Chapter

Interaction of Polyphenols with the Intestinal and Placental Absorption of some Nutrients and other Compounds

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Abstract

In this chapter, we review the existing data concerning the effect of polyphenols on the transport of some bioactive compounds (glucose, thiamine, folate and organic cations) at the intestinal and placental epithelial barriers. Important conclusions can be drawn. Firstly, different classes of polyphenols affect transport of these compounds at the intestine and placenta. Secondly, different compounds belonging to the same phenolic class often possess opposite effects upon transport of a given molecule. Thirdly, short- and long-term exposure to a given polyphenol does not produce parallel results. Fourthly, combination of distinct polyphenols, as happens in natural foods, may cause very distinct effects from the expected ones based on the effect of each of these compounds alone. Finally, ethanol is able to modify the effect of polyphenols. In summary, the studies reviewed here raise a concern about possible changes in the bioavailability of these substrates upon concomitant ingestion of polyphenols.

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... However, due to their low bioavailability, there is controversy about their mechanism of action, and direct in vivo ROS-scavenging activity is questionable [1,2]. An indirect antioxidant effect of polyphenols has been recently proposed, through the upregulation of the cellular antioxidant defense system (phase 2 enzymes), via activation of the Nrf2-Keap1 pathway [3][4][5][6]. Polyphenols may be autooxidized with the formation of H 2 O 2 and quinoidal products and both chemical species may participate in electrophilic conjugation reactions with cysteine residues of the Nrf2-Keap1 complex, allowing the translocation of Nrf2 and thus starting the pathway [6][7][8]. In this scenario, autooxidation products of polyphenols are actually mild prooxidants. ...
... An indirect antioxidant effect of polyphenols has been recently proposed, through the upregulation of the cellular antioxidant defense system (phase 2 enzymes), via activation of the Nrf2-Keap1 pathway [3][4][5][6]. Polyphenols may be autooxidized with the formation of H 2 O 2 and quinoidal products and both chemical species may participate in electrophilic conjugation reactions with cysteine residues of the Nrf2-Keap1 complex, allowing the translocation of Nrf2 and thus starting the pathway [6][7][8]. In this scenario, autooxidation products of polyphenols are actually mild prooxidants. ...
... In this scenario, autooxidation products of polyphenols are actually mild prooxidants. Alternatively, polyphenols may activate the Nrf2 pathway by mechanisms apparently independent of their electrophilic properties [6][7][8]. ...
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The protective effect of different polyphenols, catechin (Cat), quercetin (Qc) (flavonoids), gallic acid (GA), caffeic acid (CfA), chlorogenic acid (ChA) (phenolic acids), and capsaicin (Cap), against H 2 O 2 -induced oxidative stress was evaluated in rat enterocytes using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy and Fourier Transform Infrared Microspectroscopy (FTIRM), and results were compared to standard lipid peroxidation techniques: conjugated dienes (CD) and Thiobarbituric Acid Reactive Substances (TBARS). Analysis of ATR-FTIR and FTIRM spectral data allowed the simultaneous evaluation of the effects of H 2 O 2 and polyphenols on lipid and protein oxidation. All polyphenols showed a protective effect against H 2 O 2 -induced oxidative stress in enterocytes, when administered before or after H 2 O 2 . Cat and capsaicin showed the highest protective effect, while phenolic acids had weaker effects and Qc presented a mild prooxidative effect (IR spectral profile of biomolecules between control and H 2 O 2 -treated cells) according to FTIR analyses. These results demonstrated the viability to use infrared spectroscopy to evaluate the oxidant and antioxidant effect of molecules in cell systems assays.
... Quercetin is a commonly ingested flavonoid and it has been extensively investigated. This compound was previously shown to reduce glucose uptake by intestinal epithelial cells, by interfering with GLUT2-mediated, but not SGLT1-mediated, glucose transport (Ader, Block, Pietzsch, & Wolffram, 2001;Calhau, Faria, Keating, & Martel, 2014;Cermak, Landgraf, & Wolffram, 2004;Chen, Hsu, Huang, & Lin, 2007;Johnston et al., 2005;Kwon et al., 2007;Song et al., 2002). Although an effective GLUT2 inhibitor, quercetin was shown not to be a GLUT2 substrate (Kwon et al., 2007;Song et al., 2002). ...
... This result also supports the notion that combination of distinct polyphenols, as happens in natural foods, may give very distinct effects from the expected ones taking into account the effect of each of the polyphenols alone. Thus, care should be taken when speculating on the effect of a given food based on the effect of one component only (Martel et al., 2010;Calhau et al., 2014). Moreover, care should also be taken when speculating on the effect of combination of different foods, because synergistic, additive, or antagonistic interactions among food components may also exist, as demonstrated in relation to total antioxidant capacity (Wang, Meckling, Marcone, Kakuda, & Tsao, 2011). ...
... Quercetin is a commonly ingested flavonoid and it has been extensively investigated. This compound was previously shown to reduce glucose uptake by intestinal epithelial cells, by interfering with GLUT2-mediated, but not SGLT1-mediated, glucose transport (Ader, Block, Pietzsch, & Wolffram, 2001;Calhau, Faria, Keating, & Martel, 2014;Cermak, Landgraf, & Wolffram, 2004;Chen, Hsu, Huang, & Lin, 2007;Johnston et al., 2005;Kwon et al., 2007;Song et al., 2002). Although an effective GLUT2 inhibitor, quercetin was shown not to be a GLUT2 substrate (Kwon et al., 2007;Song et al., 2002). ...
... This result also supports the notion that combination of distinct polyphenols, as happens in natural foods, may give very distinct effects from the expected ones taking into account the effect of each of the polyphenols alone. Thus, care should be taken when speculating on the effect of a given food based on the effect of one component only (Martel et al., 2010;Calhau et al., 2014). Moreover, care should also be taken when speculating on the effect of combination of different foods, because synergistic, additive, or antagonistic interactions among food components may also exist, as demonstrated in relation to total antioxidant capacity (Wang, Meckling, Marcone, Kakuda, & Tsao, 2011). ...
