Ethylcoprostanol modulates colorectal cancer cell proliferation and mitigates cytotoxicity of cholesterol metabolites in non-tumor colon cells.

Sterols can be metabolized by gut microbiota. The cholesterol metabolites have been proposed as promoters of colorectal cancer (CRC), while the effect of plant sterol metabolites is unknown. This study aimed to evaluate the cytotoxicity of metabolites from cholesterol (coprostanol, cholestanol, coprostanone and cholestenone) and ß-sitosterol (ethylcoprostanol) on human colon tumor (Caco-2) and non-tumor (CCD-18Co) cells at physiological concentrations (9-300 µM) and exposure time (24 h). Ethylcoprostanol reduced the tumor cell proliferation (MTT), showing in flow cytometry assays induction of apoptosis via production of reactive oxygen species (ROS) and ceramide. Transcriptomic analysis (qPCR) showed activation of the intrinsic apoptosis pathway (BAX/BCL2 ratio and CASP9 increased), accompanied by downregulation of the p21 gene. Cholesterol metabolites, mainly the most hydrophobic, induced apoptosis and G0/G1 phase arrest in non-tumor cells through overproduction of ROS. Both the intrinsic and extrinsic (CASP8 increased) apoptosis pathways occurred. In turn, a reduction in the expression of the cyclin E1 gene confirmed the cell cycle arrest. In addition, ethylcoprostanol protected non-tumor cells from the most cytotoxic cholesterol metabolite (cholestenone). In conclusion, ethylcoprostanol is a promising candidate as a therapeutic adjuvant in CRC, while cholesterol metabolites could act as CRC promoters through their cytotoxicity.

Aging-related gastrointestinal conditions decrease the bioaccessibility of plant sterols in enriched wholemeal rye bread: in vitro static digestion.

The prevention of cardiovascular disease using foods fortified with plant sterols (PS), with a hypocholesterolemic effect, is important for the elderly population. This study aimed at identifying the different PS present in PS-enriched wholemeal rye bread (WRB) and in the ingredient source of PS, to evaluate their bioaccessibility in WRB by simulated static digestion. The gastrointestinal conditions of the elderly were adapted, and the results were compared with the adult population. Nine PS were identified, and a total amount of 2.18 g/100 g WRB was determined. Bioaccessibility was reduced in the elderly model with gastrointestinal adaptation vs. the adult model (11.2 vs. 20.3%), but no differences were observed when adapting only the gastric phase. Even though there was lower bioaccessibility of PS in the elderly, they could benefit from the consumption of WRB as it has a good nutritional profile. Further investigation including in vivo assays is needed to strengthen the results.

A Mixture of Dietary Plant Sterols at Nutritional Relevant Serum Concentration Inhibits Extrinsic Pathway of Eryptosis Induced by Cigarette Smoke Extract.

Cell death program of red blood cells (RBCs), called eryptosis, is characterized by activation of caspases and scrambling of membrane phospholipids with externalization of phosphatidylserine (PS). Excessive eryptosis confers a procoagulant phenotype and is implicated in impairment of microcirculation and increased prothrombotic risk. It has recently been reported that cigarette smokers have high levels of circulating eryptotic erythrocytes, and a possible contribution of eryptosis to the vaso-occlusive complications associated to cigarette smoke has been postulated. In this study, we demonstrate how a mixture of plant sterols (MPtS) consisting of ß-sitosterol, campesterol and stigmasterol, at serum concentration reached after ingestion of a drink enriched with plant sterols, inhibits eryptosis induced by cigarette smoke extract (CSE). Isolated RBCs were exposed for 4 h to CSE (10-20% v/v). When RBCs were co-treated with CSE in the presence of 22 µM MPtS, a significant reduction of the measured hallmarks of apoptotic death like assembly of the death-inducing signaling complex (DISC), PS outsourced, ceramide production, cleaved forms of caspase 8/caspase 3, and phosphorylated p38 MAPK, was evident. The new beneficial properties of plant sterols on CSE-induced eryptosis presented in this work open new perspectives to prevent the negative physio-pathological events caused by the eryptotic red blood cells circulating in smokers.

