In Vitro ABC Transporter Assays for Nutrient Absorption and Drug-Nutrient interaction studies

November, 2005

Table of contents:

1. Digestion 2. Transport of nutrients 3. Importance of ABC transporters in nutrition 4. SOLVO technology 5. References

1. Digestion {1}

Digestion involves the mixing of food, its movement through the digestive tract, and the chemical breakdown of the large molecules of food into smaller ones (Table 1). Digested food molecules, water and minerals are absorbed from the cavity of the upper small intestine and then carried off in the bloodstream to other parts of the body for storage or further chemical change (Figure 1). Nutrients are substances contained in foods which provide energy and raw materials for the synthesis and maintenance of living matter. Human nutrients are proteins, carbohydrates, fat, minerals, vitamins and water. All cells acquire the molecules and ions they need from their surrounding extracellular fluid (ECF). There is an unceasing traffic of molecules and ions: · In and out of the cell through the plasma membrane; · In eukaryotic cells, there is also transport in and out of membrane-bounded intracellular compartments such as the nucleus, endoplasmic reticulum, and mitochondria.

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Figure 1: The bloodstream

2. Transport of nutrients{20}

Nutrients can be transported through the cell membrane in 3 ways (Figure 2): Passive diffusion consists of the transport of water and water-soluble substances and small lipids through the lipid bilayer with a concentration gradient. In the case of a facilitated diffusion, transporter proteins create a water-filled pore through which ions and small hydrophilic molecules can pass by diffusion. Fructose, riboflavin and vitamin B12 (in combination with intrinsic factor) are among the substances absorbed by facilitated diffusion. During active transport, transmembrane proteins, called transporters, use the energy of ATP to force ions or small molecules through the membrane against their concentration gradient. These active transport mechanisms have been identified for intestinal absorption of many substances including glucose, galactose, amino acids, calcium, iron, folic acid, ascorbic acid, thiamin and bile acids. Transport proteins, embedded in lipid membranes, facilitate the import of nutrients into cells or the release of toxic products into the surrounding medium. The most important family of membrane transport proteins are the ATP-binding cassette (ABC) transporters. These ABC proteins play a central role in all living cells in the nutrient uptake, protein, drug and antibiotic excretion, osmoregulation, antigen presentation, signal transduction and other important cellular functions.

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fac diff

act diff

Figure 2: Passive diffusion, facilitated diffusion and active transport of nutrients

3. Importance of ABC transporters in nutrition {1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19}

Some studies suggest that dietary constituents regulate the expression of ABC Transporters. Changes in ABC Transporter expression may represent an important physiological response to foods containing toxins and an important component of the acute phase immune response. It has been shown that dietary phytochemicals have inhibitory effects on P-glycoprotein (MDR1) and potencies to cause drug-food interactions. Moreover, the observations in a number of studies demonstrated important roles of membrane transporters, i.e. Multidrug Resistant Protein 1 (MRP1), Multidrug Resistant Protein 2 (MRP 2), Breast Cancer Resistance Protein BCRP (MXR) and P-glycoprotein (MDR1) in the cellular accumulation, transport and potential effects of many nutrients. Since some of these nutrients are found in fruits and vegetables, their effect on MRP1, MRP2, MXR and MDR1 may be a mechanism relevant to carcinogenesis and the observed lowered cancer risk in humans with higher dietary intake of fruits and vegetables (Table 1). Table 1: Nutrient-transporter interactions

Nutrient

Membrane Transporters

Effect

Dietary phytochemicals

Ø       P-glycoprotein(MDR1)

Inhibition

Flavonoids (e.g. quercetin, Epicathechin gallate),  flavonoid glycosides (e.g. genistein-7-glucoside) and flavonoid glucuronides found in fruits and vegetables

Ø       Multidrug Resistant Protein 1 (MRP1)

Ø       Multidrug Resistant Protein 2 (MRP 2)

Ø       Breast Cancer Resistance Protein BCRP (MXR)

Ø       P-glycoprotein (MDR1)

Accumulation, Transport and potential effects

Flavonoid-like molecules (e.g. polyphenol phloretin)

Ø       Multidrug Resistant Protein 1 (MRP1)

Ø       Multidrug Resistant Protein 2 (MRP 2)

Ø       Breast Cancer Resistance Protein BCRP (MXR)

Ø       P-glycoprotein (MDR1)

Accumulation, Transport and potential effects

Extracts of bitter melon(1-monopalmitin), grape fruit (bergamottin and quercetin), soy bean, dokudami and welsh onion

Ø       P-glycoprotein (MDR1)

Inhibition

Grape seed extract (GSE)

Ø       P-glycoprotein (MDR1)

Inhibition

Sterols (e.g. Cholesterol)

Ø       ABCA1, ABCG1, ABCG5 and ABCG8

Substrate

Sesame seeds (isolated substance: lysophosphatidylcholine, linoleoyl)

Ø       Certain intestinal transporters

Inhibition

Mono-, di-, and triglutamate of folates

Ø       Breast Cancer Resistance Protein BCRP (MXR)

Ø       Multidrug Resistant Protein 1 (MRP1)

Substrate

Herb constituents

Ø       P-glycoprotein (MDR1)

Inhibition (Curcumin, ginsenosides, piperine, some catechins from green  tea, silymarin from milk thistle); Some catechins from green tea (modulation); Hyperforin, kava (Activation of pregnane X receptor, an orphan nuclear receptor acting as a key regulator of MDR1)

4. SOLVO Technology

The screening of the interaction of nutrients with ABC transporters is an excellent way to determine the transport of nutrients and also to study drug-food interactions. It is highly recommended to perform screens for MRP1, MRP2, MXR (BCRP) and MDR1. It is also an option to screen for other transporters as well. Detailed references and the description of the assays are available at www.solvo.com. A wide array of ABC transporter assays is available from SOLVO for the different ABC transporters. The patented Calcein Assay, ATPase Assay and some Vesicular transport assays are optimized for high-throughput screening on 96 well plates and widely used by pharmaceutical companies. SOLVO provides full technology transfer for in-house use as well as fee-for-service screening.

