Quantification of Lipid Phase Order of In Vivo Human Skin Using Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy and Multivariate Curve Resolution Analysis.

A new analysis methodology utilizing multivariate curve resolution (MCR) has been successfully combined with Fourier transform infrared (FT-IR) measurement of in vivo human skin to resolve lipid phase constituents in the spectra relative to high and low chain ordering. A clinical study was performed to measure lipid order through different depths of stratum corneum of human subjects. Fourier transform IR spectra were collected through the top 10 layers of the skin on four sites on the left and right forearm of 12 individuals. Depth profiling was achieved by tape stripping to remove layers of skin with 10 successive tapes from each site. In vivo ATR FT-IR spectra were collected after removing each tape. Three isolated spectral regions were analyzed, centered around 2850 cm-1 , 1460-1480 cm-1 , and 730 cm-1 , corresponding to stretching, scissoring, and rocking -CH2 vibrational modes, respectively. Both traditional lipid conformation analysis and MCR analysis were performed on the same spectral data. The lipid order ratio, expressed as the fraction of highly ordered orthorhombic (OR) lipids to the total lipids content (orthorhombic + hexagonal [HEX] + liquid crystal [LC]), was assessed as function of depth. Lipid order depth profiles (LODP) show an increase in order with the stratum corneum depth which can be adequately described by an exponential function for the data obtained in this study. The LODP derived from the three vibrational modes show very similar trends, although the absolute order ratios are somewhat different. The variance of the skin LODP across individuals is much greater than between sites within the same individual. The higher arm sites closer to the elbow on the left and right arm show no statistically significant difference and are recommended for use in comparative studies. The scissoring mode shows the highest sensitivity for determination of LODP value.