four-component systems for the optimization of barrier repair. In order to determine the optimal ratio of Cholesterol, Ceramides, Linoleic acid (EFA), and Palmitic acid (NEFA), mixtures with varying proportions were applied to the acetone-disrupted skin of hairless mice. The influence of the proportion of fatty acids has on barrier recovery was first studied. As the moles of EFA or NEFA were both individually increased, an optimal barrier recovery was noted when three parts of the fatty acids to one part of the other components was present (i.e., 1:1:3:1 or 1:1:1:3). The second group of studies examined the result of changing the proportion of either Cholesterol or Ceramides. Similarly, when the moles of either Cholesterol or Ceramides were increased from one to three, an optimization in barrier recovery occurred. However, a further increase to four or five parts of these lipids to one part of the other components led to a progressive decline in recovery rates. A preliminary study by Mao-Qing et al. (1996a) was also performed to determine whether barrier recovery in human skin can be accelerated through topical application of lipid mixtures. The optimal 3[cholesterol]:1:1:1 and 1:1:1:3[NEFA] formulas were applied to the acetone-disrupted skin of human volunteers. The extent of recovery from the use of these lipid mixtures was compared against barrier recovery after applying propylene glycol and ethanol solution. It was observed that both of these optimized ratios significantly accelerated barrier repair in human skin. Thus, Mao-Qing et al. (1996a) demonstrated that optimal recovery in an impaired skin barrier occurs when there is 3-fold increase in any one of the components that constitute the lipid mixture.

Due to the potential high cost and low availability of ceramides for ceramide-dominant 3:1:1:1 lipid mixtures, Mao-Qing et al.(1996b) investigated whether a fatty acid-dominant 1:1:3 lipid ratio could also accelerate barrier recovery. A three-component lipid mixture of Cholesterol, Ceramides and natural lipids from porcine tissue was applied to the skin of hairless mice and human skin. Prior to the application of the 1:1:3 lipid mixture, the skin barrier in test subjects was disrupted using either acetone or tape stripping. In the acetone-disrupted skin of hairless mice, barrier recovery was observed to accelerate following the application of the 1:1:3 lipid mixture, which is in agreement with the results seen in prior studies. As there was limited data available for the effect of optimal lipid mixtures on human skin, Mao-Qing et al.(1996b) analyzed barrier recovery in both acetone and taped stripped human skin following the application of the 1:1:3 lipid mixture. Treatment with the fatty acid-dominant mixture was shown to significantly accelerate barrier recovery in acetone-disrupted or tape stripped human skin.

Zetterson et al. (1997) further validated the optimal lipid ratio in four-component mixtures for accelerated barrier recovery in chronologically aged skin. In this study, lipid mixtures of Cholesterol, ceramides, palmitic acid, and linoleic acid in an equimolar ratio (1:1:1:1), in the optimal ratio (3:1:1:1), Cholesterol alone, and a 1:1:2:2 ratio of fatty acids were analyzed. Tape-stripping was employed to disrupt the skin in chronologically aged hairless mice, as well as chronologically aged human skin. It was observed that application of the cholesterol-dominant optimal ratio significantly accelerated the barrier recovery in the skin of chronologically aged hairless mice, whereas the fatty acid-dominant 1:1:2:2 ratio had the opposite effect, and barrier recovery was delayed. When the lipid mixtures were applied to disrupted chronologically aged human skin, the cholesterol-dominant 3:1:1:1 ratio significantly accelerated barrier repair.

