Sterol Oxidation Products Research Articles

Quantitative Analysis of Sterols and Oxysterols in Foods by Gas Chromatography Coupled with Mass Spectrometry

The study aimed to determine the content of sterol oxidation products (oxysterols) in selected processed food products with the use of GC-MS. It is known that an excessively high consumption of cholesterol in foods can lead to atherosclerosis and coronary heart disease and may also promote the appearance of gallstones. Cholesterol oxidation products have mutagenic, angiotoxic and cytotoxic properties and can lead to the development of atherosclerosis and promote the occurrence of some cancers. The cholesterol content in the tested products ranged from 8.72 to 2007.11 mg/100 g fat, and plant sterols were determined in 5 out of 12 tested products and their content ranged from 5.88 to 380.8 mg/100 g fat. The studies showed the presence of sterol oxidation products in each analyzed product. The total oxysterol content ranged from 0.16 to 3.95 μg/g fat in pastry products and from 0.06 to 9.72 μg/g fat in meat products. Due to the presence of sterol oxidation products in all analyzed products, their content in food products should be monitored. The presented analytical method is a proper tool for the determination of sterol oxidation products in different food matrices.

Front Cover: Single‐Cell Transcriptomics Reveals the Difference of Aortic Atherosclerosis Response to Phytosterols and Oxidation Products of Sterols

Mol. Nutr. Food Res. 2023, 67, 202200811 DOI: 10.1002/mnfr.202200811 Biological effects of dietary are evaluated for aortic cells after dietary exposure via Single-Cell Transcriptomics. Three pathways of differentiation indicate that dietary regulation affects the differentiation of aortic cells. Aortic cells are cultured by food, and healthy or unhealthy food will cause to different results. This is reported by Yao Xie, Baiyi Lu, and co-workers in article number 2200811.

Single-Cell Transcriptomics Reveals the Difference of Aortic Atherosclerosis Response to Phytosterols and Oxidation Products of Sterols.

Phytosterols (PS) and sterol oxidation products are key dietary factors influencing atherosclerosis besides cholesterol, although the mechanisms remain elusive. Recently, single-cell RNA sequencing (scRNA-seq) has revealed the heterogeneity of multiple cell types associated with complex pathogenesis in atherosclerosis development. Here, scRNA-seq is performed to investigate the alterations in the aortic cells from ApoE-/- mice induced by diet-derived PS or two sterol oxidation products, phytosterols oxidation products (POPs), and cholesterol oxidation products (COPs). The study identifies four fibroblast subpopulations with different functions, and immunofluorescence demonstrates their spatial heterogeneity, providing evidence that suggests the transformation of smooth muscle cells (SMCs) and fibroblasts in atherosclerosis. The composition and gene expression profiles of aortic cells change broadly in response to PS/COPs/POPs exposure. Notably, PS exhibits an atheroprotective effect where different gene expressions are mainly found in B cells. Exposure to COPs accelerates atherosclerosis and results in marked alternations in myofibroblast subpopulations and T cells, while POPs only alter fibroblast subpopulations and B cells. The data elucidate the effects of dietary PS/COPs/POPs on aortic cells during atherosclerosis development, especially on the newly identified fibroblast subpopulations.

Back Cover: Dietary Sterols and Sterol Oxidation Products on Atherosclerosis: An Insight Provided by Liver Proteomic and Lipidomic

Mol. Nutr. Food Res. 2021, 65, 202100516 DOI: 10.1002/mnfr.202100516 Dietary phytosterols and sterol oxidation products exhibit significant different effects on atherosclerosis, and changes in liver lipid metabolism may provide another insight for their effects. As revealed by lipidomes and proteome in this report, besides cholesterol, phytosterols and sterol oxidation products also greatly affected glycerophospholipids & glycerolipids metabolism and sphingolipids metabolism. This is reported by Xuan Yang, Baiyi Lu, and co-workers in article number 2100516.

Sterols and Sterol Oxidation Products: Effect of Dietary Intake on Tissue Distribution in ApoE-Deficient Mice.

