Phytic acid or phytin

Oxalic acid

Iron, zinc, Whole legume magnesium, and cereal crops calcium

Calcium Spinach

Sources: Adapted from References 22, 24, 21.

Hemoglobin Iron

Iron, zinc Vitamin A

Selenium Iodine

Vitamin E (a-tocopherol) Vitamin A

Animal red meats

Green and orange vegetables, red palm oil, yellow maize Animal meats Sea foods, organ meats, tropical nuts and cereals, amounts vary depending on soil levels Vegetable oils, green leafy vegetables changes are feasible for minor plant components. Genetic manipulation to augment the levels of these components would require minimal diversion of precursors and some modifications in the plant's ability to store or sequester the target micronutrients (16,33,34,52,53). Interestingly, the tolerable upper intake levels in humans for minerals fluctuate between 1- and 13-fold in their RDA values, whereas those for vitamins are higher than those for minerals, and, as it has been reviewed earlier, micronutrient intakes superior to RDAs, but not higher than those tolerable levels for humans, provide health benefits including genomic stability (Tables 5.1, 5.2) (3-7).

All this raises the opportunity for the molecular biotechnology as an emerging and powerful approach with the potential for improving nutritional quality of food plants, altering the composition, content, and bioavailability of the existing micronutrients [i.e., modifying chemical forms of the stored micronutrient, removing (or reducing the level of) antinutri-tional compounds, or elevating the amount of promoter substances] (Table 5.3), or accumulating novel and bioavailable minerals and vitamins in edible parts (i.e., the endosperm of cereals), which usually lack these components (18,33,34,41,49,50).


In developing nations, cereal grains such as wheat, maize, rice, and sorghum, and some legumes such as common bean and soybean, are the primary and cheap sources of essential minerals as iron, zinc, and calcium (20,37,43). Minerals with chemical similarities can compete for transport proteins or other uptake mechanisms, as well as for chelating organic substances, hindering absorption (Table 5.3) (32,54,55). In fact, it has also been suggested that antinutritional factors that interfere with proper nutrient absorption and bioavailability account for a large proportion of world wide micronutrient deficiencies (40,55).

Thus, increasing the amount of bioavailable micronutrients in plant foods for human consumption by molecular biotechnology is a challenge that is not only important for developing countries, but also for many industrialized countries. Theoretically, it could be achieved by increasing the total level of micronutrients in the edible part of staple crops, such as cereals and pulses, while simultaneously increasing the concentration of compounds which promote their uptake, for example ascorbic acid, and by decreasing the concentration of chemicals that inhibit their absorption, such as phytic acid or some phenolic compounds (Table 5.3) (22,41,43,50).

5.3.1 iron

Iron is both an essential micronutrient and a potential toxicant to cells; as such, it requires a highly sophisticated, coordinated, and complex set of regulatory mechanisms to meet the demands of cells as well as prevent excess accumulation (29). The human body requires Fe for the synthesis of the oxygen transport proteins hemoglobin and myoglobin and for the formation of heme enzymes and other Fe-containing enzymes that are particularly important for energy production, gene regulation, immune defense, regulation of cell growth and differentiation and thyroid function (Table 5.1) (28,29,43). The body normally regulates Fe absorption so as to replace the obligatory iron losses of about 1-1.5 mg per day. Thus, the body must be economical in its handling of iron, for example, when a red blood cell dies, its iron is reutilized, and excess level of iron can be stored by a specially designed protein, ferritin, which is used at times of increased iron metabolic requirements (28). In spite of these ingenious physiological approaches, iron deficiency is estimated to affect around 30% of the world population, making iron by far the most deficient nutrient worldwide. In general, the etiology of iron deficiency can be viewed as a negative balance between iron intake and iron loss. Whenever there is a rapid growth, as occurs during infancy, early childhood, adolescence, and pregnancy, positive iron balance is difficult to maintain (9,28). The blood volume expands in parallel with growth, with a corresponding increase in iron requirement (9).

Dietary iron is constituted by heme iron (animal origin) and nonheme iron (inorganic salts mainly from vegetal sources); the first one is absorbed by a distinct route and more efficiently than nonheme iron (Table 5.1). Usually, nonheme iron bioavailability is very low (less than 5-10%) due to its poor solubility and interaction with other diet components known as antinutrients (28,43). Inadequate absorption of this mineral will first lead to the mobilization of storage iron, and finally to lower hemoglobin levels or anemia (29). Iron deficiency is the most common cause of anemia and is usually due to inadequate dietary intake of bioavailable iron and/or excessive loss due to physiological conditions of parasitic infections (30,32). For example, dietary iron sources in developing countries consist mainly of nonheme iron. Because cereal and legume staples are rich in phytic acid, a potent inhibitor of mineral absorption, and in addition, the intake of foods that enhance nonheme iron absorption such as fruits, vegetables, and animal muscle tissues is often limited, these conditions may serve as major factors responsible for the anemia (Table 5.3) (21,22,43,47,49,55). The major consequences of this deficiency are poor pregnancy outcome, including increased mortality of mothers and children, reduced psychomotor and mental development in infants, decreased immune functions, tiredness, and poor work performance (25,26,30-32).

Increasing ferritin, the natural iron store protein, in food crops, has been suggested as an approach to raise iron levels and bioavailability. Ferritin is a multimeric iron storage protein, composed of 24 subunits, and has a molecular structure highly conserved among plants, animals, and bacteria (56,57). This protein is capable of storing up to 4500 Fe atoms in its central cavity, which are nontoxic, biologically available, and releases them when iron is required for metabolic functions. In fact, recent studies have demonstrated that iron from animal and plant ferritin can successfully be utilized by anemic rats and humans (56,58,59).

