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I. a - 1.x- 9.5 Mar/‘1 616 CH TATE UNIVERSITY UBRARIE llllllllll‘.|lillfi‘.11|1|ill“||‘.l'.|‘.i‘.‘:||lllll ' 3 1293 00629 5038 ‘ LIBRARY Michigan State University This is to certify that the thesis entitled CHOLESTEROL OXIDATION IN FOOD PRODUCTS OF ANIMAL ORIGIN presented by Karen Ruth Allen has been accepted towards fulfillment of the requirements for Master's degreein Food Science / j 61% .// Major prof or Date 7-21-89 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution _—___ . _ if: __,____.__——_.__ - - —-'- r-‘F— __ A _ * *fir—AW PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE C _, \J‘L § ‘.- ." MEN .3. :22 we I‘ t _ 1"?” u {’th ‘ ' ' MSU Is An Affirmative Actlon/Equal Opportunity Institution CHOLESTEROL OXIDATION IN FOOD PRODUCTS OF ANIMAL ORIGIN 3}! Karen Ruth Allen A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Deparment of Food Science and Human Nutrition 1989 ABSTRACT CHOLESTEROL OXIDATION IN FOOD PRODUCTS OF ANIMAL ORIGIN By Karen Ruth Allen The presence of cholesterol oxidation products (COP's) was investigated in illuminated butter, heated tallow, fat extracted from commercial french fries, powdered dairy products and spray-dried eggs. Cholesterol oxidation was not evident in the butter although the peroxide value increased slowly with time over 16 days of storage. Addition of fl-carotene to the butter resulted in lower peroxide values, while a-tocopherol and TBHQ had no effect. 7-Ketocholesterol, 7B-hydroxycholesterol and cholesterol-c-epoxide were formed in heated tallow after 24 hours, with 7-ketocholesterol dominating. The addition of 0.05% oleoresin rosemary decreased the extent of cholesterol oxidation. Small amounts of cholesterol oxidation products were detected in commercially produced french fries, but the level did not correlate well with the amount of cholesterol present. Powdered whole milk, powdered butter and powdered sour cream all contained 7-ketocholesterol. After incubation for two weeks at 38'C, eggs that had been spray-dried with air heated directly by a natural gas flame contained cholesterol- a-epoxide, while eggs spray-dried with indirectly heated air did not contain any COP's. I wish to thank Dr. J. Ian Gray for all of his help and patience as my research chairperson and major professor. The experience I gained is invaluable. The members of my guidance committee, Drs. B.R. Harte, John Partridge and Matthew Zabik earn my respect for their critical review of this thesis. Drs. Harte and Partridge deserve appreciation for obtaining packaging materials and the butter. The completion of this degree could not have possible without the help of Simin Biglari Sharifzadeh, Elizabeth Torres, Rhonda Crackel, Terri Drumm and all of my friends in the Food Science department at Michigan State University. My famdly stood by me through it all, helping me emotionally and financially. Finally I am thankful to Brian Lust for all his love and understanding. ii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES O O O O O O O O O O O O O O O O O O O v11 INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . . . . . . . 4 Health Implications of Cholesterol Oxidation Products . . . . . . . . . . . . . . . . . . 5° Oxidation of Cholesterol . . . . . 8 General Mechanism.and Resulting Oxidation Products . . . . . . . . . 8 Initiation of Cholesterol Oxidation . . . . . 13 Singlet Oxygen . . . . . . . . . . . . . . . . 13 Photosensitizers . . . . . . . . . . . . . . 14 Transition Metals . . . . . . . . . . . . . 17 Enzymes . . . . . . . . . . . . . . . . . . . 17 Heat . . . . . . . . . . . . . . . . . . . . 18 Processing of Foods . . . . . . . . . . . . . 19 Methods For Detection of Cholesterol Oxidation Products in Food . . . . . . . . . . . . . . . 20 Thin Layer Chromatography . . . . . . . . . . 20 High Performance Liquid Chromatography . . . . 22 Gas Liquid Chromatography . . . . . . . . . . 24 Mass Spectrometry . . . . . . . . . . . . . 27 Cholesterol Oxidation Products in Food . . . . . 28 Dairy Products . . . . . . . . . . . . . . . . 28 Tallow and Meat Products . . . . . . . . . . 30 Eggs and Egg Products . . . . . . . . . . . . 34 Deep-Fat Frying . . . . . . . . . . . 40 Stabilization of Frying Oils . . . . . . . . . 42 mm Rms AND “mobs O O O O O O O O O O .5 0’. Materials . . . . . . . . . . . . . . . . . . . 46 Methads O O O O O 0 O O O 0 O I O O O O O O O O 4 8 Oxidation of Cholesterol in Heated Tallow . . 48 Effects of Heating and Antioxidant Addition on Cholesterol Oxidation in Tallow . . . 48 Cholesterol Oxidation Produced in Commercial French Fries . . . . . . . . . . S3 Oxidation of Cholesterol in Dairy Products . . 54 Effect of Fluorescent Light on Cholesterol Oxidation in Butter . . . . . . . 54 Survey of Powdered Dairy Products for Cholesterol Oxidation Products . . . . . . . 57 iii Oxidation of Cholesterol in Egg Powder . . . . 57 Oxides of Nitrogen Content . . . . . . . . . 58 Sample Treatment . . . . . . . . . . . . . 58 Sample Analysis . . . . . . . . . . 58 Statistics . . . . . . . . . . . . . . . . 58 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 60 Oxidation of Cholesterol in Heated Tallow . . . 60 The Effect of Oleoresin Rosemary Addition and Heating at 180'C on the Stability of Choles- terol in Tallow . . . . . . 60 The Effect of Oleoresin Rosemary Addition and Heating at 135' C on the Stability of Choles- terol in Tallow . . . . . . . . . . . . 61 Cholesterol Oxidation in Commercial French Fries . . . . . . . . . . . . . . . . . . . . 77 Oxidation of Cholesterol in Dairy Products . . . 82 The Effects of various Antioxidants and Packaging Materials on the Oxidation of Lipids and Cholesterol in Butter Stored Under Fluorescent Light . . . . . . . . . 82 Oxidation of Cholesterol in Powdered Dairy Products . . . . . . . . . . . . . . . . . . . 88 Oxidation of Cholesterol in Egg Powder . . . . . 89 Cholesterol Loss . . . . . . . . . . . . . 90 Cholesterol Oxidation Products . . . . . . . . 91 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 95 PROPOSALS FOR FURTHER RESEARCH . . . . . . . . . . . 100 ”PENDICES O O O O O O O O O O O O O O O O O O O O O 102 “FERBNCBS O O O O O O O O O O O O O O O O O O O O O 1 1 2 iv Table 10. 11. 12. 13. 14. LIST OF TABLES Cholesterol content in organs and tissues of adult rats 0 O O O O O O O O O O O O O O O O 0 Cholesterol oxidation products which have been isolated from foods of animal origin . Sterol contents of bleached butteroils and aged Italian cheese samples . . . . . . . . . Cholesterol loss in tallow from deep-fat fryers and the intensity of cholesterol oxidation products on TLC plates . . . . . . . Cholesterol oxidation products in three year- °1d freeze-dried Pork 0 e o s o s s s e s o 0 Cholesterol oxide content of selected meat samples . . . . . . . . . . . . . . . . . . . Effects of air heating methods on cholesterol oxide formation in dehydrated egg yolks . . . Quantification of cholesterol oxidation products in scrambled egg mix . . . . . . . . The effect of Oleoresin rosemary on cholester- ol loss in continuously heated tallow . . . . The effect of Oleoresin rosemary on cholester- ol loss in intermittently heated tallow . . . The maximum concentrations of 7-ketocholester- ol, 7B-hydroxycholesterol and cholesterol-o- epoxide in heated tallow samples containing Oleoresin rosemary . . . . . . . . . . . . . . Cholesterol oxidation products in fat extract- ed from commercially produced french fries collected over a period of one week . . . . . 7-Ketocholesterol content in powdered dairy preduCt 8 O O O O O O O O O O O O O O O O O O 0 Cholesterol loss in spray-dried eggs after incubation for two weeks at 38’C . . . . . . . Page 10 29 31 33 35 37 39 69 69 74 82 89 91 Figure 2. 3. 4. 10. 11. 12. 13. 14. LIST OF FIGURES Structures of the cholesterol oxidation products commonly found in food . . . . . Major pathway for cholesterol oxidation . . Ionization of cholesterol . . . . . . . . . Two types of reactions involving triplet sensitizers O O O O O O O O O O O O O O O 0 Reactions between oxides of nitrogen and free radicals . . . . . . . . . . . . . . . Effect of oleoresin rosemary on cholesterol loss in heated tallow . . . . . . . . . . . Effect of oleoresin rosemary on the forma- tion of cholesterol oxidation products (COP's) in tallow heated at 135’C . . . . . Effect of continuous heating at 135'C on cholesterol loss in tallow containing oleoresin rosemary . . . . . . . . . . . . . Effect of intermittent heating at 135'C on cholesterol loss in tallow containing oleoresin rosemary . . . . . . . . . . . . . Effects of continuous versus intenmittent heating at 135'C on cholesterol loss in tallow O I O O O O O O O O O O O O O O O O 0 Effect of continuous heating at 135°C on the formation of cholesterol oxidation products in tallow O O O O O O O O O O O O O O O O 0 Effect of intermittent heating at 135'C on the formation of cholesterol oxidation products (COP's) in tallow . . . . . . . . . Cholesterol content in fat extracted from commercial french fries collected over a one Mk ”r 10d 0 O O O O O O O O O O O O O O 0 Total cholesterol oxidation products (COP's) in fat extracted from commercial french fries over a one week period . . . . . . . vi Page 12 14 16 37 63 64 66 67 68 71 72 78 80 15. 16. 17. Effect of illumination on the peroxide value (meg/kg) of butter packaged in various materials . . . . . . . . . . . . . . . . . 84 Effect of illumination on the peroxide value (meg/kg) of butter containing antioxidants . 86 Structure of B-carotene . . . . . . . . . . 87 vii INTRODUCTION Cholesterol is a major sterol present in food products of animal origin and is known to undergo oxidation under various conditions. Cholesterol oxidation products (COP's) are important because of their possible involvement in atherosclerosis, cancer and other deleterious conditions (Smith and van Lier, 1970; Taylor et al., 1979; and Peng and Taylor, 1984). Cholesterol oxidation usually begins with the formation of allylic hydroperoxides (Maerker, 1986) and may occur at three different locations in the cholesterol molecule: 1) on the number 3 carbon; 2) in the 8 ring at carbons 5 and 6 which are joined by a double bond and also at carbon 7, due to allylic rearrangement; and 3) in the side chain where hydroxyl groups can be formed. The cholesterol oxides most often found in food are those arising from oxidation originating in the 8 ring. The oxidation of cholesterol may be initiated by light/radiation, heat, prolonged exposure to air, enzymes and other components present in food (Smith et al., 1981). Recently, there has been interest in the capability of oxides of nitrogen in initiating cholesterol oxidation in powdered foods (Tsai and Hudson, 1985). Oxides of nitrogen may be present in directly-heated air that is used in spray-drying, originating from the combustion of natural gas used for the flame. Use of antioxidants and/or processing modifications may provide the means for control of cholesterol oxidation in such food products. The major objective of this study was to determine the extent of cholesterol oxidation in various food products including heated tallow, butter, powdered dairy products and spray-dried eggs. various physical and chemical analyses and chromatgraphic techniques were employed to assess the oxidative changes. Specific objectives included evaluating the effectiveness of oleoresin rosemary as an antioxidant in heated tallow, and evaluating a-tocopherol, B-carotene, and TBHQ as antioxidants in butter. In heated tallow, various useage levels of oleoresin rosemary were investigated to find a level that would provide maximum.protection. The effects of continuous heating were compared to those of intermittent heating to determine which was more harmful. The antioxidants in butter were each evaluated at one level. various packaging materials were compared for their ability to prevent oxidation of illuminated butter stored at 4'C. The difference in cholesterol oxidation between eggs spray-dried with indirectly heated air and those spray-dried with directly heated air was evaluated before 3 and after incubation. The purpose was to determine if oxides of nitrogen in directly heated air may have had an effect on the extent of oxidation. REVIEW OF LITERATURE Cholesterol, C27 HuO, has a cyclopentanophenanthrene ring structure with an eight carbon aliphatic side chain at C17, two angular methyl groups at C1. and C1), and a double bond between carbons 5 and 6. This white crystalline compound was first discovered in the latter half of the eighteenth century during studies on the formation of gallstones in the body. In 1816, Chevreul gave it the name ”cholesterine', coming from Greek for chole, meaning bile and stereos, meaning solid. The name was changed to cholesterol when it was shown to have an alcohol structure (Berthelot, 1859). Cholesterol is an essential metabolite as it is the 4 5 basis for families of bile acids, hormones and vitamin D, and is a vital component in cell membrane structure (Brun et al., 1985). It affects permeability and osmotic pressure in the lipid bilayer of the cell membrane. Cholesterol-poor red blood cells show increased osmotic fragility (Gibbons et al., 1982). One to two g/kg of the body weight of adults are made up of cholesterol, 5.5% of which exists in plasma (Gibbons et al., 1982). Table I shows the distribution of cholesterol in the rat, which correlates well with the distribution in other mammals with a few'minor exceptions (Gibbons et al., 1982). Rest of the cholesterol in the body is unesterified, except in plasma and skin where a greater portion exists in the ester form, The average daily metabolic requirement for an adult weighing 70 kg is approximately 350mg (Sabine, 1977). Approximately 80mg of this are excreted daily through the skin (Parsons and Goss, 1978). HEALTH IMPLICATIONS OF CHOLESTEROL OXIDATION PRODUCTS Cholesterol has, for a long time, been implicated in the development of atherosclerosis in humans. It is now thought of by many as a contributor, but not the primary cause. A.new theory is that cholesterol oxidation products (COP's) begin the atherosclerotic process. Because it is now known that cholesterol is readily oxidized in the presence of air over a period of time, it has been suggested by some, including Peng and Taylor. (1984), that cholesterol used for dietary experiments by Table 1. Cholesterol content in organs and tissues of adult rats'. Percent of n 811 W Plasma 1.7 Small intestine 4.4 Liver 10.2 Kidneys . 