Zhiqiang Cheng

Nicotianamine, a Novel Enhancer of Rice Iron Bioavailability to Humans

Background Polished rice is a staple food for over 50% of the worlds population, but contains little bioavailable iron (Fe) to meet human needs. Thus, biofortifying the rice grain with novel promoters or enhancers of Fe utilization would be one of the most effective strategies to prevent the high prevalence of Fe deficiency and iron deficiency anemia in the developing world. Methodology/Principal Findings We transformed an elite rice line cultivated in Southern China with the rice nicotianamine synthase gene (OsNAS1) fused to a rice glutelin promoter. Endosperm overexpression of OsNAS1 resulted in a significant increase in nicotianamine (NA) concentrations in both unpolished and polished grain. Bioavailability of Fe from the high NA grain, as measured by ferritin synthesis in an in vitro Caco-2 cell model that simulates the human digestive system, was twice as much as that of the control line. When added at 1∶1 molar ratio to ferrous Fe in the cell system, NA was twice as effective when compared to ascorbic acid (one of the most potent known enhancers of Fe bioavailability) in promoting more ferritin synthesis. Conclusions Our data demonstrated that NA is a novel and effective promoter of iron utilization. Biofortifying polished rice with this compound has great potential in combating global human iron deficiency in people dependent on rice for their sustenance.

Testing Iron and Zinc Bioavailability in Genetically Enriched Beans (Phaseolus Vulgaris L.) and Rice (Oryza Sativa L.) in a Rat Model

A rat model was used to determine the bioavailability of iron and zinc in bean seeds and rice grain from enriched genotypes of these globally important staple foods. Seed and grain from the genotypes tested (intrinsically radiolabelled with either 59Fe or 65Zn) were cooked, homogenized in water, and lyophilized to dryness. The dried, radiolabelled powder was fed to young male rats in single meals. Bioavailability was calculated from the amount of radiolabelled iron and zinc retained in the rats over a 10-day period as determined each day by whole-body gamma spectrometry assay. The data collected demonstrate that increasing the amount of iron or zinc in enriched rice grain and bean seed significantly increases the amount of iron or zinc bioavailable to rats. Although a rat model is not ideal for determining iron and zinc bioavailability to humans, because rats are much more efficient at absorbing iron and zinc from plant foods than humans, rats can be used to give relative estimates of bioavailable iron and zinc in plant foods. These estimates can be used to rank promising genotypes of staple foods for use in later feeding trials with humans, greatly reducing the numbers of genotypes that would have to be tested in humans without use of the rat model. Ultimately, because of the complexities of determining the bioavailability to humans of iron and zinc in plant foods, human feeding trials performed under free-living conditions should be conducted with the most promising genotypes before these genotypes are released for distribution to breeding programmes worldwide

Extrinsic Labeling Method May Not Accurately Measure Fe Absorption from Cooked Pinto Beans (Phaseolus vulgaris): Comparison of Extrinsic and Intrinsic Labeling of Beans

Isotopic labeling of food has been widely used for the measurement of Fe absorption in determining requirements and evaluating the factors involved in Fe bioavailability. An extrinsic labeling technique will not accurately predict the total Fe absorption from foods unless complete isotopic exchange takes place between an extrinsically added isotope label and the intrinsic Fe of the food. We examined isotopic exchange in the case of both white beans and colored beans (Phaseolus vulgaris) with an in vitro digestion model. There are significant differences in (58)Fe/(56)Fe ratios between the sample digest supernatant and the pellet of extrinsically labeled pinto bean. The white bean digest shows significantly better equilibration of the extrinsic (58)Fe with the intrinsic (56)Fe. In contrast to the extrinsically labeled samples, both white and red beans labeled intrinsically with (58)Fe demonstrated consistent ratios of (58)Fe/(56)Fe in the bean meal, digest, supernatant, and pellet. It is possible that the polyphenolics in the bean seed coat may bind Fe and thus interfere with extrinsic labeling of the bean meals. These observations raise questions on the accuracy of studies that used extrinsic tags to measure Fe absorption from beans. Intrinsic labeling appears necessary to accurately measure Fe bioavailability from beans.

