Caco-2 Cell Absorption Research Articles

Three alkaloids in Radix Sophorae flavescentis : metabolic rate in rat liver microsomes and absorption behavior in Caco-2 cells

Objective To investigate the absorption behaviors of three kinds of sophora alkaloids(matrine,oxymatrine and sophocarpine)in Caco-2cells and their metabolic rates in rat liver microsomes.Methods Caco-2cell absorption model was used to analyze the absorption behaviors of three sophora alkaloids and rat liver microsomal model in vitro was used to investigate the metabolic rates of three sophora alkaloids.The contents of three kinds of sophora alkaloids were determined by HPLC-MS and the permeation rates and metabolic rates were calculated.The analytical conditions were as follows:chromatographic column:Agilent Zorbax SB-C18(3.0 mm×100 mm,3.5μm);mobile phase:acetonitrile/water(60∶40,V/V)with isocratic elution;injection volume:5μL;flow rate:0.8mL/min;temperature of column:30℃;and running time:9min.The selective ion monitoring(SIM)in positive ion mode was used in mass spectrometry,with drying gas temperature:350℃;capillary voltage:4 000V;drying gas flow rate:9.0L/min;and fragmentor voltage:90eV.Results In the Caco-2cell absorption model,matrine,oxymatrine and sophocarpine were separated with good linearity(r0.999 5)between0.050 5-10.01μmol/L,with the permeation rates being:matrine 1.251,oxymatrine 0.937 5,and sophocarpine 1.424.In rat liver microsomal model,matrine,oxymatrine and sophocarpine were separated with good linearity(r0.999 8)between0.050 1-1.001μmol/L,with the half-life period being:matrine 4.331 h,oxymatrine 1.084 h,and sophocarpine 2.478 h.The intra-day and inter-day precisions were 5%,the matrix effect was 80%,and the extraction recovery rate was 88%.Conclusion The three kinds of sophora alkaloids can easily pass the Caco-2cell absorption model,with active transport being the main way,and it is difficult for them to be metabolized in rat liver microsomes.

Induction of NPC1L1 membrane translocation and cholesterol absorption by cholecystokinin

Objectives: Cholecystokinin (CCK) has been extensively studied as a gastrointestinal hormone and neuropeptide. We previously reported that intravenous injection of CCK increased mouse plasma cholesterol. The goal of the present study is to investigate the effect of CCK on cholesterol absorption in Caco-2 cells and mouse models.

Bioavailability of hydroxycinnamic acids from Crepidiastrum denticulatum using simulated digestion and Caco-2 intestinal cells.

Hydroxycinnamic acids have antioxidant properties and potentially beneficial effects on human health. This study investigated the digestive stability, bioaccessibility, and permeability of hydroxycinnamic acids from Crepidiastrum denticulatum using simulated digestion and Caco-2 intestinal cells. The major compounds of C. denticulatum were determined to be four hydroxycinnamic acids [caftaric acid, chlorogenic acid, chicoric acid, and 3,5-di-O-caffeoylquinic acid (3,5-DCQA)] and one flavonoid (luteolin-7-O-glucuronide) by high-performance liquid chromatography and electrospray ionization mass spectrometry. Hydroxycinnamic acids from C. denticulatum were rapidly released in the stomach and duodenum phase, maximizing the possibility of absorption in the intestinal Caco-2 cells. The digestive stability and bioaccessibility of hydroxycinnamic acids from C. denticulatum were markedly low after simulated digestion and remained minimal in the soluble fraction of the ileum phase. Unlike the four hydroxycinnamic acids, luteolin-7-O-glucuronide was stable in terms of digestive stability and bioaccessibility during simulated digestion. The cell permeabilities (P(app A to B)/P(app B to A)) of caftaric acid (0.054) and chlorogenic acid (0.055) were higher than those of chicoric acid (0.011) and 3,5-DCQA (0.006) in general. That of luteolin-7-O-glucuronide was not detectable, showing its low absorption in Caco-2 cells. These results indicate that the rapid release of hydroxycinnamic acids in the stomach and duodenum phase may increase the potential for absorption in Caco-2 cells, and that luteolin-7-O-glucuronide, which was stable in terms of digestive stability and bioaccessibility, has relatively low absorption compared with hydroxycinnamic acids.

