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Effectiveness of Isoascorbate versus Ascorbate as an Inducer of Collagen Synthesis in Primary Avian Tendon Cells¹

Department of Dietetics and Nutrition, University of Kansas Medical Center, Kansas City, KS 66103 ^* Division of Cell and Molecular Biology, Lawrence Berkeley Laboratory, Berkeley, CA 94720

In contrast to most biologically active molecules, the isomeric form of ascorbate retains significant biological activity. Moreover, in studies in vitro the isomer was found to be an equally effective cofactor in the enzymatic proline hydroxylation reaction. This raises questions about whether the lower biological activity in vivo results from selective transport into the cell, greater instability of the molecule, or stereospecificity by certain enzyme complexes. Distinguishing these possibilities can be accomplished most directly using a cell culture model. In this study primary avian tendon (PAT) cells were used. With PAT cells isoascorbate was shown to be three- to fivefold less active at inducing procollagen production than ascorbate. Isoascorbate was also internalized by the cell at about one-fifth the ascorbate level. In addition, isoascorbate was degraded in the medium at a slightly higher rate (half-life of 1.6 h) than ascorbate (2.1 h). The data are consistent with a model that postulates that once inside the cell isoascorbate is equally effective at inducing procollagen production but selectivity at the transport step restricts the percentage that is actually internalized. In addition, both ascorbate and isoascorbate were found to degrade very quickly inside the cell in the highly oxygenated environment of cell culture (2 h half-life). When ascorbate was added to the medium (100 µg/mL) the level inside the cell quickly reached a maximum (< 2 h) and declined rapidly. The level of ascorbate inside the cell was undetectable at 24 h (< 0.2 ng/10⁶ cells) even though these cells have previously been shown to continue to synthesize high levels of procollagen on a single dose of ascorbate for 36 h. Therefore, at the cellular level, the amount of ascorbate required for maximal procollagen production is much lower than would be predicted by extrapolation from steady state tissue levels in vivo.

KEY WORDS: • cell culture • collagen synthesis • ascorbate • isoascorbate • primary avian tendon cells

¹ Supported in part by a grant from the National Institute of General Medical Sciences (R01 GM 34274) to D. E. K. and in part by a grant to R. I. S. from the Office of Health and Environmental Research, Office of Energy, U.S. Department of Energy, under contract no. DE-ACO3-76SF00098.

Manuscript received 15 February 1989. Revision accepted 8 September 1989.