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Manganese and Iron Interrelationship in the Chick
Manganese and Iron Interrelationship in the Chick D. H. BAKER1 and K. M. HALPIN2 Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801 (Received for publication May 18, 1990) ABSTRACT Experiments were conducted to investigate tbe Mn-Fe interrelationship in the chick. Graded levels of Fe and two levels of Mn were added to a corn-soybean meal diet (157 mg Mn/kg; 372 mg Fe/kg) and to an Fe-deficient casein-dextrose diet containing 15.4 mg Mn/kg and 5.0 mg Fe/kg. Dietary Fe had little effect on the Mn status of the chick, regardless of the level of Fe supplementation. Conversely, Mn supplemented at 1,000 mg/kg reduced blood hemoglobin concentration, but only when the dietary Fe level was at or below the chick's requirement Levels of supplemental Fe up to 2,500 mg/kg had no effect on performance of chicks fed the corn-soybean diet, but a supplemental level of 5,000 mg Fe/kg diet reduced both weight gain and bone ash concentration. These data suggest that the interaction between Mn and Fe in the chick is a unidirectional phenomenon, in which excess Mn impairs Fe utilization but excess Fe does not antagonize Mn. (Key words: manganese, iron, performance, chick, hemoglobin) 1991 Poultry Science 70:146-152 INTRODUCTION
Iron and Mn share common binding sites in the gut mucosa and, therefore, are thought to compete with one another for absorption (Thomson et al, 1971; Thomson and Valberg, 1972; Davis et al, 1990). Gastrointestinal absorption of both trace elements has been shown to increase in conditions of Fe deficiency (Diez-Ewald et al., 1968; Thomson et al, 1971; Flanagan et al., 1980). Moreover, a Mn component was identified as a normal constituent of the heme molecule in human and rabbit reticulocytes (Borg and Cotzias, 1958; Thomas, 1970). Manganese incorporation into the porphyrin ring of hemoglobin was found to increase in Fe deficiency; also, Mndeficient rats fed supplemental Fe have been found to exhibit reduced Mn-dependent superoxide dismutase activity (Davis et al, 1990). Thus, an obvious rationale exists for an antagonism between Fe and Mn. Practical poultry diets are routinely supplemented with Fe and Mn, usually at levels in excess of the chick's dietary requirement. Because Fe-deficiency anemia is the most prevalent nutritional deficiency in the world,
Fe supplementation or Fe fortification of human foods is also commonplace. In addition, high levels of Fe (up to 500 mg/kg) are used to block the toxic effects of free gossypol, a toxin found in cottonseed meal (Tanksley, 1970; Smith, 1970). Thus, a Mn-Fe interrelationship may have practical significance. Although numerous studies have assessed the effect of Mn on Fe utilization (i.e., hemoglobin or hematocrit), relatively few investigators have examined the effect of excess Fe on Mn status of the animal. Iron supplementation was reported to decrease liver Mn-concentration in mice (Hurley et al, 1983) but to have no effect on tissue Mn in calves (Ho et al, 1984). The Fe-Mn interrelationship has recently taken on added significance, as Mn insufficiency has been linked to osteoporosis in humans (Strause and Saltman, 1987; Freedland-Graves et al, 1990). Because meat products are high in Fe but low in Mn, it is important to establish whether Mn utilization is reduced by ingestion of diets containing high Fe:Mn ratios. MATERIALS AND METHODS
Care of Animals s
To whom reprint requests should be addressed: 328 Mumford Hall, 1301 West Gregory Drive, Urbana, IL 61801. 2 Present address: BioKyowa Inc., 930 Roosevelt Parkway, Chesterfield, MO 63017.
Male New Hampshire X Columbian crossbred chicks were fed a standard cornsoybean meal starter diet (23% CP; 3,200 kcal MFVkg) during the first 7 days posthatching.