Bioaccessibility of Plant Sterols in Wholemeal Rye Bread Using the INFOGEST Protocol: Influence of Oral Phase and Enzymes of Lipid Metabolism.

Bioaccessibility of plant sterols (PS) in an enriched wholemeal rye bread was evaluated, for the first time, using the INFOGEST protocol without gastric lipase (GL) and cholesterol esterase (CE), with GL or GL + CE. Moreover, human chewing and an in vitro oral phase (simulated salivary fluid and α-amylase) were evaluated for this purpose. The addition of GL decreased the bioaccessibility of total PS (from 23.8 to 18.5%), whereas the use of GL + CE does not significantly affect PS bioaccessibility. The in vitro oral phase resulted in an ineffective homogenization of the fresh vs partially dried and milled bread, reducing the bioaccessibility of total (from 20.2 to 12.8%) and individual PS. The INFOGEST digestion including the use of GL and CE, as well as an oral phase with human chewing, is proposed for the assessment of PS bioaccessibility in a solid matrix such as wholemeal rye bread since it more closely approximates the in vivo situation.

Elderly gastrointestinal conditions increase sterol bioaccessibility in a plant sterol-enriched beverage: adaptation of the INFOGEST method.

Elderly people suffer from a higher cardiovascular risk. Thus, the fortification of foods with plant sterols (PSs), which have a cholesterol-lowering function, could be of great interest for this target group. To date, no studies have analyzed how the gastrointestinal conditions of the elderly affect PS bioaccessibility. Therefore, this study evaluated the impact of the adaptation of the gastric phase alone and in combination with the intestinal phase on sterol bioaccessibility. For this purpose, the standardized INFOGEST 2.0 method previously adapted for sterol bioaccessibility evaluation in healthy adults was applied to PS-enriched milk-based fruit beverages, examining changes in enzyme activity, incubation time, agitation and pH, based on elderly physiology. The results suggest that the specific gastrointestinal conditions of the elderly could increase absorption of PSs, since their bioaccessibility (%) in a PS-enriched milk-based fruit beverage was significantly increased compared with that in adults (14.95 ± 0.33 vs. 7.96 ± 0.26), also indicating that these conditions increase the bioaccessibility of the beverage's own cholesterol (61.25 ± 2.91 vs. 20.86 ± 2.79). These data support the recommendation of foods of this type for the elderly who can benefit from the increase in bioaccessibility of PSs to have an improved potential cholesterol lowering effect, thus decreasing their risk of cardiovascular disease. However, the performance of subsequent in vivo tests to confirm these results is necessary.

Anti-Eryptotic Activity of Food-Derived Phytochemicals and Natural Compounds.

Human red blood cells (RBCs), senescent or damaged due to particular stress, can be removed by programmed suicidal death, a process called eryptosis. There are various molecular mechanisms underlying eryptosis. The most frequent is the increase in the cytoplasmic concentration of Ca2+ ions, later exposure of erythrocytes to oxidative stress, hyperosmotic shock, ceramide formation, stimulation of caspases, and energy depletion. Phosphatidylserine (PS) exposed by eryptotic RBCs due to interaction with endothelial CXC-Motiv-Chemokin-16/Scavenger-receptor, causes the RBCs to adhere to vascular wall with consequent damage to the microcirculation. Eryptosis can be triggered by various xenobiotics and endogenous molecules, such as high cholesterol levels. The possible diseases associated with eryptosis are various, including anemia, chronic kidney disease, liver failure, diabetes, hypertension, heart failure, thrombosis, obesity, metabolic syndrome, arthritis, and lupus. This review addresses and collates the existing ex vivo and animal studies on the inhibition of eryptosis by food-derived phytochemicals and natural compounds including phenolic compounds (PC), alkaloids, and other substances that could be a therapeutic and/or co-adjuvant option in eryptotic-driven disorders, especially if they are introduced through the diet.

Sterol bioaccessibility in a plant sterol-enriched beverage using the INFOGEST digestion method: Influence of gastric lipase, bile salts and cholesterol esterase.

