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Bioavailability of caseinophosphopeptide-bound iron

Bioavailability of caseinophosphopeptide-bound iron Nabil Ait-Oukhatar, Jean Michel Peres, Said Bouhallab, Dominique Neuville, Francois Bureau, Gerard Bouvard, Pierre Arhan, Dominique Bougle To cite this version: Nabil Ait-Oukhatar, Jean Michel Peres, Said Bouhallab, Dominique Neuville, Francois Bureau, et al.. Bioavailability of caseinophosphopeptide-bound iron. Journal of Laboratory and Clinical Medicine, Elsevier, 2002, 140 (4), pp.290-294. 10.1067/mlc.2002.128146. hal-01568870 HAL Id: hal-01568870 https://hal.archives-ouvertes.fr/hal-01568870 Submitted on 25 Jul 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - ShareAlike| 4.0 International License Bioavailability of caseinophosphopeptide-bound iron NABIL AIT-OUKHATAR, JEAN MICHEL PERES, SAID BOUHALLAB, DOMINIQUE NEUVILLE, FRANCOIS BUREAU, GERARD BOUVARD, PIERRE ARHAN, and DOMINIQUE BOUGLE CAEN and RENNES, FRANCE Iron deficiency, one of the main worldwide nutritional deficiencies, results from the low bioavailability of most dietary iron, including cow milk. Hydrolysis of the cow milk protein casein produces low molecular weight caseinophosphopeptides (CPPs). Binding of iron to CPPs keeps it soluble in the digestive tract and prevents the formation of high molecular weight ferric hydroxides, which are poorly absorbed. Previous experimental studies have shown that iron bound to the phosphopeptide containing the first 25 amino acids of -casein, or -CN (1-25), is well absorbed and corrects efficiently iron deficiency. We sought to assess in vivo iron absorption and uptake by tissues involved in iron metabolism and storage (liver, spleen, bone marrow), using radiolabeled iron. -CN (1-25)-Fe displayed better absorption and tissue uptake by the vascularized rat loop model compared with a control sub- stance, ferric ascorbate. The metabolism of -CN (1-25)-Fe labeled with iron 59, added to cow milk, was also studied in young women. Although the absorption of -CN (1-25)-Fe was not significantly higher than that of ferrous sulfate, it displayed significantly higher tissue uptake. This increase was transient and had disappeared by the 14th day of the study, suggesting that iron was used for metabolic purposes. (J Lab Clin Med 2002;140:290-4) Abreviations: CN  casein; CPP: caseinophosphopeptide; -CN (1-25)–Fe  first 25 amino acids of -casein– bound iron; -CN (1-25)  first 25 amino acids of -casein igestion of milk proteins releases numerous bovine milk proteins keep iron soluble in the digestive bioactive peptides encoded in the native pro- tract but inhibit its absorption unless they are hydro- D teins. They display specific physiological func- lyzed. The use of CPP instead of native proteins might tions in gastrointestinal, immunological, hormonal, and improve iron absorption. 1,2 nutritional responses. The N-terminal CPP issued from the trypsin hydro- Among them, CPPs issued from casein hydrolysis lysis of -CN is one of the main CPPs produced in vivo 3,4 7 have the ability to bind and keep soluble cations; they during digestion ; previous experimental studies in rats improve calcium and zinc absorption. Indeed, intact have shown that -CN (1-25)– bound iron remains sol- uble in the digestive tract, where it escapes further 7,8 From the Laboratoire de Physiologie Digestive et Nutritionnelle, enzyme digestion. The absorption of -CN 1-25– Laboratoire de Biochimie A, and Service des Radio-Isotopes, CHU bound iron occurs partly through endocytosis and is de Caen; and the Laboratoire de Recherches de Technologie Laitie `re, more efficient than the absorption of gluconate iron ; INRA, Rennes, France. repletion with  CN (1-25)– bound iron also results in Supported in part by Die ´pal nsa, Groupe Danone. significantly greater improvement of hemoglobin and Submitted for publication January 2, 2002; revision submitted April iron concentrations in related tissues (liver, spleen) in 8, 2002; accepted May 15, 2002. iron-deficient rats than repletion with FeSO . Using Reprint requests: Dominique Bougle ´, Service de Pe ´diatrie A, CHU iron 59 –labeled iron, we assessed in vivo the metabo- Cle ´menceau, CHU de Caen F-14033, Caen Cedex, France; e-mail: bougle-d@chu-caen.fr. lism of absorbed iron in rats and compared these results Copyright © 2002 by Mosby, Inc. All rights reserved. with the metabolism of absorbed iron in human beings. 