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... PPO is an important enzyme that has numerous health-promoting properties including antiviral, anticarcinogen, antimicrobial, and antimutagenic agents [37][38][39] and can be used in several industrial applications [40,41]. However, this enzyme is also known to be responsible for the enzymatic browning of fruits [42]. ...
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... Free thiamine is converted to TPP by the action of TPK1 in the cytosol and dephosphorylation of TPP to TMP and thiamine by ACPP. At higher concentrations of thiamine, simple passive diffusion takes place [see (6) and references therein]. From blood, thiamine is taken up by either transporter (THTR1 or THTR2). ...
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... [10] Glucose homeostasis is essential to human health and any deficiency in its maintenance can increase the risk of T2D, cardiovascular events and cause a higher mortality. [11] Various evidences indicate that polyphenols can influence glucose homeostasis [12] , apparently playing a protective role in relation to T2D. [13] In fact, polyphenol-rich plant-based foods such as tea, coffee [14] and some herbal supplements [15] were found, in epidemiologic studies, to reduce the risk of T2D development. Several mechanisms can explain T2D risk reduction by dietary polyphenols: improvement of glycemic responses by inhibiting digestion or intestinal absorption of glucose, improved fasting blood glucose levels, enhanced insulin secretion and improved insulin sensitivity, mediated by stimulation of insulin secretion from pancreatic β-cells, modification of hepatic glucose release and activation of insulin receptors and glucose uptake. ...
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... The lowest CD36 expression was again shown by CEW showing 8.85 fold decrease followed by CEJ showing 6.7 fold decrease compared to control thus explaining their high foam cell preventing abilities. In cell line assays, the higher activities of wines over their respective juices could be explained by the fact that alcohols increase the cellular permeability of wines and therefore help in carrying the important nutrients into the cells thus increasing the bioavailability of important nutrients (phenolics, flavonoids, organic acids) in the wines (Conceicao et al. 2013). ...
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Proanthocyanidins (syn condensed tannins) are complex flavonoid polymers naturally present in cereals, legume seeds and particularly abundant in some fruits and fruit juices. They share some common structural features—phenolic nature and high molecular weight—with phenolic polymers found in black tea and red wine (called here tannin‐like compounds). The polymeric nature of proanthocyanidins makes their analysis and estimation in food difficult. For this reason, little is known about their consumption, although they likely contribute a large part of the daily polyphenol intake. They also share common physicochemical properties: they form stable complexes with metal ions and with proteins and are, like other polyphenols, good reducing agents. Many of their biological effects of nutritional interest derive from these properties. As metal ion chelators, they influence the bioavailability of several minerals. The nutritional significance of the non‐specific complexation of proteins is less clear. As reducing agents, they may participate in the prevention of cancers, both of the digestive tract and inner organs. They may also protect LDLs against oxidation and inhibit platelet aggregation and therefore prevent cardiovascular diseases. In vitro , animal and human studies on the prevention of these chronic diseases are reviewed with particular attention to wine and tea polyphenols. The lack of data on their bioavailability and the paucity of human studies are emphasised. © 2000 Society of Chemical Industry
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Proanthocyanidins (syn condensed tannins) are complex flavonoid polymers naturally present in cereals, legume seeds and particularly abundant in some fruits and fruit juices. They share some common structural features—phenolic nature and high molecular weight—with phenolic polymers found in black tea and red wine (called here tannin-like compounds). The polymeric nature of proanthocyanidins makes their analysis and estimation in food difficult. For this reason, little is known about their consumption, although they likely contribute a large part of the daily polyphenol intake. They also share common physicochemical properties: they form stable complexes with metal ions and with proteins and are, like other polyphenols, good reducing agents. Many of their biological effects of nutritional interest derive from these properties. As metal ion chelators, they influence the bioavailability of several minerals. The nutritional significance of the non-specific complexation of proteins is less clear. As reducing agents, they may participate in the prevention of cancers, both of the digestive tract and inner organs. They may also protect LDLs against oxidation and inhibit platelet aggregation and therefore prevent cardiovascular diseases. In vitro, animal and human studies on the prevention of these chronic diseases are reviewed with particular attention to wine and tea polyphenols. The lack of data on their bioavailability and the paucity of human studies are emphasised.© 2000 Society of Chemical Industry
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A great many epidemiological studies indicate that a diet rich in flavonoids from vegetables and fruits intake appear to be inversely related to coronary heart disease (CHD) and cancers mortality. Regular moderate consumption of wine can contribute to this phenomenon. Flavonoids in wine and food have been shown to be antioxidant and anti-aggregant in vitro and could indeed help protect against coronary disease. Thus, flavonoids may partly explain the protective effects of the Mediterranean diet, rich in vegetable, fruit and wine against CHD or cancers.
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The aim of the present study was to investigate the influence of an anthocyanin extract (extract I), and two other derivative extracts (extracts II and III), which are being developed aiming to be further applied in the food industry, on intestinal uptake of organic cations. For this purpose, the effect of these compounds on 3H-MPP+ uptake was evaluated in Caco-2 cells (an enterocyte-like cell line derived from a human colonic adenocarcinoma).Extracts I and III did not present any effect on 3H-MPP+ uptake. Extract II, an anthocyanin–pyruvic acid adduct extract, decreased organic cation uptake. Some phenolic molecules were also tested, aiming to study possible structural features responsible for these effects. The results are compatible with the hypothesis of phenolic molecules bearing a carboxylic group with extended electronic conjugation being the responsible feature for the inhibitory effect on 3H-MPP+ uptake.The use of these compounds in foodstuffs must be carefully assessed since interactions with organic cations present in the diet, as nutrients or xenobiotics, may affect their absorption and bioavailability. Type and complexity of the ingested compounds are important factors in this regard, and should be taken into consideration.
Article
The effect of feeding quercetin at the level of 1 g per kilogram of diet was studied in rats with streptozotocin-induced diabetes. The study involved a comparison between a starch-fed diabetic group and quercetin-fed diabetic group. The rats were examined for diet intake, water intake, gain in body weight, urine output, urine sugar, and fasting blood sugar. Diabetic status in rats was found to be ameliorated about 25% in rats fed with quercetin.