Stability of casein antioxidant peptide fractions during in vitro digestion/Caco-2 cell model: characteristics of the resistant peptides

Peptides with in vitro antioxidant activity have no direct reaction on in vivo organisms, because of gastrointestinal (GI) barriers in human tract. In this study, low-molecular-weight peptides of casein were acquired by hydrolysis with alcalase and isolation with Sephadex G-25 gel filtration. Each peptide fraction was performed on simulated GI digestion (stage I) and Caco-2 cell absorption (stage II) for evaluating GI stability, with molecular weight distribution, peptide nitrogen, trolox equivalent antioxidant capacity, and oxygen radical antioxidant capacity. The peptide fraction below 1,000 Da (F3) had shown weaker structural stability during stage I digestion, while it exerted excellent antioxidative stability in whole stages and finally produced the highest content of GI resistant peptides. Three resistant peptides, Ile-Glu, Ser-Asp-Lys, and Ala-Tyr-Pro-Ser, were characterized, and Ala-Tyr-Pro-Ser might strongly contribute to in vivo antioxidant activities. It is suggested that alcalase may promote the production of GI resistant peptides.

Pea protein provides a promising matrix for microencapsulating iron.

Worldwide, the most prevalent nutritional deficiency is iron. The strategies for iron supplementation often fail due to poor adherence to supplementation methods contributed to unpleasant sensory characteristics. An alternative is the use of microencapsulated nutrients for home fortification in order to mask undesirable tastes and to allow its release in strategic sites of the gastrointestinal tract. Toward this end, pea protein concentrate was tested as a natural, edible and alternative material and the spray-drying technique was utilized for the preparation of microparticles containing ferrous sulfate. Their physical and chemical characteristics were evaluated. The microparticles had a spherical shape and grooves with an average size ranging between 2and 3μm. Analysis by in vitro assays tested the release of iron in simulated salivary and gastric fluids and its intestinal absorption in Caco-2 cells. No dissolution of iron occurred in the salivary medium whereas the sensory analysis showed good acceptance of a product which incorporated 5.5mg of iron per 100g portion of food. Thus, the effectiveness of microencapsulation was demonstrated by utilizing a plant protein as an encapsulating matrix for the controlled release of iron and capable of preserving the bioaccessibility of ferrous sulfate.

Development and characterization of lutein-loaded SNEDDS for enhanced absorption in Caco-2 cells

A self-nanoemulsifying drug delivery system (SNEDDS) has been developed for enhanced oral bioavailability of lutein. Its permeation enhancement has been evaluated using monolayers of Caco-2 cells. SNEDDS is composed of a mixture of Lexol® and Emulmetik® 900, Labrasol®, and Tween 80 as oil, surfactant and co-surfactant, respectively. Upon dilution of lutein-loaded SNEDDS with water, a nanoemulsion was obtained in <10 s with spherical droplets of 40–150 nm in diameter. The zeta potential was in the range of −19 to −32 mV. Increasing the ratio of surfactant to co-surfactant decreased the mean droplet size. Dissolution studies showed that lutein was released rapidly (<5 min) from SNEDDS into 0.1 N HCl and pH 6.8 phosphate buffer solution without any aggregation. In vitro studies using Caco-2 cells revealed that lutein-loaded SNEDDS showed shorter lag time and greater (2-fold) cellular accumulation compared with the lutein dispersion.

