November 1, 1999

8 Min Read
Bioavailability Testing  with Tracer Labeling



Bioavailability Testing
with Tracer Labeling
November 1999 -- Cover Plus

By: Robert P. Heaney, M.D. and
Connie M. Weaver, Ph.D.

  In Food Product Design's March 1999 issue, the article "The Importance of Bioavailability," by Andrea Platzman, correctly stressed the importance of assessing bioavailability of calcium (or other nutrients) added as fortificants to various foods. The author favored use of in vitro methods, noting that tracer labeling was too difficult to be practical. Because of the importance of this issue, we would like to present our experience with tracer labeling and offer a somewhat different point of view.

Tracer-based labeling

  Unfortunately, in vitro assessment of such characteristics as solubility has been shown to bear little relationship to absorbability in vivo. Nor does the presence of antiabsorbers, such as phytate or oxalate, in a food reliably predict calcium absorbability. Fortunately, tracer labeling is often very feasible, and when available, it can be relatively inexpensive.

  Either stable or radioactive isotopes may be employed. Usually, the radioactive tracers will be less expensive and more precise. For calcium, they confer cumulative radiation doses on the order of only a few percent of natural background - less, for example, than the radiation dosage received flying cross-country. Radioactive tracers thus can safely be used in healthy, nonpregnant adults. Stable isotopes are needed for testing in children and pregnant or lactating women. Whichever tracer is used, the labeling of fortified foods and the interpretation of the test results are the same.

  Isotopic labeling of calcium sources is accomplished in three ways:

  1) extrinsic labeling of the end-use product;
  2) intrinsic labeling of the food source during development; and
  3) intrinsic labeling of the fortificant.

  If the source is a solution, as is the case for calcium-fortified orange juices, extrinsic labeling suffices to achieve uniform distribution of tracer through all the ions of the bulk carrier calcium. It turns out that the same extrinsic labeling approach is valid for milk, where the physical chemical state of the calcium is much more complex. For more complex foods, and for solid foods generally, extrinsic labeling does not produce uniform tracer distribution and hence may produce erroneous estimates of bioavailability (usually spuriously high).

  Intrinsic labeling of a food is accomplished by introducing the isotope into the nutrient stream of the organism concerned, such as adding tracer to the nutrient solution used for growing plants in hydroponic systems. The developing plant tissues thus incorporate the tracer along with the bulk calcium carrier into whatever calcium moieties may exist in the plant tissues. Weaver has had extensive experience using this approach with a wide range of plants, including cereals, legumes, tubers and green leafy vegetables. While it would doubtless be too expensive to set up such a system for a single application, several laboratories around the United States do this sort of labeling on a contract basis at nominal cost.

  Intrinsic labeling of the fortificant involves introducing the tracer into a calcium solution, and then precipitating the desired salt by adding reagents containing the desired anion. For example, intrinsically labeled calcium carbonate can be readily produced by addition of sodium carbonate to a solution of labeled calcium chloride. In the same way, labeled calcium phosphates, calcium citrate, calcium lactate, and even complex salts such as calcium citrate malate, can be easily synthesized. The labeled fortificant, once dried and powdered, is combined with other manufacturer-supplied ingredients to produce a reasonable facsimile of the final fortified food.

  We have had extensive experience with these methods, producing calcium-fortified yogurts, candies, breads, pastries, ready-to-eat cereals, beverages and snack bars, as well as straightforward calcium supplement tablets. Each application presents its own challenges, most related to matching the physical characteristics of the synthesized fortificant to those of the bulk material used by the food processor.

Measuring Iron Bioavailability In Vitro

    At the Agricultural Research Service's (ARS) U.S. Plant, Soil and Nutrition Laboratory in Ithaca, NY, an in vitro "artificial gut" is helping make iron-bioavailability testing of foods and crops faster and more cost-effective. Raymond P. Glahn, a human physiologist with the agency, has devised an artificial gut using caco-2 cells, which are believed to provide an accurate model of absorption due to their resemblance to epithelial cells that line the human small intestine. In the ARS's August 1999 Agricultural Research publication, Glahn says that the system "was validated by reproducing effects similar to those consistently observed in human studies. Every time, the model matched, on a relative basis, those effects known to occur in humans."

