Comparison of methods to asses mineral bioavailability (in vitro vs in vivo) Ann-Sofie Sandberg Dept of Chemical and Biological Engineering/Food Science Chalmers University of Technology Gothenburg, Sweden
Assessment of mineral bioavailability In vitro screening, predictors of absorption Animal models Humans Mineral content, inhibitors, enhancers Dialysability/solubility Caco-2 cell model Rat hemoglobin repletion (Fe) Rat 55 Fe, 59 Fe Suckling rat ( 65 Zn) Pig Hb repletion Pig radio or stable isotopes Radio isotope Stable isotope Efficacy
In vitro methods first screening Analyse content of minerals (Fe, Zn, Ca) by atomic absorption spectrophotometry or HPLC Inhibitors (polyphenols, phytate) by e.g.hplc Enhancers (ascorbic acid) by HPLC Screening of cereal, legumes and other crops
Bioavailability of minerals: the absorption and utilization of minerals for normal metabolic processes 1. Digestion (soluble/dialysable mineral) 2. Uptake (intestinal enterocytes) 3. Transport into the circulation 4. Retention, utilization, storage
Iron absorption Food factors Iron solubility/complex (phytate, polyphenols ascorbic acid, organic acids) Host factors Iron status (hepcidin) Infection, inflammation Lumen Hunt JR. Int J Vitam Nutr Res 2005;75:375-84 Food factors (Fe, AA, Ca,polyphenols) Enterocyte Blood
In vitro methods: Dialysis techniques (based on Miller et al 1981) Two step digestion at simulated physiological conditions: gastric phase (pepsin, HCl, ph2) intestinal phase ( pancreatic enzymes, bile acids, NaHCO 3, ph 7) Soluble or dialysable mineral is measured. Development of computer-controlled gastrointestinal model. ph gradient. (Minekus et al 1995) Dialysis or solubility predictor of absorption
Usefulness of iron dialysability/solubility Reproducability between labs poor, needs standardization: e.g. final ph adjustment one critical parameter. Not physiological. Usually predicts correct direction of response, but there are exceptions: small polyphenolic compounds and organic acid complexes is dialysable but not bioavailable Large molecules e.g ferritin can be absorbed but is not dialysable Useful to identify enhancers, inhibitors, (phytate and degradation products, polyphenols, ascorbic acid) but does not predict same magnitude of response as in humans.
Computer controlled model of the gastro intestinal tract (TIM). In each compartment simulation of: mixing of the meal. physiological conditions (ph regulation, secretion of digestive fluids enzymes, electrolytes, bile salts, based on literature data from humans and animals. the transport of food/digestion (gastric emptying, peristaltic movement and transit time). the diffusable minerals and other nutrients are removed through membranes.
Pea protein infant formulas. Iron availability/absorption - in vitro TIM/in vivo Human Fe absorption % Human Fe absorption % 14 12 10 8 6 4 2 0 0 10 20 30 40 50 TIM Dialysable Fe % Davidsson et al, 2001 Fredriksson et al, 2001 Hurrell et al, 1998
Dialysable Fe TIM/Fe absorption from meals with fresh/fermented vegetables 30 Percentage dialysed Fe Percentage absorbed Fe 25 20 15 10 5 TIM:Expensive time consuming, laborous, large volumes Sandberg et al, 2004
In vitro methods: Caco 2 cells Caco-2 model 1. Digestion (soluble/dialysable iron) 2. Uptake (intestinal enterocytes) 3. Transport into the circulation 4. Retention, utilization, storage Combined digestion Caco-2 cells. Uptake/absorption measured
Caco-2 cell model for iron availability/bioaccessability (Glahn R. J Nutr 2008) Food preparation Pepsin digestion 1h, 37C, ph2 Pancreatic - bile digestion 2h, ph 7 Diffusion of soluble Fe Dialysis membrane MWCO 12-14 kda Culture well Caco-2 cell monolayer Two-step in vitro digestion simulating gastric phase, intestinal phase Transfer to apical compartment Dialysis membrane Measurement of ferritin formation in Caco-2 cells after 22 h Iron uptake to predict iron bioavailability in humans
Caco-2 cell model for iron uptake Figure 1: Intestinal iron transport contains uptake and transport proteins Fe 3+ Solubility complex determinant of uptake DRA Fe 2+ haem Dcytb DMT1 HCP1 Fe 2+ HO ferritin Fe 3+ e - LIP e - Fe 2+ IREG1 Fe 3+ Fe 3+ Tf Hp Hepcidine control effects not measured lumen plasma Intracellular effects Iron,AA,Ca,polyphenols Sharp et al, 2003
Usefulness of Caco-2 cell model Benefits High through put system developed useful for screening Include iron uptake not only dialysability/solubility (and transport) Identify potential inhibitors/enhancers Molecular mechanisms of iron absorption can be studied
Usefulness of Caco-2 cell model Limitations Poor agreement between labs (Standardization. When to measure? How long for? ph?etc, Experienced labs) Does not have same magnitude of response as humans. Endpoint ferritin formation indirect measurement assume ferritin formation proportional to iron uptake. Problems with isotop measurements of transport - intracellular dilution of dietary iron Does not include hepcidin controlled transport
Differences in in vitro digestion Caco-2 cell uptake compared to human iron absorption Simulated digestion reflects in vivo situation? (No ph gradient, stomach ph2, should be 4? Digestion time?intestinal ph 6.5-7.4? No outer mucus layer (dialysis membran, cut off 15 kda) Uptake in cell, but no blood to be transported to or if transported across basolateral membrane not controlled by hepcidin. Caco-2 cells are colon cells transport rates of hydrophilic compounds paracellular lower, less leaky, less discrimination on basis of molecular size of compounds transported parallellary compaired to duodenum cells (Duizer et al 1997).
