James V Gruber, Nicole Terpak, Sebastien Massard and Elva Chen Vantage

The skin’s epidermis is not a static dead layer, but rather, a continuously replenishing, water impermeable membrane which has stimulated extensive research in this dynamic structure.^1-7 The skin lipid barrier emerges from four stages of epidermal differentiation beginning at the basal layer just above the dermis and moving through the spinous and granular layers, ultimately ending in the stratum corneum, Figure 1.

Skin lipids, comprising the stratum corneum expressed from lamellar bodies in the differentiating keratinocytes, are composed of nearly equal molar amounts of ceramides, fatty acids and cholesterol (and cholesterol esters).⁸ These lipid structural features are reproduced quite accurately in numerous Reconstructed Human Epidermal (RHE) tissue models, which help to examine ingredients that may influence the lipid barrier.^9,10 The skin’s sphingolipids and ceramides are of particular interest as they form the principal architecture of the skin’s lipid bilayer.^11-13 Within the highly crystalline ceramide bilayers, the fatty acids and cholesterol intercalate themselves, providing the skin barrier with its flexibility and impermeability. As skin ages, it produces fewer critical lipids needed to produce a strong, impermeable lipid bilayer.¹⁴ As technologies in data collection expand, and with the advent of artificial intelligence to survey larger data sets, “omics” have emerged as a new tool to study and understand skin function.¹⁵ These techniques have expanded into the study of skin lipids and the practice of such studies is called lipidomics.¹⁶

It has been reported that various lipophilic phosphatidylglycerol derivatives can influence keratinocyte differentiation and accelerate wound healing.^17,18 In particular, dioleoylphosphatidylglycerol appears to have a particular ability to improve wound healing in corneal epithelial tissues.¹⁹ This article details recent studies done on RHE tissue models (Epiderm) to examine the influence of a proprietary phosphatidylglycerol (found in Biosignal Lipid 10, INCI: Glycerin (and) Phospholipids) to stimulate the expression of skin lipids. In particular, the study focuses on skin sphingolipids and ceramides against two ingredients, ceramide and niacinamide, known to stimulate epidermal skin lipids using lipidomic HPLC/MS assay techniques.^20,21 

Materials & Methods

The tissues employed in the studies were purchased from Mattek (Epiderm, Ashland, MA). The proprietary phosphatidylglycerol was provided by Vantage absent its glycerol diluent and was used as is.  The niacinamide was provided by Sandream Impact [Fairfield, NJ] and was used as supplied.  The ceramide was provided by Vantage and used as is. DMSO (Dimethyl Sulfoxide) was purchased from Sigma-Aldrich (St. Louis, MO) and used as is.

Tissue Preparation and Treatment: Upon arrival, the Mattek Epiderm tissues were removed from the agarose shipping tray and placed into a 6-well plate containing 0.9ml assay medium (37 +/- 2oC). The tissues were allowed to incubate for one hour at 37 +/- 2 oC and 5 +/-1% CO2. Following incubation, the assay medium was replaced with fresh media and 15µl of the test materials were topically applied to the tissues. With respect to the dissolution of the test materials, niacinamide was prepared in Phosphate Buffer Solution (PBS) and tested at 1.0% while the ceramide and phosphatidylglycerol materials were prepared in DMSO and tested at 0.25%. DMSO-treated tissues served as the respective untreated control tissues. The tissues were treated in triplicate for each treatment group. Twenty-four hours after the first application of the test materials, the tissues were rinsed with PBS, blotted dry and fresh test material were applied. The assay was also replaced with fresh media and tissues were incubated for a second 24-hour interval. At the end of the second incubation, the tissues were rinsed again in PBS, blotted dry, and removed from the cell culture inserts and weighed. Individual tissues were placed into labeled 1.5 ml centrifuge tubes and stored at -70oC until tested.

