Chicken thigh, chicken liver, and iron-fortified wheat flour increase iron uptake in an in vitro digestion/Caco-2 cell model

The objective of this study was to test meat and fortified-food combinations to identify those that optimize iron uptake in an in vitro digestion/Caco-2 cell model, a proxy for iron bioavailability. Four experiments tested combinations of meats such as chicken (blood, spleen, liver, thigh), beef (cube steak), and fish (whole-fish meal) with iron-fortified foods (rice cereal, maize-soy flour, wheat flour). Chicken liver, thigh, spleen, blood, or fish meal increased the Caco-2 cell iron uptake from these combined with rice cereal (P < .05). Chicken liver, thigh, blood, and beef increased the Caco-2 cell iron uptake from these combined with wheat flour (P < .05). Chicken liver and thigh were tested further. Compared with the liver or thigh alone, adding fortified foods to these meats did not increase the Caco-2 cell iron uptake (P ≥ .05). Adding either meat to the 3 fortified foods increased the Caco-2 cell iron uptake of the fortified foods (P < .05). Chicken liver, chicken thigh, and wheat flour were selected for an infant porridge because the combinations with the highest Caco-2 cell iron uptake were chicken thigh + wheat flour, chicken liver + wheat flour, and chicken liver + maize-soy flour, and wheat flour was the least expensive fortified food sold in the target population. Per unit of iron, the chicken thigh + wheat flour and chicken liver + wheat flour combinations resulted in the highest bioavailable iron. In the proportion of 3:1 fortified food:meat examined, meat increases the bioavailability of iron-fortified foods, but iron-fortified foods do not enhance total iron bioavailability when added to meat.     

Caco-2细胞单层模型及其在毒理学中的应用

Caco 2细胞来源于人类结肠腺癌细胞 ,当其在生长达到融合后细胞可自发形成极性 ,表达出成熟肠道上皮细胞的某些形态和功能特征 ,其细胞单层模型是目前最为成熟的体外模拟肠道上皮细胞摄取和转运营养物质和外来化学物的模型 ,被广泛地应用于药理学和毒理学研究。本文介绍了Caco 2细胞单层模型的培养条件、细胞特性及常用功能指标 ,以及其在对外源化学物代谢、摄取、转运和消化道毒理学方面的应用。     

Dephosphorylation of myo-inositol phosphates in the in vitro intestinal Caco-2 cell model

Plant and microbial phytases present in raw materials can cause a dephosphorylation of phytate (myo-inositol hexakisphosphate) (InsP₆)) during food processing resulting in a broad range of different myo-inositol phosphates such as pentakisphosphate (InsP₅) and tetrakisphosphate (InsP₄) in foods. Here, we investigated whether the human intestinal epithelium is able to dephosphorylate myo-inositol phosphates (InsP₆, InsP₅-, InsP₄-, InsP₃-isomers) using an in vitro model with differentiated human Caco-2 cells cultured on semipermeable inserts. Incubation of InsP₆ and an InsP₅-isomer with cells for 3?h showed no dephosphorylation of both InsPs. Treatment of cells with a mixture of different InsP₄-isomers, however, caused a formation of about 3.5% of an InsP₃-isomer (Ins(1,5,6)P₃) and treatment with a mixture of different InsP₃-isomers caused about 20% formation of InsP₂-isomers, respectively. Thus, human intestinal cells can contribute to the dephosphorylation of myo-inositol phosphates of partly dephosphorylated forms such as InsP₃ and InsP₄.     

Ascorbic acid uptake affects ferritin, Dcytb and Nramp2 expression in Caco-2 cells

