Developing countries are undergoing an epidemiologic transition, characterized by the growing number of obesity and non-communicable diseases and a considerable reduction of malnutrition. Paradoxically, the population still suffers from micronutrient deficiency, known as hidden hunger. Poor diet leads to the deficiency of several minerals essential for human health, highlighting iron (Fe) and zinc (Zn), as well as vitamin A, which together or alone constitute public health problems [1
Iron deficiency is considered to be one of the most important nutritional problems in Brazil, resulting in iron deficiency anemia, and should be investigated early, since the deficiency occurs gradually and progressively in the body. One of the main symptoms is the delay of neurophysiological development, reduction of intellectual capacity, and physical weakness [2
]. Additionally, the subclinical and clinical deficiency of vitamin A is the most prevalent vitamin deficiency, especially in children, resulting in night blindness. It has its role in cell proliferation and differentiation, and a positive effect on iron uptake in the body [4
Strategies to combat nutritional deficiencies have been implemented along with food re-education, one of them—the biofortification of foods—developed with the intention of improving the nutritional quality of crop products, increasing the concentration of nutrients through breeding of genetically modified plants [5
Biofortification programs have been developed worldwide, and in Brazil, the BioFort network has concentrated the efforts on the enrichment of cassava, rice, beans, cowpea, potatoes, sweet potatoes, pumpkins, corn, and wheat [6
]. The cowpea is enriched with Fe and Zn [3
], and cassava with pro-vitamin A, β
Several factors influence the bioavailability, and therefore, it has been well studied to understand the metabolism, identifying factors that may have positive or negative actions in nutrients absorption [11
]. Studies indicate that there is a positive interaction of vitamin A with the iron metabolism, participating in the regulation of some proteins involved in the absorption of this mineral [13
].The mechanisms of iron homeostasis are determined by gene expression of proteins related to iron metabolism. For instance, the duodenal cytochrome b (Dcytb) acts by reducing ferric iron (Fe3+
) to the ferrous form (Fe2+
), which is transported through the enterocyte membrane by the divalent metal transporter 1 (DMT1). The Ferroportin exports iron to plasma cells, converting ferrous iron (Fe2+
) back to ferric iron (Fe3+
) through hephaestin to be bound to transferring [15
]. Therefore, it is important to evaluate this interaction effect and the factors that positively influence it, for a better comprehensiveness of this biofortification system. Thus, this study aimed to evaluate the effect of pro-vitamin A on the bioavailability of iron in biofortified cowpea and cassava mixture, compared to their conventional counterparts, by using the depletion-repletion animal model and the gene expression of proteins related to iron metabolism.
The chemical composition of the flours of the raw materials is shown in Table 3
shows iron content, β
-carotene, phenolic compounds, phytic acid, phytate/iron molar ratio, and fiber raw materials.
The iron contents of 43.83 and 52.41 g/kg were found in conventional and biofortified cowpea beans, respectively. This increase on iron content was below the target for biofortification, although it was 19.6% higher than the conventional, which may contribute to reach the daily recommended intake of iron.
Although the phytate content did not significantly differ among the cowpeas, the phytate/iron molar ratio of the biofortified cowpea was 15% higher than the conventional. Also, the phenolic content did not differ between them, but the content on the biofortified cowpea was 1.42 mg/100 g, compared to 0.04 mg/100 g on the conventional.
The content of β-carotene found was considerably high in biofortified cassava at the concentration of 7.6 μg/g.
3.1. Animal Assay
The group that consumed biofortified beans and cassava showed a significant increase in body weight gain, compared to the other groups (Table 5
There was no significant difference between the control and test groups for food consumption. The diets were formulated to provide 12mg/kg of iron, according to AOAC [16
]. It is of great importance to perform the hemoglobin regeneration efficiency, which will consider iron consumption and hemoglobin gain (Table 6
) of the animals in each group.