Sterols and sterol oxidation products (SOPs) are well-known dietary factors influencing atherosclerosis; however, their distribution in vivo after dietary sterol/SOP intake is still unclear. Here, we investigated the tissue distribution of sterols and SOPs in ApoE-/- mice after dietary exposure to diets supplemented with phytosterols (PS), phytosterol oxidation products (POPs), or cholesterol oxidation products (COPs). The results showed that PS intake reduced cholesterol in serum and the liver but increased cholesterol in the brain. PS intake increased the levels of PS in vivo and the levels of 7-keto- and triol-POPs in serum and the liver. COP intake elevated the level of all COPs in serum but did not change the 7-keto-cholesterol level in the liver and brain. All POPs in serum and parts of POPs in the liver and brain increased after dietary POP exposure. Our study indicated that dietary PS and SOPs accumulated in vivo with varying degrees and influenced cerebral cholesterol metabolism.

Dietary Sterols and Sterol Oxidation Products on Atherosclerosis: An Insight Provided by Liver Proteomic and Lipidomic

The development of atherosclerosis is closely associated with disorder of lipid metabolism. Dietary sterols and their oxidation products play a role in the pathogenesis of atherosclerosis. However, their effects on liver lipid metabolism during the atherosclerosis remain unknown. Here, we apply lipidomic and proteomic analysis on liver of ApoE-/- mice feed with phytosterols, cholesterol oxidation products (COPs), or phytosterol oxidation products (POPs) to profile lipid species and reveal the underlying mechanism. Dietary exposure of phytosterols, COPs, and POPs all reduce the accumulation of liver triglyceride (TG), but COPs and POPs accelerate the fibrosis of liver. Lipidomic analysis reveals that phytosterols mainly decrease the levels of phosphatidylinositol (PI), while COPs and POPs both increase the level of digalactosyldiacylglycerol (DGDG) and reduce TG with long-chain polyunsaturated fatty acids. Besides, COPs up-regulated levels of lipids associate with atherosclerosis risk, such as phosphatidylcholines (PC), phosphatidylethanolamine (PE) and ceramides (Cer). POPs down-regulate the level of acyl carnitine (AcCa). Furthermore, proteomic analysis shows that COPs promote oxidative phosphorylation and POPs inhibit the beta oxidation of fatty acids. This study reveals that phytosterols, COPs, and POPs differently change the composition and metabolism of glycerophospholipids, sphingolipids, and glycerolipids in liver of ApoE-/- mice.

Food Protein Sterylation: Chemical Reactions between Reactive Amino Acids and Sterol Oxidation Products under Food Processing Conditions

Sterols, especially cholesterol and phytosterols, are important components of food lipids. During food processing, such as heating, sterols, like unsaturated fatty acids, can be oxidized. Protein modification by secondary products of lipid peroxidation has recently been demonstrated in food through a process called lipation. Similarly, this study was performed to assess, for the first time, the possibility of reactions between food proteins and sterol oxidation products in conditions relevant for food processing. Therefore, reaction models consisting of oxysterol (cholesterol 5α,6α-epoxide) and reactive amino acids (arginine, lysine, and methionine) were incubated in various conditions of concentration (0–8 mM), time (0–120 min), and temperature (30–180 °C). The identification of lysine adducts through thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) with a diode array detector (DAD), and electrospray ionization (ESI) mass spectrometry (MS) evidenced a reaction with lysine. Moreover, the HPLC-ESI with tandem mass spectrometry (MS/MS) analyses allowed observation of the compound, whose mass to charge ratio m/z 710.5 and fragmentation patterns corresponded to the reaction product [M + H]+ between cholesterol-5α,6α-epoxide and the ε-amino-group of Nα-benzoylglycyl-l-lysine. Moreover, kinetic studies between Nα-benzoylglycyl-l-lysine as a model for protein-bound lysine and cholesterol 5α,6α-epoxide were performed, showing that the formation of lysine adducts strongly increases with time, temperature, and oxysterol level. This preliminary study suggests that in conditions commonly reached during food processing, sterol oxidation products could react covalently with protein-bound lysine, causing protein modifications.

