Intestinal absorption and metabolism of carotenoids: insights from cell culture

Cell culture models are useful for studying intestinal absorption and metabolism of carotenoids. The human intestinal cell line, Caco-2, has been the most widely used model for these studies. The PF11 and TC7 clones of Caco-2 exhibit beta-carotene-15,15'-oxygenase activity, a key enzyme in the conversion of carotenoids to vitamin A. Studies on the recent cloning of this enzyme are discussed. An in vitro cell culture system used to study intestinal absorption of carotenoids is presented. Under conditions mimicking the postprandial state, Caco-2 cells on membranes take up carotenoids and secrete them incorporated into chylomicrons. Both the cellular uptake and secretion of beta-carotene are saturable, concentration-dependent processes. The selective absorption of all-trans beta-carotene versus its cis isomers, the differential absorption of individual carotenoids, and the specific interactions between carotenoids during their absorption are discussed. The participation of a specific epithelial transporter in the intestinal absorption of carotenoids is proposed.     

Red wine polyphenolics suppress the secretion and the synthesis of Apo B48 from human intestinal CaCo-2 cells

Epidemiological studies suggest that the red wine consumption may reduce the risk factor of cardiovascular disease. However, the mechanisms of how the red wine phenolic components reduce the risk of cardiovascular disease is currently unknown. Our previous study demonstrated that red wine polyphenolics suppress the secretion of pro-atherogenic lipoproteins (very low density lipoproteins) from human hepatic HepG2 cells. Therefore, in this study we hypothesize that red wine polyphenolics will also attenuate the production and secretion of another pro-atherogenic lipoprotein (chylomicrons) from human intestinal CaCo-2 cells. Cultured CaCo-2 cells were incubated in the presence of dealcoholized red wine, alcoholized red wine and atorvastatin for 24 h. The apo B48 protein (marker of intestinal chylomicrons) was quantified on Western blotting and the enhanced chemiluminescence. Apo B48 levels in the cells and that secreted into the media were significantly reduced by 29% in the cells incubated with dealcoholized red wine compared with control cells. Also the similar effect was shown in the cells incubated with alcoholized red wine. The cells incubated with atorvastatin shown the significant reduction of apo B48 production compared to control cells. Collectively, this study suggests that red wine polyphenolics down regulate the production of chylomicron in intestinal CaCo-2 cells.     

Xanthophylls are preferentially taken up compared with beta-carotene by retinal cells via a SRBI-dependent mechanism

The purpose of this study was to investigate the mechanisms by which carotenoids [xanthophylls vs. beta-carotene(beta-C)] are taken up by retinal pigment epithelial (RPE) cells. The human RPE cell line, ARPE-19, was used. When ARPE-19 cells were fully differentiated (7-9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent 2-fold higher than beta-C (P < 0.05). At 9 weeks, cellular uptakes were 1.6, 2.5, and 3.2%, respectively, for beta-C, LUT, and ZEA. Similar extents were observed when carotenoids were delivered in either Tween 40 or "chylomicrons" produced by Caco-2 cells. Differentiated ARPE-19 cells did not exhibit any detectable beta-C 15,15'-oxygenase activity or convert exogenous beta-C into vitamin A. When using specific antibodies against the lipid transporters cluster determinant 36 (CD36) and scavenger receptor class B type I (SR-BI), cellular uptake of beta-C and ZEA were significantly decreased (40-60%) with anti-SR-BI but not with anti-CD36. Small interfering RNA transfection for SR-BI led to marked knockdown of SR-BI protein expression (approximately 90%), which resulted in decreased beta-C and ZEA uptakes by 51% and 87%, respectively. Thus, the present data show that RPE cells preferentially take up xanthophylls versus the carotene by a process that appears to be entirely SR-BI-dependent for ZEA and partly so for beta-C. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina.     