The hemoglobin concentration of the depletion phase did not differ significantly between the groups, since the animals were grouped to maintain the hemoglobin mean homogeneous. All animals recovered partially from the anemia caused during the depletion period. Thus, the relationship between the hemoglobin gain and the iron consumed was verified (Table 7
3.2. Biomolecular Analysis
The BBCC group (6.28 ± 2.68) had greater liver gene expression of ferritin (Figure 1
a) and the BBBC group (0.67 ± 0.5) had lower gene expression.
The transferrin transporter (Figure 1
b) was less expressed in the groups containing conventional cowpea, CBCC (0.27 ± 0.19) and CBBC (0.33 ± 0.18), suggesting lower iron release to be absorbed in enterocytes. This result was compatible with the bioavailability found in this study, which had lower absorption, although not significant in the groups with conventional cowpea, regardless of the vitamin A presence.
There was no significant difference in mRNA expression of DcytB (Figure 2
a).The iron transporter in the enterocyte, DMT-1, also did not differ between groups (Figure 2
b), although the groups with biofortified cassava, CBBC (1.69 ± 2.67) and BBBC (2.06 ± 0.99), presented numerically superior values to the control group and the groups that contained conventional cassava.
There was no significant difference between the groups fed with cowpea beans, regardless of the presence of biofortified cassava, as for the mRNA of hephaestin (Figure 2
c). However, the groups containing biofortified beans, BBCC (0.56 ± 043) and BBBC (0.47 ± 0.39), did not differ from the control (Ferrous sulfate).
As in hephaestin, in the biomolecular analysis of ferroportin (Figure 2
d), the groups with biofortified beans were more expressed, BBCC (1.01 ± 0.64) and BBBC (0.59 ± 0.37), carrying higher iron content of the enterocytes into the bloodstream, confirming the greater bioavailability of these groups.
The biofortified cowpea (BRS Arace) presented an increase of approximately 19.5% in iron content in relation to conventional cowpea (BRS Nova Era). In parallel, the biofortified cowpea had higher levels of phytates, phenolic compounds, and fibers in relation to conventional cowpea.
Mucosa of rats fed biofortified cowpea with iron was similar to the mucosa of rats fed ferrous sulfate regarding the expression of the hephaestin and ferroportin proteins, suggesting a greater efficiency in the intestinal absorption of iron. On the other hand, the expression of transferrin in the groups fed conventional cowpea was lower when compared to the control group, ferrous sulfate, which indicates lower absorption of iron in these groups. The expression of ferritin in the liver was lower in the group fed with both biofortified foods, BBBC, which may indicate a greater mobilization of hepatic iron.
There was great variation regarding protein, carbohydrate, and moisture contents between the two types of cowpeas (Table 3
). The content of protein, lipid, ashes, and moisture found in the beans were consonant to those found in the literature. Brigide [28
] found, in irradiated beans, 23.9 g/100 g of protein. A study by Bigonha [29
] evaluated biofortified BRS Agreste and Pontal varieties and found protein levels of 23.40 g/100 g and 21.01 g/100 g, respectively. Vaz-Tostes et al. [30
], in BRS Pontal (biofortified) and Perola (conventional) varieties of beans, found protein levels of 18.36 g/100 g and 21.51 g/100 g, respectively.