2.7 Adrenels 0.2 Skin 16.6 Hair 11.2 Adipose 3.9 Skeletal muscle 18.5 Bone marrow 5.4 Red cells 2.4 Nervous tissue ' 22.7 (includes brain) 'Adapted from Gibbons et a1. (1982). Anitschkow in 1913 and others following could have contained COP's. Therefore, the atherosclerotic effects originally attributed to cholesterol may actually be due to COP's. It has been suggested that COP's could replace cholesterol in aortic cell membranes, thereby decreasing their functionality and.making them abnormally susceptible to the formation of atherosclerotic deposits (Peng and Taylor, 1984). There is a large body of literature on the deleterious effects of COP's in human health. They can be regulators of sterol synthesis, immune suppressors, carcinogens, cytotoxins and mutagens (Finocchiaro et al., 1984). Twelve COP's were isolated and identified in atheromata from human aortas (Smith and van Lier, 1970). The level of cholesterol-a-epoxide in human serum.may be related to the severity of atherosclerosis (Gray et al., 1971). After feeding USP-grade cholesterol containing spontaneous oxidation products to New Zealand White rabbits, Taylor et al. (1979) reported that adverse effects were observed within 24 hours. When a similar dose of purified cholesterol was administered to rabbits, no adverse effects were observed. It was reported by Peng et a1. (1984) that, when 25-hydroxycholesterol and 5-cholestan-38,5,6B-triol were added to cultured aortic smooth muscle cells of rabbits, the activity of 3-hydroxy-3methy1-glutaryl coenzyme.a reductase (HHG CoA reductase), a regulatory enzyme in cholesterol biosynthesis, was significantly depressed. Other health implications of COP's have been suggested. The mutagenicity of air-aged samples of USP or reagent-grade cholesterol toward three strains of 5.11231113,txnnxlunlul,was demonstrated by Smith et a1. (1979). Cholesterol-a-epoxide, which is carcinogenic in rodents, can cause aberations in chromosomes of human fibroblasts (Parsons and Goss, 1978). Hyperthermia enhanced the formation of aberations induced by cholesterol-a-epoxide and ultraviolet light when used separately and in combination. Cholesterol-a-epoxide has also been found in human skin that has been irradiated with ultraviolet light (Black and Lo., 1971). Both the u- and B— epimer have been reported in breast fluid and prostatic secretions (Petrakis et al., 1981), common sites of cancer in humans. Addie et a1. (1983), suggested that oxidation products derived from cholesterol appear to have the greatest potential for health impairment of all classes of compounds isolated from rancid foods. Kandutsch (1978) has mentioned that certain sterols formed metabolically from cholesterol can exhibit the same inhibitory effect on sterol synthesis as cholesterol. OXIDATION OF CHOLESTEROL 4,.-. .4; ,4 .4 .4 .. . o _. _._.4._,_ Food products containing fat are likely to undergo some lipid oxidation, whether it is induced by radiation, heat, enzymes or free radical initiators such as metals and oxides of nitrogen. Cholesterol oxidation usually begins with the formation of allylic hydroperoxides (Haerker and Unruh, 1986). Portions of the molecule that are especially susceptible to oxidation are the unsaturated 8 ring, positions allylic to it, and the two tertiary carbons at the side chain. The structures of some COP's commonly found in foods are shown in Figure 1. Table 2 lists the common COP's found in food products. Allylic hydroperoxides are formed by oxidative attack following hydrogen abstraction (Haerker and Unruh, 1986). .9 .. HO H dxnunhbdno4¥hflrdufl. d‘“'“*5*“"*“"*‘“? <1u¢wamnydunqnunua ‘"*“W*‘““"“'*"“ HO HO HO HO 3B¢xgwuwrnhn¢45£nrJHrl £hhdtflmflanrdafihqikrufl «dumodxnonuro1) d1fllib£¥IerBfi=¥dufl H0 (25+wd13wflnhuwl1n) Figure 1. Structures of the cholesterol oxidation products commonly found in food. 10 Table 2. Cholesterol oxidation products which have been isolated from foods of animal origin. Cholesterol mideticn prouct WWW}. 74mm mm.5.éfl-triol Choleetrol-fl-qaocide Balm-«wide e W fruit anith It al., 1%1. Food tellcm, way-died eggs. cry egg neg tnix, poured scrawled egg mix . gray-cried egg yolk, butt-'- eele ee 7chhoIeeter-ol tellow. grey-cried egg yolk. pad-Fed ea'elbled egg mix, nan-Fat cry milk, m milk ‘fet tallanl, grey-cried egg yolk, poured ea'erbled egg mix, tallcu, spray-cried egg, way-#106 099 yolk. scrawled egg mix, nun-fat cry milk, m milk fat rut-fat cry milk, m milk fat, tallm, ear-ey- cried egg, pend-Fed screamed egg mix. mm cried egg yolk tellau, pad-1d screlbled '99 mix 11 The hydrogen is abstracted primarily at C7 because it is an allylic position in the plane of the double bond of the 3 ring. C4 is also in the plane of the double bond, but attack does not usually happen there, possibly due to hinderance by the C. hydroxyl group and C; which is connected to three alkyl groups (Haerker and Unruh, 1986). The major oxidative pathway involves the formation of a 7-peroxycholesterol intermediate which then abstracts a hydrogen from another species, forming the a and B isomers of 7-hydroperoxycholesterol. This pathway is shown in Figure 2. These hydroperoxides-can be reduced to 7G- and 79-hydroxycholesterol, or they can undergo dehydration to yield 7-ketocholesterol. The latter compound can undergo dehydration to form.3,5-cholestadiene-7-one (Smith, 1981). Cholesterol-epoxides are formed from secondary oxidation reactions in which cholesterol is attacked by hydroperoxides (either hydrogen peroxide or previously formed cholesterol hydroperoxides) yielding cholestan-SB,6B-epoxy-39-ol (cholesterol-fl-epoxide) and cholestan-Sa , 6o: -epoxy-3B-ol (cholesterol-e-epoxide) (Smith and Kulig, 1975). Cholestan-3B,5¢,63-triol is formed by hydration of the a or B-epoxide. 12 .Aume .hnoq aoum owudmv...1-8-an £33828 - in . a 28-7 823332.... . r. n 38:38:73.8.35338.8 -1» .c o I I\I_U 8.... . a. .1» .3 2:38.86 .8 .8 . a: Jen-1.2135323... 83.3.2-223856 o... A .8... O: .8 O.- 7 7% {Immune s.-.-s-...-§.2....-. o... o... .82. .xx .82. .2: o... 8- an :83 so- 3 :«hnug .8. .en :63 on. no .5385 \Y V/\/%9.l Iago: Azoolllxlll. .2: z/%\: dilflqg .82. .xx 8- in- -2338qu a l3 Initiation 21’ W1 __i_noxidat 0 There are several ways that cholesterol oxidation in foods can be initiated but experimental evidence is lacking in this area (Smith, 1981). The types of radiation which are suspected of initiating autoxidation of cholesterol are infrared, visible, ultraviolet, and ionizing radiation (a and T) (Smith, 1981). Irradiatation of pure cholesterol can produce energized species by increasing the rotational, vibrational or electric bond energy levels. These excited molecules could undergo bond homolysis or ionization and form known cholesterol oxidation products after reaction with molecular oxygen. Ionization of cholesterol could also be involved in the initiation of autoxidation. Radiation could cause the excited cholesterol molecule to lose or gain an electron, forming a carbocation or carbanion which can react with oxygen as shown in Figure 3. The resultant hydroperoxide anion (CHOLESTEROL-00') is protonated to form.a more stable hydroperoxide. 51W Singlet oxygen oxidation of cholesterol is used for the detenmination of '8 distribution at the C7 position in biosynthesis studies (Smith, 1981). Photosensitized formation of singlet oxygen has been demonstrated in the gas phase but direct proof of its formation in solution is lacking. This is because spectroscopic techniques used in the gas phase cannot be used in solution. Indirect methods such as chemical trapping, kinetic and quenching 14 W? W—' lpfio, mam-oo- 1*“ comm-om Fflgunelh nruxethuicfitdeIIUIwn. studies indicate the intermediacy of singlet oxygen in photooxidative reactions occurring in solutions. EDQLQQQBELLLEQIQ A.photosensitizer is a molecule which absorbs light to produce a chemical reaction which would not occur in its absence. It may or may not be chemically changed in this process. Excited nitrogen dioxide can be a photosensitizer in the gas phase (Pryor and Lightsey, 1981). Riboflavin is also a photosensitizer in simple systems (Foote, 1976). Photosensitizers can exist in two electronically excited states, singlet and triplet. Usually the triplet state is much longer lived, but the singlet state is the initial product of light absorption. Photosensitized oxidations usually proceed by way of the triplet sensitizer. Schenck and Koch (1960) and Livingston (1961) 15 classified triplet sensitizer reactions as type I and II (Figure 4). Type I reactions occur when the photosensitizer interacts with another molecule directly, to give either a hydrogen atom or electron transfer. The resulting radicals react further with oxygen or other molecules. They can extract hydrogen or an electron from other substrates, initiate free radical chain autoxidation, or initiate a back reaction. Type I sensitizer reactions are much more frequent than type II. Type II reactions take place when the photosensitizer triplet interacts with oxygen. The most common reaction of this type involves the transfer of excitation from the sensitizer to the oxygen, producing an electronically excited singlet state of oxygen (‘02). This excited oxygen can then further react with various acceptors in solution. Deactivation collisions between oxygen and most sensitizer triplets result in electron transfer less than one percent of the time. Electron transfer from sensitizer to oxygen can also occur leading to the formation of a superoxide ion (02") and an oxidized form of sensitizer, but this transfer is less efficient. The singlet oxygen formed from the type II photosensitized reaction causes a stereospecific abstraction of the quasiaxial 7c hydrogen, resulting in a shift of the 45 double bond of cholesterol to the 45 position, subsequent bonding of dioxygen at the 5G position, and hydrogen transfer to yeild Sa-hydroperoxide. Minor oxidation products formed by singlet oxygen are 16 6-hydroperoxides. TYPE I WHEN a= TRIPLEI' BBITIZER mH teenlfie triplet + .emeitizr + R ' “an" -—-I'-1eitizr + H4 emeitizr / :"\ recticne ctr- re-lgmte 02 reecticne trial-t + R———"b ”m" eI'Ieitizr + u“ —"’""i’d=" + R tizjr teeelfie ' ' eOeetten TYPE II WEN CF TRIPLEI' BBITIZER ”-191“; + a: ANNA—$102 "’ “14:13.- emsitizr Illngllt eeygln) 5. 1e7Imn + o + emeitizr- Pipe 4. Ton types of reacticne involving triplet emeitizre. 17 W1; Copper and iron which exist in two or more valence states are effective free radical oxidation catalysts because they can donate or accept a single electron (Smith, 1981). The following homolytic equations show how initiation of oxidation proceeds. 1. M" + lama—b H‘""’” + m- + l-D- 4. 2. n" + mi——"M<"'1’ 4- .Rm- 4- H The resulting radicals could react with cholesterol or other hydrocarbons, extracting R protons. This is a concern in the case of meat and meat products where iron is abundant. Sato and Hegarty (1971) and Love and Pearson (1974) presented data that suggested that non-heme iron is the major prooxidant in cooked meat. This was supported by an investigation of the influence of heme pigments, nitrite, and non-heme iron on the development of warmed-over-flavor in cooked meats (Igene et al., 1979). Enzyme: Limited experimental evidence is available concerning enzyme-initiated cholesterol oxidation in foods (Smith, 1981). According to Gumulka et a1. (1982), ground state dioxygen ('02) is involved in specific enzymic hydroxylations and the superoxide radical (0"2) is generated in some enzymic oxidations. Cholesterol-e-epoxide and 25-hydroxycholesterol are the only oxidation products that can be found in food.which are known to come from enzymic origins (Smith, 1981). It 18 has also been suggested that a 7B-hydroxylase might give rise to 7S-hydroxycholesterol (Smith, 1981). The formation of 7a- and 7B-hydroperoxides has been demonstrated with soybean lipoxygenase, horseradish peroxidase, and the microsomal NADPH- dependent lipid peroxidation system of rat liver. These enzymes are all dioxygenases, which means that they add '02 to their substrate, forming a hydroperoxide (Smith, 1981). neat Heat is known to cause extensive autoxidation of cholesterol (Smith, 1981). For this reason, the methodology used for determining the concentrations of cholesterol oxides in foods is very crucial because hot saponification of lipids can cause artifactual formation of the 7-hydroxycholesterols, the 3B,5a,69-triol, 7-ketocholesterol and 3,5-cholestadiene-7-one, the thermal degradation product of 7-ketocholesterol. The formation of high concentrations of cholesterol oxides were reported by Horvath (1966) after heating crystalline cholesterol in the dark at 60°C for 48 days, 65'C for 30 days, 70°C for 28 days, and 100'C for 42 hours. Beating the cholesterol at 105°C for a week resulted in oxidation of approximately one half of the cholesterol. Korahani et a1. (1982) indicated that autoxidation of cholesterol heated to 100'C could be due to disintegration of the protective crystalline structure at 100'C, or to the particular molecular arrangement existing at that temperature being favorable to oxidation. 