Tissue Lipid Analysis

Sample Preparation: Frozen epidermal tissues were submerged in 275µl of -20oC chilled 75% methanol and spiked with 20µl of an internal standard and calibration mixture consisting of 500 nanograms/microliter of di-myristoyl phospholipids (PG, PE, PS, PA, PC), SM (35:1), Cer(30:1) cholesterol ester (14:0) and TG(45:0). Samples were homogenized in a Fisher Scientific bead mill, transferred to 1.0ml Eppendorf Safe-Lock tubes, and an additional 100µl of methanol was added.  Then one milliliter of methyl-tert-butyl ether (MTBE) was added to each sample, and samples were vortexed for 60 minutes at room temperature. To each sample 210µl of water was added and the samples were vortexed for an additional 15 minutes. After centrifugation for 10 minutes, the supernatants were collected to new test tubes and precipitated proteins were reextracted as above. Pooled extracts were dried overnight in a speedvac and resuspended in 500µl of isopropanol containing 0.01% BHT.

Instrument and Sample Analysis Parameters: Immediately prior to analysis, aliquots of each lipid extract were diluted 20-fold into isopropanol:methanol (2:1, v:v) containing 20mM ammonium formate.  Full scan MS spectra at 100,000 resolution (defined at m/z 400) were collected on a Thermo Scientific LTQ-Orbitrap XL mass spectrometer in both positive and negative ionization modes.  Scans were collected from m/z 200 to m/z 2000.  For each analysis, 10µl of sample was directly introduced by flow injection (no LC column) at 10µl/min using an electrospray ionization source equipped with a heated ESI needle.  A Dionex Ultimate 3000 HPLC served as the sample delivery unit. The sample and injection solvent were 2:1 (v:v) isopropanol:methanol containing 20mM ammonium formate. The spray voltage was 4.5kV, ion transfer tube temperature was 275oC, and the ion trap fill time was set at 4oC. After two minutes of MS signal averaging, the LC tubing, autosampler and ESI source were flushed with 1mL of isopropanol prior to injection of the next sample. Samples were analyzed in random order, interspersed by solvent blank injections and extraction blanks. Following MS data acquisition, offline mass recalibration was performed with the Recalibrate Offline tool in Thermo Xcalibur software according to the vendor’s instructions using the theoretical computed masses for the internal calibration standard and several common endogenous mammalian lipid species. MS/MS confirmation and structural analysis of lipid species identified by database searching were performed using CID-MS/MS at 60,000 resolution and a normalized collision energy of 25 for positive ion mode, and 60 for negative ion mode. MS/MS scans were triggered by inclusion lists generated separately for positive and negative ionization modes.

Lipid Peak Finding, Identification and Quantification: Lipids were identified using the Lipid Mass Spectrum Analysis (LIMSA) v.1.0 software linear fit algorithm in conjunction with an in-house database of hypothetical lipid compounds for automated peak finding and correction of 13C isotope effects. Peak areas of found peaks were quantified by normalization against an internal standard of a similar lipid class. The top ~300 most abundant peaks in both positive and negative ionization mode were then selected for MS/MS inclusion lists and imported in Xcalibur software for structural analysis on a pooled sample as described above.  For this untargeted analysis, no attempt was made to correct for differences in lipid species ionization due to the length or degree of unsaturation of the esterified fatty acids. Therefore, lipid abundance values are inherently estimated rather than true absolute values.

QA/QC Measures: Prior to analysis, the mass spectrometer inlet capillary was removed and cleaned by sonication, and the ESI tubing and spray needle were cleaned by extensive flushing with isopropanol.  During the analysis, several injection solvent blanks and extraction blanks were interspersed between the randomized study samples to monitor for background ions and any potential sample carryover throughout the run. The offline external mass recalibration routine described above was used to eliminate in-run drift in mass calibration and improve the consistency of mass accuracy over the measured m/z range.  

Statistical Analysis: Values of each lipid expressed in ng/mg of tissues were analyzed in triplicate and standard deviations were determined using the Student Paired T-Test in Microsoft Excel.

