Transepithelial Transport of Theasinensins through Caco-2 Cell Monolayers and Their Absorption in Sprague-Dawley Rats after Oral Administration

The aim of this study is to illustrate the in vivo and in vitro absorption of theasinensins B and A that are (-)-epigallocatechin-3-O-gallate (EGCG)-(-)-epigallocatechin (EGC) dimer and EGCG dimer, respectively, and their transport pathway across the intestinal membrane. Our animal study by a single oral administration to rats demonstrated the intact absorption of theasinensins into the blood system, which was estimated to be a >10-fold lower absorption amount than EGCG. The in vitro absorption study indicated that theasinensins can be transported across Caco-2 cell monolayers, while their permeability coefficients were also >10-fold lower than those of EGCG and EGC. Transport experiments using cytochalasin D or quercetin as a tight junction (TJ) modulator and a non-saturable permeation revealed that theasinensins were transported across Caco-2 cells in a TJ paracellular diffusion route. In conclusion, the dimers of condensed catechins, theasinensins B and A, can be absorbed intact into rat blood and transported across Caco-2 cell monolayers probably through a TJ paracellular pathway.     

Recombinant human lactoferrin and iron transport across Caco-2 monolayers: effect of heat treatment on the binding to cells

Recombinant human lactoferrin (rhLF) from Aspergillus awamori bound to Caco-2 cell membranes in a saturable manner. The dissociation constant for the apo form was (Kd)=2.2 x 10(-7) M; however, the specific binding of the iron-saturated rhLF and of lactoferrin from human milk (hLF) was too low to calculate the binding parameters. Recombinant human lactoferrin subjected to heat treatment did not lose the ability to bind to cell membranes except at high temperature and long time treatments (85 and 89 degrees C for 40 min) for which there was a slight decrease in the binding. No significant differences have been found in the transport of iron bound to rhLF or to hLF across Caco-2 cell monolayers. Nevertheless, the amount of iron-saturated hLF transported across Caco-2 monolayers was significantly higher than that of rhLF. For both lactoferrins, the amount of intact protein in the lower chamber was about 4.5% of the total radioactivity transported, indicating the degradation of lactoferrin in the passage across Caco-2 cells.     

Transepithelial transport of alpha-lipoic acid across human intestinal Caco-2 cell monolayers

Alpha-lipoic acid (LA) is used in dietary supplements or food with antioxidative functions. The mechanism for the intestinal absorption of alpha-lipoic acid was investigated in this study by using human intestinal Caco-2 cell monolayers. LA was rapidly transported across the Caco-2 cell monolayers, this transport being energy-dependent, suggesting transporter-mediated transport to be the mechanism involved. The LA transport was strongly dependent on the pH value, being accelerated in the acidic pH range. Furthermore, such monocarboxylic acids as benzoic acid and medium-chain fatty acids significantly inhibited LA transport, suggesting that a proton-linked monocarboxylic acid transporter (MCT) was involved in the intestinal transport of LA. The conversion of LA to the more antioxidative dihydrolipoic acid was also apparent during the transport process.     

Transepithelial transport of microbial metabolites of quercetin in intestinal Caco-2 cell monolayers

m-Hydroxyphenylacetic acid (mHPA), 3,4-dihydroxyphenylacetic acid (DHPA), and 4-hydroxy-3-methoxyphenylacetic acid (HMPA) are major microbial metabolites of quercetin. After administration of quercetin to human subjects, these metabolites are readily detected in blood and urine. mHPA, DHPA, and HMPA are thought to exert protective biological activity within the body due to their antioxidant properties. However, very little work has been published concerning their absorption. I have examined the absorption characteristics of the quercetin metabolites in Caco-2 cells by a coulometric detection method using HPLC-ECD. All of them exhibited nonsaturable transport in Caco-2 cells up to 30 mM, whereas HMPA and mHPA also showed proton-coupled polarized absorption. The proton-coupled directional transport of HMPA and mHPA was inhibited by the substrate of the monocarboxylic acid transporter (MCT). A considerable amount of apically loaded HMPA and mHPA was taken up and transported through to the basolateral side, while almost all of the apically loaded DHPA was retained on the apical side. Furthermore, the transepithelial flux of DHPA was inversely correlated with the paracellular permeability of Caco-2 cells, although those of HMPA and mHPA were almost constant. These results indicate that transport of DHPA was mainly via paracellular diffusion, although HMPA and mHPA were absorbed to some extent by the MCT.     