19 Upon heating recrystallized cholesterol Korahani et a1. (1981) determined that 80% of the cholesterol remained unchanged after 30 days in the presence of air at 100°C. Only 6% was detected as oxidation products. They found traces of peroxides, indicating that hydroperoxides were first formed in the autoxidation reaction. These investigators also noted that 25-hydroxycholesterol is highly stable at 100°C and it did not undergo any change when heated for six months in the presence of air. Both 7a- and 7B-hydroperoxides were detected after cholesterol was stirred in an aqueous colloidal suspension dispersed with sodium stearate. It was exposed to molecular oxygen at 85°C and pH 8 for three hours (Ximura et al., 1979). The instability of these 7-hydroperoxides has been known since the early 1940's (Maerker, 1987), their products being 7-ketocholesterol and both isomers of 7-hydroxycholesterol. Kimura et al. (1979) also observed some of the 5,6-cholesterol-epoxides produced under the same heating conditions. 392W Many processed foods containing cholesterol, especially those that are spray-dried, retorted, cured or deep-fat fried may contain some COP's. Other oxidation products formed during cooking and subsequent storage may act as catalysts for further cholesterol oxidation (Pearson et al., 1983). Fieser (1953a,b) reported the presence of 5¢-cholestan-BB,5¢,6B-triol in powdered egg yolk. Dried eggs are exposed to high temperatures and 20 forced through air, two conditions favoring oxidation, especially when the large surface area of the atomized egg is considered. Further details relative to the formation of COP's in dried eggs are presented later in this review. METHODS FOR DETECTION OF CHOLESTEROL OXIDATION PRODUCTS IN FOOD There is a wide variety of analytical techniques used for the isolation and quantitation of cholesterol oxides in foods, including high performance liquid chromatography (HPLC), gas liquid chromatography (GLC), mass spectrometry (MS), and thin layer chromatography (TLC). One of the major problems in developing adequate techniques for the analysis of cholestrol oxidation products is that sterol oxides can break down during isolation (Higley et al., 1986b). Interfering substances in foods also present a problem during isolation, making it difficult to detenmine the concentrations and types of oxides present. It has already been mentioned that 7-ketocholesterol is easily degraded, and that cholesterol-e- and B-epoxides are unstable during hot saponification. All of the cholesterol oxides shown in Figure 1 can arise as artifacts from isolation and sample storage (Smith, 1981). Both 7a- and 7B-hydroxycholesterol and 7-ketocholesterol can be produced from cholesterol during hot saponification (Smith, 1981). Wang Currently, thin layer chromatography is used.most often in combination with HPLC or GLC. R; values and the 21 colors of the spots can be used for compound identifica- tion. The usual methodology includes developing the plates (silica gel type G) with heptanezethyl acetate (1:1) and spraying them with 50% sulfuric acid for visualization. After spraying, the plate is heated at 110 to 120°C for approximately ten minutes for color development. Cholesterol will appear magenta, the 7a- and B-hydroxycholesterols and the epoxides are blue, while 5-cholestan 33,56,69-triol is yellow. TLC has been used for identifying COP's from irradiated spray-dried egg yolk (Chicoye et al., 1968b), butter exposed to fluorescent light (Luby, 1982) and grated cheese and bleached butteroil (Finocchiaro et al., 1984). Recently, it has been used for analyzing dry column saponification fractions, recovering the 5,6-epoxides and 7e- and 9-hydroxycholesterols (preparative TLC) (Maerker and Unruh, 1986), and for analysis of reaction mixtures of cholesterol in aqueous dispersion with sodium.stearate or Triton surfactants treated under different conditions. Purifying or determining the purity of standards can also be done with TLC (Lee et al., 1985). There are some disadvantages in using TLC rather than other available chromatography techniques. .Alone, it is not a good method for identifying 7-ketocholesterol and cholesterol-a- and fi-epoxide because they are not well resolved. Quantitation from TLC can be inaccurate due to the necessary steps of scraping the plates and eluting and 22 transferring the oxides (Park and Addis, 1985b). TLC is also labor-intensive and time-consuming. Hi rmance l d c omato ra h High performance liquid chromatography gives higher resolution that TLC. It has been used for the analysis of cholesterol oxides by Shen and Sheppard (1983), Ansari and Smith (1979), Tsai and Hudson (1981), and Maerker and Unruh (1986), among others. Cholesterol does not have a strong absorption peak but it does have an unsaturated center and functional group, so it absorbs in the 203 to 214nm range (MCCleur, 1976). Normal phase adsorption HPLC systems where the stationary liquid phase is polar and the mobile phase is nonpolar, have given the best separation although reverse phase systems (polar mobile phase, nonpolar stationary phase) have been used (Haerker, 1987). In the normal phase, the polar cholesterol oxides are retained in the stationary phase, so that more polar substances have a longer retention time. With reverse phase HPLC, the oxides favor the mobile phase. Semipreparative HPLC can be used instead of using saponification to separate cholesterol and cholesterol oxides from other lipids (Haerker and Unruh, 1986). Haerker and Unruh (1986) eliminated the saponification step by subjecting a cholesterol-in-triolein solution to HPLC enrichment (normal phase) and then analyzing the oxide fraction by GLC. A higher amount of the heat-labile 7-ketocholesterol was found than when the solution was initially saponified with heat. A difficulty with this 23 procedure is that some cholesterol oxides which are less polar than cholesterol (such as 3,5-cholestadiene-7-one) are lost because they are eluted before cholesterol. Butylated hydroxytoluene (BHT) and other phenolic antioxidants added for protection against artifactual COP formation could also be lost. Hexanexethyl acetate (100:5) was reported by Tsai and Hudson (1981) to be a good mobile phase for compounds that elute earlier than cholesterol, which includes reduced or dehydroxylated products of cholesterol. It was unable to resolve compounds which differed in only 1 or 2 unsaturated carbons which was also a problem for 2-propanol. These compounds include 5c-cholestane (often used as an internal standard), 3,5-cholestadiene, ketones at the 3 positon and some other COP's not important for food products. .A reverse phase C1. column was used by Shen and Sheppard (1983) with methanol or methanol:water (9:1 v/v) to separate cholesterol oxides that differ in their additional functional group and also the isomers of the 5,6-epoxide. They resolved cholesterol and C1 oxide products isocratically. They found an adsorption liquid chromatographic system to be the most effective. Using a HPorasil column with hexane:isopropanol (96:4) as the solvent system, they got baseline resolution of the 5,6-epoxides and the 7-hydroxycholesterols. They were also able to resolve side chain hydroxycholesterols that differ only in the position of the hydroxyl groups. Based 24 on their results and the results of others such as Ansari and Smith (1979) and Tsai et al. (1980), Shen and Sheppard claimed that HPLC was an effective method for separation of a complex mixture of cholestrol oxides, particularly because it is operated at room temperature and uses nondestructive detection. Host sterol oxides can be detected by monitoring the effluent adsorption at 212nm (Ansari and Smith, 1979). 8 to Gas liquid chromatography offers better separation and quantitation capabilities (especially with capillary columns) than either TLC or HPLC. The detection level is easily in the parts per million range (Rrull et al., 1984; Hissler et al., 1985). GLC is usually carried out with direct on-column capillary injection on a nonpolar column with a flame ionization detector. Hass spectometry can also be used as a detection method for derivatized samples (Haerker and Bunick, 1986). Samples containing cholesterol and cholesterol oxidation products are derivatized with a silylating reagent for improved thermal stability prior to GLC analysis. Packed columns were first used for analysis of cholesterol oxidation products. Teng et al. (1973) developed adequate packed.column.methodology for separation of various hydroperoxides including the epimeric 7-hydroperoxides. However, these compounds decomposed partially to the 7-hydroxycholesterols and other minor components. The hydroperoxides also tended to 25 epimerize to a small degree. Flanagan et a1. (1975) used packed column GLC (3% OV-225) along with mass spectrometry to separate and identify 3,5-cholestadiene-7-one in anhydrous milk fat. The packed column did not give good resolution but using mass spectometry they could still identify the compound. Taylor et al. (1979) were able to quantify several cholesterol oxidation products from USP-grade cholesterol using a packed column with 3% 88-30 or 3% OV-l. They reported 25-hydroxycholesterol, 7-ketocholesterol, the a- and B- isomers of 7-hydroxychol- esterol and the triol. Tsai et a1. (1980) used packed column GLC and HPLC for the analysis of egg and egg products. They reported the lowest detectable concentration of cholesterol—e- epoxide to be 50ng/ul. A packed column with 3% SP-2100 was utilized by Luby et al. (1986) for analysis of the nonsaponifiable lipid fraction from butter exposed to fluorescent light. They observed that as the size of the peaks that eluted before cholesterol decreased, the actual number of peaks also decreased. There was a possible increase in the number of peaks eluting after cholesterol with increase in exposure time. The difficulties with the analysis were that peaks were poorly resolved and the relative quantity of oxides compared to the concentration of cholesterol was small. Capillary columns greatly improve the results obtainable with gas chromatography. The e- and B- epimers of 7-hydroxycholesterol can be separated (Teng et al., 26 1973), and Gumulka et al. (1982) reported resolution of the epimeric 5,6-epoxides using a capillary column. Hissler et al. (1985) completely resolved four common oxides in egg products along with cholesterol using on-column injection onto a bonded phase fused silica capillary column. A cold technique was used for the injection so that 76-hydroxycholesterol was not dehydrated to 7-dehydrocholesterol. An extensive study of the application of gas chromatography for the analysis of cholesterol oxides in foods was conducted by Park and Addie (1985a). They used 3 different types of columns and experimented with some 11 different sterols including cholesterol, Sa-cholestane and the common cholesterol oxides found in food products. They used derivatized and nonderivatized forms, and.mass spectometry for confirmation of the identities of the compounds. They concluded that derivatization was necessary to avoid thermal degradation of the diols. With a 08-1 column, resolution was complete. When a column more polar than DB-l was used (DB-5), 7-ketocholesterol and 25-hydroxycholesterol eluted as one peak. Using the same liquid phase on a packed column, the compounds did not separate well. There are many more examples of cholesterol/choles- terol oxidation research that have been with gas chromatography. Discussing all of them is beyond the scope of this literature review. There are some disadvantages to GLC. The sample 27 concentration is more limited (Smith, 1981), especially with capillary columns. Thermal instability of some of the compounds can lead to loss and/or artifactual formation. Some of the oxides have close structural shmilarity making their complete separation difficult (Teng, 1973), and GLC/FID is not suitable for isolation of individual compounds (Shen and Sheppard, 1983). Cholesterol oxidation products in tallow were analyzed by GLC and good resolution was obtained, but the investigators thought that TLC-FID gave better estimates for quantitation (Bascoul et al., 1986). GLC appeared to underestimate the level of oxides, which could have been a result of incomplete derivatization, irreversible absorption of highly polar compounds on the column and/or thermal degradation. HI!§.§D§§§£QE§§I¥ Mass spectrometry (MS) has been used as a confirmation tool for HPLC or GLC in the analysis of cholesterol and cholesterol oxidation products. van Lier and Smith (1970) utilized HS in a study of the thermal composition of cholesterol hydroperoxides. Flanagan et a1. (1975) used MS with packed column GLC in their analysis of milk products and components. HS was also carried out by Rorahani et al. (1981) following capillary column GLC for confirmation of cholesterol derivatives. Eggs/egg products have been analyzed using MS (Tsai et al., 1984; Hissler et al., 1985; Sugino et al., 1986; and Nourooz-zadeh and Appleqvist, 1987). Finocchiaro et 28 al. (1984) analyzed cholesterol oxides in grated cheese and bleached butteroil using MS with HPLC. Park and Addis (1985a,b, 1986a,b, 1987) have used HS to identify cholesterol oxidation products in tallow and other food products. CHOLESTEROL OXIDATION PRODUCTS IN FOODS The presence of cholesterol oxides have been reported in a variety of foods including dairy products, meats, eggs and egg products, french fries and potato chips. W To date, cholesterol oxidation products have been reported in butter (Luby et al., 1986), bleached butteroil and grated cheeses (Finocchairo et al., 1984), and dried milk products (Flanagan et al., 1975). The presence of 4-cholesten-3-one and 3,5-cholesta- diene-7-one in anhydrous milk fat (AHF) and nonfat dry milk (NFDH) were reported by Flanagan et al. (1975). They did not determine the concentration of either oxide. The isomeric 5,6-cholesterol-epoxides, 79-hydroxy- cholesterol, 7e-hydroxycholesterol and the triol in stored bleached butteroils and grated cheeses were measured by Finocchiaro et al. (1984). 