Analysis of GAG composition chnage in intrinsically aged and photoaged human skin in vivo

Abstract

Glycosaminoglycans (GAGs) are highly charged polysaccharides that are expressed in extracellular matrix and cell surface throughout the body, and participate in a variety of biological processes and functions. The objective of this study is to develop a novel easier method for determination of specific types of GAG and apply this to the investigation for the level changes of chondroitin sulfate (CS), dermatan sulfate (DS) in the dermis of intrinsically aged buttock and photoaged forearm skin. Skin samples of sun-protected buttock and sun-exposed forearm were obtained from healthy young male (21-30 years, 26.1±3.4 years, n=8) and female (20-33 years, 26.3±5.4 years, n=8), and elderly male (70-78 years, 74.4±2.6 years, n=8) and female (70-80 years, 74.5±3.6 years, n=8) volunteers. After separation of epidermis from dermis, tissue GAG samples were prepared by overnight proteinase K treatment. Thereafter, total sulfated GAG (tsGAG) contents in dermal tissue GAG samples were measured by sulfated GAG assay kit using 1,9-dimethymethylene blue, with or without enzymatic degradation by chondroitinase ABC (ChABC) or AC (ChAC) for 24 hours. Substrate specificity of ChABC and ChAC was confirmed with each type of sulfated GAG, and their complete degradation in our experimental condition was also confirmed by no further degradation between 12 hour- and 48 hour-treatment. By this method, CS (chondroitin-4 sulfate (C4S) + chondroitin-6 sulfate (C6S)), DS, and the sum of keratan sulfate (KS), heparin (HP), and heparan sulfate (HS) in tissue GAG samples could be calculated. Dermal tsGAG contents in both genders were decreased in old buttock, and those in old forearm were higher than those in old buttock. After analysis of specific GAG types using our method, dermal CS (C4S+C6S) and sum of other GAG (KS+HP+HS) contents were decreased in old buttock, and those in old forearm were higher than those in old buttock, while dermal DS contents were decreased both in buttock and forearm in old subjects. Especially, tsGAG, CS, and the sum of other GAG contents in young male forearm were higher than those in young female forearm, but not in buttock, while DS contents in male were slightly higher in both buttock and forearm than in female. Forearm/buttock ratios of tsGAG, CS, and the sum of other GAG contents were increased with aging but that of DS was not increased in aged dermis. In conclusion, using a novel easier GAG determining method, decrease of dermal CS, and the sum of other GAG contents in intrinsically aged skin and their increased levels in photoaged skin, compared to intrinsically aged skin, were observed, while DS showed a slight decrease during intrinsic aging and no increase during photoaging.