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
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Assessment of mineral bioavailability
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In vitro screening, predictors of
absorption
Animal models Humans
Mineral content, inhibitors, enhancers Dialysability/solubility Caco-2 cell model
Rat hemoglobin repletion (Fe) Rat 55Fe, 59Fe Suckling rat (65Zn) 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
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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
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Hunt JR. Int J Vitam Nutr Res 2005;75:375-84
Food factors
Iron solubility/complex (phytate, polyphenols ascorbic acid,
organic acids)
Lumen
Food factors (Fe , AA, Ca,polyphenols) Enterocyte
Host factors Iron status (hepcidin) Infection, inflammation
Blood
Iron absorption
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, NaHCO3, pH 7)
• Soluble or dialysable mineral is measured.
• Development of computer-controlled gastrointestinal model. pH gradient. (Minekus et al 1995)
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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.
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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
0 2 4 6 8 10 12 14
0 10 20 30 40 50
TIM Dialysable Fe % Human Fe absorption %Human Fe absorption %
Davidsson et al, 2001 Fredriksson et al, 2001 Hurrell et al, 1998
Dialysable Fe TIM/Fe absorption from meals with fresh/fermented vegetables
Sandberg et al, 2004 30
25 20 15 10 5
Percentage dialysed Fe Percentage absorbed Fe
TIM:Expensive time consuming, laborous, large volumes
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
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Combined digestion Caco-2 cells. Uptake/absorption measured
Caco-2 cell monolayer Dialysis membrane MWCO 12-14 kDa Culture well
Food preparation Pepsin digestion 1h, 37C, pH2 Pancreatic - bile digestion 2h, pH 7
Diffusion of soluble Fe
Caco-2 cell model for iron availability/bioaccessability
(Glahn R. J Nutr 2008)
• 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
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Intracellular effects Iron,AA,Ca,polyphenols Solubility
complex
determinant of uptake
Hepcidine control effects not
measured
Figure 1: Intestinal iron transport
LIP
e- Fe3+
ferritin
Fe2+
e-
Tf
Fe3+ Fe3+
haem
Fe2+
HO DMT1
Hp IREG1
Fe2+
Dcytb DRA
Fe3+
lumen plasma
HCP1
LIP
e- Fe3+
ferritin
Fe2+
e-
Tf
Fe3+ Fe3+
haem
Fe2+
HO DMT1
Hp IREG1
Fe2+
Dcytb DRA
Fe3+
lumen plasma
HCP1 Dcytb DRA
Fe3+
lumen plasma
HCP1
Caco-2 cell model for iron uptake
contains uptake and transport proteins
Sharp et al, 2003
Usefulness of Caco-2 cell model
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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
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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).
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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 (IP6,IP5) - 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
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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.
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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)
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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)
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Comparing human and Caco-2 cell iron bioavailability in maize ACR &TZB
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Beiseigel et al. Am J Clin Nutr 2007
Women Caco-2
Absorption relative to TZB
Caco-2 cells predict response in humans
Beiseigel et al. Am J Clin Nutr 2007
Comparing human and Caco-2 cell iron bioavailability in white & colored beans
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Beiseigel et al. Am J Clin Nutr 2007
Women Caco-2
Absorption relative to great northern
Caco-2 cells do not predict response
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.
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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.
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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.
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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.
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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.
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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.
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