7-Retocholesterol and 3,5- cholestadiene-7-one were tenatively identified but not quantified. The bleaching of the butteroil was done by benzoyl peroxide, a widely used free radical-producing agent which is used in the United States to bleach milk for Blue, Swiss and Italian cheeses. The results of their study are summarized in Table 3. 29 Table 3. Sterol contents of bleached butteroils and aged Italian cheese samples'. nggcul _§g§55;¥4 (JxHeSUI1n fine-mullbuuuetdl mm“ 906mat15°c 34001350 DD 20:3 10:3 Unmet-20°C 35001190 ”1:3 30:6- 3316 (afar-901mnnlet 15°C) c 89: no 906met15°c 3400130 3312 3012 Chetwur'et-ézrc (a-ftr90dayeet 33001300 3015 9015 60:15 15°C) Chi-IIIlIflII Elflllln bread A clear glass bottle 4100 :t 510 110:1: 10 20 1:2 20 1:2 beetlA t l I | I 41001340 30 4 101:4 103:4 heexlD turbo-H1IHNMI' 4Mx>t440 201L4 ND ND Rum-Io band A 43001570 50:5 10:2 10:2 cler gleee battle .Adapted from Finocchairo et al. (1984) I'Bleached‘with benzoyl peroxide, stored in a nitrogen atmosphere. 'Bleached with benzoyl peroxide, exposed to air. 3O Luby et al. (1986) investigated the oxidative sta- bility of cholesterol in butter exposed to fluorescent light. COP's were detected after eight days of exposure to 1500 lux at the butter surface. The oxides were identified as 7a- and 7B-hydroxycholesterol using TLC. W More information is available on the presence of cholesterol oxidation products in tallow and meat products. Ryan et al. (1981) investigated the loss of cholesterol and the formation of cholesterol oxidation products in tallow that was heated at 180°C for eight hours/day for a total of 300 hours. The cholesterol concentration decreased with time, and cholesterol oxides (3,5-cholesta- diene-7-one, 7e- and 7B-hydroxycholesterol) were detected after heating. However, Park and Addis (1986b) stated that 3,5-cholestadiene-7-one is rarley reported as an oxidation product of cholesterol, but may be a degradation product of 7-ketocholesterol. Bascoul et al. (1986) obtained samples from deep-fat fryers while they were in use and analyzed them for cholesterol and COP's. The heating time reported by the users ranged from 56 to 70 hours. Approximately 25% of the original cholesterol was destroyed during cooking as shown in Table 4. It was partially transformed into the triol, 7¢- and 7B-hydroxycholesterol, 7-ketocholesterol, 3,5-cholestadiene-7-one and both isomers of 5,6-cholesterol epoxide. The triol was the oxide formed in greatest concentration, followed by 7c- and 31 7B-hydroxycholesterol, 3,5-cholestadiene-7-one, cholesterol-B-epoxide and some a-epoxide, and 7-ketochol- esterol. Two other compounds, 20- and 25-hydroxycholes- terol, were observed only after preliminary isolation with prepatative TLC. Park and Addis (1986b) heated refined edible beef fat continuously at 155°C and 190°C. Four oxides were present in the tallow heated at 155°C; 76- and 78-hydroxycholes- terol, cholesterol-a-epoxide, and 7-ketocholesterol. Table 4. Cholesterol loss in tallow from deep-fat fryers and the intensity of cholesterol oxidation products on TLC plates‘. Blane 'finpewflmne themes-«u Amewqe inn-untycfi 1°C ) (“g/(q) Omelet-Pol loee (33:5 m 1143 (71) plate 1‘ — 17C! 11: 142 1300 24 la» 2. — 1918 15 1 a, 171 1625 a" 5a ——. 1614 ‘ 51 11 5b 173 2300 uh 4e "‘ 1336 32 Pugh 4b 146 910 5a "' 1506 10 Immune 5b 194 1475 'Adapted from Bascoul et al., 1986. After approximately 300 hours at 155°C and 200 hours at 190°C, the cholesterol concentration in the tallow was approximately 40- 45% of the initial content. At 155°C, the oxides were first detected after 36 hours. 32 7B-Hydroxycholesterol and 7-ketocholesterol appeared first. The 7B-hydroxycholesterol concentration remained at 1-2% of the original cholesterol content for the duration of the heating. Cholesterol-a-epoxide was formed and leveled off at about 4% of the initial cholesterol content. The tallow heated at 190°C contained lower levels of COP's that were sporadically formed compared to the tallow heated at 155°C. Formation of 7-ketocholesterol was not proportional to heating time at 190°C and the 7-hydroxy- cholesterols were present at approximately 1%. It was suggested that there may be less chance for oxidation to occur at an elevated temperature, or the oxides may break down soon after formation. Park and Addis (1987) also quantified the oxidation products (COP's) in meat purchased at local stores. Pork W muscle was freeze-dried and kept at 22°C for three years. It was exposed to uncontrolled conditions of relative humidity and laboratory fluorescent light. It contained 7a- and 7B-hydroxycholesterol, 7-ketocholesterol, both isomers of cholesterol-epoxide and the triol (Table 5). The total cholesterol oxidation products were almost half the cholesterol content remaining in the freeze-dried sample of pork. Park and Addis (1987) also analyzed beef which was cooked overnight in a Cryovac vacuumized bag and then sliced, wrapped in PVC film.and stored at 4°C. It was analyzed every other day for 12 days and contained no detectable amounts of 33 cholesterol oxidation products. The investigators reported almost zero thiobarbituric acid (TBA) values in the beef samples, suggesting that rancidity development was not noticable throughout the storage. Cooked ground turkey and extra lean ground beef (less than 10% fat), wrapped in PVC, and analyzed at 0, 3 and 8 days of storage at 4°C did contain COP's. 7-ketocholes- terol accounted for almost half of the total COP's formed, followed by 75- and 7a-hydroxycholesterol and both of the cholesterol epoxides C7- oxides were not detected in fried chicken meat or cooked hamburger obtained from fast food restaurants (Park Table 5. Cholesterol oxidation products in three year-old freeze-dried pork'. au.1n OumeUNHHon Hewmntcfi (an/kg) crijinel choleetrol D'Ioleetrol 959,3: 1%“ 7G-CI'I Wee: 11.8 9.4 73.04 68.44: 5.8 7.1 740mm: 259Jfl:44u1 25A; “-5901“. 12.5: 2.2 1.3 Trial 25.4: 4.9 2.9 'Pl’k I'd M711! (1W7). and Addis, 1987). Rare and well done broiled beef were also compared to raw'meat. .All steaks showed some small 34 peaks after cholesterol on a GC chromatogram. The cooked steaks appeared to contain 7B-hydroycholesterol and 7-ketocholesterol. Two general conclusions were drawn from this study. Firstly, beef, whether fresh, cooked or pre-cooked and refrigerated for several days, represents a very minor contribution of cholesterol oxides to the human diet. Secondly, COP's in cooked turkey were also low but somewhat higher than beef. Higley et al. (1986b) used HPLC to analyze ten meat samples for cholesterol oxides and found no detectable amounts in the majority of the samples (Table 6). The triol content of one duplicate determination of cooked bratwurst and raw hamburger was high compared to the other duplicate of the same sample. This was attributed to noncholesterol compounds that absorb at the 240nm absorption maximum for triol. W Dehydrated yolk products are used in the processing of bakery products, salad dressings, baby foods, cake mixes and.military rations (Tsai and Hudson, 1984). In eggs and egg products, there is a considerable potential for the formation of measurable quantities of cholesterol oxides because the cholesterol content is high. Lipids comprise 60% of the total solids in egg yolk, 4% of which is cholesterol (Tsai and Hudson, 1984). Fresh eggs have been reported to be free of COP's (Chicoye et al., 1968b; Tsai and Hudson, 1984; Neurooz- Zadeh and.Appelqvist, 1987). However, small amounts of 35 Table 6. Cholesterol oxide content of selected meat samples'. Dude Food Wm the eelple West-1453354101 Code-d brim "' hef flank: " Chick-1 roll ‘9 Cooked hm to (Jacked lem beca': 11* mum-7m RM to Chick-1 roll "‘ Fhwlwmbuwnr ‘W+ Cooked bellow-gr 30- “- Cboked 1ee1 bum ‘0 5-O'Ioleete'r-SBJB-diol km "' Omwmdtraenx1m *0 Beefivanms 3‘ Chnnmnrtnl ta Rm hfllw 0-6 S-O'nleete'r-GBJa-diol Daoked braturet use, tn hef frmke u, es Duck-1 roll tn ,tn Rush-marge' " * linkedrumou1nr 72 (immedlumon ‘B mmamaa—um Cboked bratwurst 1"! ' Jalfi'flwnhs " 'hrkeylxnogwl es Chflflunl1fl1 t. iflioeiheefloef tn Raw hamburger 189. Cooked lee-I beef ta ‘Adapted from Higley et al. (1986b). 36 25-hydroxycholesterol, 7-hydroxycholesterol and 7-ketocholesterol were reported in fresh egg yolk by Naber and Biggert (1983). These were possibly artifacts formed by the hot saponification method used in the analysis. The conditions that exist during the spray-drying of eggs are favorable for the induction of lipid oxidation (Tsai and Hudson, 1984; Hissler et al., 1985). The eggs are atomized, which increases the surface area, and then exposed to hot air. Eggs dried by a direct gas-fired system have an even greater chance of undergoing oxidation than those dried by other methods (such as steam coils) because the eggs may come into contact with oxides of nitrogen (NO.) formed during the combustion of natural gas (Table 7) (Tsai and Hudson, 1985). Some reactions between oxides of nitrogen and some free radicals are shown in Figure 5 (adapted from Foote, 1976). Nitrogen dioxide initiates lipid peroxidation by reacting with both alkanes and alkanes by free-radical mechanisms (Pryor and Lightsey, 1981). The concentration of NO. in the gas for large utility boilers can be several hundred parts per million. The air that is passed through the gas flame can pick up these stable free radicals which could initiate cholesterol oxidation. Sevanian et al. (1979) demonstrated induction of cholesterol oxide formation in the lung tissue of rats breathing 3-6.5mg/kg NO.. 37 Table 7. Effects of air heating methods on cholesterol oxide formation in dehydrated egg yolks'. Direct Bee-Fired Indirect Stun-listed Temp, .1: I'll! T"?! .c egllg s...“ 1: his: noun 0'1“" supi- e Inlet suns: one" 2 “‘* n‘ 311 1o 16;. 64 10 5 158 64 13 12 159 70 6 15 77 12 13 1“ 72 6 9 139 74 13 15 157 as 16 '-- 19 1‘7 77 41 157 68 24 w, 7., 12° 23 143 64 7 25 na na 9 50 135 68 ""‘ 23 143 67 29 35 11a- 49 —- °Adapted from Tsai and Hudson (1985). DD '0' PUT 4 m: + m- ND150°C), there may be either less chance for cholesterol oxidation to occur or a greater probability for COP's to break down quickly after formation. The variation in the results of the present study from those reported by Ryan (1982), who cited the presence of 7-ketocholesterol and 3,5-cholestadien-7-one in tallow heated at 180°C, may be explained in part by the fact that the fryers used in the two experiments were different. Ryan (1982) used large commercial fryers that had heating elements in direct contact with the fat. The fryers used in this case were of the home-type and produced no turbulence, and had a smaller tallow surface exposed to air. In addition, the heating elements were contained within the walls of the fryers, resulting in a more gradual melting of the tallow. Thus the temperature of the fat remained more constant throughout the fryers. 135;: 93 the stability 9: gh lestezgl in tgllggi- Two separate heating methods- intermittent and 62 continuous; and three levels of oleoresin rosemary (0.01%, 0.05%, and 0.1%) were examined. Cholesterol loss Cholesterol loss was monitored to determinine the amount of cholesterol broken down or oxidized during heating. Less cholesterol loss was observed in the tallow containing 0.05% oleoresin rosemary than in either the tallow containg 0.01% olesoresin rosemary or the control. Preliminary tests revealed that 0.1% oleoresin rosemary did not provide any more stability than the 0.05% (Figures 6 and 7). However no statistical analyses were performed on the 0.1% oleoresin samples due to lbmited data collection. Oleoresin rosemary did not completely inhibit the formation of COP's in heated tallow, but appeared to act more consistently than an ascorbyl paLmitate/a-tocopherol combination used by Park and Addis (1986). The level of COP's in tallow containing 0.05% oleoresin rosemary was lower than that of the control throughout the heating. These results suggest that oleoresin rosemary, which contains several compounds exhibiting antioxidant activity, is more stable at 135°C that ascorbyl palmitate/a-tocopherol. Tallow with added ascorbyl palmitate/c-tocopherol heated at 135°C for up to 70 hours did not contain any of the standard COP's (Park and Addis, 1986a). However, after 70 hours, the inhibitory effect of the antioxidant was no longer observed. At heating times of 96, 144 and 216 hours they reported similar levels of COP's in the control tallow and tallow treated with ascorbyl palmitate/a-tocopherol. They suggested .5533 03004 E moo— _o.~0amo~oeo do 5038?. 50930—0 no econom— 6 0.53...— Uéa ho mgcm 63 co“ em on av cm a 060 :l a 1 . a . Akofis ux\m8v 30 .. fl .- 234 L 25: 1 Boa-h .9580 um.- omn 2.823— 8: 1%.. - base-econ and IT as - L ad on ON A v onouumhmmoq momm R 64 .0093 as common 30:3 5 Amhouv 3260.3 doflsouo 3.50.80.vo no dofloauou one do hasaomom damage—o go «009:.— .a emu-urn “Erna be ac! can an cc 9' . ON a — T J 1 - o Beach 35:00 * so «3... .+- 4 a “O Rad ICI- .. N n n L v f: . n R (I) FOE-<4 DOG-1° 65 that at high temperature ascorbyl palmitate and a-tocopherol either do not survive sufficiently long enough to have an impact, or are consumed by the excessive quantity of free radicals formed by extended heating. The equation describing the statistical model for the levels of oleoresin rosemary as a treatment over time for the continuous heating was Y . u + a; + b; + C, while that for the intermittent heating was Y = u + a; + b, + c;, + s. The term.c;5 represents interaction between time and the oleoresin rosemary level that existed in the intermittent system but did not exist in the continuous system (p< 0.01, Appendix I). Oleoresin rosemary (0.05%) was an effective antioxidant for both the continuously and intermittently heated tallow (Figures 8 and 9). Tables 9 and 10 show the cholesterol loss in heated tallow. .A comparison was made between continuous and intermittent heating. Continuous heating caused more cholesterol loss than intermittent heating (Figure 10), except in tallow containing 0.01% oleoresin rosemary, where there was no significant difference between the two heating methods Appendix II, part B). This was not expected since repeated heating and cooling of fats is a more detrimental treatment than maintaining a constant temperature (Ryan et al., 1981). Ryan (1982) reported that intermittent heating caused more cholesterol oxidation in tallow. In this study the time periods of heating (12 hours) for the intermittently heated tallow may have been too long. The time period of heating used by Ryan 66 $83002 Hoodoo—o «dazedoo toad» 5 003 Sneeze—ode . do cor-mu as udfleod adoddfidoo uo museum .m earn ccu cfin can GEE ho gen 2: cc 0* cm a one _. A303 and: «Q. L. 63 i as i. as. E 8 \\ I. l beg R86 1*.