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5. References

1. Tissue distribution of quercetin in rats and pigs (de Boer VC., Dihal AA., van der Woude H., Arts IC, Wolffram S., Alink GM, Rietjens IM, Hollman PC). J Nutr. 2005 Jul, 135(7): 1718-25 2. Effects of dietary chemopreventive phytochemicals on P-glycoprotein function(Nabekura T, Kamiyama S, Kitagawa S.) Biochem Biophys Res Commun. 2005 Feb 18;327(3):866-70. 3. Cytoplasmic confinement of breast cancer resistance protein(BCRp/ABCG2) as a novel mechanism of adaptation to short-term folate deprivation(Ifergan I, Jansen G, Assaraf YG) Mol Pharmacol. 2005 Apr;67(4):1349-59. 4. Folate concentration dependent transport activity of the Multidrug Resistance Protein 1(ABCC1)(Hooijberg JH, Jansen G, Assaraf YG, Kathmann I, Pieters R, Laan AC, Veerman AJ, Kaspers GJ, Peters GJ) Biochem Pharmacol. 2004Apr 15; 67(8):1541-8 5. Inhibitory effect of a bitter melon extract on the P-glycoprotein activity in intestinal Caco-2 cells(Konishi T, Satsu H, Hatsugai Y, Aizawa K, Inakuma T, Nagata S, Sakuda SH, Ngasawa H, Shimizu M)br J Pharmacol. 2004 Oct;143(3):379-87 6. Folate deprivation results in the loss of breast cancer resistance protein (BCRP/ABCG2) expression. A role for BCRP in cellular folate homeostasis(Ifergan I, Shafran A, Jansen G, Hooijberg JH, Scheffer GL, Assaraf YG) J Biol Chem. 2004 Jun11;279(24):25527-34 7. Flavonoids and the brain: interactions at the blood-brain barrier and their physiological effects on the central nervous system (Youdim KA., Shukitt-Hale B., Joseph AJ) Free Radical Biology & Medicine, Vol. 37, no 11, pp. 1683-1693, 2004 ELSEVIER 8. Grape seed extract affects proliferation and differentiation of human intestinal Caco-2 cells(Laurent C, Besancon P, Auger C, Rouanet JM, Caporiccio B) J Acric Food Chem. 2004 Jun2;52(11):3301-8 9. A new method to measure intestinal activity of P-glycoprotein in avian and mammalian species(Green AK, Barnes DM, Karasov WH) J Comp Physiol . 2005 Jan;175(1):57-66. Epub 2004 Nov 25. 10. Cellular Uptake and Efflux of the Tea Flavonoid (-)-Epicathechin-3-gallate in the Human Intestinal Cell Line Caco-2 (Bharathi J., Vaidya N., Walle T.) JPET 307:745-752, 2003 11. The beta-D-glucoside and sodium-dependent glucose transporter 1 (SGLT1)-inhibitor phloridzin is transported by both SGLT1 and Multidrug Resistance Associated Proteins 1/2. (Walle T., Walle UK.) DMD 31: 1288-1291, 2003 12. The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria.(Jonker JW, Buitelaar M, Wagenaar E, Van Der Valk MA, Scheffer GL, Scheper RJ, Plosch T, Kuipers F, Elferink RP, Rosing H, Beijnen JH, Schinkel AH) Proc Natl Acad Sci U S A. 2002 Nov 26;99(24):15649-54. Epub 2002 Nov 12. 13. Evidence for an interaction between p-glycoprotein and cadmium toxicity in cadmium-resistant and -susceptible strains of Drosophila melanogaster (Callaghan A, Denny N) Ecotoxicol Environ Saf. 2002 Jul;52(3):211-3. 14. Inhibitory effect of fruit ectracts on P-glycoprotein-related efflux carriers: an in-vitro screening(Deferme S, Van Gelder J, Agustijns P) J Pharm Pharmacol. 2002 Sep;54(9):1213-9 15. Expression of P-glycoprotein in the chicken(Barnes DM) Comp Biochem Physiol A Mol Integr Physiol. 2001 Sep;130(2):301-10. 16. Identification of a taurine transport inhibitory substance in sesame seeds(Ishizuka K, Kanayama A, Satsu H, Miyamoto Y, Furihata K, Shimizu M)Biosci Biotechnol Biocehm. 2000 Jun;64 17. Transport of genistein-7-glucoside by human intestinal CACO-2 cells: potential role for MRP2 (Walle UK, French KL., Walgren RA., Walle T.) Res Commun Mol Phatol Pharmacol. 1999 Jan; 103 (1): 45-56 18. Modulation of adriamycin accumulation and efflux by flavonoids in HCT-15 colon cells. Activation of P-glycoprotein as a putative mechanism(Critchfield JW, Welsh CJ, Phang JM, Yeh GC) Biochem Pharmacol. 1994 Oct 7;48(7):1437-45. 19. Flavonol-stimulated efflux of 7,12-dimethylbenz(a)anthracene in multidrug-resistant breast cancer cells(Phang JM, Poore CM, Lopaczynska J, Yeh GC) Cancer Res. 1993 Dec 15;53(24):5977-81. 20. The Human Body: From food to Fuel, Chapter 3 (2005: Paul Insel, Stanford University, Elaine Turner, Food Science and Human Nutrition Dept., University of Florida, Dn Ross , California Institute of Human Nutrition)