Caco-2 Cell Uptake Research Articles

Enhancing the Intestinal Membrane Permeability of Zanamivir: A Carrier Mediated Prodrug Approach

The purpose of this study was to improve the membrane permeability and oral absorption of the poorly permeable anti-influenza agent, zanamivir. The poor oral bioavailability is attributed to the high polarity (cLogP ∼ -5) resulting from the polar and zwitterionic nature of zanamivir. In order to improve the permeability of zanamivir, prodrugs with amino acids were developed to target the intestinal membrane transporter, hPepT1. Several acyloxy ester prodrugs of zanamivir conjugated with amino acids were synthesized and characterized. The prodrugs were evaluated for their chemical stability in buffers at various pHs and for their transport and tissue activation by enzymes. The acyloxy ester prodrugs of zanamivir were shown to competitively inhibit [(3)H]Gly-Sar uptake in Caco-2 cells (IC(50): 1.19 ± 0.33 mM for L-valyl prodrug of zanamivir). The L-valyl prodrug of zanamivir exhibited ∼3-fold higher uptake in transfected HeLa/hPepT1 cells compared to wild type HeLa cells, suggesting, at least in part, carrier mediated transport by the hPepT1 transporter. Further, enhanced transcellular permeability of prodrugs across Caco-2 monolayer compared to the parent drug (P(app) = 2.24 × 10(-6) ± 1.33 × 10(-7) cm/s for L-valyl prodrug of zanamivir), with only parent zanamivir appearing in the receiver compartment, indicates that the prodrugs exhibited both enhanced transport and activation in intestinal mucosal cells. Most significantly, several of these prodrugs exhibited high intestinal jejunal membrane permeability, similar to metoprolol, in the in situ rat intestinal perfusion system, a system highly correlated with human jejunal permeability. In summary, this mechanistic targeted prodrug strategy, to enhance oral absorption via intestinal membrane carriers such as hPepT1, followed by activation to parent drug (active pharmaceutical ingredient or API) in the mucosal cell, significantly improves the intestinal epithelial cell permeability of zanamivir and has the potential to provide the high oral bioavailability necessary for oral zanamivir therapy.

The tandem chain extension aldol reaction used for synthesis of ketomethylene tripeptidomimetics targeting hPEPT1

The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketomethylene modified tripeptidomimetics and to investigate their binding affinity for hPEPT1. Three related tripeptidomimetics of the structure H-Phe-ψ[COCH(2)]-Ser(Bz)-X(aa)-OH were synthesized applying the tandem chain extension aldol reaction, where amino acid derived β-keto imides were stereoselectively converted to α-substituted γ-keto imides. In addition, three corresponding tripeptides, composed of amide bonds, were synthesized for comparison of binding affinities. The six investigated compounds were all defined as high affinity ligands (K(i)-values <0.5 mM) for hPEPT1 by measuring the concentration dependent inhibition of apical [(14)C]Gly-Sar uptake in Caco-2 cells. Consequently, the ketomethylene replacement for the natural amide bond and α-side chain modifications appears to offer a promising strategy to modify tripeptidic structures while maintaining a high affinity for hPEPT1.

Augmented Anticancer Efficacy of Doxorubicin-Loaded Polymeric Nanoparticles after Oral Administration in a Breast Cancer Induced Animal Model