In Vitro Studies on Atrazine Effects on Human Intestinal Cells

Considering the importance of the oral route for human exposure to atrazine, we have investigated the possible effect of this herbicide on the human intestinal cells and the integrity of the epithelial barrier, using Caco-2 cells as the intestinal model in vitro. We evaluated possibile cytotoxic and genotoxic effects of atrazine in concentrations ranging from 1 to 250 μM on the Caco-2 cells at different stages of growth after short- and long-term exposure. Results from the tetrazolium blue (MTT) test and the Trypan blue exclusion assay showed that atrazine cytotoxicity was dose- and time-dependent. Obtained data indicated that atrazine at high concentrations (50 and 250 μM) was able to induce effects on Caco-2 proliferation and viability. Moreover, it was found that the long-term exposure to atrazine at the non-cytotoxic dose caused inhibition of the intestinal cell maturation and decreased the transepithelial electrical resistance, the indicator of the epithelial barrier integrity. Studies on the atrazine genotoxicity determined using the single cell microelectrophoresis assay indicated that atrazine did not induce DNA damages in the Caco-2 cells at concentrations of up to 50 μM, whereas enhancement in the DNA damage was observed at 250 μM. Altogether, our results indicate that atrazine at expected human oral exposure concentrations is not able to induce effects on the Caco-2 cell proliferation and viability, but may suppress the intestinal cell differentiation and reduce the cell monolayer integrity. We suggest that chronic exposure on low levels of atrazine may lead to alteration in the expression of the morphological and functional features of the Caco-2 cells related to the transport and barrier function of small intestinal enterocytes. In consequence, this may lead to alterations in the intestinal absorption process.     

Transport of cranberry A-type procyanidin dimers, trimers, and tetramers across monolayers of human intestinal epithelial Caco-2 cells

A-type procyanidin oligomers in cranberries are known to inhibit the adhesion of uropathogenic bacteria. B-type procyanidin dimers and trimers are absorbed by humans. The absorption of A-type procyanidins from cranberries in humans has not been demonstrated. This study examined the transport of A-type cranberry procyanidin dimers, trimers, and tetramers on differentiated human intestinal epithelial Caco-2 cell monolayers. Procyanidins were extracted from cranberries and purified using chromatographic methods. Fraction I contained predominantly A-type procyanidin dimer A2 [epicatechin-(2-O-7, 4-8)-epicatechin]. Fraction II contained primarily A-type trimers and tetramers, with B-type trimers, A-type pentamers, and A-type hexamers being minor components. Fraction I or II in solution was added onto the apical side of the Caco-2 cell membranes. The media at the basolateral side of the membranes were analyzed using HPLC-MS(n) after 2 h. Data indicated that procyanidin dimer A2 in fraction I and A-type trimers and tetramers in fraction II traversed across Caco-2 cell monolayers with transport ratio of 0.6%, 0.4%, and 0.2%, respectively. This study demonstrated that A-type dimers, trimers, and tetramers were transported across Caco-2 cells at low rates, suggesting that they could be absorbed by humans after cranberry consumption.     

Lactic acid decreases Fe(II) and Fe(III) retention but increases Fe(III) transepithelial transfer by Caco-2 cells

Lactic acid (LA) has been proposed to be an enhancer for dietary iron absorption, but contradictory results have also been reported. In the present study, fully differentiated Caco-2 cell monolayers were used to evaluate the effects of LA (1-50 mmol/L) on the cellular retention and transepithelial transport of soluble non-heme iron (as ferric nitrilotriacetate). Our data revealed a linear decline in Fe(III) retention with respect to the concentration of LA added. In the presence of 50 mmol/L LA, retention of Fe(III) and Fe(II) decreased 57% and 58%, respectively. In contrast, transfer of Fe(III) across the cell monolayer was doubled, while Fe(II) transfer across the cell monolayer decreased 35%. We conclude that LA reduces cellular retention and transepithelial transport of Fe(II) by Caco-2 cells in a dose-dependent manner. However, while LA also reduces retention of Fe(III) by Caco-2 cells, the transfer of Fe(III) across cell monolayers is enhanced, possibly due to effects on paracellular transport.     