Identifying enhancers and inhibitors of iron absorption with Caco-2 cell model Food compound Effect Reference Ascorbic acid + Han et al. 1995, Glahn et al. 1998, Yun et al. 2004, Kalgaonkar & Lönnerdal 2007, Lei et al. 2008, Muscle tissue + Glahn et al. 1996 Inositol phosphates (IP 6, IP 5 ) - Han et al. 1994, Skoglund et al. 1999, Glahn et al. 2002, Kalgaonkar & Lönnerdal et al 2007, Jin et al. 2008, Tannic acid, polyphenols - Glahn et al. 2002, Kalgaonkar & Lönnerdal 2007, Kim et al. 2008 Calcium - Thompson et al. 2010, Kalgaonkar&Lönnerdal 2007 Organic acids (- +) Salovaara et al. 2002, Bergkvist et al. 2005 Predicts direction of response but not magnitude
Comparison meal studies Caco-2 cells human absorption studies Meals containing - - - Ascorbic acid phytic acid meat polyphenols (Au & Reddy 2000, Yun et al. 2004) Semipurified meals AA, bran, phytate, tea, different protein sources. Dose-response relationship AA, TA - replicate meals fed in human trials. Predicts direction of response but not magnitude
Comparison Caco-2 cells human iron absorption studies Vitamin A bread meal Caco-2 cells, + 2.6-fold (Gargari et al. 2006) Human absorption no effect (Walczyk et al. 2003) Oxalic acid in spinach Caco-2 cells, negative effect (Rutcke et al. 2004) Human absorption, no effect (Bonsmann et al. 2008) Caco-2 cells method did not predict direction of response but meals not identical
To make comparisons - Foods should be prepared exactly as in the human study identical meals. Only one direct comparison maize and bean meals (Beiseigel et al. 2007) Conversion to relative bioavailability Ln (Human absorption ratio) = 0.6401 x Ln (Caco- 2 absorption ratio) (Yun et al. J Nutr 2004)
Comparing human and Caco-2 cell iron bioavailability in maize ACR &TZB Absorption relative to TZB Women Caco-2 Caco-2 cells predict response in humans Beiseigel et al. Am J Clin Nutr 2007 Beiseigel et al. Am J Clin Nutr 2007
Comparing human and Caco-2 cell iron bioavailability in white & colored beans Absorption relative to great northern Women Caco-2 Caco-2 cells do not predict response Beiseigel et al. Am J Clin Nutr 2007
Conclusions In vitro Caco-2 cell method for iron correlates in most cases with human studies in prediction of direction of response But there are exceptions More direct comparisons human-caco-2 cell studies are needed Direct measurement of iron by MS (isotopes) preferable to ferritin formation, and for basolateral transport MS.
Conclusions In vitro experiments with Caco-2 cells are still important tools to understand the measurements recorded in vivo and to suggest future experiments that can be performed in the whole organism. Further developement of the Caco-2 cell model is needed to make it more closely corresponding to the in vivo situation.
Usefulness of animal models Rat: Suckling rat models useful to predict zinc absorption in humans. Less useful for iron as inhibitors (phytate polyphenols) and enhancers (ascorbic acid) have less effect. Hb repletion can be used to rank iron compounds Pig: Pig seems to be a quite good model for humans, but cannot be used for screening. Scarcity of comparisons between pig and humans.
Humans: Radioisotope methods Sensitive methods to study absorption of iron, zinc, calcium etc from single meals. Standardized for some minerals. Radiotracers added to foods (extrinsic or intrinsic labelling) Retention in blood determined by whole body counting Measurement of incorporation of isotopes after 2 weeks determined in RBC. Single meal exaggerates differences, multiple meals preferred.
Humans: Stable isotope methods Adding stable isotopes to test meals and measure incorporation into RBC after 2 weeks using mass spektrometri Safe (no radioactivity),but quite large amount of isotopes is needed. Too expensive for intrinsic labelling Not good for studies of native mineral, as added mineral changes molar ratio of food component: mineral.
Conclusions In vitro studies can make useful contributions to predict bioavailability in humans. Results from in vitro studies must allways be confirmed in vivo. Evidence generated from human studies must form the bases for policy decisions.