Wide-Bore Capillary Gas Chromatographic Method for Quantification of Cholesterol Oxidation Products in Egg Yolk Powder

A wide-bore capillary gas chromatographic (GC) method was developed for quantification of cholesterol oxidation products in egg yolk powder. Baseline separations were achieved between 7α- and 7β-hydroxycholesterol, cholesterol, cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide and 7-ketocholesterol chromatographed as the trimethylsilylated sterols. Excellent response linearity was obtained for each cholesterol oxidation product, permitting reliable quantification. Recoveries from freeze-dried egg yolk spiked with various levels of cholesterol oxidation products ranged from 78.2 to 95.1%. The coefficients of variation compared favorably to those for packed column GC methods.     

Influence of packaging atmosphere on the formation of cholesterol oxides in γ-irradiated egg powder

In a previous work the effect of ionising radiation on formation of cholesterol oxidation products (COPs) under aerobic conditions was studied. In the present work the influence of aerobic and anoxic conditions have been comparatively investigated to study the chemical changes of cholesterol in γ-irradiated egg powder. The irradiation treatment was carried out with powdered egg packed under air and also under vacuum in polyethylene (PE) bags and in laminated, oxygen impermeable three-layer (polyester-aluminium-polyethylene) foil to dosage levels 2 and 4 kGy at room temperature. The cholesterol oxidation is demonstrated by thin-layer chromatograms. In the egg powder wrapped in PE bags the predominant cholesterol derivatives?7-hydroxycholesterol isomers (α and β)—accumulated in significant amounts (increasing with dose) while vacuum-packaging in laminated foil considerably suppressed the quantity of these products and prevented the formation of cholesterol 5α, 6α-epoxide as well as 7-ketocholesterol. Little or no change was observed in non-irradiated (control) vacuum-packed egg powder stored at approximately 22°C for up to 5 months. Peroxide values showed changes parallel to the formation of COPs.     

Short communication: Cholesterol oxidation products in traditional buttermilk

The aim of this study was to quantitatively and qualitatively assess the content of cholesterol oxidation products in traditional buttermilk after butter production. Cholesterol oxidation products (COP) exhibit a wide spectrum of biological activity, including cytotoxic, carcinogenic, and pro-oxidative properties. Buttermilk has about 2 mg of COP/kg of fat, including 7β-hydroxycholesterol and 5,6α-epoxycholesterol. Buttermilk immediately after production had a relatively high level of 7β-hydroxycholesterol (1.47 mg/kg), which decreased to 0.61 mg/kg after storage for 10 h at 3°C. During storage, the content of 5,6α-epoxycholesterol increased from 0.50 to 1.40 mg/kg. After 10 h of storage, the antioxidant potential of the buttermilk decreased (expressed as radical scavenging ability; change in 1,1-diphenyl-2-picrylhydrazyl = 32.2%). This study showed the presence of COP in fresh and stored buttermilk and the influence of time on changing the direction of cholesterol oxidation.     

7α and 7β-Hydroxycholesterols Formed in a Dry Egg Nog Mix Exposed to Fluorescent Light

Light-induced generation of two cholesterol oxidation products, 7α- and 7β-hydroxycholesterol, was measured in an egg nog mix using an improved isolation procedure and high performance liquid chromatography (HPLC). Dry egg nog mix was exposed continuously to fluorescent light at room temperature for 90 days. The 7α-hydroxycholesterol was formed in consistently greater amounts than the 7β-form. Amounts of both forms increased for up to 80 days (23 ± 2°C), then declined. Mix stored in the dark did not contain detectable amounts of 7α- or 7β-hydroxycholesterol. Purification techniques enabled HPLC quantification of cholesterol oxidation products in a food.     

Effect of processing conditions on the oxidation of cholesterol in ghee

The cholesterol oxidation products (COP) in fat-rich dairy products were identified and quantified. Fresh cream contained no COP whereas fresh butter contained trace levels of 7β-hydroxy- and 5,6α-epoxycholesterol. Low levels of various major COP were present in ghee (clarified butter fat). Intermittent heating and frying of ghee induced severe oxidation of cholesterol and a number of COP were detected by GC and TLC. Most atherogenic COP. viz 25-hydroxycholesterol and cholestantriol, were formed more in intermittently heated ghee (8.1-9.2% of the total COP) than in fried ghee samples (7.1 %).     