Stabilization of oil-in-water emulsions by beta-lactoglobulin-polyethylene glycol conjugates

The disulfide bonds of beta-lactoglobulin (beta-lg) were modified by oxidative sulfitolysis to generate beta-lgSO(3). The native protein (beta-lg) and the modified protein (beta-lgSO(3)) were conjugated to activated polyethylene glycol (PEG) to generate beta-lgPEG and beta-lgSO(3)PEG, respectively. Oil-in-water (o/w) emulsions containing 1% beta-lg or beta-lg conjugates were prepared at pH 2.8, 5.0, and 7.0. Emulsion droplet diameters and zeta potentials were measured. For the same emulsifier, emulsion droplet diameters decreased when emulsion pH increased. Zeta potentials of emulsion droplets increased with pH for beta-lg and beta-lgSO(3). Zeta potentials of beta-lgPEG and beta-lgSO(3)PEG approached zero, suggesting that the protein molecule was covered by PEG chains. Accelerated and 7-day storage stabilities at 21 degrees C of the emulsions were monitored. The emulsifying activity index (EAI) of beta-lgPEG was not significantly different from the EAI of beta-lg. The EAI of beta-lg was enhanced following sulfitolysis of beta-lactoglobulin. The emulsifying activity increased more when the oxidatively modified protein was conjugated to polyethylene glycol. Emulsions made with beta-lgSO(3)PEG were more stable than emulsions made with beta-lg, beta-lgPEG, or beta-lgSO(3) under accelerated stability study and for 7 days at 21 degrees C. The stability of o/w emulsions stabilized with beta-lgSO(3)PEG increased because individual droplets were better protected, against protein bridging or coalescence, by the thick adsorbed protein-PEG layer.     

Absorption of anthocyanins from blueberry extracts by caco-2 human intestinal cell monolayers

Recent studies have shown that dietary polyphenols may contribute to the prevention of cardiovascular disease and cancer. Anthocyanins from different plant sources including blueberries have been shown to possess potential anticancer activities. One of the key factors needed to correctly relate the in vitro study results to human disease outcomes is information about bioavailability. The objectives of the current study were to evaluate the absorption of blueberry anthocyanin extracts using Caco-2 human intestinal cell monolayers and investigate the effects of different aglycones, sugar moieties, and chemical structure on bioavailability of different types of anthocyanins. The results of this study showed that anthocyanins from blueberries could be transported through the Caco-2 cell monolayers although the transport/absorption efficiency was relatively low compared to other aglycone polyphenols. The transport efficiency of anthocyanins averaged approximately 3-4% [less than 1% in delphinidin glucoside (Dp-glc)]. No significant difference in transport/absorption efficiency was observed among three blueberry cultivars. The observed trends among different anthocyanins generally agreed well with some published in vivo results. Dp-glc showed the lowest transport/absorption efficiency, and malvidin glucoside (Mv-glc) showed the highest transport/absorption efficiency. Our result indicates that more free hydroxyl groups and less OCH(3) groups can decrease the bioavailability of anthocyanins. In addition, cyanindin glucoside (Cy-glc) showed significantly higher transport efficiency than cyanidin galactoside (Cy-gal), and peonidin glucoside (Pn-glc) showed significantly higher transport efficiency than peonidin galactoside (Pn-gal), indicating that glucose-based anthocyanins have higher bioavailability than galactose-based anthocyanins.     

In vitro absorption of dietary trans-resveratrol from boiled and roasted peanuts in Caco-2 cells

Previous studies on the transport and absorption of resveratrol (3,5,4-O-trihydroxystilbene) were done using the pure compound. In this study, the absorption of resveratrol in digested peanut micellar from boiled and roasted peanuts was investigated using a human intestinal Caco-2 cell monolayer. The amount transported and rate of transport of both resveratrol glycosides and its hydrolytic product were quantified by a reverse-phase high-performance liquid chromatography method with mass spectrometric detection. Four peaks were identified in the digested peanut micellar of both boiled and roasted peanuts: two resveratrol glycosides, one resveratrol diglycoside, and possibly an acylated resveratrol glycoside. Resveratrol from roasted peanut micellar had a higher transport rate than those from the boiled peanut. This implies that resveratrol from roasted peanut is better absorbed than from boiled peanut. Also, the rate of transport and amount of resveratrol transported were higher for the hydrolytic product than the nonhydrolyzed glycosides. This has strong implications for in vivo absorption as the enzymatic activity of gut microflora could enhance the bioavailability of β-glycosides of dietary polyphenols.     

