The (193–209) 17‐residues peptide of bovine β‐casein is transported through Caco‐2 monolayer

Although the bioavailability of large peptides with biological activity is of great interest, the intestinal transport has been described for peptides up to only nine residues. β‐casein (β‐CN, 193–209) is a long and hydrophobic peptide composed of 17 amino acid residues (molecular mass 1881 Da) with immunomodulatory activity. The present work examined the transport of the β‐CN (193–209) peptide across Caco‐2 cell monolayer. In addition, we evaluated the possible routes of the β‐CN (193–209) peptide transport, using selective inhibitors of the different routes for peptide transfer through the intestinal barrier. The results showed that the β‐CN (193–209) peptide resisted the action of brush‐border membrane peptidases, and that it was transported through the Caco‐2 cell monolayer. The main route involved in transepithelial transport of the β‐CN (193–209) peptide was transcytosis via internalized vesicles, although the paracellular transport via tight‐junctions could not be excluded. Our results demonstrated the transport of an intact long‐chain bioactive peptide in an in vitro model of intestinal epithelium, as an important step to prove the evidence for bioavailability of this peptide.     

Development of a three‐tier in vitro system,using Caco‐2 cells,to assess the effects of lactate on iron uptake and transport from rye bread following in vitro digestion

A three‐tier Caco‐2 cell system was developed to assess simultaneously iron dialysability, uptake and transport across the Caco‐2 monolayer from an in vitro digested food matrix. The effect of lactate (0–200 mmol L⁻¹) on iron absorption from rye bread subjected to simulated peptic (pH 5.5) and pancreatic digestion (pH 6.5) was investigated to model absorption pre and post the sphincter of Oddi. Lactate increased dialysability (11.8%, P < 0.05) in peptic digests whereas it reduced it in pancreatic digests (4.9%, P < 0.001). Iron uptake from the peptic digests was in the region of 39–76 pmol mg⁻¹ protein whereas it decreased from 281 to 51 pmol mg⁻¹ protein in pancreatic digests. Iron transport was calculated for the peptic digests from [¹⁴C]polyethylene glycol movement and only at 200 mmol L⁻¹ lactate was there any detectable transcellular transport (180 pmol mg⁻¹ protein, P < 0.05). Iron absorption was positively correlated to dialysable iron for both digests (R² = 0.48 and 0.41, respectively, P < 0.01) and the effect of lactate was therefore associated mainly with iron bioaccessibility. The three‐tier system showed the potential to obtain detailed insight into each step involved in iron transport across the monolayer from a food mixture. Copyright © 2006 Society of Chemical Industry     

Adherence of probiotic bacteria to human colon epithelial cells and inhibitory effect against enteric pathogens – In vitro study

The aim of this study was to determine the anti‐adherence properties of three probiotic lactobacillus strains (Lb. rhamnosus 0900, Lb. rhamnosus 0908 and Lb. casei 0919), and their mixture against pathogens: Escherichia coli ATCC 10536, Salmonella enterica serovar Typhimurium ATCC 14028 and Candida albicans ATCC 10231 using Caco‐2 human colon adenocarcinoma cells. All strains of lactobacilli and the probiotic mixture to the greatest extent inhibited adherence of S. Typhimurium, up to 91%. Lb. rhamnosus 0900 inhibited E. coli by 75.9%, and Lb. casei 0919 decreased adherence of C. albicans by 49%. All pathogens activated the adherence of the mixture of probiotic bacteria.     

Bioavailability of iron from a heat treated glucose‐lysine model food system: assays in rats and in Caco‐2 cells

The influence of Maillard reaction products (MRP) from glucose–lysine on iron bioavailability was investigated. Equimolar glucose–lysine mixtures (GL, 40% moisture) were heated (150 °C, for 30, 60 and 90 min) to prepare samples GL30, GL60 and GL90, respectively, and were used to study the influence of MRP on iron solubility, iron uptake and transport in Caco‐2 cells and iron balance in rats. After an initial increase with GL30, in vitro iron solubility decreased in the presence of most of the heated samples. The Caco‐2 cell experiments showed that Fe cell content increased in the presence of heated mixtures with respect to GL, whereas transport was less affected. Iron balance was determined in rats fed diets containing the GL30 and GL90 samples (3%) for a 21 day period and compared with that measured in a control group. Consumption of glucose–lysine heated mixtures increased total iron retention, although hemoglobin values decreased. Iron accumulations in organs such as the spleen, kidney and small intestine were observed. Copyright © 2004 Society of Chemical Industry     

