The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Summary

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Abstract

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte.

Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

Introduction

To fully understand the research need and benefit of the Caco-2 cell bioassay for Fe bioavailability, one must first understand the approaches that were in place prior to the advent of this model. The measurement of Fe bioavailability from a food or meal in vivo is a challenging task, particularly when combinations of food need to be assessed in a meal or diet. Isotopic labeling has been the most common approach for the measurement of Fe bioavailability over the past 50 years¹. Isotopic labeling is used for single-meal and multiple-meal studies and is impractical for long-term studies. Stable isotopes of Fe such as ⁵⁷Fe and ⁵⁸Fe are the most commonly used; however, studies have been conducted with radioisotopes such as ⁵⁹Fe, utilizing whole-body counting². For plant foods, isotopic labeling has been done via extrinsic or intrinsic labeling. For extrinsic labeling, a known amount of isotope is added to the food or meal. The food is then mixed, and a 15-30 min equilibration period is incorporated into the protocol prior to the consumption. Hydroponic culture-adding the isotope to the nutrient solution to incorporate it into the plant while it grows and develops-is required for the intrinsic labeling of plant foods. The pros and cons of each approach are discussed below.

Extrinsic isotopic labeling
In the early to mid-1970s, human Fe absorption was studied by extrinsic labeling of Fe in foods, wherein a known amount of isotope is added to the known amount of Fe in the food or meal, mixed, and equilibrated for 15-30 min before measurements. Various amounts of extrinsic isotopes have been used, ranging from 1% to 100% of the intrinsic Fe, but most commonly in the range of 7%-30%³. Extrinsic labeling is based on the assumption that the extrinsic Fe isotope gets fully equilibrated with the intrinsic Fe of the food or meal. Extrinsic isotope absorption is then measured, and each atom of the extrinsic isotope is calculated to represent a given number of intrinsic Fe atoms. This calculation is based on the relative molar amounts. In 1983, multiple validation studies of the technique were summarized in a review paper⁴. Validation of the technique was done by simultaneously comparing the percent absorption of the extrinsic isotopic label to the percent absorption of an intrinsic isotopic label. Thus, a ratio of the extrinsic to intrinsic absorption close to 1 suggests that each pool of Fe was equally absorbed. At the time, a ratio close to 1 was also considered to represent equilibration of the extrinsic isotope with the intrinsic Fe of the food or meal. Ratios of extrinsic to intrinsic Fe absorption ranged from mean values of 0.40 to 1.62, with a mean (±SD) ratio of 1.08 ± 0.14 in 63 comparisons. It is important to note that, in all of the studies summarized in this review, none directly tested the equilibration of the extrinsic label with the intrinsic Fe. In summary, the authors of the review concluded the following:

"The extrinsic tag technique has proven valid for several foods under certain experimental conditions. But, this method cannot yet be considered proven with regards to all types of foods. The extrinsic tag method is not appropriate for monitoring iron absorption from a diet that contains insoluble forms of iron. The validity of this technique relies upon the basic assumption that the extrinsic tag exchanges completely with all endogenous nonheme food iron. At present it is not known how completely the different forms of nonheme iron are labeled by an extrinsic tag. This is important in light of studies which have suggested that iron inhibitors may affect the extrinsic tag differently than some forms of nonheme iron in foods. Research on food factors which can impair a complete isotopic exchange is scant. Thus, interpretation of bioavailability data from extrinsic tag research requires consideration of inhibitors of exchange which may be present in the food or diet."

Since 1983, only two studies have been published that evaluated the accuracy of extrinsic labeling of Fe^3,5. In both these studies, the equilibration of an extrinsic isotopic label was directly compared with the intrinsic Fe of the foods, which, in these studies, were staple food crops. White, red, and black bean varieties were tested, along with lentils and maize. Using established in vitro digestion techniques and the measurement of Fe solubility and precipitation, both studies demonstrated that extrinsic isotopic labeling does not consistently result in full equilibration, with evidence that, for some bean varieties, the misequilibration can be very high depending on the amount of extrinsic isotope and seed coat color³. Despite the conclusions of the 1983 review paper, extrinsic labeling studies of beans continued^{6,7,8,9,10,11,12}. None of these studies included testing the equilibration of the extrinsic label with the intrinsic Fe.

