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Food science

Sources and forms of iron in Nigerian foods and effects of processing on availability

Sources and forms of iron in Nigerian foods and effects of processing on availability

Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgement
References

G. Oluyemisi Latunde-Dada

The author is with the Department of Chemical Sciences of the College of Natural Sciences, University of Agriculture, in Abeokuta, Nigeria.

Abstract

The average diet in most developing countries, including Nigeria, is predominantly plant based. Cereals, legumes, tubers, and vegetables are the main food types. Although most of these food items have considerable iron, its low bioavailability is one of the factors accounting for the high incidence of iron-deficiency anaemia. The traditional processing procedures, fermentation and germination, improved the chemical and bioavailable iron in the foods studied. Techniques of measuring iron availability in vitro and in vivo were applied to a variety of foods. The advantages and disadvantages of the methods employed are discussed.

Introduction

Iron deficiency affects over 600 million people throughout the world, particularly in developing countries. The vulnerable groups are infants, young children, and women of child-bearing age [1]. The deficiency is usually due not to the absolute lack of the element in the diet but rather to its poor bioavailability. Iron availability is also influenced by the degree of iron deficiency of the individual, the adequacy of intestinal secretions, and the various components in foods that inhibit or enhance iron absorption. The majority of the population of most developing countries, such as Nigeria, rely on plant foods to provide their protein and energy requirements. The availability of iron from these plant staples is very low [2]. Traditional processing procedures also affect the availability of iron from a wide range of foods [3]. In view of the high incidence of iron-deficiency anaemia in the country [1], it is important to assess the relative contributions of different food items in the Nigerian diet to iron nutrition. Other attempts are geared towards the identification of cheap sources of meat that might further enhance the iron nutrition of the Nigerian populace.

Materials and methods

Cowpea and soya bean varieties were obtained from the International Institute of Tropical Agriculture in Ibadan, Nigeria. Other food materials were purchased from the university farm or local markets.

Total iron determination

Samples were dry ashed, and iron estimation was carried out colourimetrically as described by Hurrell et al. [4]. Non-haem iron in pigeon tissues was determined by the method described by Schricker et al. [5]. Haem iron was calculated as the difference between the total and non-haem iron in the meat samples.

In vitro availability assays

A modification of the method of Miller et al. [6] was used to estimate dialysable iron after the simulated peptic and pancreatic digestion of the food samples.

Analytical methods

Ten grams of soya bean sample was homogenized in 50 ml of deionized water. The pH of the homogenates was adjusted to 2 with 6 M HCl, and the final weight was adjusted to 100 g with 0.1 M HCl. Pepsin solution (3 ml) was added to the homogenized sample and incubated in a shaking water bath for two hours at 37°C. These 20-g aliquots of the pepsin digest were transferred into conical flasks. Segments of dialysis tubing containing 25 ml deionized water and an amount of NaHCO₃ equivalent to the titratable acidity were put into each flask [6]. The flasks were incubated in the shaking water bath for about 30 minutes or until the pH was 5. Five millilitres of the pancreatin-bile extract mixture was then added, and the incubation was continued for another two hours. After pancreatic digestion, the dialysis tubes were removed and the contents were quantitatively transferred to a 25-ml volumetric flask and brought to volume with deionized water:

TABLE 1. Iron levels of whole dehulled and cooked soya bean and cowpea varieties (mean of three samples ± SE)

Variety		Iron level (mg/100g)
		Raw whole beans	Raw dehulled beans	Cooked beans
Cowpea
	IT830-326-Z	7.15 ± 0.12	6.15 ± 0.09	5.84 ± 0.24
	Ife Brown	5.68 ± 0.17	5.19 ± 0.14	5.02 ± 0.18
	TVX 3236	4.80 ± 0.26	4.23 ± 0.30	3.20 ± 0.29
	IT82D716	6.30 ± 0.26	5.82 ± 0.35	4.57 ± 0.33
Soya bean
	TGX-996-25E	4.32 ± 0.23	3.24 ± 0.25	2.98 ± 0.30
	TGX-849-313D	2.48 ± 0.21	2.28 ± 0.20	2.90 ± 0.02
	TGX-1019-2E	4.53 ± 0.15	3.13 ± 0.15	3.28 ± 0.18
	Samsoy I	4.50 ± 0.19	3.85 ± 0.14	3.15 ± 0.16

In vivo availability assays

In vivo absorption of ⁵⁹Fe from most tissue by intragastric dosing of rats was as described previously [7]. Iron-replete male Wistar rats weighing about 250 g and caged in groups of four were used in the study. Laboratory chow (iron content 309 mg/kg) was removed from the animals overnight, and the following morning 5-ml portions of prepared pigeon test meals were given intragastrically by oral intubation [7]. Rats were killed by a sharp blow on the head and cervical dislocation 120 minutes after dosing. The abdominal cavity was opened, and ligatures were placed at the gastro-oesophageal and ileocaecal positions. Segments of the gastrointestinal tract and their contents were estimated for ⁵⁹Fe activity by removing the stomach, small intestine, and colon and carefully washing out the contents into 5-ml counting vials. The remaining gut segments were counted in 5-ml counting vials. Absorption was calculated as the difference between the ⁵⁹Fe activity administered and the ⁵⁹Fe activity recovered in the gut wall and gut contents.