Results & Discussion 

The lipidomic techniques employed in the following studies allow for examination of several skin lipid classes including: Total Phospholipids, Total Sphingolipids, Total Glycerolipids, Total Sterol Lipids, Total Non-Esterified Fatty Acids and Total Other Lipids. For the purposes of this study, the focus is on the ability of the test materials to stimulate the total lipid synthesis in the RHE skin tissue models with a focus on the ability of the materials to stimulate sphingolipids and specific ceramides including Total Ceramides, Total Ceramide-1-Phosphate, Total Hexosyl Ceramide and Total Lactosyl Ceramide.

Total Lipid Expression

Data examining the Total Lipid expression in nanogram/mg of tissues for the samples can be found in Figure 2. It can be seen from the data presented that 0.25% of phosphatidylglycerol could stimulate expression of the total skin lipids superior to DMSO or to 1.0% of niacinamide. The data against the topically applied ceramide was not statistically significant.

Total Sphingolipid Expression

The data for the Total Sphingolipid Expression can be seen in Figure 3. The data indicate that only phosphatidylglycerol showed a significant increase in expression of the sphingolipids compared against the DMSO control and ceramide treatments. While the data for the niacinamide treatment were not statistically significant, the p-value for this sample was 0.06 indicating that the phosphatidylglycerol was at 94% statistical confidence against the niacinamide. 

Total Ceramide Expression

The data for the Total Ceramides Expression can be seen in Figure 4. The data indicates that 0.25% of phosphatidylglycerol was able to significantly increase expression of the Total Ceramides in the skin compared against the DMSO control, the niacinamide and the ceramide sample.

Conclusions  

The data presented here indicate that a unique proprietary phosphatidylglycerol can stimulate not only the total expression of lipids in the RHE test tissues, but also the overall Total Sphingolipids and Total Ceramides against two ingredients known to also be effective at stimulating skin lipids.^20,21 As topical application of both niacinamide and ceramides has been shown to accelerate keratinocyte differentiation and improve wound healing, it is very likely that the unique phosphatidylglycerol presented here will function in a similar fashion. The ability of phosphatidylglycerol to stimulate keratinocyte differentiation and to accelerate wound healing was noted.^17-19 The data presented here suggest that these influences may be in part due to phosphatidylglycerol’s ability to stimulate important barrier skin lipid expression including sphingolipids and ceramides. 

Acknowledgments

The authors acknowledge the support of Bioinnovation Laboratories, Inc (Lakewood, CO) for the Mattek tissue treatments and Creative Proteomic (Shirley, NY) for the mass spectral lipid analysis. For more information, contact Vantage Specialty Chemicals, www.vantagegrp.com  