The present investigation reports an extensive evaluation of in vitro and in vivo anticancer efficacy of orally administered doxorubicin-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Dox-NPs) in a breast cancer induced animal model. Spherically shaped Dox-NPs were prepared with an entrapment efficiency and particle size of 55.40 ± 2.30% and 160.20 ± 0.99 nm, respectively, and freeze-dried with 5% trehalose using stepwise freeze-drying. Cytotoxicity, as investigated on C127I cell line, revealed insignificant differences between the IC(50) of free Dox and Dox-NPs treated cells in the first 24 h, while higher cytotoxicity was demonstrated by Dox-NPs, following 72 h of incubation. Confocal laser scanning microscopy (CLSM) imaging corroborated that nanoparticles were efficiently localized into the nuclear region of C127I cells. The cellular uptake profile of Dox-NPs revealed both time and concentration dependent increases in the Caco-2 cell uptake as compared to the free Dox solution. Further, Dox-NPs significantly suppressed the growth of breast tumor in female Sprague-Dawley (SD) rats upon oral administration. Finally, orally administered Dox-NPs showed a marked reduction in cardiotoxicity when compared with intravenously injected free Dox as also evident by the increased level of malondialdehyde (MDA), lactate dehydrogenase (LDH), and creatine phosphokinase (CK-MB) and reduced levels of glutathione (GSH) and superoxide dismutase (SOD). The reduced cardiotoxicity of orally administered Dox-NPs was also confirmed by the major histopathological changes in the heart tissue after the treatments of intravenously injected free Dox and orally delivered Dox-NPs.

Carotene and Novel Apocarotenoid Concentrations in Orange-Fleshed Cucumis melo Melons: Determinations of β-Carotene Bioaccessibility and Bioavailability

Muskmelons, both cantaloupe (Cucumis melo Reticulatus Group) and orange-fleshed honeydew (C. melo Inodorus Group), a cross between orange-fleshed cantaloupe and green-fleshed honeydew, are excellent sources of β-carotene. Although β-carotene from melon is an important dietary antioxidant and precursor of vitamin A, its bioaccessibility/bioavailability is unknown. We compared β-carotene concentrations from previously frozen orange-fleshed honeydew and cantaloupe melons grown under the same glasshouse conditions, and from freshly harvested field-grown, orange-fleshed honeydew melon to determine β-carotene bioaccessibility/bioavailability, concentrations of novel β-apocarotenals, and chromoplast structure of orange-fleshed honeydew melon. β-Carotene and β-apocarotenal concentrations were determined by HPLC and/or HPLC-MS, β-carotene bioaccessibility/bioavailability was determined by in vitro digestion and Caco-2 cell uptake, and chromoplast structure was determined by electron microscopy. The average β-carotene concentrations (μg/g dry weight) for the orange-fleshed honeydew and cantaloupe were 242.8 and 176.3 respectively. The average dry weights per gram of wet weight of orange-fleshed honeydew and cantaloupe were 0.094 g and 0.071 g, respectively. The bioaccessibility of field-grown orange-fleshed honeydew melons was determined to be 3.2 ± 0.3%, bioavailability in Caco-2 cells was about 11%, and chromoplast structure from orange-fleshed honeydew melons was globular (as opposed to crystalline) in nature. We detected β-apo-8'-, β-apo-10', β-apo-12'-, and β-apo-14'-carotenals and β-apo-13-carotenone in orange-fleshed melons (at a level of 1-2% of total β-carotene). Orange-fleshed honeydew melon fruit had higher amounts of β-carotene than cantaloupe. The bioaccessibility/bioavailability of β-carotene from orange-fleshed melons was comparable to that from carrot (Daucus carota).

Anticancer activity of the iron facilitator LS081

BackgroundCancer cells have increased levels of transferrin receptor and lower levels of ferritin, an iron deficient phenotype that has led to the use of iron chelators to further deplete cells of iron and limit cancer cell growth. As cancer cells also have increased reactive oxygen species (ROS) we hypothesized that a contrarian approach of enhancing iron entry would allow for further increased generation of ROS causing oxidative damage and cell death.MethodsA small molecule library consisting of ~11,000 compounds was screened to identify compounds that stimulated iron-induced quenching of intracellular calcein fluorescence. We verified the iron facilitating properties of the lead compound, LS081, through 55Fe uptake and the expression of the iron storage protein, ferritin. LS081-induced iron facilitation was correlated with rates of cancer cell growth inhibition, ROS production, clonogenicity, and hypoxia induced factor (HIF) levels.ResultsCompound LS081 increased 55Fe uptake in various cancer cell lines and Caco2 cells, a model system for studying intestinal iron uptake. LS081 also increased the uptake of Fe from transferrin (Tf). LS081 decreased proliferation of the PC-3 prostate cancer cell line in the presence of iron with a lesser effect on normal prostate 267B1 cells. In addition, LS081 markedly decreased HIF-1α and -2α levels in DU-145 prostate cancer cell line and the MDA-MB-231 breast cancer cell lines, stimulated ROS production, and decreased clonogenicity.ConclusionsWe have developed a high through-put screening technique and identified small molecules that stimulate iron uptake both from ferriTf and non-Tf bound iron. These iron facilitator compounds displayed properties suggesting that they may serve as anti-cancer agents.

