Anti-inflammatory activity of plant sterols in a co-culture model of intestinal inflammation: focus on food-matrix effect.

This study investigates the gut anti-inflammatory activity of a plant sterol (PS) food supplement (PS-FS), alongside PS-enriched milk-based fruit beverage and PS-enriched rye bread. A co-culture model based on a dual-chamber system with differentiated intestinal-like Caco-2 cells (apical) and RAW264.7 macrophages (basolateral) was used. The bioaccessible fractions (BF) of the samples were obtained after INFOGEST 2.0 simulated gastrointestinal digestion. The BF were added to the apical part (diluted 1/20 v/v with culture medium to avoid cytotoxicity) for 90 min, followed by stimulation with lipopolysaccharide (LPS) (1 µg mL-1, 24 h) on the basolateral side. The pharmacological interaction between samples and budesonide (1 µM, 90 min) was evaluated. Results indicate that PS-FS significantly attenuated LPS-induced secretion of IL-8 (28%) by Caco-2 cells, and TNF-α (9%) and IL-6 (54%) by RAW264.7 macrophages, whereas PS-enriched beverage and bread did not exhibit protective effects. Additionally, PS-FS demonstrated an improvement in oxidative status in Caco-2 cells, evidenced by reduced levels of reactive oxygen species (47%), iNOS protein expression (27%), and nitrite/nitrate secretion (27%). Mechanistically, PS-FS inhibited the NF-κB-COX-2-PGE2 signaling pathway in macrophages, resulting in decreased NF-κB p65 nuclear translocation (39%), COX-2 protein expression (32%), and PGE2 production (27%). Co-treatment with budesonide and PS-FS displayed an antagonistic effect (combination index 0.38-0.63). This study demonstrates the potent intestinal anti-inflammatory activity of a PS-FS, positioning it as a promising nutraceutical product for the management of inflammatory bowel diseases. However, the food matrix of the milk-based fruit beverage and rye bread appear to interfere with the anti-inflammatory activity of PS.

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.

Coupling in vitro food digestion with in vitro epithelial absorption; recommendations for biocompatibility.

As food transits the gastrointestinal tract, food structures are disrupted and nutrients are absorbed across the gut barrier. In the past decade, great efforts have focused on the creation of a consensus gastrointestinal digestion protocol (i.e., INFOGEST method) to mimic digestion in the upper gut. However, to better determine the fate of food components, it is also critical to mimic food absorption in vitro. This is usually performed by treating polarized epithelial cells (i.e., differentiated Caco-2 monolayers) with food digesta. This food digesta contains digestive enzymes and bile salts, and if following the INFOGEST protocol, at concentrations that although physiologically relevant are harmful to cells. The lack of a harmonized protocol on how to prepare the food digesta samples for downstream Caco-2 studies creates challenges in comparing inter laboratory results. This article aims to critically review the current detoxification practices, highlight potential routes and their limitations, and recommend common approaches to ensure food digesta is biocompatible with Caco-2 monolayers. Our ultimate aim is to agree a harmonized consensus protocol or framework for in vitro studies focused on the absorption of food components across the intestinal barrier.

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.

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.

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 high-pressure processing on the stability and bioaccessibility of bioactive compounds in Clementine mandarin juice and its cytoprotective effect on Caco-2 cells.

Mandarin juice is a rich source of antioxidant bioactive compounds. While the content and profile of bioactives are known, the impact of high-pressure processing (HPP) on their stability and bioaccessibility (BA) is unknown, but may allow obtaining safe, nutritious, and fresh-tasting juices with highly extractable bioactive compounds. The stability and BA of bioactive antioxidant compounds in untreated and HPP-treated (400 MPa/40 °C/1 min) Clementine mandarin juices, and the cytoprotective effect of its bioaccessible fractions (BF) obtained after simulated gastrointestinal digestion against H2O2-induced oxidative stress in differentiated Caco-2 cells were investigated. The BF of HPP-treated juices showed a better retention of carotenoids, flavonoids, ascorbic acid, total polyphenols and FRAP value, and slightly higher cytoprotection (mitochondrial membrane potential and ROS) than untreated juices. Therefore, HPP can be recommended as a suitable technology to retain or indeed increase antioxidant bioactives and their cytoprotective activity in mandarin juices after gastrointestinal digestion.