High-Resolution NMR Detection of Cholesterol Oxides in Spray-Dried Egg Yolk

A new approach, alternative to currently available methods, for studying cholesterol oxidation in egg powder is reported. The quantitative analysis of cholesterol oxides was carried out by high-resolution proton nuclear magnetic resonance (¹H-NMR). The following oxides were isolated from spray-dried egg powder by rapid chromatographic procedures and detected by selecting “marker”¹H-NMR signals: 7-ke-tocholesterol, 7α-hydroxy-cholesterol, 7β-hydroxy-cholesterol, cholesterol-α-epoxide and cholesterol-β-epoxide. The quantified cholesterol derivatives ranged from 4.9 to 9.1 ppm with a detection limit of 0.3 ppm (5 μ.g from 16g of matrix). The main usefulness of the method should be in investigating intermediates and products due to chemical transformation of cholesterol during storage and heating of foodstuffs.     

气质联用法测定反复冻融畜禽肉中胆固醇及其氧化物含量

本文以畜禽肉中胆固醇及胆固醇氧化物、水分、丙二醛指标变化,考察了反复冻融对畜禽肉品质的影响。采用气相色谱-串联质谱/质谱(GC-MS/MS)测定方法,对反复冻融7次(新鲜肉记为冻融0次)后鸡肉、猪肉和牛肉中胆固醇及5种胆固醇氧化物(25-羟基胆固醇、7β-羟基胆固醇、20α-羟基胆固醇、5α,6α-环氧化胆固醇、7-酮基胆固醇)的含量进行了测定。结果表明,随着冻融次数的增加,胆固醇含量逐渐降低,其中牛肉中的胆固醇含量减少尤为明显(减少量为32.47 μg/g)。在反复冻融的过程中,胆固醇氧化物的含量先逐渐增加,后趋于平稳,其中鸡腿肉中7β-羟基胆固醇含量增加最多(增加量为0.601 μg/g)。同时,通过对畜禽肉中脂质过氧化过程中的水分含量和丙二醛含量进行考察,鸡胸肉中的水分减少最多(减少量为14.472 g/100 g),牛肉中的丙二醛增加量最多(增量为1.004 μg/g)。反复冻融会导致畜禽肉胆固醇氧化增加,品质下降。     

Antioxidant Inhibition of Cholesterol Oxidation in a Spray-Dried Food System during Accelerated Storage

Spray-dried egg yolk was used to evaluate antioxidant inhibition of cholesterol oxidation during accelerated (CU⁺² and heat) storage. Liquid yolk was treated with equimolar amounts of BHA (0.01%, w/w of lipid), ascorbyl palmitate (0.023%), or a tocopherol blend (0.023%). Yolk batches were spray-dried, stored at 60 ± 2°C for up to 28 days, and analyzed for cholesterol oxidation products (COPS) using gas chroma-tography. COP levels generally increased during storage with 7-ketocholesterol predominant. Significant antioxidant effects were manifest in decreased levels of 7-ketocholesterol, 7α- and 7β-hydroxycholesterol; while cholestane-triol and cholesterol-5,6-epoxide levels were not affected. All antioxidants showed significant inhibitive effects relative to the control, and tocopherols were more effective than ascorbyl palmitate.     

Cholesterol Oxidation Products in Some Muscle Foods

Cholesterol oxidation products were estimated in some meat products by capillary gas chromatography after trimethylsilylation. Peak identities were confirmed by mass spectrometry. Freeze-dried pork, stored in contact with air at 22°C for ca. 3 yr, revealed 7α- and 7β-hydroxycholesterol, 7-ketocholesterol, α- and β-epoxide and cholestane-triol. The total concentration of oxidation products reached almost half of the remaining cholesterol content with 7-ketocholesterol as the predominant species. Some oxidation products were noted at a few ppm levels in broiled beef steaks, but not in precooked beef products. As rancidity development advanced in comminuted and cooked meats during storage, the oxidation of cholesterol became apparent.     

