Iron biofortification and revisiting the accuracy of extrinsic labeling in studies of iron absorption.

Extrinsic isotopic labeling of the iron in a food or meal is a method that has been used many times over the past few decades to assess iron bioavailability. The primary assumption of this methodology is that the extrinsic label exchanges or equilibrates fully with the intrinsic iron of the food or meal. In 1983, Consaul and Lee (1) published a critical review of this methodology. They concluded that “.this method cannot be considered proven with regards to all types of foods. .At present it is not known how completely the different nonheme forms of iron are labeled by an extrinsic tag.” Since 1983, only 2 studies (2, 3) have revisited the issue of validity and accuracy of extrinsic labeling of iron in foods, with both studies comparing varieties of beans. In one study (2), comparison of absorption of iron from the intrinsic iron and the extrinsic iron isotope was used as the measure of iron exchange. In this study, iron absorption was very low (i.e., 1–1.8%), and no statistical difference in absorption was observed. The authors broadly concluded that “.extrinsic labeling may be used to screen various varieties of beans for iron bioavailability in humans.” In the other study (3), in vitro digestion was used and extrinsic and intrinsic iron was directly measured in supernatant and pellet fractions of the digests. These authors found that extrinsic iron does not always equilibrate well with the intrinsic iron of beans. Taken together with evidence summarized by Consaul and Lee (1), there are clearly times when the primary assumption of extrinsic iron labeling is not met, yet since 1983, a multitude of studies have used extrinsic labeling of food iron, and there are no published results in these studies that demonstrate testing of the degree of isotopic exchange. As a scientist working in the field of iron nutrition, I am concerned by what appears to be broad acceptance of methodology based on an unreliable and improperly tested assumption. The recent publication by Petry et al. (4) is an example in which this methodology has again been used without evaluating the exchange of the extrinsic label with the intrinsic iron of the food (i.e., bean) and/or the complete meal. Petry et al. cite several references (2, 5–8) as validation of extrinsic labeling methodology. I encourage readers to review those references carefully and consider the different bean and staple crop varieties, the different meal components, and the various instances that indicate misequilibration of extrinsic and intrinsic iron. For example, in Cook et al. (5; Tables III, IV, and V), there are many results in which the extrinsic-to-intrinsic iron absorption ratios are different from a value of 1. Moreover, let us keep in mind that in these studies themeasure of iron exchange or equilibration was based solely on absorption. Absorption is an indirect measure of equilibration and clearly it is possible that similar absorption values can be obtained from intrinsic and extrinsic iron without full equilibration of the 2 sources of iron. For example, in a bean meal, the intrinsic iron could be primarily in the iron phytate form or complexed to cotyledon proteins, whereas the extrinsic iron could be bound to polyphenols emanating from the seed coat, with the end result being a similar percentage absorption of each isotope. To my knowledge, no published studies exist to demonstrate this possibility, but clearly the possibility exists. This scenario would be even more likely when iron absorption values are very low, such as <5%, which is common with human iron absorption. In a thorough search of the literature, I can find no studies other than the one by Jin et al. (3) that test equilibration directly. All published results on this subject tested iron exchange via absorption. I therefore question how extrinsic labeling can be considered valid for a broad range of foods when the primary assumption of isotopic exchange has seldom if ever been directly tested. Moreover, because equilibration has seldom been tested directly, and given the many examples of misequilibration, one could argue that there is no available literature to indicate where extrinsic labeling is a valid and accurate technique. The important evolution of the biofortification strategy for alleviating iron deficiency over the past decade warrants careful and accurate assessment of iron bioavailability from staple food crops. Numerous varieties of iron-biofortified staple food crops are being developed that can differ dramatically in phytate and polyphenolic profiles, and these lines need to be accurately evaluated and monitored in the diets of the regions for which they are targeted. Given the evidence mentioned above, it seems careless to assume that because extrinsic labeling of iron may have worked well under one set of conditions, it can be considered valid for all food combinations. Extrinsic labeling of iron can have a role in studies of iron absorption from staple food crops because it has certain practical advantages; however, this method must be validated for each application, perhaps via an in vitro digestion method (3), because the method appears to bring with it a major assumption that is not always met, and it can therefore result in misleading information.