Separation and quantitation of cholesterol oxides by HPLC with an evaporative light scattering detector in a model system

A method to analyze cholesterol and 10 of its oxidation products, ranging from the weakly polar cholest-4-ene-3,6-dione to moderately polar cholest-5-ene-3β,7α-diol, in a single run is described. The separation was achieved by normal-phase gradient high-performance liquid chromatography with an evaporative light-scattering detector. This universal mass detector does not detect changes in solvent composition; this makes it possible to employ gradients, an essential technique whenever a wide range of compounds with diverse characteristics is to be separated. Standards at concentrations from 0.1–1.0 μg were separated within 37 min on an alumina/silica column with a gradient elution system that contained dichloromethane, acetonitrile, and water.     

Oxidative decomposition of cholesterol in fish products

Cholesterol oxides in fish products popular in Japan, including salted and dried, boiled and dried and smoked products, were qualitatively and quantitatively determined as trimethylsilyl ether derivatives by gas-liquid chromatography and mass spectrometry. The level of total cholesterol oxides ranged widely between 8.3 ppm in boiled and dried shrimp and 188.0 ppm in boiled and dried anchovy. 7β-Hydroxycholesterol and 7-ketocholesterol were the most prominent oxidative decomposition products of cholesterol. The levels of epimeric epoxides, cholestane triol and 25-hydroxycholesterol were relatively low. To elucidate a mechanism of cholesterol oxidation proceeding during fish processing and subsequent preservation, four model systems, consisting of a mixture of purified cod liver triglycerides plus cholesterol, of a mixture of authentic triolein plus cholesterol, of triolein alone and of cholesterol alone, were stored separately at 25°C in dry air for up to 104 d. The residual fatty acids of the triglycerides, and the cholesterol oxides produced, were recovered and determined. Oxygen uptake remained almost unchanged for the mixture of triolein plus cholesterol. No detectable amount of cholesterol oxides was produced, and the fatty acid content of the residual oleic acid, measured by an internal standard, remained almost unchanged. For the mixture of cod liver triglycerides plus cholesterol, a remarkable increase in oxygen uptake was observed. A continuous increase in the amount of cholesterol oxides was observed, accompanied by a remarkable concurrent decrease in polyunsaturated fatty acid residues, as well as of the oleic acid naturally present. These results strongly suggest that cholesterol oxidation in fish products proceeds in conjunction with oxidative decomposition of the coexisting polyunsaturated fatty acids of fish oils.     

Effect of synthetic antioxidants on cholesterol stability during the thermal-induced oxidation of a polyunsaturated vegetable oil

As a molecule with an unsaturated bond, cholesterol is prone to oxidation. Cholesterol oxidation products (COP) are found in many common foods and have been shown to be atherogenic, cytotoxic, mutagenic, and possibly carcinogenic. Efforts to reduce the formation of oxidation products are considered important during the manufacture and processing of foods. The effect of synthetic antioxidants on cholesterol oxidation has not been extensively studied. We assayed the effect of five commonly used antioxidants—BHT, BHA, the n-propyl ester of 3,4,5-trihydroxy benzoic acid (PG), TBHQ, and 6-ethoxy-1,2-dihydro-2,4-trimethylquinoline (EQ)—on cholesterol stability when oxidation is induced in a Rancimat 679 instrument by bubbling air through the sample at 150°C. The sample consisted of 200 mg cholesterol dispersed in 100 g of a polyunsaturated vegetable oil (soybean oil). Formation of six COP was measured at the induction period, and at the 50 and 100 μS conductivity values. Under the experimental conditions, BHT and TBHQ were the most effective inhibitors of cholesterol oxidation. BHA and EQ were less effective, and PG was unable to prevent cholesterol oxidation. Synthetic antioxidants were more effective in preventing COP formation at the nucleus of the cholesterol structure than at the lateral chain.     