Enhancing effect of N-octyl-O-sulfate chitosan on etoposide absorption

P-glycoprotein (P-gp), expressed in the apical membranes of the epithelial cells of the intestine, can reduce the oral bioavailability of a wide range of drugs. Many surfactants/excipients have been demonstrated to potentially increase drug absorption by inhibiting P-gp. The purpose of the present study was to evaluate the effect of N-octyl-O-sulfate chitosan (NOSC) on the absorption of etoposide (VP16), a substrate of P-gp with low water solubility. The rat intestinal circulating perfusion in situ and Caco-2 cell uptake and monolayer membrane penetration in vitro were performed to investigate the enhancing ability of NOSC in comparison with some other P-gp inhibitors. The results indicated that various concentrations of NOSC all increased the intestinal absorption of VP16 in rat jejunum and ileum obviously and there was no significant difference in ileum between the enhancing effects of NOSC and other P-gp inhibitors. The VP16 uptake of Caco-2 cell was increased by NOSC solution with different concentrations. As the NOSC concentration was close to its critical micelle concentration (CMC), the cell uptake of VP16 reached to a maximum value. Both NOSC and verapamil (Ver) enhanced dramatically the transport of VP16 from apical side to basolateral side in Caco-2 cell monolayers. Moreover, they both decreased notably the transport of VP16 from basolateral side to apical side, but this effect of NOSC was weaker than that of Ver. However, transepithelial electrical resistance (TEER) of Caco-2 cell monolayers had no significant change during the study. These studies demonstrated that NOSC had the potential by inhibiting P-gp to improve the absorption of oral drugs which were P-gp substrates.

Propidium iodide labeling of nanoparticles as a novel tool for the quantification of cellular binding and uptake

Because nanoparticles are promising tools in drug delivery, quantification of their cellular binding and uptake is an emerging question. Therefore, rhodamine B isothiocyanate–labeled silica nanoparticles with different sizes and surface modifications were investigated concerning their uptake in Caco-2 cells. Flow cytometry studies exhibited a size- and time-dependent association for unmodified nanoparticles (50 and 77 nm), whereas larger particles (94 nm) and polyethylene glycol–modified nanoparticles showed no cellular interaction. A second approach dealt with particles with adsorbed propidium iodide (PI) to distinguish between internalized and adsorbed nanoparticles. These particles only give a fluorescence signal when associated with nucleic acids inside the cell, whereas particles adsorbed to the outer cell surface are not detected. PI-labeled nanoparticles (21 nm) showed a time-dependent uptake, exhibiting a signal in the cytoplasm but less in the nucleus. These novel PI-labeled nanoparticles in combination with flow cytometry are innovative tools for the quantification of nanoparticulate uptake. From the Clinical EditorRhodamine B isothiocyanate–labeled silica nanoparticles with different sizes and surface modifications were investigated concerning their cellular uptake. Propidium iodide containing particles only give a fluorescence signal when associated with nucleic acids and are useful in detecting internalization of the particles. These novel nanoparticles in combination with flow cytometry are innovative tools for the quantification of nanoparticulate uptake.

In-vitro Cellular Uptake and Transport Study of 9-Nitrocamptothecin PLGA Nanoparticles Across Caco-2 Cell Monolayer Model.

