"Iron deficiency is the world's most prevalent nutrient deficiency.
Even in developed countries, it remains a serious concern for women during
pregnancy and throughout their childbearing years," says ARS human
physiologist Raymond P. Glahn. He is with the agency's U.S. Plant, Soil, and
Nutrition Laboratory in Ithaca, New York. "It is extremely important that
pregnant women and children receive proper iron nutrition," he adds.
To help correct these deficiencies, Glahn has developed an in vitro
laboratory model. "This model gut is unique," he says, "because
it couples simulated food digestion with a human intestinal cell line called
Glahn says his system is the first to accurately model in the lab what
occurs in the human intestinal tract. It allows food digestion to occur at the
same time nutrients are taken up by the Caco-2 cells. He expects the model to
have broad applications for studying staple foods like rice, corn, wheat, and
beans; food supplements; pharmaceutical iron preparations; and baby foods,
including formula, cereals, and purees.
"Simply measuring the amount of iron in foods is not adequate,"
says Leon V. Kochian, who heads the Ithaca laboratory.
"To improve our food supply, we must have some measure of how much iron
we can expect to actually absorb. We can use Glahn's model as a fast,
inexpensive method for determining the relative availability of iron from
different foods or from different crop varieties used in the same food,"
For example, the model gut could be used to screen dozens of rice varieties
for high iron availability. Cost prohibits animal studies of this typenot
to mention human trials. "Human feeding trials are very expensive,"
Kochian says. "This model can help researchers refine the experimental
design of human trials so as to make more productive use of funds. And the
model can address experimental questions that are not feasible or appropriate
to address in a human study."
Caco-2 cells resemble the human intestinal epithelial cells that line the
inner surface of the small intestine and absorb nutrients from the foods we
eat," says Kochian. "Caco-2 cells have been widely accepted by
nutritionists as a model of human absorption."
To combine digestion with cell culture, Glahn needed a way to protect the
Caco-2 cells lining the bottom surface of culture wells from digestive enzymes
and microorganisms. He achieves this by dividing the well into upper and lower
chambers on the day of an experiment. As a separator, he uses a dialysis
membrane attached to a plastic insert specifically designed to fit inside each
half-dollar-sized culture well.
In the upper chamberdirectly on the membraneGlahn places a food
sample and enzymes that digest it over a period of about 3 hours. The dialysis
membrane prevents the digestive enzymes and microbes from reaching the Caco-2
cells in the lower chamber. This mimics the role of the mucus layer that
protects epithelial cells of the human digestive tract. Nutrients and minerals
pass through the membrane to the waiting Caco-2 cells.
To find out how much of the food's iron is available to the Caco-2 cells,
Glahn measures the amount of ferritinan iron storage proteinin the
cells. "We have demonstrated that ferritin formation is a highly sensitive
and accurate measure of iron uptake," he says.
According to Kochian, "Glahn's model is expected to function well as a
screening tool for plant foods and for developing food products. With this
model, scientists can measure food-iron availability directly from the producer
or supermarket shelf."
The Model's Many Advantages
Doing the studies with cells instead of animals is cheaper and faster. Glahn
can test six experimental conditions at a time, since a culture plate holds six
"The cost per culture plate is about $4.50, compared to about $54 to
purchase six rats for animal studies," he says. "Furthermore,
collecting data takes only 3 days with the model gut, compared to 10 with the
There's another advantage of the cell-based test: Currently, researchers who
conduct most human and animal feeding studies of iron uptake have to add a
minute amount of radioactive iron to a food sample so they can track absorption
of the iron in it. Radioactive iron isn't used with Glahn's model gut.
"Our model eliminates the controversy, costs, and inconvenience
associated with food-iron radiolabeling," Glahn says. "Although a
sensitive method, radiolabeling of food iron can be expensive and
controversial. It also requires extensive training and monitoring of the
radioactivity, adding further to costs."
By avoiding radioisotopes, Glahn's model is more user-friendly for
scientists in academia, government, and private industry. It may enable many
research groups to run analyses previously beyond their means.
Glahn says "estimating cost-savings of our model system versus human or
animal trials is difficult, but we estimate it to cost 20 percent as much as an
animal trial. The savings are probably greater for a human study."