Cellular uptake and efflux of trans-piceid and its aglycone trans-resveratrol on the apical membrane of human intestinal Caco-2 cells

Two stilbenes (trans-piceid and its aglycone trans-resveratrol) were investigated in the uptake across the apical membrane of the human intestinal cell line Caco-2 in order to determine their mechanisms of transport. The uptake was quantified using a reverse phase high-performance liquid chromatography method with fluorescence detection. The rate of cellular accumulation in the cells was found to be higher for trans-resveratrol than for trans-piceid. In addition, trans-resveratrol uses passive transport to cross the apical membrane of the cells, whereas the transport of trans-piceid is likely active. With regard to the mechanisms of transport, the involvement of the active transporter SGLT1 in the absorption of trans-piceid was deduced using various inhibitors directly or indirectly exploiting the activity of this transporter (glucose, phlorizin, and ouabain). Moreover, we investigated the involvement of the multidrug-related protein 2 (MRP2), an efflux pump present on the apical membrane, in stilbene efflux by Caco-2 cells. The effect of MK-571 (an MRP inhibitor) seems to implicate MRP2 as responsible for apical efflux of trans-piceid and trans-resveratrol.     

Tea polyphenols inhibit the transport of dietary phenolic acids mediated by the monocarboxylic acid transporter (MCT) in intestinal Caco-2 cell monolayers

It was previously reported that a fluorescent marker dye, fluorescein, is transported via the monocarboxylic acid transporter (MCT). Fluorescein transport was competitively inhibited by MCT substrates such as ferulic and salicylic acids. Tea polyphenols, in particular, epigallocatechin gallate (EGCg) and epicatechin gallate (ECg), inhibited the transport of fluorescein. Tea polyphenols also inhibited the transport of salicylic and ferulic acids, suggesting tea polyphenols might be substrates of MCT. However, the transepithelial flux of tea polyphenols was much lower than that of the MCT substrates and was inversely correlated with the paracellular permeability of Caco-2 cell monolayers. These findings suggest that tea polyphenols are not substrates but inhibitors of MCT. Furthermore, the transepithelial transport of these polyphenols is mainly via paracellular diffusion. However, directional transport of ECg and EGCg from the basolateral to the apical side was observed, indicating that the behavior of tea polyphenols in the intestinal epithelium is complex.     

Effect of food models and low-temperature storage on the adhesion of Lactobacillus rhamnosus GG to Caco-2 cells

This study evaluated the effects of fat and sugar levels on the surface properties of Lactobacillus rhamnosus GG during storage in food model systems, simulating yogurt and ice cream, and related them with the ability of the bacterial cells to adhere to Caco-2 cells. Freeze-dried L. rhamnosus GG cells were added to the model food systems and stored for 7 days. The bacterial cells were analyzed for cell viability, hydrophobicity, ζ potential, and their ability to adhere to Caco-2 cells. The results indicated that the food type and its composition affected the surface and adhesion properties of the bacterial cells during storage, with yogurt being a better delivery vehicle than ice cream in terms of bacterial adhesion to Caco-2 cells. The most important factor influencing bacterial adhesion was the storage time rather than the levels of fats and sugars, indicating that conformational changes were taking place on the surface of the bacterial cells during storage.     