Cholesterol oxidation in heated lard enriched with two levels of cholesterol

Cholesterol oxidation in lard containing two levels of added cholesterol was monitored using capillary gaschromatography. Loss of cholesterol and formation of cholesterol oxidation products (COPs) were measured. Lard samples with 10 times (Test I) and 2 times (Test II) the amount of cholesterol originally found in each batch of lard were heated at 180°C for 10 hr a day for 240 and 160 hr, respectively. Cholesterol steadily decreased throughout the heating period in both tests. Cholesterol loss followed a first-order reaction rate, with a rate constant (k) of −1.18×10⁻³ h⁻¹ for Test I and −9.45×10⁻³ h⁻¹ for Test II. The COPs accumulated during both heating tests. But the amount of COPs formed did not total the amount of cholesterol lost. During heating, thermal degradation of cholesterol likely occurred, and those products were not detected. During cooling, hydroperoxides formed, which further oxidized into the COPs that were detected. The 7-ketocholesterol and 5α,6α-epoxycholesterol were the predominant COPs formed. The isomeric 7α-and 7β-hydroxycholesterols also accumulated in the heating tests. The 3β,5α,6β-cholestantriol was found in very small amounts and the 25-hydroxycholesterol was not detected. Presented in part at the 80th AOCS Annual Meeting, Cincinnati, OH, in May, 1989.     

Effects of frying and storage on cholesterol oxidation in minced meat products

The presence of cholesterol oxidation products (COP) in the diet is a health concern for their various known adverse effects. It is important that the generation of COP be assessed during different stages of production, handling, and storage of meats and meat products so that relevant measures can be taken to minimize the production of COP. In a preliminary study, we investigated the content of COP in the lipids of raw meatballs (50% prok+50% beef), prefried meatballs (50% pork +50% beef), raw hamburger (100% beef), and prefried burger (50% pork+50% beef). Six of the common COP, viz 7α- and 7β-hydroxycholesterol, 7-ketocholesterol, 5α,6α-epoxycholestanol, 5β,6β-epoxycholestanol, and cholestanetriol, were analyzed by gas chromatography (GC) and GC-mass spectroscopy. The total content of these COP was in the range of 7 to 10 μg/g lipids in raw meatballs, prefried meat balls, and raw hamburger, after frying these samples for consumption. The prefried hamburger had ca. 8μg/g lipids of the total COP before frying, and this amount increased to 29 μg/g lipids after frying. During the storage of this fried sample, the total COP increased to 42 and 50 μg/g lipids, after 1 and 2 wk of storage, respectively. The results of this study show that freshly prepared meat products are a minor source of COP in the diet. However, if semiprepared frozen meat products are fried once and then stored for future consumption, the levels of COP can increase considerably, and this may be of concern for certain groups of consumers. Presented in part as a poster at the 88th AOCS Annual Meeting and Exposeattle, WA, May 11–14, 1997.     

Inhibition of cholesterol autoxidation by the nonsaponifiable fraction in rice bran in an aqueous model system

The inhibition of cholesterol autoxidation by nonsaponifiable fraction from rice bran oil (700, 1400, and 2100 ppm) was studied in an aqueous model system for 16 h at pH 5.5 and 80°C. Antioxidant effectiveness was investigated by following the loss of cholesterol and the formation of 7-keto-cholesterol. The changes in levels of vitamin E vitamers and γ-oryzanol in the system were determined during cholesterol autoxidation. The 2100 ppm treatment produced a 92% reduction of 7-ketocholesterol, 1400 ppm an 82% reductions and 700 ppm a 64% reduction after 16 h, whereas without the nonsaponifiable fraction, the samples showed almost complete degradation of cholesterol. Vitamin E vitamers decreased (P<0.05) in all treatments, but γ-oryzanol was not significantly (P<0.05) reduced in the 2100 ppm treatment.     