Polyphenols and phenolic acids from strawberry and apple decrease glucose uptake and transport by human intestinal Caco‐2 cells

The effect of polyphenols, phenolic acids and tannins (PPTs) from strawberry and apple on uptake and apical to basolateral transport of glucose was investigated using Caco‐2 intestinal cell monolayers. Substantial inhibition on both uptake and transport was observed by extracts from both strawberry and apple. Using sodium‐containing (glucose transporters SGLT1 and GLUT2 both active) and sodium‐free (only GLUT2 active) conditions, we show that the inhibition of GLUT2 was greater than that of SGLT1. The extracts were analyzed and some of the constituent PPTs were also tested. Quercetin‐3‐O‐rhamnoside (IC₅₀=31 μM), phloridzin (IC₅₀=146 μM), and 5‐caffeoylquinic acid (IC₅₀=2570 μM) contributed 26, 52 and 12%, respectively, to the inhibitory activity of the apple extract, whereas pelargonidin‐3‐O‐glucoside (IC₅₀=802 μM) contributed 26% to the total inhibition by the strawberry extract. For the strawberry extract, the inhibition of transport was non‐competitive based on kinetic analysis, whereas the inhibition of cellular uptake was a mixed‐type inhibition, with changes in both V_max and apparent K_m. The results in this assay show that some PPTs inhibit glucose transport from the intestinal lumen into cells and also the GLUT2‐facilitated exit on the basolateral side.     

Bioavailability of zinc from infant foods by in vitro methods (solubility,dialyzability and uptake and transport by Caco‐2 cells)

The aim of this study was to evaluate the bioavailability of zinc from infant foods (adapted, follow‐up and toddler milk‐based formulas and fruit juices containing milk and cereals, FMC) using solubility, dialyzability and a model combining simulated gastrointestinal digestion and zinc uptake and transport by Caco‐2 cells. The greater solubility of zinc from infant formulas compared with fruit juices (FMC) could be due to the greater casein phosphopeptide content resulting from casein hydrolysis. The highest zinc dialysis percentage corresponded to FMC, which on the other hand had the lowest zinc contents of the analyzed samples. The presence of organic acids in samples of this kind favors the formation of soluble low molecular weight complexes with zinc, thereby increasing the solubility of the latter. Bifidobacterium addition exerted no effect upon zinc bioavailability. Transport and uptake efficiency in Caco‐2 cells were significantly greater for toddler formulas, which presented the highest casein contents. The greater efficiency in zinc transport and uptake from the powdered toddler formula compared with the liquid formulation could be explained by the effect of Maillard reaction products. Copyright © 2006 Society of Chemical Industry     

Monocarboxylate transporter expression is associated with the absorption of benzoic acid in human intestinal epithelial cells

A diet rich in fruit and vegetables is associated with a decreased risk of developing a number of chronic diseases including colon cancer. The active components of the diet which exert these protective effects are unknown but may include small aromatic and phenolic acids. To elicit a physiological response, these acids must first be absorbed by the intestinal epithelium and the present study has focussed on one potential absorptive pathway—the monocarboxylate transporter MCT1. Using Caco‐2 cells (a human intestinal cell line) we have established that benzoic acid uptake is pH‐dependent (resulting in intracellular acidification) and is enhanced in cells expressing higher levels of MCT1 protein. A number of monohydroxybenzoic acids (including salicylic acid) also induced a decrease in intracellular pH which was of a similar magnitude to that elicited by benzoic acid itself, suggesting that they may all be substrates for the same transport pathway. The role of these small organic acids in maintaining normal cell physiology remains to be fully explored. Copyright © 2006 Society of Chemical Industry     