Effect of eicosapentaenoic acid on triacylglycerol transport in CaCo-2 cells

The human intestinal cell line, CaCo-2, was used to study the effect of the n-3 fatty acid, eicosapentaenoic acid, on triacylglycerol secretion. In cells incubated with 250 microM eicosapentaenoic acid, the incorporation of [3H]glycerol into triacylglycerols secreted into the medium was decreased by 58% compared to cells incubated with 250 microM oleic acid. The incorporation of [3H]glycerol into cellular triacylglycerols was decreased 32% in cells incubated with eicosapentaenoic acid. In cells preincubated with [3H]glycerol to label existing triacylglycerols, the rates of secretion of preformed triacylglycerols were similar in response to the addition of either fatty acid. Initial uptake rates of the n-3 fatty acid were higher than for oleic acid. Both eicosapentaenoic acid and oleic acid were minimally oxidized to CO2. Oleic acid was predominantly incorporated into cellular triacylglycerols (62% vs. 47%), whereas more eicosapentaenoic acid was incorporated into cellular phospholipids (46% vs. 30%). Phospholipids of microsomes prepared from cells incubated with eicosapentaenoic acid were enriched in this fatty acid. The rate of synthesis of triacylglycerol and diacylglycerol acyltransferase activities were significantly less in microsomes prepared from cells incubated with eicosapentaenoic acid. Triacylglycerol mass secreted by CaCo-2 cells incubated with either fatty acid was similar. In CaCo-2 cells, eicosapentaenoic acid decreases the synthesis and secretion of newly synthesized triacylglycerol without decreasing the secretion of triacylglycerol mass. Modification of microsomal membrane phospholipid fatty acid composition is associated with a decrease in microsomal triacylglycerol synthesis and diacylglycerol acyltransferase activities.     

Mechanisms of provitamin A (carotenoid) and vitamin A (retinol) transport into and out of intestinal Caco-2 cells

The purpose of this study was to compare the mechanisms of intestinal retinol (ROL) and carotenoid transport. When differentiated Caco-2 cells were incubated with ROL for varying times, cellular ROL plateaued within 2 h, whereas retinyl ester (RE) formation increased continuously. ROL and RE efflux into basolateral medium (BM) increased linearly with time, ROL in the nonlipoprotein fraction and REs in chylomicrons (CMs). In contrast to carotenoids, ROL uptake was proportional to ROL concentration (0.5-110 microM). ROL efflux into BM occurred via two processes: a) a saturable process at low concentrations (<10 microM) and b) a nonsaturable process at higher concentrations. When ROL-loaded cells were maintained on retinoid-free medium, free ROL, but not REs, was secreted into BM. Glyburide significantly reduced ROL efflux but not ROL uptake. Inhibition of ABCA1 protein expression by small interfering RNAs decreased ROL efflux but not carotenoid efflux. Scavenger receptor class B type I (SR-BI) inhibition did not affect ROL transport but decreased carotenoid uptake. The present data suggest that a) ROL enters intestinal cells by diffusion, b) ROL efflux is partly facilitated, probably by the basolateral transporter ABCA1, and c) newly synthesized REs, but not preformed esters, are incorporated into CM and secreted. In contrast to ROL transport, carotenoid uptake is mediated by the apical transporter SR-BI, and carotenoid efflux occurs exclusively via their secretion in CM.     

Influence of eicosapentaenoic acid (20:5, n-3) on secretion of lipoproteins in CaCo-2 cells.

CaCo-2 cells, grown on filter membranes, were used to study the effects of fatty acids on cellular metabolism of triacylglycerol and phospholipids. The rate of triacylglycerol secretion was enhanced more than 2-fold, from 1 to 2 weeks after reaching confluency, in the presence of 0.6 mM fatty acids. Triacylglycerol secretion and oxidation of oleic acid increased 2- and 9-fold, respectively, with this culture system, as compared to cells grown on conventional plastic dishes. Eicosapentaenoic acid (20:5 n-3), when compared to oleic acid, did not reduce formation of triacylglycerol or enhance phospholipid synthesis in CaCo-2 cells during short term (less than 24 h) experiments, when the cells resided on membranes, regardless of what type of radioisotopes were used as precursors in the incubation media. However, the n-3 fatty acid was preferentially incorporated into phosphatidylinositol, lysophosphatidylcholine, and sphingomyelin, as compared to oleic acid. The disappearance from the apical medium and cellular uptake of labeled eicosapentaenoic and oleic acid were similar during incubations up to 24 h, and the metabolism of these fatty acids to acid-soluble materials and CO2 was equal. Light scattering analysis indicated that secreted lipoproteins of density less than 1.006 g/ml were in the same size-range as chylomicrons derived from human plasma. Assessment of secreted apolipoprotein B showed that by incubating CaCo-2 cells with oleic acid, apolipoprotein B levels increased approximately 1.4-fold when compared to cells incubated with eicosapentaenoic acid, whereas the amount of triacylglycerol and size-range of particles were similar for the two fatty acids. Our data indicate that CaCo-2 cells grown on filter membranes exhibit enterocyte-like characteristics with the ability to synthesize and secrete chylomicrons. Eicosapentaenoic acid and oleic acid are absorbed, metabolized, and influence secretion of lipoprotein particles in a similar way, except for some differences in incorporation of the fatty acids into certain phospholipid classes and a reduced secretion of apolipoprotein B. The culture conditions, including time after confluency and cellular support, are critical for the rate of secretion in the presence of eicosapentaenoic acid and oleic acid.     