0022-2143/2002 $35.00  0 5/1/128146 We compared the vascularized duodenal rat loop doi:10.1067/mlc.2002.128146 model with iron absorption on the basis of the deter- 290 J Lab Clin Med Volume 140, Number 4 Ait-Oukhatar et al 291 tory disease, or infection; were taking no medication or min- mination of radioactivity incorporated into circulating eral supplementation; were using a chemical method of con- red blood cells, as well as tissue uptake, in young traception; and had negative results on pregnancy testing. women. Before the administration of the first test meal, blood was drawn for measurement of red blood cell count, serum fer- METHODS ritin, C-reactive protein (a marker of recent inflammation or Preparation of -CN (1-25)– bound iron. -CN (1-25) infection), and background radioactivity. (purity  85%) was purified from tryptic hydrolysate of The test subjects drank radioisotopically labeled milk after -CN as previously described. an overnight fast; nothing but water was allowed for the next We bound iron by mixing -CN (1-25) with a FeCl 4 hours. All subjects drank 250 mL of sterilized milk con- solution for 1 hour at 37°C and a pH of 6.5. The resulting taining 3 mg iron (12 mg/L), either as FeSO or -CN solutions were then ultrafiltered and diafiltered on a 3-kD (1-25)-Fe, labeled with Fe. The two meals were given at membrane to remove free minerals. The amount of iron random. The first was given on day 1; on day 14, blood was complexed to phosphopeptides and the presence of traces of drawn to measure the increase in red blood cell radioactivity. calcium and sodium were determined with the use of atomic- External counting of liver, spleen, and blood marrow (sa- absorption spectrometry (Model AA 1275; Varian; Les Ulis, crum) areas was performed on days 7 and 14 with the use of France) on freeze-dried samples. The final product had an a solid scintillation counter equipped with a thick crystal for iron/phosphopeptides molar ratio of 4 and contained less than external isotope tissue measurement (CGR Nucle ´aire Me ´- 1 mg sodium and 0.1 mg calcium/g. decine GM2C; Paris, France); counting lasted 10 minutes. Labeling of iron. -Emitting Fe was purchased from The heart area was used as control, and background radioac- Amersham (Amersham Pharmacia Biotech, Orsay; specific tivity was subtracted. activity 3.7 MBq/mL). Perfusion solutes were extrinsically The second meal was given after a washout period of 2 labeled in the laboratory with FeCl and mixed with cold weeks (day 28), after residual red blood cell and body radio- ferric ascorbate at a ratio of 0.001%, whereas the complex of activity had been determined. External counting was per- -CN (1-25)– bound iron was intrinsically labeled during its formed on days 35 and 42; erythrocyte radioactivity was preparation. measured on day 42. For the human study, milk was labeled either with extrin- Percentage absorption of iron was calculated on the basis sically tagged FeSO with Fe citrate or with the intrinsically of blood volume estimated from weight and height and an labeled -CN (1-25)–Fe. assumed hemoglobin incorporation of absorbed iron of Each 250-mL meal contained the same amount of radioac- 80%. In addition, data were corrected for background ra- tivity (74 kBq). dioactivity and for the radioisotope decay of the residual Experimental study. In an experimental study of perfused radioactivity of tissues and red blood cells. rat duodenal loop, four groups (each group n  6) of adult Statistical methods. We used Student’s t test to analyze Sprague-Dawley rats were studied after being fasted over- data from the experimental study. Human data were subjected night, as previously described. They were perfused with to analysis of variance and and Fisher’s exact test. P values of ferric ascorbate or -CN (1-25)-Fe. We tagged iron with Fe less than .05 were considered statistically significant. so that we might assess iron tissue uptake in addition to its RESULTS absorption. The composition of the perfusion solute was adapted from Experimental study. The absorption of ferric ascorbate Ringer-Lavoisier solute; its pH was adjusted to that of the and -CN (1-25)-Fe by isolated perfused rat duodenal proximal duodenum (5.5) and contained 100 mol/L iron in loop system is shown in Table I. -CN (1-25)-Fe dis- either form. played greater gut uptake and net absorption, greater Duodenum of anaesthetized rats was perfused at a delivery spleen uptake, and increased blood radioactivity com- rate of 0.16 mL/min; every element of the