Caco-2 Cell and Animal Model Studies (Gallus gallus) Are Effective at Screening and Developing Staple Food Crops with Improved Fe Bioavailability

Transcription

1 Caco-2 Cell and Animal Model Studies (Gallus gallus) Are Effective at Screening and Developing Staple Food Crops with Improved Fe Bioavailability Elad Tako * and Raymond Glahn * Senior Scientist Physiologist and Courtesy Assistant Professor USDA-ARS, Robert W. Holley Center for Agriculture and Health, Cornell University, Ithaca NY, ASA, CSSA, and SSSA International Annual Meeting Synergy in Science: Partnering for Solutions, November Minneapolis, Minnesota C09 Biomedical, Health-Beneficial, and Nutritionally Enhanced Plants 347 Symposium--Improving Pulse Crops for Nutrition and Health

2 The world as we know it? Source: World Health Organization (2005) World Health Report: Make every mother and child count. WHO; Geneva. Most affected regions: South East Asia Central/South America Africa Territories are sized in proportion to the absolute number of people who died from iron deficiency anemia in one year. Iron-deficiency anemia caused 0.24% of all deaths worldwide in 2012, an average of 22 deaths per million people per year (WHO, 2013).

3 Challenge To seek innovative solutions for better health. Poor nutrition, specifically Fe and Zn deficiencies are identified as one of the major global health problems to be addressed (WHO, Micronutrient initiative) Problem Low Fe bioavailability from grain/plant based diets that are low in absorbable Fe Strategy Biofortification Conventional plant breeding to increase concentrations and/or bioavailabilities of micronutrients in foods (Welch et al., 2002) Goal To create a full range of optimal, bioavailable nutrients in a single staple plant species. Iron Bioavailability Research: Developing screening tools for Fe bioavailability, and guiding the plant breeding process Improving Fe bioavailability by breeding towards increased Fe content : India Pearl Millet (Tako et al., 2015) Mexico Black Beans (Tako et al., 2014) Rwanda Carioca Beans (Tako et al., 2015)

4 In Vitro Digestion/Caco-2 Cell Culture Model: Fe Bioavailability (US patent # , Glahn et al., 1998, 2002, 2005; Siem et al., 2013; Hart et al., 2015; Tako et al., 2007ab, 2008abc, 2009ab, 2010abc, 2011abc, 2012abcd, 2013abc, 2014abcd, 2015abc) The in vitro assay allows the analysis of hundreds of samples (Bean, Wheat, Rice, Pearl Millet, Sorghum, Sweet Potatoes, Maize, and various dietary combinations), prior the selection of the most promising lines to be assessed in vivo. This is a cost effective, accurate and rapid screening tool. Culture well Insert ring Food Preparation Pepsin Digestion ph 2, 1 h, 37 C (50 ml tube) Pancreatin-Bile Digestion ph , 2 h, 37 C Dialysis membrane 15K MWCO Soluble iron Caco-2 cells Harvest cells for ferritin analysis 24 h post start of Panc/Bile digestion

5 In vivo model (Gallus gallus) The broiler chicken (Gallus gallus) model exhibits the appropriate responses to Fe deficiency and can serve as a model for Fe bioavailability (Tako et al., 2010; 2011 abc; 2012 abc; 2013 abcd; 2014 ab,; Mahler et al., 2012; Reed et al., 2014; Reed et al., 2015ab; Hartono et al., 2015; Tako et al., 2015abc) 1. Fast growing animal (4-6 weeks) 2. Sensitive for dietary mineral deficiencies. 3. Dietary manipulation 4. >85% homology between gene sequences of human and chicken intestinal DMT1, DcytB, ZnT1 and Ferroportin 5. In vivo Fe bioavailability assessment: Intra amniotic administration (The in ovo feeding technique, US patent # ) Long term feeding trial