Reduction in cholesterol absorption in Caco-2 cells through the down-regulation of Niemann-Pick C1-like 1 by the putative probiotic strains Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74 from fermented foods

Hypercholesterolaemia is a major risk factor related to atherosclerosis, and it may be influenced by our diet. This study addresses the impact of Lactobacillus rhamnosus BFE5264 (isolated from Maasai fermented milk) and Lactobacillus plantarum NR74 (from Korean kimchi) on the control of cholesterol absorption through down-regulation of Niemann-Pick C1-like 1 (NPC1L1) expression. Caco-2 enterocytes were treated with the live, heat-killed (HK) bacteria, bacterial cell wall extracts and metabolites; mRNA level and protein expression were measured. Caco-2 cells showed lower NPC1L1 expression in the presence of the live test strains than the control, elucidating down-regulation of cholesterol uptake, and were compared well with the positive control, L. rhamnosus GG. This effect was also observed with HK bacteria and cell wall fractions but not with their metabolites. The potential of some Lactobacillus strains associated with traditional fermented foods to suppress cholesterol uptake and promote its efflux in enterocytes has been suggested from these data.

Assessment of Extracts from Red Yeast Rice for Herb-Drug Interaction by in-vitro and in-vivo assays

Red yeast rice (RYR) is made by fermenting the yeast Monascus purpureus over rice. It is a source of natural red food colorants, a food garnish and a traditional medication. Results of the current study demonstrated that polar fractions of the RYR preparations contained herbal-drug interaction activity, which if left unremoved, enhanced P-glycoprotein activity and inhibited the major drug metabolizing cytochromes P450, i,e, CYP 1A2, 2C9 and 3A4. The data from Caco-2 cell absorption and animal model studies further demonstrated that the pharmacokinetic modulation effect by RYR preparations containing the polar fractions (“untreated” preparation) was greater than that from RYR preparations with the polar fractions removed (“treated” preparation). The data indicates a potential for herb-drug interactions to be present in RYR commonly sold as nutritional supplements when the polar fractions are not removed and this should be taken into consideration when RYR is consumed with medications, including verapamil.

New Zealand green-lipped mussels (Perna canaliculus) enhance non-haem iron absorptionin vitro

Fe bioavailability can be manipulated by the nutritional composition of a meal. Ascorbic acid and unidentified components of meat, fish and poultry, but particularly beef, all appear to enhance the absorption of non-haem Fe. The aim of the present study is to identify whether extracts of green-lipped mussels (GLM; Perna canaliculus) enhance non-haem Fe absorption in Caco-2 cells and to compare the effect with that of beef. Raw GLM and raw beef homogenates were digested in vitro with pepsin at pH 2, and pancreatin and bile salts at pH 7. Tracer ⁵⁵Fe was used to measure cellular Fe uptake. Ascorbic acid was used as a positive control and egg albumin, exposed to the same in vitro digestion process, was used as a negative control. Caco-2 cell monolayers were incubated with treatments for 60 min. All values were standardised per μg of GLM, egg albumin, beef or ascorbic acid. The results showed that ascorbic acid enhanced non-haem Fe absorption to the highest degree. Beef and GLM digestates both significantly enhanced Fe absorption compared with egg albumin. In conclusion, GLM digestate significantly enhances non-haem Fe uptake in Caco-2 cells with a similar magnitude to that of beef.

Predicting Phenolic Acid Absorption in Caco-2 Cells: A Theoretical Permeability Model and Mechanistic Study

There is a considerable need to rationalize the membrane permeability and mechanism of transport for potential nutraceuticals. The aim of this investigation was to develop a theoretical permeability equation, based on a reported descriptive absorption model, enabling calculation of the transcellular component of absorption across Caco-2 monolayers. Published data for Caco-2 permeability of 30 drugs transported by the transcellular route were correlated with the descriptors 1-octanol/water distribution coefficient (log D, pH 7.4) and size, based on molecular mass. Nonlinear regression analysis was used to derive a set of model parameters a', β', and b' with an integrated molecular mass function. The new theoretical transcellular permeability (TTP) model obtained a good fit of the published data (R² = 0.93) and predicted reasonably well (R² = 0.86) the experimental apparent permeability coefficient (P(app)) for nine non-training set compounds reportedly transported by the transcellular route. For the first time, the TTP model was used to predict the absorption characteristics of six phenolic acids, and this original investigation was supported by in vitro Caco-2 cell mechanistic studies, which suggested that deviation of the P(app) value from the predicted transcellular permeability (P(app)(trans)) may be attributed to involvement of active uptake, efflux transporters, or paracellular flux.

