Lipolysis and Sterol Stability and Bioaccessibility of Wholemeal Rye Bread Enriched with Plant Sterols Subjected to Adult and Elderly Digestion Conditions.

This study evaluated the impact of different digestion conditions (adult and senior) on lipolysis and bioaccessibility of plant sterols (PS) and phytosterol oxidation products (POPs) in PS-enriched wholemeal rye bread. Under adult digestion conditions, the addition of gastric lipase (GL) reduced lipolysis products (by 6.1% for free fatty acids and 11.7% for monoacylglycerols) and the bioaccessibility of PS by 6.7%, compared to the control. In digestion with both GL and cholesterol esterase (CE), these reductions were 12.9, 20.1, and 11.3%, respectively. Both modifications (GL and GL + CE) increased the bioaccessibility of POPs by 4.5-4.0%. When simulating the elderly digestion, the modified gastric and intestinal phases did not alter PS bioaccessibility but decreased POPs bioaccessibility by 21.8% compared to control, along with reduced lipolysis. Incorporating GL and CE thus approached physiological conditions and influenced lipid digestion. Elderly simulated digestion conditions resulted in a positive outcome by maintaining PS bioaccessibility while reducing potentially harmful POPs.

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

SCOPE: 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. METHODS AND RESULTS: 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. CONCLUSION: The data elucidate the effects of dietary PS/COPs/POPs on aortic cells during atherosclerosis development, especially on the newly identified fibroblast subpopulations.

Serum Concentration of Plant Sterol Oxidation Products (POP) Compared to Cholesterol Oxidation Products (COP) after Intake of Oxidized Plant Sterols: A Randomised, Placebo-Controlled, Double-Blind Dose‒Response Pilot Study.

Plant sterols (PS) are oxidized to PS oxidation products (POP). This study quantified the change in serum POP compared to cholesterol oxidation products (COP) after the intake of increasing POP doses. This was a double-blind, randomized, placebo-controlled, dose‒response pilot study with healthy individuals in four groups (15 per group). The control group received products with no added PS or POP and treatment groups received daily 20-25 g margarine with added PS (mean 3 g/d) and two cookies (~28 g) for six weeks. Cookies delivered 8.7 (low-dose), 15.2 (medium-dose), or 37.2 (high-dose) mg/d POP. Fasting serum POP and COP were measured at the baseline, days 14, 28, and 42 in all participants and days 7, 21, and 35 in a subset. Sixty individuals completed the study; 52 were included in per protocol analysis. Serum POP increased with increasing POP intake and plateaued at dose >15 mg/d. Stabilized POP concentrations were (mean ± SD) 38.9 ± 6.9, 91.0 ± 27.9, 144.4 ± 37.9 and 203.0 ± 63.7 nmol/L, for control, low-, medium-, and high-dose POP groups, respectively. For all groups, the serum COP ranged from 213 to 262 nmol/L and the average POP/COP ratio was <1. Serum POP concentrations increased non-linearly, reaching stabilized concentrations in <7 days, and remained below COP concentrations after the intake of increasing POP doses.

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.

Thermal stability of plant sterols and formation of their oxidation products in vegetable oils and margarines upon controlled heating.

Fat-based products like vegetable oils and margarines are commonly used for cooking, which may enhance oxidation of plant sterols (PS) present therein, leading to the formation of PS oxidation products (POP). The present study aims to assess the kinetics of POP formation in six different fat-based products. Vegetable oils and margarines without and with added PS (7.5-7.6% w/w) in esterified form were heated in a Petri-dish at temperatures of 150, 180 and 210°C for 8, 12 and 16min. PS and POP were analysed using GC-FID and GC-MS-SIM, respectively. Increasing PS content, temperature and heating time led to higher POP formation in all tested fat-based products. PS (either naturally occurring or added) in margarines were less susceptible to oxidation as compared to PS in vegetable oils. The susceptibility of sitosterol to oxidation was about 20% lower than that of campesterol under all the applied experimental conditions. During heating, the relative abundance of 7-keto-PS (expressed as% of total POP) decreased in all the fat-based products regardless of their PS contents, which was accompanied by an increase in the relative abundance of 7-OH-PS and 5,6-epoxy-PS, while PS-triols were fairly unchanged. In conclusion, heating time, temperature, initial PS content and the matrix of the fat-based products (vegetable oil vs. margarine) showed distinct effects on POP formation and composition of individual POP formed.

Oxysterols formation: A review of a multifactorial process.

Dietary sterols are nutritionally interesting compounds which can suffer oxidation reactions. In the case of plant sterols, they are being widely used for food enrichment due to their hypocholesterolemic properties. Besides, cholesterol and plant sterols oxidation products are associated with the development of cardiovascular and neurodegenerative diseases, among others. Therefore, the evaluation of the particular factors affecting sterol degradation and oxysterols formation in foods is of major importance. The present work summarizes the main results obtained in experiments which aimed to study four aspects in this context: the effect of the heating treatment, the unsaturation degree of the surrounding lipids, the presence of antioxidants on sterols degradation, and at last, oxides formation. The use of model systems allowed the isolation of some of these effects resulting in more accurate data. Thus, these results could be applied in real conditions.