Haemoglobin regeneration assays

The bioavailability of iron in some tropical cereals and legumes was evaluated by the haemoglobin regeneration technique using FeSO₄ as a reference standard [8]. All experimental diets were based on the low-iron diet, with the test material substituting for casein and maize starch to obtain the desired protein and iron concentrations. Weanling rats with initial haemoglobin of 5.92 to 6.78 g/100 ml were assigned to groups of four rats per test diet [8].

Results

Iron content of different sources of foods and their processing

The iron levels in soya bean and cowpea varieties and the different tissues of pigeon meat are presented in tables 1 and 2. Significant amounts of iron were lost as a result of dehulling and cooking the legumes. The iron content also varied with the different tissues in pigeon meat [9,10].

In vitro dialysable iron in processed food samples

Cooked cowpeas had a significantly higher percentage of dialysable iron than uncooked cowpeas (4.74% vs 2.41%; p <.05)[9].

The traditional practice of adding kanwa (an alkaline salt) to food during cooking resulted in reduced iron availability. Germination and fermentation enhanced the availability of iron from soya beans. The percentage of dialysable iron in Nigerian home-made soya products ranged from 3.91% to 13.6% (table 3). Blanching in boiling water for five minutes and squeeze washing (homogenization) of some Nigerian vegetables resulted in significant (p <.05) losses of iron (table 4). Ascorbic acid and meat enhanced the diffusibility of iron across the dialysis membranes from the tropical foods studied.

TABLE 2. Non-haem iron levels of pigeon (Columba L.) tissues using the method of Schricker et al. [5] and haem iron obtained by difference (mean of three samples ± SE)

Tissue	Non-haem iron (mg/100g)	Iron (%)	Haem iron (mg/100g)	Iron (%)
Breast	1.77 ± 0.06	36.3 ± 1.2	3.1 ± 0.09	63.7 ± 1.8
Leg	0.96 ± 0.04	39.9 ± 1.8	1.44 ± 0.02	60.1 ± 0.7
Liver	14.1 ± 0.51	74.1 ± 2.7	4.91 ± 0.58	25.9 ± 3.1

TABLE 3. Iron content and dialysable iron from some soya products (mean of three samples ± SE)

Soya product	Iron (mg/100g)	Dialysable iron (% of total)
Defatted flour a	10.5 ± 0.96	2.26 ± 0.12
Soya concentrate	10.1 ± 0.81	2.12 ± 0.08
Soya isolate	9.30 ± 0.25	2.51 ± 0.91
Soya maize snack	5.85 ± 0.29	5.06 ± 0.23
Soya cassava biscuits	6.30 ± 0.29	4.20 ± 0.1
Soya wheat biscuits	6.21 ± 0.05	3.91 ± 0.15
Soya candies	5.13 ± 0.29	5.62 ± 0.10
Extruded full fat flour	6.03 ± 0.12	6.95 ± 0.19
Soya cereal (“Soyogi”)	6.11 ± 0.06	7.95 ± 0.19
Extruded defatted flour	6.35 ± 0.18	5.11 ± 0.17
Defatted flour b	6.06 ± 0.03	5.00 ± 0.32
Fermented soya beans	4.93 ± 0.02	13.6 ± 0.15
Germinated soya beans	4.49 ± 0.01	11.0 ± 0.50

a. Solvent-extracted soya flour.
b. Screw-pressed soya flour.

The bioavailability of iron in some tropical cereals and legumes was evaluated by the haemoglobin regeneration technique using FeSO₄ as a reference standard. The relative biological values ranged from 15% to 30% for the staples (table 5). The variation in the bioavailability of iron from these cereals and legumes was presumably due to the presence within each food item of different levels of tannins, polyphenols, and fibre components.