Transport of vitamin E by differentiated Caco-2 cells

In hepatocytes, vitamin E is secreted via the efflux pathway and is believed to associate with apolipoprotein B (apoB)-lipoproteins extracellularly. The molecular mechanisms involved in the uptake, intracellular trafficking, and secretion of dietary vitamin E by the intestinal cells are unknown. We observed that low concentrations of Tween-40 were better for the solubilization and delivery of vitamin E to differentiated Caco-2 cells, whereas high concentrations of Tween-40 and sera inhibited this uptake. Vitamin E uptake was initially rapid and then reached saturation. Subcellular localization revealed that vitamin E primarily accumulated in microsomal membranes. Oleic acid (OA) treatment, which induces chylomicron assembly and secretion, decreased microsomal membrane-bound vitamin E in a time-dependent manner. To study secretion, differentiated Caco-2 cells were pulse-labeled with vitamin E and chased in the presence and absence of OA. In the absence of OA, vitamin E was associated with intestinal high density lipoprotein (I-HDL), whereas OA-treated cells secreted vitamin E with I-HDL and chylomicrons. No extracellular transfer of vitamin E between these lipoproteins was observed. Glyburide, an antagonist of ABCA1, partially inhibited its secretion with I-HDL, whereas plasma HDL increased vitamin E efflux. An antagonist of microsomal triglyceride transfer protein, brefeldin A, and monensin specifically inhibited vitamin E secretion with chylomicrons. These studies indicate that vitamin E taken up by Caco-2 cells is stored in the microsomal membranes and secreted with chylomicrons and I-HDL. Transport via I-HDL might contribute to vitamin E absorption in patients with abetalipoproteinemia receiving large oral doses of the vitamin.     

Processing of vegetable-borne carotenoids in the human stomach and duodenum

Carotenoids are thought to diminish the incidence of certain degenerative diseases, but the mechanisms involved in their intestinal absorption are poorly understood. Our aim was to obtain basic data on the fate of carotenoids in the human stomach and duodenum. Ten healthy men were intragastrically fed three liquid test meals differing only in the vegetable added 3 wk apart and in a random order. They contained 40 g sunflower oil and mashed vegetables as the sole source of carotenoids. Tomato purée provided 10 mg lycopene as the main carotenoid, chopped spinach (10 mg lutein), and carrot purée (10 mg beta-carotene). Samples of stomach and duodenal contents and blood samples were collected at regular time intervals after meal intake. all-trans and cis carotenoids were assayed in stomach and duodenal contents, in the fat and aqueous phases of those contents, and in chylomicrons. The cis-trans beta-carotene and lycopene ratios did not significantly vary in the stomach during digestion. Carotenoids were recovered in the fat phase present in the stomach during digestion. The proportion of all-trans carotenoids found in the micellar phase of the duodenum was as follows (means +/- SE): lutein (5.6 +/- 0.4%), beta-carotene (4.7 +/- 0.3%), lycopene (2.0 +/- 0.2%). The proportion of 13-cis beta-carotene in the micellar phase was significantly higher (14.8 +/- 1.6%) than that of the all-trans isomer (4.7 +/- 0.3%). There was no significant variation in chylomicron lycopene after the tomato meal, whereas there was significant increase in chylomicron beta-carotene and lutein after the carrot and the spinach meals, respectively. There is no significant cis-trans isomerization of beta-carotene and lycopene in the human stomach. The stomach initiates the transfer of carotenoids from the vegetable matrix to the fat phase of the meal. Lycopene is less efficiently transferred to micelles than beta-carotene and lutein. The very small transfer of carotenoids from their vegetable matrices to micelles explains the poor bioavailability of these phytomicroconstituents.     