Article
The aim of this study was to characterize the intestinal absorption of thiamine, by investigating the hypothesis of an involvement of Organic Cation Transporter (OCT) family members in this process. [(3)H]-T(+) uptake was found to be: 1) time-dependent, 2) Na(+)- and Cl(-)-dependent, 3) pH-dependent, with uptake increasing with a decrease in extracellular pH and decreasing with a decrease in intracellular pH, 4) inhibited by amiloride, 5) inhibited by the thiamine structural analogues oxythiamine and amprolium, 6) inhibited by the unrelated organic cations MPP(+), clonidine, dopamine, serotonin, 7) inhibited by the OCT inhibitors decynium22 and progesterone. Moreover, the dependence of [(3)H]-T(+) uptake on phosphorylation/dephosphorylation mechanisms was also investigated and [(3)H]-T(+) uptake was found to be reduced by PKA activation and protein tyrosine phosphatase and alkaline phosphatase inhibition. In conclusion, our results are compatible with the possibility of thiamine being transported not only by ThTr1 and/or ThTr2, but also by members of the OCT family of transporters (most probably OCT1 and/or OCT3), thus sharing the same transporters with several other organic cations at the small intestinal level.
Article
Appropriate foetal growth and development is dependent on adequate placental glucose uptake. Oxidative stress regulates glucose uptake in various tissues. The effect of oxidative stress on placental glucose transport is not known. Thus, the aim of this study was to determine the effect of oxidative stress on glucose uptake and glucose transporters (GLUTs) in human placenta. Human placenta was incubated in the absence or presence of 0.5 mM hypoxanthine+15 mU/ml xanthine oxidase (HX/XO) for 24 h. Gene and protein expressions of the GLUTs were analysed by quantitative RT-PCR and western blotting respectively. Glucose uptake was measured using radiolabelled ((14)C) glucose. HX/XO significantly decreased GLUT1 gene and protein expression and resultant glucose uptake. There was no effect of the antioxidants N-acetylcysteine, catalase and superoxide dismutase or the NF-κB inhibitor BAY 11-0782 on HX/XO-induced decrease in glucose uptake. However, HX/XO treatment significantly decreased both gene and protein expression of SIRT1. In the presence of the SIRT1 activator resveratrol, the decrease in GLUT1 expression and glucose uptake mediated by HX/XO was abolished. Collectively, the data presented here demonstrate that oxidative stress reduces placental glucose uptake and GLUT1 expression by a SIRT1-dependent mechanism.
Article
To examine in mice the acute effects of epigallocatechin gallate (EGCG), a green tea bioactive polyphenol on substrate metabolism with focus on the fate of dietary lipids. Male C57BL/6 mice were fed high-fat diets supplemented with EGCG extracted from green tea (TEAVIGO, DSM Nutritional Products Ltd, Basel, Switzerland) at different dosages up to 1% (w/w). Effects of EGCG on body composition (quantitative magnetic resonance), food intake and digestibility, oxidation and incorporation of exogenous lipids (stable isotope techniques: (13)C-labeled palmitate and diet supplemented with corn oil as a natural source of (13)C-enriched lipids) as well as gene expression (quantitative real-time PCR) in liver and intestinal mucosa were investigated. Short-term supplementation (4-7 days) of dietary EGCG increased energy excretion, while food and energy intake were not affected. Fecal energy loss was accompanied by increased fat and nitrogen excretion. EGCG decreased post-prandial triglyceride and glycogen content in liver, increased oxidation of dietary lipids and decreased incorporation of dietary 13C-enriched lipids into fat tissues, liver and skeletal muscle. EGCG dose dependently reversed high-fat diet-induced effects on intestinal substrate transporters (CD36, FATP4 and SGLT1) and downregulated lipogenesis-related genes (ACC, FAS and SCD1) in liver in the post-prandial state. Anti-obesity effects of EGCG can be explained by a decreased food digestibility affecting substrate metabolism of intestinal mucosa and liver, leading to increased post-prandial fat oxidation and reduced incorporation of dietary lipids into tissues.
Article
The effect of polyphenols, phenolic acids and tannins (PPTs) from strawberry and apple on uptake and apical to basolateral transport of glucose was investigated using Caco-2 intestinal cell monolayers. Substantial inhibition on both uptake and transport was observed by extracts from both strawberry and apple. Using sodium-containing (glucose transporters SGLT1 and GLUT2 both active) and sodium-free (only GLUT2 active) conditions, we show that the inhibition of GLUT2 was greater than that of SGLT1. The extracts were analyzed and some of the constituent PPTs were also tested. Quercetin-3-O-rhamnoside (IC₅₀ =31 μM), phloridzin (IC₅₀=146 μM), and 5-caffeoylquinic acid (IC₅₀=2570 μM) contributed 26, 52 and 12%, respectively, to the inhibitory activity of the apple extract, whereas pelargonidin-3-O-glucoside (IC₅₀=802 μM) contributed 26% to the total inhibition by the strawberry extract. For the strawberry extract, the inhibition of transport was non-competitive based on kinetic analysis, whereas the inhibition of cellular uptake was a mixed-type inhibition, with changes in both V(max) and apparent K(m) . The results in this assay show that some PPTs inhibit glucose transport from the intestinal lumen into cells and also the GLUT2-facilitated exit on the basolateral side.
Article
Natural polyphenols are chemically and biologically active. This study aimed at examining the physiological effects of high doses of polyphenol extracts from green tea and new polyphenol-rich sources (chokeberry and honeysuckle fruits) on nutrient absorption. 32 male Wistar rats were divided into four groups and fed a diet supplemented with one of the three polyphenolic extracts (at 0.4%) or a control diet for 4 weeks. A perfusion technique was used to study the effects at intestinal level. Pure polyphenols from the three sources were introduced into perfusion fluid at a concentration of 0.4% and allowed to cross the intestinal tract in 1.5 h. In the perfusion experiment, addition of the extracts caused a strong and statistically significant reduction in absorption of the selected nutrients (water, glucose, cholesterol, amino acids and minerals) compared to control animals. In the nutritional experiment, we recorded a slight decrease in diet utilization and growth in rats on polyphenolic diets relative to control group. In the same experiment, we observed a reduction of Zn and Cu absorption, but this was not accompanied by diminished concentrations in the bone femur. The presence of the polyphenolic extracts in the perfusion liquids significantly reduced absorption from the small intestine, but the nutritional experiment did not confirm deleterious consequences of the consumption of high extract doses.