146
MANGANESE AND IRON INTERRELATIONSHIP
147
were bled by cardiac puncture, and blood was pooled by replicate. The chicks were then killed by cervical dislocation, and the right tibia as well as bile and liver samples were removed. The tibia, bile, and livers were pooled by replicate, dried, and analyzed for Mn. Iron concentration in the liver was also determined. The tibia were ashed 24 h at 650 C before analysis, and bile and liver samples were dried overnight at 100 C, weighed, and then wet ashed with HNO3 and H2Q2 (30%). Manganese and Fe concentrations in diets and tissues were quantified by atomic absorption spectrophotometry.3 Blood samples Experimental Diets were analyzed immediately after bleeding for The basal diet (Table 1) used in Experiment 1 hemoglobin content (Crosby et al., 1954) and was a typical corn-soybean meal diet formulated hematocrit (Cohen, 1967). to meet or exceed all nutrient requirements of the growing chick (National Research Council, 1984). It was analyzed to contain 157 mg Mn/kg Statistical Analyses and 372 mg Fe/kg. Chicks in Experiment 2 were All data were analyzed by analysis of fed an Fe-deficient casein-dextrose diet. The diet variance procedures appropriate for factorial was analyzed to contain 5 mg/kg Fe and the arrangement of treatments (Steel and Torrie, minimal Mn requirement of 15.4 mg/kg 1980). Orthogonal as well as meaningful nonor(Southern and Baker, 1983a; Halpin and Baker, thogonal single-degree-of-freedom comparisons 1986) for chicks fed this phytate and fiber-free were used to test treatment differences. In basal diet. Additions to the basal diets were Experiment 1, Fe effects tested were 0 to 2,500 made at the expense of cornstarch (Experiment 1) or dextrose (Experiment 2). Inorganic Fe and versus 5,000 mg/kg, and for liver Fe, linear and Mn were provided as reagent-grade quadratic effects were evaluated as well. In FeS04-7H20 and MnS0 4 H 2 0, respectively. Experiment 2, the Fe effects tested were 0 versus 40 to 2,000 mg/kg and 0 and 40 mg/kg versus 100 to 2,000 mg/kg. For liver Fe, linear and Experimental Protocol quadratic effects were tested. The Mn main A 6 x 2 factorial arrangement of treatments effect and Mn x Fe interactions were also was employed in both experiments to assess the evaluated. Mn-Fe interrelationship. Six levels of supplemental Fe up to a maximum of 5,000 mg Fe/kg RESULTS were added to the corn-soybean meal diet (157 Supplemental Fe up to 2,500 mg/kg did not mg Mn/kg; 372 mg Fe/kg) in the presence and absence of 1,000 mg/kg supplemental Mn. affect chick performance or bone ash concenBecause excess Fe is more growth depressing in tration when added to a com-soybean meal semipurified diets than in grain-based diets diet (Table 2). Iron supplemented at 5,000 mg/ (Southern and Baker, 1982), lower levels of kg, however, depressed (P<.01) gain, gain: supplemental Fe (up to 2,000 mg Fe/kg) were feed, and bone ash concentration. Although added to the casein-dextrose diet (15.4 mg Mn/ Mn supplementation had little effect on the kg; 5.0 mg Fe/kg), again in the presence or rate or efficiency of growth at lower levels of absence of 1,000 mg/kg supplemental Mn. dietary Fe, it depressed performance when coadministered with 5,000 mg/kg supplemental Fe. This resulted in a Fe by Mn interaction Tissue Analyses (P<.01) for gain and gain:feed. At the termination of each experiment, the Supplemental Mn markedly enhanced three median-weight chicks within a replicate (P<01) Mn deposition in tissues (Table 2). However, the effect of Fe supplementation on tissue Mn levels was minimal. Similarly, 3 Model 306, Peridn-Elmer Corporation, Norwalk, CT supplemental Fe increased (P<.01) Fe deposi06856. tion in liver, but Mn supplementation was Following an overnight fast, the chicks were weighed and allotted to experimental groups so that each group had a similar mean initial weight and weight distribution. Three replicate groups of five chicks were fed the experimental diets from 8 to 22 days posthatching. The chicks were housed in thermostatically controlled, wirefloored starter batteries, and a 24-h constant light schedule was maintained. Feed and water were provided for ad libitum consumption throughout each assay period.
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BAKER AND HALPIN TABLE 1. Composition of basal diets
Diet
Ingredient
Corn-soybean meal1
Cornstarch Com Soybean meal Com oil Dicalcium phosphate Limestone, ground Iodized salt Manganese sulfate Ferric citrate Zinc carbonate Vitamin mixture2 Choline chloride (60%) DL-methionine Selenium premix3 Dextrose Casein Com oil Mineral mixture (Mn- and Fe-free)5 Glycine DL-methionine L-arginine NaHC0 3 Vitamin mixture6 Choline chloride DL-cc-tocopheryl acetate (20 mg/kg) Manganese sulfate
Casein-dextrose4
(%) to 100.00 49.73 38.40 5.00 2.20 1.00 .40 .04 .01 .01 .10 .10 .20 .10 to 100.00 20.00 3.00 5.25 2.00 .50 1.00 1.00 .20 .20 + .0043
Contained 157 mg Mn/kg and 372 mg Fe/kg. Vitamin mixture provided per kilogram of diet: vitamin A, 4,400 IU; vitamin D3,1,000 ICU; vitamin E, 11IU; vitamin Bj 2 , -01 mg; riboflavin, 4.41 mg-, d-pantothenic acid, 10.0 mg; niacin, 22.0 mg; menadione sodium bisulfite, 2.33 mg. Contributed .1 mg Se/kg diet. 4 Contained 15.4 mg Mn/kg and 5.0 mg Fe/kg. 5 Mineral mixture provided per kilogram of diet: CaC03, 3.0 g; Ca 3 (P04) 2 , 28.0 g; K2HPO4, 9.0 g; NaCl, 8.8 g; MgS04-7H 2 0,3.5 g; ZnC0 3 ,100.0 mg; CuSC>4-5H20,20.0 mg; H ^ O ^ 9.0 mg; Na 2 Mo0 4 -2H 2 0, 9.0 mg; KI, 40.0 mg; CoS0 4 -7H 2 0, 1.0 mg; Na^eC^, .215 mg. ^Vitamin mixture provided per kilogram of diet thiamine HC1,20 mg; niacin, 50 mg; riboflavin, 10 mg; Ca-pantothenate, 30 mg; vitamin B j 2 , .04 mg; pyridoxine-HCl, 6 mg; biotin, .6 mg; folic acid, 4 mg; inositol, 100 mg; para-aminobenzoic acid, 2 mg; vitamin K, 2 mg; ascorbic acid, 250 mg; cholecalciferol (200,000 IU/g), 600 IU; retinyl acetate (650,000 IU/g), 5,200 IU.
without effect. Addition of Fe or Mi to the nutritionally adequate basal diet did not significantly affect blood hemoglobin concentration or hematocrit. In Experiment 2, supplemental Fe enhanced (P<01) growth when added to the Fe-deficient casein-dextrose diet (Table 3). However, no further improvement in performance was observed beyond the first level (40 mg/kg) of Fe supplementation, nor was growth depressed at higher levels. Manganese supplemented at 1,000 mg/kg did not affect chick performance, regardless of dietary Fe level. Supplemental Mn, however, increased bone ash concentration (P<01).