Safety of the extension of use of plant sterol esters as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of an extension of use of the novel food ‘plant sterol esters’ when added to vegetable fat spreads and to liquid vegetable fat‐based emulsions for cooking and baking purposes pursuant to Regulation (EU) 2015/2283. Member States expressed concerns in relation to plant sterol oxidation products (POP) and consumption by non‐target population groups. The median (0.5%) and P90 (2.28%) value of the oxidation rates of plant sterols determined by a wide range of cooking experiments were used together with exposure estimates for plant sterol when added and cooked with vegetable fat spreads and liquids. The no‐observed adverse effect level (NOAEL) of a subchronic rat study and an applied default uncertainty factor of 200 served to derive levels (i.e. 0.64 mg POP/kg body weight (bw) per day) considered safe for humans. This safe level of exposure would be exceeded at the P95 by all age groups when considering the P90 oxidation rate and using EFSA's comprehensive food consumption database for assessing the potential exposure. When considering the median oxidation rate, the safe level of 0.64 mg POP/kg bw per day would be exceeded at the highest P95 intake estimates in children below 9 years of age. When considering an intake of the maximum authorised use level of 3 g plant sterols/person per day and oxidation rates of 0.5% and 2.28%, the resulting daily POP intakes per kg bw by an adult weighing 70 kg would be 0.21 and 0.98 mg/kg bw per day, respectively, the latter value exceeding 0.64 mg/kg bw per day. The Panel concludes that the safety of the intended extension of use of plant sterol esters under the proposed conditions of use has not been established.

Oxidation of sitosterol and campesterol in foods upon cooking with liquid margarines without and with added plant sterol esters

Plant sterol (PS) oxidation products (POP) derived from sitosterol and campesterol were measured in 15 foods cooked with liquid margarine without (control) and with added 7.5% PS. POP were analyzed using a GC–MS method. PS liquid vs. control margarine resulted in a higher median POP content per food portion (1.35mg, range 0.08–13.20mg versus 0.23mg, 0.06–0.90mg), a lower PS oxidation rate (0.63 vs. 1.29%) and lower oxidation susceptibility of sitosterol vs. campesterol. POP formation was highest in shallow-fried potatoes with PS liquid margarine (64.44mg per portion food plus residual fat). Mean relative abundances of epoxy-, 7-keto-, 7-hydroxy- and triol-PS derived from sitosterol and campesterol were 40.0, 34.4, 21.5 and 4.0% with control vs. 44.1, 23.8, 29.6 and 2.4% with PS liquid margarine. In conclusion, PS liquid margarine increased POP content in foods with a POP profile characterized by a higher ratio of epoxy- to 7-keto-derivatives.

The effects of amoxicillin and vancomycin on parameters reflecting cholesterol metabolism

BackgroundChanges in the microbiota composition have been implicated in the development of obesity and type 2 diabetes. However, not much is known on the involvement of gut microbiota in lipid and cholesterol metabolism. In addition, the gut microbiota might also be a potential source of plasma oxyphytosterol and oxycholesterol concentrations (oxidation products of plant sterols and cholesterol). Therefore, the aim of this study was to modulate the gut microbiota by antibiotic therapy to investigate effects on parameters reflecting cholesterol metabolism and oxyphytosterol concentrations. DesignA randomized, double blind, placebo-controlled trial was performed in which 55 obese, pre-diabetic men received oral amoxicillin (broad-spectrum antibiotic), vancomycin (antibiotic directed against Gram-positive bacteria) or placebo (microcrystalline cellulose) capsules for 7days (1500mg/day). Plasma lipid and lipoprotein, non-cholesterol sterol, bile acid and oxy(phyto)sterol concentrations were determined at baseline and after 1-week intervention. ResultsPlasma secondary bile acids correlated negatively with cholestanol (marker for cholesterol absorption, r=−0.367; P<0.05) and positively with lathosterol concentrations (marker for cholesterol synthesis, r=0.430; P<0.05). Fasting plasma secondary bile acid concentrations were reduced after vancomycin treatment as compared to placebo treatment (-0.24±0.22μmol/L vs. −0.08±0.29μmol/L; P<0.01). Vancomycin and amoxicillin treatment did not affect markers for cholesterol metabolism, plasma TAG, total cholesterol, LDL-C or HDL-C concentrations as compared to placebo. In addition, both antibiotic treatments did not affect individual isoforms or total plasma oxyphytosterol or oxycholesterol concentrations. ConclusionDespite strong correlations between plasma bile acid concentrations and cholesterol metabolism (synthesis and absorption), amoxicillin and vancomycin treatment for 7days did not affect plasma lipid and lipoprotein, plasma non-cholesterol sterol and oxy(phyto)sterol concentrations in obese, pre-diabetic men.

