In the human absorption study, we used a randomized, crossover, single-blind study design. The order of the test meal and reference meal administration was randomized. The primary outcome was FAFe, which was determined as incorporation of isotopic iron labels into erythrocytes 14 to 32 days after the administration of iron-stable isotope-labeled fortified test meals to fasting Ghanaian children (n = 26).

Each participant consumed six different kinds of isotopically labeled test meals, and one kind of meal was provided in a series of 10 servings over five consecutive days (two meals per day) followed by the administration of a different kind of meal on days 8, 14, 36, 43, 50 following the same regimen (Fig. 1), resulting in a total number of 60 servings per participant. The six different meals were based on rice extruded with (i) 57FePP and ZnSO4 (57FeZnSO4); (ii) 54FePP and ZnO (54FeZnO); (iii) 57FePP, ZnSO4, CA, and TSC (57FeZnSO4CT); (iv) 54FePP, ZnO, CA, and TSC (54FeZnOCT); (v) 58FePP, ZnO, CA, and EDTA (58FeZnOCE); and (vi) no iron or zinc, but fortified with 58FeSO4 added as solution before consumption (reference). Each extruded rice batch also contained a vitamin premix (vitamin A, folic acid, and vitamins B1, B3, B6, and B12; table S2B), and the meals were accompanied with a bean or a tomato sauce. Each meal contained 2 mg of iron as labeled FePP or FeSO4 and 3 mg of zinc as ZnO or ZnSO4. As the fortification level was consistent with a moderate dietary iron content, we did not expect changes of iron status during the duration of the feeding study. Furthermore, isotopic signals in RBC are constant after approximately 10 days of incorporation (consistent with RBC precursor maturation in the bone marrow) and are then invariant for the duration of RBC life span (120 days on average).

A randomization schedule was designed allocating a random number to a list of possible meal administration sequences. This took into account that meal sequences administered with the same stable isotopes had to be administered at least 21 days apart. Eligible children were individually randomly assigned a number corresponding to one of the generated meal administration sequences.

Inclusion criteria for the human absorption study assessed at baseline were as follows: (i) ages 6 to 8 years and (ii) presence of IDE [defined as erythrocyte zinc protoporphyrin/heme (ZnPP/H) >43 μmol/mol] with or without anemia [defined as hemoglobin (Hb) <11.5 g/dl]. Exclusion criteria were as follows: (i) severe underweight or wasting (Z score weight-for-age and weight-for-height < −3), (ii) chronic or acute illness, (iii) regular intake (>2 days) of iron-containing mineral and vitamin supplements within 2 months before onset of the study, and (iv) blood donation or comparable blood loss in the 4 months preceding the study.

On study days, the first serving was administered in the morning between 0700 and 0800 after an overnight fast and the second serving was administered ~3.5 hours after completion of the first serving. All meal administrations were supervised. After meal consumption, the empty bowls were rinsed with a total of 20 ml of water and the participants received 250 ml of water; all water had to be consumed by the participants to ensure complete intake of isotopes. Participants were not allowed to eat or drink between the test meals. Approximately 1.5 hours after the second serving, the participants received a standardized snack (wheat flour cookies, Mass Industries Ltd., Tema, Ghana) and had to refrain from eating and drinking for another 1.5 hours. Thereafter, they were allowed to eat and drink ad libitum. If the next day was a study day, ad libitum eating and drinking were allowed until 2000 and 0000, respectively.

During screening (baseline measurements), body weight and height of the participants were measured and blood samples were collected to assess iron status [Hb, ZnPP/H, plasma ferritin (PF), plasma CRP, soluble transferrin receptor (sTfR), body iron stores (BIS), hepcidin, AGP, and retinol binding protein (RBP)] and malaria parasitemia. Before each venipuncture, the body temperature of the children was measured with a digital thermometer (OMRON Healthcare Europe B.V., Nigeria) to help identify children who may have symptomatic malaria. All participants received anthelmintic treatment (albendazole, 400 mg), and participants positive for malaria antigens received antimalarial treatment (arthesunate-amodiaquine, 100 mg/270 mg). Measurements for body weight and height, body temperature, as well as blood collections were repeated on days 33 and 68.

The study test meals were designed on the basis of commonly consumed rice meals in the study area and consisted of two meals per day (50 g of raw rice each) served alternately with either a tomato sauce or a cowpea-based sauce. The composite test meals were prepared at the school kitchen of SOS children’s village in Tamale, Ghana, and contained 49.5 g of raw unfortified rice and about 500 mg of raw extruded rice (433 ± 1.4, 529 ± 0.5, 508 ± 0.6, 481 ± 0.5, 509 ± 0.6, and 498 ± 2.0 mg of extruded rice for meals 54FeZnO, 57FeZnSO4, 54FeZnOCT, 57FeZnSO4CT, 58FeZnOCE, and reference, respectively). Raw unfortified rice and extruded rice were mixed and cooked in a small industrial oven for 37 min at ~150°C in separate glass bowls for each participant. After cooking the rice, 499 ± 4.0 μl of a FeSO4 solution containing 2 mg of Fe labeled with 0.2 mg 58Fe was added to each reference meal. The solution was produced from enriched elemental iron (99.86% 58Fe enrichment), as previously described (23).

Tomato and bean sauces were prepared in three and two batches, respectively (table S3), and kept frozen until 1 day before a meal administration when they were thawed. On each administration day, the sauces were reheated and 50 g of sauce was added to each cooked rice meal. Each sauce was given once per day (with either the first or second serving). The meals were transported in cooling boxes to Dungu Primary School, where each meal was administered to its corresponding participant.

Iron contents in the unfortified rice, extruded rice, and vegetable sauce were analyzed by atomic absorption spectrophotometry (Agilent Technologies GTA 120 or AA240FS) after mineralization by microwave digestion (TurboWave, MLS GmbH) using nitric acid. PA and PP contents were determined as previously described (40) with the only difference that fat was extracted from the sauces with petroleum ether before PA determination. The AA content in the vegetable sauce was analyzed by high-performance liquid chromatography (Acquity H-Class UPLC System, Waters AG) after stabilization and extraction in metaphosphoric acid and reduction by dithiothreitol.

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