Free Radical Scavenging of Lutein in Vitro

PDFlib PLOP: PDF Linearization, Optimization, Protection Page inserted by evaluation version www.pdflib.com – sales@pdflib.com zy zy zyxwv zy Free Radical Scavenging of Lutein in Vitroa M. CHOPRA,bp' R. L. WILLSON,' AND D. I. THURNHAMd bDunn Nutritional Laboratory Medical Research Council Downhams Lane Milton Road Cambridge CB4 IXJ, United Kingdom CDepartmentof Biochemistry Brunel University Uxbridge, Middlesex UB8 3PH, United Kingdom dHuman Nutrition Group Department of Biology and Biomedical Sciences University of Ulster at Coleraine Coleraine BTS2 I SA, Northern Ireland zyxw INTRODUCTION Lutein is one of the most common xanthophyll carotenoids in our diet and is as abundant as p-carotene in most green and yellow vegetables.' In the British population, lutein forms 20-40% of the total carotenoid components in the plasma.2 Its structure is very similar to that of a-carotene except that it has two hydroxyl groups in the terminal ionone rings. The presence of these functional groups should make it slightly more hydrophilic and may influence its antioxidant properties. In the present study we have looked at the oxy, sulfur (RS.)and peroxy (ROO.) radical scavenging activity of lutein in vitro. METHODS zyxw zyx zyx Oxy and Sulfur Radical Scavenging Activity of Lutein A gamma radiolysis study was undertaken in which radicals were generated using a cobalt 60 type source at 1 K rad/min. The solvent was 75 : 25 methanol : water. Under our experimental conditions, mainly methanol radicals and a small amount of hydroxyl and superoxide ion will be produced. These would lead to the generation of RS. in the presence of sulfur compounds. Lutein was exposed to gamma radiolysis for one hour in the presence and absence of glutathione (GSH). Aliquots were removed every 15 min and the decrease in absorbance at 445 nm was measured spectrophotometrically. This study was funded by Nestec Switzerland. Address correspondence to Dr. M. Chopra, Human Nutrition Group, Department of Biological and Biomedical Sciences, University of Ulster at Coleraine, Coleraine BT52 ISA, Northern Ireland. a 246 zyxwvutsr zy CHOPRA eta/.:LUTEIN 247 zyxwvut zyxw zyxw Effect of Lutein on Azo-Initiated Peroxidation of Linoleic Acid Azo-initiated peroxidation of methyl linoleic acid (LA) was followed by measuring oxygen consumption in the presence and absence of antioxidants. Final concentrations in the electrode cell were 133 mmol/l LA, 13.3 mmol/l AMVN and 10 and 25 pmol/l lutein. For comparison the effect of p-carotene and Trolox on azoinitiated oxidation of LA was also investigated. zyx 2.0 Luteinonly 0 Lutein + 10 uM GSH Lutein + 20uM GSH Q Lutein + 25uM GSH 1.5 E E zyxwv 3 1.0 Y cp 8 4 0.5 0.0 T 0 15 30 45 60 Time (minutes) FIGURE 1. Gamma radiolysis of lutein in methanol : water (75 : 25). Final concentration of lutein was 10 pmol/l, and that of GSH was 10, 20 and 25 wmol/l. RESULTS AND DISCUSSION Results of this study show that lutein can scavenge toxic oxygen species in uifro. Under our experimental conditions it reacts faster with sulfur radicals than oxy radicals (FIG.1). It inhibited the azo-initiated peroxidation of LA at concentra- z zyxwvu zyx z L 2.0 -A- -+- LA + AMVN + 1OuM Car LA+AMVN+lOuMLut 1.5 - + LA + AMVN + 50uM Trolox -A- AMVNonly - f * LA+AMVN LA + A W N + lOuM Lut --*-LA + AMVN + 25uM Lut FIGURE 2. The effect of lutein on AMVN-initiated peroxidation of LA followed by measuring (a) oxygen consumption and (b) diene conjugate formation. Final concentration of LA was 133 mmol/l, and that of AMVN was 13.3 mmolll. Incubation temperature was 50°C. s m 2 R 3 E zyxwvutsr zyxwvut zy CHOPRA et al.: LUTEIN 249 zyx zyx tions as low as 10 pmol/l, when oxidation was followed by measuring oxygen consumption (FIG.2a) and diene conjugate formation at 234 nm (FIG. 2b). p-Carotene (10 pmol/l final) showed a small inhibitory effect on oxygen consumption. Trolox (vitamin E analogue) showed no effect at 10 pmol/l and significant inhibition at 50 pmol/l. Stoichiometry of peroxy radical scavenging of a-tocopherol and its hydrophilic homolog Trolox has been reported to be 2 : 1.3.4 From this the rate of peroxy radical generation by AMVN can be calculated to be approximately 25 x mol/minute in our assay system. Since we were measuring oxygen consumption every minute and Trolox reacts very fast with peroxy radicals this can explain why Trolox showed no significant effect on oxygen consumption at the concentration of 10 pmol/l. Lutein inhibited the oxygen consumption at a constant rate in our assay system. This suggests that lutein scavenges radicals more slowly than Trolox but can inhibit peroxidation for a longer time and at lower concentrations. ACKNOWLEDGMENT Lutein was a gift from Linexa, Quito, Ecuador. zyxwvut REFERENCES HEINONEN, M. I., V. OLLILAINEN, E. K. LINKOLA, P. T. VARO& P. E. KOIVISTOINEN. 1989. Carotenoids in Finnish foods: vegetables, fruits, and berries. J. Agric. Food Chem. 37: 655-659. 2. THURNHAM, D. I. & P. S . FLORA.1988. Do higher vitamin A requirements in men explain the difference between the sexes in plasma provitamin A carotenoids and retinol? Roc. Nutr. SOC.47: 181A. 3. N I K I ,E., T. SAITO,A. KAWAKAMI & Y. KAMIYA.1984. Inhibition of oxidation and methyl linoleate in solution by vitamin E and vitamin C. J. Biol. Chern. 259: 4177-4182. 4. TSUCHIYA, M., G. SCITA,H.-J. FREISLEBEN, V. E. KAGAN& L. PACKER.1992. Antioxidant radical-scavenger activity of carotenoids and retinoids compared to a-tocopherol. Methods Enzymol. 213: 460-472. 1.