Article
Quercetin is a food component that may ameliorate the diabetic symptoms. We examined hepatic gene expression of BALB/c mice with streptozotocin (STZ)-induced diabetes to elucidate the mechanism of the protective effect of dietary quercetin on diabetes-associated liver injury. We fed normal and STZ-induced diabetic mice with diets containing quercetin for 2 wk and compared the patterns of hepatic gene expression in these groups of mice using a DNA microarray. Diets containing 0.1 or 0.5% quercetin lowered the STZ-induced increase in blood glucose levels and improved plasma insulin levels. A cluster analysis of the hepatic gene expressions showed that 0.5% quercetin diet suppressed STZ-induced alteration of gene expression. Gene set enrichment analysis (GSEA) and quantitative RT-PCR analysis showed that the quercetin diets had greatest suppressive effect on the STZ-induced elevation of expression of cyclin-dependent kinase inhibitor p21(WAF1/Cip1) (Cdkn1a). Quercetin also suppressed STZ-induced expression of Cdkn1a in the pancreas. Dietary quercetin might improve liver and pancreas functions by enabling the recovery of cell proliferation through the inhibition of Cdkn1a expression. Unexpectedly, in healthy control mice the 0.5 and 1% quercetin diets reduced the expression of ubiquitin C (Ubc), which has heat-shock element (HSE) in the promoter region, in the liver.
Article
Yerba maté (Ilex paraguariensis) is rich in polyphenols, especially chlorogenic acids. Evidence suggests that dietary polyphenols could play a role in glucose absorption and metabolism. The aim of this study was to evaluate the antidiabetic properties of yerba maté extract in alloxan-induced diabetic Wistar rats. Animals (n = 41) were divided in four groups: nondiabetic control (NDC, n = 10), nondiabetic yerba maté (NDY, n = 10), diabetic control (DC, n = 11), and diabetic yerba maté (DY, n = 10). The intervention consisted in the administration of yerba maté extract in a 1 g extract/kg body weight dose for 28 days; controls received saline solution only. There were no significant differences in serum glucose, insulin, and hepatic glucose-6-phosphatase activity between the groups that ingested yerba maté extract (NDY and DY) and the controls (NDC and DC). However, the intestinal SGLT1 gene expression was significantly lower in animals that received yerba maté both in upper (p = 0.007) and middle (p < 0.001) small intestine. These results indicate that bioactive compounds present in yerba maté might be capable of interfering in glucose absorption, by decreasing SGLT1 expression.
Article
Fetal oxygen consumptions ranging from 6 to 9 ml STPxmin-1xkg-1 have been measured in fetuses of different species. Fetuses of small mammals apparently do not have a higher rate of O2 consumption per unit weight than fetuses of large mammals, in contrast to the behavior of the standard metabolic rate in adult animals. In man and larger mammals, the supply of calories needed to sustain fetal growth is small in comparison with that needed to sustain fetal oxygen consumption. In the ovine fetus the principal exogenous substrates of oxidative metabolism are glucose, lactate, and amino acids. The supply of glucose to the fetus decreases during maternal fasting and requires a reorientation of fetal metabolism toward an increased amino acid degradation. There is contradictory evidence in the literature concerning the rate of fetal gluconeogenesis and its regulation. The placenta supplies neutral and basic amino acids to the fetus. In sheep, the acidic amino acids glutamate and aspartate are produced within the fetus and there is excretion of glutamate from fetus to placenta. There is also evidence that in man and other species the placenta does not supply acidic amino acids to the fetus in the amount needed for growth and catabolism. All mammalian fetuses studied thus far are capable of synthesizing lipids but differ markedly in the amount and origin of the fat stored before birth and the permeability of the placenta to fatty acids. Fetuses are also capable of oxidation of FFA and ketoacids but it is not clear whether these substances are an important source of energy in those species whose placenta is permeable to fatty acids. According to indirect evidence, the fetal heart consumes predominantly glucose under aerobic conditions. This is in contrast to the adult heart, for which FFA is the principal substrate. The fetal cerebral oxygen and glucose utilization rates per unit brain weight are comparable with those observed in adult mammals.
Article
We have previously investigated the normal characteristics of thiamine intestinal transport in rats and found that a very low concentrations (0.06 to 2.0 muM) thiamine transport is a saturable, carrier-mediated, active process while at high concentrations (greater than 2.0 muM) transport proceeds by simple diffusion. The present studies were undertaken to characterize the effect of ethanol on thiamine transport. Intact isolated loops were used to measure rates of 35S-thiamine hydrochloride absorption into the circulation in vivo, and everted jejunal segments to measure net transmural flux, unidirectional uptake, and cellular exit of 14C-thiamine hydrochloride in vitro. Intragastric administration of ethanol (50 to 750 mg. per 100 grams of weight) reduced absorption of low thiamine concentration in vivo to 65.44 per cent of control value. A similar inhibition was noted after intravenous ethanol. Once attained, the inhibition of thiamine absorption was not related to the ethanol dose or to ethanol concentration in the blood or in the intestinal lumen; this inhibition was reversible. In contrast, ethanol did not affect absorption of high concentrations of thiamine. These findings were confirmed by the in vitro results. In transmural flux studies, the movement of low, but not high, thiamine concentration against a concentration gradient was inhibited by ethanol, so that the normal serosal/mucosal ratio of 1.5 was reduced to 1.0. Ethanol did not affect unidirectional uptake into the mucosa of either low or high thiamine concentrations, but blocked cellular exit of low thiamine concentrations from the cells into the serosal compartment. Exit of high thiamine concentrations was not affected. Ouabain, like ethanol, markedly reduced cellular exit but did not influence uptake of low thiamine concentrations. The present studies suggest that ethanol adversely affects the active, but not the passive, component of thiamine transport. Moreover, ethanol appears to block thiamine exit from the cells but does not affect cellular uptake of thiamine. The similarity to ouabain action suggests that ethanol may impair active thiamine transport by inhibiting Na-K ATPase activity.
