Phytosterol Oxidation Research Articles

Phytosterol oxidation products: state of the art

This editorial gives an overview of the current status of the research on the phytosterol oxidation products. The most relevant studies on their in vivo origin, biological effects and analysis in foodstuffs and biological samples are critically reviewed, in order to give concluding remarks on and to establish the future research needs in this field.

Gas chromatographic properties of common cholesterol and phytosterol oxidation products

The most common cholesterol and phytosterol oxidation products found in foodstuffs or biological matrices are the 7α- and 7β-hydroxysterol, 7-ketosterol, 5α,6α- and 5β,6β-epoxysterol, and triol derivatives of sterols. This study focused on the preparation and purification of such products derived from campesterol, stigmasterol and β-sitosterol. The identity of the substances was confirmed by mass spectroscopic analysis. The elution order of a complex mixture composed of the 7α- and 7β-hydroxysterol, 7-ketosterol, 5α,6α- and 5β,6β-epoxysterol, and triol derivatives of cholesterol, campesterol, stigmasterol and β-sitosterol was recorded on an apolar as well as a medium-polarity capillary column in relation to two commonly used internal standards, i.e. α-cholestane and 19-hydroxy cholesterol. Flame-ionization detector as well as mass spectrometry response factors were derived from a gravimetrically prepared mixture of commercially available cholesterol oxide standards. It was proven that the ionization efficiency of cholesterol and phytosterol oxides are very similar and that response factors obtained for cholesterol oxidation products are also valid for quantitative work regarding phytosterol oxidation products.

Effects of sterol structure, temperature, and lipid medium on phytosterol oxidation.

Factors contributing to the oxidative stability of phytosterols were studied. Unsaturated stigmasterol and saturated sitostanol were used as model compounds and were heated at different temperatures in different lipid matrices for various periods of time. Accumulations of the major secondary oxidation products were used as a marker of the stability of heated compounds, and the products were analyzed by gas chromatography-mass spectrometry. The results showed that both temperature and heating time, as well as sterol structure and lipid matrix composition, affected phytosterol oxidation. In particular, the interactions between different lipid matrices and temperatures had drastic effects on the total contents of the phytosterol oxides formed and also on the reaction pathways of oxidation. During heating at high temperatures for prolonged periods, >20% of stigmasterol was oxidized. At moderate temperatures the oxidation of stigmasterol was rather slow. Sitostanol oxide contents were low under all heating conditions studied.

Phytosterol Oxidation Products Are Absorbed in the Intestinal Lymphatics in Rats but Do Not Accelerate Atherosclerosis in Apolipoprotein E–Deficient Mice

Phytosterol oxidation products (oxyphytosterols) are formed during the processing and storage of foods. However, it is unknown whether oxyphytosterols affect human health. To address these issues, we prepared beta-sitosterol and campesterol oxides, evaluated their lymphatic absorption in rats, and examined the effect of an oxyphytosterol diet on atherosclerosis in apolipoprotein (apo) E-deficient mice. The lymphatic absorption of cholesterol and 6 oxyphytosterols (7alpha-hydroxy, 7beta-hydroxy, beta-epoxy, alpha-epoxy, dihydroxy, and 7-keto) of beta-sitosterol or campesterol was assessed in thoracic duct-cannulated rats fed an AIN-93G-based diet containing 2.5 g of cholesterol, oxyphytosterols, or intact phytosterols per kg. Lymphatic recoveries (on a mass basis) of oxycampesterols (15.9 +/- 2.8%, n = 10) and oxysitosterols (9.12 +/- 1.77%, n = 10) were higher than for campesterol (5.47 +/- 1.02%, n = 12, P < 0.05) and beta-sitosterol (2.16 +/- 0.37%, n = 12, P < 0.05), but lower than for cholesterol (37.3 +/- 8.3%, n = 6, P < 0.05). Apo E-deficient mice were fed an AIN-93G-based diet containing 0.2 g oxyphytosterols or intact phytosterols per kg for 9 wk. Diet-derived oxyphytosterols accumulated in the serum, liver, and aorta. Furthermore, the oxyphytosterol diet increased oxycholesterol in the serum compared to the phytosterol diet. However, there was no significant difference between the 2 groups in the serum and aortic cholesterol concentration, the lesion area in the aortic root, or 8-iso-prostaglandin F2alpha concentration in the urine. These results indicate that exogenous oxyphytosterols are well-absorbed and accumulate in the body, but do not promote the development of atherosclerosis in apo E-deficient mice.

