Palmitoyl Ascorbate: Selective Augmentation of Procollagen mRNA Expression Compared With L -Ascorbate in Human Intestinal Smooth Muscle Cells Gennady Rosenblat, 1 Amy Willey, 2 Ya-N an Zhu, 2 Adi Jonas, 1 Robert F. Diegelmann, 3 Ishak N eeman, 1 and Martin F. Graham 2 * 1 Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa 32000, Israel 2 Department of Pediatrics, Laboratory of Tissue Repair, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia 23298–0529 3 Department of Surgery, Laboratory of Tissue Repair, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia 23298–0529 Abstract The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion wasinvestigated and compared with the effect of L-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3 H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesisat 2.5 and 5 μM. By contrast, L-ascorbate wasrequired at 4–5 timesthe concentration for the same response. However, at 20 μM, both palmitoyl and L-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levelsof procollagen I and III mRNA levels respectively, whereas L-ascorbate had no effect. Palmitoyl ascorbate and L-ascorbate induced similar increasesin the amountsof newly synthesized procollagen secreted into the medium and in the amountsof collagen typesI, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or L-ascorbate on the activity of a procollagen  2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than L-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studiesof collagen synthesisin vitro. J. Cell. Biochem. 73:312–320, 1999.  1999 Wiley-Liss, Inc. Key words: collagen; HISM cell; ascorbic acid; tissue repair; intestine; gene expression; collagen secretion; collagen synthesis Ascorbic acid is a critical component for the biological expression, synthesis, and secretion of collagen. This essential vitamin acts as a cofactor in the hydroxylation of prolyl and lysyl residues in procollagen molecules by the en- zyme prolylhydroxylase (lysylhydroxylase) [Jimenez et al., 1973; Berg and Prockop, 1973]. Ascorbic acid also increases steady-state levels of procollagen mRNA in some fibroblast lines [Geesin et al., 1988; Tajima and Pinnell, 1996], but not in human intestinal smooth muscle (HISM) cells [Graham et al., 1995b]. A major problem in cell culture studies, and therapeutic applications, of ascorbic acid is the extreme instability of the compound resulting from its oxidation [Peterkofsky, 1972; Geesin et al., 1993; Austria et al., 1997]. In order to over- come this problem, attempts have been made to use various esters of ascorbic acid such as the fatty acid ester palmitoyl ascorbate and the phosphated, inorganic water-soluble salt of ascorbic acid, L-ascorbate phosphate. Intestinal smooth muscle cells play a major role in the maintenance and repair of the intes- tinal wall. Defective extracellur matrix and in- adequate repair in the intestine lead to sponta- neous perforation, as seen in Ehlers Danlos type IV [Prockop and Kivirikko, 1984], and to morbid pathology such as fistula formation in Crohn’s disease [Graham et al., 1992]. Studies Grant sponsor: National Institutes of Health; Grant num- ber: DK34151; Grant number: GM20298. *Correspondence to: Martin F. Graham, Laboratory of Tis- sue Repair, Box 529, MCV Station, Richmond, VA 23298. E-mail: mgraham@hsc.vcu.edu Received 31 July 1998; Accepted 17 November 1998 Journal of Cellular Biochemistry 73:312–320 (1999)  1999 Wiley-Liss, Inc.