perfusion device pared with ferric ascorbate. had been washed with a solution of Triton X-100 (1 g/L) to Human study. Hematologic data and results of iron- prevent contamination. We kept the perfusion solute at 37°C with a thermostatic control and added a nonabsorbable absorption testing are given in Table II. Three subjects marker (polyethylene glycol 4000) to assess actual net water were iron-deficient (ferritin concentration  12 g/L). flux. After 2 hours of perfusion, the animal was killed with an Mean iron absorption was similar in the two groups, overdose of Dole ´thal; then the perfused loop was withdrawn yet paired comparisons showed a nonsignificant trend and washed with saline solution. toward better absorption of -CN (1-25)-Fe. Tissues were digested in nitric acid. The radioactivity of Results of external counting are given in Fig 1. A gut mucosa, blood, liver, and spleen was measured on a significant difference was displayed at day 7 between scintillation counter. FeSO and iron-labeled CPP for liver (P  0.05), Human study. Our human study was approved by the local spleen (P  0.05), and sacrum (P  0.03); the total committee of ethics. Ten subjects gave their written informed radioactivity of these organs was different between consent to participate: All were 20- to 30-year-old female groups (P  0.01) at day 7. No difference was observed students of the university, and all stated that they were in good health, had no recent history of digestive or inflamma- at day 14. J Lab Clin Med 292 Ait-Oukhatar et al October 2002 Table I. Absorption and tissue uptake of radiolabeled iron, given as iron ascorbate or -CN (1-25)-bound iron, by rat duodenal loop Test substance Mucosal uptake* Mucosal retention* Net absorption* Liver uptake* Spleen uptake* Blood activity* Sum of tissue activity Iron ascorbate 14.1  1.0* 6.2  0.5 7.9  1.0 4.7  0.3 0.02  0.00 3.0  0.6 13.3  0.8 CN (1-25)- 18.2  1.2 7.0  0.2 11.2  1.1 4.8  0.3 0.03  0.01 3.6  0.1 15.4  0.3 Fe P value .001 .006 .001 .49 .022 .001 .001 *Percent of dose perfused; mean  SD, n  6 per group. Initial iron concentration-100 mol. Sum of mucosa, liver, spleen, and blood activity. DISCUSSION of -CN (1-25) bound iron supports the results of tissue 9,10 analysis after a 4-week repletion period. Iron deficiency remains a worldwide health problem, Results varied between the rat and human groups. mainly involving growing infants and women of child- Differences in experimental methods could explain the bearing age. In addition, the side effects of iron isotopic data in the human group, in which iron-absorp- supplementation limit adherence to its prescription, tion rates were close to those obtained from studies that necessitating a search for highly bioavailable sources of 6,20-23 employed cow milk as source of iron. As expect- iron, free of digestive interactions. ed, these results differed quantitatively from those of CPPs are produced during digestion of CN; they bind the rat group but displayed the same trends; some divalent cations and keep them soluble at luminal 7,16,17 differences between the two experiments could explain pH. The strength of iron binding to CPP is about the lower significance in the human trial. First, iron was 100 times greater than that of calcium and others cat- 3,18 given in milk and not in a pure solution as in the rat ions. group. This decreases iron absorption and blunts dif- When assessed in rats on the basis of metabolic ferences between dietary sources of iron. Second, balance and direct measurement of tissue storage, the physiologic changes, such as the onset of menses, could bioavailability of iron bound to -CN (1-25) is higher have occurred during the time elapsed between the two than that of reference iron salts gluconate or ferrous 55 59 9,10 tests in the human group. Use of Fe and Fe on sulfate. consecutive days could have alleviated these potential In this study, the absorption rate and tissue uptake of 59 19 Fe were similar to those noted in recent reports. Our variations, but the use of -emitting Fe precludes findings showed that absorption, blood content, and external counting. spleen uptake of -CN (1-25)– bound iron by duodenal Although stable isotopes are valid tools in the assess- 25,26 rat loop during the experiment were better than that of ment of iron absorption from food, they do not inorganic iron. The better absorption and tissue uptake supply information on tissue kinetics, as radioisotopes Table II. Clinical details