Background  Ascorbic acid (vitamin C) enhances iron uptake in human intestinal cells. It is commonly believed that the enhancement is due to the capacity of ascorbic acid to reduce ferric iron to ferrous iron. Other suggestions have recently been made about the effects of ascorbic acid on the cellular metabolism of iron. These effects must be investigated for several reasons. One important issue is to study whether ascorbic acid has effects on iron metabolism in the absence of extracellular iron in the intestinal lumen. Aim of the study  The aim of this investigation was to determine whether cellular uptake of ascorbic acid affects iron acquisition in the Caco-2 cell line. The possible event was investigated by studying the expression of the iron storage protein ferritin, the iron uptake protein Nramp2 and a duodenal ferric reductase Dcytb after incubating ascorbic acid deficient or ascorbic acid fed cells with iron and/or ascorbic acid. Methods  The above stated interactions were studied in the human Caco-2 cell model. Cell lysates were collected and subjected to SDS-PAGE and Western blotting. The blotted samples were stained with specific antibodies (Rabbit α-human-Nramp2 and Goat α-human Dcytb) against the respective proteins and the bands achieved were analysed by reflective density measurements. The cellular ferritin content was analysed with a commercial kit and the intracellular ascorbic acid concentration was measured by HPLC. Results  The results indicate that ascorbic acid uptake induces both iron independent and iron dependent ferritin formation, but the effect on iron dependent ferritin expression was significantly greater (470% compared to 19%). Western Blot analyses revealed a long term down-regulating effect of ascorbic acid on iron independent and iron dependent Nramp2 and Dcytb expression. However, the down-regulation of Dcytb was in general more extensive than that of Nramp2 (31–50% compared to 8–29%). In a second study of short term Nramp2 and Dcytb expression, the results suggested that both proteins were significantly up-regulated by ascorbic acid, regardless of intracellular ascorbic acid status. However, the impact of iron alone on Nramp2 up-regulation seems to be greater in the absence of ascorbic acid. Conclusions  The influence of intracellular ascorbic acid status on ferritin formation must be considered in iron uptake studies in Caco-2 cells. This could be a cause of diverging inter-laboratory results. The long term down-regulation of Nramp2 and Dcytb seems to correlate with results of human studies, where long term ascorbic acid supplementation does not affect iron status. Similarly, the short term up-regulation of Nramp2 and Dcytb seems to agree with the improvement in iron uptake shown in humans when single doses of ascorbic acid were administrated. These results are important for the understanding of the impact of ascorbic acid on iron status and will hopefully lead to further investigations on the matter.     

体外消化/Caco-2细胞方法评价富铁小麦生物利用率

目的通过研究生物强化富铁小麦面粉的铁生物利用率,筛选出最具潜力的富铁小麦品种。方法采用体外消化/Caco-2方法对10种生物强化富铁面粉进行铁生物利用率比较,以人Caco-2细胞铁蛋白生成量来反映铁生物利用率。结果发现不同面粉间铁生物利用率差异有显著性,出粉率78%的安阳中优9507和京冬8号面粉的铁生物利用率最高。结论生物强化富铁小麦安阳中优9507和京冬8号是最具潜力品种。     

Increased intake of fruit and vegetables and a low-fat diet, with and without low-fat plant sterol-enriched spread consumption: effects on plasma lipoprotein and carotenoid metabolism

BACKGROUND: Regular intake of plant sterol (phytosterol)-enriched foods enhances the cholesterol lowering effect of diets. One side effect associated with plant sterol consumption is a modest reduction in plasma carotenoid concentrations. This study investigated the effect of consuming a low-fat National Cholesterol Education Programme (NCEP) Step 1 diet, including a low-fat plant sterol ester (PSE)-enriched spread on cholesterol metabolism to determine if specific dietary advice to increase daily fruit and vegetable intake could prevent reduced plasma carotenoid concentrations. MATERIALS AND METHODS: In this randomised, crossover double-blind trial, 48 hypercholesterolaemic men received 21 g day(-1) of a low-fat PSE-enriched spread or placebo for 3 weeks, interrupted by 3 weeks washout. Individuals also adhered to a NCEP Step 1 diet and repeated 3-day food diaries monitored adherence. Specific advice was provided to increase dietary fruit and vegetable intakes. Fasting blood samples were collected at pre- and post-intervention for lipoprotein and carotenoid analysis. RESULTS: Plasma total and low-density lipoprotein (LDL) cholesterol concentrations were significantly (P <0.05) reduced, by 4.6 and 7.1%, respectively, after the PSE-enriched low-fat spread. Plasma apo B concentrations were significantly (P <0.0005) lower after the PSE spread. PSE consumption was also associated with significantly (P <0.05) lower total plasma beta-carotene concentrations, but this change was not significant after lipid standardisation. PSE consumption had no effect on retinol, alpha-carotene, gamma-tocopherol, alpha-tocopherol, lutein, zeaxanthin, beta-crypyoxanthin or lycopene concentrations. CONCLUSION: Dietary advice to increase daily fruit and vegetable consumption may be effective in preventing a reduction in plasma carotenoid concentrations previously associated with PSE consumption. Further, PSE incorporated in a low-fat spread and consumed as part of a NCEP Step 1 diet are effective in reducing total and LDL cholesterol.     

