Riego localizado

The study was carried out at the Division of Human Nutrition, Wageningen University, The Netherlands, among sixteen apparently healthy young women. Participants were recruited from the student population of Wageningen University through advertisements. The inclusion criteria were: age 18-40 y; body weight < 65 kg; non-pregnant, non-lactating and not planning to become pregnant; free of chronic illnesses and not taking chronic medication except oral contraceptives; not taking supplemental iron; no intake of vitamin and mineral supplements in the last two 2 wk prior to the study, or willingness to discontinue; no blood donation in the last 6 mo preceding the study, and no participation in studies that administered enriched stable iron isotope labels. Eligible participants were invited for information sessions where the study, requirements for participation, and risks were explained after which an informed consent was obtained. A sample size of sixteen participants was estimated to be adequate to detect an intra-individual variation in log (iron absorption) of 0.3 ⁽¹⁶⁾ and a 35% increase in iron bioavailability with a power of 0.8 and a significance level of 0.05. Ethical approval was obtained from the Medical Research Ethics Committee of Wageningen University, the Netherlands, and the Internal Review Board of Noguchi Memorial Institute for Medical Research, Ghana.

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Study design

A paired crossover study design was used to allow within-participants comparisons. Cowpea based test meals (Tubani) labelled with ⁵⁷Fe or ⁵⁸Fe were administered over two pairs of consecutive days. Therefore each participant consumed a total of four test meals (Table 1) in a randomized fashion. On d 0, participants were invited to the study location, a baseline blood sample (7 mL) was collected, and weight and height were measured following standard procedures ⁽¹⁷⁾. On d 1 and d 2, the test meals were administered. Fourteen days later, on d 15, a second blood sample was collected after which a third (d 16) and fourth (d 17) test meals were administered. Fourteen days later, on d 30 of the investigation, a final blood sample was collected.

Table 1. Description of cowpea test meals consumed by participants¹

Test meal Description

WC-EDTA White cowpea meal (low polyphenol) + 4 mg ⁵⁷Fe as NaFeEDTA WC-FS White cowpea meal (low polyphenol) + 4 mg ⁵⁸Fe as FeSO4

RC-EDTA Red cowpea meal (high polyphenol) + 4 mg ⁵⁷Fe as NaFeEDTA RC-FS Red cowpea meal (high polyphenol) + 4 mg ⁵⁸Fe as FeSO₄

1 RC-EDTA, red cowpea plus NaFeEDTA; RC-FS, red cowpea plus ferrous sulphate; WCEDTA, white cowpea plus NaFeEDTA; WC-FS, white cowpea plus ferrous sulphate

All meal ingredients were purchased and cooked in bulk and used for the entire study. The

test meal portions were kept frozen until use. Participants consumed the four test meals as breakfast after an overnight fast each time and were not allowed to eat or drink until 3 h post feeding. All test meals were served between 0700 and 0900 h under close supervision of investigators. Each portion was defrosted for 1.5 min in a microwave oven on the day of feeding. The isotopically labelled iron compounds were added quantitatively to the test meals

~ 5 min prior to consumption. Isotope labels were carefully spread on the surface of test meals. After consuming the entire portion of the test meal, the glass bowl used to serve the test meal was rinsed with Milli-Q water (Millipore SAS, Molsheim, France) for the

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participants to drink. Test meals were served with a maximum of 300 mL of Milli-Q water (Millipore SAS, Molsheim, France).

Test meals

The test meal served to all participants was Tubani: a local Ghanaian dish made from cowpea (Vigna unguiculata (L.) Walp) flour. There were two different Tubani test meals: one Tubani test meal was made from cowpea known to have high concentration of polyphenols (red variety, “San i Zei”) and the other made from cowpea known to have low concentration of polyphenols (white variety, “Kom tuya”). The white variety was chosen because it is widely cultivated and most preferred for consumption, and the red variety was chosen because of its higher concentration of PP. Each portion (150 g) of test meal contained 66 g of whole cowpea flour, 13 mL of 5% (w/v) food grade sodium bicarbonate solution (serves as softener and rising agent) mixed into a paste in 71 mL of Milli-Q water (Millipore SAS, Molsheim, France). The resulting paste was wrapped in aluminium foil and steamed at 100^oCfor 45 min.

Each Tubani was served with a standard amount (31 ± 1 g) of sauce made up of groundnut oil, salt, fried onions, chili and “false sesame seeds” (Ceratotheca sesamoides). In each type of test meal 4 mg of ⁵⁷Fe or ⁵⁸Fe was added as either NaFeEDTA or FeSO₄, respectively.

