Darrell Van Campen’s research while affiliated with Cornell University and other places

Within the scientific agricultural community it is widely known that the total micronutrient content of soils is not a useful measure of the amount of `available' micronutrients to plants. Thus, soil tests have been developed to determine the amounts of micronutrients in soils available to plants for growth. This same concept applies to plant foods eaten by humans because not all of the micronutrients in plant foods are available (i.e. bioavailable) for absorption and or utilization. Antinutrients and promoter substances within plant foods that can either inhibit or enhance the absorption and/or utilization of micronutrients when eaten. As a result, numerous techniques have been developed to determine the amounts of bioavailable micronutrients present in plant foods when consumed in mixed diets with other dietary constituents that can interact and affect the micronutrient bioavailability. Unfortunately, micronutrient bioavailability to humans fed mixed diets is still a confusing and complex issue for the human nutrition community. Our understanding of the processes that control micronutrient bioavailability from mixed diets containing plant foods is relatively limited and still evolving. It remains the subject of extensive research in many human nutrition laboratories globally. This article reviews some of the numerous methodologies that have arisen to account for the bioavailability of micronutrients in plant foods when eaten by humans.

We have applied an in vitro digestion/Caco-2 cell culture model to the assessment of iron availability from human milk and a generic cow's milk-based infant formula. Experiments were designed to determine the availability of iron from human milk relative to infant formula and whether known promoters of iron absorption would increase Caco-2 cell iron uptake and availability from the infant formula. In addition, we sought to determine if decreasing the citrate concentration in the infant formula would increase the iron uptake. Although approximately twice as much iron was in solution from digests of the infant formula relative to that of human milk, smaller or equal amounts of iron were taken up from the infant formula relative to the human milk digest. These results are qualitatively similar to in vivo studies. Addition of known iron uptake promoters to infant formula did not enhance Caco-2 cell iron uptake from the infant formula digest, indicating that the iron in the infant formula existed predominantly in a tightly bound unavailable form(s). Enzymatic pretreatment of the infant formula with citrate lyase and oxalacetate decarboxylase decreased the citrate concentration by 67% and resulted in a 64% increase of iron in solution, which corresponded to a 46% increase in the cell iron uptake. Iron uptake from the "low citrate" formula plus cysteine was 102% greater relative to the nontreated formula. The results indicate that too much citrate can reduce iron uptake, particularly if it is present at concentrations greater than promoters such as ascorbic acid and cysteine.

Human and animal studies have shown that amino acids and peptides influence iron absorption from the intestinal lumen. This study was conducted using Caco-2 cell monolayers as the experimental model to determine whether similar effects on iron absorption occur. Conditions were chosen to mimic the pH of the intestinal lumen and the most likely order whereby ferric and ferrous forms of iron would combine with various amino acids and dipeptides resulting from protein digestion. We demonstrated the enhancing effect of cysteine and reduced cysteinyl glycine on iron uptake by Caco-2 cells. The addition of glutathione to the transport media had no effect on uptake from ferrous or ferric iron complexes, nor did it affect iron solubility. Cysteine and reduced cysteinyl glycine increased iron solubility when added to a solution containing insoluble iron. This effect is different from that of ascorbate, which must be combined with soluble ferric iron at pH 2 to reduce and solubilize iron. Taken together, these observations are evidence that cysteine and reduced N-terminal cysteine peptides are capable of enhancing iron uptake from soluble and insoluble ferric iron. These results qualitatively reflect those observed in human studies. Our results indicate that glutathione requires digestion to Cys or Cys-Gly in order to promote iron uptake. The similarity between this study and human studies further reinforces that the Caco-2 cell model is a useful tool in studies of iron absorption and bioavailability.