Article
This review deals with cell culture models for studies of drug absorption across the intestinal mucosa. The selection of appropriate cells and cell culture conditions is discussed, guidelines for the characterization of the cell models are presented, and the intestinal barriers to drug absorption are discussed and compared with those in the cell culture models. Finally, recent applications of the cell culture models in drug and peptide absorption and metabolism studies are reviewed.
Article
Rat everted jejunal sacs were incubated for 15 and 30 min at 37 degrees C in oxygenated Krebs-Henseleit buffer, pH 7.4, containing 0.2 microM [3H]-thiamin (3H-T) or [3H]-thiamin monophosphate (3H-TMP) with and without 10 mM 1-phenylalanine (PAL) or 2.5 mM levamisole (LEV). The concentrations of 3H-T and its phosphoesters in sac wall and serosal fluid were determined by a radiometric method after electrophoretic separation. In separate experiments, thiamin pyrophosphokinase (TPKase) and thiamin pyrophosphatase (TPPase) activities were determined in mucosal scrapings, with and without PAL or LEV, by using a radiometric and a colorimetric method, respectively. 3H-TMP was transported partly unchanged by an active mechanism similarly to 3H-T, but less efficiently. During transport, 3H-TMP was also enzymatically transformed to thiamin (T) and thiamin pyrophosphate, which accumulated in the tissue. In the serosal fluid, the concentration of 3H-TMP exceeded that of 3H-T. Presence of PAL or LEV with 3H-T or 3H-TMP in the incubation medium reduced the serosal transport and the tissue content of T compounds. LEV caused a dose-dependent inhibition of TPKase without affecting TPPase, whereas PAL inhibited both activities to about the same extent. These results indicate that the transport of TMP involves a number of different processes similar to those responsible for T transport. The effects of PAL and LEV underline the importance of phosphorylation-dephosphorylation coupling.
Article
The various biochemical mechanisms considered to explain the selective dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are reviewed. MPTP is metabolized by monoamine oxidase in the brain, ultimately yielding 1-methyl-4-phenylpyridinium cation (MPP+), which is accumulated in dopamine cells by the high-affinity dopamine uptake pump. Cell death appears to reflect a compromise in energy production arising as a result of the Nernstian concentration of MPP+ inside mitochondria and persistent inhibition of Site 1 of the respiratory chain. The structural features underlying each biochemical step involved in the expression of neurotoxicity are described, and the implications of the MPTP phenomenon to efforts aimed at elucidating the pathogenesis of idiopathic parkinsonism are discussed.
Article
These studies assessed the role of biotransformation in the rapid elimination of MPP+ from the central nervous system (CNS) compartment of mice. Mice were given either MPP+ via the intracerebroventricular (i.c.v.) route, or MPTP intraperitoneally. The elimination of MPP+ from the brain over 10 h was similar in both groups and followed exponential kinetics. Using liquid scintillation counting, high-pressure liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) analysis, no compounds other than MPP+ were detected in brain 2-10 h after the i.c.v. administration of radiolabelled [14C/3H]MPP+. MPP+ was also unchanged after incubation with brain homogenates. These data indicate that MPP+ is not removed from the CNS compartment via biotransformation. Because of its positive charge and kinetics of elimination, the possibility of an active transport system is suggested.
Article
The effect of ethanol and other aliphatic alcohols on the intestinal transport of 5-methyltetrahydrofolate and folic acid was examined using everted sacs from rat jejunum. Ethanol added to the mucosal medium inhibited the transport of both folate compounds in a parallel manner, and the inhibition increased with increasing ethanol concentration (0.5-10% v/v). Ethanol at 3% v/v in the mucosal medium caused: depression in the pH dependency of the active transport of 5-methyltetrahydrofolate; higher inhibition in the transport of low concentration (0.1 microM) than high concentration (10 microM) of 5-methyltetrahydrofolate, and inhibition in the active accumulation against a concentration gradient of 5-methyltetrahydrofolate and L-leucine. Methanol, propanol and butanol also inhibited the transport of the folate compounds; and in general, the inhibitory effect increased with the increase in the number of carbon atoms in the hydrophobic chain. This study indicates that ethanol and other alcohols inhibit the intestinal transport of folates, that the degree of inhibition is related to the concentration and chain length of the alcohol, that the inhibition is not specific for folates and finally that the mechanism of inhibition is multifactorial.
Article
Transfer of glucose from maternal to fetal circulations requires transport across both the microvillous (maternal-facing) and basal (fetal-facing) plasma membranes of the placental syncytium. We have previously reported transport properties of the microvillous membrane and we now report those of the basal membrane. Basal plasma membrane vesicles were prepared by selective sonication and density gradient centrifugation. Glucose or glucose analogues were rapidly transported across these membranes by facilitated diffusion. Transport was inhibited by cytochalasin B, phloretin and phloridzin. L-Glucose at 1 mM was transferred at only 1/700 of the rate of D-glucose, which indicated an insignificant nonspecific diffusion component. Transport was independent of sodium gradients, and kinetic studies under equilibrium-exchange conditions demonstrated a Km of 23 mM. Competition studies demonstrated that aldohexoses in the C-1 chair conformation were the preferred substrates. Placental steroids estriol and progesterone inhibited transport. In contrast to other polarized epithelia, the basal and microvillous membranes of the human placental syncytium possess transport systems with similar properties. Thus, the directionality and rate of transfer of glucose across the intact syncytium are likely to be direct functions of the materno-fetal concentration gradient and the total transport capacities of the two plasma membranes.