Effect of soluble soybean protein hydrolysate-calcium complexes on calcium uptake by Caco-2 cells

Soybean protein hydrolysates (SPHs) bind with calcium, forming soluble SPH-calcium complexes via the carboxyl groups of glutamic and aspartic acid residues. However, their effect on calcium uptake is still unclear. In this study, Caco-2 cells were used to estimate the effect of SPH-calcium complexes with different molecular weights on calcium uptake in vitro. The changes in intracellular calcium ion concentration were measured by Fura-2 loading and expressed in fluorescence intensity. SPH-calcium complexes could promote calcium uptake. Improved fluorescence intensity was significantly different in SPH-calcium complexes (10 to 30 kDa), SPH-calcium complexes (3 to 10 kDa), and SPH-calcium complexes (1 to 3 kDa). The maximum levels of relative fluorescence intensity (18.3) occurred with SPH-calcium complexes (10 to 30 kDa). The effect of SPH-calcium complexes (10 to 30 kDa) on Ca(2+) increase was determined to be concentration dependent in the range of 0.5 to 4 mg/mL. Our results indicate that soybean protein itself might be responsible for promoting calcium absorption.     

Molecular mechanisms of the naringin low uptake by intestinal Caco-2 cells

Naringin, the main flavanone of grapefruit, was reported to display numerous biological effects: antioxidant, hypocholesteremic, anti-atherogenic and favoring drug absorption. Naringin absorption mechanisms were studied in Caco-2 cells (TC7 clone). We investigated the possible involvement of several membrane transporters implicated in polyphenolic compounds intestinal transport (sodium-dependent glucose transporter 1, monocarboxylate transporter, multidrug-associated resistance proteins 1 and 2, and P-glycoprotein). Naringin was poorly absorbed by Caco-2 cells, according to its low value of apparent permeability coefficient (P(app) = 8.1 +/- 0.9 x 10(-8) cm/s). In the presence of verapamil, a specific inhibitor of P-glycoprotein, cellular uptake was increased by almost threefold after 5 min, and P(app) was doubled after 30 min. Our results indicated the involvement of P-glycoprotein, an ATP-driven efflux pump, capable of transporting naringin from the Caco-2 cell to the apical side. This phenomenon could explain, at least in part, the low absorption of this flavanone at the upper intestinal level.     

Flavonoids alter P-gp expression in intestinal epithelial cells in vitro and in vivo

Flavonoids are secondary plant metabolites included in our diet but are also provided in a growing number of supplements. They are suggested to interact with intestinal transport systems including phospho-glycoprotein (P-gp) which mediates the efflux of a variety of xenobiotics back into the gut lumen. In human intestinal Caco-2 cells, we tested the effects of 14 different flavonoids on P-gp expression in vitro. Protein expression levels were quantified by Western blotting, flow cytometry, and real-time PCR. Except apigenin, all flavonoids at concentrations of 10 microM increased P-gp expression in Western blotting experiments when cells were exposed to the compounds over 4 wk. Flavone was one of the most effective P-gp inducers in Caco-2 cells and its effects were, therefore, also assessed for changes in P-gp in vivo in the gastrointestinal tract of C57BL/6 mice. P-gp expression was significantly increased by flavone (400 mg/kg body weight x day over 4 wk) in the small intestine but not in the colon which displayed intrinsically the highest expression level. In conclusion, the increase in P-gp expression caused by flavonoids in intestinal epithelial cells in vitro and also in vivo may serve as an adaptation and defense mechanism limiting the entry of lipophilic xenobiotics into the organism.     