Hepatotoxic pyrrolizidine alkaloids—Bioavailability and cellular effects on human HEPG2 cells

Purpose: 1,2-Unsaturated pyrrolizidine alkaloids (PA) belong to the most toxic compounds. These substances are found in several plants such as Asteraceae and Boraginaceae families. Acute PA poisoning via food contamination causes severe damage to the liver; long-term, sub-lethal doses may cause cumulative damage or cancer. Methods: In this study, we analyzed the toxicological effects (cytotoxicity, ATP-content, and oxidative stress) of selected PA (retrorsine, senecionine, and seneciphylline) on the hepatocarcinoma cell line HepG2 by using a bioactivating system (S9-fraction). Furthermore, we characterized the uptake of senecionine and its N-oxidated form across the intestinal barrier by using the well-characterized human Caco-2 cell Transwell-model as an in vitro system for the human gut barrier. The PA content was analyzed by LC-MS/MS. Results: The metabolic bioactivation of PA with the S9-fraction of rat liver homogenate resulted in a weak increase of cellular cytotoxicity including a slight reduction of cellular ATP-content, as well as in a weak increase of the apoptosis rate and of oxidative stress. The Caco-2 cell absorption studies revealed a fast passage of senecionine from the apical into the basolateral compartment suggesting that active transport mechanisms are involved in the uptake and excretion of the substance. In contrast, the oxidized metabolite senecionine-N-oxide did not cross the differentiated Caco-2 monolayer from the apical to the basolateral side. In addition, we observed the conversion of senecionine into the oxidated form, as well as the reduction of senecionine-N-oxide. This indicates that Caco-2 cells are able to metabolize this compound.

Permeation enhancer effect of chitosan and chitosan derivatives: Comparison of formulations as soluble polymers and nanoparticulate systems on insulin absorption in Caco-2 cells

In this study four quaternized derivatives of chitosan: trimethyl chitosan (TMC), dimethylethyl chitosan (DMEC), diethylmethyl chitosan (DEMC) and triethyl chitosan (TEC) with degree of substitution of approximately 50 ± 5% were synthesized and their effect on the permeability of insulin across intestinal Caco-2 monolayers was studied and compared with chitosan both in free-soluble form and in nanoparticulate systems. Transepithelial electrical resistance (TEER) studies revealed that all four chitosan derivatives in free-soluble forms were able to decrease the TEER value in the following order TMC > DMEC > DEMC = TEC > chitosan, indicating their abilities to open the tight junctions. Recovery studies on the TEER showed that the effect of the polymers on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all polymers. A similar rank order was also observed when measuring the zeta-potentials of the various polymers in solution form. Transport studies of insulin together with the soluble polymers across Caco-2 cell layers showed the following ranking: TMC > DMEC > DEMC > TEC > chitosan which is in agreement with the strength of the cationic charge of the polymer. In comparison to the free-soluble polymers, the nanoparticles prepared by ionic gelation of the chitosan and its quaternized derivatives had much lower effect on decreasing the TEER by opening of the tight junctions. This can be explained by the reduced available amount of positive charge at the surface of the nanoparticles. In accordance with these results, the insulin loaded nanoparticles showed much less permeation across the Caco-2 cell monolayer in comparison to the free-soluble polymers. Mass balance transport studies revealed that a substantial amount of the nanoparticles has been entrapped into the Caco-2 monolayer or attached to the cell surface. It can thus be stated that while free-soluble polymers can reversibly open the tight junctions and increase the permeation of insulin, the nanoparticles had basically only a low effect on the opening of the tight junction and the paracellular transport of insulin across the Caco-2 cell monolayer. These data convincingly show that nanoparticles consisting of chitosan and its quaternary ammonium derivatives loaded with insulin are less effective in facilitating paracellular transport across Caco-2 cell monolayers than the corresponding free polymers.