Test meal analysis

Iron concentration of cowpea seeds, cowpea flour and cowpea meal were measured using inductively coupled plasma atomic emission spectrophotometer (ICP-AES, Varian Vista-Pro, Palo Alto, CA, USA) after digestion with HNO3-HF-H2O2. Phytic acid determination was done using a modified Makower method ⁽¹⁸⁾ in which the released inorganic phosphate is measured according to the van Veldhoven’s method ⁽¹⁹⁾ and expressed as inositol hexaphosphate (IP6). A modified Folin-Ciocalteau method ⁽²⁰⁾ was used to measure total polyphenol concentration of the cowpea meal and expressed as gallic acid equivalent (GAE).

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Preparation of stable isotope labels

Isotopically labelled ⁵⁸FeSO4 was prepared from isotopically enriched elemental iron (⁵⁸Fe, enrichment 99.6%, Chemgas Boulogne, France) by dissolution in diluted sulphuric acid. The solutions were stored in Teflon® containers and flushed with nitrogen to keep the Fe in the +II oxidation state. Isotopically labelled Na⁵⁷FeEDTA (⁵⁷Fe enrichment 97.6%, Chemgas Boulogne, France) was prepared according to the method described by Loots et al. ⁽²¹⁾.

Blood analysis and iron isotope measurements

Haemoglobin concentration in whole blood was measured on the day of blood collection using a Beckman-Coulter LH750 HmX haematology analyzer (Beckman Coulter, Miami, FL, USA). Serum ferritin (SF), soluble transferrin receptors (sTfR), C-reactive protein (CRP) and α1-acid glycoprotein (AGP) were measured simultaneously using an in-house sandwich ELISA technique ⁽²²⁾. All measurements were done in duplicates and if CVs were ≥10%

measurements were repeated. The CVs (inter-assay) for the various indicators were: SF, 2.6%; sTfR, 2.4%; CRP, 7.2% and AGP, 2.9%. Certified quality control samples from the CDC/Atlanta and Bio-Rad Liquicheck (Bio-Rad, Munich, Germany) were used.

Whole blood samples were mineralized and separated as described by Schoenberg and von Blanckenburg ⁽²³⁾. Iron isotopic analyses were performed employing a high-resolution, multicollector-inductively coupled plasma-mass spectrometer (Thermo-Finnigan Neptune, University of Bonn, Germany; see ⁽²⁴⁾ for details). Copper was added (1 µg/g) to the solution immediately prior to analysis to correct for mass bias ^{(23, 25)}. Each isotopically enriched solution was measured in triplicate using standard sample bracketing (23, 25, 26)

. One third of the samples were re-measured as external duplicates for quality control. Analysis was done under chemical blank monitoring using ⁵⁷Fe or ⁵⁸Fe indicator solutions as an external quality control.

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Calculation of iron absorption

Iron absorption measurement was based on erythrocyte incorporation of iron stable isotope labels 14 d after intake of the labelled test meals ⁽²⁷⁾. Circulating iron was calculated on the basis of blood volume, which was estimated from the participant’s height and weight ⁽²⁸⁾. Calculation of iron absorption was based on the shift in the isotopic ratios after a 14-d incorporation period as previously described ⁽²⁷⁾ taking in to account that isotopic labels are not monoisotopic ⁽²⁹⁾. An incorporation rate of 80% of the absorbed iron into red blood cells was assumed.

Statistical analysis

Data were analyzed in Excel (Microsoft Office 2007, Microsoft, Seattle, USA) and GraphPad Prism version 5.04 for Windows (GraphPad Software, San Diego, CA, USA) and SPSS (version 18.0, Armonk, NY, USA). Variables were checked for normality and if not normally distributed were log transformed before use in analysis. Student’s t test was used to compare iron, PA and PP concentration of the red and white cowpea meals. A general linear model (Two-Way ANOVA) for repeated measures was used to evaluate the main effects of variety and fortificant as well as their interaction (V x F). Correlations between ln(SF) and ln(% iron absorption) were checked using Pearson correlation. The level of statistical significance was set at P < 0.05 for all analysis. Summary values of iron status and iron absorption are reported as geometric means. Fractional iron absorption data for participants was standardized to an SF value of 15 µg/L with the method proposed by Cook et al.⁽³⁰⁾.

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RESULTS