Intestine-specific MTP and global ACAT2 deficiency lowers acute cholesterol absorption with chylomicrons and HDLs

Intestinal cholesterol absorption involves the chylomicron and HDL pathways and is dependent on microsomal triglyceride transfer protein (MTP) and ABCA1, respectively. Chylomicrons transport free and esterified cholesterol, whereas HDLs transport free cholesterol. ACAT2 esterifies cholesterol for secretion with chylomicrons. We hypothesized that free cholesterol accumulated during ACAT2 deficiency may be secreted with HDLs when chylomicron assembly is blocked. To test this, we studied cholesterol absorption in mice deficient in intestinal MTP, global ACAT2, and both intestinal MTP and global ACAT2. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their transport with chylomicrons. In contrast, global ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption with chylomicrons and increased cellular free cholesterol. Their combined deficiency reduced cholesterol secretion with both chylomicrons and HDLs. Thus, contrary to our hypothesis, free cholesterol accumulated in the absence of MTP and ACAT2 is unavailable for secretion with HDLs. Global ACAT2 deficiency causes mild hypertriglyceridemia and reduces hepatosteatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in global ACAT2-deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid hepatosteatosis in cholesterol-fed animals. Therefore, ACAT2 inhibition might avert hepatosteatosis associated with high cholesterol diets by increasing hepatic MTP expression and lipoprotein production.     

Inhibition of heat-induced aggregation of beta- and gamma-crystallin by alpha-crystallin evaluated by gel permeation HPLC

The capability of alpha-crystallin (alpha-C), a known molecular chaperon, of protecting beta-C and gamma-C against heat-induced aggregation was studied by gel permeation high performance liquid chromatography. The activity was calculated using a formula based on the changes in the areas under the chromatographic peaks of these proteins, which appeared well separated. When heat-induced aggregation was studied in the range 22-90 degrees C, beta-C appeared more stable than gamma-C. The activity of alpha-C in stabilizing gamma-C but not beta-C was already relevant at 60 degrees C, but the maximum activity was higher (about 35%) for beta-C than for gamma-C. This method could be useful for studying the effect of drugs with potential anti-cataract activity on heat-induced aggregation of individual lens proteins.     

Intestinal absorption of menhaden and rapeseed oils and their fatty acid methyl and ethyl esters in the rat

The relative cellular uptake and incorporation into prechylomicrons and chylomicrons was investigated for the menhaden and rapeseed oil fatty acids, when given by stomach tube as the original oils or the corresponding methyl and ethyl esters. The intermediates and final products of cellular acylation were determined by chromatographic methods at various times over a period of 1-24 h. There was little selectivity in the uptake among the oligo- and poly-unsaturated fatty acids of menhaden oil, when either oil or esters were fed. In contrast, the long-chain saturated and monounsaturated fatty acids of rapeseed oil were discriminated against during both cellular uptake and reacylation (60% overall reduction in utilization). Also, there was detectable discrimination against the long-chain polyunsaturated monoacylglycerols of menhaden oil and against the long-chain saturated and monounsatured monoacylglycerols of rapeseed oil during both cellular uptake and reacylation (30% overall reduction in utilization). Evidence was obtained for an indiscriminate cellular uptake of variable amounts (4-22%) of intact dietary methyl and ethyl esters of fatty acids, which, however, appeared in the chylomicrons only to a very limited extent (0.1-1.0% of total lipid). During peak absorption the cellular and lymphatic appearance of fatty acids from the digestion and absorption of the alkyl esters was nearly 50% lower than that from the corresponding triacylglycerols. The slower absorption of the fatty acids from the alkyl ester feeding is hypothetically attributed to a lower efficiency of the phosphatidic acid pathway, which is required in the absence of dietary 2-monoacylglycerols, but other mechanisms cannot be excluded.     