Evaluation of Metallothionein formation as a proxy for zinc absorption in an in vitro digestion/Caco‐2 Cell culture Model

Caco-2 cell metallothionein (MT) formation was studied to determine if MT could be used as a proxy for zinc (Zn) bioavailability in an in vitro/cell culture model. The intracellular concentration of MT was determined using a cadmium/hemoglobin affinity assay. Total cellular Zn uptake was determined on acid treatment (5% HNO3) using inductively-coupled argon-plasma emission spectroscopy (ICPES). The effect of phytic acid (PA) on cellular MT formation was also studied. Caco-2 cells were treated with media containing 0, 2, 5, 10, 25, 50, and 75 μmol/L Zn (as ZnCl2). Some Zn absorption studies employed radio-Zn (65ZnCl2). The effect of varying Zn:PA molar ratios (i.e., 1:0, 1:1, 1:5, 1:10, and 1:20) on Zn uptake was determined. A positive correlation between Zn-media concentrations and cellular 65Zn uptake and cellular MT formation was observed. The concentrations of Zn and MT in cells treated with increasing levels of non-radioactive Zn (i.e., 25, 50 or 75 μmol/L Zn) increased as Zn concentrations in the media increased. Zn and MT concentrations in cells incubated with non-radioactive Zn (2, 5 or 10 μmol/L) did not differ from that of cells receiving no added Zn. The presence of PA significantly lowered the cellular MT concentration. When Zn:PA molar ratios were at or above 1:5, cellular MT concentrations were not different from that of cells receiving no added PA. The results of the study suggest that the measurements of cellular Zn and MT concentration have some limitations at or below 10 μmol/L Zn. The initial Zn status of cells may play a role in cellular MT synthesis. Further studies are required to validate the usefulness of using MT as a proxy for Zn absorption in Caco-2 cells. Funded by HarvestPlus.

Effect of the main dietary antioxidants (carotenoids, γ-tocopherol, polyphenols, and vitamin C) on α-tocopherol absorption

(R,R,R)-alpha-tocopherol is a fat-soluble antioxidant vitamin generally ingested with other dietary antioxidants. The objective of this study was to assess whether the main dietary antioxidant classes, that is carotenoids, polyphenols, vitamin C and gamma-tocopherol, affect the intestinal absorption of alpha-tocopherol. METHODS, DESIGN AND SUBJECTS: We evaluated first the effect of different combinations of antioxidants on (R,R,R)-alpha-tocopherol absorption by a human intestinal cell line (Caco-2 clone TC7). Then we compared the effect of two doses of a dietary antioxidant (lutein) on the postprandial chylomicron alpha-tocopherol responses to an alpha-tocopherol-rich meal. Eight healthy men ate two similar meals in a random order at a 1 month interval. The meals contained 24 mg alpha-tocopherol in sunflower oil plus either 18 or 36 mg lutein. Blood samples were collected during the postprandial periods to compare chylomicron alpha-tocopherol responses. A mixture of polyphenols (gallic acid, caffeic acid, (+)-catechin and naringenin) and a mixture of carotenoids (lycopene, beta-carotene and lutein) significantly impaired alpha-tocopherol absorption in Caco-2 cells (P<0.001 and P<0.0001, respectively). The inhibitory effect of gamma-tocopherol was close to significance (P=0.055). In contrast, vitamin C had no significant effect (P=0.158). Naringenin was the only polyphenol that significantly impaired alpha-tocopherol absorption. Postprandial alpha-tocopherol response was weakest at the highest dose of lutein (616+/-280 nmol/l h vs 1001+/-287 nmol/l h). The observed extent of reduction (-38%, P=0.069) supported the inhibitory effect of carotenoids observed in the Caco-2 experiments. Naringenin, carotenoids and probably gamma-tocopherol can impair alpha-tocopherol absorption whereas vitamin C and phenolic acids have no effect.