Analysis of Sterol Oxidation Products in Foods

The main aspects related to the analysis of sterol oxidation products (SOP) in foods are comprehensively reviewed. Special emphasis is placed on the critical and controversial points of this analysis because these points affect crucial analytical parameters such as precision, accuracy, selectivity, and sensitivity. The effect of sample preparation and the conditions of quantification by gas chromatography and liquid chromatography on these parameters are also reviewed. The results show that, in order to choose an adequate method to analyze SOP in a certain food, the analyst must consider its SOP concentration and matrix complexity. The term SOP includes both cholesterol oxidation products (COP) and phytosterol oxidation products (POP). The state of the art of COP and POP analysis is quite different; many more studies have dealt with the analysis of COP than of POP. However, most of the results presented here about COP analysis may be extrapolated to POP analysis because both groups of compounds show similar structures and characteristics.

Safety evaluation of phytosterol esters. Part 8. Lack of genotoxicity and subchronic toxicity with phytosterol oxides

Vegetable oil spreads containing phytosterol-esters are marketed as a cholesterol-lowering functional food in more than 20 countries worldwide. An extensive package of safety data has shown phytosterol-esters to be safe for human use. However, even though phytosterols are very stable molecules, oxidation may occur at low levels under extreme heating conditions, resulting in phytosterol oxides. As there is some suggestion of adverse biological effects in the literature for the related cholesterol oxidation products, safety data have been generated for phytosterol oxides. A phytosterol oxide concentrate (POC) was generated by prolonged heating of phytosterol-esters in the presence of oxygen. The genotoxicity and subchronic toxicity of this mixture was assessed in a series of in vitro genotoxicity assays (bacterial mutation, chromosome aberration and micronucleus) and a subchronic feeding study in the rat. Results showed that a phytosterol oxide concentrate containing approximately 30% phytosterol oxides did not possess genotoxic potential and no obvious evidence of toxicity when administered in the diet of the rat for 90 consecutive days. In the latter study, a NOEL was established at an estimated dietary level of phytosterol oxides of 128 mg/kg/day for males and 144 mg/kg/day for females. In conclusion, these materials have been shown to raise no obvious concerns for human safety.

GC‐MS method for characterization and quantification of sitostanol oxidation products

AbstractA new GC‐MS method for characterization and quantification of phytosterol oxidation products was developed. Applicability of this method was tested by characterizing sitostanol oxides formed in bulk and then quantifying selected oxides in purified rapeseed oil and tripalmitin matrices in which the complex matrix made oxide analysis difficult. In bulk, nine different sitostanol oxides were characterized, including epimers of 7‐ and 15‐hydroxysitostanol and 6‐ and 7‐ketositostanol. In both lipid matrices, the amounts of sitostanol oxides generated in thermo‐oxidation were very low. According to statistical analyses, depending on the oxide, the GC‐MS results were the same or slightly higher than those quantified by the more common GC‐FID method. Thus, GC‐MS provides a powerful alternative for characterization and quantification of phytostanol oxides found in low amounts in complex matrices and is a promising method for future phytosterol oxide studies.

