European Journal of Nutrition IRON BIOAVAILABILITY AND IRON STATUS OF FOUR IRON SOURCES USED TO FORTIFY INFANT CEREALS, USING ANEMIC WEANING PIGS AS A MODEL. --Manuscript Draft-- Manuscript Number: EJON-D-18-00043 Full Title: IRON BIOAVAILABILITY AND IRON STATUS OF FOUR IRON SOURCES USED TO FORTIFY INFANT CEREALS, USING ANEMIC WEANING PIGS AS A MODEL. Article Type: Original Contribution Keywords: iron fortification; bioavailability; iron status; weaned piglets; iron salts. Corresponding Author: Carmen Martínez Graciá, PhD.M.D Universidad de Murcia Murcia, SPAIN Corresponding Author Secondary Information: Corresponding Author's Institution: Universidad de Murcia Corresponding Author's Secondary Institution: First Author: Ana María Caballero Valcárcel, M.D. First Author Secondary Information: Order of Authors: Ana María Caballero Valcárcel, M.D. Carmen Martínez Graciá, PhD., M.D Silvia Martínez Miró, M.D., Ph.D Josefa Madrid Sánchez, PhD., M.D Carlos Alberto González Bermúdez, PhD., M.D Guillermo Domenech Asensi, PhD., M.D Rubén López Nicolás Marina Santaella Pascual Order of Authors Secondary Information: Funding Information: Secretaría de Estado de Investigación, Desarrollo e Innovación (AGL2013-40617-R) Mrs. Carmen Martínez Graciá Abstract: Abstract Purpose Iron (Fe) deficiency anemia is a global health concern in young children which can be reduced by Fe fortification of foods. Cereal is often one of the first foods given to infants, providing adequate quantities of Fe during weaning. In this work, we have compared the iron bioavailability and iron status of four iron sources used to fortify infant cereals, employing piglets as an animal model. Methods The study was conducted on 36 piglets, 30 of them with induced anemia. From day 28 of life the weaned piglets were fed with four experimental diets (n=6) each fortified with 120 mg Fe/kg by ferrous sulfate heptahydrate (FSH), electrolytic iron (EI), ferrous fumarate (FF), or micronized dispersible ferric pyrophosphate (MDFP) for another 21 days. In addition, one group of six anemic piglets fed with the basal diet with no iron added (Control-) and a Control+ group of non-anemic piglets (n=6) were also studied. Blood indicators of iron status were measured after depletion and during the repletion period. The Fe content in organs, hemoglobin regeneration efficiency (HRE), and relative bioavailability (RBV) were also determined. Results The Fe salts were adequately utilized, allowing the animals to recover from the anemic state, although EI was less efficient with regard to filling Fe stores giving lower concentrations of serum ferritin and of iron in the spleen, liver, lung, and kidney. In Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation addition, the RBV of EI was 88.27% with respect to the reference iron salt (FSH). Conclusions In the animal model chosen, FF and MDFP were equally as bioavailable as the reference salt, and were used significantly better than EI. These results improve the scientific evidence that can be used to choose the best fortificant for infant cereals. Suggested Reviewers: Carmen Frontela Saseta, PhD Assistant Professor, Universidad de Murcia carmenfr@um.es Expert in iron bioavailability Reyes Barberá, PhD Professor, Universitat de Valencia reyes.barbera@uv.es Expert in iron bioavailability Francisco Rincón León, PhD Professor, Universidad de Cordoba Facultad de Veterinaria bt1rilef@uco.es Expert in iron bioavailability Susan Fairweather-Tait, PhD Professor, University of East Anglia Norwich Medical School s.fairweather-tait@uea.ac.uk Expert in micronutrients bioavailability Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript Click here to download Manuscript MANUSCRIPT.docx Click here to view linked References 1 2 3 Iron bioavailability and iron status of four iron sources used to fortify infant cereals, using anemic weaning pigs as a model. 4 5 6 Ana María Caballero Valcárcel1. CarmenMartínez Graciá1*. Silvia Martínez Miró2. Josefa Madrid Sánchez2. González Bermúdez Carlos Alberto1. Guillermo Domenech Asensi1. Rubén López Nicolás1 .Santaella Pascual Marina1 7 8 9 10 11 1Food Science and Nutrition Department. 2 Animal Production Department. Veterinary Faculty. 30100, University of Murcia, Murcia, Spain. 12 13 * To whom correspondence should be addressed TLF +34 868 88 8263 14 Fax +34 868 88 4748 15 E-mail address: mamen@um.es 16 Running tittle: Assessing iron fortificants for infant cereals 17 18 1 19 Abstract Abstract 20 Purpose Iron (Fe) deficiency anemia is a global health concern in young children which 21 can be reduced by Fe fortification of foods. Cereal is often one of the first foods given to 22 infants, providing adequate quantities of Fe during weaning. In this work, we have 23 compared the iron bioavailability and iron status of four iron sources used to fortify infant 24 cereals, employing piglets as an animal model. 25 Methods The study was conducted on 36 piglets, 30 of them with induced anemia. From 26 day 28 of life the weaned piglets were fed with four experimental diets (n=6) each 27 fortified with 120 mg Fe/kg by ferrous sulfate heptahydrate (FSH), electrolytic iron (EI), 28 ferrous fumarate (FF), or micronized dispersible ferric pyrophosphate (MDFP) for 29 another 21 days. In addition, one group of six anemic piglets fed with the basal diet with 30 no iron added (Control-) and a Control+ group of non-anemic piglets (n=6) were also 31 studied. Blood indicators of iron status were measured after depletion and during the 32 repletion period. The Fe content in organs, hemoglobin regeneration efficiency (HRE), 33 and relative bioavailability (RBV) were also determined. 