It is now recognized that for optimal barrier repair to occur in damaged skin, there must be a 3-fold increase in any one of the components that constitute the Lipid mixture. Due to a reduction in the Ceramide, Cholesterol, and Fatty acid content in compromised skin, Elias et al. (2019) recently indicated that the optimal lipid mixture should be present at a minimum concentration of 5%. The lipid mixture should also be Ceramide-dominant so that three parts of ceramides to one part of the other components are present. This ceramide-dominant lipid mixture will then prompt an increase in lipid production in the stratum corneum. Normal lamellar bodies can then be produced, and the structure of the lipid matrix is corrected. Clinical evidence has demonstrated that ceramide-dominant 3:1:1 topical emulsions with Cholesterol and Fatty acids are efficacious in treating dry skin conditions. Notably, the United States FDA approved a barrier repair emulsion cream employing this specific ratio in 2006 for the management of symptoms associated with dry skin conditions. Kircik et al. (2011) conducted a study to evaluate the ceramide-dominant barrier repair emulsion cream as monotherapy or as part of combination topical therapy in subjects with mild-to-moderate atopic dermatitis. The study concluded that the lipid mixture was an effective therapy that could be used independently or in combination with additional atopic dermatitis treatments. As the lipid mixture targets the structural abnormalities in a defective skin barrier, Kircik et al. (2011) concluded that it may be advantageous to employ a ceramide-dominant 3:1:1 topical emulsion for the initial management of dry skin disorders.

Limitations in Current Research

• The limitations in skin barrier studies lie in the models that are selected and how they are used. In order to generalize data to human skin, studies need to create experimental conditions or models that mimics (or preferably - is identical) to human skin.
• Most studies utilize murine models which present several issues in translational research. Mice have thinner and loose skin compared to the thick and firm skin of humans. Specifically, the epidermis in mice contains 2-3 times less skin cell layers, making it more susceptible to barrier damage, and more effective at percutaneous absorption (Zomer & Trentin, 2018).
• The study of barrier recovery requires methods that initially disrupt the skin barrier. Studies such as the one conducted by Man et al. (1993) utilized acetone to perturb the skin of mice. Limitations lie in whether this form of skin perturbation successfully depicts the full picture of how skin works. Although it is not ethically possible to recruit human participants and study their skin by damaging it, there does exist specific non-invasive methods such as tape-stripping in humans or mice, as well as ex vivo and in vitro models. Regardless of the model chosen, each has its own limitations which could be counteracted by using several different models within the same study. However, this is rarely done due to time, resource and funding constraints.
• The other limitation is the lack of moisturizer controls in the studies. Most of the published research did not compare the efficacy of their test product to the efficacy of a standard over-the-counter moisturizer. One study with 39 subjects showed that petrolatum-based moisturizers could achieve similar clinical efficacy as barrier repair moisturizers. Our recommendation is for future clinical studies to include a comparison with standard moisturizers.
• Limitations also lie in the preparation of the lipid mixtures for topical application. In the following studies, a 7:3 vol/vol solution of propylene glycol: ethanol was employed to solubilize the lipid mixtures:

The four-component lipid mixtures prepared by Zetterson et al. (1997) employed a 7:3 vol/vol solution of propylene glycol:n-propanol. Propylene glycol and alcohols (ethanol and n-propanol) are penetration enhancers which penetrate into the skin by disrupting the lipid bilayer.

It is also important to note the percentage of the lipid mixtures solubilized in each solution. It is now known that the optimal lipid mixture should be present at a minimum concentration of 5% to facilitate barrier repair; however, none of skin barrier studies were performed using the lipid mixture at this percentage. As seen below, the percentage of the lipid mixture varied between studies:

Overall, the 3:1:1:1 ratio has been widely accepted as an effective ratio for promoting accelerated barrier recovery. However, it is not clear whether other ratios may be more effective. Future studies should investigate whether different Ceramides, Cholesterol and Fatty acids ratios can further accelerate barrier recovery. Additionally, the accuracy of studies may be improved by using a minimum lipid concentration of 5% using moisturizer as a vehicle. Regimen Lab is currently conducting studies to help close this gap in understanding, and determine whether there could be more effective ratios while addressing many of the limitations learned from prior studies.

