Iron Bioavailability from Common Raisin-containing Foods Assessed with an In Vitro Digestion/Caco-2 Cell Culture Model: Effects of Raisins

The effects of raisins on iron bioavailability from wheat bran cereal, bread, rice pudding, and granola bars were studied. Iron bioavailability was assessed with an in vitro digestion/Caco‐2 cell culture model. Raisins reduced iron bioavailability from all foods except granola bars. Raisins also reduced iron bioavailability from samples of wheat bran cereal and bread fortified with elemental iron or ferrous sulfate, but this inhibitory effect was less pronounced in samples fortified with sodium iron ethylenediaminetetraacetate (NaFeEDTA). Iron bioavailability was markedly higher for samples fortified with NaFeEDTA, suggesting that iron in the form of NaFeEDTA is more bioavailable than elemental iron or ferrous sulfate in raisin‐containing foods.     

体外消化/Caco-2细胞模型分析发酵对面粉铁生物利用率的影响

中国居民膳食以谷物为主食,谷物中铁吸收率低是造成营养性贫血的主要原因,适当的加工方式可缓解谷物中植酸、多酚等物质对铁生物利用率(Fe bioavailability,FeBV)的影响。为考察酵母发酵对面粉FeBV的影响,采用体外消化/Caco-2细胞模型。结果表明,面团在发酵后pH值呈下降趋势,酸度呈上升趋势;多酚、植酸含量降低,植酸酶活性升高,以上各指标在发酵前后差异均具有统计学意义(P0.05)。发酵后面粉样品的FeBV增加约5%~38%,大多数面粉FeBV发酵前后比较,差异具有统计学意义(P0.05)。结论:发酵可降低面粉的pH值,增加酸度,有利于面粉多酚和植酸的降解以及植酸酶活力的升高,可有效提高面粉FeBV。     

体外消化/Caco-2 细胞模型评价面粉锌生物利用率

采用体外消化/Caco-2 细胞模型对10 种小麦粉进行锌生物利用率比较。以锌吸收率(Caco-2 细胞中的锌含量占加入到细胞的面粉消化液总锌量比例)反映锌生物利用率(Zn bioavailability,Zn BV)。发现不同面粉间锌生物利用率差异具有统计学意义,出粉率78% 的安阳中优9507 是面粉锌吸收率和可吸收锌量都高的品种,它将进入下一步人体实验进行营养功效的验证。     

In Vitro Digestion/Caco-2 Cell Culture Model to Determine Optimal Ascorbic Acid to Fe Ratio in Rice Cereal

The objectives were to determine if adding ascorbic acid (AA) to rice cereal enhanced iron availability, and if so, the best ratio of AA to Fe to maximize Fe availability. Also, we sought to determine whether mixing rice cereal with apple juice fortified with AA, compared to mixing it with water, would increase Fe availability. An in vitro digestion/cell culture model was used. Iron availability was increased at an AA:Fe molar ratio of 0.8:1 and maximal at 1.6:1. Mixing apple juice with rice cereal to approximate an AA:Fe ratio of 1.2:1 did not enhance Fe availability. Results indicate that factors in the apple juice offset any enhancement effects of AA on Fe availability.     

基于体外模拟消化/Caco-2细胞模型测定大米中铅的生物有效性

为考察铅污染大米对人体的潜在健康风险,研究建立体外模拟消化/Caco-2细胞模型来测定大米中铅的生物有效性。结果表明：在模拟大米中铅的胃肠消化过程中,生米和熟米在胃消化阶段铅的生物利用率为61.34%～70.59%和39.69%～47.48%,肠消化阶段分别为24.39%～41.79%和13.57%～15.13%,经胃肠消化后籼米中铅的生物利用率均高于粳米,生米经胃、肠消化生物有效性程度均高于蒸煮后的大米。建立并利用成熟Caco-2细胞模型对大米中铅的生物有效性进行测定,使用四乙基铅与无机铅对大米加标,生米的生物有效性为3.32%和7.0%,熟米的生物有效性为2.65%和5.7%。体外模拟消化/Caco-2细胞模型是一种评价大米中铅生物有效性的有效手段。     

Comparison of the Availability of Various Iron Fortificants in Bread and Milk Using an In Vitro Digestion/Caco-2 Cell Culture Method

ABSTRACT: The availabilities and dialyzabilities of various iron fortificants in bread and milk were compared using an in vitro digestion/Caco-2 cell culture model. In white bread, availability and dialyzability of electrolytic iron were lower than that of ferrous fumarate, Ferrochel®, and FeSO₄. NaFe(III)EDTA was also lower in availability than ferrous fumarate, Ferrochel®, and FeSO₄ but had the highest dialyzability. In 2% fat milk, NaFe(III)EDTA was again the highest in dialyzability but was similar in availability to ferrous fumarate, encapsulated ferrous fumarate, Ferrochel®, and FeSO₄. The results suggest that iron from NaFe(III)EDTA and electrolytic iron does not completely exchange with intrinsic iron in foods.     