34 Results The Fe salts were adequately utilized, allowing the animals to recover from the 35 anemic state, although EI was less efficient with regard to filling Fe stores giving lower 36 concentrations of serum ferritin and of iron in the spleen, liver, lung, and kidney. In 37 addition, the RBV of EI was 88.27% with respect to the reference iron salt (FSH). 38 Conclusions In the animal model chosen, FF and MDFP were equally as bioavailable as 39 the reference salt, and were used significantly better than EI. These results improve the 40 scientific evidence that can be used to choose the best fortificant for infant cereals. 41 Keywords iron fortification, bioavailability, iron status, weaned piglets, iron salts. 42 43 Introduction 44 Iron deficiency (ID) is considered to be the most common mammalian nutritional 45 deficiency in both developing and industrialized countries, and is the primary cause of 46 anemia in young children because their rapid growth leads to high iron (Fe) requirements 47 [1]. ID adversely affects the cognition and physical development of infants and children, 48 immune status, and morbidity of infections. Iron deficient anemic children are reported 49 to have lower mental, social, emotional, and motor development scores than infants with 50 normal iron status, which can result in lower school performance at 11-14 years of age 51 [2]. 52 Food fortification with iron has been recommended as one of the preferred 53 approaches in the prevention and eradication of Fe deficiency. It is considered the most 54 cost-effective population-based strategy in the long-term to combat anemia, being 55 effective with no side effects or with fewer than for supplementation [3, 4]. However, 56 fortification with sources of bioavailable iron often presents multiple challenges in 57 product acceptance, product shelf life, and effectiveness in terms of improving Fe status 58 and cost [5-8]. Infant cereals fortified with Fe are often the first supplementary food given 59 to children and can be the major source of Fe from the weaning period until  18 mo of 60 age [9, 10]. Industrially produced infant formulas are usually fortified with soluble iron 61 compounds, such as ferrous sulfate, which has been designated as the reference standard 62 source with a relative bioavailability of iron (RBV) of 100. However, in contrast with 63 infant formulas, infant cereals are difficult to fortify with soluble iron compounds because 64 of unacceptable organoleptic changes such as rancidity and color and flavor changes in 65 the products during storage. This has led to the use of less soluble, and hence less 66 bioavailable iron compounds, such as elemental iron, ferric pyrophosphate, or ferrous 67 fumarate [11]. These iron salts are recommended for the fortification of cereals-based 68 complementary foods in the WHO Guidelines on Food Fortification [12]. Due to its 69 chemical stability and low cost, electrolytic iron is commonly used to fortify commercial 70 infant cereals in the United States, whereas European manufacturers usually add ferrous 71 fumarate or ferric pyrophosphate [13], which have relatively higher bioavailability than 72 electrolytic iron. Micronized dispersible ferric pyrophosphate (MDFP) is a novel iron 73 source developed for food fortification in recent years. The main advantage of this 74 compound is the very small average size of the particles (0.5 m), and also the coating of 75 monoglycerides and diglycerides that minimize aggregation. However, the bioavailability 76 of this compound seems to depend on the food matrix [14, 15] and it is still necessary to 77 further compare iron absorption and iron efficacy for foods fortified with these 78 compounds. 79 The hemoglobin repletion efficiency (HRE) assay in animals is one of the tests 80 used most frequently to assess the relative bioavailability of an iron compound [16]. 81 Using this technique, experimental animals are made iron deficient, after which they are 82 randomly allocated into comparable groups which receive diets containing the iron 83 compounds under study. Blood is sampled at the initiation and at the end of the repletion 84 period to obtain the initial and final Hb concentrations. The feed intake is measured 85 throughout the period, to calculate the iron intake, and the blood volume is estimated from 86 the body weight; it is used to calculate the HRE, which is an indicator of iron 87 bioavailability [17, 18]. Young pigs are an excellent model for the study of human iron 88 nutrition because of similarities in the anatomy of the gastrointestinal tract, digestive 89 physiology, and diet between the two species [17, 19, 20]. The iron status of young pigs 90 can be readily manipulated by adjusting the dosage of the iron injections routinely given 91 shortly after birth. The tremendous growth rate of weaning pigs, the small iron store in 92 their body, and their low iron intake from sow´s milk allow these animals to develop an 93 “iron deficient state” in a relatively short period of time [21, 22]. 94 The aim of this study was to assess the bioavailability of four iron compounds 95 used to fortify infant cereals: electrolytic iron, ferrous sulfate heptahydrate, ferrous 96 fumarate, and micronized dispersible ferric pyrophosphate, using anemic weaning pigs 97 as a model. 98 99 Materials and Methods 100 The trial was performed at the Animal Nutrition Experimental Unit of the Farm of the 101 University of Murcia, and received prior approval from the University Ethical Committee 102 for Animal Experimentation and the Autonomic Community of Murcia Region Ethical 103 Committee (Study protocol number: A13150101). The study was carried out in 104 accordance with EU Directive 2010/63/EU for the care and use of animals for scientific 105 purposes. 