Protective effect of antioxidants contained in milk-based fruit beverages against sterol oxidation products

Sterol oxidation products (SOPs) have shown cytotoxic effect in human intestinal cells; however, their effect within a food matrix has not been assayed yet. This study evaluated the possible cytotoxic effect of SOPs within bioaccessible fractions (BFs) of two milk-based fruit beverages with (BFA)/without (BFB) plant sterols in differentiated Caco-2 cells and if the BFs counteracted the cytotoxic effect induced by COPs mixture (30 and 60μM). BFs did not evoke cytotoxic effect in any of the tests carried out and they protected against the loss of intestinal cohesion, mitochondrial depolarization and necrosis induced by COPs mixture. Moreover, BFB sample protected from cell cycle arrest, lessened lysosomal injury and overproduction of ROS at 30μM. Therefore, the formation of SOPs within milk-based fruit beverages with/without plant sterols does not contribute to intestinal epithelium damage and these types of food matrices confer additional protection against COPs at dietary concentrations.

Oxysterols in cosmetics—Determination by planar solid phase extraction and gas chromatography–mass spectrometry

Sterol oxidation products (SOPs) are linked to several toxicological effects. Therefore, investigation of potential dietary uptake sources particularly food of animal origin has been a key issue for these compounds. For the simultaneous determination of oxysterols from cholesterol, phytosterols, dihydrolanosterol and lanosterol in complex cosmetic matrices, planar solid phase extraction (pSPE) was applied as clean-up tool. SOPs were first separated from more non-polar and polar matrix constituents by normal phase thin-layer chromatography and then focussed into one target zone. Zone extraction was performed with the TLC–MS interface, followed by gas chromatography-mass spectrometry analysis. pSPE showed to be effective for cleaning up cosmetic samples as sample extracts were free of interferences, and gas chromatographic columns did not show any signs of overloading. Recoveries were between 86 and 113% with relative standard deviations of below 10% (n=6). Results of our market survey in 2016 showed that some cosmetics with ingredients of plant origin contained phytosterol oxidation products (POPs) in the low ppm range and therefore in line with levels reported for food. In lanolin containing products, total SOPs levels (cholesterol oxidation products (COPs), lanosterol oxidation products (LOPs), dihydrolanosterol oxidation products (DOPs)) being in the low percent range exceeded reported levels for food by several orders of magnitudes.

Current Knowledge about Oxysterols: A Review.

For years food consumers have been warned that a cholesterol-rich diet may result in atherosclerosis. It is also well known that consumption of large amounts of phytosterols decreases concentration of low-density lipoproteins (LDLs) in blood (LDLs are regarded a key risk factor in development of cardiovascular diseases). However, no scientific evidence has unambiguously proved any direct connection between amount of consumed cholesterol and LDL level in blood. On the other hand, concentration of cholesterol oxidation products, oxysterols, seems to be indeed relevant; for example, they significantly impact appearance of atherosclerotic lesions (plaques). Phytosterols (like sitosterol or campasterol) decrease LDL level in blood, but on the other hand products of their oxidation are toxic. Therefore, it is worth to know influence of phytosterols on living organisms, processes which lead to their formation, and their levels in popular foodstuffs. This paper is an attempt to review literature data on the above aspects, as well as on impact on living organisms of oxidation products of popular sterols.

Effect of Transition Metal Ions on the B Ring Oxidation of Sterols and their Kinetics in Oil-in-Water Emulsions.