6 Presentation outline Research Area Focus Source Emphasis Citation Crop Biofortification Beans Increased Fe content 1. Red Mottled beans In vitro Fe bioavailability In vivo Fe Bioavailbility Tako E, Blair MW, Glahn RP. Biofortified red mottled beans (Phaseolus vulgaris L.) in a maize and bean diet provide more bioavailable iron than standard red mottled beans: studies in poultry (Gallus gallus) and an in vitro digestion/caco-2 model. Nutr J. 2012, 14;10:113. Lentil Maize Pearl Millet Wheat Sorghum Rice 2. Black beans In vitro Fe bioavailability In vivo Fe Bioavailbility Fe bioavailability potential inhibitors Tako E, Beebe SE, Reed S, Hart JJ, Glahn RP. Polyphenolic compounds appear to limit the nutritional benefit of biofortified higher iron black bean (Phaseolus vulgaris L.). Nutr J. 2014, 5(9);19. Hart JJ, Tako E, Kochian LV, Glahn RP. Identification of Black Bean (Phaseolus vulgaris L.) Polyphenols that Inhibit and Promote Iron Uptake by Caco-2 Cells. J Agric Food Chem. 2015, 63(25): Increased Fe bioavailability 3. Cream Seeded Carioca beans In vitro Fe bioavailability In vivo Fe Bioavailbility Fe bioavailability potential inhibitors Tako, Anandaraman, Hart, Beebe and Glahn, Studies of cream seeded Carioca Beans (Phaseolus vulgaris L.) from a Rwandan efficacy trial: In vitro and In vivo screening tools reflect human studies and predict beneficial results from iron biofortified beans. PLoS One Sep 18;10(9):e doi: /journal.pone

7 Increasing Fe bioavailability by increasing Fe content * : Red Mottled 1 and Black Beans 2 In vitro and In vivo (Gallus gallus) Models 1 Tako E, Blair MW, Glahn RP. Biofortified red mottled beans (Phaseolus vulgaris L.) in a maize and bean diet provide more bioavailable iron than standard red mottled beans: studies in poultry (Gallus gallus) and an in vitro digestion/caco-2 model. Nutr J. 2012, 14;10: Tako E, Beebe SE, Reed S, Hart JJ, Glahn RP. Polyphenolic compounds appear to limit the nutritional benefit of biofortified higher iron black bean (Phaseolus vulgaris L.). Nutr J. 2014, 5(9);19. * In collaboration with HarvestPlus and CIAT ( International Center for Tropical Agriculture, Cali, Colombia

8 In vitro assessment of Fe bioavailability Red Mottled Bean Black Bean Fe content in the beans (μg Fe/g) Biofortified High Standard Low Red-mottled bean lines used in the study were Biofortified High Standard Low Black bean lines used in the study were a developed71±0.6 from an African commercial 49±0.4variety: commercial 88±0.9variety for South America: 59±0.7 Dietary Composition 1. Standard 60% Fe (CAL96, 49µg/g Fe). 60% 1. Standard 40% Fe (DOR500, 59µg/g 40% Fe). 2. Biofortified Fe (NUA35, 71µg/g Dietary Fe]. Fe content 2. (μg Biofortified Fe/g) Fe (MIB465, 88µg/g Fe). 54.6± ± ± ±0.2 Phytate : Fe Molar Ratio Source: 8.28 Darien, ± 9.2 Colombia 8.59 ± 1.06 Source: 8.25 Palmira, ± Colombia 8.95 ± 0.72 * Beans were grown under fertile soil conditions and standard 2.5 agronomic practices and shipped to 2Ithaca, New York in sealed containers (imported as grain) Upon arrival, beans were rinsed in ultra-pure (18Ω) water Cell and baselinethen MIB465 cooked (high Fe) (autoclaved based DOR500 (low for Fe) based 45 diet diet minutes in water and until soft). Beans were then freeze-dried and milled prior to the mixing the Figure diets (for 1.Summary all processing of vitro bioavailability stainless steel results appliances in Caco-2 cells were exposed used). to samples (ng of ferritin/mg of protein). Values are means±sd. ng Ferritin/mg protein

9 In vivo assessment Gallus gallus (n=15) 4-6 weeks long term feeding trial (ad libitum) Weekly: Blood, BW, Fe intakes Tako et al., 2012 End: Duodenal and hepatic tissue samples Tako et al., 2014 Hemoglobin (g/l) Total Body Hb-Fe (mg) Hemoglobin Maintenance Efficiency (%) Figure 2. Fe-related parameters assessed during studies. (A) Blood hemoglobin concentration (g/l), (B) Total body Hb-Fe (mg), (C) Hemoglobin maintenance efficiency (%). * P < 0.05 between treatment groups.