A whole-body radioassay method was used to assess effects of supplemental dietary methionine (Met) on absorption by rats of zinc (Zn) provided in test meals labeled either extrinsically or intrinsically with 65Zn. Intrinsically labeled meals contained kernels harvested from two genotypes of corn (Zea mays L.) grown in 65Zn-labeled nutrient solutions. One genotype had kernels with normal Met content (Nor-Met corn) (2.0 g Met per kg) and the other had Met-rich kernels (High-Met corn) (3.2 g Met per kg). Rats fed extrinsically labeled meals absorbed about 57, 67, 73 and 71% of the 65Zn dose when a soy-based diet contained either 0, 1, 2 or 3 g of Met added per kg diet, respectively. Absorption of extrinsic 65Zn from test meals was not affected by additional Met when dietary protein was provided by egg whites. Supplemental Met enhanced absorption by rats of intrinsic 65Zn in kernels of both the Nor-Met and High-Met corn genotypes. As indicated by absorption of 65Zn, the bioavailability to rats of intrinsic Zn in the kernels was similar regardless of the Met content of the corn. However, the Zn concentration in High-Met kernels (30 μg/g) was greater than that in the Nor-Met corn (22 μg/g) so that rats fed High-Met corn ingested and absorbed more Zn from the test meals than did rats fed the Nor-Met corn. Zinc absorption was depressed in rats fed a soy-based diet without added Met, and these rats were marginally deficient in Met. The Met status of experimental animals may be an important consideration in studies conducted to assess Zn bioavailability. Selection of seeds and grains with increased amounts of Met may enhance Zn bioavailability and thereby increase the nutritional value of plant foods. Copyright © 1996 Elsevier Science Inc.

Caco-2 cells in culture provide an attractive model for the study of human iron absorption. Because iron status has a marked effect on human iron absorption, we devised serum-free growth conditions that allow manipulation of Caco-2 cell iron stores while maintaining growth. Caco-2 cells were cultured in serum-free media containing 0-20 micromol/L added iron. Intracellular ferritin, measured by radioimmunoassay, increased 100-fold with the addition of 20 micromol/L iron to the serum-free growth medium. Iron uptake and transfer across Caco-2 cell monolayers were measured from balanced salt solutions of ferrous and ferric forms of iron. Uptake from ferrous, but not ferric, iron was inversely related to cell ferritin concentration and culture medium iron concentration. Kinetic analysis of uptake data from solutions of ferrous and ferric iron revealed saturable and nonsaturable components for ferrous iron, but only a nonsaturable component for ferric iron. Uptake by the nonsaturable pathway was not affected by cell ferritin concentration for either form of iron. Maximal uptake from a ferrous iron solution via the saturable pathway was nearly 100% greater in cells cultured under low compared with high iron conditions. Iron transfer across Caco-2 monolayers was not proportional to iron uptake, but was related to monolayer permeability. Iron uptake by Caco-2 cells was a reliable indicator of relative iron availability. We observed no difference in iron transfer that was related to the iron status of the cell monolayer. The lack of this effect suggests that this model may be inadequate for studies of iron transfer.

A model for the rapid assessment of iron availability was developed that combines in vitro digestion with iron uptake by Caco-2 cell monolayers. In this method, samples (beef, ascorbic acid, or citric acid) were adjusted to pH 2, labeled with 59Fe, and subjected to pepsin digestion (pH 2, 37°C) for 1 h to simulate gastric digestion. Next, a dialysis bag (12,000–14,000 molecular weight cutoff) containing 150 mM PIPES buffer (pH 6.7) was placed in the digest and incubation continued for 30 min. Then, a pancreatin-bile mixture was added, and incubation was continued for an additional 2 h. The contents of the dialysis bag were removed and an aliquot applied to Caco-2 cell monolayers. After a 60 min incubation, iron that was non-specifically bound to the surface of the monolayer was removed by rinsing with a solution containing bathophenanthrolinedisulfonic acid and sodium dithionite. Cells were then counted for 59Fe activity to measure uptake. Beef and ascorbic acid enhanced Caco-2 cell iron uptake, whereas citric acid had no effect. These results compare favorably with literature reports of human studies and suggest that a dialyzable factor(s) less than 14,000 daltons, released during beef digestion, was responsible for the iron absorption-enhancing properties of beef. We believe that this system will be useful for studying basic mechanisms of iron absorption and for in vitro estimation of iron bioavailability.

We developed a model for assessing iron bioavailability from foods which combines simulated peptic and intestinal digestion with measurement of iron uptake by Caco-2 cell monolayers. Our objective was to further validate this model by determining if meat enhances Caco-2 cell iron uptake relative to casein. Caco-2 cell monolayers were covered with Hank's balanced salt solution (HBSS) buffered with HEPES, pH 7.4. An upper chamber was created over the cells by fitting the bottom of a Costar Transwell insert with a 12,000-14,000 molecular weight cut-off dialysis membrane. This membrane allowed low molecular weight iron complexes to diffuse into the media bathing the cells and prevented damage to the cells from the digestive enzymes. Prior to digestion, each sample (homogenate of beef, chicken, fish or casein) was mixed with 59FeCl3 to achieve an iron concentration of 10 mumol/L. Following pepsin digestion (pH2), pH was adjusted to 7.4, pancreatic enzymes and bile extract were added to each digest, and an aliquot was then introduced into the upper chamber of the culture dish. During this intestinal digestion period, 59Fe uptake occurred from iron that dialyzed into the lower chamber. The 59Fe uptake from beef, chicken and fish digests was 300-400% of the 59Fe uptake from a casein digest. Our results parallel human absorption studies indicating that meat enhances iron absorption. The results suggest that digestion products of the meat proteins were at least partially responsible for the enhancement of iron uptake. Overall, this study supports the usefulness of our model as a means of assessing iron bioavailability.