The uptake and transport of 9-nitrocamptothecin (9-NC), a potent anticancer agent, across Caco-2 cell monolayers was studied as a free and PLGA nanoparticle loaded drug. Different sizes (110 to 950 nm) of 9-nitrocamptothecin nanoparticles using poly (lactic-glycolic acid) were prepared by via the nanoprecipitation method. The transport of nanoparticles across the Caco-2 cell monolayer as a function of incubation time and concentration was evaluated for each different nanoparticle formulation. The amount of 9-NC transported from the apical to the basolateral side and the uptake of the drug into the cells was determined by HPLC. The uptake of intact nanoparticles into Caco-2 cells was visualized by confocal laser scanning microscopy using 6-coumarin as a fluorescent marker. The study demonstrated that Caco-2 cell uptake and transport of encapsulated 9-nitrocamptothecin is significantly affected by the diameter of the carrier and incubation time. In addition it was shown to be independent of concentration. The results indicated a significant accumulation of the drug in the cell membrane and an enhanced diffusion across the cell membrane. There was also a sustained release of characteristics pertaining to polymeric carriers that provided prolonged drug availability for absorptive cells.

Flavonoid-rich berry-extract treatment decreases glucose uptake in human intestinal Caco-2 cells

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The Use of Caco-2 Cells to Estimate Fe Absorption in Humans - a Critical Appraisal

The Caco-2 cell model is widely used to assess the bioaccessibility/availability of iron from foods and diets. Analysis of iron uptake in this human epithelial cell line is usually preceded by a two-step digestion to simulate the conditions in the stomach and small intestine. Moreover, culturing the cells on inserts permits the measurement of iron transport. The cellular iron uptake is determined by direct measurements using radioisotopes, or indirectly by measurement of ferritin, the intracellular storage form of iron. There is a good correlation between Caco-2 cell uptake and human iron bioavailability for a number of dietary factors known to affect iron absorption. However, recent data suggest that in some cases there is no correlation. Possible reasons for such discrepancies, the benefits, and limitations of the Caco-2 cell model are discussed. In conclusion, in vitro experiments with Caco-2 cells are important tools for ranking foods with respect to bioavailability, for mechanistic studies of iron absorption, and for studies of dietary factors influencing absorption. The results need to be confirmed in humans.

Dietary Ligands as Determinants of Iron–Zinc Interactions at the Absorptive Enterocyte

Iron and zinc interact at the enterocyte and influence the absorption of one another. We have previously reported that zinc noncompetitively inhibits iron uptake in Caco-2 cells, a widely accepted model of the absorptive enterocyte. However, the determinants of this interaction, such as the effect of dietary ligands, remain uncharacterized. Dietary ligands selectively chelate iron and zinc in definite stoichiometric proportions and thus alter the bioavailability from food matrices. Here, we have used common dietary ligands, such as ascorbic acid, phytic acid, tannic acid, tartaric acid, cysteine, histidine, and methionine to characterize iron, zinc uptake individually and in combination, using Caco-2 cells. Selective chelation of zinc, using cysteine, decreased the magnitude of inhibition of iron uptake but could not reverse the inhibition. On the other hand, selective increase in iron uptake in the presence of methionine resulted in increased zinc uptake, rather than inhibition. Taken together, these in vitro results suggest that dietary ligands can modulate iron-zinc interaction and that zinc cannot competitively inhibit iron uptake.