This study evaluates the influence of increasing bile salts and the addition of key enzymes of the lipidic metabolism in the INFOGEST digestion method on sterol bioaccessibility from a plant sterol (PS)-enriched beverage. The assayed modifications were increasing concentration of bovine bile salts (10 vs. 17.5 mM), and addition of gastric lipase (GL) (60U/mL), cholesterol esterase (CE) (0.075 or 2U/mL) or both. Compared to the original method (10 mM bile salts without enzymes), the assayed conditions significantly reduced bioaccessibility of individual (from 11.3 to 19.7 to 5.1-16.6%) and total PS (13.7 to 6.9-8.0%), and cholesterol (52.8 to 20.9-26.1%), except only when CE is added not allowing cholesterol quantification. The bioaccessibility achieved when lipolytic enzymes were tested was similar for all sterols. For a more physiological approach to in vivo conditions, incorporation of bile salts (10 mM), GL (60U/mL) and CE (0.075U/mL) to the INFOGEST method is proposed, although it increases the cost compared to the established method.

Influence of Galactooligosaccharides on the Positive Effect of Plant Sterol-Enriched Beverages on Cardiovascular Risk and Sterol Colon Metabolism.

In the present study, the impact of galactooligosaccharide (GOS) addition to a plant sterol (PS)-enriched beverage on the hypocholesterolemic effect and on the bioavailability and colonic metabolization of sterols was evaluated. A crossover trial was undertaken in postmenopausal women who intook a PS-enriched (2 g PS/day) or PS-GOS-enriched beverage (2 g PS/day and 4.3 g GOS/day) for 6 weeks. The presence of GOS did not modify the hypocholesterolemic effect of the PS-enriched beverage (total- and low-density lipoprotein-cholesterol reductions) or sterol bioavailability (increments of serum markers of dietary PS intake and of cholesterol synthesis). The consumption of both beverages led to an increase of sterol and metabolite excretion (with the exception of coprostanol, which decreased) and to slight changes in women's capacities for sterol conversion, regardless of the GOS presence. This study demonstrates the suitability of simultaneous enrichment with PS and GOS in milk-based fruit beverages, considering their hypocholesterolemic effect.

Enrichment of Wholemeal Rye Bread with Plant Sterols: Rheological Analysis, Optimization of the Production, Nutritional Profile and Starch Digestibility.

Bread is one of the staple foods of many countries, and its enrichment with bioactive compounds is trending. This phenomenon is focused on breads with a good nutritional profile, such as wholemeal rye bread (WRB), in which enrichment with plant sterols (PSs) is allowed in accordance with European regulations. The objective of the present study was to optimize the production of a WRB enriched with PS (PS-WRB) and to evaluate the proximate composition and starch digestibility as an indicator of nutritional quality. The rheological analysis showed that the bread dough presents satisfactory farinographic properties (dough development time 6 min; stability 4 min; degree of softening 100 Brabender units) but high water absorption (67%). The PS-WRB is high in dietary fiber and low in protein (20.4 and 7.7% w/w, dry basis, respectively) compared with other cereals reported in the scientific literature. In turn, a low starch proportion was hydrolyzed during the simulated digestion (59.9% of total starch), being also slowly hydrolyzed, as deduced from the rapidly digestible starch value (56.5% of total starch). In conclusion, WRB is a suitable matrix for PS enrichment, which allows for obtaining a product with a good nutritional profile and potential health benefits.

Hypercholesterolemic patients have higher eryptosis and erythrocyte adhesion to human endothelium independently of statin therapy.