Lipid and cholesterol oxidation in chicken meat are inhibited by sage but not by garlic

The effects of the addition of sage and garlic in chicken meat on lipid and cholesterol oxidation, having as prooxidant factors the addition of salt, thermal treatment, and frozen storage, were evaluated. The content of unsaturated fatty acids did not change in the presence of sage; on the contrary, with garlic, the content of these fatty acids decreased after cooking and storage. Hexanal and pentanal contents were lower in patties containing sage, and higher in those with garlic. The 7-ketocholesterol was the cholesterol oxide found in higher amount in raw chicken on day 0, while the formation of 7β- and 7α-hydroxycholesterol was verified only from day 30 on. Cooking and storage resulted in increase of total cholesterol oxides and decrease of α- and γ-tocopherol. Sage was effective in controlling lipid and cholesterol oxidation, minimizing the prooxidant effects of salt, cooking, and storage. However, garlic presented no effect as antioxidant and accelerated lipid oxidation. PRACTICAL APPLICATION: The addition of sage to chicken meat (0.1 g/100 g) is a good alternative to prevent and delay the formation of compounds derived from lipid oxidation that are responsible for off-flavors and loss of nutritional quality during long-term frozen storage. Care must be taken when using garlic to seasoning chicken meat products, such as hamburgers and meatballs, especially cooked or precooked due to its potential to promote lipid oxidation and consequently raising the risk of having the product rejected by the consumer.     

Effect of cooking and storage on lipid oxidation and development of cholesterol oxidation products in water buffalo meat

Buffalo meat was subjected to two cooking methods viz. broiling and pressure cooking and two storage procedures viz. refrigerated (4 °C) storage for six days and frozen (-10 °C) storage for 90 days. Changes in lipid oxidation and development of cholesterol oxidation products were studied in raw as well as cooked meat samples. Total lipid, phospholipid, cholesterol, free fatty acid, glycolipid and glyceride contents increased significantly on cooking of meat but did not show any significant changes during either refrigerated or frozen storage except for free fatty acid content which showed an increase. The TBA values also increased during storage but not to the extent of indicating rancidity. Cholesterol oxidation products separated by thin layer chromatography were: cholestanetriol, 7-α-hydroxycholesterol, 19-hydroxycholesterol, 7-ketocholesterol, cholesterol-α-epoxide, cholesterol-β-epoxide and an unidentified fraction. All these fractions, except for the unidentified fraction, increased on cooking and storage. The cholesterol-β-epoxide fraction was resistant to changes. Changes in broiled meat were more pronounced compared to pressure cooked meat. Frozen storage did not prevent the development of cholesterol oxidation products in buffalo meat.     

LIPID AND CHOLESTEROL OXIDATION,COLOR CHANGES,AND VOLATILE PRODUCTION IN IRRADIATED RAW PORK BATTERS WITH DIFFERENT FAT CONTENT1

An emulsion-type product was prepared to determine the effect of irradiation on lipid and cholesterol oxidation, color change, and volatile production in raw pork with different fat contents. Lipid oxidation increased with an increase in fat content or irradiation dose. Irradiated batters had higher cholesterol oxides than nonirradiated, and the major cholesterol oxides formed in irradiated pork batters were 7α- and 7β-hydroxycholesterol. Hunter color a- and b-values of raw pork batters were decreased by irradiation regardless of fat content. Irradiation increased the amount ofvolatiles significantly. Although lipid oxidation of high fat products (10 and 15% fat) was higher than that of low fat products (4%), high fat products did not always produce greater amount of volatiles. In summary, irradiation increased lipid and cholesterol oxidation, volatiles production and had detrimental effects on the color of raw pork batters under aerobic condition.     