Evaluating the 3:1:1:1 ratio

Due to the limitations in existing research, we developed studies to decipher whether other ratios may be more or less effective at promoting barrier recovery using the tape-stripping method on human skin. The purpose of these studies is to test various ratios of Ceramides, Cholesterol and Fatty acid cream formulations to identify which ratios help repair (or hinder the repair) of the skin barrier. We hypothesize that testing various ratios in the form of moisturizers can be more useful in investigating their real-world applicability.

In this experiment, we wanted to find out the real-world applicability of the ratios so we tested them on human skin using a cream as a vehicle rather than propylene glycol: ethanol solution. 6 human volunteers were tape stripped using D-squames until the TEWL is between 30-40 g/m2/h. 0.010g of creams with various ratios were applied and the TEWL was measured after 8 hours (compared to 2 hours in literature). The following Ratios were tested:

 

Results

The results we got are similar to the results obtained by Mao-Quiang et. al. The lowest TEWL results were obtained with 3:1:1:1, followed by 1:3:1:1. It seems that 3:1 ratio works the best as long as Ceramide or Cholesterol is the dominant specie. However, the ratios tested here are mostly focused on single-specie-dominant ratios. What if there are two dominant species for example 3:3:1:1? Such ratios haven't been tested so these are worth further exploring.

Beyond 3:1:1:1

We decided to explore ratios with Ceramide as one of the dominant species. We looked into the possible ratios with Ceramides as 3, and Cholesterol, Fatty acids with either 3, 2, or 1. Six human volunteers were tape stripped until the TEWL is at 30-40 g/m2/h. 0.010g of cream samples were applied and the TEWL was measured after 2, 4, 6, 8, and 24 hours. Percent barrier repair is calculated as: 1-(Measured TEWL-Baseline TEWL)/(TEWL after Stripping-Baseline TEWL) x 100

Results

There are some ratios that we tested that don't exactly fit the 3:1 ratio. Technically ratios must always be expressed in its lowest terms. In this case, it should always have 1 in the ratio. There 25 ratios and among them are 7 ratios that aren't expressed in their lowest terms. Interestingly, these 7 are among the lowest Barrier Repair Percentage in the group. Anything below the no lipids means that they performed worse than the vehicle and 3 of them performed worse than without any cream at all. 

There are 5 ratios that surpassed 90% barrier repair after 8 hours and they are not statistically different from each other. The green ratios are the only ones that consistently surpassed the No Lipid Control while the yellow ratios are the ones that at least once delayed the barrier repair more than No Lipid Control. The red ones are those that delayed the barrier more than the No Lipid Control and the black ones are those that delayed barrier repair even lower than without any product at all.

The results indicate that there might be other ratios that could be at least as good as the golden 3:1:1:1 ratio. We decided to investigate these five ratios and because we only have 5, we can perform triplicate sites for the measurements hoping we get enough power to differentiate the barrier ability of these 5. As a control we included a cream with no barrier lipids. TEWL measurement was assessed at 1, 2, 3, 4, 5, 6, 7, 8 Hours. 

Results

As you can see in the graph, these 5 ratios accelerated barrier repair compared to the carrier cream. In terms of barrier repair at 8 hours there is no significant difference between the 5 ratios in this experiment. It is most likely that the experiment lacks enough power to differentiate the 5 ratios but those tiny differences would not translate to any significant change in barrier repair ability as we are talking only of a few percentage. Nevertheless it is still worth looking into. 

Limitations

• Previous studies used 6 to 10 human volunteers for tape stripping. In this study we have 6 volunteers as well, but we need more subjects to increase the power of the study. The problem lies in the invasiveness and tediousness of the procedure. Essentially, the subjects have to be tape stripped with each site taking around 10-20 minutes, multiple that with 20 sites per forearm resuting to 6 hours of just tape stripping. (If you read this, email us for a 15% discount to CREAM 2.0) Afterwards, TEWL needs to me measured at the exact time after product application. It usually takes about 14 hours for the whole procedure where the subjects must stay immobile (bathroom and lunch breaks allowed of course). It's actually borderline unethical if we were to do these to other people. 
• Aside from the tediousness, tape stripping leaves scars as we remove the layers of the skin up to the stratum granulosum. Some of the sites turn red and become hyperpigmented after a few days. It takes at least a month before they eventually fade away thanks to Level serum. 
• The other limitation is that the study is specific to our CREAM as that is the only vehicle we used. 
• We also have not yet determined barrier repair ability after SLS exposure. 