Antiproliferative activity of green, black tea and olive leaves polyphenols subjected to biosorption and in vitro gastrointestinal digestion in Caco-2 cells.

Olive (Olea europaea L.) leaves and tea (Camellia sinensis) are rich sources of bioactive compounds, especially polyphenols. Our previous studies have evidenced the potential use of Saccharomyces cerevisiae as a natural delivery system for these antioxidants and a means to improve their bioaccessibility in the human gut. In the present work, the antiproliferative effect of green tea (GT), black tea (BT) and olive leaves (OL) infusions and suspensions of S. cerevisiae were evaluated, for the first time, in human colon cancer cells (Caco-2) after biosorption and in vitro gastrointestinal digestion. The bioaccessible fractions (BF) were not overtly cytotoxic, not affecting cell viability. ROS and mitochondrial membrane potential changes (Δψm) values were reduced compared with control cells. Moreover, all the BF after biosorption induced a significant (p < 0.05) increase in cell proportions in S-phase. The arrest of the cell cycle was reversible without induction of apoptosis, suggesting that the biosorbed phenolics in both infusions and suspensions act as cytostatic agents.

Antiproliferative Effect of Bioaccessible Fractions of Four Brassicaceae Microgreens on Human Colon Cancer Cells Linked to Their Phytochemical Composition.

The antiproliferative effect of the bioaccessible fractions (BFs) of four hydroponic Brassicaceae microgreens (broccoli, kale, mustard and radish) was evaluated on colon cancer Caco-2 cells vs. normal colon CCD18-Co cells after 24 h treatment with BFs diluted 1:10 v/v in cell culture medium. Their bioactivity was compared with the digestion blank, while the colon cancer chemotherapeutic drug 5-fluorouracil was used as a positive control. Cell viability (mitochondrial enzyme activity assay (MTT test) and Trypan blue test) and mechanisms related to antiproliferative activity (cell cycle, apoptosis/necrosis, mitochondrial membrane potential, reactive oxygen species (ROS) production, Ca2+ and glutathione (GSH) intracellular content) were studied. All microgreen BFs increased ROS and decreased GSH, altering the redox status and causing mitochondrial membrane dissipation followed by a general cell cycle arrest in G2/M and apoptotic cell death via a Ca2+-independent mechanism. As a result, the antioxidant bioactive compounds present in these microgreen species reduced the proliferation of tumoral cells (10 to 12.8% -MTT or 20 to 41.9% -Trypan blue), showing lesser effects with broccoli microgreens, in line with their lower ascorbic acid content and total antioxidant capacity. Therefore, the daily intake of microgreens within a balanced diet could be a preventive nutritional strategy to reduce the burden of chronic degenerative diseases such as colon cancer.

Anti-Inflammatory and Cytoprotective Effect of Plant Sterol and Galactooligosaccharides-Enriched Beverages in Caco-2 Cells.

Plant sterol (PS) (1 g/100 mL) enriched milk-based fruit beverages with or without galactooligosaccharides (GOS) (1.8 g/100 mL) were used in differentiated Caco-2 cells. Their potential cytopreventive effect against oxidative stress induced by cholesterol oxidation products (COPs) and their anti-inflammatory properties were evaluated. Preincubation (24 h) with bioaccessible fractions from beverages without and with GOS (MfB and MfB-G) completely prevented the COPs (60 µM/4 h) induced oxidative stress independent to GOS presence with exception to calcium influx and GSH content, where a partial protective effect was observed. Besides, MfB produced a significant (p < 0.05) reduction of IL-8 (40%) and IL-6 (50%) after IL-1ß-induction (1 ng/mL/24 h) through the inhibition of NF-κB p65 translocation into the nucleus (16%) compared to control cells, while GOS presence compromised their anti-inflammatory effect. Therefore, PS-enriched milk-based fruit beverage could be an interesting strategy to prevent intestinal injury produced by COPs and to attenuate the pro-inflammatory process in intestinal human diseases. GOS addition had no extra beneficial antioxidant effect and even reduced their anti-inflammatory properties.

