Effects of Riboflavin and Fatty Acid Methyl Esters on Cholesterol Oxidation during Illumination PO CHEN HU AND BING HUEI CHEN* Department of Nutrition and Food Science, Fu Jen University, Taipei, Taiwan 242 The effect of riboflavin or fatty acid methyl esters on cholesterol photooxidation was studied. Samples containing cholesterol, either alone or in combination with riboflavin or fatty acid methyl esters, were illuminated at 25 °C in an incubator for 28 days. The various cholesterol oxidation products (COPs) and cholesterol were analyzed by gas chromatography-mass spectrometry (GC-MS), and riboflavin was determined by HPLC. Results showed that the presence of riboflavin or fatty acid methyl esters facilitated production of COPs and degradation of cholesterol, and the degradation fits a first-order model. The COPs formed during light storage included 7R-OH, 7-OH, 7-keto, 3,5-cholestadien-7- one, 5,6R-EP, and 5,6-EP. The addition of riboflavin caused formation of 3,5-cholestadien-7-one through dehydration of 7-keto, whereas in the presence of docosahexaenoic acid methyl ester, the formation of 5,6R-EP or 5,6-EP was favored. Riboflavin was more effective for generation of COPs than fatty acid methyl esters. KEYWORDS: Riboflavin; fatty acid methyl ester; cholesterol photooxidation; GC-MS INTRODUCTION Cholesterol, an important class of neutral lipids, is widely present in food products of animal origin. As an essential nutrient for maintaining normal physiological function, choles- terol often exists in free form or in ester linkage with long chain fatty acids in foods (1). However, because of the presence of one unsaturated double bond, cholesterol may undergo oxidation to form cholesterol oxidation products (COPs) when exposed to light or heat (2, 3). It has been well established that the consumption of COPs in excess may induce mutagenicity and carcinogenicity (4, 5). Thus, the variety and amount of COPs formed in model systems or food products during illumination or heating has to be explored. The formation mechanism of COPs during heating has been well documented and illustrated (3, 6, 7). However, the cholesterol photooxidation was not often studied. In the presence of light the triplet oxygen could be converted to singlet oxygen and in turn react with cholesterol to form 5,6R-EP or 5,6-EP (8). However, in the presence of some other components such as fatty acids, some more varieties of COPs were formed during light storage (2). For instance, several reports have shown that during illumination of egg powder or cheese powder, some COPs such as 5,6-EP, 7-keto, triol, 7R-OH, and 7-OH were formed (2). The presence of unsaturated fatty acids or photo- sensitizers such as chlorophyll have been reported to accelerate the cholesterol oxidation during heating or illumination (9- 11). In a review dealing with photosensitized oxidation of lipids, Girotti (8) reported that both 7R-OOH and 7-OOH were formed as reactive intermediates in the presence of triplet oxygen, whereas in the presence of singlet oxygen both 5R- OOH and 5-OOH were formed. As chlorophyll is not com- monly found in meat and egg products, the effect of some other photosensitizers, such as riboflavin, on cholesterol photooxi- dation has to be investigated. Also, the effect of riboflavin on the degradation of cholesterol during illumination remains unknown. Furthermore, it will be difficult to assess the formation mechanism of COPs during light storage in food systems because of possible interference by the presence of other components such as protein, carbohydrate, and fat. Using a model system to study the phenomenon of cholesterol photo- oxidation would make it easier to interpret results. Fatty acid methyl esters were selected instead of triglycerides because the former should be more susceptible to promoting cholesterol oxidation than the latter (9). The objectives of this study were to develop a model system for illumination and to determine the effect of riboflavin or fatty acid methyl esters on cholesterol photooxidation. MATERIALS AND METHODS Materials. Cholesterol and cholesterol oxidation products (COPs) standards, including cholestan-5R,6R-epoxy-3-ol (5,6R-EP), cholestan- 5,6-epoxy-3-ol (5,6-EP), 5-cholesten-3-ol-6-one (6-keto), 5-cho- lesten-3-ol-7-one (7-keto), cholestane-3,5R,6-triol (triol), 5-cholesten- 3,20R-diol (20-OH), 5-cholestane-3,25-diol (25-OH), and internal standard 5R-cholestane were purchased from Sigma Chemical Co. (St. Louis, MO). 5-Cholesten-3,7R-diol (7R-OH), 5-cholesten-3,19-diol (19-OH), 5-cholesten-3,7-diol (7-OH), and 3,5-cholestadien-7-one were obtained from Steraloids Co. (Wilton, NH). Fatty acid methyl ester standards, including stearic acid methyl ester, linoleic acid methyl ester, and cis-4,7,10,13,16,19-docosahexaenoic acid methyl ester were * To whom correspondence should be addressed. Phone: 886-2- 29031111-3626. Fax: 886-2-29021215. E-mail: nutr1007@mails.fju.edu.tw. 3572 J. Agric. Food Chem. 2002, 50, 3572-3578 10.1021/jf020003q CCC: $22.00 © 2002 American Chemical Society Published on Web 05/10/2002