Bioavailability of iron and zinc from multiple micronutrient fortified beverage premixes in Caco-2 cell model

Abstract: Iron and zinc deficiencies are the most prevalent nutrient deficiencies worldwide. They often coexist as the dietary factors, especially phytate, which impairs iron absorption also affects zinc absorption. Therefore, suitable strategies are required to control multiple micronutrient deficiencies in populations that subsist on high‐phytate foods such as the whole wheat flour based Indian bread (chapatti). The objective of the study, therefore, was to test the bioavailability of iron and zinc in 2 multiple micronutrient beverage premixes in the absence and presence of chapatti. The premix‐1 contained iron, zinc, and vitamin A while premix‐2 contained all micronutrients in premix‐1, plus folic acid and ascorbic acid. Ferritin induction and ⁶⁵Zn uptake were assessed using coupled in vitro digestion/Caco‐2 cell line model as the surrogate markers of iron and zinc bioavailability, respectively. The results show that iron bioavailability from premixes‐1 and 2 was similar in the absence of chapatti. However, premix‐2 showed significantly higher iron bioavailability compared to premix‐1 in the presence of chapatti. In contrast, the zinc uptake was similar from both premixes‐1 and 2 in the absence or presence of chapatti. These results suggest that both the premixes provide bioavailable minerals, but premix‐2 appears to be promising in the presence of foods that have high phytate.     

Iron bioavailability in iron-fortified cereal foods: The contribution of in vitro studies

Iron deficiency anemia is the most common nutritional deficiency in humans. Not all dietary ingested iron, heme or nonheme, will be available to absorption and negative imbalance between iron requirements and absorption leads to iron deficiency and/or anemia. The recommended iron values usually are based on the genetic and on diet iron-bioavailability, which can be considered as the principal factor that change among the cultures and influences the distinct levels of recommendation among countries. Dietary changes present practical limitations due to be difficult to change food habits. The iron food fortification is considered more cost effective and economically more attractive than iron supplementation. There are many iron compounds available to be used in iron fortification. Cereals represent a target food group to iron fortification programs due to high consumption and the in vitro studies can be useful to estimate the relative iron bioavailability in large number of products in short time and with a low cost. Wheat flour baked into bread or not was the main product tested in in vitro bioavailability studies and ferrous sulfate was the principal iron compound used in the fortification studies. However, iron bioavailability from ferrous sulfate is lower than from other compounds, such FeNaEDTA or ferric pyrophosphate. The variables level of fortification, storage, level of extraction, baking and also the association or not with other chemical compound seems to influence the results obtained.     

Purified Glycosaminoglycans from Cooked Haddock May Enhance Fe Uptake Via Endocytosis in a Caco-2 Cell Culture Model

ABSTRACT:  This study aims to understand the enhancing effect of glycosaminoglycans (GAGs), such as chondroitin/dermatan structures, on Fe uptake to Caco-2 cells. High-sulfated GAGs were selectively purified from cooked haddock. An  in vitro  digestion/Caco-2 cell culture model was used to evaluate Fe uptake (cell ferritin formation) from a Fe⁺³-containing solution, and Fe⁺³/ascorbic acid (AA) and Fe⁺³/GAGs mixtures. Mitochondria (MTT test) and endosomal/lysosomal activities (neutral red uptake, NR), intracellular accumulation of reactive oxygen species, and GSH concentration were monitored as biomarkers of the changes of cellular metabolism. Changes in mRNA expression of Fe transporters, divalent metal transporter-1 (DMT1), and duodenal cytochrome-b (DcytB) were also evaluated. The Fe uptake from Fe⁺³/GAGs mixture was up to 1.8-fold higher than from Fe⁺³ alone. Both Fe⁺³ alone and Fe⁺³/AA mixture produced highest increase in MTT conversion. In contrast, cell cultures exposed to the Fe⁺³/GAGs mixture exhibited highest NR uptake values. All Fe-containing solutions tested caused a sharp intramitochondrial accumulation of reactive oxygen species. Cell cultures exposed to the Fe⁺³/GAGs mixture exhibited a more preserved (by 8%) intracellular GSH concentration compared to cultures exposed to Fe⁺³ or Fe⁺³/AA mixture. In addition to cell responses, the mRNA expression of Fe transporters may suggest that Fe could also be internalized into cells by endocytosis in addition to via DMT1 in Fe⁺³/GAGs mixtures. These aspects need to be confirmed in  in vivo  experiments to better establish nutritional interventional strategies.     