Intrinsic labeling
Intrinsic labeling of plant food for the assessment of Fe bioavailability eliminates the accuracy issues of equilibration in extrinsic labeling. However, this approach cannot yield large amounts of material because of the requirement of greenhouse space for hydroponic culture. Hydroponic culture is labor-intensive, requires a high quantity of expensive stable isotope, and often results in plant growth different in terms of yield and seed Fe concentration. Due to the cost, intrinsic labeling is only suitable for small-scale studies aimed at understanding mechanisms underlying Fe uptake or factors influencing Fe uptake from foods. Production of 1-2 kg of a staple food crop costs approximately $20,000-$30,000 for materials alone, depending on the isotope and hydroponic approach^13,14.

Given the challenges associated with isotopic labeling, investigators sought to develop in vitro approaches. Early methods utilized simulated gastric and intestinal food, coupled with the measurement of Fe solubility or Fe dialyzability as an estimate of bioavailability¹⁵. Such studies quickly found that Fe dialyzability was not a consistent measure of bioavailability as Fe can be soluble, tightly bound to compounds and, therefore, not exchangeable, leading to the overestimation of bioavailability. To address these issues, methodology to utilize a human intestinal cell line evolved, thereby adding a living component and enabling the measurement of Fe uptake¹⁶. The human intestinal cells-Caco-2 cells-originated from a human colon carcinoma and have been widely used in nutrient uptake studies. This cell line is useful as, in culture, the cells differentiate into enterocytes that function similarly to the brush border cells of the small intestine. Studies have shown that Caco-2 cells exhibit the appropriate transporters and response to factors that influence Fe uptake^17,18.

The initial studies, utilizing radioisotopes to measure Fe uptake in Caco-2 cells, were refined to measure Fe uptake based on Caco-2 cell ferritin formation. Caco-2 cell ferritin measurement enhanced sample throughput and negated issues of radioisotope handling and the equilibration of extrinsic Fe with intrinsic Fe^19,20. Measurement of Fe uptake via ferritin formation enabled researchers to study a broad range of foods, including complex meals²¹. Thus, simulated (in vitro) digestion coupled with Caco-2 cell Fe uptake provided a better physiological assessment of Fe uptake from foods. It is important to note that this model primarily determines relative differences in Fe bioavailability. Like many useful cell lines, Caco-2 cells also have shown variability in responsiveness but have maintained consistent relative differences in Fe uptake between foods. Proper technique and careful attention to detail can improve consistent cell ferritin formation response in Caco-2 cells.

The in vitro digestion/Caco-2 cell model is also known as Caco-2 cell bioassay. This assay has been thoroughly validated via direct comparison to human and animal studies²². In addition to the direct parallel comparison of the bioassay to human efficacy trials, this model has been shown to exhibit a qualitatively similar response in Fe uptake to that of humans^18,19,23. Therefore, as an in vitro approach, the Caco-2 cell bioassay warrants high credibility as a screening tool for evaluating Fe nutrition from foods. It has been widely applied to numerous foods and food products^{21,24,25,26,27,28}.

Since its inception in 1998, the Caco-2 cell bioassay has advanced the field of Fe nutrition as it has helped identify factors that influence intestinal Fe uptake. In so doing, this model has developed and refined research objectives for more definitive and less costly human studies. One could also argue that the use of the model negates the need for some human trials.

In summary, the relative delivery of Fe from a food or meal can be measured with the Caco-2 cell bioassay. Regardless of the amount of Fe in the test meal, the bioassay defines the relative amount of Fe taken up into the enterocyte-the first step of the absorption process. This is the most important step in defining Fe bioavailability, as most often the goal is to measure with the intent to improve or, at the very least, monitor the nutritional quality of Fe in a food. Given that iron status is regulated by absorption, and thus Fe uptake is upregulated in Fe-deficient individuals to meet nutritional needs, the standard conditions of the model are designed so that Fe uptake by the cells is maximal. In this way, the bioassay provides a true measure of the potential of the food to deliver Fe.

