The Hydroxyl Free Radical Reactions of Ascorbyl Palmitate as Measured in Various in Vitro Models N. Perricone,* K. Nagy,† F. Horva ´ th,† G. Dajko ´,‡ I. Uray,‡ and I. Zs.-Nagy† ,1 *Department of Dermatology, Yale School of Medicine, New Haven, Connecticut; †Department of Gerontology (VILEG, Hungarian Section), University Medical School, POB 50, H-4012 Debrecen, Hungary; and ‡Institute of Nuclear Research of the Hungarian Academy of Sciences, H-4001 Debrecen, Hungary Received August 4, 1999 The OH • free radical scavenging properties of ascor- byl palmitate (AP), water-solubilized in the presence of a surfactant (Brij 35), were tested in various sys- tems: (1) The inhibition of polymerization of bovine serum albumin by OH • free radicals generated by the Fenton reaction indicated AP exerts a considerable protective effect against polymerization by scaveng- ing the OH • free radicals. (2) ESR spin trapping com- parisons of DMPO with AP were conducted. Using the Fenton reaction as a source of OH • free radicals, AP was 1 order of magnitude faster in scavenging these radicals than DMPO. (3) Oxidative modification of BSA by 60 Co-gamma irradiation of 80 krad, results in a strong increase in protein carbonyl content. AP inhib- its carbonyl formation very efficiently, indicating that AP may be utilized as a biological OH • free radical scavenger in human therapy. © 1999 Academic Press L-Ascorbyl-6-palmitate (AP) is a fat-soluble syn- thetic ester of ascorbic acid. It has been used exten- sively as an antioxidant in foods, pharmaceuticals and cosmetics, particularly as a preservative for various edible oils and waxes (1–5). Its FDA status is “gener- ally recognized as safe” (GRAS) with no limitations on levels used in food or cosmetics. In the past ten years, AP has been in the focus of various areas of research such as: 1. The anti-inflammatory and photoprotective ef- fects (6, 7). 2. General toxicology (8 –11). 3. Carcinogenesis and mutagenicity in various mod- els (12–17). 4. The controlled insulin release from chitosan mi- croparticles (18, 19). 5. Protection of free fatty acids against oxidation in low density lipoproteins (20). 6. Protection against cytotoxicity of nitrogen oxides in combination with beta-carotene (21). 7. Protection of erythrocytes from oxidative stress (22). These studies appear promising and suggest wider pharmacological applications of AP. A greater under- standing of the free radical scavenging activity of this compound, particularly of the OH • free radical, will lend deeper insight into the mode of action of this agent. The experimental results obtained in these studies are presented. MATERIALS AND METHODS AP was purchased from Roche Vitamins and Fine Chemicals Di- vision of Hoffmann-La Roche, Inc. (340 Kingsland Street, Nutley, NJ 07110, USA), manufactured on 16 December, 1997. Purity was as- sayed at 99.2% by Hoffmann-La Roche, Inc. In order to conduct these experiments, it was necessary to water solubilize AP by using very low concentrations of the surfactant (Brij 35) at a concentration of 0.025% weight per volume. Mild ultrasoni- cation of the mixture led to a stable, homogeneous stock solution of a 50 mM concentration of AP. The OH • free radical scavenging ability of this substance was tested by various methods as we de- scribe below. Inhibition of protein cross linking by AP. Bovine serum albumin (BSA), a completely water-soluble protein, is polymerized by the OH • free radical, which results in loss of solubility. The OH • free radicals are generated by the Fenton reaction (23), and if an OH • free radical scavenger is added to the system, a reduction of polymerization results in a quantifiable decrease of water insoluble protein fraction (24, 25). The final volume of the reaction mixture was of 0.5 ml, and it was composed as follows (the sequence of additions of various compo- nents should be strictly respected): (a) 0.1 ml aqueous solution of BSA (0.4%). (b) Water to reach 0.5 ml total volume (see items d and e). (c) 0.21 ml of NaCl (0.1 M)-NaHCO 3 (0.025 M) buffer, pH 7.1. (d) 0.1 ml AP solution of various dilutions, resulting in final con- centrations of 1, 2.5 and 5 mM (in controls only water is added). In all 1 Address of corresponding author. Fax: (36-52) 418-470. E-mail: izsnagy@jaguar.dote.hu. Biochemical and Biophysical Research Communications 262, 661– 665 (1999) Article ID bbrc.1999.1277, available online at http://www.idealibrary.com on 661 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.