This study investigated the effect of metal ions on the oxidation of sterols and their kinetics in oil-in-water emulsions. Sterol substrates were added with different metal ions (Cu2+, Fe2+, Mn2+, Zn2+, Na+, and Mg2+) of five concentrations and investigated after 2 h of heating at 90 °C. The substrates added with Fe2+ and Cu2+ were heated continuously to evaluate the kinetics of four sterols and their corresponding sterol oxidation products (SOPs). Sterol oxidation increased as the metal ion concentration increased and the heating time was prolonged. The capability of the metal ions oxidizing sterols ranked as followed: Fe2+ > Cu2+ > Mn2+ > Zn2+ > Mg2+ ≈ Na+. 7-Ketosterol, 7β/7α-Hydroxysterol, 5β,6β/5α,6α-Epoxysterol, and Triols were the main oxides on the B ring, whereas 6β-Hydroxysterol was not or only slightly influenced. The acceleration of sterol degradation induced by Fe2+ and Cu2+, as well as the formation of oxidation products, followed first-order formation/elimination kinetics. The acceleration effect may be partly ascribed to the increase in elimination rate constant and formation rate constant. Transition metal ions can significantly induce sterol oxidation, which reduces food nutritional quality and triggers the formation of undesirable compounds, such as SOPs.

Normal-phase liquid chromatography–atmospheric-pressure photoionization–mass spectrometry analysis of cholesterol and phytosterol oxidation products

During thermal processing of sterols, complex mixtures of sterol oxidation products may be formed. Here, a new method for the separation and detection of such products is described. The method is based on normal-phase liquid chromatography (NPLC) for separation and atmospheric-pressure photoionization–mass spectrometry (APPI–MS) for detection. The method was optimized using commercial cholesterol oxidation products and tested on an experimentally derived mixture of phytosterol oxidation products. The investigated parameters include solvent and dopant selection, dopant concentration, polar modifiers, the type of stationary phase, and flow rate.Best chromatographic separation and highest sensitivity were achieved using a diol-bonded silica column, employing a solvent system consisting of hexane and isopropanol. The dopant of choice was chlorobenzene, added post-column to the solvent stream at 10% of the flow rate.The developed NPLC–APPI–MS method proved to be a valuable tool for the separation and detection of sterol oxidation products.

Cholesterol and stigmasterol within a sunflower oil matrix: Thermal degradation and oxysterols formation

The characteristics of the lipid matrix surrounding sterols exert a great influence in their thermal oxidation process. The objective of this work was to assess the oxidation susceptibility of equal amounts of cholesterol and stigmasterol within a sunflower oil lipid matrix (ratio 1:1:200) during heating (180°C, 0–180min). Remaining percentage of sterols was determined and seven sterol oxidation products (SOPs) were analysed for each type of sterol along the heating treatment. Evolution of the fatty acid profile and vitamin E content of the oil was also studied. Overall oxidation status of the model system was assessed by means of Peroxides Value (PV) and TBARS. PV remained constant from 30min onwards and TBARS continued increasing along the whole heating treatment. Degradation of both cholesterol and stigmasterol fitted a first order curve (R2=0.937 and 0.883, respectively), with very similar degradation constants (0.004min−1 and 0.005min−1, respectively). However, higher concentrations of oxidation products were found from cholesterol (79μg/mg) than from stigmasterol (53μg/mg) at the end of the heating treatment. Profile of individual oxidation products was similar for both sterols, except for the fact that no 25-hydroxystigmasterol was detected. 7α-Hydroxy and 7-keto-derivatives were the most abundant SOPs at the end of the treatment. PUFA and vitamin E suffered a significant degradation along the process, which was correlated to sterols oxidation.

Postprandial plasma oxyphytosterol concentrations after consumption of plant sterol or stanol enriched mixed meals in healthy subjects

Epidemiological studies have reported inconsistent results on the relationship between increased plant sterol concentrations with cardiovascular risk, which might be related to the formation of oxyphytosterols (plant sterol oxidation products) from plant sterols. However, determinants of oxyphytosterol formation and metabolism are largely unknown. It is known, however, that serum plant sterol concentrations increase after daily consumption of plant sterol enriched products, while concentrations decrease after plant stanol consumption. Still, we have earlier reported that fasting oxyphytosterol concentrations did not increase after consuming a plant sterol- or a plant stanol enriched margarine (3.0g/d of plant sterols or stanols) for 4weeks. Since humans are in a non-fasting state for most part of the day, we have now investigated effects on oxyphytosterol concentrations during the postprandial state. For this, subjects consumed a shake (50g of fat, 12g of protein, 67g of carbohydrates), containing no, or 3.0g of plant sterols or plant stanols. Blood samples were taken up to 8h and after 4h subjects received a second shake (without plant sterols or plant stanols). Serum oxyphytosterol concentrations were determined in BHT-enriched EDTA plasma via GC–MS/MS. 7β-OH-campesterol and 7β-OH-sitosterol concentrations were significantly higher after consumption of a mixed meal enriched with plant sterol esters compared to the control and plant stanol ester meal. These increases were seen only after consumption of the second shake, illustrative for a second meal effect. Non-oxidized campesterol and sitosterol concentrations also increased after plant sterol consumption, in parallel with 7β-OH concentrations and again only after the second meal. Apparently, plant sterols and oxyphytosterols follow the same second meal effect as described for dietary cholesterol. However, the question remains whether the increase in oxyphytosterols in the postprandial phase is due to absorption or endogenous formation.