106 Animal management and experimental design 107 The experimental study design is shown in Fig. 1. 108 Thirty-six newborn male Landrace piglets were selected from twelve different litters of 109 the same farm. Six of them, chosen randomly, were given an intramuscular injection of 110 iron dextran in the usual amount (180 mg Fe) on the second day of life, representing the 111 positive control treatment (Control+), and 30 were given an injection containing 30 mg 112 of Fe, so that they would develop iron deficiency by the time they were weaned without 113 markedly affecting growth [23]. The piglets were weaned at 21 days. After weaning, all 114 piglets were housed in an environmentally controlled nursery. 115 The diets were formulated to meet the National Research Council nutrient requirements 116 [24] for piglets, except for iron. The low iron basal diet consisted of basal cereal and 117 skimmed milk powder as main ingredients (Table 1), to reproduce a diet similar to that 118 of infants at weaning. The diet was presented in mash form. When the piglets reached 21 119 days of age (with an average body weight (BW) of 6.58 ± 0.15 kg), hematological 120 parameters were measured (Day 0 of the experiment). The anemic state (Hgb ≤ 8g/dl) 121 was confirmed at weaning in the piglets challenged with iron deficiency. During the first 122 week after weaning (21 to 28 days old), in order to set the same basal iron status, all 123 piglets (except the Control+ group) were fed the basal low iron diet (Table 1) in groups 124 2of six animals housed in pens of 6 m , containing two feeders and two cup waterers to 125 provide ad libitum access to feed and water. At the age of 28 days, the piglets were 126 randomly allocated to five groups of treatments (n=6). This day (the seventh of the 127 experiment) was the beginning of the repletion period (see Fig. 1). The diet treatments 128 were: the basal diet supplemented with 120 mg Fe/ kg in the form of Fe-sulfate 129 heptahydrated (FSH) supplied by Merck Millipore, (Darmstadt, Germany); Electrolytic 130 iron (EI) supplied by Transaco (Madrid, Sapin); Ferrous fumarate (FF) supplied by 131 Quimivita (Barcelona, Spain); Micronized Dispersible Ferric Pyrophosphate (MDFP) 132 supplied by SunActive-Fe-P80E (Taiyo Kagaku, Yokkaichi-shi, Japan); and the negative 133 control (Control-), in which the piglets remained on the iron-deficient diet throughout the 134 study period. In addition, a sixth treatment was set up with the non-anemic piglets 135 (Control+ group) receiving the basal diet fortified with ferrous sulfate heptahydrated for 136 the entire study period, and serving as the iron-replete reference group. The iron 137 compounds were added to the basal feed and then blended using a solids mixer (V-BL 8 138 mixer, Lleal S.A., Badalona, Barcelona, Spain). The basal diet (without iron fortificant) 139 had an intrinsic iron content of 109 mg/kg. 140 During the three weeks of the repletion period (28 to 49 days old), all piglets were housed 141 individually in pens of 1 m 2 containing a feeder and a cup waterer to provide ad libitum 142 access to feed and water. Six piglets were set per diet treatment. The feed intake of 143 individual piglets was recorded daily, by weighing the feed supplied and the leftovers; 144 moreover, the body weight of individual piglets was measured initially and then weekly 145 in order to estimate iron intake and the performance parameters (average daily intake: 146 ADI, average daily weight gain: ADWG, and feed to gain ratio F:G). 147 Sampling 148 Blood samples of all individual piglets (fasted overnight for 8 h) were collected weekly 149 from the jugular vein, (Vena jugularis externa) using5-mL EDTA tubes (Vacutainer, 150 Becton, Dickinson, and Company, Franklin Lakes, NJ), to assay iron status parameters. 151 Serum was separated immediately by centrifugation (3000g for 15 min at 4ºC) and stored 152 at -80ºC until analysis. After three weeks of the repletion period (day 21 of the 153 experiment), all piglets were euthanized by i.v. injection of sodium thiopental (50mg/kg 154 BW, Tiobarbital Braun Medical S.A., Barcelona, Spain). They were bled and the thorax 155 and abdomen were opened. The full gastrointestinal tract and organs (liver, spleen, heart, 156 lungs, pancreas, and leg muscle) were quickly removed, weighed immediately to obtain 157 the fresh weight, and then stored in a -80ºC freezer until iron analysis. 158 159 Blood analyses 160 The blood samples were transported from the farm to the laboratory refrigerated and were 161 immediately processed. The EDTA blood samples were analyzed for hematological 162 indices: erythrocyte count (RBC), Hb, Hct, MCV, MCH, and mean corpuscular Hb 163 concentration (MCHC). Reticulocyte indices were also determined, including 164 reticulocyte count, reticulocyte hemoglobin content (CHr), and hemoglobin in mature red 165 blood cells (CHm). The hematological measurements were made with a Bayer Advia 120 166 (Siemens Diagnostics, Tarrytown, NJ). 167 The serum iron concentration (OSR6186, Beckman Coulter) and unsaturated iron 168 binding capacity (UIBC) (OSR6124, Beckman Coulter) were determined via quantitative 169 methods. To determine ferritin in serum, a commercial kit (Olympus) was used. All 170 determinations were carried out in an Olympus AU600Biochemical auto analyzer 171 (Olympus Europa GMbH) at the University Hospital of the Veterinary Faculty in Murcia. 172 The serum total iron binding capacity (TIBC) was calculated as the sum of UIBC and 173 serum iron (TIBC= UIBC + serum iron) [25]. Transferrin saturation (TS) was calculated 174 using the following formula: TS (%) = (serum iron/TIBC) x 100 [26]. 