Introduction
Accumulation of damaged macromolecules in the skin, including oxidatively damaged (carbonylated) proteins, is a hallmark of oxidative stress and accelerated skin aging. However, due to technical limitations for their assessment, the quantification of decreased carbonylated skin proteins has not been widely used as a read-out of efficacy in dermo-cosmetics clinical studies. The objective of this research project was to: (i) validate a novel targeted proteomics approach to assess protein carbonylation on the stratum corneum from tape strips, and (ii) to investigate whether absolute quantification of carbonylated proteins could be used in studies on volunteers not only to support an anti-oxidant/ proteome protection effect of formulated products, but also to evaluate its prognostic and predictive value by studying its association with known skin physical parameters, such as brown or red spots and skin texture.
Methods
Optimization of protein extraction methods from tape strips using different combinations of chaotropic agents and detergents has been performed. Protein carbonylation detection was performed using a specific functionalized fluorescence probe targeting carbonyls moieties. Absolute protein carbonylation quantification (Carbonyl Score) was obtained upon fluorescent signal integration of carbonylated proteins/total proteins after protein separation. In-situ detection of carbonylated proteins by epi-fluorescence microscopy on tape strips was also validated for imaging approaches. For final validation of the approach, an in vivo study on 23 volunteers was performed using a formulation containing the anti-carbonylation / proteome protectant active ingredient compared to a placebo (same formulation without the active ingredient), in a double-blind, upon hemi-face topical application, twice a day, for 28 days. Protein carbonylation was assessed at D0, D14 and D28. In addition, biometrological analyses were performed using the VISIA’s multi-point positioning system. Statistical analyses were performed using GraphPad software.
Results
Optimized and validated protocols for protein carbonylation assessment (absolute quantification & epi-fluorescence imaging) on stratum corneum stripped samples were developed. A remarkable sensitivity and quantitative dynamic range of carbonylated proteins signal was obtained. Indeed, the positive effects (inhibition of protein carbonylation) of a formulated anti-carbonylation compound was already detected upon only 14 days of topical application on 61% of volunteers. Moreover, at D28 a significant decrease in protein carbonylation (17%, p-value <0.05) was observed when compared to DO. The placebo did not show any beneficial effect. In addition, the decrease in protein carbonylation inhibition was positively correlated with a significantly greater decrease in brown spotting of the skin at D28 on the active ingredient side than on the placebo side. Another remarkable positive correlation of protein carbonylation attenuation was with the improvement of skin texture (smoothing effect) in more than 70% of the volunteers (12% on average), which was also significantly greater.
Discussion and Conclusion:
In this study, a reliable method for protein carbonylation assessment on stratum corneum has been developed and validated. This method can be used as an early read-out of efficacy in anti aging studies, since the beneficial effects are already observed upon 14 days of treatment. In addition, the benefits to be gained from increased protection or the stimulation of carbonylated proteins elimination were evidenced by the amelioration of parameters related to skin appearance. Taken together, these results underscore the prognostic and predictive value of carbonylated protein assessment for in vivo dermo-cosmetics efficacy evaluation on aging.