Article
Malnutrition with folate deficiency is frequently found among alcoholics, and could be caused in part by decreased intestinal absorption. To evaluate the relationship of nutrition and absorption in alcoholics, intestinal absorption was studied in patient volunteers placed on a folate-deficient diet with ethanol. Intestinal absorption was measured by conventional means and by the technique of triple lumen perfusion of the jejunum. In 2 patients who ingested ethanol, 200 g per day with a low folate diet, the dietary induction of folate deficiency was followed by decreased absorption of d-xylose, labeled folic acid (³H-pteroylglutamic acid), glucose, fluid, and sodium. Mild net secretion of fluid and sodium into the intestinal lumen was observed in a patient who remained sober on the low folate diet and in a patient who ingested ethanol, 300 g per day, with a regular diet. The morphology of the jejunal mucosa was not affected. These preliminary data suggest that the combination of dietary folate deficiency and prolonged ethanol intake results in intestinal malabsorption of several water-soluble substances, which may account in part for the poor nutrition often found in binge drinkers.
Article
The jejunal uptake of labeled folic acid (3H-PGA) was measured in chronic alcoholic patients without definite liver disease. The intestine was perfused through a triple lumen tube with a solution containing 3H-PGA in a concentration of 25 ng per milliliter. Uptake was low in eight patients who gave a history of poor diet during the alcoholic binge that preceded admission (20.4 per cent ± 4.16 S.D.). A significantly greater value was obtained in nine abstinent subjects fed a hospital diet for two weeks (36.5 per cent ± 5.75). The subsequent administration of ethanol for two weeks to seven patients did not change the mean uptake of 3H-PGA (34.8 per cent ± 9.87). This study confirms that the absorption of folic acid is decreased in malnourished, actively drinking alcoholics. The data suggest that the functional defect is caused by poor nutrition rather than a toxic effect of ethanol on the jejunum.
Article
Using 6-deoxy-d-glucose and d-xylose as substrates, it is shown that phlorizin, but not its aglycon, phloretin, is a fully competitive inhibitor of sugar active transport in hamster small intestine. The results indicate that: (1) phlorizin competes with glucose and its analogs for a common membrane binding site; and (2) phloretin inhibits sugar transport allosterically through binding to a different,albeit closely associated binding site. To explain these findings, a kinetic model has been constructed in which the carrier is assumed to possess two different sites, namely a sugar- and a phenol-binding site. Phlorizin binds simultaneously to both of these sites. The synergistic effect of this double binding causes the overall affinity of phlorizin for the sugar carrier to be three to four orders of magnitude higher than that of the physiological substrate, glucose. Phloretin binds only to the phenol site. The differences among tissues in their relative responses to phlorizin and phloretin are attributed to differing spatial relationships between the sugar site and the phenol site.
Article
In the United States and other developed countries thiamin deficiency is often related to chronic alcoholism. A number of mechanisms may be involved in the pathogenesis of thiamin deficiency in the alcoholic population. An important cause is inadequate intake of thiamin. Moreover, there may be decreased converstion of thiamin to the active coenzyme, reduced hepatic storage of the vitamin in patients with fatty metamorphosis, ethanol inhibition of intestinal thiamin transport, and impaired thiamin absorption secondary to other states of nutritional deficiency. The present discussion focuses on the mechanism of ethanol-related thiamin malabsorption. Under normal conditions thiamin transport in animals and humans is biphasic. At low or physiological thiamin concentrations, transport is a saturable, carrier-mediated, active process; but at higher concentrations, the transport of thiamin is predominantly passive. Ethanol reduces the rate of intestinal absorption and the net transmural flux of thiamin. Furthermore, ethanol inhibits only the active and not the passive component of thiamin transport by impeding the cellular exit of thiamin across the basolateral or serosal membrane. The impairment of thiamin movement out of the enterocyte correlates with a fall in the activity of Na-K ATPase. Bound to the basolateral membrane, Na-K ATPase is believed to be involved in the kinetics of active transport. Ethanol also increases the fluidity of enterocyte brush border and basolateral membranes. Since ethanol increases membrane fluidity it is possible that tahe impairment of thiamin transport and the diminution of Na-K ATPase activity may be related, at least partly, to a physical perturbation of the enterocyte membrane.
Article
Transport through the microvillous membrane of the syncytium is the first step in placental transfer of nutrients. We have therefore studied glucose transport by isolated microvillous membrane vesicles. Transport occurred by selective and rapid facilitated diffusion, which was inhibited by phloridzin, phloretin, cytochalasin B, and HgCl2. Nonmediated diffusion of the substrate was found by three independent methods to be very slow. Competition studies showed that aldohexoses in the C-1 chair conformation were the preferred substrates. Transport was independent of sodium gradients and was not modulated by insulin. However, several steroids inhibited transport including estriol and progesterone, which are abundant in utero. Kinetic analysis by equilibrium exchange demonstrated a Km of 31 mM and a Vmax of 120 nmol . s-1 . mg protein-1. The Km and Vmax suggest a large capacity in relation to calculated fetal needs. In consequence of this capacity, intrasyncytial concentrations of glucose are probably maintained near those of maternal blood. Augmentation of transport at this membrane by hormones or other agents is unlikely to increase fetal supply of glucose, but down regulation by steroids may serve a regulatory function.
Article
(1)'Uptake' of phlorizin by intestinal brush border membrane vesicles is stimulated, much as that of D-glucose, by the simultaneous presence of Naout+ and delta psi much less than 0. However, phlorizin contrary to D-glucose, fulfills all criteria of a non-translocated ligand (i.e., of a fully competitive inhibitor) of the Na+,D-glucose cotransporter. (2) The stoicheiometry of Na+/phlorizin binding is 1, as shown by a Hill coefficient of approx. 1 in the Naout+-dependence of phlorizin binding. (3) The preferred order of binding at delta psi much less than 0 is Na+ first, phlorizin second. (4) The velocity of association of phlorizin to the cotransporter, but not the velocity of its dissociation therefrom, responds to delta psi. These observations, while agreeing with the effect of delta psi much less than 0 on the Kd of phlorizin binding in the steady-state time range, also confirm that the mobile part of the cotransporter bears a negative charge of 1. (5) A model is proposed describing the Na+, delta psi-dependent interaction of phlorizin with the cotransporter and agreeing with a more general model of Na+, D-glucose cotransport. (6) The kon, koff and Kd constants of phlorizin interaction with the Na+,D-glucose cotransporter are smaller in the kidney than in the small-intestinal brush border membrane, which results in a number of quantitative differences in the overall behaviour of the two systems.