Variations in the compositions may occur because of the location in which they were grown and the environmental conditions. According to a study by Marinho, Pereira, and Costa [31
], cowpea beans have a slightly higher protein content than common beans (Phaseolus vulgaris
), varying from 20% to 30%. It is important to evaluate the protein content, as it may increase the bioavailability of iron due to the need of iron transporters during absorption [32
There was no great variation in protein, lipid, ash, and moisture contents between the two types of cassava (Table 3
). The protein content found in cassava flour was higher than the studies of Feniman [33
] (0.70 g/100 g) and Dias and Leonel [34
] (0.71 g/100 g). There was variation between the carbohydrate contents: the conventional cassava presented a higher content in relation to the biofortified one, such difference is due to the high total fiber content of the biofortified cassava (Table 4
Tako et al. [35
] found higher contents of iron, 78.8 mg/kg iron in red beans, whereas Correa [36
] found results similar to this study, with 57.8 mg/kg in biofortified Aracê
cowpea. In addition to the antinutritional factors, there may be a change in the composition of the food, due to the cooking process, reducing the content of minerals such as iron and zinc [37
Vaz-Tostes et al. [30
] used conventional and biofortified beans to evaluate the bioavailability of iron and found iron contents similar to this study, 60.52 and 52.43 mg/kg of Fe, biofortified and conventional, respectively. Different from Pachón et al. [38
], that between biofortified and conventional beans does not present significant difference.
Mezette et al. [39
] found a similar result in cooked cassava roots, with iron contents of 6.2 to 10.9 mg/kg, the variation occurred due to the different genotypes. A component that reduces the bioavailability of iron is phytate, which acts to form insoluble complexes and, thereby, decreases absorption [40
Tako et al. [41
] also found higher phytate content in biofortified beans, but due to the higher amount of iron in the biofortified ones, the phytate:iron ratio was higher in conventional beans [42
]. The phytate:Fe molar ratio indirectly evaluates the bioavailability of iron: when the result is greater than 1, it means that there may be low bioavailability. In this study, all foods were greater than 1. For the studied raw material, the values were between 4 and 7, except for conventional cassava, where the result was 25.41. The diets containing biofortified cassava (CBBC) and (BBBC), had a lower phytate/Fe molar ratio, 4.22 and 4.68respectively, in relation to diets containing conventional cassava (CBCC, 6.67) and (BBCC, 6.84). Thus, lower interference in the bioavailability of iron in diets with higher carotenoid content. According to Correa [36
], the molar ratio found for conventional and biofortified cowpea beans suggests that phytate may compromise the bioavailability of iron.
Another factor that negatively influences iron absorption is the phenolic compounds. One of the factors that increase the content of these compounds is the maceration and the cooking of the grains, because with the solubility of the phenolic compounds, they are released [43
].In addition to the iron content, its bioavailability should be considered, and may be influenced by several factors, mainly by other food components present in the diet [32
].Fibers can also exert negative effects on the absorption of iron, it can bind to the ions of minerals and prevent this absorption. In addition, the fiber, by exerting influence of intestinal transit and decreasing the time, can reduce the absorption of minerals [45
The content of β-carotene found in this study was similar to the results obtained by EMBRAPA with a content of 8.73 μg/g [46
], possibly due to a greater presence of trans β-carotene, which is predominant in biofortified cassava [47
]. Berni et al. [47
] found similar values, 6.4 μg/g, even in cooked biofortified cassava, with the same processing performed in the raw material of this study.Silva et al. [48
] conducted a study with conventional Cerrado
cassava, finding a value of 2.31 and 3.40 μg/g respectively, in the Pretinha
and BRS Dourada
varieties, respectively. In comparison, the β-carotene content used as a reference in this study was 9 μg/g, a target of the cultivars of the BioFORT biofortification network. Reduction of pro-vitamin A carotenoids may be a consequence of the exposure time on the heat treatment and the presence of oxygen to which they were subjected, such as drying [49
The group that consumed biofortified beans and cassava showed a significant increase in body weight gain, compared to the other groups (Table 5
). This difference shows that the biofortified food diet meets the needs of animals, providing a more effective weight gain. According to Toaiari et al. [50
], this difference in weight gain may be due to the bioavailability of iron in the diets’ tests, which is requested for young animal growth. Iron deficiency, resulting from the long period of depletion, can lead to an absorption difficulty, resulting in less weight gain in animals [51
]. Food consumption of the BBBC group was similar to the other groups, but FER was higher than the control ferrous sulfate group, which means a better conversion of the energy consumed intobody weight gain in animals fed biofortified foods [52
The literature shows that vitamin A helps iron absorption, but in this study, there was no significant difference (p
< 0.05) in hemoglobin gain per iron consumed in the different groups. The efficiency of hemoglobin regeneration showed no significant difference between the groups, regardless of the presence of cassava (Table 7
). Bigonha [29
] evaluated that bioavailability of the cultivars BRS Agreste (84.6 mg/kg) and BRS Pontal (96.4 mg/kg) biofortified beans was not significant in relation to the control group (ferrous sulfate), demonstrating efficiency of the beans in the hemoglobin recovery.
