Abstract
The outermost layer of the skin is the stratum corneum (SC), which is mainly comprised of solid proteins and lipids. Minor amounts of mobile proteins and lipids are crucial for the macroscopic properties of the SC, including softness, elasticity and barrier function. Still this minor number of mobile components are not well characterized in terms of structure or amount. Conventional quantitative direct polarization (Q-DP) 13C solid-state NMR gives signal amplitudes proportional to concentrations, but fails to quantify the SC mobile components because of spectral overlap with the overwhelming signals from the solids. Spectral editing with the INEPT scheme suppresses the signals from solids, but also modulates the amplitudes of the mobile components depending on their values of the transverse relaxation times T2, scalar couplings JCH, and number of covalently bound hydrogens nH. This study describes a quantitative INEPT (Q-INEPT) method relying on systematic variation of the INEPT timing variables to estimate T2, JCH, nH, and amplitude for each of the resolved resonances from the mobile components. Q-INEPT is validated with a series of model systems containing molecules with different hydrophobicity and dynamics. For selected systems where Q-DP is applicable, the results of Q-INEPT and Q-DP are similar with respect to the linearity and uncertainty of the obtained molar ratios. Utilizing a reference compound with known concentration, we quantify the concentrations of mobile lipids and proteins within the mainly solid SC. By melting all lipids at high temperature, we obtain the total lipid concentration. These Q-INEPT results are the first steps towards a quantitative understanding of the relations between mobile component concentrations and SC macroscopic properties.
Highlights
The outer layer of the human skin, stratum corneum (SC), has a vital function of being an efficient permeability barrier.[1,2] SC consists of dead keratin-filled cells, corneocytes, embedded in a multilamellar lipid matrix (Fig. 1)
Typical 13C nuclear magnetic resonance (NMR) experiments on 13C natural abundance sample relies on 1H - 13C cross polarization (CP)[9] or insensitive nuclei enhanced by polarization transfer (INEPT)[10] to increase the signal
The results obtained on both the 500 and 800 MHz NMR spectrometers are similar in respect of the spreading of the normalized molar ratios over different segments (Fig. 4A and Table 1) and the evolution of the signal intensities in quantitative INEPT (Q-INEPT) experiments (Fig. 5)
Summary
The outer layer of the human skin, stratum corneum (SC), has a vital function of being an efficient permeability barrier.[1,2] SC consists of dead keratin-filled cells, corneocytes, embedded in a multilamellar lipid matrix (Fig. 1). Typical 13C NMR experiments on 13C natural abundance sample relies on 1H - 13C cross polarization (CP)[9] or insensitive nuclei enhanced by polarization transfer (INEPT)[10] to increase the signal These polarization transfer steps have been utilized to identify rigid and mobile 13C molecular segments in many different systems, including SC (Fig. 1C and D).[4,11,12,13] Site-specific qualitative information about molecular mobility can be obtained by PT ssNMR (polarization transfer solid-state NMR),[11,14] wherein comparing signal intensities acquired with DP, CP, and INEPT yields information about the rate and anisotropy of the C–H bond reorientation
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