175 176 Hemoglobin Repletion Efficiency assay 177 The total body hemoglobin Fe for each pig was calculated from the hemoglobin 178 concentration and estimated blood volume using the following formula, as described by 179 Yasuda et al. [22]: 180 Hb Fe (mg) = (BW (g) x 0.067 mL of blood/g of BW) x (Hb (g/mL of blood)) x (3.35 mg 181 of Fe/g of Hb), where Hb Fe = the total body hemoglobin iron; Hb = hemoglobin; BW = 182 body weight. 183 The hemoglobin repletion efficiency (HRE) was calculated as follows: HRE = (Hb Fe, 184 mg (final) – Hb Fe, mg (initial))/ total Fe intake, mg x 100. 185 The HRE values were calculated using data collected on days 21 and 28 of the 186 experimental period. At each time point, the value for the initial Hb Fe was the value 187 determined on day 0; that is, the HRE values are cumulative, not weekly values [27]. 188 189 Diets and organs analysis 190 The concentration of iron was determined in the diets and organs (liver, heart, kidney, 191 spleen, pancreas, lungs, and leg muscle), after microwave acidic digestion, by plasma 192 optical emission spectroscopy (iCAP6500 ICP-OES Duo, Thermo Scientific, Waltham, 193 MA, USA). 194 195 Statistical analysis 196 The data obtained in the animal study were expressed as the means  SEM of six 197 determinations. For normally distributed data, a 1-way ANOVA test was used at a 198 significance level of p< 0.05, together with a by-couples multiple comparison test using 199 a Tukey analysis to ascertain the effect of the iron source on the different responses. For 200 the non-normal variables, a non-parametric test (the Kruskal-Wallis test) was used and, 201 in case of significance (p< 0.05), pairwise comparisons were made using the Wilcoxon 202 rank sum test. Pearson´s correlations were determined between variables indicating iron 203 status. The statistical analyses were carried out using the SPSS software package 204 version19 (Chicago, IL, USA). 205 206 Results 207 Performance 208 Table 2 displays the effects of the four iron fortificants on piglets performance. The 209 average daily intake (ADI) of Control+ piglets (851.8g) was significantly higher (p<0.05) 210 than for the rest of the treatments. The piglets grew slowly in the Control-group (GMD 211 148.3 g), while in anemic piglets, even when supplemented with the different iron 212 sources, the values of GMD did not reach those of the Control+ piglets (on average, 213 478.19 g), although these treatments resulted in significantly increased weaning weights 214 compared to that of the pigs that did not receive the trace element. The gain to feed ratio 215 (GF) was significantly higher in the Control-group (p<0.05) than in all other groups. 216 Regarding the body weight (see Table 3), the experiment started with similar values for 217 all treatments (day 0), but at the end of the three-week repletion period (day 28) the 218 Control+ piglets gave the highest values (19.50  1.08 kg), the weight of the Control219 piglets was only half that of the Control+ values, and the piglets treated with the different 220 iron sources showed intermediate results, ranging from 13.14  0.64 to 14.081.22 kg 221 without statistical differences among the four groups. 222 223 Blood parameters 224 Hematological indices of the piglets at different stages of the experiment are presented in 225 Table 3. The hemoglobin concentration did not differ among the five anemic groups at 226 baseline (day 0), ranging from 5.13 0.55 to 6.02  0.50 g/dL. Neither were differences 227 observed for RBCs, HCT, MCV, MCH, or MCHC, so the different groups started the 228 assay under the same conditions. As expected, Control+ piglets showed Hb values (10.66 229  0.22 g/dL) above the limit described for anemia (≤8 g/dL). Significant changes in the 230 hematological indices were observed in the four iron-fortified groups of piglets following 231 the iron-repletion period. Hence, after two weeks of treatment (day 21 of the experiment) 232 no differences were found in the Hb, HCt, or CHr values with respect to the Control+ 233 group, showing a recovery from the anemic state. In addition, a positive and significant 234 correlation was detected between the weight of the piglets and both the Hb values (0.767, 235 p<0.001) and the HTC content in the blood (0.675, p< 0.001). At the end of the 236 experiment the FF and FSH piglets presented higher values than their EI and MDFP 237 counterparts for most of the measured parameters, although without statistical 238 differences. The Control-piglets (remaining on the non-fortified diet) showed the lowest 239 values of the blood indicators (p<0.05) throughout the feeding trial, indicating the 240 occurrence of severe iron deficiency anemia. 241 The serum iron, TIBC, TS, and UIBC results obtained during the study are presented in 242 Table 4, while the variations in serum ferritin are shown in Fig. 2. These parameters were 243 examined to evaluate the iron status together with the iron concentrations in the organs. 244 A great variation in the data was observed among individuals of the same group: the 245 concentrations of serum iron (61.36 μg/dL) and TS (20.48 %) were significantly higher 246 in the Control+ piglets, compared to the anemic groups (serum iron ranged from 8.57 to 247 18.97 μg/dL and TS from 1.65 to 4.41 %), at the beginning of the treatment, while the 248 value of UIBC was lower (295.21 μg/dL). At the end of the repletion period the diets 249 fortified with iron caused these parameters to remain within a range similar to the value 250 of the Control+ group except in the case of MDFP, for which serum iron and TS were 251 slightly lower than for the other groups of feed-fortified piglets. In the same way, the iron 252 fortificants enhanced the final serum ferritin concentration, compared to the Control253 group (3.2 ± 1.2 g/dL), although in this case the iron stores did not recover so efficiently 254 in EI piglets (around 4 times less) compared with the other groups treated with iron. 255 256 Iron in organs 257 The iron concentration in most of the organs and tissues of the piglets treated with the 258 different iron sources improved significantly with respect to that of the Control-animals; 259 however, it only reached values similar to those of the Control+ group in heart and leg 260 muscle (Fig. 3). The main differences between the anemic and Control+ animals were 261 found in the spleen (64.69 ± 8.00 versus 145.27 ± 22.71 mg/kg w.w.) and liver (18.67 ± 262 2.48 versus 83.67 ± 6.51 mg/kg w.w.) (p< 0.05). The spleen, liver, lung, and kidney iron 263 concentrations were higher in FF and FSH with respect to EI and MDFP, although without 264 statistical differences. 