and iron absorption rate by human subjects given 3 mg of iron tagged with Fe, either bound to -CN (1-25) or as FeSO in 250 mL milk Iron absorption (%) Ratio of -CN (1-25)-Fe Subject Ferritin (g/L) Hemoglobin (g/dL) -CN (1-25)-Fe FeSO to FeSO 4 4 1 36 14.4 24.3 19.6 1.24 2 24 13.2 4.4 6.9 0.65 3 45 12.4 19.8 19.3 1.03 4 5 14.8 14.6 18.4 0.80 5 48 13.3 4.2 2.1 2.05 6 31 14.1 10.2 9.5 1.08 7 11 11.6 9.7 26.5 0.37 8 40 12 7.7 1.8 4.37 9 4 11.4 22.6 16.6 1.36 10 33 14.3 5.3 2.8 1.92 MeanSD 27.7  16.1 13.1  0.4 9.8  6.1 9.9  7.1 1.49  0.36* *Wilcoxon’s test not significant. J Lab Clin Med Volume 140, Number 4 Ait-Oukhatar et al 293 findings of external counting of organs was temporarily enhanced in human subjects after intake of  CN (1- 25)– bound iron. These beneficial results may partly explain the high iron bioavailability of breast milk, which is rich in -CN. Further human studies are needed to precisely assess the bioavailability of -CN (1-25)– bound iron, partic- ularly the lack of interaction with other minerals pre- viously shown in experimental studies. REFERENCES 1. Bos C, Gaudichon C, Tome ´ D. Nutritional and physiological criteria in the assessment of milk protein quality for humans. J Am Coll Nutr 2000;19:191S-205. 2. Clare DA, Swaisgood HE. Bioactive milk peptides: a prospectus. J Dairy Sci 2000;83:1187-95. 3. Brule ´ G, Fauquant J. Interaction des prote ´ines du lait et des oligo-e ´le ´ments. Lait 1982;62:323-31. 4. Bouhallab S, Le ´onil J, Maubois JL. Complexation du fer par le phosphopeptide (1-25) de la case ´ine : action de l’alcalase et de la phosphatase acide. Lait 1991;71:435-43. 5. Hansen M, Sandstro ¨mB,Lo ¨ nnerdal B. The effect of casein phosphopeptides on zinc and calcium absorption from high phytates infant diets assessed in rat pups and Caco-2 cells. Pediatr Res 1996;40:547-52. 6. Hurrell RF, Lynch SR, Trinidad PT, Dassenko SA, Cook JD. Fig 1. External counting of liver, spleen, sacrum, and myocardial Iron absorption in humans as influenced by bovine milk proteins. areas of human subjects given 3 mg iron tagged with Fe, either Am J Clin Nutr 1989;49:546-52. bound to -CN (1-25) or as FeSO , in 250 mL milk. 7. Naito H, Suzuki H. Further evidence for the formation in vivo of *Analysis of variance and Fisher’s exact test, FeSO vs -CN (1-25) phosphopeptide in the intestinal lumen from dietary -casein. at day 7 (P  .05). Agr Biol Chem 1974;38:1543-5. 8. Bouhallab S, A¨ ıt-Oukhatar N, Molle ´ D, Henry G, Maubois JL, Arhan P, et al. Sensitivity of -casein phosphopeptide-iron com- plex to digestive enzymes in ligated segment of rat duodenum. J Nutr Biochem 1999;10:723-7. do. Our results suggest that tissue uptake of -CN 9. Pe ´re `s JM, Bouhallab S, Bureau F, Neuville D, Maubois JL, (1-25)– bound iron was significantly enhanced; this as- Arhan P, et al. Mechanisms of absorption of caseinophosphopep- tide bound iron. J Nutr Biochem 1999;10:215-22. sumption is supported by the findings of our experi- 10. A¨ ıt-Oukhatar N, Bouhallab S, Arhan P, Maubois JL, Dros- mental study of the rat group, in which tissues were dowsky M, Bougle ´ D. Iron tissue storage and hemoglobin levels assessed immediately after absorption. The transient of deficient rats repleted with iron bound to the casein phos- increase of iron concerned every organ involved in iron phopeptide 1-25 of  casein. J Agric Food Chem 1999;47:2786-90. metabolism (bone marrow, liver, and spleen). Yet the 11. Najean Y. Etude de l’absorption du fer. In: Najean Y, Ardeillon N, Dresch C, eds. Utilisation des techniques isotopiques en function of iron uptake remains unclear; it may merely he ´matologie. Paris: Baille `re Eds, 1969:83-91. reflect the difference of radioactivity in the blood be- 12. Brown E, Hoper J Jr, Hodges JL Jr, Bradley B, Wennesland R, cause blood was not removed before counting; how- Yamauchi H. Red cell, plasma, and blood volume in healthy ever, counting of the blood-filled myocardial tissue did women measured by radiochromium cell-labelling and hemato- not display significant differences. Stored iron was crit. J Clin Invest 1962;41:2182-90. 