Capabilities of different cooking oils in prevention of cholesterol oxidation during heating

The potential of various cooking oils to prevent cholesterol degradation and/or oxidation, as measured by the production of 7-ketocholesterol during heating at different temperatures, was studied using a cholesterol model system. In the control group (without cooking oil), cholesterol was relatively stable, and 73% of its initial concentration was present after 30 min of heating at 125°C. Less than 30 and 10% of cholesterol remained at 150 and 175°C after 30 min, respectively, and 10% at 200°C after 10 min. In the treatment group, cholesterol mixed with corn, canola, soybean, or olive oil had significantly improved thermal stability. More than 60 and 40% of cholesterol remained at 150 and 175°C after 30 min, respectively. In the control group, 7-ketocholesterol was produced when samples were heated above 150°C, and levels increased consistently during 30 min of heating. At 175 or 200°C, the level of 7-ketocholesterol did not increase further after reaching the highest level after 10 min of heating. 7-Ketocholesterol is not stable above 175°C, and its degradation rate could be much faster than its production at 200°C. 7-Ketocholesterol was not found in samples of cholesterol mixed with corn oil or laboratory-prepared soybean and rice bran oils until the heating temperature was raised to 175°C for 20 min. The levels of 7-ketocholesterol in those treatment groups were greater than that in the control group at 175°C for 30 min. These oils may increase the thermal stability of 7-ketocholesterol and retard its degradation rate.     

Active oxygen from CeO2 and its role in catalysed soot oxidation

An advanced TAP reactor is used for the first time to study CeO₂ catalysed soot oxidation using labelled oxygen. In the absence of catalyst oxidation takes place above 500°C and mainly labelled oxidation species (CO and CO₂) were observed. In the presence of catalyst it is shown that the gas-phase labelled oxygen replaces non-labelled lattice oxygen of CeO₂ creating highly active oxygen. This highly active non-labelled oxygen reacts with soot giving CO and CO₂. The creation of such active oxygen species starts from 400°C and it will decrease the soot oxidation temperature. The rate of gas-phase oxygen exchange by the CeO₂ lattice oxygen and the rate of this very active lattice oxygen with soot are much faster than the reaction rate of ¹⁸O₂ reacting with soot directly. Similarly the rate of active oxygen reaction with soot is much faster than the rate of its combination giving gas-phase O₂.     

Linalyl acetate and methyl silicone effects on cholesterol and triglyceride oxidation in heated lard

The effectiveness of linalyl acetate (LA) at 0.02 and 0.04% as a high-temperature antioxidant was tested in lard with two levels (2 times and 10 times) of added cholesterol. Lard samples with and without added LA or methyl silicone (MS, 0.2 and 1.0 ppm) and a control with no additives were heated at 180°C for 10 hr/day for up to 240 hr. The loss of cholesterol, the formation of cholesterol oxidation products (COP) and the percentage fatty acid retention were measured during the heating periods. Addition of 1.0 ppm MS was significantly effective in reducing the loss of fatty acids (p<0.0005) in lard with 2 times the added cholesterol, but MS at 0.2 ppm was not effective in lard with 10 times the added cholesterol. Addition of LA at 0.02 and 0.04% had little effect at reducing changes in cholesterol loss, COP formation and fatty acid loss in lard samples with both cholesterol levels. But there was a tendency for MS and 0.04% LA to reduce formation of some COP in lard containing 2 times the added cholesterol and to reduce the loss of cholesterol. Presented in part at the 80th AOCS Annual Meeting, Cincinnati, OH, in May, 1989.     

Liquid-phase oxidation of anthracene in acetic acid with an oxygen/Nitric acid system

Oxidation of anthracene in acetic acid by the oxygen/nitric acid system has been studied and an attempt has been made to ascertain a practical limit of the amount of solvent keeping the commercial prospects in view. The effects of other reaction parameters such as flow rate, amount of nitric acid and water, residence time, etc., have been investigated. While opting for a solvent/substrate ratio of 10, the optimum conversion of anthracene to anthraquinone free from nitro-compounds has been found to be 91% with purity of 98.7%, acceptable to dye-stuff industries.     