Effect of 17β-oestradiol on transepithelial calcium transport in human intestinal-like Caco-2 cells and its interactions with 1,25−dihydroxycholecalciferol and 9-cis retinoic acid

Oestrogen therapy helps prevent bone loss in postmenopausal women and corrects a decline in Ca absorption efficiency at the onset of menopause. However, the mechanism by which 17beta-oestradiol (17beta-E2) stimulates Ca absorption is unclear. Oestrogen may exert its effect indirectly via increasing 1,25-dihydroxycholeciferol (1,25 (OH)2D3) or its receptor, or act more directly on the intestines via the oestrogen receptor (OR). Since oestrogen also increases retinol levels, this may influence Ca absorption. To investigate the effect of 17beta-E2 alone and in combination with 1,25 (OH)2D3 on intestinal Ca uptake and absorption in Caco-2 cells cultured under deplete- and replete-9-cis retinoic acid (9-cis RA) conditions. Twenty-one day-old Caco-2 cell monolayers (n 9 wells per treatment) were exposed to 9-cis RA-deplete and -replete media containing dimethyl sulfoxide (control), 10 nM-1,25 (OH)2D3, 10 nM-17beta-E2, or 10 nM-1,25 (OH)2D3 plus 10 nM-17beta-E2, for 48 h. 1,25 (OH)2D3 stimulated Ca uptake, total Ca transport, calbindin D(9K) and CaT1 mRNA levels, while 17beta-E2 and 9-cis RA had no effect on Ca absorption or uptake. Nor did they augment the stimulatory effect of 1,25 (OH)2D3. These in vitro findings suggest that oestrogen does not have a direct effect on intestinal Ca absorption.

Absorption and lipoprotein transport of sphingomyelin

Dietary sphingomyelin (SM) is hydrolyzed by intestinal alkaline sphingomyelinase and neutral ceramidase to sphingosine, which is absorbed and converted to palmitic acid and acylated into chylomicron triglycerides (TGs). SM digestion is slow and is affected by luminal factors such as bile salt, cholesterol, and other lipids. In the gut, SM and its metabolites may influence TG hydrolysis, cholesterol absorption, lipoprotein formation, and mucosal growth. SM accounts for approximately 20% of the phospholipids in human plasma lipoproteins, of which two-thirds are in LDL and VLDL. It is secreted in chylomicrons and VLDL and transferred into HDL via the ABCA1 transporter. Plasma SM increases after periods of large lipid loads, during suckling, and in type II hypercholesterolemia, cholesterol-fed animals, and apolipoprotein E-deficient mice. SM is thus an important amphiphilic component when plasma lipoprotein pools expand in response to large lipid loads or metabolic abnormalities. It inhibits lipoprotein lipase and LCAT as well as the interaction of lipoproteins with receptors and counteracts LDL oxidation. The turnover of plasma SM is greater than can be accounted for by the turnover of LDL and HDL particles. Some SM must be degraded via receptor-mediated catabolism of chylomicron and VLDL remnants and by scavenger receptor class B type I receptor-mediated transfer into cells.