13. Hosain F, Marsaglia G, Finch CA. Blood ferrokinetics in normal quickly used: No difference was found 1 week later. man. J Clin Invest 1967;46:1-9. However, these data suggest that measuring only iron 14. Hurrell RF. Bioavailability of iron. Eur J Clin Nutr 1997; radioactivity incorporated into circulating erythrocytes 51(suppl 1):S4-8. could miss some differences in iron bioavailability, as 15. Frykman E, Bystrom M, Hansen T. Side effects of iron supple- noted by Fomon in newborn infants ; it is not known mentation in blood donors: superior tolerance of heme iron. J Lab Clin Med 1994;123:561-4. whether the observed differences concern iron absorp- 16. Berrocal R, Chanton S, Juillerat MA, Pavillard B, Scherz JC, Jost tion or kinetics. R. Tryptic phosphopeptides from whole casein. II. Physicochem- Our findings show that in experimental models, iron ical properties related to the solubilization of calcium. J Dairy bound to -CN (1-25) displayed better absorption and Res 1989;56:335-41. better tissue uptake than inorganic salts; similarly, the 17. Sato R, Shindo M, Gunshin H, Noguchi T, Naito H. Character- J Lab Clin Med 294 Ait-Oukhatar et al October 2002 ization of phosphopeptide derived from bovine -casein: an two different distributions of daily calcium intake. Am J Clin inhibitor to intra-intestinal precipitation of calcium phosphate. Nutr 1995;61:97-104. Biochim Biophys Acta 1991;1077:413-5. 23. Hurrell RF, Davidsson L, Reddy M, Kastenmayer P, Cook JD. A 18. Emery T. Iron oxidation by casein. Biochem Biophys Res Com- comparison of iron absorption in adults and infants consuming mun 1991;182:1047-52. identical infant formulas. Br J Nutr 1998;79:31-6. 19. Benito P, House W, Miller D. Influence of iron supplementation 24. Reddy MB, Cook JD. Assessment of dietary determinants of frequency on absorption efficiency and mucosal ferritin in anae- nonheme-iron absorption in humans and rats. Am J Clin Nutr mic rats. Br J Nutr 1997;78:469-77. 1991;54:723-8. 20. Deehr MS, Dallal GE, Smith KT, Taulbee JD, Dawson-Hugues 25. Barrett JFR, Whittaker PG, Fenwick JD, Williams JG, Lind T. B. Effects of different calcium sources on iron absorption in Comparison of stable isotopes and radioisotopes in the measurement postmenopausal women. Am J Clin Nutr 1990;51:95-9. of iron absorption in healthy women. Clin Sci 1994;87:91-5. 21. Galan P, Cherouvrier F, Preziosi P, Hercberg S. Effects of the 26. Aggett PJ. Iron, copper, and zinc absorption and turnover: the use increasing consumption of dairy products upon iron absorption. of stable isotopes. Eur J Pediatr 1997;156(Suppl 1):S29-34. Eur J Clin Nutr 1991;45:553-9. 27. Fomon SJ, Ziegler EE, Serfass EE, Nelson SE, Rogers RR, 22. Gleerup A, Rossander-Hulthe ´n L, Gramatkovski E, Hallberg L. Frantz JA. Less than 80% of absorbed iron is promptly incorpo- Iron absorption from the whole diet: comparison of the effect of rated into erythrocytes of infants. J Nutr 2000;130:45-52. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Laboratory and Clinical Medicine Unpaywall

Bioavailability of caseinophosphopeptide-bound iron

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Nabil Ait-Oukhatar, Jean Michel Peres, Said Bouhallab, Dominique Neuville, Francois Bureau, Gerard Bouvard, Pierre Arhan, Dominique Bougle To cite this version: Nabil Ait-Oukhatar, Jean Michel Peres, Said Bouhallab, Dominique Neuville, Francois Bureau, et al.. Bioavailability of caseinophosphopeptide-bound iron. Journal of Laboratory and Clinical Medicine, Elsevier, 2002, 140 (4), pp.290-294. 10.1067/mlc.2002.128146. hal-01568870 HAL Id: hal-01568870 https://hal.archives-ouvertes.fr/hal-01568870 Submitted on 25 Jul 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - ShareAlike| 4.0 International License Bioavailability of caseinophosphopeptide-bound iron NABIL AIT-OUKHATAR, JEAN MICHEL PERES, SAID BOUHALLAB, DOMINIQUE NEUVILLE, FRANCOIS BUREAU, GERARD BOUVARD, PIERRE ARHAN, and DOMINIQUE BOUGLE CAEN and RENNES, FRANCE Iron deficiency, one of the main worldwide nutritional deficiencies, results from the low bioavailability of most dietary iron, including cow milk. Hydrolysis of the cow milk protein casein produces low molecular weight caseinophosphopeptides (CPPs). Binding of iron to CPPs keeps it soluble in the digestive tract and prevents the formation of high molecular weight ferric hydroxides, which are poorly absorbed. Previous experimental studies have shown that iron bound to the phosphopeptide containing the first 25 amino acids of -casein, or -CN (1-25), is well absorbed and corrects efficiently iron deficiency. We sought to assess in vivo iron absorption and uptake by tissues involved in iron metabolism and storage (liver, spleen, bone marrow), using radiolabeled iron. -CN (1-25)-Fe displayed better absorption and tissue uptake by the vascularized rat loop model compared with a control