鳕鱼皮胶原蛋白肽在Caco-2细胞单层模型中的吸收机制

目的：鳕鱼皮胶原蛋白肽（cod skin collagen peptide,CSCP）对肝损伤具有较好的保护作用,但其吸收机制尚不明确,本实验拟采用人结肠腺癌Caco-2细胞单层模型对CSCP在肠道中的吸收机制进行研究,为CSCP在动物肠道内的吸收机制提供依据。方法：在进行转运实验前,先对CSCP在人工胃、肠液、不同pH值条件及在Caco-2细胞单层中的稳定性进行评价;采用CCK-8（cell counting kit-8）法确定CSCP在转运实验中的最高质量浓度,然后建立Caco-2细胞单层模型并测定跨膜电阻值和碱性磷酸酶活力以检验细胞单层的致密性、完整性和极化现象;考察转运时间、CSCP质量浓度、维拉帕米、MK-571、氧化苯砷和去氧胆酸钠对转运的影响,利用高效液相色谱法检测CSCP质量浓度并计算累积转运量和表观渗透系数。结果：在3 h内,CSCP在人工胃、肠液及接近胃肠道pH值环境中基本保持稳定,转运过程中未见多肽发生水解。CSCP的转运具有时间和浓度依赖性,不受维拉帕米和氧化苯砷的影响,去氧胆酸钠和MK-571对CSCP的转运具有非常显著的促进作用（P＜0.05）。结论：CSCP跨Caco-2细胞单层转运的机制为细胞旁路途径,其外排受到多药耐药蛋白的介导。     

Flavonoids from a grape seed extract interact with digestive secretions and intestinal cells as assessed in an in vitro digestion/Caco-2 cell culture model

Although the bioactivity of flavonoids is related to their bioavailability, little is known about pre-absorption events in the gastrointestinal tract and their possible interactions with digestive constituents and intestinal cells. Using an in vitro digestion/Caco-2 cell culture model, we investigated the effect of digestive secretions on the stability of (+)-catechin (CAT), (−)-epicatechin (EC) and B2 and B3 dimers from a procyanidin-rich grape seed extract (GSE). The availability of phenolic compounds was not affected by salivary and gastric incubations but decreased during intestinal digestion in the absence of Caco-2 cells due to interactions with pancreatic proteins, unmasked by acetonitrile extraction. Then, in the presence of cells, about 43.9% of CAT, 85.3% of EC and all dimers disappeared at the end of 2 h of intestinal incubation. The stability of all compounds at intestinal pH was demonstrated as well as interactions with proteins, associated with a decrease of some cells enzyme activities, e.g., alkaline phosphatase (−79.8%), sucrase-isomaltase (−60.9%) and aminopeptidase N (−60.7%). Moreover, no compounds were detected in the basal compartment of transwells or in cell monolayers.     

金针菇蛋白聚糖对脂多糖诱导的Caco-2/RAW264.7细胞共培养模型炎症的抑制作用

随着生活节奏的加快和生活方式的改变,炎症性肠病的发病率不断增加,从饮食中寻找抑制炎症成分是防控炎性肠病的有效途径。本实验以金针菇蛋白聚糖（Flarnmulina velutipes proteoglycans,FPG）1-1为原料,建立脂多糖（lipopolysaccharide,LPS）诱导的人结肠腺癌细胞（Caco-2）、巨噬细胞（RAW264.7）共培养炎症模型,测定FPG1-1对一氧化氮（nitrite oxide,NO）、活性氧（reactive oxygen species,ROS）及细胞因子的影响,以分析其对炎症的作用。结果显示：与模型组相比,质量浓度为200 μg/mL FPG1-1可以极显著降低RAW264.7 NO、ROS的生成（P＜0.01）,同时可以上调白细胞介素（interleukin,IL）-10、下调肿瘤坏死因子（tumor necrosis factor,TNF-α）、IL-1β、IL-6的分泌。以上结果表明,FPG1-1能够有效抑制LPS诱导的Caco-2/RAW264.7共培养模型炎症反应。本研究结果可为金针菇功能成分利用和抑制炎性肠病功能食品开发提供理论依据。     

Digestion stability and evaluation of the metabolism and transport of olive oil phenols in the human small-intestinal epithelial Caco-2/TC7 cell line

The aims of this study were to investigate (i) the metabolism of olive oil phenolics by intestinal epithelial cells and (ii) their transport across epithelial cell monolayers. The various conjugates and derivatives produced by the intestinal epithelial cells were identified following separation by ultra-performance liquid chromatography (UPLC), using a combination of UV/visible spectra, mass spectrometry and specific enzyme treatments (β-glucuronidase and aryl-suphatase). Limited metabolism of olive oil phenolics was observed using Caco-2/Tc7 cells as a model of the human intestinal epithelium, and the methyslated conjugates were the major metabolites detected. The results of the transport rate data for phenols and their metabolites to the apical, cellular, and basolateral compartments after apical loading of the phenol at 100 μM showed a time-dependent efflux of various free and conjugated forms of phenols.     