Article
Glucose uptake into plasma membrane vesicles from the maternal surface of the human placenta was measured with the Millipore filtration technique. Uptake off D-glucose was dependent on the osmolarity of the incubation medium surrounding the vesicles. Uptake of D-glucose exceeded that of L-glucose. The uptake of D-glucose was not enhanced by placing 100 mM NaCl or NaSCN in the medium outside the vesicles (none inside) at the onset of uptake determinations. D-glucose transport was inhibited by cytochalasin B; phloretin, phlorizin, and 1-fluoro-2,4-dinitrobenzene. D-glucose uptake was inhibited by 2-deoxy-D-glucose, 3-O-methyl-D-glucose and to a lesser extent by D-galactose. It was not inhibited by alpha-methyl-D-glucoside. Cytochalasin B binding to the vesicles was 30% inhibited in the presence of 80 mM D-glucose. The results indicate that the system for facilitated transport of D-glucose at the maternal face of the placenta is distinctly different from that on the brush-border membrane of intestine or renal tubule and more closely resembles that of human erythrocyte.
The effect of tea extract on the intestinal absorption of glucose and sodium was studied in rats. Tea extract reduced the mucosal uptake of glucose and its portal plasma concentration, but was without any effect on its serosal transport. The mucosal uptake of sodium was similarly inhibited. The tea extract reduced also the in vitro activity of the Na(+)-K+ ATPase in an intestinal mucosal homogenate. These data are consistent with the hypothesis that an active ingredient in tea reduced sodium extrusion from the enterocytes by inhibiting the Na(+)-K+ pump, thus destroying the gradient needed for the mucosal transport of glucose.
Article
In the present study, the facilitative D-glucose transporter protein GLUT 1 was localised by immunohistochemistry in the placenta of human, marmoset (Callithrix jacchus) and rat at different developmental stages. A polyclonal antiserum against a 13-amino-acid peptide of the GLUT 1 carboxy terminus was used. It identified a protein of around 50 kDa molecular weight in immunoblotting of the placental tissues. GLUT 1 was located in the syncytiotrophoblast, in cytotrophoblast cells and in fetal endothelium. Similar staining patterns, except in human extravillous cytotrophoblast cells, were observed at all differentiation stages, despite differences in the internal placental architecture of the species. In the marmoset placenta, GLUT 1 was undetectable in endothelial cells of maternal vessels. In rat placentae, trophoblastic giant cells, epithelial cells of both visceral and parietal yolk sac, yolk sac vessels and the stratum spongiosum were stained. Reichert's membrane did not immunoreact. Preadsorption of the antiserum with a 13-amino-acid peptide resulted in the loss of immunoreactivity. The results suggest that GLUT 1 is a prominent isoform of glucose transporters in mammalian placentae. It is generally abundant in placental cell populations bordering on the maternal and fetal circulations and may therefore facilitate an effective glucose supply to the fetus and placenta.
Article
Phenylglucosides are transported by the intestinal Na+/glucose cotransporter (SGLT1) and phlorizin, the classical competitive inhibitor of SGLT1, is also a phenylglucoside. To investigate the structural requirements for binding of substrates to SGLT1, we have studied the interactions between phenylglucosides and the cotransporter expressed in Xenopus oocytes using tracer uptake and electrophysiological methods. Some phenylglucosides inhibited the Na+-dependent uptake of 14C-α-methyl-d-glucopyranoside (αMDG) with apparent K is in the range 0.1 to 20 mm, while others had no effect. Electrophysiological experiments indicated that phenylglucosides can act either as: (1) transported substrates, e.g., arbutin; (2) nontransported inhibitors, e.g., glucosylphenyl-isothiocyanate; or (3) noninteracting sugars, e.g., salicin. The transported substrates (glucose, arbutin, phenylglucoside and helicin) induced different maximal currents, and computer simulations showed that this may be explained by a difference in the translocation rates of the sugar and Na+-loaded transporter. Computational chemistry indicated that all these β-phenylglucosides have similar 3-D structures. Analysis showed that among the side chains in the para position of the phenyl ring the -OH group (arbutin) facilitates transport, but the-NCS (glucosylphenyl-isothiocyanate) inhibits transport. In the ortho position, -CH2OH (salicin) prevents interaction, but the aldehyde (helicin) permits the molecule to be transported. Studies such as these may help to understand the geometry and nature of glucoside binding to SGLT1.
Article
The subcellular distributions of the mammalian passive glucose transporter isoforms GLUT1, GLUT3 and GLUT4, in the human placenta, were investigated using isoform-specific anti-peptide antibodies. On western blots of both basal and brush-border plasma membranes isolated from the syncytiotrophoblast, antibodies specific for GLUT1 labelled a broad band (apparent Mr 55,000) that co-migrated with the human erythrocyte GLUT1 glucose transporter. In contrast, no labelling was detectable when blots were probed with antibodies specific for the GLUT3 or GLUT4 isoforms. Densitometric analysis of blots showed that GLUT1 accounts for approximately 90 and 65 per cent of the D-glucose-sensitive cytochalasin B binding sites present in brush-border and basal membranes, respectively. Confocal immunofluorescence microscopy of fixed placental tissue showed that GLUT1 is abundant at both maternal- and fetal-facing surfaces of the syncytiotrophoblast whereas it was undetectable at the fetal capillary endothelium. In parallel experiments, no staining by antibodies against either the GLUT3 or the GLUT4 isoforms was detected in placental tissue. These results indicate that GLUT1 is the major isoform responsible for glucose transfer from mother to fetus. The absence of GLUT4 is consistent with the lack of insulin-sensitive glucose transport across the placenta.