10 Regulation of Intestinal Fe absorption DcytB Reductase Ferritin American Journal of Physiology - Gastrointestinal and Liver Physiology, 2005 Vol. 289 no. 6, G981-G986

11 Expression of duodenal Fe related proteins 1.2 High Fe 1.2 High Fe mrna expression (relative value, AU) Low Fe mrna expression (relative value, AU) Low Fe 0 DMT-1 DcytB Ferroportin 0 DMT-1 DcytB Ferroportin Tako et al., 2012 Tako et al., 2014 Figure 3. Changes in mrna expression are shown relative to expression of 18S rrna in arbitrary units (AU, P <.05, n=15)

12 Table 1. Concentrations 1 of the most prevalent polyphenols observed in the black beans (Phaseolus Vulgaris L., nmol/g, n=6, p<0.05) Compound Biofortified Fe Standard Fe Caffeic acid 6.0 ± 0.9b 25.8 ± 4.7a Gallic acid ± 8.8a ± 17.8a Ferulic acid ± 11.1a ± 20.1a Kaempferol 5.0 ± 0.1a 0.00 ± 0.00b Catechin ± 31.1a ± 25.5b Myricetin 24.0 ± 1.7a 12.5 ± 4.0b Kaempferol 3-glucoside ± 10.7a 19.2 ± 5.6b Quercetin 3-glucoside 239 ± 20.3a 45.8 ± 7.9b 1 Extracts were analyzed by LC-MS with an Acquity UPLC coupled to a Xevo G2 QTof spectrometer (Waters Corp. Milford, MA). Tako et al., 2014

13 Figure 4. In vitro ferritin formation in response to Epicatechin and Myricetin Hart et al., 2015

14 Figure 5. In vitro ferritin formation in response to Kaempferol, Kaempferol 3-glucoside, Quercetin and Quercetin 3-glucoside Caco-2 ferritin formation in response to kaempferol, kaempferol 3-glucoside, quercetin and quercetin 3-glucoside Kaempferol ng ferritin / mg protein Kaempferol 3-glucoside ng ferritin / mg protein 20 Quercetin 3-glucoside No Fe Quercetin Polyphenol concentration ( M) Cell baseline + 10 M FeCl M asc. acid 10 0 Hart et al., 2015

15 Conclusions: Bean biofortification Minor increase in absorbable-fe human efficacy study (Black Beans Mexico, La Frano et al., 2014). Breeding for increased Fe-concentration elevated the levels of polyphenolic-compounds that can reduce the Fe biofortified bean nutritional benefit. Bean polyphenol profile should be further evaluated and modified if possible in order to improve the nutritional quality of higher-fe beans. Tako et al. Biofortified red mottled beans (Phaseolus vulgaris L.) in a maize and bean diet provide more bioavailable iron than standard red mottled beans: studies in poultry (Gallus gallus) and an in vitro digestion/caco-2 model. Nutrition J Oct 14;10:113. Tako et al. Polyphenolic compounds appear to limit the nutritional benefit of biofortified higher iron black bean (Phaseolus vulgaris L.). Nutrition J Mar 26;13:28.

16 Increasing Fe bioavailability by increasing Fe content: Cream Seeded Carioca Beans 1 In vivo (Gallus gallus) and in vitro Models Tako E, Reed S, Anandaraman A, Beebe SE, Hart JJ, Glahn RP (2015) Studies of Cream Seeded Carioca Beans (Phaseolus vulgaris L.) from a Rwandan Efficacy Trial: In Vitro and In Vivo Screening Tools Reflect Human Studies and Predict Beneficial Results from Iron Biofortified Beans. PLoS ONE 10(9): e doi: /journal.pone In collaboration with HarvestPlus and CIAT

17 Table 1. Composition of the experimental bean based diets 1-2 The specific Rwandese dietary formulation that was used in the study was achieved by a close consultation with and approval of the HarvestPlus nutritionist team, and was based on the menus that were used during the human efficacy trial Fe bioavailability in Fe biofortified beans human efficacy study, Kigali University, Rwanda