265 266 Iron bioavailability 267 Table 5 shows the Fe bioavailability (measured as HRE) in piglets fed the four iron 268 fortificants, after two or three weeks of treatment. This parameter reflects the ratio of the 269 dietary Fe converted into Hb to the amount of Fe ingested over the course of the repletion 270 period. The EI animals showed lower HRE values (4.98 ± 0.51 %) compared to those in 271 the MDFP group (8.33± 0.66 %) (p< 0.05) after 14 days of treatment (day 21 of the 272 experiment). One week later, FF and MDFP showed the best results (7.5 and 7.8 %, 273 respectively), even slightly higher than the reference salt (FSH = 6.99 %). Food intake 274 showed a positive correlation with Fe intake (mg) (0.741, p< 0.001) and Hb gain (g/dL) 275 (0.554, p<0.001). When the relative iron bioavailability (RBV) was calculated, 276 considering FSH as 100%, we did not find differences among the fortificants; however, 277 EI gave an average value of 88.27 %, again lower than the rest of the salts (see Fig. 4). 278 279 Discussion 280 The purpose of the present study was to assess the Fe bioavailability of four fortificants 281 added to infant cereals, using anemic piglets as a model because of their similarities to 282 humans during early development. The resemblance of the pig’s digestive system and 283 metabolic processes to those of humans makes the pig a species suitable for the study of 284 the effects of iron deficiency [28], as piglets have limited iron stores at birth and high iron 285 requirements [29, 30]. The anemic state of the piglets, caused by the administration of a 286 minimum dose of iron in the first few days of life and the supply of fodder without added 287 iron, allowed us to compare the absorption and bioavailability of different iron sources 288 during the repletion period of the study. 289 The anemic state was present at day 0, in all groups except Control+, considering 290 a reference level for anemia of ≤ 8 g/dL in piglets [31, 32]. As stated by Windsch [33], 291 the bioavailability of a trace element can be assessed only under the conditions of an 292 insufficient supply because absorption is down-regulated by homeostatic control in the 293 case of a supply in excess of the requirement. Anemic piglets remaining on the basal diet 294 (the Control-group) showed the lowest values of the red cell parameters at the end of the 295 repletion period; therefore, the iron present intrinsically in the feed (109 mg/kg) was not 296 enough to improve the erythropoiesis. In this regard, we observed how anemia was 297 associated with lower growth rates post-weaning, as other authors have reported 298 previously [34]. Even after receiving the basal diet fortified with 120 mg Fe/kg feed for 299 three weeks the piglets did not recover a normal weight (weighing around 14 kg at day 300 28), the reference (Control+) being 19.5 ±1.08 kg. The efficacy of the fortification was 301 tested in the present study against a background of two groups of piglets of contrasting 302 iron status: deficient (Control-) and replete (Control+). In a recent study, Ventrella et al. 303 [35] emphasized the large differences in the values of blood parameters between healthy 304 piglets of two different ages (newborns and 30 days old). The reference intervals proposed 305 by these authors are extremely wide for non-anemic piglets; that is why we decided to 306 include both Control+ and Control-groups, to be able to compare the results with anemic 307 and non-anemic animals growing in the same conditions. 308 The different iron sources added to the basal diet led to a consistent increase in 309 the circulating erythrocyte mass and volume (higher values of Hb, HCT, RBC count, and 310 MCV) of the piglets. In the same way, the erythrocyte hemoglobin indices (CHr, CHm, 311 and MCH) tended to increase. Therefore, all treatments were adequately utilized, enabling 312 the animals to recover from the anemic state, in almost the same manner. 313 To evaluate differences in iron status we examined the serum iron level, TIBC, 314 TS, UIBC, and serum ferritin, as well as the iron levels in organs. The main differences 315 were found in serum iron and TS, but these parameters did not change in the untreated 316 groups (Control+ and Control-) throughout the study. In the treated piglets, iron in serum 317 increased approximately four times from day 0 until the end of the study, although the 318 MDFP group had lower values than the other three treated groups (without statistical 319 differences, probably due to the great intra-group variability). In this regard, Fiesel et al. 320 [26] considered that plasma concentrations of most trace elements are influenced not only 321 by the dietary supply but also by several metabolic factors, and thus are compromised by 322 a lack of specificity. Logically, the TS increased during the repletion period, 323 demonstrating that iron from the different fortificants was transferred adequately to the 324 blood and mobilized towards the different organs and tissues by the transporting protein 325 transferrin. On the other hand, with the exception of the Control-group, the EI group 326 exhibited the lowest values for serum Ferritin (8 g/L). Serum ferritin responds quickly 327 to iron treatment or iron deficiency, is less likely to be influenced by other factors, and 328 correlates with the iron in the liver and spleen [36]; therefore, electrolytic iron seems to 329 be as efficient as the other salts with regard to recovering Hb levels, but not iron stores. 