A simple and non-invasive visualization for assessment of carbonylated protein in the stratum corneum

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Abstract

Background/purpose

Stratum corneum (SC) is the interface of body and environment and is continuously exposed to oxidative stress, resulting in oxidative modification of proteins. Consequent carbonylated proteins (CPs) have so far been labeled with 2,4-dinitrophenyl (DNP) hydrazine and subsequently detected with anti-DNP antibody. We developed a simpler, non-invasive method to assess CP level in the SC and applied it to following research.

Methods

SC was collected by adhesive tape stripping and its carbonyl groups were labeled with fluorescein-5-thiosemicarbazide (FTZ). The staining image was observed by fluorescence microscopy and the average fluorescence intensity of the SC extracted from the image was calculated as stratum corneum carbonylated protein (SCCP) level.

Results

By reaction with FTZ, carbonyl groups in the SC could be detected easily. Relatively strong fluorescence was observed in exfoliating scales. Lipid removal from the SC in vitro or in vivo did not show any change in fluorescence intensity, suggesting that carbonyl groups were mainly derived from proteins, not from lipids. SCCP level was higher in the upper layer than the lower layer, and higher in the cheek (sun-exposed) than the inside of upper arm (unexposed), positively correlated with age especially in male cheek, positively correlated with transepidermal water loss, negatively correlated with water content, and showed a subtle correlation with sebum level. On the other hand, SC collected by cyanoacrylate resin and labeled with FTZ revealed strong fluorescence around the pores in the cheek and on the grooves in the upper arm, suggesting the role of sebum in the generation of SCCP.

Conclusion

SCCP was assessed in a simple and non-invasive method, and suggested to be a novel indicator that reflects some aspect of skin condition.

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