亚油酸乳状液体系氧化模型与求解

研究了恒表面氧压静置式无限氧补偿条件下乳状液中亚油酸的氧化,通过综合考虑气液边界传质阻力、水相扩散、油水边界乳化剂膜边界层阻力、多不饱和脂肪酸(PUFA)自催化氧化反应动力学,建立了扩散-氧化数学模型。采用非对称正交配置法处理特殊边界,求解偏微分方程组,计算了氧化过程中乳状液中氧和亚油酸在垂直于液膜方向的浓度分布,计算方法快捷、有效;结合数学模拟试验和实验值,确定了气液界面氧的液膜传质系数和水相中氧的扩散系数。实验验证该模型能较好地拟合静置式无限氧补偿条件下乳状液中氧的扩散和亚油酸的氧化过程。     

有氧补偿乳状液中多不饱和脂肪酸氧化模型

研究了恒表面氧压搅拌式有氧补偿条件下乳状液中多不饱和脂肪酸(PUFA)的氧化。通过综合考虑气液边界传质阻力、油水边界乳化剂形成的液膜边界层阻力、PUFA自催化氧化反应动力学,建立了相应的扩散-氧化数学模型。实验验证该模型能较好地拟合搅拌式有氧补偿条件下乳状液中氧的扩散和亚油酸的氧化过程,并在乳化剂浓度变化对乳状液中PUFA氧化的影响方面表现出很好的适用性。研究对揭示乳状液中PUFA的氧化机制最终有效控制PUFA的氧化具有重要的理论研究价值。     

Electrochemical and spectral studies on the catalytic oxidation of nitric oxide and nitrite by high-valent manganese porphyrins at an ITO electrode

Catalytic oxidation of nitric oxide and nitrite by water-soluble manganese(III) meso-tetrakis(N-methylpyridinium-4-yl) porphyrin (Mn(III)(4-TMPyP) was first studied at an indium-tin oxide (ITO) electrode in pH 7.4 phosphate buffer solutions. A stepwise oxidation of Mn(III)(4-TMPyP) through high-valent manganese porphyrin species has been observed by electrochemical and spectroelectrochemical (OTTLE) techniques. The formal potential of 0.63 V for the formation of OMn(IV)(4-TMPyP) has been estimated from OTTLE data. The product, oxoMn(IV) porphyrin, was relatively stable decaying slowly to Mn(III)(4-TMPyP) with a first-order rate constant of 3.7 × 10⁻³ s⁻¹. OMn(IV)(4-TMPyP) has been found to oxidize NO catalytically at potentials about 70 mV more negative than that previously reported for OFe(IV)(4-TMPyP) with good selectivity against nitrite. Nitrite was catalytically oxidized at potentials higher than 1.1 V presumably by OMn(V)(4-TMPyP). OMn(IV)(4-TMPyP) was observed as an intermediate species. Nitrate has been confirmed to be a final product of the electrolysis at 1.2 V, while at 0.8 V nitrite left unchanged, demonstrating that OMn(IV)(4-TMPyP) could not oxidize nitrite. A possible schemes of the catalytic oxidation of NO by OMn^IV(4-TMPyP) and NO₂⁻ by OMn(V)(4-TMPyP) have been proposed.     

Identification and quantification of cholesterol oxidation products in canned tuna

Cholesterol oxidation in tuna canned in brine was studied. Gas chromatography-mass spectrometry was used for the detection of the seven major cholesterol oxidation products originating from both direct and indirect oxidation. The total amount of cholesterol oxidation products in the analyzed samples varied considerably, ranging between 40 and 350 μg/g lipids, with the exception of an anomalous sample, that reached a 1600 μg/g level. The lipid content ranged between 0.5 and 1 g/100 g wet product. As most samples did not exceed 100 μg/g lipids, it is possible to assume that the total content of cholesterol oxidation products can be kept below this value if good manufacturing conditions are used, together with a careful choice of the best tuna cuts. The application of principal component analysis to the detected variables confirmed that 7-ketocholesterol is a useful index of the whole oxidation process.     