References

1. Rudan, M.V., Watt, F.M. (2022). Mammalian epidermis: A compendium of lipid functionality. Front Physiol. 12 804824. DOI:10.3389/phys.2021.804824. doi:10.3389/fphys.2021.804824.
2. Holleran, W.M., Takagi, Y. (2006). Stratum corneum lipid processing: The final steps in barrier formation. In: Skin Barrier. Elias, P.M. and Feingold, K.R, eds. Taylor & Francis, New York. Pages 231-259.
3. Pappas, A. Epidermal surface lipids. (2009). Dermato-Endocrin. 1:2 72-76. 
4. Feingold, K.R., Elais, P.M. (2014). Role of lipids in the formation and maintenance of the cutaneous permeability barrier. Biochima Biophysica Acta. 1841 280-294. Dx.doi.org.10.1016/j.bbalip.2013.11.007.
5. Lundborg, M., Narangifard, A., Wennberg, C.L., Lindahl, E., Daneholt, B., Norlen, L. (2018). Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation. J Struct Biol. 203 149-151. DOI.org/10.1016/j.jsb.2018.04.005.
6. Norlen, L., Lundborg, M., Wennberg, C., Narangifard, A., Daneholt, B. (2022) The skin’s barrier: A cryo-EM based overview of its architecture and stepwise formation. J Invest Dermatol. 142 285-292. DOI:10.1016/j.jid.2021.06.037.
7. Norlen, L. (2023). Molecular organization of the skin barrier. Acta Derm Venereal. 103 adv13356. DOI:10.2340/actadv.v103.13356.
8. Mojumdar, E.H., Gooris, G.S., Barlow, D.J., Lawrence M.J., Deme, B., Boustra, J.A. (2015). Skin lipids: Localization of ceramide and fatty acids in the unit cell of the long periodicity phase. Biophysical J. 108 2670-2679. Dx.doi.org/10.1016/j.bpj.2015.04.030.
9. Ponec, M., Boelsma, E., Weerheim, A., Mulder, A., Bouwstra, J., Mommaas, M. (2000). Lipid and ultrastructural characterization of reconstructed skin models. Int J Pharmaceutics 203 211-225.
10. Ponec, M., Weerheim, A., Lankhorst, P., Wertz, P. (2003). New acylceramide in native and reconstructured epidermis. J invest Dermatol. 120 581-588.
11. Holleran, W.M., Mao-Qiang, M., Gao, W.N., Menon, G.K., Elias, P.M., Feingold, K.R. (1991). Sphingolipids are required for mammalian epidermal barrier function. J Clin Invest. 88 1338-1345.
12. Rabionet, M., Gorgas, K., Sandhoff, R. (2014). Ceramide synthesis in the epidermis. Biochima Biophysica Acta. 1841 422-434. Dx.doi.org/10.1016/jbbalip.2013.08.011.
13. Cha, H.J., He, C., Zhao, H., Dong, Y., An, I.S., An, S. (2016). Intercellular and intracellular functions of ceramides and their metabolites in skin (review). Int J Mol Med. 38 16-22. DOI:10.3892/ijmm.2016.2600. 
14. Barland, C.O., Elias, P.M., Ghadially, R. (2006). The aged epidermal permeability barrier. Basis for functional abnormalities. In: Skin Barrier. Elias, P.M. and Feingold, K.R, eds. Taylor & Francis, New York. Pages 535-552.
15. He, J., Jia, Y. (2022). Application of omics technologies in dermatological research and skin management. J Cosmet Dermatol.  21 451-460. DOI:10.1111/jocd.14100.
16. Gruber, F., Kremslehner, C., Narzt, M.S. (2019). The impact of recent advances in lipidomics and redox lipidomics on dermatological research. Free Rad Biol Med. 144 256-265. Doi.org/10.1016/j.freeradbiomed.2019.04.019.
17. Xie, D., Seremwa, M., Edwards, J.G., Podolsky, R., Bollag, W.B. (2014). Distinct effects of different phosphatidylglycerol species on mouse keratinocyte proliferation. PLoS One 9(9) e107119. DOI:10.1371/journal.pone.0107119.
18. Luo, Y., Marrero, E.V., Choudhary, V., Bollag, W.B. (2023). Phosphatidylglycerol to treat chronic skin wounds in diabetes. Pharmaceutics 15 1497. Doi.org/10.3390/pharmaceutics 15051497.
19. Bollag, W.B., Olala, L.O., Xie, D., Lu, X., Qin, H., Choudhary, V., Patel, R., Bogorrad, D., Estes, A., watsky, M. (2020). Dioleoylphosphatidylglycerol accelerates corneal epithelial wound healing. Invest Ophthamol Vis Sci. 61 29, doi.org/10.1167/lovs.61.3.29.
20. Gehring, W. (2004). Nicotinic acid/niacinamide and the skin. J Cosmet Dermatol. 3 88-93.
21. Shin, K.O., Miharra, H., Ishida, K., Uchida, Y., Parl, K. (2022). Exogenous ceramide serves as a precursor to endogenous ceramide synthesis and as a modulator of keratinocyte differentiation. Cells 11 1742. Doi.org/10.3390/cells11111742.