330 Considering the iron concentrations in organs, the main differences between anemic and 331 iron-replete piglets were in the spleen and liver, so both organs can be routinely used as 332 indicators of body Fe status, and their Fe can be mobilized in the case of an insufficient 333 supply, as was reported by Linder [37], Fiesel et al. [26], and Zhang et al. [38]. In general, 334 more than two-thirds of body iron is incorporated into hemoglobin, and the liver serves 335 as a major storage site for the remainder [39]. Taking this fact into consideration, the liver 336 Fe concentration in treated piglets did not reach that of the Control+ group, which 337 indicates that a longer repletion period with fortified diets could be needed for a complete 338 recovery, as the piglets still exhibited a marginal iron-deficient state. Indeed, the iron 339 status was not fully replenished. 340 The fact that the lowest Fe value in the liver (28.33 2.7 mg/kg) occurred for the 341 EI group, in parallel to its low serum ferritin concentration (8g/L), indicates a worse 342 efficiency in the utilization of this iron salt or a slower response with respect to filling 343 iron stores. On the other hand, the median values of iron in the whole organs of FF piglets 344 were similar to or higher than those of the FSH group (the standard salt), which suggests 345 that FF is more effective at improving iron status than EI or MDFP. However, this 346 statement should be interpreted with caution since no statistical differences were found. 347 The differences in the results between the ferrous and ferric salts could be due to 348 differences in their uptake into the intestinal cells: Fe3+ must be reduced previously to 349 Fe2+ by a reducing substance (such as cytochrome b or another reductase on the brush 350 border membrane, or by reductants in the food or gastrointestinal secretions) in order to 351 be transported by DMT1 to the enterocyte. The iron is stored in ferritin, transported out 352 of cells by ferroportin, and distributed by transferrin [40]. 353 With regard to iron bioavailability (HRE), we observed no differences in 354 hemoglobin repletion efficiency among FF, MDFP, and FSH (the control salt). In a 355 previous study performed by our research group [6], we observed a similar bioavailability 356 of MDFP, with respect to ferrous sulfate, when added to a fruit juice, using the same 357 method but a different animal model (weaned rats). In the current study, the different 358 matrix could have changed the solubility and thus the absorption of this compound, but 359 the final result was similar. In addition, we have used piglets this time, a better model 360 than weaned rats to assess iron bioavailability in infants, and, as mentioned above, the 361 two salts were equally well absorbed. We have to take in mind that the diet of the piglets 362 was formulated to be as similar as possible to infant cereals; therefore, the phytate content 363 could have inhibited iron absorption partially. We aimed also to compare the encapsulated 364 ferric pyrophosphate with a soluble iron form in diluted acid, ferrous fumarate, as 365 previously investigated by [41] in infants, when the salts were added to infant cereals. 366 However, these authors used neither a micronized nor an encapsulated form of the ferric 367 pyrophosphate; presumably, that is why they found better bioavailability for ferrous 368 fumarate, while in the present study the two salts provided data comparable to those of 369 the soluble iron salt ferrous sulfate. 370 371 Conclusion 372 The major finding from our study is the effectiveness of the four iron sources in 373 alleviating the anemic status of weaned piglets, especially for the repletion of Hb 374 synthesis. However, our objective was to assess the differences in iron bioavailability, 375 using the parameters HRE and RBV%, among these salts that are to be used in infant 376 cereals. These analyses were based on the effectiveness of the incorporation of the iron 377 absorbed from the diet into Hb. Therefore, we may conclude that FF and MDFP are 378 equally as bioavailable as FSH (the reference salt), and significantly more so than EI. 379 Also, the restoration of the iron status in the initially-anemic piglets was faster for FF and 380 FSH (ferrous iron sources) than for MDFP and EI (ferric iron sources). 381 382 Acknowledgements 383 This work was supported by the Ministry of Science and Innovation of the Spanish 384 Government (project Ref. AGL2013-40617-R). 385 386 Conflict of Interest 387 No conflict of interest exists in the submission of this manuscript, and the 388 manuscript was approved by all authors for publication. The work described is original 389 research that has not been published previously and is not under consideration for 390 publication elsewhere. 391 References 392 1. World Health Organization (2001) Iron Deficiency Anemia. 