As in Experiment 1, supplemental Fe had little effect on Mn status of the chick (Table 3). Manganese supplementation markedly increased (P<.01) tissue Mn concentrations, but supplemental Fe was without effect. However, 40 mg/kg Fe supplementation increased (P<01) blood hemoglobin, but higher levels of Fe did not increase these criteria further, liver Fe concentration increased linearly (P<.01) with increasing levels of dietary Fe. Manganese supplementation reduced hemoglobin concentration when added to diets containing 0 or 40 mg/kg supplemental Fe, but had little effect when coadministered witii higher levels of Fe. This resulted in a Fe x Mn interaction
MANGANESE AND IRON INTERRELATIONSHIP
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0 40 100 500 1,000 2,000 0 40 100 500 1,000 2,000 Pooled SEM
Supplemental Fe
225 262 246 248 256 247 218 252 249 266 258 253 6
(el
Gain -
1 2
1
(g/kg) 649 665 666 658 668 679 645 662 633 676 656 639 13
Gaiiufeed
Perf
46.5 46.8 45.8 46.6 46.6 45.9 45.6 47.1 47.7 47.4 49.0 48.2 .6
(%)
Ash3-4 4.1 3.9 2.9 2.4 2.4 2.5 32.1 39.0 34.4 36.0 31.1 29.0 7
Mn3-4
Tibia
4.1 4.9 3.1 4.7 3.8 4.5 113 143 140 193 140 131 7
Mh -
3 4
Bile
Ws'8/ 6.1 10.3 8.8 7.8 8.4 8.9 24.9 35.6 29.0 37.8 35.3 34.9 2.5
Mh -
3 4
120 386 317 443 755 1,638 153 401 423 547 696 2,187 7
Fe -
3 5
Liver -
(g/100 mL) 5.1 8.8 8.3 8.9 8.6 8.9 4.4 8.0 8.9 9.0 8.8 8.9 2
Treatment variance was heteroscedastic for tibia Mn, bile Mn, and liver Fe.
7
Significant Mh x Fe (0 and 40 versus 100 to 2,000 mg Fe/kg) interaction (P<05).
Significant Fe linear effect (P<01).
Significant Mh main effect (P<01).
^Data are means of three samples, each sample representing pooled tissue from the three median-weight chicks within a replicate.
Significant Fe level (0 versus 40 to 2,000 mg Fe/kg) effect (P<01).
3 6
Hemoglobin '
'Data are means of three replicate groups of five male chicks during the period 8 to 22 days posthatching; mean initial weight was 76 g.
1,015.4
15.4
Supplemental Mn
18.3 25.5 28.2 27.8 27.7 27.6 .8
19.3 27.7 27.0 28.0 27.8 28.1
(%)
Hematocrit3
Blood
TABLE 3. Assessment of manganese and iron status of chicks fed a casein-dextrose diet containing variable levels of manganese and iron (Experiment 2)
MANGANESE AND IRON ESTTERRELAITONSHIP
(P<.05) for blood hemoglobin. Supplemental Mn did not affect Fe deposition in the liver. DISCUSSION
Chick performance was depressed when 5,000 mg Fe/kg were added to the cornsoybean meal diet containing 372 mg Fe/kg, regardless of dietary Mn level. These data are in agreement with the observations of Deobald and Elvehjem (1935) that approximately 4,500 mg Fe/kg diet causes anorexia in chickens fed grain-based diets. Farrow et al. (1982) also observed a growth depression in chicks fed a milo-soybean meal diet containing 5,000 mg Fe/mg. It was surprising that growth was not depressed when Fe was supplemented up to 2,000 mg/kg in the casein-dextrose diet (Experiment 2). McGhee et al. (1965) reported that 400 to 800 mg Fe/kg (depending on the level of Cu) was toxic to chicks fed a semipurified skim milk-sucrose diet. Maximum improvement in performance was observed at the lowest level (40 mg/kg) of Fe supplementation. Southern and Baker (1982) determined that the minimal Fe requirement was 40 mg/kg in chicks fed a caseindextrose diet. Supplemental Mn markedly enhanced Mn deposition in tibia, bile, and liver in both experiments. Excess Fe ingestion had no effect on Mn uptake by these tissues. Previous studies have shown that tibia Mn is a sensitive indicator of Mn status (Halpin et al., 1986; Fly et al, 1989; Wedekind et al, 1989). Thus, even with the corn-soybean diet containing a surfeit level of Mn (i.e., 157 mg/kg), excess supplemental Fe up to 5,000 mg/kg would have been expected to depress tibia Mn if, in fact, the Fe load were interfering with Mn absorption. In the corn-soybean diet series, the Fe:Mn ratio (wt/wt) at its extreme was 34:1 whereas in the casein diet series the highest Fe:Mn ratio was 130:1. In both diet treatments, these extreme Fe:Mn ratios did not decrease Mn levels in any of the tissues evaluated. Manganese supplemented at 1,000 mg/kg reduced blood hemoglobin concentration of chicks fed the casein-dextrose diet but only when the level of dietary Fe was at or below the chick's requirement. The interference of excess Mn with hemoglobin formation has previously been shown in a variety of animal species. Hemoglobin concentration was depressed in chicks fed an iron-adequate diet
151