sub- stance, ferric ascorbate. The metabolism of -CN (1-25)-Fe labeled with iron 59, added to cow milk, was also studied in young women. Although the absorption of -CN (1-25)-Fe was not significantly higher than that of ferrous sulfate, it displayed significantly higher tissue uptake. This increase was transient and had disappeared by the 14th day of the study, suggesting that iron was used for metabolic purposes. (J Lab Clin Med 2002;140:290-4) Abreviations: CN  casein; CPP: caseinophosphopeptide; -CN (1-25)–Fe  first 25 amino acids of -casein– bound iron; -CN (1-25)  first 25 amino acids of -casein igestion of milk proteins releases numerous bovine milk proteins keep iron soluble in the digestive bioactive peptides encoded in the native pro- tract but inhibit its absorption unless they are hydro- D teins. They display specific physiological func- lyzed. The use of CPP instead of native proteins might tions in gastrointestinal, immunological, hormonal, and improve iron absorption. 1,2 nutritional responses. The N-terminal CPP issued from the trypsin hydro- Among them, CPPs issued from casein hydrolysis lysis of -CN is one of the main CPPs produced in vivo 3,4 7 have the ability to bind and keep soluble cations; they during digestion ; previous experimental studies in rats improve calcium and zinc absorption. Indeed, intact have shown that -CN (1-25)– bound iron remains sol- uble in the digestive tract, where it escapes further 7,8 From the Laboratoire de Physiologie Digestive et Nutritionnelle, enzyme digestion. The absorption of -CN 1-25– Laboratoire de Biochimie A, and Service des Radio-Isotopes, CHU bound iron occurs partly through endocytosis and is de Caen; and the Laboratoire de Recherches de Technologie Laitie `re, more efficient than the absorption of gluconate iron ; INRA, Rennes, France. repletion with  CN (1-25)– bound iron also results in Supported in part by Die ´pal nsa, Groupe Danone. significantly greater improvement of hemoglobin and Submitted for publication January 2, 2002; revision submitted April iron concentrations in related tissues (liver, spleen) in 8, 2002; accepted May 15, 2002. iron-deficient rats than repletion with FeSO . Using Reprint requests: Dominique Bougle ´, Service de Pe ´diatrie A, CHU iron 59 –labeled iron, we assessed in vivo the metabo- Cle ´menceau, CHU de Caen F-14033, Caen Cedex, France; e-mail: bougle-d@chu-caen.fr. lism of absorbed iron in rats and compared these results Copyright © 2002 by Mosby, Inc. All rights reserved. with the metabolism of absorbed iron in human beings. 0022-2143/2002 $35.00  0 5/1/128146 We compared the vascularized duodenal rat loop doi:10.1067/mlc.2002.128146 model with iron absorption on the basis of the deter- 290 J Lab Clin Med Volume 140, Number 4 Ait-Oukhatar et al 291 tory disease, or infection; were taking no medication or min- mination of radioactivity incorporated into circulating eral supplementation; were using a chemical method of con- red blood cells, as well as tissue uptake, in young traception; and had negative results on pregnancy testing. women. Before the administration of the first test meal, blood was drawn for measurement of red blood cell count, serum fer- METHODS ritin, C-reactive protein (a marker of recent inflammation or Preparation of -CN (1-25)– bound iron. -CN (1-25) infection), and background radioactivity. (purity  85%) was purified from tryptic hydrolysate of The test subjects drank radioisotopically labeled milk after -CN as previously described. an overnight fast; nothing but water was allowed for the next We bound iron by mixing -CN (1-25) with a FeCl 4 hours. All subjects drank 250 mL of sterilized milk con- solution for 1 hour at 37°C and a pH of 6.5. The resulting taining 3 mg iron (12 mg/L), either as FeSO or -CN solutions were then ultrafiltered and diafiltered on a 3-kD (1-25)-Fe, labeled with Fe. The two meals were given at membrane to remove free minerals. The amount of iron random. The first was given on day 1; on day 14, blood was complexed to phosphopeptides and the presence of traces of drawn to measure the increase in red blood cell radioactivity. calcium and sodium were determined with the use of atomic- External counting of liver, spleen, and blood marrow (sa- absorption spectrometry (Model AA 1275; Varian; Les Ulis, crum) areas was performed on days 7 and 14 with the use of France) on freeze-dried samples. The final product had an a solid scintillation counter equipped with a thick crystal