Development and validation of a gas chromatography-mass spectrometry method for determination of sterol oxidation products in edible oils

An efficient GC-MS method for determination of sterol oxidation product profiles in edible oils was established by combination with optimized silylation, and validated using the standards of sitosterol oxidation products.

Relative expression of cholesterol transport-related proteins and inflammation markers through the induction of 7-ketosterol-mediated stress in Caco-2 cells

Human diets contain sterol oxidation products that can induce cytotoxic effects, mainly caused by cholesterol oxides. However, phytosterol oxides effects have been less extensively investigated. This study evaluates the production of inflammatory biomarkers (IL-1β, IL-8, IL-10, TNFα) and the influence of gene expression transporters and enzymes related to cholesterol absorption and metabolism (NPC1L1, ABCG5/8, HMGCoA, ACAT) produced by 7-ketosterols (stigmasterol/cholesterol) in Caco-2 cells. These effects were linked to intracellular signaling pathways by using several inhibitors. Results showed 7-ketostigmasterol to have a greater proinflammatory potential than 7-ketocholesterol. In non-pre-treated cells, only efflux transporters were down-regulated by 7-ketosterols, showing a greater influence upon ABCG5 expression. Cell-pre-incubation with bradykinin induced changes in ABCG expression levels after 7-ketostigmasterol-incubation; however, the energetic metabolism inhibition reduced NPC1L1 expression only in 7-ketocholesterol-incubated cells. In non-pre-treated cells, HMG-CoA was up-regulated by both 7-ketosterols. However, exposure to inhibitors down-regulated the expression levels, mainly in 7-ketocholesterol-incubated cells. While ACAT expression values in non-pre-treated cells were unchanged, exposure to inhibitors caused down-regulation of mRNA levels. These results suggest that internalization and excretion of 7-ketostigmasterol is probably influenced by [Ca]i, which also could mediate HMGCoA activity in POPs metabolism. However, energetic metabolism and reducing equivalents exert different influences upon the 7-ketosterol internalization.

Plant sterol oxidation products – Analogs to cholesterol oxidation products from plant origin?

Cholesterol and plant sterols are lipids which are abundantly present in a western type diet of animal and plant origin, respectively. The daily intake averages 300mg/day each. Over the past decades, a steadily increasing consumption of plant sterol enriched dairy products (2-3g/day) took place to lower circulating LDL cholesterol concentrations. Like all unsaturated components, plant sterols can be attacked by reactive oxygen species resulting in plant sterol oxidation products (POPs). The most widespread methods for POP determination are high-performance liquid chromatography and gas-liquid chromatography. Yet, based on the low plasma POP concentrations in normophytosterolemic subjects (POPs: ∼0.3-4.5ng/mL), a reliable quantification yielding an appropriate limit of detection remains a challenge. While the more abundantly present cholesterol oxidation products (COPs) have elaborately been studied, research on the metabolism and biological effects of POPs is only emerging. In relation to atherogenity, biological effects including modulation of cholesterol homeostasis, membrane functioning, and inflammation are attributed to POPs. Although mostly supra-physiological concentrations are applied in invitro assays, anti-tumor activity, cytotoxicity and estrogen-competition have been attributed to specific POPs. However, it is not obvious, if and how POPs may exert invivo adverse or beneficial health effects similar to those attributed to COPs. In the field of nutritional science, standardized methods for the determination of POPs are required to perform relevant biological studies and to assess their presence in complex foods or biological tissues and fluids. The aim of this review is to provide an overview and evaluation of the published methods and an update on the biological effects attributed to POPs.