Cooking significantly (p <.01) reduced the absorption of ⁵⁹Fe from pigeon meat (table 6). The absorption of ⁵⁹Fe from cooked and raw liver was not significantly different (p >.05). On the basis of the total iron determinations of labelled pigeon tissues, test meals containing 8 g iron per millilitre were prepared as follows: (1) A slurry was prepared by homogenizing a known weight of raw pigeon breast meat in distilled water. (2) A known weight of pigeon meat was cooked in distilled water at 90°C for 30 minutes and then homogenized. (3) Raw pigeon meat soluble extract was prepared by homogenizing a known weight of pigeon meat in distilled water and centrifuging the slurry at 8,000 rpm for 15 minutes. (4) Cooked pigeon meat extract was prepared by heating the soluble extract obtained, as for test meal 3, to 90°C for 30 min and rehomogenizing. (5) Raw pigeon insoluble residue was prepared by the same extraction procedure as in test meal 3, and a known weight of the insoluble residue was rehomogenized in distilled water. (6) Cooked pigeon insoluble residue was prepared by cooking the weighed extracted residue (test meal 3) in distilled water for 30 minutes at 90°C, followed by homogenization. (7) Pigeon meat haemoproteins were prepared by following the extraction procedure as for test meal 3; the soluble extract was heated in a water bath at 90°C for 30 minutes to precipitate the haemoproteins, and after cooking, the heat coagulum (haemoproteins) was rehomogenized in distilled water at the correct iron level. (8) Muscle ferritin was assumed to be present in the supernatant fraction after the heat precipitation of the haemoproteins from the soluble extract. The low-molecular-weight iron compounds were removed from this solution by dialysis against 10 times its volume of distilled water by stirring for 72 hours at 3°C and changing the water every 2 hours; the retentate was regarded as ferritin. (9 and 10) Raw and cooked liver meals were prepared in the same way as for test meals 1 and 2 for pigeon whole meat.

TABLE 4. Effects of processing on dialysable iron from 12 Nigerian vegetables (mean of three samples ± SE)

Vegetable	% total iron
	Vegetable alone	Vegetable plus kanwa a	Vegetable plus ascorbic acid b	Vegetable after squeeze washing	Vegetable after blanching c
Ewedu (Corchorus olitorrius)	6.26 ± 0.14	2.87 ± 0.36	12.30 ± 0.41	-	-
Ebolo (Crassocephalum crepidivides)	5.03 ± 0.58	1.78 ± 0.24	6.62 ± 0.22	-	3.18 ± 0.38
Green soko (Celocia argentea)	9.34 ± 0.64	4.57 ± 0.18	12.6 ± 1.00	3.26 ± 0.41	3.38 ± 0.42
Red soko (Celocia argentea)	7.79 ± 0.20	6.20 ± 0.33	10.8 ± 0.27	1.89 ± 0.06	2.74 ± 0.26
Okra (Hibiscus esculentus)	2.77 ± 0.17	1.88 ± 0.18	6.14 ± 0.45	-	-
Tete (Amaranthus hybridus)	10.1 ± 0.23	5.94 ± 0.47	16.9 ± 0.53	3.20 ± 0.48	4.73 ± 0.30
Gbure (Talinum triangulae)	1.84 ± 0.07	0.93 ± 0.06	6.84 ± 0.08	1.56 ± 0.17	1.39 ± 0.07
Igbo leaves (Solanum macrocapon)	5.09 ± 0.56	3.05 ± 0.31	7.14 ± 0.25	1.84 ± 0.08	4.67 ± 0.11
Igba fruit (Solanum melongena)	5.67 ± 0.08	4.92 ± 0.12	12.2 ± 0.40	3.49 ± 0.32	2.50 ± 0.06
Ewuro (Vernonia amygdalina)	3.49 ± 0.25	2.16 ± 0.45	7.09 ± 0.18	1.20 ± 0.11	2.22 ± 0.13
Ugu (Telfairia occidentalis)	9.93 ± 0.53	2.13 ± 0.02	16.5 ± 2.66	2.74 ± 0.20	4.49 ± 0.25
Ukali (Gnetum bucholzianum)	4.36 ± 0.26	1.87 ± 0.21	10.2 ± 1.17	-	-

a. Kanwa (0.05 g) was added to 10 g of vegetable.
b. Ascorbic acid (0.01 g) was added to 10 g of vegetable.
c. In boiling water for five minutes and squeeze washed.

Discussion

Traditional processing procedures affect the content and availability of iron from a wide range of staple foods in Nigeria. Precipitation and insolubilization of iron in meat during cooking have been shown to decrease availability [11]. Differences in the iron content of plant foods may be due to variety, soil conditions, and maturity at harvest. Iron in animal foods, however, is influenced by age, tissue, sex, nutrition, breed, and amount of activity performed by an animal. Significant amounts of iron will be conserved in cowpea and soya bean recipes incorporating the hulls and cooking broth [9, 10]. Most vegetables are cooked before consumption in Nigeria, which reduces the level of iron intake from these foods. Significant losses of iron occur during the processes of blanching and squeeze washing [11] of the vegetables.