Absorption and metabolism of the mycotoxin zearalenone and the growth promotor zeranol in Caco-2 cells in vitro

Scope: Zearalenone (ZEN) and α‐zearalanol (α‐ZAL, zeranol) were studied in differentiated Caco‐2 cells and in the Caco‐2 Millicell^® system in vitro to simulate their in vivo intestinal absorption and metabolism in humans. Methods and results: In addition to metabolic reduction/oxidation, extensive conjugation with glucuronic acid and sulfate of the parent compounds and their phase I metabolites was observed. The positional isomers of the glucuronides and sulfates were unambiguously identified: Sulfonation occurred specifically at the 14‐hydroxyl group, whereas glucuronidation was less specific and, in addition to the preferred 14‐hydroxyl group, involved the 16‐ and 7‐hydroxyl groups. Using the Caco‐2 Millicell^® system, an efficient transfer of the glucuronides and sulfates of ZEN and α‐ZAL and their phase I metabolites into both the basolateral and the apical compartment was observed after apical administration. The apparent permeability coefficients (P_app values) of ZEN, α‐ZAL and the ZEN metabolite α‐zearalenol were determined, using an initial apical concentration of 20 μM and a permeation time of 1 h. Conclusion: According to the P_app values, the three compounds are expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycones and as glucuronide and sulfate conjugates in humans.     

Verbascosides from olive mill waste water: assessment of their bioaccessibility and intestinal uptake using an in vitro digestion/Caco-2 model system

Olive mill wastewater (OMWW) is an agricultural waste material produced in high quantities in the Mediterranean basin. OMWW may be an inexpensive source of health promoting phytochemicals with potential economic value including many low molecular weight compounds such as verbascosides. While promising as antioxidants in vitro, little information is available on the potential absorption of verbascosides by humans. The main objective of the present study was to characterize the verbascoside content and potential for their bioavailability from a partially purified phenolic fraction (IP) of OMWW. The IP was obtained after ultrafiltration step at 5000 Dalton and gel filtration low-pressure chromatography (LH20) of OMWW. RP-HPLC analysis identified several soluble phenolics compounds including verbascoside and isoverbascoside as major components of OMWW fractions. The potential for bioavailability of these polyphenols was estimated by using both in vitro digestion and Caco-2 human intestinal cell models. In vitro digestive recoveries (bioaccessibility) were found to be 35.5%± 0.55% for verbascoside and 9.2% ± 0.94% for isoverbascoside highlighting potential sensitivity of these phenolics to gastric and small intestinal digestive conditions. Accumulation of verbascosides by highly differentiated Caco-2 monolayers was linear between 10 and 100 μM of verbascoside and isoverbascoside from IP extract. Uptake of verbascoside and isoverbascoside was rapid with peak accumulation occurring after 30 min with total accumulation efficiency of 0.1% and 0.2% providing intracellular levels of 130 and 80 pmol/mg cell protein for verbascoside and isoverbascoside, respectively. Combined, these data suggest that verbascosides present in OMWW are bioaccessible and provides a rationale for subsequent in vivo studies on the bioavailability and bioactivity of OMWW components.     

Bioavailability Assessment of Copper,Iron, Manganese,Molybdenum, Selenium,and Zinc from Selenium‐Enriched Lettuce

Cu, Fe, Mn, Mo, Selenium (Se), and Zn bioavailability from selenate‐ and selenite‐enriched lettuce plants was studied by in vitro gastrointestinal digestion followed by an assay with Caco‐2 cells. The plants were cultivated in the absence and presence of two concentrations (25 and 40 µmol/L of Se). After 28 days of cultivation, the plants were harvested, dried, and evaluated regarding the total concentration, bioaccessibility, and bioavailability of the analytes. The results showed that biofortification with selenate leads to higher Se absorption by the plant than biofortification with selenite. For the other nutrients, Mo showed high accumulation in the plants of selenate assays, and the presence of any Se species led to a reduction of the plant uptake of Cu and Fe. The accumulation of Zn and Mn was not strongly influenced by the presence of any Se species. The bioaccessibility values were approximately 71%, 10%, 52%, 84%, 71%, and 86% for Cu, Fe, Mn, Mo, Se, and Zn, respectively, and the contribution of the biofortified lettuce to the ingestion of these minerals is very small (except for Se and Mo). Due to the low concentrations of elements from digested plants, it was not possible to estimate the bioavailability for some elements, and for Mo and Zn, the values are below 6.9% and 3.4% of the total concentration, respectively. For Se, the bioavailability was greater for selenite‐enriched than selenate‐enriched plants (22% and 6.0%, respectively), because selenite is biotransformed by the plant to organic forms that are better assimilated by the cells.