- h—eg Reed IT .. :3 ug II: cu A OIO-Iflmt-HGO-I H6010) ii; 67 53503.. 50030—0 ududueedoo 55:3 5 003 38832—0 :0 0035 as «03.2. 8328.35 .0 53:".— .a 23E 0.58: so was: can ova and can co co ow om o “dc HI! 5 4 . 4 4 q A . Ase—1.. EQHS \ ARV - n m fi :4 S .1 - 2 c A .. 3 .— 88 .. m 1 ON U a one .. - am a - a bug use... into on m 1. one 58883. 5: [T o 8:5 .6880 IT. on 8* Ar .1 3 68 93 as can: .53 $3 $33 5 mmo— Eocene—ode do Donna as mdfleod «doeamduuoadw ode-dob adoddfidoo uo 300:".— .3 0g..— "EEJBHOQSEu co 3. cm a ; lv 1 d 258.. 88:83... IT - 25.3 335-8 1:.- l an A v omouumhumoa uomm N 69 Table 9. The effect of oleoresin rosemary on cholesterol loss in continuous 1y heated tal low . mmn Dialect-Pol Gleam-1n mm l-batinq WY Lon M109 My Loos Tim- (tr) (7:) (10 Tim- (hr) (71) (X) 24 0.0 3.9 94, 0.0 29.6 o. 01 4' 2 o. 01 m. 2 o. o: 9. 5 0. (b 23. 7 48 O. O 17. 6 1m 0, o 41 . 7 0. 01 16' 3 o. 01 we 0 0. w 14. 3 0. w 24. 4 72 0.0 27-8 144 0.0 43.0 0. 01 Z. 3 0. O1 43, 0 0. 05 16. 1 0. CB 29. 6 Table 10. The effect of loeoresin rosemary on cholesterol loss in intermittently heated tal low . mm" mm macro-in mm listing m Lal- M199 Mary Lou Tim- (hr) (2) (X) Tin! (hr) (2) (X) 24 0.0 2.9 96 0.0 27. 1 O. 01 10. 7 0. 01 27- 8 0| w 6- O 0. w 120 6 4a 0. 0 9. B 120 0. 0 SS. 3 O. 01 13. 3 0. 01 35. 0 0.05 9.7 0-05 19' 1 7'2 0. 0 1B. 3 144 O. O 34. 9 O. 01 26. 0 o. 01 34. 7 o. (5 9. 6 0. (B 30. 4 70 was eight hours. If the fryers had been turned on and off more frequently, the tallow may have undergone more extensive oxidation. Comparing treatment means using the Tukey multiple comparison procedure revealed that the largest difference in cholesterol loss was between the tallow containing 0.01% oleoresin rosemary heated intermittently and the tallow containing 0.05% oleoresin rosemary heated intermittently after 120 hours. The difference was 82.9 t 3.4 mg/kg cholesterol (Appendix IV). The addition of 0.01% oleoresin rosemary to the tallow did not stabilize the cholesterol and may have acted as a prooxidant, since the control tallow had a smaller cholesterol loss. Formation of cholesterol oxidation products The effects of continuous and intermittent heating on the formation of COP's in tallow are shown in Figures 11 and 12. Formation of COP's was very random.which indicated that compounds may have formed and then reacted further to form other products. In the continuously heated tallow. the addition of 0.051 oleoresin rosemary lowered the formation of COP's throughout the heating period. In the intermittently heated tallow, after 72 hours there was a substantial formation of cholesterol oxidation products in the sample containing 0.01% oleoresin rosemary. The total COP's amounted to 7.3t of the original cholesterol, while the COP's in the control tallow’amounted to 1.7§ of the original cholesterol. The most prevelant COP in the heated tallow system was 71 53—3 5 3269:— dofisguo 3533—vo no doflsfiuou 25 so Donna as good 2595.33 no woman .3 as GEE no «“8! can an am God 8 co 3 ca 6 H + T fi d u . . . ‘ .3 ban-8:33 *. 3 ban-88:3... IT 533.0 ICI- as BOG-dd OOfln- I 72 £23 5 3.38 3033.3 dofisguo ~ou33—oda uo dofisauou 2: no oonfl s. 258.. 3328.35 3 ocean .3 955 02:4: .56 gal 8n ov— emu Sq 8 co 3 8 o 1 J 3 JG q u 4 a game... *1 bails—us... IT FOE-c.9581?! 1N 1‘. r/\ % ‘ .0 Lo A N V HOS-04A GOD" I 73 tentatively identified as 7-ketocholesterol (Table 11). It was identified by TLC and capillary column gas liquid chromatography by comparison of retention times with a standard. The standard COP was also added to some of the samples to observe the change in peak size of the suspected COP. Tallow contained an average of 3.2% 7-ketocholesterol before heating (six samples were analyzed). .After heating, the concentration range was 1.3% - 9.7%, with the exception of one sample of the tallow containing 0.01% oleoresin rosemary that had been heated continuously for 96 hours and which contained 19% 7-ketocholesterol. Park and Addis (1986) reported that the formation of 7-ketocholesterol was almost linear with heating time, and after 376 hours of heating at 155’C, its concentration was approximately 10% of the initial cholesterol content. They also reported concentrations of 4.6% 7-ketocholesterol in tallow which was heated at 135'C for 216 hours. The results shown in Table 11 suggest that due to the cycling nature of lipid oxidation reactions, new compounds are being formed and broken down continuously. Thus COP concentration does not increase over time but fluctuates. Even though the COP concentration was random, the highest concentration in the tallow containing 0.05% oleoresin rosemary was always lower than that of the control and tallow containing 0.01% oleoresin rosemary. In the majority of tallow samples, 73-hydroxycholesterol was observed after 48 hours of heating (Table 11). It was identified in the same manner as the 7-ketocholesterol. The 74 highest level of 7B-hydroxycholesterol (8%) was found in the same sample that contained 19% 7-ketocholesterol (heated Table 11. The maximum.concentrations of 7-ketocholester- ol, 7B-hydroxycholesterol and cholesterol-«- epoxide in heated tallow samples containing oleoresin rosemary. lkrmdnummanflung Influmdthmfl:Hlflnng OR Hiding zqaflp OR “guano 74G¢o m “rm (hr) (x) (x) Ting (hr) 01) 0.0 24 9.7 0-0 24 5.6 0.01 96 19 0.01 49 8.6 o.“ n 6s1 Ola % 4.5 73-04 79-0-1 m (x) 0.0 96 3"“ 0.0 120 2.6 0.01 96 9-0 0.01 72 3.2 0.05 96 2.9 0.05 149 2.3 O43=K m4iru (x) m 0.0 72 16.2 0'0 I” 1.1 0.01 72 7.3 0-01 48 6.0 0.05 72 1.1 0.05 149 1.3 continuously for 96 hours, containing 0.01% oleoresin rosemary). Excluding this sample, the range of 7B-hydroxy- cholesterol concentration for all samples heated intermittently and continuously was 0 - 6.2% of the original cholesterol. 75 The highest level of 7B-hydroxycholesterol detected in the continuously heated sample containing 0.05% oleoresin roseemary was 2.9%, after 96 hours of heating. For the intermittently heated tallow containing 0.05% oleresin rosemary, the highest level of 7B-hydroxycholesterol detected was 2.3%, and occurred after 148 hours of heating. The control tallow contained a maximum.of 3.4% and 2.6% 76-hydroxycholesterol after 96 hours of continuous heating and 120 hours of intermittent heating. The continuously heated tallow containing 0.01% oleoresin rosemary contained more 7B-hydroxycholesterol than its intermittently heated counterpart, with 3.2% after 72 hours of intermittent heating, and 8.0% after 72 hours of continuous heating. Since there was less cholesterol loss in the intermittently heated tallow, it follows that fewer cholesterol oxidation products would be formed. In most of the samples that contained 7B-hydroxycholes- terol, 7a-hydroxycholesterol was also tentatively identified by TLC. Both 7a- and 76-hydroxycholesterol were reported by Park and Addis (1986) in the 1 - 2% range throughout the heating of tallow at 155'C. It could not be quantified in this study with capillary gas chromatography due to incomplete resolution with cholesterol. When sample size was decreased in attempt to resolve the peaks, the concentration of 7a-hydroxycholesterol was too low for detection. Cholesterol-c-epoxide was also observed in approximately 25% of the tallow samples, usually at the beginning and reappearing again toward the end of the heating. The highest 76 amount detected was 16.2%, in the control sample heated continuously for 72 hours. Excluding this sample, the range of cholesterol-a-epoxide was 0 - 7.3%. The continuously heated sample that contained 19% 7-ketocholesterol and 8% 7B-hydroxycholesterol contained no cholesterol-a-epoxide. In both the continuously and intermittently heated samples containing 0.05% oleoresin rosemary, only the samples heated for 148 hours contained any cholesterol-d-epoxide. The oleoresin rosemary may have prevented the oxidation of 7B-hydroxycholesterol to the epoxide. The intermittently heated sample contained 1.3% and the continuous sample contained 1.1%. The maximum level of cholesterol-a-epoxide (1.1%) in the control tallow heated intermittently occurred after 120 hours of heating. After 48 hours, 6.0% cholesterol-a-epoxide was detected in the 0.01% oleoresin rosemary sample heated intermittently, which was the highest level detected in that sample. The tallow containing 0.01% oleoresin rosemary and continuously heated contained a maximum of 7.3% cholesterol-a—epoxide after 72 hours. The results obtained in this study are generally consistent with those of Park and.Addis (1986a) in that the same four COP's (7a-hydroxycholesterol, 7B-hydroxycholes- terol, 7-ketocholesterol and cholesterol-a-epoxide) were present and the 7-ketocholesterol was present in the greatest amount. However, unlike Park and Addis (1986a), linearity was not obtained for the formation of any of the COP's with time. This could be due to several factors. The initial tallow’may have been dissimilar from that used by Park and 77 Addis (1986a). The cholesterol content of tallow has been reported to be as low as 7051 12 mg/kg (Park and Addis, 1986) and as high as 16202 236 mg/kg (Bascoul et al., 1986). Rosemary may interact with the free radicals differently depending on the temperature stages, possibly even acting as a prooxidant at some times. This may be the case with the low level (0.01%) of rosemary, since the control tallow appeared to be slightly more stable when heated intemittently. Frying fat darkens with increased use. The french fries that yielded the darkest fat also had higher fat contents. This indicates that used fat is absorbed by the french fries more readily than fresh fat. variations in the color of the fat may be attributed to the addition of fresh fat to the fryers, changing of the fat in the fryers and length of use. The fat content of the french fries (measured from day 2 through day 7) ranged from 14.7 to 16.8%. This range is lower than that reported by Ryan (1982) who found 16.4 to 20.9%, but higher than that reported by Slover et al. (1980), which was 11.94 to 13.76%. Figure 13 shows the cholesterol content of the fat extracted from the french fries. .A comparison of the F- ratios from the analysis of the variance indicated a significant difference (p< 0.01) in the cholesterol content of the frying media from the two restaurants (Appendix V). It is possible that they have different tallow suppliers, since there was always less cholesterol present in the fat as; 81/2111 as pessadea. 'popad Ileana sea 3 .1060 pmoanoo sap} qouan [010100111100 mos} panama 1s; 11; 111011100 [03013010113 '81 9mm 78 i film-12°C BONE-NDHHOIO °$¥$%i§¥%§ . \\\\§\\\\\ ~ \\ \.\L\.\\ \\\\\\>. W \\.\\\\.\ m» V \\\\\\\\\\§\~}\\> \\.\\\\\\1\\§.\>3§\\\\\\\\\\ \\.\\\‘ NfiNNNWNNNfi)iii .\.~.\_\\\\ \\ \.\\‘\>.\\f\ \ \\“ \\~\ \7 ‘ “N“\\\.\\\\§\\\\\\‘x§\§\\x\\\\\\\\\\‘\\&\\\\§5\\\§3‘ \ \ « m \\\ Ml \\\\\\\\\\\\ \\\ .1 .\\ CA6 {\\\\\\'\\\(\\ ~va N n~~\‘~.\'\\\\\\\\\\.\\\\\\\\"~~,\\\\\\\\\\\\\x I 1031““! - E g 79 samples from restaurant 2. There was a significant difference (p< 0.05) in cholesterol levels between at least two of the seven sampling days for each restaurant (Appendix V). Using Duncan's multiple range test for restaurant 1 it was determined that there was no significant difference between the cholesterol content in the fats extracted from the fries on days 2, 3 and 7 (p< 0.05). There was also no significant difference between days 4 and 5. This indicates that the condition of the frying media was similar on days 2, 3 and 7 and also on days 4 and 5. The days in between similar days possibly represent a replacement of the fat or addition of new fat. For restaurant 2 there was no significant difference between days 2, 3 and 7 and also between days 4, 5 and 6. Figure 14 shows the total cholesterol oxidation products found in the fat extracted from the french fries. The F-ratio from the analysis of the variance showed that there was no significant difference (p< 0.01) in total COP's between restaurants 1 and 2 (Appendix VI). There was a significant difference in the content of cholesterol oxidation products between at least two of the seven sampling days for both restaurants (p< 0.01, Appendix VI). The Duncan multiple range test was used to determine which days were not significantly different from each other. The concentration of cholesterol oxidation products tended to be similar during the middle days and during the last two sampling days. This indicates that there was a lag time in formation of cholesterol oxidation products, such that the lipids and 'pouad aaau one a .1360 pazoauoo cam qonaz; [2101311111100 1110.!) pagosnxa is; n! (0,303) [nonponl nonnpgxo [0.101831qu 1010; '91 0.111813 nu am. 10 AVG « «(ax/am) 80 dZH-BZOO “BOO 1.0 O c I § § - 093 ‘008 \\‘\\\\\\\\\\\\‘\ 1'8 \ uns \ \ g \\‘.\\‘\\.\\\\‘ ‘... ...‘\\‘. \_\\j - 1 \ \\\\\\x\\\ \\ \\\\ u\\\\\‘m\\ \ .‘v .\. ,\ \‘.‘ A..\\ ‘1 "'(;\'\.\'.v_-\\ ;\\.\ \~. \ \ \“n \\ mm):5:)::i\&%&\\§§\\)\)§\\\h\)\\\h\\\\ik\ “(mg w W \m :\:(\x»‘_\‘1\\.\~\ . . 1 . \\\\\\\ \\\\\\\\\\.\\\\\\\\.\,\\\\\\\\\\\\\\.\\“.\\\.\\\.\\\\.\\\\\‘WM“,‘MX‘ \ \::\\x\\:\\. \\.\>:\.).\,\\\>\\ \‘-.\ ‘\\\\\ \\«\>\\>«\\\\)\\\\\ 1m ,g\i<\\&\\\\\\\\\\\\\ ‘.\‘\\ \ \\.\\‘ \\\|\‘\ \\ \x \‘K‘\‘\. \\ \‘\\\\ X\\\\\\\\\‘,\\\\\\.\\.\‘ \ \\ w s»\x'\ékx\>x&>8&x\§sr~$$\\\&\\;~.