supplemented with 3,000 mg/kg Mn (Southern and Baker, 1983b). Moreover, Matrone et al. (1959) showed that hemoglobin regeneration was markedly retarded in anemic rabbits and baby pigs fed excess (2,000 mg/kg) Mn. They suggested that dietary Mn concentrations as low as 50 to 125 mg/kg might interfere with hemoglobin formation. Similarly, hemoglobin and serum iron were depressed in young lambs fed 45 mg/kg supplemental Mn, whereas higher levels of Mn also reduced Fe concentrations in liver, spleen, and kidney (Hartman et al, 1955). Maines (1980) suggested that excess Mn reduces heme synthesis by inhibiting 5-aminolevulinate synthase. In treatments with the casein-dextrose diet, liver Fe concentration increased linearly with increasing levels of dietary Fe, but 1,000 mg/ kg supplemental Mn did not affect Fe deposition in the liver. In previous research with chicks from the authors' laboratory, liver Fe has been shown to be an unreliable index of Fe status (Bafundo et al, 1984; Southern and Baker, 1982), whereas blood hemoglobin has proven to be a sensitive indicator of Fe adequacy (Southern and Baker, 1982; Parsons et al, 1989). The data herein suggest that the interaction between Mn and Fe is a unidirectional phenomenon, in which excess Mn impairs Fe utilization but excess Fe does not antagonize Mn. This unidirectional interaction is similar to that which occurs between zinc and Fe when these trace elements are added to phytate- and fiber-containing diets; i.e., excess Fe has no effect on zinc, but excess zinc strongly inhibits nonheme-Fe utilization (Bafundo et al, 1984; Anonymous, 1985). Zinc also has been shown to antagonize copper, whereas excess copper has little effect on zinc utilization (O'Dell et al, 1976; Southern and Baker, 1983c,d). Recent evidence suggests that phosphate supplements used in poultry rations can antagonize Mn if excess levels of the supplements are used (Wedekind and Baker, 1990a,b). Phosphorous supplements are high in bioavailable Fe (Czarnecki-Maulden, 1990), but the data herein suggest that the Fe contained in phosphorus supplements such as dicalcium phosphate and defluorinated rock phosphate is not the factor antagonizing Mn. Indeed, Wedekind and Baker (1990a) have shown that phosphorus per se is the Mnantagonizing factor in commercial phosphorus supplements.
152
BAKER AND HALPIN REFERENCES
Anonymous, 1985. Interactions of dietary iron and zinc in the chick. Nutr. Rev. 43:121-122. Bafundo, K. W., D. H. Baker, and P. R. Fitzgerald, 1984. The iron-zinc interrelationship in the chick as influenced by Eimeria acervulina infection. J. Nutr. 114:1306-1312. Borg, D. C, and G. C. Cotzias, 1958. Incorporation of manganese into erythrocytes as evidence for a manganese porphyrin in man. Nature 182: 1677-1678. Cohen, R. R., 1967. Anticoagulation, centrifugation time and sample replicate number in the microhematocrit method for avian blood. Poultry Sci. 46:214-218. Crosby, W. H., J. I. Munn, and F. W. Furth, 1954. Standardizing a method for clinical hemoglobinometry. US Armed Forces Med. J. 5:693-703. Czarnecki-Maulden, G. L., 1990. Iron bioavailability. Pages 73-84 in: Proceedings of the Guelph Nutrition Conference, Guelph, Ontario, Canada. Davis, C. D., D. M. Ney, and J. L. Greger, 1990. Manganese, iron and lipid interactions in rats. J. Nutr. 120:507-513. Deobald, H. J., and C. A. Elvehjem, 1935. The effect of feeding high amounts of soluble iron and aluminum salts. Am. J. Physiol. 111:118-123. Diez-Ewald, M., L. R. Weintraub, and W. H. Crosby, 1968. Interrelationship of iron and manganese metabolism. Proc. Soc. Exp. Biol. Med. 129: 448-451. Farrow, G., A. S. Glassman, P. Vohra, and F. H. Kratzer, 1982. Effect of high fat and iron levels on the growth and mortality of chickens. Poultry Sci. 62:85-90. Flanagan, P. R., J. Haist, and L. S. Valberg, 1980. Comparative effects of iron deficiency induced by bleeding and a low iron diet on the intestinal absorptive interactions of iron, cobalt, manganese, zinc, lead and cadmium. J. Nutr. 110:1754-1763. Fly, A. D., O. A. Izquierdo, K. R. Lowry, and D. H. Baker, 1989. Manganese bioavailability in a Mnmethionine chelate. Nutr. Res. 9:901-910. Freedland-Graves, J., Y. X. Li, X. H. Wir, R. Tesar, and R. Pobocik, 1990. Manganese status of osteoporotics and age-matched healthy women. Fed. Am. Soc. Exp. Biol. J. 4:A777. (Abstr.) Halpin, K. M, and D. H. Baker, 1986. Long-term effects of corn, soybean meal, wheat bran, and fish meal on manganese utilization in the chick. Poultry Sci. 65: 1371-1374. Halpin, K. M., D. G. Chausow, and D. H. Baker, 1986. Efficiency of manganese absorption in chicks fed corn-soy and casein diets. J. Nutr. 116:1747-1751. Hartman, R. H., G. Mattone, and G. H. Wise, 1955. Effect of high dietary manganese on hemoglobin formation. J. Nutr. 55:429-439. Ho, S. Y., W. J. Miller, R. P. Gentry, M. W. Neathery, and D. M. Blackmail, 1984. Effects of high but nontoxic dietary manganese and iron on their metabolism by calves. J. Dairy Sci. 67:1489-1495. Hurley, L. S., C. L. Keen, and B. Lonnerdal, 1983. Aspects of trace element metabolism during development. Fed. Proc. 42:1735-1739. Maines, M. D., 1980. Regional distribution of the enzymes of haem biosynthesis and the inhibition of 5-aminolevulinate synthase by manganese in the rat brain. Biochem. J. 190:315-321.