for iron/phosphopeptides molar ratio of 4 and contained less than external isotope tissue measurement (CGR Nucle ´aire Me ´- 1 mg sodium and 0.1 mg calcium/g. decine GM2C; Paris, France); counting lasted 10 minutes. Labeling of iron. -Emitting Fe was purchased from The heart area was used as control, and background radioac- Amersham (Amersham Pharmacia Biotech, Orsay; specific tivity was subtracted. activity 3.7 MBq/mL). Perfusion solutes were extrinsically The second meal was given after a washout period of 2 labeled in the laboratory with FeCl and mixed with cold weeks (day 28), after residual red blood cell and body radio- ferric ascorbate at a ratio of 0.001%, whereas the complex of activity had been determined. External counting was per- -CN (1-25)– bound iron was intrinsically labeled during its formed on days 35 and 42; erythrocyte radioactivity was preparation. measured on day 42. For the human study, milk was labeled either with extrin- Percentage absorption of iron was calculated on the basis sically tagged FeSO with Fe citrate or with the intrinsically of blood volume estimated from weight and height and an labeled -CN (1-25)–Fe. assumed hemoglobin incorporation of absorbed iron of Each 250-mL meal contained the same amount of radioac- 80%. In addition, data were corrected for background ra- tivity (74 kBq). dioactivity and for the radioisotope decay of the residual Experimental study. In an experimental study of perfused radioactivity of tissues and red blood cells. rat duodenal loop, four groups (each group n  6) of adult Statistical methods. We used Student’s t test to analyze Sprague-Dawley rats were studied after being fasted over- data from the experimental study. Human data were subjected night, as previously described. They were perfused with to analysis of variance and and Fisher’s exact test. P values of ferric ascorbate or -CN (1-25)-Fe. We tagged iron with Fe less than .05 were considered statistically significant. so that we might assess iron tissue uptake in addition to its RESULTS absorption. The composition of the perfusion solute was adapted from Experimental study. The absorption of ferric ascorbate Ringer-Lavoisier solute; its pH was adjusted to that of the and -CN (1-25)-Fe by isolated perfused rat duodenal proximal duodenum (5.5) and contained 100 mol/L iron in loop system is shown in Table I. -CN (1-25)-Fe dis- either form. played greater gut uptake and net absorption, greater Duodenum of anaesthetized rats was perfused at a delivery spleen uptake, and increased blood radioactivity com- rate of 0.16 mL/min; every element of the perfusion device pared with ferric ascorbate. had been washed with a solution of Triton X-100 (1 g/L) to Human study. Hematologic data and results of iron- prevent contamination. We kept the perfusion solute at 37°C with a thermostatic control and added a nonabsorbable absorption testing are given in Table II. Three subjects marker (polyethylene glycol 4000) to assess actual net water were iron-deficient (ferritin concentration  12 g/L). flux. After 2 hours of perfusion, the animal was killed with an Mean iron absorption was similar in the two groups, overdose of Dole ´thal; then the perfused loop was withdrawn yet paired comparisons showed a nonsignificant trend and washed with saline solution. toward better absorption of -CN (1-25)-Fe. Tissues were digested in nitric acid. The radioactivity of Results of external counting are given in Fig 1. A gut mucosa, blood, liver, and spleen was measured on a significant difference was displayed at day 7 between scintillation counter. FeSO and iron-labeled CPP for liver (P  0.05), Human study. Our human study was approved by the local spleen (P  0.05), and sacrum (P  0.03); the total committee of ethics. Ten subjects gave their written informed radioactivity of these organs was different between consent to participate: All were 20- to 30-year-old female groups (P  0.01) at day 7. No difference was observed students of the university, and all stated that they were in good health, had no recent history of digestive or inflamma- at day 14. J Lab Clin Med 292 Ait-Oukhatar et al October 2002 Table I. Absorption and tissue uptake of radiolabeled iron, given as iron ascorbate or -CN (1-25)-bound iron, by rat duodenal loop Test substance Mucosal uptake* Mucosal retention* Net absorption* Liver uptake* Spleen uptake* Blood activity* Sum of tissue activity Iron ascorbate 14.1  1.0* 6.2  0.5 7.9  1.0 4.7  0.3 0.02  0.00 3.0  0.6 