Quantitation of cholesterol oxidation products in unirradiated and irradiated meats

A method to detect 7-ketocholesterol, cholesterol-5β,6β-epoxide, cholesterol-5α,6α-epoxide, 4-cholesten-3-one, 4,6-cholestadien-3-one and 4-cholestene-3,6-dione in unirradiated and irradiated beef, pork and veal was developed by use of chloroform-methanol-water extraction, solid-phase extraction, column separation, thin-layer chromatography and gas chromatography. This method recovered 78–88% of the cholesterol oxidation products and detected the cholesterol oxidation products at 10 ppb or higher. Irradiation of the meats to a dose of 10 kGy increased these compounds, except 4,6-cholestadien-3-one for all three types of meat, over unirradiated, and except cholesterol-5α,6α-epoxide and 4-cholesten-3-one for the pork. All the cholesterol oxidation products in the unirradiated meats increased during storage at 0–4°C for 2 wk with some exceptions for the pork. The increases of cholesterol oxidation products in stored irradiated meats were greater than those in the unirradiated.     

Electrochemical oxidation of cholesterol in acetonitrile leads to the formation of cholesta-4,6-dien-3-one

Cholesterol was shown to be oxidized at the glassy carbon electrode in an acetonitrile–2-propanol mixture and this oxidation reaction was applied to the determination of serum total cholesterol by high-performance liquid chromatography with electrochemical detection (K. Hojo, H. Hakamata, A. Ito, A. Kotani, C. Furukawa, Y.Y. Hosokawa, F. Kusu, J. Chromatogr. A 1166 (2007) 135–141). To gain insight into the detection mechanisms of cholesterol, an electrolytic product of cholesterol was collected and characterized by infrared spectroscopy, one- and two-dimensional nuclear magnetic resonance, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The three techniques, together with comparisons of literature spectral data, confirmed the formation of cholesta-4,6-dien-3-one. The conversion of cholesterol to cholesta-4,6-dien-3-one, a four-electron, four-proton electrochemical process, has been proposed as an electrochemical oxidation mechanism of cholesterol in acetonitrile.     

Effects of phospholipids on lipid oxidation of a salmon oil model system

Total lipid (TL), neutral lipid (NL), and phospholipid (PL) fractions were extracted from bluefish (Pomatomus saltatrix) white and dark muscle with skin. The effects of each fraction on the oxidative stability of a refined salmon oil model system was measured by monitoring changes in the 2-thiobarbituric acid assay and decreases in the ratio of docosahexaenoic acid (DHA) to palmitic acid (C22:6/C16:0) following incubation at 55°C or 180°C. Phospholipid fractions at 2.5% and 5.0% (wt/wt) of oil improved the oxidative stability of oils incubated at both temperatures compared to controls, TL- and NL-supplemented oils at similar concentrations. Phospholipid fractions exhibiting antioxidant properties contained an average of 34% DHA as compared to only 15% in the NL and TL fractions.     

Enhancing effect of S and F moieties on the performance of Fenton system in the selective oxidation of propane

The selective oxidation of propane to oxygenated products (isopropanol, n-propanol, propionic aldehyde and acetone) mediated by the Fe(II)/H₂O₂ Fenton system at 80 °C in the presence of solid acid and superacid promoters containing S and F moieties has been studied. The occurrence of a radical reaction pathway accounting for the activation of the CH bonds of the propane molecule by OH radicals has been proved by assessing the inhibiting effect of both Cl⁻ and NO₃⁻ radical scavengers and organic (CH₃COOH, CH₃CN, DMSO) reaction media on the reaction pattern. S and F functionalities of several solid agents promote the electron transfer processes controlling the H₂O₂ activation. Any effect of the Brönsted acid features of the solid promoters on the reaction kinetics and pathway has been disregarded.     

Reactivities of active oxygen species and their roles in the catalytic oxidation of inactive hydrocarbon

Reactivities of active oxygen species with hydrocarbons and their roles in the catalytic oxidation are discussed. The concepts and recent developments including the activation of dioxygen molecule with reducing agents are summarized with emphasis on the catalytic oxidation of inactive hydrocarbons.