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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.

Analytical Studies on Hyaluronic Acid Synthesis by Normal Human Epidermal Keratinocytes Cultured in a Serum-Free Medium.