Matrone, G., R. H. Hartman, and A. J. Clawson, 1959. Studies of a manganese-iron antagonism in the nutrition of rabbits and baby pigs. J. Nutr. 67: 309-317. McGhee, F., C. R. Creger, and J. R. Couch, 1965. Copper and iron toxicity. Poultry Sci. 44:310-312. National Research Council 1984. Page 12 in: Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. O'Dell, B. L., P. G. Reeves, and R. F. Morgan, 1976. Interrelationships of tissue copper and zinc concentrations in rats nutritionally deficient in one or the other of these elements. Pages 411-421 in: Trace Substances in Environmental Health. University of Missouri, Columbia, MO. Parsons, C. M., D. H. Baker, and C. C. Welch, 1989. Effect of excess zinc on iron utilization by chicks fed a diet devoid of phytate and fiber. Nutr. Res. 9: 227-231. Smith, K. J., 1970. Practical significance of gossypol in feed formulation. J. Am. Oil Chem. Soc. 47: 448-450. Southern, L. L., and D. H. Baker, 1982. Iron status of the chick as affected by Eimeria acervulina infection and by variable iron ingestion. J. Nutr. 112: 2353-2362. Southern, L. L., and D. H. Baker, 1983a. Eimeria acervulina infection in chicks fed deficient or excess levels of manganese. J. Nutr. 113:172-177. Southern, L. L., and D. H. Baker, 1983b. Excess manganese ingestion in the chick. Poultry Sci. 62: 642-646. Southern, L. L., and D. H. Baker, 1983c. Eimeria acervulina infection and the zinc-copper interrelationship in the chick. Poultry Sci. 62:401-404. Southern, L. L., and D. H. Baker, 1983d. Zinc toxicity, zinc deficiency and the zinc-copper interrelationship in Eimeria acervulina-infected chicks. J. Nutr. 113: 688-696. Steel, R.GX>., and J. H. Torrie, 1980. Pages 137-171 in: Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed. McGraw-Hill Book Co., New York, NY. Strause, L., and P. Saltman, 1987. Role of manganese in bone metabolism. Page 146 in: Nutritional Bioavailability of Manganese. C. Kies, ed. American Chemistry Society Symposium Series 354. American Chemistry Society Publishing Co., Washington, DC. Tanksley, T. D., Jr., 1970. Use of cottonseed meal in swine rations. Feedstuffs 42:22. Thomas, J. W., 1970. Metabolism of iron and manganese. Jf. Dairy Sci. 53:1107-1123. Thomson, A S * . , D. Olatunbosun, and L. S. Valberg, 1971. Interrelation of intestinal transport system for manganese and iron. J. Lab. Clin. Med. 78:642-655. Thomson, A3JR., and L. S. Valberg, 1972. Intestinal uptake of iron, cobalt, and manganese in the irondeficient rat Am. J. Physiol. 223:1327-1329. Wedekind, K. J., and D. H. Baker, 1990a. Manganese utilization in chicks as affected by excess calcium and phosphorus ingestioa Poultry Sci. 69:977-984. Wedekind, K. J., and D. H. Baker, 1990b. Effect of varying calcium and phosphorus level on manganese utilization. Poultry Sci. 69:1156-1164. Wedekind, K. J., M R. Murphy, and D. H. Baker, 1989. Influence of manganese status and excess dietary phosphorus on manganese turnover in chicks. Poultry Sci. 68(Suppl. 1):157. (Abstr.)