Evaluation of the Bioaccessibility of Antioxidant Bioactive Compounds and Minerals of Four Genotypes of Brassicaceae Microgreens.

Microgreens constitute an emerging class of fresh, healthy foods due to their nutritional composition. In this study the content of minerals and antioxidant bioactive compounds, and for the first time bioaccessibility, were evaluated in broccoli (Brassica oleracea L. var. italica Plenck), green curly kale (Brassica oleracea var. sabellica L.), red mustard (Brassica juncea (L.) Czern.) and radish (Raphanus sativus L.) hydroponic microgreens. Macro- (K, Ca, Mg) and oligo-elements (Fe, Zn), ascorbic acid, total soluble polyphenols, total carotenoids, total anthocyanins, total isothiocyanates and total antioxidant capacity (Trolox Equivalent Antioxidant Capacity and Oxygen Radical Absorbance Capacity) were determined before and after the standardized simulated gastrointestinal digestion process. All microgreens provided relevant amounts of vitamin C (31-56 mg/100 g fresh weight) and total carotenoids (162-224 mg ß-carotene/100 g dry weight). Mineral content was comparable to that normally found in hydroponic microgreens and the low potassium levels observed would allow their dietetic recommendation for patients with impaired kidney function. Both total soluble polyphenols and total isothiocyanates were the greatest contributors to the total antioxidant capacity after digestion (43-70% and 31-63% bioaccessibility, respectively) while macroelements showed an important bioaccessibility (34-90%). In general, radish and mustard presented the highest bioaccessibility of bioactive compounds and minerals. Overall, the four hydroponic Brassicaceae microgreens present a wide array of antioxidant bioactive compounds.

Effect of a Milk-Based Fruit Beverage Enriched with Plant Sterols and/or Galactooligosaccharides in a Murine Chronic Colitis Model.

The potential anti-inflammatory effect of plant sterols (PS) enriched milk-based fruit beverages (PS, 1 g/100 mL) (MfB) with/without galactooligosaccharides (GOS, 2 g/100 mL) (MfB-G) in an experimental mice model of chronic ulcerative colitis was evaluated. Beverages were orally administered to mice every day by gavage to achieve PS and GOS doses of 35 and 90 mg/kg, respectively, and experimental colitis was induced by giving mice drinking water ad libitum containing 2% (w/v) dextran sulphate sodium (DSS) for 7 days, alternating with periods without DSS up to the end of the study (56 days). MfB beverage showed significant reduction of symptoms associated to ulcerative colitis and improved the colon shortening and mucosal colonic damage, but it was not able to reduce the increase of myeloperoxidase levels produced by DSS. MfB-G showed higher incidence of bloody feces and loss of stool consistency than MfB, as well as high levels of immune cells infiltration in colon tissue and myeloperoxidase. Therefore, PS-enriched milk-based fruit beverage could be an interesting healthy food to extend the remission periods of the diseases and the need to evaluate, in a pre-clinical model, the anti-inflammatory effect of the combination of bioactive compounds in the context of a whole food matrix.

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.

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.

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.

Protective effect of bioaccessible fractions of citrus fruit pulps against H2O2-induced oxidative stress in Caco-2 cells.

Fruit pulps from Navel (N) and Cara Cara (CC) oranges, and Clementine mandarin freshly harvested (M) and refrigerated stored (M12) were used to evaluate the cytoprotective effect of their bioaccessible fractions (BF) against H2O2-induced oxidative stress in Caco-2 cells. BF of samples preserved viability vs. H2O2 treated cells, reaching values similar to controls. Lipid peroxidation was reduced to levels of control cells, but M did not reach control values. ROS and mitochondrial membrane potential changes (Δψm) values were reduced compared with H2O2 treated cells, but without achieving control levels. A significant reduction in cell proportions in G1 phase and a significant increase in sub-G1 phase (apoptosis) of cell cycle was shown in H2O2 treated cells, and BF allowed a recovery close to control levels. Thus, BF of samples protect the cells from oxidative stress by preserving cell viability, mitochondrial membrane potential and correct cell cycle progression, and diminishing lipid peroxidation and ROS.