Measuring iron and zinc bioavailability in humans

Iron and zinc deficiencies are common in populations dependent on cereal-based diets because of the poor bioavailability of these minerals in those foods. Selective breeding of high-mineral grains can improve the total intake of iron and zinc. However, the additional iron and zinc from those grains may not be available for absorption because of the high phytate content of cereals. Iron and zinc bioavailability needs to be measured before the high-mineral crops are promoted. Iron or zinc bioavailability can be measured from the response of a physiological variable, assessment of body retention, tissue or blood uptake, changes in pool size, or rates of absorption. Iron bioavailability is preferentially measured from erythrocyte uptake of oral radioactive or stable iron tracers; zinc bioavailability is measured from the rate of absorption of an oral isotopic tracer compared with an intravenous tracer. The oral label, which is required for studies of both iron and zinc, may be intrinsically added to the plant during growth or extrinsically added before feeding. Iron and zinc bioavailability from intrinsically and extrinsically labelled normal and high-mineral common bean varieties was tested in young women with low iron stores. The absorption of intrinsic and extrinsic labels of iron and zinc did not differ. The bioavailability of iron and zinc from both varieties was low, about 1.5% and 13%, respectively. Methods to improve the bioavailability of iron and zinc from plant foods need to be developed.

Extrinsic Labeling of Staple Food Crops with Isotopic Iron Does Not Consistently Result in Full Equilibration: Revisiting the Methodology

Extrinsic isotopic labeling of food Fe has been used for over 50 years to measure Fe absorption. This method assumes that complete equilibration occurs between the extrinsic and the intrinsic Fe prior to intestinal absorption. The present study tested this assumption via in vitro digestion of varieties of maize, white beans, black beans, red beans, and lentils. Prior to digestion, foods were extrinsically labeled with (58)Fe at concentrations of 1, 10, 50, and 100% of the intrinsic (56)Fe. Following an established in vitro digestion protocol, the digest was centrifuged and the Fe solubilities of the extrinsic (58)Fe and the intrinsic (56)Fe were compared as a measure of extrinsic/intrinsic equilibration. In the beans, significantly more of the extrinsic Fe (up to 2-3 times, p < 0.001) partitioned into the supernatant. The effect varied depending upon the seed coat color, the harvest, and the concentration of the extrinsic Fe. For lentils and maize the extrinsic Fe tended to partition into the insoluble fraction and also varied depending on variety and harvest. There was no crop that consistently demonstrated full equilibration of the extrinsic Fe with the intrinsic Fe. These observations challenge the accuracy of Fe absorption studies in which isotopic extrinsic Fe was used to evaluate Fe absorption and bioavailability.

Evaluation of metallothionein formation as a proxy for zinc absorption in an in vitro digestion/Caco-2 cell culture model

Caco-2 cell metallothionein (MT) formation was studied to determine if MT could be used as a proxy for zinc (Zn) absorption in a cell culture model. The MT intracellular concentration was determined using a cadmium/hemoglobin affinity assay. The cellular Zn uptake was determined by acid digests (5% HNO(3)) using inductively-coupled argon-plasma emission spectroscopy. The effect of phytic acid (PA) on cellular Zn and MT concentrations was also studied. Cells were treated with a media containing 0, 2, 5, 10, 25, 50, 75 μmol L(-1) Zn (ZnCl(2)). The effect of varying the Zn:PA molar ratios (1:0, 1:1, 1:5, 1:10, 1:20) on the Zn uptake and MT formation was determined. The results showed a positive linear correlation between Zn-media concentrations and cellular Zn uptake, and MT formation was observed. Zn and MT concentrations in the cells treated with increasing levels of Zn (>25 μmol L(-1) Zn) were elevated. The Zn and MT concentrations in the cells incubated with Zn (when <10 μmol L(-1)) were similar to the untreated cells. PA significantly lowered the cellular Zn and MT concentrations. When the Zn:PA molar ratios were >1:5, cellular MT concentrations were no different to untreated cells. When a combined in vitro digestion/cell model was used, the cellular MT concentrations in white or red beans and fish samples were no different to the cell baseline. This study suggests that measurements of cellular Zn and MT concentrations have some limitations (<10 μmol L(-1) Zn). PA was observed to be a potent inhibitor of Zn uptake. Under the conditions of this in vitro model, Caco-2 cell monolayers are not useful for evaluating the Zn availability from foods.

Enhancing Beans (Phaseolus Vulgaris L.) as Sources of Bioavailable Iron through Genetic Selection

Iron (Fe) deficiency is the most prevalent nutrient deficiency in humans. Globally, this disorder adversely affecting the health, well being, and livelihood of over 2 billion people, especially those living in developing countries. Significantly increasing the bioavailable amount of Fe in staple plant foods could contribute greatly to reducing the incidence of Fe deficiency, particularly among those at greatest risk (i.e., poor women, infants and children). Fe-enriched genotypes of beans could be selected for in plant breeding programs to improve human nutrition and thereby enhance nutritional health in a sustainable manner.