Effect of Protein Fermentation Products on Gut Health Assessed in an In Vitro Model of Human Colon (TIM-2)

Scope

Western type of diets are characterized by high animal protein intake and are associated with various chronic inflammatory diseases. With a higher protein consumption, excess undigested protein will reach the colon and be subsequently metabolized by gut microbiota. Depending on the type of protein, fermentation in the colon generates different metabolites with varying biological effects. This study aims to compare the impact of protein fermentation products from different sources on gut health.

Methods and results

Three high protein diets (vital wheat gluten [VWG], lentil, or casein) are submitted to the in vitro model of colon. Fermentation of excess lentil protein for 72 h results in highest production of short-chain fatty acids and lowest production of branched-chain fatty acids. Exposure of Caco-2 monolayers or Caco-2 monolayers co-cultured with THP-1 macrophages to luminal extracts of fermented lentil protein results in less cytotoxicity of Caco-2 monolayers and less damage to barrier integrity, when compared to VWG and casein. Lowest induction of interleukin-6 is observed in THP-1 macrophages after treatment with lentil luminal extracts, which is identified to be regulated by aryl hydrocarbon receptor signaling.

Conclusion

The findings indicate that protein sources affect the health effects of high protein diet in the gut.     

Anti-inflammatory effect and modulation of cytochrome P450 activities by Artemisia annua tea infusions in human intestinal Caco-2 cells

In an attempt to understand the beneficial health effects of Artemisia annua other than its anti-malaria properties, extracts from different cultivars prepared as tea infusions were investigated using Caco-2 cells on the intestinal inflammation and cytochrome P450 (CYP) activities. The characterisation of their phenolic compound (PC) profile revealed rosmarinic and chlorogenic acids as the main PCs. The extracts, assayed on Caco-2 cells at a plausible intestinal concentration, significantly decreased the secretion of pro-inflammatory cytokines, IL-8 and IL-6. This effect could be attributable at least to their content in rosmarinic acid, detected as a potent anti-inflammatory compound. The extracts also inhibited the activity of CYP3A4, whose expression was induced by 1,25-dihydroxyvitamin D₃, and of CYP1A1, induced by benzo(a)pyrene. Our results highlight the advantage of drinking A. annua infusions for their potent anti-inflammatory effect, linked to PC content, which could synergise their antimalarial activity.     

食用酸枣仁多糖的小鼠肠道菌群培养上清液对Caco-2细胞吸收相关蛋白表达的影响

为了探究食用酸枣仁多糖(ZSSP)的小鼠肠道菌群培养上清液是否会调节Caco-2细胞中外排转运蛋白、紧密连接蛋白和Ⅱ相代谢酶的表达水平。将20只C57BL/6小鼠随机分为对照组和ZSSP组,分别灌胃超纯水和酸枣仁多糖水溶液(100 mg/kg·d^-1)14 d,收集小鼠粪便制备肠道菌群培养上清液。采用噻唑蓝(methyl thiazolyl tetrazolium,MTT)法检测肠道菌群培养上清液对Caco-2细胞活力的影响,采用免疫印迹(western blot,WB)法检测Caco-2细胞中P-糖蛋白(P-glycoprotein,P-gp)、多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)、咬合蛋白(Occludin)和闭合蛋白1(Claudin-1)的表达水平,用酶联免疫检测(enzyme linked immunosorbent assay,ELISA)试剂盒测定磺酸基转移酶(sulfotransferase,SULT)和尿苷二磷酸葡萄糖醛酸基转移酶(UDP-glucuronosyltransferase,UGT)的含量。结果表明,ZSSP组的肠道菌群培养上清液能显著降低Caco-2细胞中外排转运蛋白(P-gp和MRP2)的表达水平(P<0.05),显著升高UGT的表达水平(P<0.05),但对紧密连接蛋白(Occludin和Claudin-1)和SULT没有显著影响(P>0.05)。由此可知,食用酸枣仁多糖的小鼠肠道菌群培养上清液能调节Caco-2细胞中的外排转运蛋白和Ⅱ相代谢酶的表达水平。