Hyaluronic acid (HA) synthesized by normal human epidermal keratinocytes cultured in a serum-free medium was monitored by a highly sensitive HPLC method, which was established by us for the simultaneous determination of HA, chondroitin sulfate (ChS) and dermatan sulfate (DS) as their unsaturated disaccharides. The major glycosaminoglycan (GAG) in the medium of the keratinocytes was HA, and the ability of the cells to synthesize HA increased relatively with an increase in cell numbers during the logarithmic phase and reached a maximum level after the cells became confluent. HA synthesis by the keratinocytes was inhibited by the addition of calcium chloride to the culture medium, and was strongly stimulated by the addition of retinoic acid (RA), respectively. It was shown that the ability of the cells to synthesize HA exists in the spinous cell stage. Furthermore, we found that HA synthesis by the cells was slightly increased by the addition of dibutyryl cyclic AMP (dbcAMP). Our results indicate that the measurement of time-course levels of HA in the culture medium is useful for the screening of active substances for proliferation and differentiation of the keratinocytes.

A gas chromatography-mass spectrometry procedure for the determination of subnanomolar amounts of uronic acids derived from glycosaminoglycans (GAGs) is described. It involves stoichiometric reduction of carboxyl groups by sodium borodeuteride coupled with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and methanolysis in methanolic hydrogen chloride (1.0 M) at 80°C for 24 h followed by trimethylsilylation. The uronic acid compositions of commercially available GAGs, such as dermatan sulfate from pig skin, heparan sulfate from bovine kidney, and heparin from bovine intestinal mucosa, were investigated by the present method. The results were confirmed by the quantitative 1H-NMR spectroscopy . The method is also applicable to complex samples such as human urinary GAGs.

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Dermatan sulfate-proteoglycans (DS-PGs) were extracted from rabbit, rat and bovine defatted livers by magnesium chloride extraction and DEAE-cellulose chromatography, and then submitted successively to Asahipak GS-520 gel filtration chromatography, Asahipak ES-502N anion exchange chromatography, and cellulose acetate membrane electrophoresis. The disaccharide composition of the glycosaminoglycan chains was determined by differential digestion by chondroitinase ABC, AC, ACII and/or B followed by HPLC for analysis of the resulting unsaturated disaccharides. The hepatic dermatan sulfate chains contained disulfated disaccharide units; Di-diSB and Di-diSE. The hepatic DS-PGs were divided into two groups; Di-diSE-poor DS-PGs and Di-diSE-rich DS-PGs. The iduronic acid content of Di-diSE-poor dermatan sulfate chains was higher than that of Di-diSE-rich ones.

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