Dielectric study of heat-denatured ovalbumin in aqueous solution by time domain reflectometry method

The dielectric behavior of native and heat-denatured ovalbumins (OVAs) from three avian species in aqueous solution was examined over a frequency range of 100 kHz to 20 GHz, using the time domain reflectometry (TDR) method. For the native OVA solutions, three kinds of relaxation processes were observed at around 10 MHz, 100 MHz, and 20 GHz, respectively; these could be assigned to the overall rotation of protein molecules, the reorientations of the bound water, and the free water molecules, respectively. For the heat-denatured samples, three relaxation processes were also observed. However, the relaxation process at approximately 100 MHz originated via a different mechanism other than the reorientation of bound water, namely, the micro-Brownian motion of peptide chains of heat-denatured protein. From the observed relaxation process at approximately 100 MHz, the relaxation strength of heat-denatured OVA solution for duck was higher than that of OVA solutions for hen and guinea fowl and showed the pH dependency from pH 7.0 to 8.0 for OVAs obtained from all three species. Furthermore, the results demonstrated that the relaxation strength was closely related to surface hydrophobicity of protein molecules and gel rheological properties. It was suggested that the difference in the surface hydrophobicity of protein influenced the dielectric behavior of water around denatured protein, whereas the dielectric behavior of denatured protein could be an indication of the gel rheological properties. Such studies can aid in the understanding of the different network structures of OVA gels from three avian species.     

Pressure-induced conformational changes of beta-lactoglobulin by variable-pressure Fourier transform infrared spectroscopy.

Pressure-induced conformational changes in D(2)O solutions of the two genetic variants of beta-lactoglobulin A (beta-lg A) and beta-lactoglobulin B (beta-lg B) and an equal mixture of both variants (beta-lg A+B) were studied by employing variable-pressure Fourier transform infrared (VP-FTIR) spectroscopy. Changes in the secondary structure of beta-lg A were observed at lower pressure compared to beta-lg B, indicating that beta-lg A had a more flexible structure. During the decompression cycle beta-lg A showed protein aggregation, accompanied by an increase in alpha-helical conformation. The changes in the secondary structure of beta-lg B with the pressure were minor and for the most part reversible. Upon decompression no aggregation in beta-lg B was observed. Increasing the pressure from 0.01 to 12.0 kbar of a solution containing beta-lg A+B resulted in substantial broadening of all major amide I bands. This effect was partially reversed by decreasing the hydrostatic pressure. beta-lg A+B underwent less aggregate formation than beta-lg A, possibly as a result of protein-protein interactions between beta-lg A and beta-lg B. Hence, it is likely that the functional or biological attributes of beta-lg proteins may be affected in different ways by hydrostatic pressure.     

Heat-induced aggregation of beta-lactoglobulin as a function of pH.

The effect of pH in the range 6.0-8.0 on the denaturation and aggregation of beta-lactoglobulin (beta-lg) was investigated. Results were interpreted in terms of the reaction scheme for the denaturation and aggregation of beta-lg proposed by Roefs and De Kruif (Eur. J. Biochem. 1994, 226, 883-889). The rate of conversion of native beta-lg increased strongly at higher pH values, whereas the molecular mass of the aggregates decreased strongly. In the pH range 6.4-8.0 aggregates were formed mainly by intermolecular disulfide bonds, but even at pH 6.0, thiol/disulfide exchange reactions were involved, although to a lesser extent. The time course of the exposure of the thiol group in native beta-lg upon heating and the subsequent disappearance of this group through the formation of disulfide-linked aggregates was investigated by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and varied strongly with pH. These observations could be used, in combination with the reaction steps of the reaction scheme, to describe qualitatively the strongly pH-dependent isothermal calorimetry curves, measured at 65 degrees C.     

Uptake of quercetin and quercetin 3-glucoside from whole onion and apple peel extracts by Caco-2 cell monolayers

Evidence suggests that regular consumption of fruits and vegetables may reduce the risk of chronic diseases, and phytochemicals from fruits and vegetables may be responsible for this health benefit. However, there is limited knowledge on the bioavailability of specific phytochemicals from whole fruits and vegetables. This study used Caco-2 cells to examine uptake of quercetin aglycon and quercetin 3-glucoside as purified compounds and from whole onion and apple peel extracts. Pure quercetin aglycon was absorbed by the Caco-2 cells in higher concentrations than quercetin 3-glucoside (p < 0.05). Caco-2 cells treated with quercetin 3-glucoside accumulated both quercetin 3-glucoside and quercetin. Caco-2 cells absorbed more onion quercetin aglycon than onion quercetin 3-glucoside (p < 0.05), and the percentage of onion quercetin absorbed was greater than that of pure quercetin, most likely due to enzymatic hydrolysis of quercetin 3-glucoside and other quercetin glucosides found in the onion by the Caco-2 cells. Caco-2 cells absorbed low levels of quercetin 3-glucoside from apple peel extracts, but quercetin aglycon absorption was not detected. Caco-2 cell homogenates demonstrated both lactase and glucosidase activities when incubated with lactose and quercetin 3-glucoside, respectively. This use of the Caco2 cell model appears to be a simple and useful system for studying bioavailability of whole food phytochemicals and may be used to assess differences in bioavailability between foods.     