Preparation and mass spectrometry of 14 pure and 18O 2-labeled oxidation products from the phytosterols β-sitosterol and stigmasterol

To lower cholesterol, phytosterols are currently introduced as food additives, where they may become oxidized. In addition, specific biological effects of oxyphytosterols are suggested by the recent molecular clarification of the phytosterol storage disease as a dysfunctional mutation of an active sterol reexporter potentially regulated by oxidized phytosterols. We therefore studied the hydroxybenzotriazole-mediated PbO 2-driven oxidation of phytosterols and compared it to the oxidation of cholesterol. We prepared, identified, and purified standards of 14 oxidation products of two major phytosterols. The gas chromatographic mass spectrometric characteristics of the 7α- and 7β-hydroxy-, 5α,6α-epoxy, 5β,6β-epoxy, 7keto-, 3β,5α,6β-trihydroxy-, 3keto-, and 7-dehydro-derivatives of β-sitosterol and stigmasterol are presented. The method also provided a convenient access to prepare 18O-labeled oxyphytosterols of high chemical and isotopic purity and can easily be extended to further phytosterols and -stanols. This enables the gas chromatography–mass spectrometry analysis of oxyphytosterols and the study of their biological effects.

Characterization of side‐chain oxidation products of sitosterol and campesterol by chromatographic and spectroscopic methods

AbstractBased on the cholesterol‐lowering effects of phytosterols, known since the 1950s, extra phytosterol amounts have been added to certain food products for almost 10 yr. Literature reports on oxidation of phytosterols in general are limited, and data on side‐chain oxidation of these compounds are scarce. The aim of this study was to investigate and characterize autoxidation products from a mixture of the two phytosterols sitosterol and campesterol. A commercial mixture of the two sterols was oxidized for 72 h at 120°C in an air‐ventilated oven. The oxidation products were separated by preparative TLC and identified and characterized with GC‐MS. The following oxidation products were identified from the two phytosterols: 24‐methylcholest‐5‐en‐3β,24‐diol, 24‐methylcholest‐5‐en‐3β,25‐diol, 24‐methylcholest‐4‐en‐6α‐ol‐3‐one, 24‐methylcholest‐4‐en‐6β‐ol‐3‐one, 24‐ethylcholest‐5‐en‐3β,24‐diol, 24‐ethylcholest‐5‐en‐3β, 25‐diol, 24‐ethylcholest‐4‐en‐6α‐ol‐3‐one, 24‐ethylcholest‐4‐en‐6β‐ol‐3‐one. Full‐scan mass spectra of these compounds are reported for the first time as their trimethylsilyl‐ether derivatives.

Side‐chain autoxidation of stigmasterol and analysis of a mixture of phytosterol oxidation products by chromatographic and spectroscopic methods

AbstractAlthough the structure of phytosterols is closely related to cholesterol, there is a gap in knowledge concerning the formation and occurrence of oxidation products from phytosterols. The main objective of this study was to isolate and characterize some side‐chain oxidation products formed after autoxidation of stigmasterol. Another objective was to highlight the difficulties in the analysis of phytosterol and a mixture of their oxidation products by GC. Pure stigmasterol was oxidized at 120°C for 72 h in an air‐ventilated oven. Preparative TLC separated the oxidation products, and the products were characterized with GC‐MS and NMR. In addition to the common ring‐structure oxidation compounds, three semipolar oxidation products—24‐ethylcholest‐5,22‐dien‐3β,25‐diol, 24‐ethylcholest‐5,22‐dien‐3β,24‐diol, and 24‐ethyl‐5,22‐choladien‐3β‐ol‐24‐one—were characterized for the first time by TLC, GC‐MS, and NMR. Moreover, the results of the analysis of a large number of oxidation products from sitosterol, campesterol, and stigmasterol by capillary column GC indicated that further efforts and optimization are required in this area.

Presence of Phytosterol Oxides in Crude Vegetable Oils and Their Fate during Refining