Chylomicrons promote intestinal absorption of lipopolysaccharides

Recent data suggest that dietary fat promotes intestinal absorption of lipopolysaccharides (LPS) from the gut microflora, which might contribute to various inflammatory disorders. The mechanism of fat-induced LPS absorption is unclear, however. Intestinal-epithelial cells can internalize LPS from the apical surface and transport LPS to the Golgi. The Golgi complex also contains newly formed chylomicrons, the lipoproteins that transport dietary long-chain fat through mesenteric lymph and blood. Because LPS has affinity for chylomicrons, we hypothesized that chylomicron formation promotes LPS absorption. In agreement with our hypothesis, we found that CaCo-2 cells released more cell-associated LPS after incubation with oleic-acid (OA), a long-chain fatty acid that induces chylomicron formation, than with butyric acid (BA), a short-chain fatty acid that does not induce chylomicron formation. Moreover, the effect of OA was blocked by the inhibitor of chylomicron formation, Pluronic L-81. We also observed that intragastric triolein (TO) gavage was followed by increased plasma LPS, whereas gavage with tributyrin (TB), or TO plus Pluronic L-81, was not. Most intestinally absorbed LPS was present on chylomicron remnants (CM-R) in the blood. Chylomicron formation also promoted transport of LPS through mesenteric lymph nodes (MLN) and the production of TNFalpha mRNA in the MLN. Together, our data suggest that intestinal epithelial cells may release LPS on chylomicrons from cell-associated pools. Chylomicron-associated LPS may contribute to postprandial inflammatory responses or chronic diet-induced inflammation in chylomicron target tissues.     

Insulin decreases the secretion of apoB-100 from hepatic HepG2 cells but does not decrease the secretion of apoB-48 from intestinal CaCo-2 cells

We compared the acute effect of insulin on the human colonic intestinal epithelial cell line CaCo-2 and the transformed human hepatic cell line HepG2. Over 24 h, 100 nM and 10 µM insulin significantly inhibited the secretion of apolipoprotein (apo) B-100 from HepG2 cells to 63 and 49% of control, respectively. Insulin had no effect on the secretion of apoB-48 from CaCo-2 cells. There was no effect of insulin on the cholesterol ester or free cholesterol concentrations in HepG2 or CaCo-2 cells. HepG2 and CaCo-2 cells bound insulin with high affinity, leading to similar stimulation of insulin receptor protein tyrosine kinase activation. Protein kinase C or mitogen-activated protein kinase activity in the presence or absence of insulin was not correlated with apoB-48 production in CaCo-2 cells. Therefore, insulin acutely decreases the secretion of apoB-100 in hepatic HepG2 cells, but does not acutely modulate the production or secretion of apoB-48 from CaCo-2 intestinal cells.     

Chylomicrons Promote Intestinal Absorption and Systemic Dissemination of Dietary Antigen (Ovalbumin) in Mice

Background

A small fraction of dietary protein survives enzymatic degradation and is absorbed in potentially antigenic form. This can trigger inflammatory responses in patients with celiac disease or food allergies, but typically induces systemic immunological tolerance (oral tolerance). At present it is not clear how dietary antigens are absorbed. Most food staples, including those with common antigens such as peanuts, eggs, and milk, contain long-chain triglycerides (LCT), which stimulate mesenteric lymph flux and postprandial transport of chylomicrons through mesenteric lymph nodes (MLN) and blood. Most dietary antigens, like ovalbumin (OVA), are emulsifiers, predicting affinity for chylomicrons. We hypothesized that chylomicron formation promotes intestinal absorption and systemic dissemination of dietary antigens.

Methodology/Principal Findings

Absorption of OVA into MLN and blood was significantly enhanced when OVA was gavaged into fasted mice together with LCT compared with medium-chain triglycerides (MCT), which do not stimulate chylomicron formation. The effect of LCT was blocked by the addition of an inhibitor of chylomicron secretion, Pluronic L-81. Adoptively transferred OVA-specific DO11.10 T-cells proliferated more extensively in peripheral lymph nodes when OVA was gavaged with LCT than with MCT or LCT plus Pluronic L-81, suggesting that dietary OVA is systemically disseminated. Most dietary OVA in plasma was associated with chylomicrons, suggesting that these particles mediate systemic antigen dissemination. Intestinal-epithelial CaCo-2 cells secreted more cell-associated, exogenous OVA when stimulated with oleic-acid than with butyric acid, and the secreted OVA appeared to be associated with chylomicrons.