The effect of two dietary and a synthetic phytoestrogen on transepithelial calcium transport in human intestinal-like Caco-2 cells

Summary Background Recently, dietary phytoestrogens (PEs) have been suggested as possible alternatives to estrogen therapy, as a means of preventing bone loss associated with ovarian hormone deficiency. PEs are non-steroidal, plant-derived compounds that exhibit some estrogen-like activity in some tissues, and which appear to prevent postmenopausal bone loss. While PEs act directly on bone cells, their protective effect on bone may be partly due to their ability to enhance Ca absorption. Aim of the study Therefore, the aim of this study was to investigate the effect of two dietary PEs (coumestrol and apigenin) as well as a synthetic PE, ipriflavone, on Ca absorption in human Caco-2 intestinal-like cells. Methods Caco-2 cells were seeded onto permeable filter supports and allowed to differentiate into monolayers. On d 21, the Caco-2 monolayers (n 10–16 per treatment), grown in estrogen-free or low-estrogen media, were then exposed to 10 nM-1,25 (OH)₂ D₃, or 50 µM ipriflavone, -coumestrol or -apigenin for 48 hours. After exposure, transepithelial and transcellular transport of ⁴⁵Ca and fluorescein transport (a marker of paracellular diffusion) were measured. Results As expected, 1,25 (OH)₂ D₃ stimulated Ca absorption. Treatment with coumestrol or apigenin had no effect on Ca transport. On the other hand, ipriflavone increased total Ca transport (by about 1.5-fold, P < 0.05) under lowestrogen conditions, but not under estrogen-free conditions. This increase in total Ca transport by ipriflavone was via an increased transcellular Ca transport (by about 2-fold, P < 0.05) relative to control. Conclusion In conclusion, the protective effect of dietary PE on bone mass would appear to be due to their direct effect(s) on bone cells, as opposed to an indirect effect on bone by stimulation of intestinal Ca absorption.     

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.     

Digestion and absorption of an egg white ACE-inhibitory peptide in human intestinal Caco-2 cell monolayers

The objective of this study was to investigate the digestion and absorption of egg white-derived angiotensin I-converting enzyme (ACE)-inhibitory peptide TNGIIR in human intestinal Caco-2 cell monolayers. Results showed that the digestion of TNGIIR to simulated gastrointestinal enzymes and brush border membrane peptidases were 5.87% ± 1.92% and 17.17% ± 0.64%, respectively (p?app) of TNGIIR from the apical to basolateral side in Caco-2 cell monolayers was determined to be (4.92?±?0.40) × 10^?6 cm/s, indicating that TNGIIR can transport across Caco-2 cell monolayers in intact form. In addition, only cytochalasin D, a disruptor of tight junctions (TJs), changed TNGIIR transport rate significantly (p?via TJs.     

Modulation of folate uptake in cultured human colon adenocarcinoma Caco-2 cells by dietary compounds

Folate is a water-soluble B vitamin with a crucial role in the synthesis and methylation of DNA and in the metabolism of several amino acids. In the present study we investigated whether beverages like wine, beer and tea, or some of their specific constituents, affect the intestinal uptake of ³H-folic acid or ³H-methotrexate (an antifolate). All tested beverages significantly inhibited the uptake of ³H-folic acid by Caco-2 cells. Most of these beverages, with the exception of wines (not tested), also inhibited ³H-methotrexate uptake in these cells. Additionally, ethanol, when tested separately, inhibited the uptake of both compounds. Some of the tested phenolic compounds, namely myricetin, epigallocatechin gallate (EGCG) and isoxanthohumol, markedly inhibited ³H-folic acid uptake. Myricetin and EGCG also had a concentration-dependent inhibitory effect upon the uptake of ³H-methotrexate by Caco-2 cells. Resveratrol, quercetin and kaempferol were able to inhibit the transport of both compounds, but only in the concentration of 100 μM. In conclusion, dietary constituents may impact on intestinal folate uptake, as here shown for phenolic compounds.     

Effects of yoghurt enriched with free plant sterols on the levels of serum lipids and plant sterols in moderately hypercholesterolaemic subjects on a high-fat diet

Apparently, one of the primary reasons for purchasing organic food is the perception that it is more nutritious than conventional food. Given the increasing interest towards organic food products, it is imperative to review the existing literature concerning the nutritional value of the produce, and to determine to what extent are consumer expectations met. There are only few well-controlled studies that are capable of making a valid comparison and, therefore, compilation of the results is difficult and generalisation of the conclusions should be made with caution. In spite of these limitations, however, some differences can be identified. Although there is little evidence that organic and conventional foods differ in respect to the concentrations of the various micronutrients (vitamins, minerals and trace elements), there seems to be a slight trend towards higher ascorbic acid content in organically grown leafy vegetables and potatoes. There is also a trend towards lower protein concentration but of higher quality in some organic vegetables and cereal crops. With respect to the rest of the nutrients and the other food groups, existing evidence is inadequate to allow for valid conclusions. Finally, animal feeding experiments indicate that animal health and reproductive performance are slightly improved when they are organically fed. A similar finding has not yet been identified in humans. Several important directions can be highlighted for future research; it seems, however, that despite any differences, a well-balanced diet can equally improve health regardless of its organic or conventional origin.     