The content of phytosterol oxidation products was determined in samples of crude vegetable oils: peanut, sunflower, maize, palm nut, and lampante olive oils that were intended for refining and not for direct consumption. The 7 alpha- and 7 beta-hydroxy derivatives of beta-sitosterol, stigmasterol, and campesterol and the 7-keto-beta-sitosterol were the principal phytosterol oxides found in almost all of the oils analyzed. In some oils, the epoxy and dihydroxy derivatives of beta-sitosterol were also found at very low levels. The highest total concentrations of phytosterol oxides, ranging from 4.5 to 67.5 and from 4.1 to 60.1 ppm, were found in sunflower and maize oils, respectively. Lower concentrations were present in the peanut oils, 2.7-9.6 ppm, and in the palm nut oil, 5.5 ppm, whereas in the lampante olive oils, only three samples of the six analyzed contained a low concentration (1.5-2.5 ppm) of oxyphytosterols. No detectable levels of phytosterol oxides were found in the samples of palm and coconut oils. Bleaching experiments were carried out on a sample of sunflower oil at 80 degrees C for 1 h with 1 and 2% of both acidic and neutral earths. The bleaching caused a reduction of the hydroxyphytosterol with partial formation of steroidal hydrocarbons with three double bonds in the ring system at the 2-, 4-, and 6-positions (steratrienes). The same sunflower oil was deodorized at 180 degrees C under vacuum for 1 h, and no dehydration products were formed with a complete recovery of the hydroxyphytosterols. A bleaching test with acidic earths was carried out also with an extra virgin olive oil fortified with 7-keto-cholesterol, dihydroxycholesterol, and alpha-epoxy-cholesterol. There was no formation of steratrienes from these compounds, but dihydroxycholesterol underwent considerable decomposition and alpha-epoxycholesterol underwent ring opening with formation of the dihydroxy derivative, whereas 7-ketocholesterol was rather stable

Oxidation of phytosterols in a test food system

AbstractThe oxidative stability of phytosterols in canola, coconut, peanut, and soybean oils was examined under simulated frying conditions of 100, 150, and 180°C for 20 h. The degree of oxidative decomposition was assessed by the loss of phytosterols, accumulation of phytosterol oxides, and the change in fatty acid profiles. The phytosterol oxides produced in the oils were identified using mass spectroscopy. Oils with higher levels of polyunsaturated fatty acids showed greater amounts of sterol loss; however, the sterol loss was less complete than in the more saturated oils. A greater variety of sterol oxides was observed at the lower temperatures of 100 and 150°C compared to 180°C. This study demonstrates that under conditions similar to frying, there is a loss of phytosterols and polyunsaturated fatty acids. The accumulation of phytosterol oxides may be temperature‐limited because of further break‐down into products not measurable by typical gas chromatography‐mass spectrometry techniques.

Lymphatic absorption of phytosterol oxides in rats.

Two of the main classes of oxyphytosterols (7-keto and epoxides) were synthesized from sitosterol and campesterol and given to mesenteric duct-cannulated adult male rats. Lymph was collected during 24 h and was analyzed for oxysterols. The results showed that the lymphatic recovery of the phytosterol oxides was low: 4.7% of the given dose for epoxy derivatives and 1.5% for 7-keto compounds. The campesterol oxides presented a better absorption than the sitosterol oxides. During the process of absorption, the epoxyphytostanols were also partly transformed in campestanetriol and stigmastanetriol.

Sterol oxidation in infant milk formulas and milk cereals.

7-Ketocholesterol and 7-ketositosterol were chosen as reliable markers of the oxidation of cholesterol and phytosterols in infant milk formulas and infant milk cereals. A reversed-phase HPLC method was developed to measure them simultaneously in infant formulas. This method was then tested on a wide range of infant milk formulas and milk cereals on sale in Italy whose lipid composition is representative of the most common commercial formulas. The analytical results revealed no significant differences in the extent of oxidation of cholesterol and sitosterol. As the level of 7-ketocholesterol often followed the cholesterol level, a cholesterol content similar to that of human milk produced amounts of cholesterol oxides with possible negative effects on infant health. In contrast, the low cholesterol content of milk cereals never produced amounts of cholesterol oxides high enough to cause concern. The contents of phytosterols and hence their oxides were always low.