Iron uptake by Caco-2 cells following in vitro digestion: Effects of heat treatments of pork meat and pH of the digests

The present in vitro studies report on iron uptake by Caco-2 cells from pepsin and pepsin + pancreatin-digested pork meat proteins at pH values between 4.6 and 7 mimicking conditions in the duodenum and the proximal jejunum, respectively. Heat treatment of the pork meat resulted in increased iron uptake from pepsin-digested samples to Caco-2 cells at pH 4.6. The major enhancing effects on iron uptake by Caco-2 cells were observed after pepsin digestion in the pH range 4.6–6.0, whereas the pepsin + pancreatin-digested samples resulted in negligible iron uptake in Caco-2 cells at pH 7. Thus, the results emphasize the importance of separating pepsin-digested and pepsin + pancreatin-digested proteins during in vitro studies on iron availability. Furthermore, the present results showed the pH dependency of iron uptake anticipated. The enhancing effect of ascorbic acid was verified by increased iron uptake from pepsin-digested pork meat samples at pH 4.6, while no effect of ascorbic acid was observed at pH 7 in pepsin + pancreatin-digested samples.

The proton‐coupled amino acid transporter, SLC36A1 (hPAT1), transports Gly‐Gly, Gly‐Sar and other Gly‐Gly mimetics

BACKGROUND AND PURPOSE The intestinal proton-coupled amino acid transporter, SLC36A1, transports zwitterionic α-amino acids and drugs such as vigabatrin, gaboxadol and δ-aminolevulinic acid. We hypothesize that SLC36A1 might also transport some dipeptides. The aim of the present study was to investigate SLC36A1-mediated transport of Gly-Gly and Gly-Gly mimetics, and to investigate Gly-Sar transport via SLC36A1 and the proton-coupled dipeptide/tripeptide transporter, SLC15A1 in Caco-2 cells. EXPERIMENTAL APPROACH Transport of a compound via SLC36A1 was determined by its ability to induce an increase in the inward current of two-electrode voltage clamped SLC36A1 cRNA-injected Xenopus laevis oocytes. SLC36A1-mediated L-[³H]Pro uptake in Caco-2 cells was measured in the absence and presence of Gly-Gly or Gly-Sar. In addition, apical [¹⁴C]Gly-Sar uptake was measured in the absence and presence of the SLC36A1 inhibitor 5-hydroxy-L-tryptophan (5-HTP) or the SLC15A1 inhibitor L-4,4'-biphenylalanyl-L-proline (Bip-Pro). KEY RESULTS In SLC36A1-expressing oocytes, an inward current was induced by Gly-Sar, Gly-Gly, δ-aminolevulinic acid, β-aminoethylglycine, δ-aminopentanoic acid, GABA, Gly and Pro, whereas Val, Leu, mannitol, 5-HTP and the dipeptides Gly-Ala, Gly-Pro and Gly-Phe did not evoke currents. In Caco-2 cell monolayers, the apical uptake of 30 mM Gly-Sar was inhibited by 20 and 22% in the presence of 5-HTP or Bip-Pro, respectively, and by 48% in the presence of both. CONCLUSION AND IMPLICATIONS Our results suggest that whereas Gly-Gly amid bond bioisosteres are widely accepted by the hPAT1 carrier, dipeptides in general are not; and therefore, Gly-Sar might structurally define the size limit of dipeptide transport via SLC36A1.

Fundamental studies on the inhibitory action of Acanthopanax senticosus Harms on glucose absorption

Acanthopanax senticosus Harms extract (ASE) is used as an ingredient of over-the-counter drugs and functional foods, such as health supplements, in Japan. ASE exhibits a hypoglycemic effect; however, the mechanism of the hypoglycemic effect is not clear. In the present study, we investigated whether ASE has a glucose absorption inhibitory action. We examined the effects of ASE on α-amylase and α-glucosidase activities, and on glucose uptake in Caco-2 cells. We also examined the effects of ASE oral administration on glucose tolerance in type 2 diabetes mellitus model db/db mice. The addition of ASE inhibited α-glucosidase activity but not α-amylase activity. The α-glucosidase inhibitory activity of ASE was approximately 1/13 of that of acarbose. The addition of ASE inhibited 2'-deoxy-D-glucose (DG) uptake in human intestinal Caco-2 cells, and the inhibitory activity of ASE was approximately 1/40 of that of phloretin. Kinetic analysis of glucose uptake indicated that ASE has no effects on DG uptake through passive diffusion, but that ASE inhibits intracellular DG uptake chiefly by inhibiting transport via a glucose transporter. In the glucose tolerance study, db/db mice orally administered ASE for 3 days showed significantly lower plasma glucose level than the control group 30 min after sucrose loading, without affecting plasma insulin levels. In addition, ASE oral administration significantly inhibited α-glucosidase activity in the small intestine mucosa extirpated from the mice. These findings indicate that ASE may be useful as an ingredient of functional foods to improve postprandial hyperglycemia and prevent type II diabetes mellitus.