\&\‘x\\\l\$,\\$\\\\‘e I Inn‘W‘l - / ' . £21: a imam-ea 81 cholesterol had to break down to a certain point before cholesterol oxidation products were formed. It is also possible that there was total replacement of the frying medium soon after the fat reached the stage where COP's were formed. When the frying media was replaced the total cholesterol oxidation products may have been lower than at other points during use because they cycle, being broken down and formed continuously. The actual level of COP's detected in the french fries was higher than those reported by Lee et al. (1985) and Park and Addis (1985). Lee et al. found up to 81 mg/kg 7B-hydroxycholesterol (the COP in greatest quantity) and Park and Addis reported up to 58.8 mg/kg 7B-hydroxycholesterol. In this investigation 76-hydroxycholesterol was also found in some of the samples, but the predominant COP was 7-ketocho- lesterol (Table 12). It is possible that some 7-ketocholes- terol was formed during saponification and isolation, even though cold saponification was used. Ryan et al. (1982) detected 78-hydroxycholesterol, 3B-hydroxycholesterol, and 3,5-cholestadiene-7-one in the lipids extracted from french fries from a fast food franchise. The 3,5-cholestadiene-7-one could have been a degradation product of 7-ketocholesterol. 82 Table 12. Cholesterol oxidation products in fat extracted from commerciallt produced french fries collected over a period of one week. Rmstmrmt 1 letmrrlt 2 7m + 7-Iata + 74:616. 7a L 7m. 73-0-1 79.1.14 my (mg/kg) 1% of d'Dl.) (mu/km (x of M.) 1 108 0 20.2 133 16 30.3 2 167 17 27.4 147 15 27.4 3 256 0 36.9 214 0 33.8 4 226 3 35.9 233 0 43.2 5 10 37.0 170 O 31.5 176 7 30.9 202 2 37.6 170 10 26.7 142 0 24.7 OXIDATION OF CHOLESTEROL IN DAIRY PRODUCTS 2: T-, = t8 0 -_ 0-3 5.1 0- (12-1 ;_ <19. -, 1-0.10. .L'.’ - - 1: 0H2: on Of 0,9; -1-. 1° =9 : 0 1, .0. ; ; 0 =1! on under fluorescent light Lipid oxidation in.butter The effects of various packaging materials and antioxidants on the oxidative stability of butter stored under fluorescent lights were investigated. Comparison of packaging materials Light in the range of 300- 700 nm has been reported to cause oxidation in butter (Sattar and denan, 1975), however no measurements of light intensity or wavelength distribution were made in this study. The effect of packaging materials on the stability of butter with added salt stored under fluorescent light was investigated. The results are shown in 83 Figure 15. As expected, aluminum foil prevented light exposure of the butter and in three replications there was no evidence of lipid oxidation in any of the foil-wrapped samples after 16 days of illumination. Similar results for samples wrapped in aluminum.foil have been reported by Wilster (1957), Downey and Murphy (1968), Emmons et al. (1981) and Luby et al. (1986). The peroxide values of the butter samples wrapped in margarine wrap were similar to those of the butter wrapped in polyethylene. At a wavelength of 300 nm, the percent light transmission for polyethylene is 88%, while that for margarine wrap is only 48% (Luby et al., 1986). Hewever, at 400 nm, their light transmission is equal and at wavelengths above 400 nm.margarine wrap has a slightly higher transmission than polyethylene. Therefore, it was not unexpected that the samples in margarine wrap and polyethylene would undergo similar levels of oxidation. Statistical analysis (Appendices VII and VIII) indicate that there was no significant difference between the overall means of the peroxide values of the butter in polyethylene and margarine wrap. A factor that could have influenced the rate of oxidation in the margarine wrap sample is water permeability, although this is unlikely since the samples were stored at 4'6. The difference between the overall means of the peroxide values for foil and margarine wrap was 12.8: 3.2 while that for foil and polyethylene was 9.82 3.2, (p< 0.05) (Appendix VIII). Therefore, the peroxide values of the butter samples 84 div—363 moot!» 5 cones—o:— uofisa no Ana—\aoav ogdb ~33qu 23 do nevi—1:3: no vacuum .3 0.5-:— log .3 :3 cm on cu n c J a q q + a HI 6 all d fl-IIOHHDU #439. 85 in foil were significantly lower than those in both the margarine wrap and the polyethylene wrapped samples. Unpackaged butter stored under fluorescent lights was analyzed by Luby et al. (1986), and was reported to contain peroxides and cholesterol oxidation products. Effect of antioxidants on lipid oxidation in butter The effects of antioxidant addition on the stability of lipids was determined in butter which was packaged in polyethylene and exposed to fluorescent light (Figure 16). Two natural antioxidants (a-tocopherol and fl-carotene) were compared to TBHQ (tertiary butylhydroxyquinone), a syntheic antioxidant. fl-carotene significantly lowered the peroxide value, while a-tocopherol and tertiary butlyhydroquinone (TBHQ) did not (Figure 16, Appendix VIII). T880 and a-tocopherol are phenols which function as free radical chain-breaking antioxidants. In photooxidation, B-carotene (Figure 17) is expected to be a more effective antioxidant than the free radical chain-breaking phenols because of its quenching effect on singlet oxygen (Trappel, 1980). It has been demonstrated that B-carotene minimizes the oxidation of soybean oil stored under light by quenching singlet oxygen (Lee and Min, 1988). Cholesterol oxidation.prodncts in butter Butter was analyzed for the presence of cholesterol oxidation products using thin layer chromatography and a capillary gas chromatography system with a detection limit of 0.2 mg/kg. However, no COP's were present in the butter samples. Several small peaks appeared on the GLC 86 defiance»: 2553.30 .53.:— uo Aua\qoav 9N egab oomuouom 25 no fleas—=33: no vacuum .3 0.3.:— zoEzioqn no 53 ea ed a c . . I. N \ 1 .v xx I. c n m 3.... .m: 1 g *1 on a 3828.88 + 3849631384 I... L 3 33 «53.835 a - 3 QIIONMBI >449" \ \ \ \ \\ \\ Figure 17. Stuntm- of B—crot-u. chromatograms near the cholesterol peak in most of the samples, but did not have retention times corresponding to those of known COP standards. Contrary to these results, Luby et al. (1986) detected 73- and 7B-hydroxycholesterol in unpackaged butter after eight days of exposure to light. Two weeks of exposure to fluorescent light produced very low concentrations of compounds in some packaged butter samples that were tentatively identified as 7c- and 7B-hydroxycholesterol (Luby et al., 1986). The samples were packaged in polyethylene, margarine wrap, opaque parchment and wet strength dry wax paper. A possible explanation for the difference in the results of the present study and the results obtained by Luby et al. (1986) is that less oxidation of the lipids in the butter occurred in this study. Peroxides are the main initial products of lipid oxidation. The peroxide values of the butter when cholesterol oxidation products were first detected by Luby et al. ranged from 18.2 to 49.7 meq/kg after 10 to 15 days of exposure to fluorescent light. The peroxide 88 values in this study were between 4.2 and 19.7 meg/kg, after the same storage period. Of the ten samples that Luby et al. (1986) reported to contain COP's, only two of them had peroxide values below 20 when the COP's were first detected. Five of them had peroxide values greater than 36. Lower peroxide values in this study indicated that conditions were not as favorable for oxidation. It would be expected that if peroxide formation was low, the rate of cholesterol oxidation would also be low. As the overall rate of lipid oxidation was lower in this case, there would be less free radicals formed that could initiate cholesterol oxidation. Four commercial powdered dairy products (sour cream, cheddar cheese, butter and whole milk) of unknown history were analyzed for cholesterol oxidation products. The sour cream, butter and whole milk were all found to contain 7-ketocholesterol. The results are shown in Table 13. Since powdered milk contains less cholesterol than powdered sour cream.and butter, it would be expected to also contain fewer cholesterol oxidation products. The fact that the powdered cheddar cheese did not contain any COP's may have been due to the addition of an antioxidant by the processor. These levels of 7-ketocholesterol have not been previously reported in powdered dairy products, but 3,5-cholestadien-7-one, a thermal degradation product of 7-ketocholesterol, was reported by Flanagan et al. (1975) in 89 This 13. 7mm cent-1t in padrsd dairy prairie. Phamxx; 74G¢cd1flashrtn “EZEE’ 45 aux-Cremn * W m Chmthrliwlnl amour-d 43 lint-r Retired 16 Ffllk «NIk-notckflcCUII anhydrous milkfat. Cholesterol-a-epoxide, 7e- and 79-hydroxycholesterol and the triol were reported by Finocchairo et al. (1984) in bleached butteroils and aged Italian cheese samples (Table 3). It is possible that the oxidative conditions during the drying of the dairy products in this study were such that the initially formed 7a- and 7B-hydroperoxides were directly hydrated to form.7-ketocholesterol, rather than being reduced to the diols (Figure 2). OXIDATION OF CHOLESTEROL IN EGG POWDER Powdered egg samples prepared in Fermoy, Ireland were analysed for cholesterol loss and the formation of cholesterol oxidation products during storage. A.direct heating method of the drying air was used for half of the samples, while an indirect method was used for the other half. The directly heated air contained 7-8 mg/kg oxides of 9O nitrogen at the dryer inlet, while the indirectly-heated air contained less than 1 mg/kg. It is important to note that the indirectly-heated air was measured closer to the heating source than was the directly heated air which was measured away from the flame. Thus, the concentration of the oxides of’nitrogen in the drying chamber would have been much lower for the indirectly heated system. Cholesterol loss There was a significant difference in cholesterol content (p< 0.05, analyzed in duplicate) between the sample dried with indirectly heated air and the sample dried with directly heated air (Table 14, Appendix X). Commercial spray-dried eggs have previously been reported to contain ’ 1570: 130mg/kg cholesterol (Sugino et al., 1986). This higher level may have been due to a difference in.moisture of the product, a difference in the biological make-up of the eggs used in this experiment, or a difference in methodology. After incubation at 50°C for 40 days, those investigators reported 1590: l40mg/kg cholesterol indicating experimental error or error in calculations. The sample dried with directly heated air may have come into contact with oxides of nitrogen which are formed during the combustion of natural gas (Tsai and Hudson, 1985). Figure 5 shows some reactions that can occur between oxides of nitrogen and free radicals. The lower cholesterol content of the egg sample dried with indirect heat indicates that more cholesterol oxidation occurred in the sample dried with directly heated air. 91 This 14. Ouisstm'ol loss in gray-(rid sggs sftm' inabstim flu'twotllksln:aaflm lur'nunnng lawn-su-1u.camunm QEVBQ) """'°" W ”tw— Dirsct 9123 7575 nrursct 8574 8585 In this study, cholesterol was oxidized by the oxides of nitrogen in the directly heated air. In an investigation of dehydrated egg yolk, Tsai and Hudson (1985) reported concentrations of 8 to 311ppm total COP's in samples dried with directly heated air, while those dried with indirectly heated air contained 10ppm.and less. After incubation the drop in cholesterol content was slightly greater in the sample dried with directly heated air than in the sample dried by indirectly heated air (6.7% versus 5.78). Cholesterol oxidation products Several cholesterol oxidation products have been reported in spray-dried eggs and egg products. These include 7e- and 7B-hydroxycholesterol, 25-hydroxycholesterol, cholesterol-a-epoxide, cholesterol-B-epoxide, the triol and 7-ketocholesterol (Chicoye et al., 1968; Tsai and Hudson, 1984; Herian and Lee, 1985; Hissler et al., 1985; Naber and Biggert, 1985; and Nourooz-Zadeh and Appelqvist, 1987). In the present study, no cholesterol oxidation products were detected before incubation of the spray-dried eggs, and 92 cholesterol-a-epoxide (187 mg/kg) was the only COP detected after two weeks of incubation at 38'C in the egg sample spray-dried with air heated directly. The egg sample spray-dried with indirectly heated air did not contain any COP's. The concentration of cholesterol-o-epoxide was high, especially considering that none of the other common COP's were present on TLC plates or detected with GLC. Several investigators have reported the presence of cholesterol-e- epoxide in freshly spray-dried eggs. In freshly dehydrated commercial whole eggs and yolk powders Tsai et al. (1980) reported the range of cholesterol-a-epoxide to be from undetectable to 62 mg/kg. .A concentration of 17.4: 1.9 mg/kg cholesterol-s-epoxide was reported by Sugino et al. (1986), 50 and 21.5mg/kg for eggs dried with directly and indirectly heated air respectively was reported by Missler et al. (1985) and a range of l- 30 mg/kg was reported by Tsai and Hudson (1985). .After storage for nine months Tsai and Hudson (1985) reported 33 mg/kg cholesterol-a-epoxide. After strorage for five months at ambiant temperature (22.5’C) Nourooz-Zadeh and Appelqvist (1987) reported 2.4 mg/kg cholesterol~¢-epoxide in dehydrated yolk. The particular conditions in this study may have been favorable for the formation of cholesterolus-epoxides, while in most cases several different Cholesterol oxidation products are formed. After incubation for 70 days at 50'C, Sugino et al. (1986) reported 31.9: 3.3 mg/kg cholesterol-c- epoxide in the spray-dried egg which previously contained 93 17.4: 1.9 mg/kg. They also detected cholesterol-B-epoxide, but their HPLC methodology was not specific for other oxidation products that were possibly present. The presence of cholesterol oxidation products prior to incubation may have altered the effect of the incubation compared to the present study, where cholesterol oxidation products