TABLE 5. Iron availability from some tropical foods fed to anaemic rats for 14 days (mean ± SE for four rats)

Food	Weight gain a (g)	Food intake (g)	Haemoglobin iron gain (mg)	Bioavailability (%)
Basal b	32.6 ± 1.46	85.6 ± 1.30	0.27 ± 0.02	38.4 ± 2.26
Millet	30.1 ± 0.70	85.9 ± 1.60	0.54 ± 0.03	14.7 ± 0.73
Cowpeas	29.6 ± 0.67	86.0 ± 1.40	0.78 ± 0.06	21.0 ± 1.37
Finger millet	31.0 ± 0.87	83.3 ± 1.01	0.60 ± 0.03	17.9 ± 1.00
Sorghum	19.5 ± 0.64	66.3 ± 1.18	0.54 ± 0.05	21.3 ± 2.05
Bambra nuts	18.8 ± 0.94	66.6 ± 1.98	0.64 ± 0.10	24.5 ± 3.84
Peanuts	19.4 ± 0.71	64.7 ± 1.27	0.58 ± 0.05	24.6 ± 2.23
Maize	19.7 ± 0.51	66.7 ± 1.32	0.78 ± 0.05	30.4 ± 2.49
FeSO4 c (standard meal)	19.0 ± 0.61	99.6 ± 3.72	2.11 ± 0.06	60.7 ± 2.42

a. Weight gain for experimental period.

b. Basal diet contained the following (g/kg diet): casein, 150; dextrinized starch, 300; maize starch, 389; salt mixture, 50; maize oil, 50; B vitamin mixture, 11.

c. Basal diet plus FeSO₄.

Test meal (40g)	% 59Fe absorption
Raw meat	21.9 ± 2.90 a
Cooked meat	10.9 ± 2.10 a
Raw soluble extract	16.5 ± 1.90
Cooked soluble extract	8.98 ± 2.40
Raw insoluble residue	8.0 ± 1.60
Cooked insoluble residue	10.5 ± 2.40
Haemoproteins	7.48 ± 0.95
Ferritin	3.13 ± 0.70
Raw liver	13.9 ± 2.00
Cooked liver	16.7 ± 3.40

a. Eight rats were used.

Dietary practices that would increase the content and the availability of iron in foods include the following:

· incorporating the hulls of seeds in recipes
· incorporating broth in recipes,
· steaming vegetables rather than blanching and squeeze washing them (homogenization),
· eating rare or medium-cooked meat,
· germinating legumes and cereals,
· fermenting legumes and cereals,
· using alkali salts, such as kanwa, sparingly in recipes,
· using mixtures of foods incorporating known enhancers of iron availability.

Iron availability can be estimated in humans, in animals, or in vitro. Each of these methods has advantages and disadvantages. Studies in humans provide information most directly applicable to human populations. However, the lack of facilities for the use of radioisotopes limits such studies in developing countries. Moreover, human studies are very expensive and time-consuming. Experimental subjects object to the inconvenience and hesitate to risk exposure to radioisotopes. For these reasons, laboratory animals, such as rats, are commonly used in iron availability studies in developing countries.

However, rat studies are not easily extrapolatable to humans because of species differences in food iron bioavailability. Studies have shown that whereas humans absorb the ferrous (Fe²⁺) better than the ferric (Fe³⁺) form [15, 16], rats, guinea pigs, and dogs absorb both equally well [17]. Furthermore, rats are less sensitive than humans to dietary factors that influence the absorption of non-haem iron [18]. The authors therefore concluded that rodents should not be used to assess the quantitative importance of dietary factors in human iron nutrition. However, the rat can be used to estimate at low cost the relative in vivo bioavailabilities of iron from different foods, particularly those of plant origin.

The chemistry of iron digestion in the intestinal tract ultimately determines the bioavailability of food iron to the organism. Simulated in vitro digestion and the chemical analysis of partially digested samples from humans and animals are the only practical means of approaching the underlying chemical mechanisms that are manifested in iron bioavailability.

In vitro techniques allow studies on the chemical mechanisms influencing iron availability. They also have the advantages of cost, speed, and reduced variability for screening large numbers of samples. The relationship of iron chemistry and bioavailability, with particular relevance to contributions of in vitro studies, has been reviewed [19].

Solubility of iron is a prerequisite for absorption. Fe²⁺ is better absorbed than Fe³⁺ in solutions. Hence, factors that maintain Fe²⁺ in the soluble phase of the digestive milieu enhance iron bioavailability. Reduction of Fe³⁺ enhances iron availability, as opposed to oxidation, absorption, precipitation, and polymerization.

Although the commercial fortification of food with iron salts is the practice employed to combat anaemia in the developed world, tropical foods can also be manipulated at the home level to promote iron availability. In summary, therefore, the availability of iron from foods is influenced by a wide range of factors, which include the physiological state of the individual, the interactions of inhibitors and enhancers, and the various processing procedures employed in the preparation of the food items.

Acknowledgement

The International Foundation for Science, Sweden, funded the research reported in this article.

References

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