Conclusions/Significance

Postprandial chylomicron formation profoundly affects absorption and systemic dissemination of dietary antigens. The fat content of a meal may affect immune responses to dietary antigens by modulating antigen absorption and transport.     

Scavenger receptor class B type I reduces cholesterol absorption in cultured enterocyte CaCo-2 cells

Scavenger receptor class B type I (SR-BI) mediates selective uptake of cholesteryl esters from HDL as well as efflux of cellular free cholesterol to HDL. It is unclear whether the receptor is involved in intestinal cholesterol absorption. We addressed this issue by studying [3H]cholesterol flux in differentiated CaCo-2 cells incubated at their apical side with mixed taurocholate/phosphatidylcholine/cholesterol micelles. Biotinylation and HDL binding experiments showed predominant apical expression of endogenous and overexpressed SR-BI. Mixed micellar cholesterol saturation affected the magnitude and direction of cholesterol flux with significant net uptake only from supersaturated micelles and net efflux from unsaturated micelles. Incubation with micelles that depleted cellular cholesterol resulted in a decrease of SR-BI protein, whereas incubation with cholesterol-loading micelles resulted in a significant increase of SR-BI protein. Apical cholesterol uptake by CaCo-2 cells was increased in the presence of a SR-BI-blocking antibody and by partial inhibition of SR-BI expression with small inhibitory RNA. Adenovirus-mediated overexpression of apical SR-BI did not affect cholesterol uptake but stimulated apical cholesterol efflux, even to supersaturated mixed micelles. Partial inhibition of SR-BI with small inhibitory RNA reduced apical cholesterol efflux. Our data argue against a direct role for SR-BI in micellar cholesterol uptake. However, SR-BI might be involved in cholesterol absorption by facilitating cholesterol efflux to micelles.     

Proteins involved in uptake, intracellular transport and basolateral secretion of fat-soluble vitamins and carotenoids by mammalian enterocytes

Our understanding of the molecular mechanisms responsible for fat-soluble vitamin uptake and transport at the intestinal level has advanced considerably over the past decade. On one hand, it has long been considered that vitamin D and E as well as β-carotene (the main provitamin A carotenoid in human diet) were absorbed by a passive diffusion process, although this could not explain the broad inter-individual variability in the absorption efficiency of these molecules. On the other hand, it was assumed that preformed vitamin A (retinol) and vitamin K1 (phylloquinone) absorption occurred via energy-dependent processes, but the transporters involved have not yet been identified. The recent discovery of intestinal proteins able to facilitate vitamin E and carotenoid uptake and secretion by the enterocyte has spurred renewed interest in studying the fundamental mechanisms involved in the absorption of these micronutrients. The proteins identified so far are cholesterol transporters such as SR-BI (scavenger receptor class B type I), CD36 (cluster determinant 36), NPC1L1 (Niemann–Pick C1-like 1) or ABCA1 (ATP-Binding Cassette A1) displaying a broad substrate specificity, but it is likely that other membrane proteins are also involved. After overviewing the metabolism of fat-soluble vitamins and carotenoids in the human upper gastrointestinal lumen, we will focus on the putative or identified proteins participating in the intestinal uptake, intracellular transport and basolateral secretion of these fat-soluble vitamins and carotenoids, and outline the uncertainties that need to be explored in the future. Identifying the proteins involved in intestinal uptake and transport of fat-soluble vitamins and carotenoids across the enterocyte is of great importance, especially as some of them are already targets for the development of drugs able to slow cholesterol absorption. Indeed, these drugs may also interfere with lipid vitamin uptake. A better understanding of the molecular mechanisms involved in fat-soluble vitamin and carotenoid absorption is a priority to better optimize their bioavailability.     