Studies on phytosterol oxides. I: Effect of storage on the content in potato chips prepared in different vegetable oils

AbstractPotato chips fried in palm oil, sunflower oil, and high‐oleic sunflower oil were studied for the content of different phytosterol oxides during 0 to 25 weeks of storage in the dark. Oxidation products of sitosterol (24α‐ethyl‐5‐cholesten‐3β‐ol) and campesterol (24α‐methyl‐t‐cholesten‐3β‐ol) were synthesized to help identify the phytosterol oxides. The oxides of phytosterols were analyzed by preparative thin‐layer chromatography, solid‐phase extraction, capillary column gas chromatography (GC), and GC‐mass spectrometry. Epimers of 7‐hydroxysitosterol and 7‐hydroxycampesterol; 7‐ketositosterol and 7‐ketocampesterol; epimers of 5,6‐epoxy‐sitosterol and 5,6‐epoxy‐campesterol; 24α‐ethylcholestane‐3β,5,6β‐triol (dihydroxysitosterol) and 24α‐methylcholestane‐3β,5,6β‐triol (dihydroxycampesterol) were detected and quantitated in the samples of chips fried in different vegetable oils. Potato chips fried in palm oil had the lowest level of total sterol oxides, ranging from 5 to ca. 9 ppm in the lipids from time 0 to 25 wk of storage. The level of total sterol oxides in chip samples fried in sunflower oil ranged from 46 to 47 ppm, and the lipids in samples fried in high‐oleic sunflower oil ranged from 35 to 58 ppm from 0 time to 25 wk of storage. During 25 wk of storage no considerable increase in sterol oxides was observed in the samples of chips fried in palm oil and sunflower oil. The chip samples fried in high‐oleic sunflower oil had slightly higher levels of sterol oxides after 10 and 25 weeks of storage. In addition to the levels of individual sterol oxides, a new method for enrichment of phytosterol oxides from the unsaponifiables and full‐scan mass spectra of various oxidation products of sitosterol and campesterol are reported in this paper.

Studies on phytosterol oxides. II: Content in some vegetable oils and in French fries prepared in these oils

AbstractHydrogenated rapeseed oil/palm oil blend, sunflower oil and high‐oleic sunflower oil, and French fries fried in these oils were assessed for contents of sterol oxidation products. Different oxidation products of phytosterols (7α‐ and 7β‐hydroxy‐sito‐and campesterol, 7‐ketosito‐ and 7‐ketocampesterol, 5α,6α‐epoxy‐sito‐ and campesterol, 5β,6β‐epoxy‐sito‐and campesterol, dihydroxysitosterol and dihydroxycampesterol) were identified and quantiated by gas chromatography (GC) and GC‐mass spectroscopy. Rapeseed oil/palm oil blend contained 41 ppm total sterol oxides before frying operations. After two days of frying, this level was increased to 60 ppm. Sunflower oil and high‐oleic sunflower oil had 40 and 46 ppm sterol oxides, respectively, before frying operations. After two days of frying operations, these levels increased to 57 and 56 ppm, respectively. In addition to campesterol and sitosterol oxidation products, small amounts of 7α‐ and 7β‐hydroxystigmasterol were detected in the oil samples. Total sterol oxides in the lipids of French fries fried at 200°C in rapeseed oil/palm oil blend, sunflower oil, and high‐oleic sunflower oil were 32, 37, and 54 ppm, respectively. The levels of total oxidized sterols, calculated per g sample, ranged from 2.4 to 4.0 ppm. In addition to the content of phytosterol oxides, full scan mass spectra of several oxidation products of stigmasterol are reported for the first time.

Oxidized phytosterols increase by ageing in photoautotrophic cell cultures of Chenopodium rubrum

Young and old cell cultures of Chenopodium rubrum were investigated for their content of phytosterol derivatives by gas chromatography-mass spectrometry. The ratio of the main phytosterol constituents, sitosterol, stigmasterol and campesterol (1.2:2.4:1.0) remained approximately constant, but their overall content (402.5 μg g −1 dry wt) decreased from 11 days to 8 weeks old cell cultures by about 64% (260.1 μg g −1 dry wt); in constrast the content of corresponding epoxides increased from traces at day 11 to 11.1 μg g −1 (dry wt) at the 8th week. A comparable increase was observed for 3,5,6-trihydroxysteranes (2.8 μg g −1 dry wt), day 11; 27.5 μg g −1 dry wt), 8 weeks). The content of sterols oxidized in position C-7 showed also an increase from 1.9 μg g −1 dry wt) (11 days) to 51.7 μg g −1 dry wt) (8 weeks). These results show that the increase of phytosterol oxidation products correlated with age of the cell culture. Therefore, the sterol epoxide content and related oxidation products may be used as a measure for ageing.