Article
Nutrient supply to the fetus is a key factor in the regulation of fetal growth. However, the direct supply of nutrients to provide building blocks for tissue growth is likely to be only a minor component of this regulation. The indirect effects of nutrition on fetal endocrine and metabolic status, and on the interaction between the fetus, placenta and mother all of which must be coordinated to allow fetal growth are also important. Maternal undernutrition may alter the growth of the fetus and its different component tissues in a way which cannot be explained solely on the basis of reduced substrate supply during the rapid growth phase of the tissues involved. Adaptation to altered substrate supply, during both undernutrition and refeeding, involves sequential changes in the metabolic and endocrine interactions between the fetus and the placenta. In addition, undernutrition has long-term consequences for the fetus. There is evidence for nutritional programming of fetal endocrine and cardiovascular systems before birth. Nutritional effects may also persist over more than one generation. The effects of nutrition on fetal growth are far more complex than simply those of substrate deprivation.
Article
To evaluate and optimize the use of Caco-2 cell monolayers to predict the in vivo absorption of a broad range of compounds in man. Caco-2 cells are derived from human adenocarcinoma colon cells and spontaneously differentiate when grown on porous polyethylene terephthalate membranes (PETP) in a 12 well format to form monolayers of polarized cells possessing function similar to intestinal enterocytes. Transport experiments were conducted using 21 day cultured cells in a shaking water bath at 37 degrees C. Radiolabeled mannitol was used to determine monolayer integrity. Apparent permeability coefficient (Papp) was calculated from the appearance of drug in the receiver side. A strong correlation was observed between in vivo human absorption and in vitro Papp for a variety of compounds (R = 0.95, N = 35). For compounds that are substrates of p-glycoprotein (Pgp), use of a Pgp inhibitor resulted in a better estimate of absorption in humans. The results of this study suggest that the overall ranking of compounds with Papp < 1 x 10(-6) cm/sec, between 1-10 x 10(-5) cm/ sec, and > 10 x 10(-6) cm/sec can be classified as poorly (0-20%), moderately (20-70%) and well (70-100%) absorbed compounds, respectively. These data suggest that Caco-2 cells developed under the culturing and transport conditions defined herein can be used to predict in vivo human absorption of compounds regardless of transport mechanism, viz., transcellular, paracellular and carrier-mediated.
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Riboflavin (RF), a water-soluble vitamin, is essential for normal cellular functions, growth, and development. Normal RF body homeostasis depends on intestinal absorption and recovery of the filtered vitamin in renal tubules. The mechanism and cellular regulation of the RF renal reabsorption process, especially in the human situation, are poorly understood. The aim of this study was therefore to address these issues, using a recently established human normal renal epithelial cell line, HK-2, as a model. Uptake of RF by HK-2 cells was found to be 1) linear with time for 5 min of incubation and occurring with minimal metabolic alterations, 2) temperature dependent, 3) Na+ independent, 4) saturable as a function of concentration [apparent Michaelis constant (K(m)) of 0.67 +/- 0.21 microM and maximal velocity (Vmax) of 10.05 +/- 0.87 pmol.mg protein-1.3 min-1], 5) inhibited by structural analogs and anion transport inhibitors, and 6) energy dependent. Protein kinase C-, protein kinase A-, and protein tyrosine kinase-mediated pathways were found to have no role in regulating RF uptake. On the other hand, a Ca2+/calmodulin-mediated pathway appeared to play a role in the regulation of RF uptake by HK-2 cells via an effect on the Vmax, as well as on the apparent K(m) of the RF uptake process. The uptake process of RF was also found to be adaptively regulated by the level of the substrate in the growth medium, with the effect being mediated through changes in the apparent K(m) and the Vmax of the uptake process. These results demonstrate that RF uptake by the human-derived renal epithelial cell line HK-2 is via a carrier-mediated system that is temperature and energy dependent and appears to be under the regulation of a Ca2+/calmodulin-mediated pathways and substrate level in the growth medium.
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Organic cation transporters are critical in drug absorption, targeting, and disposition. It has become increasingly clear that multiple mechanisms are involved in organic cation transport in the key tissues responsible for drug absorption and disposition: the kidney, liver, and intestine. In this review, we discuss current models of transepithelial flux of organic cations in these three tissues. Particular emphasis is placed on the more recent molecular studies that have paved the way for a more complete understanding of the physiological and pharmacological roles of the organic cation transporters. Such information is essential in predicting pharmacokinetics and pharmacodynamics and in the design and development of cationic drugs.
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3, 4-methylenedioxymethamphetamine (MDMA or Ecstasy) is a substituted amphetamine whose acute and long-term effects on the serotonin system are dependent on an interaction with the 5-HT uptake transporter (SERT). Although much of the work dedicated to the study of this compound has focused on its ability to release monoamines, this drug has many important metabolic consequences on neurons and glial cells. The identification of these physiological responses will help to bridge the gap that exists in the information between the acute and neurotoxic effects of amphetamines. Substituted amphetamines have the ability to produce a long-term translocation of protein kinase C (PKC) in vivo, and this action may be crucial to the development of serotonergic neurotoxicity. Our earlier results suggested that PKC activation occurred through pre- and postsynaptic mechanisms. Because the primary site of action of these drugs is the 5-HT transporter, we now expand on our previous results and attempt to characterize MDMA's ability to translocate PKC within cortical 5-HT nerve terminals. In synaptosomes, MDMA produced a concentration-dependent increase in membrane-bound PKC (as measured by 3H-phorbol 12, 13 dibutyrate, 3H-PDBu) bindings sites. This response was abolished by cotreatment with the specific serotonin reuptake inhibitor (SSRI), fluoxetine, but not by the 5-HT2A/2C antagonist, ketanserin. In contrast, full agonists to 5-HT1A and 5-HT2 receptors did not produce significant PKC translocation. MDMA-mediated PKC translocation also requires the presence of extracellular calcium ions. Using assay conditions where extracellular calcium was absent prevented in vitro activation of PKC by MDMA. Prolonged PKC translocation has been hypothesized to contribute to the calcium-dependent neurotoxicity produced by substituted amphetamines. In addition, many physiological processes within 5-HT nerve terminals, including 5-HT reuptake and vesicular serotonin release, are susceptible to modification by PKC-dependent protein phosphorylation. Our results suggest that prolonged activation of PKC within the 5-HT nerve terminal may contribute to lasting changes in the homeostatic function of 5-HT neurons, leading to the degeneration of specific cellular elements after repeated MDMA exposure.