Regulated vectorial secretion of cholesteryl ester transfer protein (LTP-I) by the CaCo-2 model of human enterocyte epithelium

We have investigated the human CaCo-2 enterocyte model for secretion of the plasma cholesteryl ester transfer protein, LTP-I. CaCo-2 cells secrete a cholesteryl ester transfer protein which possesses molecular identity with plasma LTP-I, demonstrated by anti-LTP-I immunoblot analysis and immunoinhibition of all cell-secreted cholesteryl ester transfer activity. When CaCo-2 are cultured on permeable membranes, cholesteryl ester transfer activity is detected only in the lower culture compartment. Thus, CaCo-2 vectorially sort and secrete LTP-I, as well as the intestinal apolipoproteins, from the basolateral cellular domain. Over a 24-h period, CaCo-2 secrete cholesteryl ester transfer activity in a time-dependent manner, at approximately twice the rate of HepG2. Furthermore, CaCo-2 enterocytes, but not HepG2 hepatocytes, regulate LTP-I secretion in response to fatty acid concentration in the culture medium. Based on these observations, we speculate that the intestine may be the principal regulated source of human plasma LTP-I.     

The evaluation of protective effect of lycopene against genotoxic influence of X-irradiation in human blood lymphocytes

Many studies suggest that exogenous antioxidants may protect cells against DNA damage caused with ionizing radiation. One of the most powerful antioxidants is lycopene (LYC), a carotenoid derived from tomatoes. The aim of this study was to investigate, using the comet assay, whether LYC can act as protectors/modifiers and prevent DNA damage induced in human blood lymphocytes, as well as to mitigate the effects of radiation exposure. In this project, LYC, dissolved in DMSO at a concentration of 10, 20 or 40 μM/ml of cell suspension, was added to the isolated lymphocytes from human blood at appropriate intervals before or after the X-irradiation at doses of 0.5, 1 and 2 Gy. Cell viability in all groups was maintained at above 70%. The results showed the decrease of DNA damage in cells treated with various concentrations of LYC directly and 1 h before exposure to X-rays compared to the control group exposed to irradiation alone. Contrary results were observed in cells exposed to LYC immediately after exposure to ionizing radiation. The studies confirmed the protective effect of LYC against DNA damage induced by ionizing radiation, but after irradiation the carotenoid did not stimulate of DNA repair and cannot act as modifier. However, supplementation with LYC, especially at lower doses, may be useful in protection from radiation-induced oxidative damage.     

Discrimination between forms of vitamin E by humans with and without genetic abnormalities of lipoprotein metabolism.

To study the mechanisms of discrimination between various forms of vitamin E, four normal subjects, one patient with lipoprotein lipase deficiency, and three patients with abnormal apolipoprotein B-100 production were given an oral dose containing three tocopherols labeled with differing amounts of deuterium (2R,4'R,8'R-alpha-(5,7-(C2H3)2)tocopheryl acetate (d6-RRR-alpha-tocopheryl acetate), 2S,4'R,8'R-alpha-5-(C2H3)tocopheryl acetate (d3-SRR-alpha-tocopheryl acetate), and 2R,4'R,8'R-gamma-(3,4-2H)tocopherol (d2-RRR-gamma-tocopherol). The tocopherol contents of plasma, red cells, and lipoproteins were measured up to 76 h after the dose. In normal subjects all three tocopherols were absorbed and secreted in chylomicrons with equal efficiencies. Both d2-gamma- and d3-SRR-alpha-tocopherols peaked at similar concentrations in the other lipoprotein fractions, then decreased similarly, but 2-4 times more rapidly than did d6-RRR-alpha-tocopherol. A lipoprotein lipase-deficient patient and a patient with prolonged production of chylomicrons with absent apolipoprotein B-100 also demonstrated the lack of discrimination between tocopherols during absorption. Despite abnormal apolipoprotein B-100 production in two patients, the "VLDL" was preferentially enriched in d6-RRR-alpha-tocopherol. Our results show that there is no discrimination between the three tocopherols during absorption and secretion in chylomicrons, but subsequently there is a preferential enrichment of very low density lipoprotein (VLDL) with RRR-alpha-tocopherol. Catabolism of this VLDL results in the maintenance of plasma RRR-alpha-tocopherol concentrations.