were not detectable prior to incubation. The temperature used in the present study (38’C) was lower than that used by Sugino et a1. (1986), and more likely to be similar to temperatures encountered in warehouse storage. Higher temperature may cause oxidation reactions to occur more randomly, producing a wider range of products. The specific conditions that existed during the spray-drying of the samples used by Sugino et al. were also unknown. variations from the conditions used for this study may have caused oxidation to proceed differently. Spray-dried egg yolk stored in closed plastic bags at 4'C for 12 months contained 2.5 mg/kg cholesterol-¢-epoxide, as reported by Hourooz-Zadeh and Appelqvist (1987). Powdered scrambled egg mix contained 50 mg/kg cholesterol-a-epoxide (Hissler et al., 1985). None of the other investigators mentioned above reported the high levels of cholesterol-a-epoxide found in this study, however the total cholesterol oxidation products reported were often in the range of the 187 mg/kg cholesterol-a-epox- ide reported here. Several samples of dehydrated yolk contained between 150 and 188 mg/kg total cholesterol oxidation products as reported by Tsai and Hudson (1985), and 94 one sample contained 311 mg/kg cholesterol oxidation products. There was a small peak that eluted after cholesterol in the sample containing cholesterol-a-epoxide wich did not correspond to any of the standards available for this study. It was possibly 20¢-hydroxycholesterol which Nourooz-Zadeh and Appelqvist (1987) reported in dehydrated egg yolk, or an artifactual cholesterol degradation product. SUHHBR!.AND CONCLUSIONS The oxidation of cholesterol in heated tallow, several dairy products, and spray-dried eggs was investigated. The effectiveness of oleoresin rosemary as an antioxidant in tallow was evaluated, as well as the efficacy of fl-carotene, TBHQ and a-tocopherol as antioxidants in butter. Several packaging materials were tested for their ability to prevent oxidation caused by illumination of lipids and cholesterol in butter. .A survey of powdered dairy products was also completed to identify COP's present and an indirect heating system was compared to direct air-heating methods for spray-drying eggs. Analysis by TLC and GLC showed that no COP's were present in tallow and tallow containing oleoresin rosemary which was heated at 180'C for up to 240 hours. Tallow heated at 135'C did contain COP's and it was determined that the cholesterol content decreased with heating. Less cholesterol loss was observed in tallow containing 0.05% oleoresin rosemary than in the control tallow or tallow containing 0.01% oleoresin rosemary. The addition of 0.1% oleoresin rosemary did not provide any greater protection against cholesterol loss than the 0.05& level. Continuous heating of the tallow caused more 95 96 cholesterol loss than intermittent heating. This observation was attributed to the duration of the heating times employed in the intermittent heating study. Several COP's were formed randomly in the tallow which were identifiable after 24 hours of heating. These COP's were identified by TLC and GLC as 7-ketocholesterol, 7a- and 7B-hydroxycholesterol, and cholesterol-a-epoxide. The predominant COP was 7-ketocholesterol. Tallow containing 0.05% oleoresin rosemary contained less total COP's than the control tallow or tallow containing 0.01% oleoresin rosemary (analyzed every 24 hours) during the total 144 hours of continuous heating. In the intemmittent heating experiment, there were generally less total COP’s in the 0.05% oleoresin rosemary sample than in the control tallow samples. Commercial french fries were obtained daily from two fast food restaurants on seven consecutive days to investigate the cholesterol and COP concentrations in the extracted fat extracted from them. There was a significant difference in the cholesterol contents of the extracted fats obtained from the two restaurants. This indicates that the two restaurants may not have had the same tallow suppliers, since the cholesterol content of the fat from restaurant 2 was always lower than that of the fat extracted from the french fries obtained from restaurant 1 on all seven days. The cholesterol content of the extracted fat also varied from day to day. There was a significant difference between at least two of the 97 seven sampling days for each restaurant. The predominant COP in the fat extracted from the french fries was 7-ketocholesterol, indicating that initially formed 7a- and 7B-hydroperoxides were directly hydrated to form 7-ketocholesterol. Some samples also contained 7B-hydroxycholesterol. There was no significant difference in total COP's between the two restaurants. The effects of various antioxidants and packaging materials on the oxidation of lipids, including cholesterol, in butter stored under fluorescent light at 4'C for 16 days were investigated. Analysis with TLC and GLC showed that none of the butter samples contained any COP's. Data obtained from peroxide values every four days revealed that aluminum foil effectively protected the - butter from oxidation. Substantial oxidation occurred in butter packaged in margarine wrap and polyethylene. B-carotene significantly lowered the peroxide value of butter wrapped in polyethylene exposed to fluorescent light, while a-tocopherol and TBHQ did not. In photooxidation, B-carotene has a quenching effect on singlet oxygen and is expected to be a more effective antioxidant than free radical chain-breaking phenols. Four commercial powdered diary products (sour cream, cheddar cheese, butter and whole milk) of unknown history were analysed for COP's. The powdered sour cream, butter and whole milk all contained 7-ketocholesterol. The powdered cheddar cheese did not contained any COP's Cholesterol loss and formation of COP's were 98 investigated in two powdered egg samples prepared in Fermoy, Ireland that were incubated at 38°C for two weeks. One sample was spray-dried with indirectly heated air, while the other was spray-dried using directly heated air. After spray-drying, the direct-heat sample initially contained less cholesterol than the sample dried with indirectly heated air, possibly indicating that there was greater cholesterol oxidation in the direct-heat sample. However, this could be due to variability in the original eggs. There were no COP's in either sample prior to incubation. Cholesterol-a-epoxide was identified by TLC and GLC in the direct-heat sample after the incubation period. Conclusions derived from this study are summarized as follows: 1) At extremely high temperatures, COP's are not formed in tallow or are broken down too quickly after formation to be detected. 2) ‘With moderate heating, 7-ketocholesterol, 7a- and 78-hydroxycholesterol and cholesterol-c-epoxide will form in tallow, 7-ketocholesterol being predominant. 3) Oleoresin rosemary (0.05%) is an effective antioxidant for reducing cholesterol oxidation and COP formation in heated tallow. 4) Commercial french fries may contain 7-ketocholes- terol and 7B-hydroxycholesterol, 7-ketocholesterol being predominant. 5) The concentration of COP's in french fries cannot 99 be correlated to the concentration of cholesterol. 6) Packaging materials that prevent light transmission effectively reduces the extent of lipid oxidation occurring in butter. B-carotene also slows down the oxidation process. 7) Cholesterol oxidation due to illumination does not easily occur in butter and would not be expected under normal conditions of production, distribution and storage of butter. 8) Direct heating of air for spray-drying eggs is more likely to cause cholesterol oxidation than indirect heat. Cholesterol-c-epoxide, which was present in eggs dried with directly heated air after incubation, has been implicated as having carcinogenic properties. 100 PROPOSALS FOR FURTHER RESEARCH Since this study was first initiated, several questions have been raised that may be worthy of further research. 1) An investigation of the relationship between the extent of lipid oxidation in butter measured by peroxide value and the extent of cholesterol oxidation. 2) The analysis of the effectiveness of B-carotene as an antioxidant in products other than butter which are exposed to illumination. 3) Acceptance tests of the sensory qualities of foods fried in tallow containing oleoresin rosemary. 4).A concentrated study of the effects of continuous versus intermittent heating of frying media. 5) Cholesterol oxidation products appeared to be formed erratically in french fry oil. An investigation of frying temperature, frying time, and fry load may provide insight to the cause. 6) Further work needs to be done to establish components of cheddar cheese and other dairy products which may initiate or retard cholesterol oxidation. 7) Cholesterol oxidation could be monitored in spray-dried eggs under various storage and packaging since literature currently offers conflicting observations 101 8) Continuing medical research must attempt to define the relationship between cholesterol, diet, exercise, cardiovascular disease and cholesterol oxidation products. APP.ICBS 102 Appendix I Analysis of the variance for the effect of oleoresin rosemary (OH) on cholesterol loss in heated tallow. A. Continuously heated tallow Source W961... W OR 27234.4 2 Time 255246.5 5 Interaction 19011.8 10 Residual 13040.0 18 B. Intermittenly heated tallow Sarcoma... OR 42592.0 2 Time 215810.1 5 Interaction 22396.0 10 Residual 8210.5 . 18 Critical F values: 83—9415. (2, 18) 3.6 (5, 18) 2.8 (10, 18) 2.4 *Indicates significance at as 0.05 **Indicates significance at a- 0.01 13617.2 51049.3 1901.2 724.4 8222492222 21296.0 43162.0 2239.6 456.1 Emits). 18.8** 70.5** 2.6* 103 Appendix II Analysis of the variance for the effect of continuous versus intermittent heating on cholesterol loss in tallow. A. Pure tallow Sgugce 52; of sggageg g.g. aggn_§ggg;§ F ratio Heating 15453.4 1 15453.4 23.2** method Residual 800.8 12 667.3 8. Tallow containing 0.01% oleoresin rosemary 5.9322 Wit. W Larisa Heating 570.4 1 570.4 .94 method Residual 7244.5 12 603.7 C. Tallow containing 0.05% oleoresin rosemary 593m W def: More £143.12 Heating 10375.0 1 10375.0 20.8** method Residual 5998.5 12 499.9 Critical F values: 1:12.115 1:01.01 (1, 12) 4.8 9.3 104 Appendix III Tukey multiple comparison of the means for cholesterol loss in continuous versus intermittently heated tallow. A. Pure tallow 50.7: 37.2 mg/kg 50.7: 41.7 mg/kg B. Tallow containing 0.05% oleoresin rosemary 41.5: 32.1 mg/kg 41.5: 36.1 mg/kg 105 Appendix IV Tukey multiple comparison of the means for cholesterol loss in heated tallow. A. Intermittently heated tallow 0.01% OR: control: 0.05% OR: control: 0.05% OR: 0.01% OR 1 - a 28.5: 3.4 mg/kg 82.9: 3.4 mg/kg 54.4: 3.4 mg/kg B. Continuously heated tallow 0.01% OR: control: 0.05% OR: control: 0.05% OR: 0.01% OR 12.4: 3.4 mg/kg 63.6: 3.4 mg/kg 51.2: 3.4 mg/kg 0 28.5: 3.7 mg/kg 82.4: 3.7 mg/kg 54.4: 3.7 mg/kg 12.4: 3.7 mg/kg 63.6: 3.7 mg/kg 51.2: 3.7 mg/kg 106 Appendix V Analysis of the variance for the cholesterol content of oil extracted from french fries obtained from two fast food restaurants over seven consecutive days. Sogrce Sum of squares g.g. Mean square 1;;gtig Restaurant 21607.1 1 21607.1 51.0** Day 21506.4 6 3584.4 8.5* Residual 2543.9 6 424.0 Critical F values IL:_0.:_9_5. s._-_0_,_9,1 (1, 13) 6.0 13.7 (6, 13) 4.3 8.5 Q2nQQBLE.HMLL£21§.B§BQ§.I§§E* W W Day Day 1c 1c 2a 2a 3a 3a 4b 4b 5b 5b 6d 6b 7a 7a *Days with the same letter were not significantly different from each other. 107 Appendix VI Analysis of the variance for the total cholesterol oxidation products in oil extracted from french fries obtained from two fast food restaurants over seven consecutive days. SQBIEQ. SEE_QI_§QE§£§§. 92:2. H§§§_§92££§ E.£§212 Restaurant 2716.1 1 2716.1 4.1 Day 44743.4 6 7457.2 11.3** Residual 3945.4 6 657.6 Critical F values KLLJLS. 3.3—0.1.9.1 (1, 13) 6.0 13.7 (6, 13) 4.3 8.5 DnnEQQL§_!3151212_3589§_1§§§* Beatanrant_l Restaurant_z 1c 1d 2a 2a 3b 3b,c 4b 4b 5b 5c 6a 6c 7a 7a *Days with the same letter were not significantly different from each other. 108 Appendix VII Analysis of the variance for the effect of antioxidants and packaging materials on the peroxide values of butter stored under fluorescent light. .A. Antioxidants mwmwm Time 155.0 3 51.7 12.3** Antioxidants 365.8 3 121.9 29.0** Interaction 186.2 9 20.7 4.9** Residual 133.0 32 4.2 Critical F values g a 9,! g - Q 95 (3, 32) 2.28 2.92 (9, 32) 1.84 2.20 8. Packaging materials 5.911222 W Li... 822nm 2.14219 Time 337.3 3 112.4 21.6** Packaging 1075.6 2 537.8 103.4** Interaction 171.8 6 28.6 5.5** Residual 124.3 24 5.2 Critical F values SILL; LLLJLE (3, 24) 2.3 3.0 (2, 24) 2.5 3.4 (6, 24) 2.0 2.5 **Indicates significance at as 0.1 and as 0.05. 109 Appendix VIII Tukey multiple comparison of means of the peroxide values of butter with various antioxidants and packaging materials. A. Antioxidants e-tocopherol : B-carotene 3 . 9: e-tocopherol: TBHQ o-tocopherol: salt only B-carotene: TBHQ B-carotene: salt only TBHQ: salt only B. Packaging materials Foil: margarine wrap Foil : polyethylene 0.91 2.81 3.0: 6.7: 3.71 3.2 3.2 3.2 3.2 3.2 3.2 meg/kg meq/kg Iraq/kg mes/k9 meg/k9 meq/kg 12.8: 2.9 9.8: 2.9 Margarine wrap: polyethylene 3.0: 2.9 3.91 0.9: 2.8: 3.0: 6.7: 3.7: 3.4 3.4 3.4 3.4 3.4 3.4 meg/k9 meg/kg meg/kg meq/kg meg/kg meg/k9 12.8: 3.2 9.8: 3.2 3.0: 3.2 110 Appendix IX Equations for Tukey's multiple comparison of treatment means. D:thfl)) iaEE n D-Yi'J.‘-Y1'J' “T‘- qti-oq as, (n-nasn 2 1-lmloffm8 s-Mbroflsvslso-F J-lsvsloffmztcra fm“ b-rumbro-Flmlso-F flour-B w-totalmbrofpossiblstrsstmts n-mrbrofmitsrscsivingagimtrsstm uniladngtmpal) 111 Appendix X Analysis of the variance for the effects on cholesterol content of eggs spray-dried with directly or indirectly heated air, and after two weeks of incubation at 38°C. Sooroo 522.21.2322222 elf: 8222.292222 z_rooio Drying 593670.3 1 573670.3 1561.3* method Incubation 279312.3 1 279312.3 734.6* Residual 380.3 1 380.3 Critical F values s_:_929§. 