Iron and Mn share common binding sites in the gut mucosa and, therefore, are thought to compete with one another for absorption (Thomson et al, 1971; Thomson and Valberg, 1972; Davis et al, 1990). Gastrointestinal absorption of both trace elements has been shown to increase in conditions of Fe deficiency (Diez-Ewald et al., 1968; Thomson et al, 1971; Flanagan et al., 1980). Moreover, a Mn component was identified as a normal constituent of the heme molecule in human and rabbit reticulocytes (Borg and Cotzias, 1958; Thomas, 1970). Manganese incorporation into the porphyrin ring of hemoglobin was found to increase in Fe deficiency; also, Mndeficient rats fed supplemental Fe have been found to exhibit reduced Mn-dependent superoxide dismutase activity (Davis et al, 1990). Thus, an obvious rationale exists for an antagonism between Fe and Mn. Practical poultry diets are routinely supplemented with Fe and Mn, usually at levels in excess of the chick's dietary requirement. Because Fe-deficiency anemia is the most prevalent nutritional deficiency in the world,
Fe supplementation or Fe fortification of human foods is also commonplace. In addition, high levels of Fe (up to 500 mg/kg) are used to block the toxic effects of free gossypol, a toxin found in cottonseed meal (Tanksley, 1970; Smith, 1970). Thus, a Mn-Fe interrelationship may have practical significance. Although numerous studies have assessed the effect of Mn on Fe utilization (i.e., hemoglobin or hematocrit), relatively few investigators have examined the effect of excess Fe on Mn status of the animal. Iron supplementation was reported to decrease liver Mn-concentration in mice (Hurley et al, 1983) but to have no effect on tissue Mn in calves (Ho et al, 1984). The Fe-Mn interrelationship has recently taken on added significance, as Mn insufficiency has been linked to osteoporosis in humans (Strause and Saltman, 1987; Freedland-Graves et al, 1990). Because meat products are high in Fe but low in Mn, it is important to establish whether Mn utilization is reduced by ingestion of diets containing high Fe:Mn ratios. MATERIALS AND METHODS
Care of Animals s
To whom reprint requests should be addressed: 328 Mumford Hall, 1301 West Gregory Drive, Urbana, IL 61801. 2 Present address: BioKyowa Inc., 930 Roosevelt Parkway, Chesterfield, MO 63017.
Male New Hampshire X Columbian crossbred chicks were fed a standard cornsoybean meal starter diet (23% CP; 3,200 kcal MFVkg) during the first 7 days posthatching.
146
MANGANESE AND IRON INTERRELATIONSHIP
147
were bled by cardiac puncture, and blood was pooled by replicate. The chicks were then killed by cervical dislocation, and the right tibia as well as bile and liver samples were removed. The tibia, bile, and livers were pooled by replicate, dried, and analyzed for Mn. Iron concentration in the liver was also determined. The tibia were ashed 24 h at 650 C before analysis, and bile and liver samples were dried overnight at 100 C, weighed, and then wet ashed with HNO3 and H2Q2 (30%). Manganese and Fe concentrations in diets and tissues were quantified by atomic absorption spectrophotometry.3 Blood samples Experimental Diets were analyzed immediately after bleeding for The basal diet (Table 1) used in Experiment 1 hemoglobin content (Crosby et al., 1954) and was a typical corn-soybean meal diet formulated hematocrit (Cohen, 1967). to meet or exceed all nutrient requirements of the growing chick (National Research Council, 1984). It was analyzed to contain 157 mg Mn/kg Statistical Analyses and 372 mg Fe/kg. Chicks in Experiment 2 were All data were analyzed by analysis of fed an Fe-deficient casein-dextrose diet. The diet variance procedures appropriate for factorial was analyzed to contain 5 mg/kg Fe and the arrangement of treatments (Steel and Torrie, minimal Mn requirement of 15.4 mg/kg 1980). Orthogonal as well as meaningful nonor(Southern and Baker, 1983a; Halpin and Baker, thogonal single-degree-of-freedom comparisons 1986) for chicks fed this phytate and fiber-free were used to test treatment differences. In basal diet. Additions to the basal diets were Experiment 1, Fe effects tested were 0 to 2,500 made at the expense of cornstarch (Experiment 1) or dextrose (Experiment 2). Inorganic Fe and versus 5,000 mg/kg, and for liver Fe, linear and Mn were provided as reagent-grade quadratic effects were evaluated as well. In FeS04-7H20 and MnS0 4 H 2 0, respectively. Experiment 2, the Fe effects tested were 0 versus 40 to 2,000 mg/kg and 0 and 40 mg/kg versus 100 to 2,000 mg/kg. For liver Fe, linear and Experimental Protocol quadratic effects were tested. The Mn main A 6 x 2 factorial arrangement of treatments effect and Mn x Fe interactions were also was employed in both experiments to assess the evaluated. Mn-Fe interrelationship. Six levels of supplemental Fe up to a maximum of 5,000 mg Fe/kg RESULTS were added to the corn-soybean meal diet (157 Supplemental Fe up to 2,500 mg/kg did not mg Mn/kg; 372 mg Fe/kg) in the presence and absence of 1,000 mg/kg supplemental Mn. affect chick performance or bone ash concenBecause excess Fe is more growth depressing in tration when added to a com-soybean meal semipurified diets than in grain-based diets diet (Table 2). Iron supplemented at 5,000 mg/ (Southern and Baker, 1982), lower levels of kg, however, depressed (P<.01) gain, gain: supplemental Fe (up to 2,000 mg Fe/kg) were feed, and bone ash concentration. Although added to the casein-dextrose diet (15.4 mg Mn/ Mn supplementation had little effect on the kg; 5.0 mg Fe/kg), again in the presence or rate or efficiency of growth at lower levels of absence of 1,000 mg/kg supplemental Mn. dietary Fe, it depressed performance when coadministered with 5,000 mg/kg supplemental Fe. This resulted in a Fe by Mn interaction Tissue Analyses (P<.01) for gain and gain:feed. At the termination of each experiment, the Supplemental Mn markedly enhanced three median-weight chicks within a replicate (P<01) Mn deposition in tissues (Table 2). However, the effect of Fe supplementation on tissue Mn levels was minimal. Similarly, 3 Model 306, Peridn-Elmer Corporation, Norwalk, CT supplemental Fe increased (P<.01) Fe deposi06856. tion in liver, but Mn supplementation was Following an overnight fast, the chicks were weighed and allotted to experimental groups so that each group had a similar mean initial weight and weight distribution. Three replicate groups of five chicks were fed the experimental diets from 8 to 22 days posthatching. The chicks were housed in thermostatically controlled, wirefloored starter batteries, and a 24-h constant light schedule was maintained. Feed and water were provided for ad libitum consumption throughout each assay period.