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Am J Clin Nutr 71(6): 1597-1602 521 522 523 524 525 526 527 528 529 530 Figure 1 Treatments (30 piglets) Weaning 30mg Fe dextran, via intramuscular Phase 1 Phase 2 Adaptation period (feed base without iron added) Repletion period Diets (FF, EI, MDFP, FSH and C-) Day 0 Day 7 Randomization Day 7 Day 14 Day 21Day 28 Sample collection Sample collection Positive control 6 piglets Weaning 180 mg Fe dextran, via intramuscular Adaptation period (feed base + FSH) Repletion period (feed base + FSH) Day0 Day 7 Day 7 Day 14 Day 21Day 28 Sample collection Sample collection Fig. 1. Experimental study design. FF= Ferrous Fumarate, EI= Electrolytic Iron powder, MDFP= Micronized Dispersible Ferric Pyrophosphate, FSH= Ferrous Sulfate Heptahydrated. Figure 2 Fig 2 Plasma Ferritin (μg/L) in each day of sampling according to different sources added to diet. Values are expressed as means± SEM (standard error of the mean); n= 6 per treatment. FF= Ferrous Fumarate, EI= Electrolytic Iron powder, MDFP= Micronized Dispersible Ferric Pyrophosphate, FSH= Ferrous Sulfate Heptahydrated. Different letters for the same day indicate statistically significant differences at p<0. 05 Figure 3 abbabababaababababbabbbbbacbcabcabaaaababacababbababbcababbcababbcb Fig. 3 Fe concentrations (mg/kg) in organs according to the source of Fe added to the diet. Values are expressed as means± SEM (standard error of the mean); n= 6 per treatment. FF= Ferrous Fumarate, EI= Electrolytic Iron powder, MDFP= Micronized Dispersible Ferric Pyrophosphate, FSH= Ferrous Sulfate Heptahydrated. Figure 4 Fig. 4. Relative Bioavailability Values, expressed as a percentage of the response to Ferrous Sulfate Heptahydrated (FSH). There were no statistically significant differences between the different iron sources. FF= Ferrous Fumarate, EI= Electrolytic Iron powder, MDFP= Micronized Dispersible Ferric Pyrophosphate. Table 1 Table 1 Feed base composition. Items Ingredients Percent Mix cooked cereals 28.0000 Raw corn 15.0000 FULL-FAT soybean 9.9898 Raw barley 9.5624 Sweet milk whey 7.5000 SPC 62% Select 5.0000 Flour 48% soybean 5.0000 Monocalcium phosphate 1.2242 Soya oil 1.0000 Calcium carbonate 0.9977 Lysine 0.6393 Corrective 0.4000 Common salt 0.3500 Threonine 0.2233 Methionine DL 0.2163 Tryptophan 0.0397 Lohmann sweetener 0.0175 Analyzed composition Percent Dry matter 90.4114 Crude protein 17.5000 Crude fat 5.9939 Lysine 1.3500 Calcium 0.7400 Native iron 0.0109 The feed base was fortified with 120mg/kg of each iron supplement. Table 2 Table 2 Parameters of growth obtained with different sources of iron. Treatment FF EI MDFP FSH Control Control + SEM1 p value ADI (g) 547.84bc 569.64bc 571.47b 564.68bc 380.62c 851.80a 25.499 0.001 ADWG (g) 265.20bc 286.54b 293.77b 293.75b 148.29c 478.190a 15.493 <0.001 F:G (kg/kg) 2.06b 2.12b 2.03b 1.99b 3.01a 1.79b 0.098 0.046 1SEM (standard error of the mean) Values are expressed as means; n= 6 per treatment. ADI= Average daily intake, ADWG =Average daily weight gain, F:G= Feed to gain ratio, FF= Ferrous Fumarate, EI= Electrolytic Iron powder, MDFP= Micronized Dispersible Ferric Pyrophosphate, FSH= Ferrous Sulfate Heptahydrated. Different letters in a row indicate statistically significant differences at p <0.05. Table 3 Table 3 Blood parameters of piglets fed with different irons sources on day 0, 14, 21 and 28 Treatment FF EI MDFP FSH Control -Control + Day 0 Weight(kg) 6.53±0.38a 6.50±0.38a 6.35±0.37a 6.69±0.41a 6.20±0.23a 7.16±0.40a Hb(g/dL) 5.13±0.55b 5.66±0.54b 5.73±0.39b 5.44±0.59b 6.02±0.50b 10.66±0.22a RBC(x106cells/μL) 5.45±0.38a 5.70±0.51a 5.62±0.23a 5.47±0.59a 5.72±0.40a 5.89±0.15a HCt(%) 19.46±1.84b 20.26±1.47b 20.60±0.93b 18.64±1.67b 21.26±1.20b 33.11±0.79a MCV(fL) 35.40±0.95b 35.43±1.92b 36.81±1.75b 36.40±1.57b 37.46±1.82b 56.26±1.56a MCH(pg) 9.35±0.41b 9.96±0.65b 10.18±0.62b 10.00±0.55b 10.48±0.51b 18.16±0.68a MCHC(g/dL) 26.38±0.87b 28.08±0.54b 27.63±0.71b 27.44±0.52b 28.10±0.86b 32.28±0.72a Reticulocyte(x106cells/μL) 0.20±0.02a 0.20±0.03a 0.15±0.02ab 0.18±0.02ab 0.25±0.04ab 0.05±0.02b CHr(pg) 10.63±0.29b 11.13±0.28b 11.31±0.29b 11.00±0.38b 11.68±0.35b 17.66±0.82a CHm(pg) 12.55±0.28b 12.35±0.62b 12.90±0.47b 12.80±0.32b 13.12±0.50b 19.05±0.63a Day 14 Weight(kg) 8.06±0.47 b 8.07±0.69 b 7.83±0.55 b 8.5±0.49 b 6.04±0.24 b 12.22±0.79 a Hb(g/dL) 7.78±0.49 b 7.43±0.23 b 7.60±0.46 b 7.20±0.66 b 6.54±0.62 b 9.93±0.25 a RBC(x106cells/μL) 7.14±0.34 a 7.02±0.26 a 7.13±0.26 a 6.83±0.35 a 7.22±0.59 a 5.93±0.20 a HCt(%) 27.65±1.38ab 25.86±0.87 a-b 26.70±1.70 a-b 25.68±1.82 a-b 22.84±1.96 b 31.86±0.75a MVC(fL) 39.05±2.36b 37.01±1.73 b 37.85±3.09b 37.56±1.85b 31.58±1.29b 53.81±1.25a MCH(pg 11.05±0.86b 10.63±0.51 b 10.76±0.86b 10.54±0.76b 9.02±0.44b 16.80±0.43a MCHC(g/dL) 28.10±0.78 b 28.80±0.67a 28.50±0.48b 27.90±0.74b 28.56±0.35b 31.23±0.14a Reticulocyte(x106cells/μL) 0.23±0.04 a 0.25±0.07 a 0.19±0.03 a 0.20±0.01 a 0.24±0.04 a 0.18±0.04 a CHr(pg) 15.10±0.67 ab 14.78±0.60 ab 14.20±1.19 bc 14.66±1.10 abc 11.22±0.34c 18.08±0.43 a CHm(pg) 12.73±0.63 b 11.96±0.44 b 12.31±0.75 b 12.18±0.60 b 10.98±0.32 b 17.71±0.44 a Day 21 Weight(kg) 9.75±0.79 b 10.14±0.87 b 10.39±0.88 b 10.53±1.07 b 7.57±0.66 b 16±1.06 a Hb(g/dL) 10.21±1.53 a 8.33±0.21 a 8.40±0.78 a 8.70±0.86 a 7.12±0.52 a 10.55±0.24a RBC(x106cells/μL) 8.44±1.26a 7.00±0.22a 7.02±0.25a 7.37±0.46a 7.80±0.44 a 6.26±0.22 a HCt(%) 35.70±5.32a 28.35±0.81a 29.38±2.69a 30.54±2.68a 24.82±1.5 a 34.16±1.06a MVC(fL) 42.70±2.46b 41.21±1.14bc 42.00±3.78b 41.44±2.14bc 31.58±1.43c 53.88±1.23 a MCH(pg) 12.28±0.92b 11.98±0.42 bc 11.98±1.08 bc 11.80±0.78 bc 9.04±0.51c 16.95±0.37 a MCHC(g/dL) 28.56±0.69b 29.01±0.36b 28.56±0.42b 28.36±0.47b 28.58±0.48b 31.40±0.16 a Retyculocyte(x106cells/μL) 0.25±0.07 a 0.22±0.07 a 0.24±0.04 a 0.24±0.07 a 0.27±0.07 a 0.21±0.04 a CHr(pg) 16.53±0.66 a 15.63±0.61 a 15.46±1.24 a-b 16.58±1.09 a 12.00±0.54 b 18.38±0.35 a CHm(pg) 13.83±0.67 b 13.18±0.39 bc 13.53±0.91 b 13.20±0.67 bc 10.64±0.35 c 17.61±0.40 a Day 28 Weight(kg) 