Effects of Plant Sterols or ß-Cryptoxanthin at Physiological Serum Concentrations on Suicidal Erythrocyte Death.

The eryptotic and hemolytic effects of a phytosterol (PS) mixture (ß-sitosterol, campesterol, stigmasterol) or ß-cryptoxanthin (ß-Cx) at physiological serum concentration and their effect against oxidative stress induced by tert-butylhydroperoxide (tBOOH) (75 and 300 µM) were evaluated. ß-Cryptoxanthin produced an increase in eryptotic cells, cell volume, hemolysis, and glutathione depletion (GSH) without ROS overproduction and intracellular Ca2+ influx. Co-incubation of both bioactive compounds protected against ß-Cx-induced eryptosis. Under tBOOH stress, PS prevented eryptosis, reducing Ca2+ influx, ROS overproduction and GSH depletion at 75 µM, and hemolysis at both tBOOH concentrations. ß-Cryptoxanthin showed no cytoprotective effect. Co-incubation with both bioactive compounds completely prevented hemolysis and partially prevented eryptosis as well as GSH depletion induced by ß-Cx plus tBOOH. Phytosterols at physiological serum concentrations help to prevent pro-eryptotic and hemolytic effects and are promising candidate compounds for ameliorating eryptosis-associated diseases.

Influence of orange cultivar and mandarin postharvest storage on polyphenols, ascorbic acid and antioxidant activity during gastrointestinal digestion.

Polyphenols, ascorbic acid content and antioxidant activity of two sweet oranges (Navel-N and Cara Cara-CC) and mandarin (Clementine-M) as well as their bioaccessibilities were evaluated in pulps and compared to those in fresh juice. Thus, pulps of oranges and mandarins displayed higher hesperidin (HES), narirutin (NAR), total flavonoids (TF), total phenols (TP) and antioxidant activity (AAC) than their corresponding juices. Also, CC products presented higher bioactive compounds content than N ones. Bioaccessibility of bioactive compounds and AAC were higher in pulps of both oranges and mandarin than in their corresponding juices. Oranges (N and CC) pulps and juices presented higher bioaccessibilities than mandarin ones. The postharvest storage of mandarin at 12°C during 5weeks not only produced a significant increase of the bioactive compounds but also an increase of their bioaccessibility. The bioaccessibility of Citrus bioactive compounds is necessary for calculating more accurately their daily intake amount.

Iron bioavailability in iron-fortified cereal foods: The contribution of in vitro studies.

Iron deficiency anemia is the most common nutritional deficiency in humans. Not all dietary ingested iron, heme or nonheme, will be available to absorption and negative imbalance between iron requirements and absorption leads to iron deficiency and/or anemia. The recommended iron values usually are based on the genetic and on diet iron-bioavailability, which can be considered as the principal factor that change among the cultures and influences the distinct levels of recommendation among countries. Dietary changes present practical limitations due to be difficult to change food habits. The iron food fortification is considered more cost effective and economically more attractive than iron supplementation. There are many iron compounds available to be used in iron fortification. Cereals represent a target food group to iron fortification programs due to high consumption and the in vitro studies can be useful to estimate the relative iron bioavailability in large number of products in short time and with a low cost. Wheat flour baked into bread or not was the main product tested in in vitro bioavailability studies and ferrous sulfate was the principal iron compound used in the fortification studies. However, iron bioavailability from ferrous sulfate is lower than from other compounds, such FeNaEDTA or ferric pyrophosphate. The variables level of fortification, storage, level of extraction, baking and also the association or not with other chemical compound seems to influence the results obtained.