DIETARY OILS AND TOCOPHEROL SUPPLEMENTATION ON CHOLESTEROL OXIDE FORMATION IN FREEZE-DRIED CHICKEN MEAT DURING STORAGE

The influence of dietary oils [fish (FO), flax (LO), palm (PO) and sunflower (SO) oil] and tocohperol supplementation on the oxidation of cholesterol and lipids of chicken meat was investigated. Lipid oxidation in chicken meat powders was determined by thiobarbituric acid reactive substances (TBARS) and cholesterol oxidation products were estimated by capillary gas chromatography (GC) after trimethylsilylation. Peak identities from GC were confirmed by mass spectrometry. Freeze-dried chicken meat powder, stored in contact with air at room temperature for four months showed the presence of 7α- and 7β-hydroxycholesterol, 7-ketocholesterol, α and β-epoxide, and cholestane-triol. Total cholesterol oxides contents significantly (P < 0.05) increased with storage. The amount of cholesterol oxides in the meat powder was the highest in FO and SO group, and lowest in PO group. Lipid and cholesterol oxidation were accelerated by the presence of long-chain PUFA. Lipid and cholesterol stability of chicken meat were improved by the dietary supplementation of tocopherol.     

Separation and Identification of Cholesterol Oxidation Products in Dried Egg Preparations

A technique is described for the simultaneous determination of a wide range of angiotoxic sterols in foods produced by the oxidation of cholesterol. Gas chromatography using on-column injection onto a bonded phase fused silica capillary column gave excellent separation of the oxides examined. Combined gas chromatography- mass spectrometry was used to confirm the identities of the trimethylsilyl ether derivatives of the various sterols and 6-ketocholestanol was found to be an acceptable internal standard for determination. Analysis of powdered scrambled egg mix showed the presence of several cholesterol oxides, with cholesterol-5α,6α-epoxide, cholesterol-5β,6β-epoxide and 7-ketocholesterol predominating. Other atherogenic sterols identified included 25-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol and 5α-cholestane-3β,5,6β-triol.     

Effect of different cooking methods on lipid oxidation and formation of free cholesterol oxidation products (COPs) in Latissimus dorsi muscle of Iberian pigs

The aim of this work was to study the influence of different cooking methods (grilled (GR), fried (FP), microwave (MW) and roasted (RO)) on lipid oxidation and formation of free cholesterol oxidation products (COPs) of meat from Iberian pigs that have been fed on an intensive system. Moisture and total lipid content, TBARs, hexanal and COPs were measured in Latissimus dorsi muscle samples. Cooking did not produce changes in total lipid content in meat but induced significantly higher lipid oxidation (TBARs and hexanal values) (p < 0.001) and cholesterol oxidation (COPs) (p < 0.01). When the different cooking methods were studied, the grilled method was the least affected by lipid oxidation (TBARs and hexanal) compared to the others. There were no significant differences among different cooking methods on COPs values. The most abundant cholesterol oxides were both 7α-hydroxycholesterol and 7β-hydroxycholesterol in all groups studied.     

Influence of carcass weight on instrumental and sensory lamb meat quality in intensive production systems

The effects of cooking viz. pressure-cooking and broiling and storage at 4 °C for six days and -10 °C for 90 days on lipid oxidation and development of cholesterol oxidation products in mutton were studied. Results revealed that cooking of meat significantly increased the total lipids, phospholipids, cholesterol, glycolipids, free fatty acids and glycerides, but they did not change during refrigerated and frozen storage. The TBA values increased on cooking and during storage. However, the values were below the threshold level for rancidity development. The following cholesterol oxidation products were separated by thin layer chromatography cholestanetriol, 7-α-hydroxy cholesterol, 19-hydroxycholesterol, 7-ketocholesterol, cholesterol-α-epoxide, cholesterol-β-epoxide and an unidentified fraction. All these fractions except the unidentified fraction increased on cooking. On refrigerated and even on frozen storage all these fractions increased except the unidentified fraction, which showed a concomitant reduction. The changes in broiled meat were more pronounced compared to pressure-cooked meat. Results clearly indicated that even frozen storage of cooked meat did not prevent the development of cholesterol oxidation products.     