13.14±0.64 bc 14.08±1.22 b 13.97±1.11 bc 13.99±1.20 bc 9.46±0.98 c 19.50±1.08 a Hb(g/dL) 10.13±0.45 a 9.15±0.24 a 8.93±0.52 a 9.58±0.57 a 6.50±0.49 b 10.73±0.32 a RBC(x106cells/μL) 7.62±0.45 a 7.18±0.34 a 7.14±0.36 a 7.44±0.30 a 7.31±0.34 a 6.48±0.26 a HCt(%) 34.70±1.29 a 31.38±1.09 a 31.26±1.61 a 33.46±1.60 a 23.36±1.58 b 34.53±1.34 a MCV(fL) 45.36±1.52 ab 43.43±0.92 b 43.13±3.22 b 45.08±1.96 ab 31.98±1.86 c 53.30±0.78 a MCH(pg) 13.25±0.61 b 12.80±0.39 b 12.75±1.13 b 12.90±0.81 b 8.86±0.53 c 16.63±0.34a MCHC(g/dL) 29.18±0.47 ab 29.46±0.33 ab 29.45±0.57 ab 28.52±0.67 b 27.74±0.33 b 31.16±0.32a Reticulocyte(x106cells/μL) 0.22±0.02 a 0.20±0.05 a 0.18±0.06 a 0.33±0.04 a 0.23±0.05 a 0.20±0.04a CHr(pg) 17.38±0.39 a 16.86±0.41 a 16.38±0.95 a 17.42±0.92 a 12.50±0.94 b 18.48±0.23a CHm(pg) 15.18±0.65ab 14.16±0.38 b 13.90±0.92 b 14.32±0.76 b 10.62±0.42 c 17.50±0.38 a Values are expressed as means ± SEM (standard error of the mean); n= 6 per treatment. FF= Ferrous Fumarate, EI= Electrolytic Iron powder, EMFP= Encapsulated and Micronized Ferric Pyrophosphate, FSH= Ferrous Sulfate Heptahydrated. Different letters in a row indicate statistically significant differences at p<0. 05 RBC= Erythrocyte count; Hb= hemoglobin; HCt= hematocrite; MCV= Mean Corpuscular Volume; MCH=Mean Corpuscular Hemoglobin; MCHC= Mean Corpuscular hemoglobin concentration; CHr=reticulocyte hemoglobin content; CHm=Hemoglobin in mature red blood cells. Table 4 Table 4 Unsaturated Iron-Blinding Capacity(UIBC). Serum Iron (Iron). Total Iron-Blinding Capacity (TIBC). and Transferrin Saturation (ST) of piglets during the period of study. Values are expressed as means[Min-Max]; n= 6 per treatment Treatment FF EI MDFP FSH Control Control + Day 0 Iron (μg/dL) TIBC (μg/dL) TS (%) UIBC (μg/dL) 14.90 (2.0 – 39.70)a 391.19 (379.33 – 418. 23) a 3.65 (0.53 – 9.49)b 378.55 (378.23 -379.46) b 18.97 (1.40 -61.20)a a391.34 (374.49 – 441.05) 4.41 (0.37 – 13.88)b 378.69 (377.76 – 379.85) b a6.41 (2.70 – 13.10) a385.01 (381.37–390.82) 1.65 (0.71 – 3.35)b 378.59 (377.26-379.97) b 8.57 (1.50 – 20.70) a 384.86 (379.07 -397.30) a 2.18 (0.40 -5.21)b 378.00 (376.60 – 379.40) b 8.80(2.70 – 17.60)a 385.59(378.98 – 397.18)a 2.23 (0.70-4. 43)b 380.31(378.98 -384.77)b 61.36 (10.40 – 109.20) a 356.57 (238.96 – 451.35) a 20.48 (12.83 – 27.37) a a295.21 (182.76 – 357.33) Day 14 Iron (μg/dL) TIBC (μg/dL) TS (%) UIBC (μg/dL) 19.22 (8.20 – 45.50) a a381.56 (336.97 – 422.36) 4.96 (2.12 – 10.77) a a362.34 (317.07 – 379.20) a26.71 (7.50 – 50.40) a393.75 (372.80 – 426.60) a6.69 (1.94– 12.80) a367. 04 (343.46 – 378.58) a30.75 (3.00 – 99.90) a399.48 (343.46 – 479.17) a7.17 (0.78 – 20.85) a368.73(317.56 – 380.18) 24.16 (8.0 – 39.60) a 396.37 (387.01 – 411.05) a 6.05 (2.07 -10.09) a 372.21 (352.86 – 379.01) a 21.62 (3.0 – 65.10) a 386.01(360.04 – 441.03)a 5.14 (0.78 – 14.76) a 368.71(353.38 – 379.84)a a38.88 (26.10 – 57.0) a409.83 (378.74 434.27) 15.25 (12.32 – 17.56) a 377.43 (376.83 – 378. 74) a Day 21 Iron (μg/dL) TIBC (μg/dL) TS (%) UIBC (μg/dL) 32.08 (11.0 – 46.40)abc 395.78 (364.89 – 424.34)abc 7.90 (3.01 – 11.60)abc 369.05 (352.15 -378.73) a 23.10 (6.0 – 76.90)bc 369.92 (307.12 – 452.89)bc 5.82 (1.56 – 16.98)bc a346.82 (291.32 – 377.51) 19.93 (1.0 – 55.10)bc 393.96(378.42 – 408.59)bc 4.95 (0.26 – 13.49)bc a374.04 (353.49 – 379.96) 24.66 (3.0 – 39.90)abc 397.37(378.91 – 418.25)abc 6.11 (0.79 – 9.54)bc 372.71 (354.51 – 378.35) a 12.00 (1.40 – 21. 80)c 376.66(355.13 – 392.55)c 3.14 (0.37 – 5.81) c 369.46(353.10 – 381.16)a 50.06 ( 29.10 – 64.10) a a423.78 (406.76 – 439.94) 15.09 (11.35 – 19.75) a a373.71 (359.74 – 377.66) Day 28 Iron (μg/dL) TIBC (μg/dL) TS (%) UIBC (μg/dL a56.68 (10 – 130.0) 423.70 ( 386.78 – 478.43) ab 12.84 (2.59 – 27.17) ab a367.02 (343.60 – 378.97) a48.11 (10.80 – 121.70) 415.24 (378.77 – 473.59) ab 11.03 (2.79 – 25.70)abc a376.13 (342.69 – 378.24) a26.70 (4.30 – 80.10) 398.78 (382.24 – 421.95)bc 6.50 (1.12 – 18.98)bc a372.08 (341.85 – 378.78) 54.78 (3.0 – 151.10) a 423.74 (363.70 – 504.63) ab 29.94)abc 11.77 (0.82 – 368.96 (353.53 – 377.88) a 9.55 (4.60 – 18.50) a 375.04(351.18 – 386.59)c 2.54 (1.31 – 4.96) c 367.40(346.58 – 380.39)a a67.33 (24.90 – 101.0) a443.25 (401.13 – 475.48) a18.16 (14.40 – 21.24) a375.91 (374.48 – 376.74) FF= Ferrous Fumarate. EI= Electrolytic Iron powder. MDFP= Micronized Dispersible Ferric Pyrophosphate. FSH= Ferrous Sulfate Heptahydrated. Different letters in a row indicate statistically significant differences at p <0.05 on the different days of sampling. Table 5 Table 5 Food intake, hemoglobin regeneration efficiency(HRE), Iron (Fe) intake, hemoglobin (Hb) gain, iron hemoglobin gain, and hemoglobin regeneration efficiency (HRE) in the medium-term (day 21) and longterm (day 28) during the experiment Treatments FF EI MDFP FSH Day 21 Food intake (kg) 7.14±1.50a 7.68±1.64a 7.08±1.29a 6.58±1.57a Fe intake (mg) 1718.50±209.89a 1989.00±305.75a 1510.66±224.35a 1790.20±324.09a Hb gain (g/dL) 5.17±1.49a 2.67±0.49a 2.83±0.65a 3.20±0.58a Fe Hb gain (mg) 132.50±28.61a 99.09±18.64a 110.00±26.44a 120.94±33.44a HRE (%) 6.31±0.58ab 4.98±0.512b 8.33±0.66a 6.85±1.19ab Day 28 Food intake (kg) 11.06±1.92a 11.78±2.14a 11.38±1.63a 10.81±2.09a Fe intake (mg) 2733.00±223.15a 3256.01±366.89a 2524.68±354.14a 3002.41±405.098a Hb gain (g/dL) 5.00±0.68a 3.00±0.816a 3.17±0.477a 4.20±0.583a Fe Hb gain (mg) 205.66±31.95a 189.44±26.97a 180.66±27.91a 205.20±33.29a HRE (%) 7.50±0.37a 5.00±0.32b 7.80±0.62a 6.99±0.91ab Values are expressed as means ± SEM (standard error of the mean); n= 6 per treatment. FF= Ferrous Fumarate, EI= Electrolytic Iron powder, MDFP= Micronized Dispersible Ferric Pyrophosphate, FSH= Ferrous Sulfate Heptahydrated. Different letters in a row indicate statistically significant differences at p < 0.05.