While staple food, such as corn and wheat flours are usually fortified with iron, rice grains present much hard problems and challenges. In addition, whole brown rice is barely consumed, and its commercial milling (polishing) produces considerable loss of micronutrients, up to 30% and 67% for zinc and iron, respectively, by eliminating its outer layers where these metals are accumulated (27).

With the aim of increasing the iron content and its bioavailability in rice, two different research groups have overexpressed a ferritin gene into its endosperm isolated from either common bean or soybean (Table 5.4) (60-62). In both cases the plant ferritin is produced at high levels and correctly accumulated in the cereal endosperm. Notably, the iron content of bean ferritin rice is 22.1 p,g/g dry weight whereas soybean ferritin rice stores up to 31.8 p,g/g dry weight, resulting in two- and threefold greater levels, respectively, than that of the corresponding untransformed crop (10-11 p,g/g dry weight). A two to three times extra iron enrichment in ferritin in transgenic grains would appear to be of nutritional significance. In fact, a daily consumption of about 300 g of the iron-rich rice by an adult would be sufficient to provide 50-75% of the daily adult requirements for this mineral, which is about 13-15 mg (Table 5.1).

Recently, Vansconcelos et al. (63), a third distinct research group, also reported the expression of soybean ferritin gene, driven by the endosperm-specific glutelin promoter, leading to higher iron accumulation in transgenic indica rice seed than control grains, even after commercial milling. They selected as target the indica rice line IR68144-3B-2-2-3, an elite line, which presents high tolerance for tungro virus and an excellent grain quality, good yield and resistance for growing in mineral-poor soils, and high iron level in the crop (15-17 p,g/g untransformed brown rice). Transgenic rice lines were obtained containing as much as 71 p,g iron/g dry weight unpolished rice. This accounts for a 4.4-fold increase in iron compared with that of the control; a two- to threefold extra iron content of transformed rice with plant ferritins would already be of nutritional relevance as noted earlier (Tables 5.1, 5.4) (60-62). But when the iron levels of the rice grains (untransformed and transgenic) were assessed after the seeds were polished, they indicated that the highest iron content of transgenic lines ranged from 19-37 p,g/g milled rice, versus control material of only 10 p,g/g milled rice. This is the first report which shows that after commercial milling the iron concentration remains higher than that of that milled negative control, and even that of untransformed brown crop. These results with transgenic rice expressing a ferritin from either soybean or bean would imply that low iron concentration in food seeds may not result from low iron availability for transport, but rather from a lack of sequestering and storing capacity in the seeds.

In order to explore and test the potential benefit of iron-improved transgenic rice incorporating soybean ferritin in its edible tissue (60), a standard hemoglobin depletion bioassay was employed with anemic rats followed by complete diets having equivalent quantities of either iron as FeSO4 (a popular compound used in anemic human beings in medical treatments) or bioengineered ferritin rice. Iron-rich rice diet was as effective as the diet containing FeSO4, and it was shown that full recovery of anemia in rats occurred after 28 days of treatment with any of the iron sources (Table 5.4) (64).

It is generally agreed that nutrients are effectively utilized from breast milk and that breast-fed infants possess a lower prevalence of infections than those fed with commercial formula. Breast milk not only provides the infant with a well balanced supply of nutrients, but also several unique components that facilitate nutrient digestion and absorption, protection against pathogenic microorganisms, and promotion of healthy growth and development. It is believed that those benefits are due in part to milk proteins (41,65,66). One of such bioactive proteins is lactoferrin. Lactoferrin is an 80 kDa iron-binding glycoprotein belonging to the transferrin family and is found in elevated levels (1-2 g/l) in human milk. Proposed biological activities for this protein include antimicrobial properties, regulation and facilitating iron absorption, immune system modulation, cellular growth activity, and antivirus and anticancer activities (41).

Rice was used as a useful bioreactor to produce, in its edible endosperm, recombinant human lactoferrin to infant food because it presents a low allergenicity, and is likely a vehicle safer than transgenic microorganisms or animals. Therefore, a human milk lactoferrin linked to a rice glutelin 1 promoter was inserted into rice cells and a very high expression level was reached in a large scale field trial (5 g of recombinant human lactoferrin per kilogram of dehusked transgenic rice), being stable for four generations. In fact, the boosting expression of lactoferrin in rice endosperm turned this cereal grains pink, as a consequence of iron bound to lactoferrin (65). The gross nutrient composition of transgenic cereal was similar to that of nontransformed rice, except for a twofold increase in iron content (negative control, 5.7 p,g/g dehusked rice; transgenic rice, 19.3 p,g/g dehusked rice) probably because each molecule of lactoferrin is able to bind two Fe31 ions (Table 5.4) (65).

Additionally, the lactoferrin purified from transgenic rice exhibited similar pi, antimicrobial activity against a human pathogen (i.e., inhibition of growth of enteropathogenic Escherichia coli, one of the most common causes of diarrhea in infants and children) and bind and release iron capacity at acidic gastric pH as those of native human lactoferrin (Table 5.4) (65,66). Lactoferrin-rich rice crops (as lactoferrin is bioactive and therefore, has the ability to store iron in a bioavailable manner) can be incorporated directly into infant formula or baby foods, and even to be consumed by people at any age; without purification

Table 5.4

Manipulation of selected micronutrients for human nutrition and nutraceutical uses by molecular biotechnology.

Table 5.4

Manipulation of selected micronutrients for human nutrition and nutraceutical uses by molecular biotechnology.

Micronutrient Molecular Approach

Improved Plant

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