E Vitamer Fraction in Rice Bran Inhibits Autooxidation of Cholesterol and Linoleic Acid in Emulsified System during Incubation

ABSTRACT: The antioxidant activities of E vitamer fraction extracted from rice bran were investigated and compared with α-tocopherol at 2 concentration levels (16 ppm and 160 ppm) in the emulsified cholesterol and linoleic acid systems. 7-Ketocholesterol, 7α-hydroxycholesterol (7α-OH), 7β-hydroxycholesterol (7β-OH), 25-hydroxycholesterol (25-OH), and 5,6-epoxycholesterol (5,6-EP) were found at different incubation times (0, 6, 12, 24, and 48 h) in the emulsified cholesterol system accelerated by using 2, 2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) at 80°C and pH 5.5. Samples treated with 160 ppm of E vitamer fraction exhibited a significant antioxidant activity by inhibiting the formation of cholesterol oxidation products (COPs). Addition of E vitamer fraction to the emulsified linoleic acid model at 37°C significantly inhibited the hydroperoxide formation and decomposition more than that of α-tocopherol. The former was more effective in inhibiting the primary and secondary oxidations of the polyunsaturated fatty acid (PUFA). No significant differences ( P < 0.05) were found in inhibition of the linoleic acid autoxidation between the E vitamer fraction and the α-tocopherol at 16 and 160 ppm, respectively. E Vitamer fraction was most effective in inhibiting both emulsified cholesterol and linoleic acid. These results suggested that compounds inhibiting cholesterol autoxidation and PUFA autoxidation in both food and biological systems were present in the rice bran extract. Thus, the addition of E vitamer fraction to animal- derived muscle food may be a more compelling way to replace the synthetic antioxidants currently used in the food industry because the former is both natural and effective in inhibiting both cholesterol and PUFA autoxidation.     

Oxidation of cholesterol in mayonnaise during storage

The oxidative stability of cholesterol in commercial mayonnaise under different storage conditions was evaluated by measuring cholesterol oxides (COs), 7-ketocholesterol (7-Keto), 25-hydroxycholesterol (25-OH), 7α-hydroxycholesterol (7α-OH) and 7β-hydroxycholesterol (7β-OH) using HPLC. Oxidation of cholesterol was indicated within about 15 days after manufacture by the presence of 7-Keto. Oxidation increased during storage at 4 and 25 °C (being greater at 25 °C) for 165 days in darkness, as indicated by the presence of 7-Keto, 25-OH, 7α-OH and 7β-OH. There was a strong correlation between COs and PV (peroxide value) [r²=0.95 (4 °C) and r²=0.96 (25 °C)] during the process of oxidation. The pattern of fatty acids was not affected during the period of the experiment. Temperature and time were important factors in the oxidative stability of cholesterol. Total formation of COs during 165 days was 20.3 μg/g at 4 °C and 30.2 μg/g at 25 °C.     

Quantification of 5α-Cholestane-3β,5,6β-triol and Other Cholesterol Oxidation Products in Fast Food French Fried Potatoes

Concerns about toxicity of cholesterol oxidation products (COPS) prompted this study. Two restaurants were selected which use animal-vegetable (A/V) shortening for deep-frying. The survey of COPS for 30 days indicated values for total COPS in French fried potatoes were 20 ± 9 ppm to 24 ± 6 ppm. 5α-Cholestane-3β,5,6β-triol (triol) was identified in French fried potatoes from one restaurant. The mean for triol was 9 ± 3 ppm. Triol, 7α-hydroxycholesterol, 7β-hydroxy-cholesterol and 7-ketocholesterol were confirmed by co-chromatography and mass spectrometry. Triol is one of the most potent of angiotoxic COPS. This and other studies suggested French fried potatoes and other deep fried foods cooked in A/V shortening are a major source of COPS.     

Cholesterol oxides and atherosclerosis: A review

Cholesterol is an important constituent of animal food products and has often been implicated in the etiology of atherosclerosis and coronary heart diseases. Recent reports have, however, shown the possible role of cholesterol oxidation products (COP), rather than cholesterol, in the initiation of atherosclerotic plaque formation. Cholestan-3β,5α,6β-triol and 25-hydroxycholesterol have been reported as the most potent atherogenic agents. Inhibition of HMG-CoA reductase (EC 1.1.1.34) enzyme activity and cholesterol biosynthesis by cholesterol oxidation products has also been thoroughly investigated in cultured cells. Various animal food products, viz meat products, egg products and dairy products (especially butter, butter oil, ghee, cheese etc) have been reported to contain various COP developed during certain processing treatments. The literature on the presence of COP in food products and their cytotoxic and atherogenic effects is reviewed.