Alendronate-loaded microparticles for improvement of intestinal cellular absorption

This study examined a novel alendronate formulation that was developed to overcome the shortcomings of alendronate, such as its low bioavailability and gastric adverse effects. Alendronate microparticles were prepared using mucoadhesive polymers such as chitosan for improving the intestinal cellular absorption of alendronate and also using a gastric-resistant polymer such as Eudragit L100-55 for reducing the gastric inflammation of alendronate. Alendronate microparticles including chitosan showed a threefold increase in alendronate uptake (6.92 ± 0.27%) in Caco-2 cells when compared with the uptake of alendronate solution (2.38 ± 0.27%) into Caco-2 cells. Most interestingly, alendronate microparticles including chitosan showed 2.80 × 10−6 cm/s of an apparent permeability coefficient across Caco-2 cells and caused a significant 42.4% enhancement compared with that of alendronate solution across Caco-2 cells. The morphology of the Caco-2 cells treated with alendronate microparticles including chitosan was similar to that of the untreated cells and alendronate microparticles exhibited a negative effect to propodium iodide with some annexin-V fluorescence isothiocyante positive effect. It was proposed that the novel alendronate microparticles could possess the potential of an increased intestinal absorption and fewer adverse effects of alendronate.

Polyphenol‐Rich Beverages Enhance Zinc Uptake and Metallothionein Expression in Caco‐2 Cells

The effect of red wine (RW), red grape juice (RGJ), green tea (GT), and representative polyphenols on Caco-2 cell (65)Zn uptake was explored. RW, RGJ, and GT enhanced the uptake of zinc from rice matrix. Fractionation of RW revealed that enhancing activity of zinc uptake was exclusively resided in the polyphenol fraction. Among the polyphenols tested, only tannic acid and quercitin stimulated the uptake of zinc while others did not influence the uptake. In tune with these results, only tannic acid and quercitin competed with zinquin (a zinc selective fluorophore) for zinc in vitro. Although all the polyphenols tested appear to enhance the expression of metallothionein (MT), the induction was higher with tannic acid, quercitin, and RW extract. Furthermore, phytic acid abrogated the tannic acid-induced MT expression. These results suggest that polyphenol-rich beverages, tannic acid, and quercitin bind and stimulate the zinc uptake and MT expression in Caco-2 cells.

Cellular Entry of G3.5 Poly (amido amine) Dendrimers by Clathrin- and Dynamin-Dependent Endocytosis Promotes Tight Junctional Opening in Intestinal Epithelia

This study investigates the mechanisms of G3.5 poly (amido amine) dendrimer cellular uptake, intracellular trafficking, transepithelial transport and tight junction modulation in Caco-2 cells in the context of oral drug delivery. Chemical inhibitors blocking clathrin-, caveolin- and dynamin-dependent endocytosis pathways were used to investigate the mechanisms of dendrimer cellular uptake and transport across Caco-2 cells using flow cytometry and confocal microscopy. Dendrimer cellular uptake was found to be dynamin-dependent and was reduced by both clathrin and caveolin endocytosis inhibitors, while transepithelial transport was only dependent on dynamin- and clathrin-mediated endocytosis. Dendrimers were quickly trafficked to the lysosomes after 15 min of incubation and showed increased endosomal accumulation at later time points, suggesting saturation of this pathway. Dendrimers were unable to open tight junctions in cell monolayers treated with dynasore, a selective inhibitor of dynamin, confirming that dendrimer internalization promotes tight junction modulation. G3.5 PAMAM dendrimers take advantage of several receptor-mediated endocytosis pathways for cellular entry in Caco-2 cells. Dendrimer internalization by dynamin-dependent mechanisms promotes tight junction opening, suggesting that dendrimers act on intracellular cytoskeletal proteins to modulate tight junctions, thus catalyzing their own transport via the paracellular route.

Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression

BackgroundCurcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1) protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells.MethodsCaco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification.ResultsWe found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 μM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 μM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively.ConclusionCurcumin inhibits cholesterol uptake through suppression of NPC1L1 expression in the intestinal cells.

Generating Ceramide from Sphingomyelin by Alkaline Sphingomyelinase in the Gut Enhances Sphingomyelin-Induced Inhibition of Cholesterol Uptake in Caco-2 Cells

Background Sphingomyelin (SM) is present in dietary products and cell plasma membranes. We previously showed that dietary SM inhibited cholesterol absorption in rats. In the intestinal tract, SM is mainly hydrolyzed by alkaline sphingomyelinase (alk-SMase) to ceramide.Aims We investigated the influence of SM and its hydrolytic products ceramide and sphingosine on cholesterol uptake in intestinal Caco-2 cells.Methods Micelles containing bile salt, monoolein, and (14)C-cholesterol were prepared with or without SM, ceramide,or sphingosine. The micelles were incubated with Caco-2 cells, and uptake of radioactive cholesterol was quantified.Results We found that confluent monolayer Caco-2 cells expressed NPC1L1, and the uptake of cholesterol in the cells was inhibited by ezetimibe, a specific inhibitor of NPC1L1. Incorporation of SM in the cholesterol micelles inhibited cholesterol uptake dose-dependently; 38% inhibition occurred at an equal mole ratio of SM and cholesterol.The inhibition was further enhanced to 45% by pretreating the cholesterol/SM micelles with recombinant alk-SMase, which hydrolyzed SM in the micelles by 85%, indicating ceramide has stronger inhibitory effects on cholesterol uptake. To confirm this, we further replaced SM in the micelles with ceramide and sphingosine, and found that at equal mole ratio to cholesterol, ceramide exhibited stronger inhibitory effect (50% vs 38%) on cholesterol uptake than SM, whereas sphingosine only had a weak effect at high concentrations.Conclusion Both SM and ceramide inhibit cholesterol uptake, the effect of ceramide being stronger than that of SM. Alk-SMase enhances SM-induced inhibition of cholesterol uptake by generating ceramide in the intestinal lumen.

Development and validation of a GC/IT-MS method for simultaneous quantitation of para and meta-synephrine in biological samples

After the FDA's ban of ephedrine-containing supplements, Citrus aurantium appeared as an alternative to ephedra in herbal weight loss products. Synephrine, the most abundant active component of C. aurantium, exists in three different structural or positional isomeric forms (ortho-o-, meta-m-, and para-p-). Dietary supplements contain m- and p-synephrine, both alpha-adrenergic agonists,while the m-isoform is the most potent at alpha(1)-adrenoreceptors. In spite of the pharmacokinetic and toxicological interest in the study of these compounds, adequate methods for their quantification in biological samples are yet to be developed. Thus, in the present study, a sensitive gas chromatography-ion trap mass spectrometry (GC/IT-MS) method was developed and validated for the simultaneous quantitation of m- and p-synephrine in a cellular matrix after solid phase extraction (SPE). The validation of the method was performed through the evaluation of the following parameters: selectivity, linearity, specificity, precision, accuracy, limit of detection, limit of quantification, and recovery. The method's applicability was studied in two different biological matrices by evaluating p- and m-synephrine uptake in Caco-2 cells and also in freshly isolated cardiomyocytes from adult rat. The developed GC/IT-MS method was shown to be selective, accurate, precise, and valid for simultaneous determination of p- and m-synephrine in biological samples.