Profile and bioavailability analysis of myo-inositol phosphates in rye bread supplemented with phytases: a study using an in vitro method and Caco-2 monolayers

Commercial preparations of 6-phytase A alone and in combination with phytase B were used in rye breadmaking. Determination of bioavailability of myo-inositol phosphates from bread was performed by an in vitro digestion method followed by the measurement of an uptake by Caco-2 cells in culture. In bread supplemented with a combination of 6-phytase A and phytase B, a significant reduction in phytate content was observed from 3.62?μmol/g in the control to 0.7?μmol/g. Bioavailability of phytate estimated by an in vitro method simulating digestion in the human alimentary tract was 9% in the bread supplemented with phytase B, 7% (6-phytase A) and 50% in the control bread. In cell culture, the bioaccessibilities of inositol triphosphates from bread baked with the addition of 6-phytase A was higher by 36% as compared to the samples baked with phytase B and by 32% in breads baked with combination of both phytases.     

Calcium,iron and zinc uptakes by Caco-2 cells from white beans and effect of cooking

White beans (Phaseolus vulgaris L.) have an interesting content of essential elements, calcium, iron and zinc, but they content also phytates, oxalates, proteins, polyyphenols and complex polysaccharides that are known to interact with minerals and to affect their bioavailability. The bioavailability of calcium, iron and zinc from raw and cooked white beans was estimated using their uptake by Caco-2 cells as the criteria. Previously, the mineral fraction (soluble or dialysable) to be added to the Caco-2 cell monolayer was selected. The results obtained show that cooking increases the Caco-2 cells’ uptake percentages (calcium, 18.8 versus 3.6; iron, 33.7 versus 1.7; and zinc, 17.2 versus 2.1) and improved the value of beans as a dietetic source of minerals.     

Homogenization, lyophilization or acid-extraction of meat products improves iron uptake from cereal-meat product combinations in an in vitro digestion/Caco-2 cell model

The effect of processing (homogenization, lyophilization, acid-extraction) meat products on iron uptake from meat combined with uncooked iron-fortified cereal was evaluated using an in vitro digestion/Caco-2 cell model. Beef was cooked, blended to create smaller meat particles, and combined with electrolytic iron-fortified infant rice cereal. Chicken liver was cooked and blended, lyophilized, or acid-extracted, and combined with FeSO4-fortified wheat flour. In the beef-cereal combination, Caco-2 cell iron uptake, assessed by measuring the ferritin formed by cells, was greater when the beef was blended for the greatest amount of time (360 s) compared with 30 s (P < 0.05). Smaller liver particles (blended for 360 s or lyophilized) significantly enhanced iron uptake compared to liver blended for 60 s (P < 0.001) in the liver-flour combination. Compared to liver blended for 60 s, acid-extraction of liver significantly enhanced iron uptake (P = 0.03) in the liver-flour combination. Homogenization of beef and homogenization, lyophilization, or acid-extraction of chicken liver increases the enhancing effect of meat products on iron absorption in iron-fortified cereals.     

Comparison of the in vitro toxicity of ancient Triticum monococcum varieties ID331 and Monlis

Abstract

Triticum monococcum L. is one of the oldest ancestors of wheat. There is some evidence that einkorn encloses forms of gliadin-deriving peptides which may potentially exert a reduced toxicity to consumers with gluten-related disorders. Accordingly, ID331 and Monlis lines were comparatively investigated in this study. The biological effects of gastro-resistant peptides deriving from an in vitro simulated digestion were evaluated on 21 d differentiated Caco-2 cells. Triticum aestivum digested gliadin was included as the positive control. ID331 neither enhanced cell permeability nor induced zonulin release in Caco-2 monolayers. Monlis exerted a detectable toxicity as confirmed by the reorganisation of enterocyte cytoskeleton, in addition to changes both in monolayers permeability and apical release of zonulin. Differences in patterns of gastro-resistant prolamins may account for the differences. Outcomes support the use of ID331 as a prospective candidate for the development of innovative approaches to reduce wheat flour toxicity.     

