日本香粧品学会誌–105（2017）Vol. A
41,
Novel
No. 2,Image
pp. 101
Analysis
Method to Evaluate Carbonylated Proteins in the Stratum Corneum

101〈Regular Article〉

A Novel Image Analysis Method to Evaluate Carbonylated Proteinsin the Stratum Corneum
Taeko MIZUTANI*, Toshiyuki MIZUTANI, Chiho IMAMURA, Yuri OKANO, Hitoshi MASAKI(Accepted: February 7, 2017)

Abstract
We established an algorithm for the image analysis of carbonylated proteins (CPs) in the stratum corneum (SC) by eliminating the multiplex SC area using Threshold-based image Processing (TBIP) of image histograms. CPs are produced byvarious aldehydes yielded by reaction with lipids and reactive oxygen species. The carbonyl modiﬁcations are therefore irreversible and CPs are markers of oxidative stress. Recent studies suggested that CPs in the SC are not only markers of oxidative stress but also might be triggers leading to alterations of skin conditions. The content of CPs in the SC (SCCP level) canbe visualized by labelling with ﬂuorescent hydrazide derivatives after collecting the SC non-invasively using the adhesivetape-stripping method. However, the strong ﬂuorescence emitted from the multiplex SC area interferes with the comprehensive evaluation of SCCP levels by image analysis. Therefore, a more reliable and accurate method to measure SCCP levels isrequired. Image histograms of ﬂuorescence-labeled CPs in the SC were obtained. The TBIP method simply separates each
image into monolayer and multiplex areas based on the gradation value which is decided by the image histogram shape.
SCCP levels were calculated by the image of the selected monolayer area and were compared with simple SCCP levels calculated without image separation of monolayer and multiplex areas of the SC. The comparison result of SCCP levels withand without the TBIP method indicates that the TBIP method reduces not only the value of the SCCP level but also minimizes the deviation in SCCP levels in the ﬂuorescence images. Thus, the TBIP method provides sensitive results to evaluate levels of CPs in the SC.
Key words:
1.

carbonylated proteins, stratum corneum, image analysis.

Introduction

Carbonylated proteins (CPs) are oxidized proteins that areproduced during lipid oxidation, and are biomarkers reﬂecting
oxidative stress1)–3). In the skin, CPs can be readily seen in theentire epidermis, especially in the stratum corneum (SC), andin the dermis2), 4). Considering the mechanism, lipid peroxidation is an initial reaction of CP formation. Sebum is found at
the skin surface and cell membranes of keratinocytes consistof several lipids. These facts indicate that the carbonyl modiﬁcation of SC proteins is recognized as an integrated result ofoxidative stress in living cell layers and on the skin surface.
On the other hand, a few recent studies have indicated that
CPs in the SC inﬂuence the physical and physiological properties of the skin, such as lowering its optical transparency, altering the skin colour to yellowish dark and decreasing its water holding capacity4)–6). In light of these facts, it is understoodthat CPs in the SC (SCCPs) have a signiﬁcant impact affecting substances important to skin conditions and not only asend products of oxidative stress. Therefore, an appropriate
and objective measurement method to accurately determine
the level of SCCPs is required to investigate oxidative stressin the skin. Few reports have been published describing techniques to measure CPs by chemical or immunological reac* Corresponding author

School of Bioscience and Biotechnology, Tokyo University of Technology（1404–1 Katakura-machi, Hachioji, Tokyo 192–0982, Japan）
E-mail: g12110085b@edu.teu.ac.jp

tions based on the labelling of aldehyde groups in CPs2), 4), 7), 8).
Among them, the analysis of ﬂuorescence microscopic images
produced by the reaction of ﬂuorescein-5-thiosemicarbazide(FTSC) with aldehyde groups in SCCPs is a simple and useful
method, since it is a one-step reaction with aldehyde groups4).
The ﬂuorescence intensity of microscopic images of tapestripped SC visualized by FTSC labelling has been employed
as a measure of SCCP levels4)–6). However, almost all of theSC obtained by the tape-stripping method contains multiplex
areas, for instance, multilayer desquamation and peripheral
overlapping of neighbouring SCs. The multiplex areas of theSC emit a strong intensity of ﬂuorescence when labelled withFTSC. It is considered that the multiplex SC area shows different ﬂuorescence characteristics from the monolayer SC due
to light scattering and emitting ﬂuorescence. The average ﬂuorescence intensity, including the strong ﬂuorescence, results
in a possible interference with the comprehensive evaluationof SCCP levels. Also, the average ﬂuorescence value calculated without distinction of ﬂuorescence characteristics is questionable from the accuracy of the data. To measure SCCP levels comprehensively requires that the ﬂuorescence intensity
originating from corneocytes is extracted by eliminating theﬂuorescence from the multiplex areas using a technique ofimage analysis. The purpose of this study was to establish a