148
BAKER AND HALPIN TABLE 1. Composition of basal diets
Diet
Ingredient
Corn-soybean meal1
Cornstarch Com Soybean meal Com oil Dicalcium phosphate Limestone, ground Iodized salt Manganese sulfate Ferric citrate Zinc carbonate Vitamin mixture2 Choline chloride (60%) DL-methionine Selenium premix3 Dextrose Casein Com oil Mineral mixture (Mn- and Fe-free)5 Glycine DL-methionine L-arginine NaHC0 3 Vitamin mixture6 Choline chloride DL-cc-tocopheryl acetate (20 mg/kg) Manganese sulfate
Casein-dextrose4
(%) to 100.00 49.73 38.40 5.00 2.20 1.00 .40 .04 .01 .01 .10 .10 .20 .10 to 100.00 20.00 3.00 5.25 2.00 .50 1.00 1.00 .20 .20 + .0043
Contained 157 mg Mn/kg and 372 mg Fe/kg. Vitamin mixture provided per kilogram of diet: vitamin A, 4,400 IU; vitamin D3,1,000 ICU; vitamin E, 11IU; vitamin Bj 2 , -01 mg; riboflavin, 4.41 mg-, d-pantothenic acid, 10.0 mg; niacin, 22.0 mg; menadione sodium bisulfite, 2.33 mg. Contributed .1 mg Se/kg diet. 4 Contained 15.4 mg Mn/kg and 5.0 mg Fe/kg. 5 Mineral mixture provided per kilogram of diet: CaC03, 3.0 g; Ca 3 (P04) 2 , 28.0 g; K2HPO4, 9.0 g; NaCl, 8.8 g; MgS04-7H 2 0,3.5 g; ZnC0 3 ,100.0 mg; CuSC>4-5H20,20.0 mg; H ^ O ^ 9.0 mg; Na 2 Mo0 4 -2H 2 0, 9.0 mg; KI, 40.0 mg; CoS0 4 -7H 2 0, 1.0 mg; Na^eC^, .215 mg. ^Vitamin mixture provided per kilogram of diet thiamine HC1,20 mg; niacin, 50 mg; riboflavin, 10 mg; Ca-pantothenate, 30 mg; vitamin B j 2 , .04 mg; pyridoxine-HCl, 6 mg; biotin, .6 mg; folic acid, 4 mg; inositol, 100 mg; para-aminobenzoic acid, 2 mg; vitamin K, 2 mg; ascorbic acid, 250 mg; cholecalciferol (200,000 IU/g), 600 IU; retinyl acetate (650,000 IU/g), 5,200 IU.
without effect. Addition of Fe or Mi to the nutritionally adequate basal diet did not significantly affect blood hemoglobin concentration or hematocrit. In Experiment 2, supplemental Fe enhanced (P<01) growth when added to the Fe-deficient casein-dextrose diet (Table 3). However, no further improvement in performance was observed beyond the first level (40 mg/kg) of Fe supplementation, nor was growth depressed at higher levels. Manganese supplemented at 1,000 mg/kg did not affect chick performance, regardless of dietary Fe level. Supplemental Mn, however, increased bone ash concentration (P<01).
As in Experiment 1, supplemental Fe had little effect on Mn status of the chick (Table 3). Manganese supplementation markedly increased (P<.01) tissue Mn concentrations, but supplemental Fe was without effect. However, 40 mg/kg Fe supplementation increased (P<01) blood hemoglobin, but higher levels of Fe did not increase these criteria further, liver Fe concentration increased linearly (P<.01) with increasing levels of dietary Fe. Manganese supplementation reduced hemoglobin concentration when added to diets containing 0 or 40 mg/kg supplemental Fe, but had little effect when coadministered witii higher levels of Fe. This resulted in a Fe x Mn interaction
MANGANESE AND IRON INTERRELATIONSHIP
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0 40 100 500 1,000 2,000 0 40 100 500 1,000 2,000 Pooled SEM
Supplemental Fe
225 262 246 248 256 247 218 252 249 266 258 253 6
(el
Gain -
1 2
1
(g/kg) 649 665 666 658 668 679 645 662 633 676 656 639 13
Gaiiufeed
Perf
46.5 46.8 45.8 46.6 46.6 45.9 45.6 47.1 47.7 47.4 49.0 48.2 .6
(%)
Ash3-4 4.1 3.9 2.9 2.4 2.4 2.5 32.1 39.0 34.4 36.0 31.1 29.0 7
Mn3-4
Tibia
4.1 4.9 3.1 4.7 3.8 4.5 113 143 140 193 140 131 7
Mh -
3 4
Bile
Ws'8/ 6.1 10.3 8.8 7.8 8.4 8.9 24.9 35.6 29.0 37.8 35.3 34.9 2.5
Mh -
3 4
120 386 317 443 755 1,638 153 401 423 547 696 2,187 7
Fe -
3 5
Liver -
(g/100 mL) 5.1 8.8 8.3 8.9 8.6 8.9 4.4 8.0 8.9 9.0 8.8 8.9 2
Treatment variance was heteroscedastic for tibia Mn, bile Mn, and liver Fe.