  In Glahn's system, caco-2 cells are placed in the bottom of culture wells, then covered by a dialysis membrane that functions like the mucus layer that protects human intestinal cells. Food and digestive enzymes are placed on top of the membrane. This prevents the enzymes from reaching the cells, while nutrients and minerals pass through. Next, the amount of ferritin in the caco-2 cells is measured. "We have demonstrated that ferritin formation is a highly sensitive and accurate measurement of iron uptake," says Glahn. Finally, comparing the amount of absorbed iron to the amount originally present in the food yields the iron bioavailability for that food.

  One of the uses for this system is measuring the iron availability in food products. So far, Glahn and his team have investigated rice cereal, breast milk, infant formulas and iron supplements for available iron. In one study, they determined that when vitamin C is added to infant rice cereal (in a 2:1 ratio of vitamin C to iron), the amount of available iron increases. In another study, the researchers determined that human milk contains a lower level of citric acid than cow's milk. This citric acid decreases iron availability, and Glahn says that even when a iron-uptake promoter is added, the effect of citric acid is difficult to overcome. He suggests that if manufacturers could find an economical means to decrease citric-acid concentration, the iron availability of cow's-milk-based formulas could be improved.

  The system might also measure bioavailability on the crop level. Researchers could screen varieties of rice, beans, wheat and corn for improved iron availability, for example. "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," says Glahn. "Our goal is to use agriculture to its fullest extent to alleviate iron deficiency."

- Heidi L. Kreuzer

Data interpretation

  Interpretation of tracer-based bioavailability data is based on the fact that the tracer is not normally present in the body, and when introduced into the food, it enters the body along with the bulk carrier. Thus, any tracer found in body fluids (serum, urine, or even whole body regions, the latter measurable with gamma-emitting isotopes), will be there as a direct result of absorption. For stable calcium isotopes, which are always present wherever the carrier is found, it is the excess tracer over the natural percent abundance of the isotope that serves to reflect the amount of absorbed calcium.

  Various algorithms are used to adjust measured levels of tracer for differences in body size, as well as various sampling times (e.g., from one to five hours after ingestion). In general, early times reflect mainly the active transport component of absorption, while later time points better capture total absorption, a quantity more closely related to the notion of bioavailability. Generally, a single blood sample is all that is required, but in those instances where classic pharmacokinetic data may be desired, multiple blood samples can be taken, and quantities such as AUC (area under curve) calculated and compared.

  When absorbability relative to some reference calcium source is the desired outcome variable, a cross-over design is usually employed. Each subject is tested two (or more) times, and under these circumstances, no body size adjustment is needed, since relative blood tracer levels provide the desired information.

  Once a suitably labeled product has been prepared, a good estimate of its bioavailability can be provided by testing as few as 12 to 18 subjects, and a two-way comparison can be accomplished by paired testing at two- to four-week intervals.

  No robust theory for calcium bioavailability exists. Hence, it is very hard to predict whether a calcium salt introduced into a given food matrix will exhibit the desired bioavailability. Under these circumstances, there is little substitute for direct testing. The tracer-based methods are a cost-effective way to do this.

  Robert P. Heaney, M.D., F.A.C.P., F.A.I.N., is a professor at Creighton University, Omaha, NE. He is an expert in the field of calcium nutrition research, and has worked for over 40 years in the study of osteoporosis and calcium physiology. A member of the Panel on Calcium and Related Nutrients, he was involved in the development of the new Dietary Reference Intakes for these nutrients, and his laboratory has done bioavailability testing for many of the major food companies in North America.

  Connie M. Weaver, Ph.D., is a professor and head of the department of foods and nutrition at Purdue University, West Lafayette, IN. She was a member of the National Academy of Sciences' Food and Nutrition Board panel that developed new recommendations for calcium and related-mineral requirements. She serves as immediate past president of the American Society for Nutritional Sciences, is a scientist advisor to NASA, and is on the board of trustees of the International Life Sciences Institute.

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