BACKGROUND: Phosphatidylserine (PS) externalization out of the membrane facilitates the eryptotic erythrocytes (EE) binding to endothelial cells (EC), potentially leading to atherosclerosis. Thus, the levels of eryptosis and interactions of EE-EC in hypercholesterolemic patients, either non-medicated or medicated, compared with healthy subjects were studied. METHODS: A total of 56 subjects clustered into three groups: (control (n = 20), hypercholesterolemic non-treated (HCNT) (n = 15), and statin-treated (HCT) (n = 21)) were enrolled in this cross-sectional study. Biochemical parameters were determined with validated and standard methods. PS exposure was estimated from annexin-V-binding, cell volume from forward scatter (FSC), and GSH from CMFDA fluorescence by flow cytometry. The erythrocyte-EC adhesion assay was performed by using the parallel-plate flow chamber technique. RESULTS: Higher PS externalization and adhesion of erythrocytes to EC (P < .05) was found in hypercholesterolemic subjects, regardless of statin treatment, compared with the control group. Although no correlation between FSC and PS externalization with other parameters was found, GSH was inversely correlated with erythrocyte adhesion, which was significantly correlated with total cholesterol, LDL-c, and apolipoprotein B. CONCLUSION: The link between hypercholesterolemia and eryptosis suggests a possible detrimental impact of this binomial on endothelial function with possible further development of atherosclerosis and microcirculation problems in hypercholesterolemic patients, independently of statin therapy.

Impact of a Plant Sterol- and Galactooligosaccharide-Enriched Beverage on Colonic Metabolism and Gut Microbiota Composition Using an In Vitro Dynamic Model.

A beverage enriched with plant sterols (1 g/100 mL) and galactooligosaccharides (1.8 g/100 mL) was subjected to a dynamic gastrointestinal and colonic fermentation process to evaluate the effect on sterol metabolism, organic acid production, and microbiota composition. Production of sterol metabolites (coprostanol, methylcoprostanol, ethylcoprostenol, ethylcoprostanol, and sitostenone) was observed in the transverse colon (TC) and descending colon (DC) vessels in general, from 24 and 48 h, respectively. Microbial activity was assessed through the production of organic acids, mainly acetate in all colon vessels, lactate in the AC, and butyrate and propionate in the TC and DC. A higher diversity in the microbial community was found in the TC and DC, in accordance with a higher sterol metabolism and organic acid production. Although the prebiotic effect of galactooligosaccharides was not detected, changes in microbiota composition (an increase in the Parabacteroides genus and the Synergistaceae and Lachnospiraceae families) indicated an enhancement of sterol metabolism.

First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs.

Apoptotic effect of a phytosterol-ingredient and its main phytosterol (ß-sitosterol) in human cancer cell lines.

Dietary interventions may effectively control cancer development, with phytosterols (PS) being a class of cancer chemopreventive dietary phytochemicals. The present study, for the first time, evaluates the antiproliferative effects of a PS-ingredient used for the enrichment of several foods and its main PS, ß-sitosterol, at physiological serum levels, in the most prevalent cancer cells in women (breast (MCF-7), colon (HCT116) and cervical (HeLa)). In all three cell lines, these compounds induced significant cell viability reduction without a clear time- and dose-dependent response. Moreover, all treatments produced apoptotic cell death with the induction of DNA fragmentation through the appearance of a sub-G1 cell population. Thus, the use of PS as functional ingredients in the development of PS-enriched foods could exert a potential preventive effect against human breast, colon and cervical cancer, although further in vivo studies are required to confirm our preclinical findings.

Oat and lipolysis: Food matrix effect.

Oat is rich in a wide range of phytochemicals with various physico-chemical, colloidal and interfacial properties. These characteristics are likely to influence human lipid metabolism and the subsequent effect on health following oat consumption. The aim of this work was to investigate the impact of oat materials varying in complexity on the lipolysis process. The composition, structure and digestibility of different lipid systems (emulsions, oil bodies and oil enriched in phytosterols) were determined. The surface activities of phytosterols were examined using the pendant drop technique. Differences in lipid digestibility of the oat oil emulsions and the oil bodies were clearly seen. Also, the digestion of sunflower oil was reduced proportionally to the concentration of phytosterols present. This may be due to their interfacial properties as demonstrated by the pendant drop experiments. This work highlights the importance of considering the overall structure of the system studied and not only its composition.

Sterols in human milk during lactation: bioaccessibility and estimated intakes.