Analysis of Polar Cholesterol Oxidation Products: Evaluation of a New Method Involving Transesterification, Solid Phase Extraction, and Gas Chromatography

Monitoring cholesterol oxidation products is important in the evaluation of the potential health risks associated with lipid oxidation. In the present study, a method allowing quick and reliable analysis of polar cholesterol oxidation products was evaluated. After Soxhlet-lipid extraction, the fat was transesterified under mild conditions, thereby minimizing degradation and allowing determination of the free and esterified cholesterol oxides. Sample fractionation was achieved with aminopropyl solid phase extraction cartridges and a stepwise elution with hexane, hexane/methyl tert-butyl ether, and acetone to separate polar cholesterol oxides from cholesterol and other lipid products. Further analysis of the trimethylsilyl derivatives was performed by gas chromatography with detection by flame ionization or mass spectrometry. A phytosterol oxide such as sitosterol α-epoxide (24α-ethyl-5α,6α-epoxycholestan-3β-ol) was employed for the first time as an internal standard for the quantification of cholesterol ox...

Oxisterol determination in selected coffees.

The main aim of green-coffee processing techniques, such as decaffeination and roasting, is always to maintain a very high level of quality in taste and flavor, the beverage's most important characteristics to consumers. Oxidative alterations of coffee lipids, which can occur in roasting, exert a very marked influence on these quality traits. Determining the extent of oxidation thus can provide an indication of the product's potential shelf-life and reveal traces of any newly-formed oxidative products that might prove nutritionally unsafe. Yet, while much attention has recently been focused on certain by-products induced by cholesterol oxidation and their proven toxicity as risk factors in atherosclerosis and cancer, oxidated phytosterols have largely gone unnoticed, being considered along with beta-sitosterol as not very dangerous in that neither is absorbed by the intestinal tract. The present study investigates the substances derived from phytosterol oxidation (oxisterols) in samples of regular and decaffeinated commercial coffees. The findings show that oxisterols were absent in some samples and that the traces of oxidate phytosterols detected in others were well below the threshold considered as toxicologically active.

AOCS method Ce 1c‐89 underestimates thetrans‐octadecenoate content in favor of thecis isomers in partially hydrogenated vegetable oils

Journal of the American Oil Chemists' SocietyVolume 69, Issue 2 p. 192-192 Letter to the Editor AOCS method Ce 1c-89 underestimates thetrans-octadecenoate content in favor of thecis isomers in partially hydrogenated vegetable oils W. M. N. Ratnayake, W. M. N. Ratnayake Bureau of Nutritional Sciences, Food Directorate, Health and Welfare Canada, Ottawa, Ontario, K1A 0L2 CanadaSearch for more papers by this author W. M. N. Ratnayake, W. M. N. Ratnayake Bureau of Nutritional Sciences, Food Directorate, Health and Welfare Canada, Ottawa, Ontario, K1A 0L2 CanadaSearch for more papers by this author First published: 01 February 1992 https://doi.org/10.1007/BF02540576Citations: 6AboutRelatedInformationPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessClose modalShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume69, Issue2February 1992Pages 192-192 RelatedInformation RecommendedStudies on phytosterol oxides. II: Content in some vegetable oils and in French fries prepared in these oilsParesh Chandra Dutta, Journal of the American Oil Chemists' SocietyTriterpene alcohols and sterols of vegetable oilsE. Fedeli, A. Lanzani, P. Capella, G. Jacini, Journal of the American Oil Chemists' SocietyHydrogenated vegetable oils as candle waxKaramatollah Rezaei, Tong Wang, Lawrence A. Johnson, Journal of the American Oil Chemists' Society