13.3  0.8 CN (1-25)- 18.2  1.2 7.0  0.2 11.2  1.1 4.8  0.3 0.03  0.01 3.6  0.1 15.4  0.3 Fe P value .001 .006 .001 .49 .022 .001 .001 *Percent of dose perfused; mean  SD, n  6 per group. Initial iron concentration-100 mol. Sum of mucosa, liver, spleen, and blood activity. DISCUSSION of -CN (1-25) bound iron supports the results of tissue 9,10 analysis after a 4-week repletion period. Iron deficiency remains a worldwide health problem, Results varied between the rat and human groups. mainly involving growing infants and women of child- Differences in experimental methods could explain the bearing age. In addition, the side effects of iron isotopic data in the human group, in which iron-absorp- supplementation limit adherence to its prescription, tion rates were close to those obtained from studies that necessitating a search for highly bioavailable sources of 6,20-23 employed cow milk as source of iron. As expect- iron, free of digestive interactions. ed, these results differed quantitatively from those of CPPs are produced during digestion of CN; they bind the rat group but displayed the same trends; some divalent cations and keep them soluble at luminal 7,16,17 differences between the two experiments could explain pH. The strength of iron binding to CPP is about the lower significance in the human trial. First, iron was 100 times greater than that of calcium and others cat- 3,18 given in milk and not in a pure solution as in the rat ions. group. This decreases iron absorption and blunts dif- When assessed in rats on the basis of metabolic ferences between dietary sources of iron. Second, balance and direct measurement of tissue storage, the physiologic changes, such as the onset of menses, could bioavailability of iron bound to -CN (1-25) is higher have occurred during the time elapsed between the two than that of reference iron salts gluconate or ferrous 55 59 9,10 tests in the human group. Use of Fe and Fe on sulfate. consecutive days could have alleviated these potential In this study, the absorption rate and tissue uptake of 59 19 Fe were similar to those noted in recent reports. Our variations, but the use of -emitting Fe precludes findings showed that absorption, blood content, and external counting. spleen uptake of -CN (1-25)– bound iron by duodenal Although stable isotopes are valid tools in the assess- 25,26 rat loop during the experiment were better than that of ment of iron absorption from food, they do not inorganic iron. The better absorption and tissue uptake supply information on tissue kinetics, as radioisotopes Table II. Clinical details and iron absorption rate by human subjects given 3 mg of iron tagged with Fe, either bound to -CN (1-25) or as FeSO in 250 mL milk Iron absorption (%) Ratio of -CN (1-25)-Fe Subject Ferritin (g/L) Hemoglobin (g/dL) -CN (1-25)-Fe FeSO to FeSO 4 4 1 36 14.4 24.3 19.6 1.24 2 24 13.2 4.4 6.9 0.65 3 45 12.4 19.8 19.3 1.03 4 5 14.8 14.6 18.4 0.80 5 48 13.3 4.2 2.1 2.05 6 31 14.1 10.2 9.5 1.08 7 11 11.6 9.7 26.5 0.37 8 40 12 7.7 1.8 4.37 9 4 11.4 22.6 16.6 1.36 10 33 14.3 5.3 2.8 1.92 MeanSD 27.7  16.1 13.1  0.4 9.8  6.1 9.9  7.1 1.49  0.36* *Wilcoxon’s test not significant. J Lab Clin Med Volume 140, Number 4 Ait-Oukhatar et al 293 findings of external counting of organs was temporarily enhanced in human subjects after intake of  CN (1- 25)– bound iron. These beneficial results may partly explain the high iron bioavailability of breast milk, which is rich in -CN. Further human studies are needed to precisely assess the bioavailability of -CN (1-25)– bound iron, partic- ularly the lack of interaction with other minerals pre- viously shown in experimental studies. REFERENCES 1. Bos C, Gaudichon C, Tome ´ D. Nutritional and physiological criteria in the assessment of milk protein quality for humans. J Am Coll Nutr 2000;19:191S-205. 2. Clare DA, Swaisgood HE. Bioactive milk peptides: a prospectus. J Dairy Sci 2000;83:1187-95. 3. Brule ´ G, Fauquant J. Interaction des prote ´ines du lait et des oligo-e ´le ´ments. Lait 1982;62:323-31. 4. Bouhallab S, Le ´onil J, Maubois JL. Complexation du fer par le phosphopeptide (1-25) de la case ´ine : action de l’alcalase et de la phosphatase acide. Lait 1991;71:435-43. 5. Hansen M, Sandstro ¨mB,Lo ¨ nnerdal B. The effect of casein phosphopeptides on zinc and calcium absorption from high phytates infant diets assessed in rat pups and Caco-2 cells. Pediatr Res 1996;40:547-52. 6. 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