Human milk (HM) is the exclusive food during the first 4-6 months of an infant's life. Breastfeeding has been related to significant health benefits for infants, and hence it is of interest to study the bioactive compounds present in HM, such as sterols (cholesterol being the most abundant). The aim of this study was to determine the contents of sterols (cholesterol, desmosterol, lathosterol, lanosterol, campesterol, stigmasterol and ß-sitosterol) in 10 pools of colostrum, transitional milk, and 1, 3 and 6 month HM obtained from Spanish volunteers from two different geographical areas (coastal and central) and to estimate the intake and bioaccessibility (BA) of sterols in order to ascertain the fate of sterols after digestion. The results showed that the total sterol contents decreased to half the initial level during lactation (24-11 mg per 100 mL) and was significantly higher in samples from the coastal area. Total and animal sterol intakes were between 200 and 400 times higher than plant sterol intakes and were significantly higher in samples from the coastal area. However, no statistically significant differences were found in cholesterol and plant sterol intakes between areas. The BA of total sterols ranged from 45% to 69% and was higher in the first month, which coinciding with the highest fat content of milk. In conclusion, the sterol content varies depending on the lactation stage and the geographical area, and the BA of sterols can be positively affected by a higher lipid content. All these data may contribute to the development of infant formulas that are more similar to HM in terms of composition and behaviour after digestion, according to the lactation stage involved.

A positive impact on the serum lipid profile and cytokines after the consumption of a plant sterol-enriched beverage with a milk fat globule membrane: a clinical study.

The hypocholesterolemic effect and the modification of serum biomarkers of a dietary plant sterol (PS) intake, cholesterol precursors and cytokines after the consumption of milk-based fruit beverages with a milk fat globule membrane were evaluated by a randomized, double-blind, crossover, multiple dose bioavailability study. Postmenopausal women (n = 38) consumed daily 250 mL of a beverage with or without 2 g of PS added during 6 weeks in each of the study periods. With the intake of the PS-added beverage, significant decreases (mg dL-1) in serum total cholesterol (pre-treatment: 220.0 ± 27.8 vs. post-treatment: 212.9 ± 25.8; p < 0.05) and LDL-cholesterol (129.4 ± 28.5 vs. 121.7 ± 24.4; p < 0.05) were detected. The cholesterol precursor lathosterol (11.2%), markers of the dietary PS intake (campesterol 43.1% and ß-sitosterol 32.5%), and anti-inflammatory IL-10 cytokine (22.5%) increased significantly, with a concomitant significant reduction in pro-inflammatory IL-1ß (6.7%). No variations in HDL-cholesterol, other sterols (desmosterol and stigmasterol) or cytokines (IL-6, IL-8, IL-12p70 and TNF-α) were detected. These results indicated that this kind of PS-enriched milk-based fruit beverage is suitable during the period of clinical intervention, and its consumption may be an adequate way to improve PS functionality since a significant reduction in cholesterol levels has been observed. Therefore, the intake of this beverage could contribute to reduce the risk of cardiovascular disease also obtaining a beneficial effect on the serum inflammatory status in postmenopausal women.

International descriptive and interventional survey for oxycholesterol determination by gas- and liquid-chromatographic methods.

Increasing numbers of laboratories develop new methods based on gas-liquid and high-performance liquid chromatography to determine serum concentrations of oxygenated cholesterol metabolites such as 7α-, 24(S)-, and 27-hydroxycholesterol. We initiated a first international descriptive oxycholesterol (OCS) survey in 2013 and a second interventional survey 2014 in order to compare levels of OCS reported by different laboratories and to define possible sources of analytical errors. In 2013 a set of two lyophilized serum pools (A and B) was sent to nine laboratories in different countries for OCS measurement utilizing their own standard stock solutions. In 2014 eleven laboratories were requested to determine OCS concentrations in lyophilized pooled sera (C and D) utilizing the same provided standard stock solutions of OCS. The participating laboratories submitted results obtained after capillary gas-liquid chromatography-mass selective detection with either epicoprostanol or deuterium labelled sterols as internal standards and high-performance liquid chromatography with mass selective detection and deuterated OCS as internal standard. Each participant received a clear overview of the results in form of Youden-Plots and basic statistical evaluation in its used unit. The coefficients of variation of the concentrations obtained by all laboratories using their individual methods were 58.5-73.3% (survey 1), 56.8-60.3% (survey 2); 36.2-35.8% (survey 1), 56.6-59.8, (survey 2); 61.1-197.7% (survey 1), 47.2-74.2% (survey 2) for 24(S)-, 27-, and 7α-hydroxycholesterol, respectively. We are surprised by the very great differences between the laboratories, even under conditions when the same standards were used. The values of OCS's must be evaluated in relation to the analytical technique used, the efficiency of the ample separation and the nature of the internal standard used. Quantification of the calibration solution and inappropriate internal standards could be identified as major causes for the high variance in the reported results from the different laboratories. A harmonisation of analytical standard methods is highly needed.