A whole-body radioassay method was used to assess effects of supplemental dietary amino acids on absorption by rats of zinc in two varieties of corn kernels labeled either intrinsically or extrinsically with 65Zn. Corn genotypes were a normal type (Nor-corn) with one row of cells in the aleurone layer of the kernel and a variety (Mal-corn) that had multiple rows of cells in the aleurone. In experiment 1, rats fed a basal diet containing 15% soy protein absorbed 64% of the 65Zn provided extrinsically in test meals. When provided test meals that contained intrinsically labeled Nor-corn or Mal-corn kernels, the rats absorbed 64 and 57% of the intrinsic 65Zn, respectively. In rats fed diets that contained supplemental lysine, methionine, or both lysine and methionine, absorption of extrinsic 65Zn averaged 69, 82 and 86% of the dose, respectively. In experiment 2, rats fed the basal diet absorbed 57 and 53% of the intrinsic 65Zn in Nor-corn and Mal-corn kernels, respectively, but rats fed supplemental methionine or cysteine absorbed 73% of the intrinsic 65Zn in either the Nor-corn or Mal-corn kernels. Supplemental methionine or cysteine fed to rats that were marginally zinc deficient enhanced the absorption of 65Zn provided in test meals in the form of either intrinsically labeled corn kernels or as 65Zn-labeled zinc sulfate.

Iron absorption is inversely related to iron stores in animals. Our objective was to determine whether iron absorption in a Caco-2 cell model also responds to iron stores. Cells were cultured on microporous membranes in standard serum-based medium for 13 d. The medium was then replaced with serum-free (SF) media, containing either 0 or 20 uM iron, and were cultured for an additional 22 d. The media were removed and iron uptake from 59Fc-labeled solutions of 10 uM ferrous ascorbate, ferric NT A, or FeCl3 was determined from 59Fe counts in the cells following 1 hr exposure. Iron transfer was determined from counts that passed through the monolayer to the bottom chamber. Monolayer integrity and permeability were assessed by transepithelial electrical resistance (TEER) and 14C-mannitol transfer measurements. Iron uptake from FeCl3 and ferric NTA was not affected by iron concentration in the culture media. However, iron uptake from ferrous ascorbate was greater in cells grown in the low iron medium (1.4410.16 pmol·μg-1·h-1) than in the high iron medium (0.73±0.09 pmol·μg-1·h-1) Transfer across Caco-2 monolayers was not affected by iron concentration in the culture media for any of the iron sources These data suggest that iron uptake by Caco-2 cells is better than iron transfer for modeling iron absorption in animals.

Iron uptake by Caco-2 cell monolayers is commonly assessed by incubating the cells under radiolabeled iron solutions, removing the radiolabeled solution, rinsing to stop uptake and measuring the radioactivity retained by the cells. It is therefore essential to differentiate between iron that is nonspecifically bound to the cell surface from that which has been taken up by the cell. We report here on a method for removal of surface-bound iron from Caco-2 cell monolayers. We used a 140 mmol/L NaCl, 10 mmol/L PIPES, pH 6.7 solution containing 5.0 mmol/L sodium dithionite (Na2S2O4) and 5.0 mmol/L bathophenanthroline disulfonic acid to reduce, remove and chelate iron bound to the cell surface. We validated our method by demonstrating the removal of 97% of an insoluble iron complex from the apical surface of Caco-2 cell monolayers. Our data indicate that the removal solution does not damage the apical membrane and thereby does not have access to intracellular iron; thus only surface bound iron is removed. The remaining cell-associated iron represents that which has been transported into the cell. We present data on the uptake and nonspecific binding of iron from iron complexes of both ferrous and ferric forms, and show that iron removal treatment resulted in uptake measurements that agree more closely with accepted principles of iron uptake by intestinal epithelium. The iron removal method used in this study should provide investigators with a valuable tool for accurately determining iron uptake by epithelial cells in culture.