A Novel Protein C-Phycocyanin Plays a Crucial Role in the Hypocholesterolemic Action of Spirulina platensis Concentrate in Rats

This study was designed to clarify the mechanisms of the hypocholesterolemic action of Spirulina platensis concentrate (SPC) and identify the novel hypocholesterolemic protein derived from SPC. We investigated the effects of casein or SPC on the solubility of cholesterol, taurocholate binding capacity in vitro, cholesterol absorption in Caco-2 cells, and cholesterol metabolism in rats for 10 d. We also evaluated the effects of SPC, C-phycocyanin (PHY), and PHY residue on cholesterol metabolism in rats fed a high-cholesterol diet for 5 d, and SPC or SPC-acetone extract for 10 d. SPC had a significantly greater bile acid-binding capacity than casein in vitro. Micellar cholesterol solubility and cholesterol uptake by Caco-2 cells was significantly lower in the presence of SPC compared with casein. Fecal excretion of cholesterol and bile acids was significantly greater in rats fed the SPC-supplemented diet than in those fed the casein control diet. Serum and liver cholesterol concentrations were significantly lower in rats fed SPC than in those fed casein. Thus, the hypocholesterolemic action of SPC may involve the inhibition of both jejunal cholesterol absorption and ileal bile acid reabsorption. Although no studies to date have found a hypocholesterolemic protein among the algal proteins, we report here the discovery of a hypocholesterolemic effect in the novel protein C-phycocyanin. This study provides the first direct evidence that PHY, a novel hypocholesterolemic protein derived from Spirulina platensis, can powerfully influence serum cholesterol concentrations and impart a stronger hypocholesterolemic activity than SPC in animals.

Lack of dose-responsive effect of dietary phyto-oestrogens on transepithelial calcium transport in human intestinal-like Caco-2 cells.

Ca absorption has been shown to be unaffected by high luminal concentrations of two commonly consumed soyabean phyto-oestrogens (PO) (genistein and daidzein) in Caco-2 cells grown under oestrogen-depleted conditions. However, these compounds exhibit dose-dependent biphasic effects in some tissues, such as reproductive tissue and bone. Thus, in light of this biphasic activity, the effect of lower concentrations of genistein and daidzein on Ca absorption requires further investigation. Therefore, the aim of the present study was to investigate the effect of a range of concentrations of genistein and daidzein on Ca absorption in the human Caco-2 intestinal-like cell model. Caco-2 cells were seeded onto permeable filter supports and allowed to differentiate into monolayers. On day 21, the Caco-2 monolayers (n 12 per treatment), grown in oestrogen-deplete media, were then exposed to 10 nm-1,25-dihydroxycholecalciferol (1,25 (OH)2D3), or 1, 10 and 50 microm-genistein or -daidzein for 24 h. After exposure, transepithelial and transcellular transport of (45)Ca and fluorescein transport were measured. As expected, 1,25 (OH)2D3 stimulated Ca absorption in Caco-2 cells, by up regulating transcellular transport. Ca absorption was unaffected by either PO at luminal concentrations of 1, 10 or 50 microm, typical of intakes by Western and Asian populations as well as supplemental levels, respectively. The results of this model suggest that the proposed beneficial effects of supplemental levels of these PO compounds on bone mass in postmenopausal women more probably arise from direct effects on bone cells, and not by an indirect effect of these compounds on Ca absorption.

Inhibitory effect of wheat fibre extract on calcium absorption in Caco-2 cells: evidence for a role of associated phytate rather than fibre per se.