1.2.9101 (1, 3)‘ 161.0 4052 Addis, P.B. 1983. Some lipid oxidation products as xenobiotics. ACS Symposium Series 254. J.W. Finley, Ed. Addis, P.B. 1986. Occurence of lipid oxidation products in foods. Fd. Chem. Toxic. 24:1021. Ansari, G.A.S. and Smith, L.L. 1979. High performance liquid chromatography of autoxidation products. J. Chromatog. 175:307 Baeuerlen, R., Brody, H. and Erickson, D. 1968. Frying fats and their uses. Bakers Digest 12:51 Barbut, 8., Josephson, D. and Maurer, A.J. 1985. Antioxidant properties of rosemary oleoresin in turkey sausage. J. Food Sci. 50:1356. Bascoul, J., Domerque, N. and Crastes de Paulet, A. 1983. Pre aration of autoxidized cholesterol mixtures from (4- ‘0) cholesterol and cholesteryl fatty acid esters. J. Steroid Biochem. 19:1779. Bascoul, J., Domerque, R., Olle, H. and Crastes de Paulet, A. 1986. Autoxidation of cholesterol in tallows heated under deep frying conditions: evaluation of oxysterols by GLC and TLC-FID. Lipids 21:383. Berner, D.L. and Jacobson, G.A. 1973 0.8. patent 3,732,111. Black, H.S. and Lo, W.-B. 1971. Formation of a carcinogen in human skin irradiated with ultraviolet light. Nature 234:306. Bracco, 0., Loliger, J. and Viret, J.L. 1981. Production and uses of natural antioxidants. J. Amer. Oil Chem. Soc. 58:686. Brun, J.P., Burt, R. and Nicole, B.W. 1985. "Role of Fats in Human Nutrition", P.B. Padley and J. Podmore, Eds. Ellis Horwood Ltd. Chichester, England. Burton, G.W. and Ingold, K.U. 1984. B-Carotene: An unusual type of lipid andtioxidant. Science 224:569. 112 113 Chang, S.S., Biserka, O.M., Hsieh, O.A.L., Huang, C.L. 1977. Natural antioxidants from rosemary and sage. J. Food Sci. 42:1102. Chicoye, E., Powrie, W.D. and Fennema, O. 1968a. Isolation and characterization of cholesterol- 56,68-oxide from an aerated aqueous dispersion of cholesterol. Lipids 3:335. Chicoye, E., Powrie, W.D. and Fennema, O. 1968b. Photoxidation of cholesterol in spray-dried egg yolk upon irradiation. J. Food Sci. 33:581. Chipault, J.R., Hizuno, G.R., Hawkins, J.H. and Lundberg, W.O. 1952. The antioxidant properties of natural spices. Food Res. 17:46. Csiky, I. 1982. Trace enrichment and separation of cholesterol oxidation products by adsorption high performance liquid chromatography. J. Chromatog. 241:381. Fractionated beef tallow as a deep fat frying medimm for french fries. J. Food Sci. 46:452. Draper, H.H. 1980. Nutrient interrelationships. In "Vitamin E- A.Comprehensive Treatise", Part 5D. L.J. Hachlin, Ed. In: "Basic and Clinical Nutrition", Marcel Dekker, Inc, New‘York, V61. 1, pp. 272-288. Fieser, L.F. 1953a. Cholesterol and companions III. Cholestanol, lathosterol and ketone 104. J. Am. Chem. Soc. 75:4395. Fieser, L.F. 1953b. Cholesterol and companions IV. Steroid dibromides. J. Am. Chem. Soc. 75:5422. Finocchiaro, E.T., Lee, E. and Richardson, T. 1984. Identification and quantitation of cholesterol oxides in grated cheese and bleached butteroil. J. Am. Oil Chem. Soc. 61(5):877. Finocchiaro, E.T. and Richardson, T. 1983. Sterol oxides in foodstuffs: a review. J. Food Prot. 46(10):917. Flanagan, V.P., Ferretti, A., Schwartz, D.P. and Ruth, J.H. 1975. Characterization of 2 steroidal ketones and 2 isoprenoid alcohols in dairy products. J. Lipid Res. 16:97. Foote, C.S. 1976. Photosensitized oxidation and singlet oxygen: consequences in bioloical systems. Ch. 3. In ”Free Radicals in Biology“, vol. II,‘H.A. Pryor, Ed. Academic Press, New York. 114 Fritsch, C.W. 1981. Measurements of frying fat deterioration: A brief review. J. Am. Oil Chem. Soc. 58(3):272. Fritsch, C.W., Egberg, D.C. and Magnuson, J.S. 1979. Changes in dielectic constant a measure of frying oil deterioration. J. Am. Oil Chem. Soc. 56(8):?46. Gemert, L.J. van, Hoekman, H. 1986. Effects of type and duration of use of frying fat on the sensory quality of croquettes and french fried potatos. Vbedingsmiddelentechnologie l9(1):28. Gibbons, G.F., Mitropoulos, K.A. and Myant, N.B. 1982. "Biochemistry of Cholesterol". Elsevier Biomedical Press, New York. Gray, J.I. 1978. Measurement of lipid oxidation: A review. J..Am. Oil Chem. Soc. 55:539. Gray, M.F., Lawrie, T.D.V. and Brooks, c.r.w. 1971. Isolation and identification of cholesterol-a-epoxide and other minor sterols in.human serum. Lipids 6:836 Gummlka, J., St. Pyrek, J. and Smith, L.L. 1982. Interception of discrete oxygen species in aqueous media by cholesterol: Formation of cholesterol epoxides and secosterols. Lipids l7(3):197. Haumann, B.F. 1987. Fast foods: trends in frying fat usage. J. Am. Oil Chem. Soc. 64(6):789. Herian, A.M. and Lee, K. 1985. 7G- and.7B-hydroxychol- esterols formed in a dry egg nog mix exposed to fluorescent light. J. Food Sci. 50:276. Higley, N.A. and Taylor, S.L. 1986a. Cholesterol oxides in processed meats. Meat Sci. 16:175. Higley, N.A., Beery, J.T., Taylor, S.L., Porter, J.w., Dziuba, J.A. and Lalich, J.J. 1986b. Comparative atherogenic effects of cholesterol and cholesterol oxides. In “Atherosclerosis”. Elsevier Scientific Publishers, Ireland, Ltd. pp. 91-104. Horvath, C. 1966. Quantitative determination of cholesterol in autoxidation mixtures by thin-layer chromatography. J. Chromatog. 22:52. Houlihan, C.M., Ho, C.-T. and Chang, 8.8. 1984. Elucidation of the chemical structure of a novel antioxidant, rosmaridiphenol, isolated from rosemary. J. Am. Oil. Chem. Soc. 61(6):1036. 115 Houlihan, C.M., Ho, C.T. and Chang, 8.8. 1985. The structure of rosemariquinone- a new antioxidant- isolated from Rosmarinus officinalis L. J. Am Oil Chem. Soc. 62(1):96. Hurst, H.J., Aleo, M.D. and Marir, R.A., Jr. High performance liquid chromatographic analysis of cholesterol in milk. J. Dairy Sci. 66:2192. Igene, J.O., King, J.A., Pearson, A.M. and Gray, J.I. 1979. Influence of heme pigments, nitrite, and non-heme iron on development of warmed-over flavor (WOF) in cooked meat. J. Agric. Food Chem. 27(4):838. Imai, H., Wertheson, N.T., Subramanyam, V., LeQuesne, P.W., Soloway, A. and Kanisawa, M. 1980. Angiotoxicity of oxygenated sterols and possible precursors. Science 207(2):651. Inatani, R., Nakatani, N. and Fuwa, H. 1983. Antioxidant effect of the constituents of rosemary (Rosmarinus officinalis L.) and their derivatives. Agric. Biol. Chem. 47(3):521. Kandutsch,.A., Chen, H.H. and Heiniger, H.J. 1978. Biological activity of some oxygenated sterols. Science 201:498. Kimura, M., Jin, Y. and Sawaya, T. 1979. Autoxidation of cholesterol and behavior of its hydroperoxide in aqueous medium. Chem. Pharm. Bull. (Tokyo) 27:710. Korahani, V. Bascoul, J. and Crastes de Paulet,.A. 1981. Capillary column gas-liquid chromatographic analysis of cholesterol derivatives. Application to the autoxidation products of cholesterol. 1981. J. Chromatog. 211:392. Korahani, V., Bascoul, J. and Crastes de Paulet, A. 1982. Autoxidation of fatty acid esters in solid state and aqueous dispersion. Lipids 17:703. 'Kou, I.L. and Holmes, R.P. 1985. The analysis of 25-hydroxycholesterol in plasma and cholesterol containing foods by high performance liquid chromatography. J. Chromatog. 330:339. Krull, H.J., Thompson, M. and Arya,.A. 1984. Chromatographic analysis of phospholipid and cholesterol oxidation. Talanta 31:489. 116 Lee, E.C. and Min, D.B. 1988. Quenching mechanism of B-carotene on the chlorophyll sensitized photooxidation of soybean oil. J. Food Sci. 53(6):1894. Lee, R., Herian, A.M. and Higley, N.A. 1985. Sterol oxidaiton products in french fries and stored potato chips. J. Food Prot. 48(2):158. Livingston, R. 1961. Photochemical autoxidation. In: "Autoxidation and Antioxidants”, W.O. Lundberg, Ed., Interscience Publishers, John Wiley and Sons, New York, vol. 1, pp. 249-298. Love, J.D. and Pearson, A.M. Netmyoglobin and nonheme iron as prooxidants in egg-yolk phospholipid dispersions and cooked meat. J. Agric. Food Chem. 24:494. Luby, J.M. 1982. Photooxidation of cholesterol in butter. H.S. Thesis, Michigan State University, East Lansing, MI. Luby, J.M., Gray, J.I., Harte, B.R. and Ryan, T.C. 1986. Effects of packaging and light source on the oxidative stability of cholesterol in butter. J. Food Sci. 51:904. MacNeil, J.H., Dimick, P.S. and Mast, M.G. 1973. Use of chemical compounds and a rosemary spice extract in quality maintenance of deboned poultry meat. J. Food Sci. 38:1080. Maerker, G. and Unruh, J. Jr. 1986a. Cholesterol oxides 1. Isolation and determination of some cholesterol oxidation products. J. Am. Oil Chem. Soc. 63:767. Maerker, G. and Bunick, F.J. 1986b. Cholesterol oxides II. Measurement of the 5,6-epoxides during cholesterol oxidation in aqueous dispersions. J. Am. Oil Chem. Soc. 63(6):771. Maerker, G. 1987. Cholesterol autoxidation- current status. J. Am. Oil Chem. Soc. 64(3):388. McCay, P.B. and King, H.H. 1980. Biochemical funtion. In "Vitamin E-.A Comprehensive Treatise", L.J. Machlin, Ed. Part 5E. In “Basic Clinical Nutrition“, Marcel Dekker, Inc. New York. vol. 1. pp. 289-317. MbCleur, R.H. and Jungalwala, P.B. 1976. High performance liquid chromatographic analysis of glycossphingolipids and phospholipids. Adv. Exp. 117 McGill, B.A. 1980. The chemistry of frying. Baker's Digest 6:38. Missler, S.R., wasilchuh, B.A. and Merrit, C. Jr. 1985. Separation and identification of cholesterol oxidation products in dried egg preparations. J. Food Sci. 50:595. Naber, E.C. and Biggert, M.D. 1983. Analysis for and generation of cholesterol oxidation products in egg yolk by heat treatment. Poultry Sci. 64:341. Nourooz-zadeh, J. and Appelqvist, L.A. 1987. Cholesterol oxides in Swedish foods and food ingredients: Fresh eggs and dehydrated egg products. J. Food Sci. 52(1):57. Park, S.W. and Addis, P.B. 1985a. Capillary column gas-liquid chromatographic resolution of oxidized cholesterol derivatives. Anal. Biochem. 149(3):275. Park, S.W. and Addis, P.B. 1985b. HPLC determination of C-7 oxidized derivatives in foods. J. Food Sci. 50:1437. Park, S.W. and Addis, P.B. 1986a. Further investigation of oxidized cholesterol derivatives in heated fats. J. Food Sci. 51(5):1380. Park, S.W. and Addis, P.B. 1986b. Identification and quantitative estimation of oxidized cholesterol derivatives in heated tallow. J. Agric. Food Chem. 34(4):653. Park, s.w. and Addis, P.B. 1987. Cholesterol oxidation products in some muscle foods. J. Food Sci. 52(6):1500. Parsons, P.G. and Goss, P. 1978. Chromosome damage and DNA repair induced in human fibroblasts by UV and cholesterol oxide. Aust. J. Exp. Biol. Med. Sci. 56(3):287. Pearson, A.M., Gray, J.I., Helzak, A.M. and Horenstein, N.A. 1983. Safety implications of oxidized lipids in muscle foods. Food Technol. 37:121. Peng, 8.x. and Taylor, C.B. 1984. Cholesterol autoxidation, health and arteriosclerosis. ‘Wld. Rev. Nutr. Diet. 44:117. Petrakis, N.L., Gruenke, L.D., Craig, J.C. 1981 Cancer Res. 41:2563. 118 Pryor, W.A. and Lightsey, J.W. 1981. Mechanisms of nitrogen dioxide reactions: initiation of lipid peroxidation and the production of nitrous acid. Science 214(10):435. Ryan, T.C. 1982. Oxidation of cholesterol in heated tallow. M.S. Thesis, Michigan State University, East Lansing, MI. Ryan, T.C., Gray, J.I. and Morton, I.D. 1981. Oxidation of cholesterol in heated tallow. J. Sci. Food Agric. 32:305. Sabine, J.R. 1977. "Cholesterol”. Marcel Dekker Inc., New York. Sato, K. and Hegarty, G.R. 1971. Warmed-over flavor in cooked meats. J. Food Sci. 36:1098. Schenk, G.O. and Koch, E. 1960. Elektrochem. 64:170. Sevanian, A-, Mead, J.F. and Stein, R.A. 1979. Epoxides as products of lipid autoxidaiton in rat lungs. Lipids 14:634. Shen, C.S.J. and Sheppard, A.J. 1983. High performance liquid chromatography of some biologically important cholesterol oxides. Chromatographia 17(9):469. Smith, L.L., Smart, V.B. and.Ansari, G.A.S. 1979. Mutagenic cholesterol preparations. Mut. Res. 68:23. Smith, L.L. and vanLier, J.E. 1970. Sterol metabolism- part 9. 26-Hydroxycholesterol levels in the human aorta. Atherosclerosis 12:1. Smith, L.L. 1981. "Cholesterol Autoxidation“. Plenum Press, New York. Smith, L.M.,'Clifford,.A.J., Hamblin, C.L. and Creveling, R.H. 1986. Changes in physical and chemical properties of shortenings used for commercial deep-fat frying. J. Am. Oil Chem. Soc. 63(8):1017. Smith, L.L., Rulig, M.J. 1975. On the derivation of carcinogenic sterols from cholesterol. Cancer Biochem. Biophys. 1:79. Sugino, R., Terao, J., Murakami, H. and Matsushita, S. 1986. High performance liquid chromatographic method for the quantification of cholesterol epoxides in spray-dried egg. J. Agric. Food Chem. 34(1):37. 119 Taylor, C.B., Peng, S.K., worthessen, N.T., Tham, P. and Lee, E.T. 1979. Spontaneously occuring angiotoxic derivatives of cholesterol. Am. J. Clin. Nutri. 32(1):40. Teng, J.I., Xulig, M.J. and Smith, L.L. 1973. Sterol metabolism. XXII gas chromatographic differentiation among cholesterol hydroperoxides. J. Chromatog. 75:108. Tsai, L.S., Ijichi, R., Hudson, C.A. and Meehan, J.J. 1980. A method for the quantitative estimation of cholesterol a-oxide in eggs. Lipids 15:124. Tsai, L.S. and Hudson, CMA. 1981. HPLC of oxygenated cholesterols and related compounds. J. Am. Oil Chem. Soc. 58(10):931. Tsai, L.S. and Hudson, C.A. 1984. Cholesterol oxides in commercial dry egg products: Isolation and Identification. J. Food Sci. 49:1245. Tsai, L.S. and Hudson, C.A. 1985. Cholesterol oxides in commercial dry egg products: Quantitation. J. Food Sci. 50:229. van Lier, J.E. and Smith, L.L. 1968. Sterol metabolism II. Gas chromatographic recognition of cholesterol metabolites and artifacts. Anal. Biochem. 24:419. van Lier, J.E. and Smith, L.L. 1970. Sterol metabolism XI. Thermal decomposition of some cholesterol hydroperoxides . Steroids 15(4) : 485 . weiss, T.J. 1983. ”Food Oils and Their Uses”. Avi Publishing Company, Inc., westport, Conn. Wu, J.H., Lee, H.H., Ho, C.T. and Chang, S. Elucidation of the chemical structures of natural antioxidant isolated from rosemary. J. Am. Oil Chem. Soc. 59(8):339. "I71111111171411.1111