Iron for Infants
So far, Glahn and coworkers have used the cell-model system to investigate
iron availability of rice cereal, infant formulas, and iron supplements. The
coworkers are Cornell University technician Jean S. Hsu, retired ARS biochemist
John F. Thompson, and Cornell University undergraduate students Jennifer Cha,
Matt Goldman, Cindy Lai, and Olivia Lee.
For their first application of the model, they found that adding vitamin C
to infant rice cereal increased the amount of available iron. At current levels
of iron fortification, the team found that a 2-to-1 ratio of vitamin C to iron
was necessary to maximize availability of iron from the cereal.
The in vitro culture model is ideally suited for determining the relative
availability of iron in foods. It can be used to evaluate products such as
infant formula and cereals, staple foods, and dietary supplements.
In a related study, they determined that mixing the infant rice cereal with
water fortified with vitamin C increased iron availability more than mixing it
with vitamin C-fortified apple juice. "Our findings suggest apple juice
contains one or more substances that offset the vitamin's beneficial effects on
iron uptake," Glahn says.
"It's an excellent example of how the food-product industry can use
this model system to improve nutrient content and offer consumers a better
product," he says. The research was funded in part by a grant from Gerber
Products Company of Fremont, Michigan, a major baby food manufacturer.
For their next study, Glahn's team assessed iron uptake from human breast
milk and from infant formula made with cow's milk.
"Citric acid, a natural organic acid present at levels several times
higher in cow's milk than human milk, decreased iron availability," he
says. "Unless citric acid levels are significantly reduced, adding an
iron-uptake promoter, such as vitamin C, to the formula at a palatable level
would not overcome the effect of citric acid."
Glahn says manufacturers of cow's-milk-based infant formula could improve
iron availability by decreasing the citric acid concentration. "The
question is whether a company could do this cost-effectively," he says.
Plant Breeders Could Bolster World Health
The most far-reaching application of Glahn's model involves its use as a
screening tool for identifying breeding lines of staple food crops such as
rice, wheat, maize, and beans for improved iron availability. Glahn's model gut
was developed with precisely this application in mind.
"With over 2 billion people in the world suffering from micronutrient
malnutrition, we need to find sustainable ways to improve absorption of iron,
zinc, vitamin A, and iodine. One way to do this is to produce crops from which
more of these micronutrients can be absorbed.
"However, because the cost of animal and human trials limits their use
in this task," Glahn says, "we haven't had a method for screening the
hundreds of varieties that plant physiologists have identified for this
Initial screenings of rice and bean varieties indicate that this model gut
is well-suited to the task. Glahn says there's now a team using this model to
identify lines of staple foods with improved iron availability.
"This application of the model is part of a multisystem approach aimed
at disrupting the devastating effects of micronutrient malnutrition in target
populations and developing countries," he says. "Our goal is to use
agriculture to its fullest extent to alleviate iron deficiency."
Glahn says his model "was validated by reproducing effects similar to
those consistently observed in human studies. This was achieved many times
under different experimental conditions. Every time, the model matched, on a
relative basis, those effects known to occur in humans."
Glahn has applied for a U.S. patent on his model gut. He is currently
negotiating a license with a private laboratory to produce an iron
"The kit would make the unique components of this model available to
other investigators," he says.
Meanwhile, Glahn and his research team continue to apply, adapt, and refine
their model. Glahn believes it can eventually be used to measure
bioavailability of other micronutrients, such as vitamin A, zinc, selenium, and
"Humans need these micronutrients in much smaller quantities than
macronutrients such as carbohydrates, fats, and proteins," he says.
"Yet, they are critical to good health." By
Hank Becker, Agricultural
Research Service Information Staff.
This research is part of Human Nutrition Requirements, Food Composition,
and Intake, an ARS National Program described on the World Wide Web at:
Raymond P. Glahn is at
the USDA-ARS U.S. Plant,
Soil, and Nutrition Laboratory, Tower Rd., Cornell University, Ithaca, NY,
14853-2901; phone (607) 255-2452, fax (607) 255-1132.
"A Gut IssueMeasuring Iron Bioavailability" was
published in the August 1999
issue of Agricultural Research magazine.