Protocol

NOTE: As a convenient point of reference for readers, the following methodology describes the specific culture conditions and materials required for the measurement of Fe bioavailability from 20 experimental samples, plus the required quality controls, in a run of the bioassay. Increasing the number of samples beyond this capacity is not recommended due to the time required for various cell culture and in vitro digestion steps within the bioassay. 1. Choosing the amount of sampl…

Representative Results

Identification and measurement of Fe bioavailability in staple food crops One of the primary reasons for developing this model was to identify factors that influence Fe bioavailability in staple food crops and provide a tool for plant breeders that would enable them to identify and develop varieties with enhanced Fe bioavailability. The common bean (Phaseolus vulgaris) has been targeted globally as a crop for Fe biofortification; thus, the model has been applied extensively to evaluate the…

Discussion

Since its inception, numerous studies have been published that describe this method for the Caco-2 cell bioassay. The basic conditions have remained relatively unchanged since the initial publication in 1998¹⁸. However, over the past 20 years, numerous technical details have been refined and standardized to yield unprecedented consistency in the response of the bioassay. Careful and precise adherence to the cell culture and in vitro digestion conditions are the key to the consistent and s…

Materials

0.5 M HCl	Fisher Scientific	A508-4 Hydrochloric Acid TraceMetal Grade
18 megaohm water			Also known as distilled, deionized water
3,3′,5-Triiodo-L-thyronine sodium salt	Sigma Aldrich Co	T6397
6-well plates	Costar	3506	Use for bioassay experiments
ascorbic acid	Sigma Aldrich Co	A0278
bile extract	Sigma Aldrich Co	B8631
Caco-2 cells	American Type Culture Collection	HTB-37	HTB-37 is a common variety.
Cell culture flasks T225	Falcon	353138
Cell culture flasks T25	Corning	430639
Cell culture flasks T75	Corning	430641U
Chelex-100	Bio-Rad Laboratories Inc	142832	Known as the weak cation exchange resin in the protocol
collagen	Corning	354236
dialysis membrane	Spectrum Laboratories	Spectra/Por 7 Pretreated RC Dialysis Tubing 15,000 MWCO	Spectra/Por 7 Pretreated RC Dialysis Tubing 15,000 MWCO
Dulbecco’s Modified Eagle’s Medium	Gibco	12100046	DMEM
epidermal growth factor	Sigma Aldrich Co	E4127-5X.1MG
Ferritin ELISA Assay Kit	Eagle Biosciences	FRR31-K01
fetal bovine serum	R&D Systems	S12450	Optima
HEPES	Sigma Aldrich Co	H3375
Hydrocortisone-Water Soluble	Sigma Aldrich Co	H0396
insert ring	Corning Costar	not sold	Transwell, for 6 well plate, without membrane
insulin	Sigma Aldrich Co	I2643
KCl	Sigma Aldrich Co	P9333
large column	VWR International	KT420400-1530
Minimum Essential Medium	Gibco	41500034	MEM
NaCl	Fisher Scientific	S271
pancreatin	Sigma Aldrich Co	P1750
PIPES disodium salt	Sigma Aldrich Co	Piperazine-1,4-bis(2-ethanesulfonic acid) disodium salt P3768
porcine pepsin	Sigma Aldrich Co	P6887 or (P7012-25G Sigma
protein assay kit	Bio-Rad Laboratories Inc	Bio-Rad DC protein assay kit 500-0116	Measurement of Caco-2 cell protein
silicone o rings	Web Seal, Inc Rochester NY	2-215S500
sodium bicarbonate	Fisher Scientific	S233
Sodium selenite	Sigma Aldrich Co	S5261
ZellShield	Minerva Biolabs	13-0050	Use at 1% as antibiotic/antimycotic ordered through Thomas Scientific