References

Biochemistry of Human Stratum Corneum Lipids. (2006). In Skin Barrier. CRC Press.

Bouwstra, J. A., & Ponec, M. (2006). The skin barrier in healthy and diseased state. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1758(12), 2080–2095. https://doi.org/10.1016/j.bbamem.2006.06.021

Elias, P. M., Wakefield, J. S., & Man, M-Q. (2019). Moisturizers versus Current and Next-Generation Barrier Repair Therapy for the Management of Atopic Dermatitis. Skin Pharmacology and Physiology, 32, 1-7.

Feingold, K. R. (2007). Thematic review series: Skin Lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis. Journal of Lipid Research, 48(12), 2531–2546. https://doi.org/10.1194/jlr.R700013-JLR200

Kircik, L. H., Del Rosso, J. Q., & Aversa, D. (2011). Evaluating Clinical Use of a Ceramide-dominant, Physiologic Lipid-based Topical Emulsioin for Atopic Dermatitis. Journal of Clinical Aesthetic Dermatology, 4 (31), 34-40.

Man, M.-Q., Feingold, K. R., & Elias, P. M. (1993). Exogenous Lipids Influence Permeability Barrier Recovery in Acetone-Treated Murine Skin. Archives of Dermatology, 129(6), 728–738. https://doi.org/10.1001/archderm.1993.01680270066008

Mao-Qiang, M., Feingold, K. R., Thornfeldt, C. R., & Elias, P. M. (1996). Optimization of Physiological Lipid Mixtures for Barrier Repair. Journal of Investigative Dermatology, 106(5), 1096–1101. https://doi.org/10.1111/1523-1747.ep12340135

Mao-Qiang, M., Feingold, K. R., Thornfeldt, C. R., Elias, P. M. (1996). Optimization of Physiological Lipid Mixtures for Barrier Repair. The Journal of Investigative Dermatology, 106 (5), 1096-1101.

Mao-Qiang, M., Feingold, K. R., Wang, F., Thornfeldt, C. R., & Elias, P. M. (1996). A Natural Lipid Mixture Improves Barrier Function and Hydration in Human Skin. Journal of the Society of Cosmetic Chemists, 47, 157-166.

van Smeden, J., & Bouwstra, J. A. (2016). Stratum Corneum Lipids: Their Role for the Skin Barrier Function in Healthy Subjects and Atopic Dermatitis Patients. Current Problems in Dermatology, 49, 8–26. https://doi.org/10.1159/000441540

YANG, L., MAO‐QlANG, M., TALJEBINI, M., ELIAS, P. M., & FEINGOLD, K. R. (1995). Topical stratum corneum lipids accelerate barrier repair after tape stripping, solvent treatment and some but not all types of detergent treatment. British Journal of Dermatology, 133(5), 679–685. https://doi.org/10.1111/j.1365-2133.1995.tb02738.x

Zetterson, E. M., Ghadially, R., Feingold, K. R., Crumrine, D., & Elias, P. M. (1997). Optimal Ratio of Topical Startum Corneum Lipids Improve Barrier Recovery in Chronologically Aged Skin. Journal of the American Academy of Dermatology, 37 (3), 403-408.

Zomer, H. D., & Trentin, A. G. (2018). Skin wound healing in humans and mice: Challenges in translational research. Journal of Dermatological Science, 90(1), 3–12. https://doi.org/10.1016/j.jdermsci.2017.12.009