Cholesterol Content in Human Milk during Lactation: A Comparative Study of Enzymatic and Chromatographic Methods.

This study validates a gas chromatography (GC) method for determining the sterol profile of human milk (HM) and compares it with an enzymatic-spectrophotometric (E-S) method. Good linearity ( r > 0.97) and low limits of detection and quantification were obtained with the GC method (<1.8 and <6 µg/100 g of HM, respectively). Suitable intra- and interassay precisions (all <18%) and satisfactory recovery percentages (80-109%) were obtained for both methods. In addition, both methodologies were used to assess cholesterol evolution in HM during lactation, showing a 50% decrease at 6 months versus colostrum. The E-S method overestimated cholesterol content by <20% versus the GC method. The results indicate that both methods may be used by the industry and in research to better understand the differences between the sterol profiles of infant formulas and HM.

The impact of galactooligosaccharides on the bioaccessibility of sterols in a plant sterol-enriched beverage: adaptation of the harmonized INFOGEST digestion method.

The effect of the addition of galactooligosaccharides (GOS) on sterol bioaccessibility in three plant sterol (PS)-enriched milk-based fruit beverages (without GOS addition (MfB) and with 2.5 g (MfB-G2) and 5.0 g (MfB-G5) GOS per 250 mL) was evaluated after micellar gastrointestinal digestion. Cholesterol bioaccessibility was very similar among beverages, though a slight significant increase (from 80% to 85%) was observed by the addition of 5.0 g GOS. The addition of GOS did not affect total PS bioaccessibility (≈37%). Based on the results obtained after micellar digestion, it has been demonstrated that these beverages could be a suitable food matrix for simultaneous enrichment with PS and GOS. The harmonized in vitro digestion model INFOGEST was applied to the MfB beverage, but the cholesterol content could not be quantified due to its contribution of bile salts. Hence, it was proposed: (i) a change in porcine bile salt concentration from 10 mM to 1.4 mM (in order to compare with micellar digestion); or (ii) a change of bile salt origin (bovine instead of porcine), maintaining physiological concentration (10 mM, INFOGEST condition). Both options allowed cholesterol quantification, with bioaccessibilities of 62% (reduction of bile salts) and 38% (replacement of the bile salt source), whereas plant sterol bioaccessibilities were 22% and 14%, respectively. Therefore, the change of bile salt origin maintaining INFOGEST concentration is proposed as a method to evaluate sterol (cholesterol and PS) bioaccessibility in these beverages, demonstrating the need for the selection of appropriate conditions of the INFOGEST harmonized method according to the food matrix and compounds to be determined.

Sterols in Infant Formulas: A Bioaccessibility Study.

The design of infant formulas (IFs) seeks to resemble human milk (HM) composition and functionality. The fat sources used usually comprise vegetable oil blends to mimic the fatty acid composition of HM and introduce changes in the animal/plant sterol ratio. In contrast, the use of milk fat globule membrane (MFGM)-rich ingredients could improve this aspect by increasing the ratio. The present study evaluates the bioaccessibility (BA) of sterols (cholesterol, desmosterol, brassicasterol, campesterol, stigmasterol, and ß-sitosterol) in three IFs (with or without MFGM) using an in vitro digestion method simulating infant conditions. Analytical parameters confirmed the suitability of the method for all of these sterols. Results showed the presence of MFGM to increase cholesterol content (6-7 vs 2 mg/100 mL), this being the most bioaccessible sterol in the IFs. Although the BA of cholesterol was reduced in MFGM-enriched IF (65.6-80.4% vs 99.7%), the intake of bioaccessible cholesterol from these IFs was higher.