Beta-carotene micellarization during in vitro digestion and uptake by Caco-2 cells is directly proportional to beta-carotene content in different genotypes of cassava

Cassava, a staple food in sub-Saharan Africa, does not provide adequate amounts of pro-vitamin A (VA) carotenoids and has been targeted for biofortification (i.e. selectively breeding cultivars of increased nutrient density with agroeconomically acceptable characteristics). However, the accessibility of pro-VA carotenoids for absorption in different cultivars of cassava remains unknown. Here, we used the coupled in vitro digestion/Caco-2 cell uptake model to screen the relative accessibility of beta-carotene (betaC) in 10 cultivars of cassava with varying concentrations of betaC. After cooking (boiled for 30 min), the betaC concentration in tubers from different cultivars ranged from less than detectable to 6.9 microg betaC/g cassava. Samples were subjected to simulated oral, gastric, and small intestinal digestion to determine stability and micellarization of betaC. All-trans betaC, 9-cis betaC, and 13-cis betaC were the most abundant carotenoids in cooked cassava and recoveries after digestion exceeded 70%. Efficiency of micellarization of total betaC was 30 +/- 2% for various cultivars with no significant difference in isomers and linearly proportional to concentration in cooked cassava (r = 0.87; P < 0.001). Accumulation of all-trans betaC by Caco-2 cells incubated with the diluted micelle fraction for 4 h was proportional (R(2) = 0.99; P < 0.001) to the quantity present in micelles. These results suggest that all-trans betaC content appears to provide the key selection marker for breeding cassava to improve VA status and that the more complicated screening procedure using in vitro digestion coupled to cell uptake does not provide additional information on potential bioavailability.     

用体外法研究锰对脂蛋白脂酶活性的影响

目的:使用纯化的脂蛋白脂酶,用体外法研究锰对脂蛋白脂酶活性的影响。方法:采用3×4双因子安排的完全随机设计,4种锰源为无机硫酸锰(MnSO4.H2O)、氨基酸锰A(Mn-AA A)和氨基酸锰B(Mn AAB),3个锰添加水平为0、0.02、0.04和0.08μg/ml,在tris培养液中分别加入以上浓度的锰和纯化的脂蛋白脂酶(LPL),共组成10个处理组。结果表明:锰源、锰水平及锰源与锰水平互作对LPL活性均无显著影响(P>0.58)。结论:锰在体外培养条件下不能直接抑制纯化的LPL活性。     

Effects of in vitro digestion and storage on the phenolic content and antioxidant capacity of a red grape pomace

Red grape pomace (RGP) is a major winery by-product with interesting applications due to its high phenolic content and antioxidant capacity. Effects of in vitro gastrointestinal digestion and storage on the phenolic content and antioxidant capacity of RGP were studied. RGP polyphenols were stable under stomach-mimicking conditions and more sensitive to small intestine conditions, reducing anthocyanins and flavonols. After 3- and 6-month storage, at either 4 or 25?°C, there were no changes in the total phenolic and condensed tannin content, or antioxidant capacity (evaluated by ABTS, FRAP, ORAC assays); however, after 9 months these parameters decreased. Contrarily, chromatic b* values were higher, thus the samples had more intense red color, which may be related to the increased condensed tannin content. Storage time or temperature induced no changes in microbiological load. RGP preserves high antioxidant capacity after storage and in vitro digestion and thus presents potential as a functional ingredient or nutraceutical.     

Effect of a low molecular weight,high-purity β-glucan on in vitro digestion and glycemic response

β-Glucans are believed to lower postprandial glycemia due to their ability to increase viscosity and slow down gastric emptying. The effect of high-purity barley β-glucan (Glucagel?) was tested on in vitro starch digestibility and glycemic response of chapattis. In a randomized controlled crossover trial, 10 healthy human subjects consumed chapattis containing 0, 4 and 8% β-glucan on different occasions. Capillary blood samples were collected before and at 0, 15, 30, 45, 60, 90 and 120 min after consuming the chapattis. There was no significant difference either in the amount of glucose released after in vitro digestion or in the glycemic response to chapattis with 0, 4 and 8% β-glucan (P>0.05). It may be concluded that low molecular weight barley β-glucan, although of 75% purity, was not effective in lowering glycemic response possibly due to its inability to influence starch digestion and particle breakdown during in vitro digestion.