One factor that positively influences the evaluation of iron bioavailability in rats is the ability to synthesize ascorbic acid, which may lead to higher absorption of iron. Another factor is the presence of the enzyme phytase, which increases the bioavailability of minerals, due to the capacity to undo the phytate chelates with minerals. Although this animal model may overestimate the bioavailability, it is favorable because of the easy handling and allocation, besides the low cost, as well as the aid in the definition of genotypes that can be improved in biofortification programs [53
Murray-Kolb et al. [54
] used the depletion/repletion method in piglets, with diets based on white and red beans, and they did not obtain a significant difference in the final concentration of hemoglobin between the groups. Arruda et al. [13
] observed that diets deficient in vitamin A for 57 days influenced lower weight gain and higher concentration of hemoglobin in animals.
The BBCC group (6.28 ± 2.68) demonstrates that there was a greater accumulation of iron in the organism, possibly associated with the lower pro-vitamin A carotenoid content of the conventional cassava (Figure 1
a). On the contrary, the BBBC group (0.67 ± 0.5) had lower gene expression and may be associated with vitamin A activity in the release of iron from the hepatic reserve (Figure 1
a). According to Martini [55
], there is an inverse relationship in the presence of retinol and iron in the liver Ferritin is responsible for iron storage, when there is iron excess in the cells, being able to avoid oxidative damages.
Dias et al. [56
], when evaluating the ferritin gene expression, found lower expression of ferritin in the liver of animals fed with iron-biofortified beans (BRS Pontal), without addition of pumpkin or sweet potato biofortified with beta-carotene, contrasting the results of this study. They also found greater expression of DcytB in the group that contained biofortified beans associated with vitamin A, contrasting the findings of this study.
The iron transporter in the enterocyte, DMT-1, also did not differ between groups (Figure 2
b). It is possible that the higher carotenoid content may have aided in the mobilization of iron, as pointed out in the studies that affirm the role of vitamin A in iron metabolism [57
]. Moreira [60
] did not find significant differences between the groups that were treated with 35mg of a Fe/kg diet with and without vitamin A, suggesting that vitamin A does not act directly at the levels of DMT-1.
The hephaestin of the groups containing biofortified cowpeas, BBCC (0.56 ± 043) and BBBC (0.47 ± 0.39) (Figure 2
c), did not differ significantly from the control, so it is assumed that ferrous iron was converted to ferric in the basolateral membrane, resulting in greater iron uptake in these groups. In the study by Dias et al. [56
] on the other hand, the presence of carotenoids in the bean diet promoted greater gene expression of hephaestin, observing the influence of vitamin A on iron absorption.
Ferroportin was more expressed in the groups fed with biofortified cowpeas, BBCC (1.01 ± 0.64) and BBBC (0.59 ± 0.37) (Figure 2
d). Silva [10
] found lower expression of ferroportin in the group that contained Chia, responsible for its higher iron content. In the study by Dias et al. [56
], the higher pro-vitamin A carotenoid content in the diet influenced the induction of ferroportin.
The expression of these proteins depends on the stage of depletion of the animal, the iron intake, and its bioavailability. Therefore, the values may have been lower because the analyses are done at the end of the experiment, when the animals already have hemoglobin levels recovered, even if these proteins are partially associated with the iron deficiency compensation in the body. As the animals were not anemic in the collection of organs, they may not report the effective function of these transporters [56