18 Table 2. Ferritin concentration in Caco-2 cells exposed to samples of beans only (whole bean), additional meal plan components and bean-based diets 1-2. Tako et al., 2015

19 Figure 1. Fe-related parameters assessed during the study. (1A) Blood hemoglobin concentration (g/l), (1B) Total body Hb-Fe (mg), (1C) Hemoglobin maintenance efficiency (%). * P < 0.05 between treatment groups. ** P < 0.01 between treatment groups, n=14. 1A B Biofortified Standard * Hemoglobin (g/l) Total Body Hb-Fe (mg) Start Day 7 Day 14 Day 21 Day 28 Day 35 Day C 40 * 0 Start Day 7 Day 14 Day 21 Day 28 Day 35 Day HME (%) Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Tako et al., 2015

20 Figure 2. Duodenal mrna gene expression of Fe-related proteins collected on day Standard * Biofortified 1.5 Relative Expression (AU) DMT-1 DcytB Ferroportin 1 Changes in mrna expression are shown relative to expression of 18S rrna in arbitrary units (AU, * P < 0.05, n=14). Tako et al., 2015

21 Table 3. Ferritin protein and Fe concentration in the liver Tako et al., 2015

22 Table 4. Concentrations 1 (µmol/g, n=6) of prevalent polyphenols observed in the cream seeded Carioca Beans seed coats 1 Seed coat extracts and polyphenol standards were analyzed with an Agilent 1220 Infinity UPLC coupled to an Advion expressionl compact mass spectrometer (CMS). In vivo results suggests a benefit to the Fe biofortified bean variety, it is possible that the Fe bioavailability was limited due to increased polyphenolic content (Tako et al., 2014; 2015; Hart et al., 2015). Tako et al., 2015

23 Conclusions Overall in vitro and in vivo tools to aid biofortification efforts in staple food crops Minor increase in absorbable-fe human efficacy study (Rwanda, Petry et al.,2014; Haas et al., 2014) Further utilize our screening tools (in vitro and in vivo) to guide future studies aimed to assess biofortified staple food crops, as this approach will allow proceeding to human efficacy studies Phytic acid and polyphenols, may be responsible for limiting the nutritional effects of more theffectively Fe biofortified. beans. The capacity to cost-effectively monitor Fe biofortified crops once they are released to farmers and dispersed into the food system. Such monitoring will likely be needed to ensure the biofortification effect. Further evaluation and if possible modification of the polyphenol profile of the biofortified cream seeded carioca bean. Overall, we conclude that Fe biofortified bean varieties remain a promising vehicle for increasing intakes of bioavailable Fe. References : Tako E, Blair MW, Glahn RP. Biofortified red mottled beans (Phaseolus vulgaris L.) in a maize and bean diet provide more bioavailable iron than standard red mottled beans: studies in poultry (Gallus gallus) and an in vitro digestion/caco-2 model. Nutr J. 2012, 14;10:113. Tako E, Beebe SE, Reed S, Hart JJ, Glahn RP. Polyphenolic compounds appear to limit the nutritional benefit of biofortified higher iron black bean (Phaseolus vulgaris L.). Nutr J. 2014, 5(9);19. Tako et al., Studies of cream seeded Carioca Beans (Phaseolus vulgaris L.) from a Rwandan efficacy trial: In vitro and In vivo screening tools reflect human studies and predict beneficial results from iron biofortified beans. PLoS One Sep 18;10(9):e doi: /journal.pone

24 Dr. Ray Glahn Dr. Leon Kochian Dr. Jon Hart Dr. Matthew Blair Dr. Steve Beebe Peipei Chang Mary Bodis Dr. Shree Giri Dr. Steve Beebe Dr. Jere Haas Dr. Erick Boy Spenser Reed Karen Hartono Amrutha Anandaraman Nadia Putri Sarina Pacifici Jaehong Song Thank you H arvestplus CIAT Session title: Biomedical, Health-Beneficial & Nutritionally Enhanced Plants: II (Poster #1102 ): Developing Staple Food Crops for Improved Nutritional Quality: Identification of Compounds in Bean Seed Coats That Inhibit and Enhance Fe Absorption.