Fibril assemblies in aqueous whey protein mixtures

Fibril formation in mixtures of whey proteins upon heating at pH 2 was investigated. Fibrils were found to coexist with other structures, such as spherulites. These spherulites consist of radially oriented fibrils. At total protein concentrations above 6 wt %, transparent gels were formed. Changing the ratio between the various whey proteins did not affect this gelation concentration as long as beta-lactoglobulin (beta-lg) was present, suggesting that beta-lg was dominant in the gelation. Pure alpha-lactalbumin and pure bovine serum albumin did not form fibrils, nor did they gel upon heating at pH 2 and 80 degrees C for up to 10 h. They did however induce a decrease in the beta-lg concentration needed for gel formation upon heating at pH 2. Our results suggest that beta-lg is the only fibril forming protein at the conditions used and that no mixed fibrils are formed.     

Inhibitory action of soybean beta-conglycinin hydrolysates on Salmonella typhimurium translocation in Caco-2 epithelial cell monolayers

Soybean protein hydrolysates are widely used as functional foods as they have antioxidative properties able to enhance immune responses in humans. The alcalase enzymatic hydrolysates of beta-conglycinin were fractionated by ultrafiltration, and two main fractions, SP1 (<10 kDa) and SP2 (10-20 kDa), were obtained. The effects of these two fractions on the growth, development of epithelial cells, and formation of intercellular tight junctions were tested on an in vitro Caco-2 cell culture system. The inhibitory effects of SP1 and SP2 on the penetration of Salmonella typhimurium into Caco-2 epithelial cells were also examined. The results showed that the addition of >0.05 g/L of SP2 improved epithelial cell growth and that a concentration of 0.5 g/L of SP2 increased intercellular tight junction formation, which resulted in increased of transepithelial monolayer resistance (TER) values. Moreover, a lower S. typhimurium count compared to control was obtained when Caco-2 cells were grown in 0.05 and 0.5 g/L of SP2. These results show that beta-conglycinin hydrolysates play an important role in resisting S. typhimurium penetration into intestinal epithelial cells and that high molecular mass peptides (10-20 kDa) were more effective overall than low molecular mass peptides.     

Absorption of black currant anthocyanins by monolayers of human intestinal epithelial Caco-2 cells mounted in ussing type chambers

Anthocyanins (ACNs) have been reported to have multiple biological properties imparting benefits to human health. Their role in human nutrition, however, needs to be related to biokinetic data, such as bioavailability. The purpose of the present study was to focus on the potential absorption of black currant ( Ribes nigrum L.) ACNs. Caco-2 monolayers were used as an in vitro model of the absorptive intestinal epithelium. For absorption studies, Caco-2 cells grown on permeable filters were mounted into Ussing type chambers. The monolayer integrity was monitored by measuring the transepithelial electrical resistance (TEER). Luminal to serosal transport of ACNs was examined by comparing ACN disappearance from the luminal solution of Ussing chambers not containing any inserts (control chambers) with that of Ussing chambers containing inserts. ACNs (C total ACN approximately 180 microM) were not detected in any serosal solution. However, it was shown that ACNs disappeared from the luminal side, not due to ACN degradation processes but rather--at least in part--due to physiological actions of the cells. The luminal net disappearance of ACNs was calculated (max(t20 min) approximately 11% for total ACNs) and labeled as "absorption efficiency". This apical transport might occur to a much larger extent than the further translocation across the basolateral membrane. Thus, cell metabolism and translocation across the basolateral membrane may be the key determinants of ACN absorption and bioavailability.