In spite of the strong evidence for the beneficial health effects of dietary fibres, one of the potential nutritional disadvantages of high fibre diets is the adverse effect on the bioavailability of micronutrients, especially minerals and trace elements. With regard to Ca, there is considerable evidence that phytate, which is associated with fibre in many foods, such as cereals and soya products, inhibits Ca absorption. However, there is some doubt as to whether fibres per se have an influence on Ca absorption. Therefore, the purpose of this study was to investigate the effect on Ca absorption of two cereal-based fibre extracts (wheat bran and barley hull). In addition, in order to distinguish between the effect of the fibre components per se and the associated phytate content in these fibre extracts, we investigated the effect of dephytinised wheat and barley fibre extracts and the effect of phytate, as sodium phytate at levels present in the fibre extracts, on Ca absorption. Ca absorption was assessed in Caco-2 cells, as a model for studying Ca absorption in humans. The effect of wheat and barley fibre extracts, dephytinised wheat and barley fibre extracts, cellulose, and of sodium phytate on transepithelial 45Ca transport and 45Ca uptake was studied in differentiated Caco-2 cells grown on permeable filter supports. Wheat and barley fibre extracts were dephytinised with wheat phytase. Wheat fibre extract had a 3.2-fold higher phytate content (48.0 mmol/kg) than barley fibre extract (15.1 mmol/kg). Enzymatic dephytinisation of both fibre extracts reduced the phytate content to undetectable levels. The rate of transepithelial 45Ca transport across Caco-2 cell monolayers and the uptake of 45Ca into Caco-2 cells were unaffected by cellulose or barley fibre extract. On the other hand, inclusion of wheat fibre extract in the Ca transport buffer (50 g fibre/l) significantly reduced the rate of 45Ca transport (by 17 and 19% respectively) and the uptake of 45Ca (by 24 and 25% respectively) relative to a fibre-free buffer and a control fibre (cellulose) transport buffer. Increasing the phytate concentration of the transport buffer from 0 to 2 mM (a level close to that in the wheat fibre containing buffer) significantly reduced the rate of 45Ca transport (by 16%) and 45Ca uptake (by 26%). Dephytinisation of the wheat fibre extract removed its inhibitory effects on 45Ca transport and e.g. 45Ca uptake. The results from the present study in Caco-2 cells suggest that it is the phytate in wheat fibre extract which is the major inhibitory factor of Ca absorption and that wheat fibre per se has little if any inhibitory effect on Ca absorption. In addition, the results of this study support the usefulness of Caco-2 cells for investigating the effects of dietary factors on the cellular uptake and transepithelial intestinal transport of Ca.

Development of a rectal nicotine delivery system for the treatment of ulcerative colitis

The aims of this investigation were: i. to develop a rectal nicotine delivery system with bioadhesives for the treatment of ulcerative colitis and ii. to evaluate nicotine transport and cytotoxicity of the delivery system using Caco-2 cell culture systems. Rectal nicotine suppository formulations were prepared in semi-synthetic glyceride bases (Suppocire AM and AI, Gattefosse Inc.) by fusion method. The in vitro release of nicotine was carried out in modified USP dissolution apparatus 1. Differential scanning calorimetry (DSC) and powder X-ray diffraction were used to study the polymorphic changes if any in the formulations. An LC method was used for the assay of nicotine. The effect of bioadhesives (glyceryl monooleate (GMO), and Carbopol) on the nicotine flux was evaluated using Caco-2 cell permeability studies and Caco-2 cell viability was determined using the MTT toxicity assay. In vitro release studies indicated that the low melting AI base was superior to that of the AM base. Presence of GMO in the formulation enhanced the release of nicotine whereas Carbopol showed an opposite effect. The enhanced release of nicotine in the presence of GMO was found to be partly due to the melting point lowering effect of this compound. Caco-2 cell absorption studies showed that there was a decrease in the flux of nicotine in the presence of both the bioadhesives. The flux of the fluorescein marker which is used to study the integrity of the cell monolayers was found to be slightly higher only in the presence of 10% (w/w) Carbopol. Nicotine, Carbopol, and GMO do not have any cytotoxic effect on these cell monolayers within the concentration range used in the formulations. Rectal nicotine formulations containing bioadhesives were developed and characterized. Both in vitro release and cell culture studies have indicated that one can manipulate the nicotine release from these rectal delivery systems by incorporation of various bioadhesives or the use of different bases in the formulation. Nicotine concentration below 2% (w/v) and bioadhesive concentration below 10% (w/w) do not have any cytotoxic effect on Caco-2 cells.