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.

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.

Unsaturated lipid matrices protect plant sterols from degradation during heating treatment.

The interest in plant sterols enriched foods has recently enhanced due to their healthy properties. The influence of the unsaturation degree of different fatty acids methyl esters (FAME: stearate, oleate, linoletate and linolenate) on a mixture of three plant sterols (PS: campesterol, stigmasterol and ß-sitosterol) was evaluated at 180 °C for up to 180 min. Sterols degraded slower in the presence of unsaturated FAME. Both PS and FAME degradation fit a first order kinetic model (R(2)>0.9). Maximum oxysterols concentrations were achieved at 20 min in neat PS and 120 min in lipid mixtures and this maximum amount decreased with increasing their unsaturation degree. In conclusion, the presence of FAME delayed PS degradation and postponed oxysterols formation. This protective effect was further promoted by increasing the unsaturation degree of FAME. This evidence could help industries to optimize the formulation of sterol-enriched products, so that they could maintain their healthy properties during cooking or processing.

Formation of Plant Sterol Oxidation Products in Foods during Baking and Cooking Using Margarine without and with Added Plant Sterol Esters.

Plant sterols (PS) in foods are subject to thermal oxidation to form PS oxidation products (POP). This study measured POP contents of 19 foods prepared by typical household baking and cooking methods using margarines without (control) and with 7.5% added PS (as 12.5% PS-esters, PS-margarine). Median POP contents per portion size of cooked foods were 0.57 mg (range 0.05-1.11 mg) with control margarine versus 1.42 mg (range 0.08-20.5 mg) with PS-margarine. The oxidation rate of PS (ORP) was 0.50% (median) with the PS-margarine and 3.66% with the control margarine. Using the PS-margarine, microwave-cooked codfish had the lowest POP content, with 0.08 mg per portion, while shallow-fried potatoes had the highest POP content, 20.5 mg per portion. Median POP contents in cookies, muffins, banana bread, and sponge cake baked with the control or PS-margarine were 0.12 mg (range 0.11-0.21 mg) and 0.24 mg (range 0.19-0.60 mg) per portion, with a corresponding ORP of 1.38% and 0.06%, respectively. POP contents in all the cooked and baked foods did not exceed 20.5 mg per typical portion size. A wide variation in the distribution of individual POP among different foods existed, with 7-keto-PS and 5,6-epoxy-PS being the major oxidation products.

Plant sterol oxides in functional beverages: influence of matrix and storage.

Three plant sterol (PS)-enriched beverages, milk based fruit juice (MFJPS), fruit juice (FJPS) and milk beverage (MPS), were stored at 4, 24, or 37 °C and analysed at regular time intervals of 2 months until 6 months. PS stability was analysed from the production of phytosterol oxidation products (POPs). The ß-sitosterol oxides (7α/7ß-hydroxy, ß/α-epoxy, triol, and 7-keto) and campesterol oxides (ß/α-epoxy, and 7-keto) were detected in all beverages and at all storage times and temperatures. Total POP contents followed the order MPS≫FJPS>MFJPS. In general, the beverages showed low PS oxidation levels (<0.17%). Predictive models of POP content versus storage time were established. These models explain total POP content by over 75% and individual POP content by over 50%. We propose 7-ketositosterol and 7-ketocampesterol as PS oxidation markers during storage of beverages of this kind.

Plant sterols from foods in inflammation and risk of cardiovascular disease: a real threat?

High dietary intakes of cholesterol together with sedentary habits have been identified as major contributors to atherosclerosis. The latter has long been considered a cholesterol storage disease; however, today atherosclerosis is considered a more complex disease in which both innate and adaptive immune-inflammatory mechanisms as well as bacteria play a major role, in addition to interactions between the arterial wall and blood components. This scenario has promoted nutritional recommendations to enrich different type of foods with plant sterols (PS) because of their cholesterol-lowering effects. In addition to cholesterol, PS can also be oxidized during food processing or storage, and the oxidized derivatives, known as phytosterol oxidation products (POPs), can make an important contribution to the negative effects of both cholesterol and cholesterol oxidation oxides (COPs) in relation to inflammatory disease onset and the development of atherosclerosis. Most current research efforts have focused on COPs, and evaluations of the particular role and physiopathological implications of specific POPs have been only inferential. Appreciation of the inflammatory role described for both COPs and POPs derived from foods also provides additional reasons for safety studies after long-term consumption of PS. The balance and relevance for health of all these effects deserves further studies in humans. This review summarizes current knowledge about the presence of sterol oxidation products (SOPs) in foods and their potential role in inflammatory process and cardiovascular disease.