7
Significant Mh x Fe (0 and 40 versus 100 to 2,000 mg Fe/kg) interaction (P<05).
Significant Fe linear effect (P<01).
Significant Mh main effect (P<01).
^Data are means of three samples, each sample representing pooled tissue from the three median-weight chicks within a replicate.
Significant Fe level (0 versus 40 to 2,000 mg Fe/kg) effect (P<01).
3 6
Hemoglobin '
'Data are means of three replicate groups of five male chicks during the period 8 to 22 days posthatching; mean initial weight was 76 g.
1,015.4
15.4
Supplemental Mn
18.3 25.5 28.2 27.8 27.7 27.6 .8
19.3 27.7 27.0 28.0 27.8 28.1
(%)
Hematocrit3
Blood
TABLE 3. Assessment of manganese and iron status of chicks fed a casein-dextrose diet containing variable levels of manganese and iron (Experiment 2)
MANGANESE AND IRON ESTTERRELAITONSHIP
(P<.05) for blood hemoglobin. Supplemental Mn did not affect Fe deposition in the liver. DISCUSSION
Chick performance was depressed when 5,000 mg Fe/kg were added to the cornsoybean meal diet containing 372 mg Fe/kg, regardless of dietary Mn level. These data are in agreement with the observations of Deobald and Elvehjem (1935) that approximately 4,500 mg Fe/kg diet causes anorexia in chickens fed grain-based diets. Farrow et al. (1982) also observed a growth depression in chicks fed a milo-soybean meal diet containing 5,000 mg Fe/mg. It was surprising that growth was not depressed when Fe was supplemented up to 2,000 mg/kg in the casein-dextrose diet (Experiment 2). McGhee et al. (1965) reported that 400 to 800 mg Fe/kg (depending on the level of Cu) was toxic to chicks fed a semipurified skim milk-sucrose diet. Maximum improvement in performance was observed at the lowest level (40 mg/kg) of Fe supplementation. Southern and Baker (1982) determined that the minimal Fe requirement was 40 mg/kg in chicks fed a caseindextrose diet. Supplemental Mn markedly enhanced Mn deposition in tibia, bile, and liver in both experiments. Excess Fe ingestion had no effect on Mn uptake by these tissues. Previous studies have shown that tibia Mn is a sensitive indicator of Mn status (Halpin et al., 1986; Fly et al, 1989; Wedekind et al, 1989). Thus, even with the corn-soybean diet containing a surfeit level of Mn (i.e., 157 mg/kg), excess supplemental Fe up to 5,000 mg/kg would have been expected to depress tibia Mn if, in fact, the Fe load were interfering with Mn absorption. In the corn-soybean diet series, the Fe:Mn ratio (wt/wt) at its extreme was 34:1 whereas in the casein diet series the highest Fe:Mn ratio was 130:1. In both diet treatments, these extreme Fe:Mn ratios did not decrease Mn levels in any of the tissues evaluated. Manganese supplemented at 1,000 mg/kg reduced blood hemoglobin concentration of chicks fed the casein-dextrose diet but only when the level of dietary Fe was at or below the chick's requirement. The interference of excess Mn with hemoglobin formation has previously been shown in a variety of animal species. Hemoglobin concentration was depressed in chicks fed an iron-adequate diet
151
supplemented with 3,000 mg/kg Mn (Southern and Baker, 1983b). Moreover, Matrone et al. (1959) showed that hemoglobin regeneration was markedly retarded in anemic rabbits and baby pigs fed excess (2,000 mg/kg) Mn. They suggested that dietary Mn concentrations as low as 50 to 125 mg/kg might interfere with hemoglobin formation. Similarly, hemoglobin and serum iron were depressed in young lambs fed 45 mg/kg supplemental Mn, whereas higher levels of Mn also reduced Fe concentrations in liver, spleen, and kidney (Hartman et al, 1955). Maines (1980) suggested that excess Mn reduces heme synthesis by inhibiting 5-aminolevulinate synthase. In treatments with the casein-dextrose diet, liver Fe concentration increased linearly with increasing levels of dietary Fe, but 1,000 mg/ kg supplemental Mn did not affect Fe deposition in the liver. In previous research with chicks from the authors' laboratory, liver Fe has been shown to be an unreliable index of Fe status (Bafundo et al, 1984; Southern and Baker, 1982), whereas blood hemoglobin has proven to be a sensitive indicator of Fe adequacy (Southern and Baker, 1982; Parsons et al, 1989). The data herein suggest that the interaction between Mn and Fe is a unidirectional phenomenon, in which excess Mn impairs Fe utilization but excess Fe does not antagonize Mn. This unidirectional interaction is similar to that which occurs between zinc and Fe when these trace elements are added to phytate- and fiber-containing diets; i.e., excess Fe has no effect on zinc, but excess zinc strongly inhibits nonheme-Fe utilization (Bafundo et al, 1984; Anonymous, 1985). Zinc also has been shown to antagonize copper, whereas excess copper has little effect on zinc utilization (O'Dell et al, 1976; Southern and Baker, 1983c,d). Recent evidence suggests that phosphate supplements used in poultry rations can antagonize Mn if excess levels of the supplements are used (Wedekind and Baker, 1990a,b). Phosphorous supplements are high in bioavailable Fe (Czarnecki-Maulden, 1990), but the data herein suggest that the Fe contained in phosphorus supplements such as dicalcium phosphate and defluorinated rock phosphate is not the factor antagonizing Mn. Indeed, Wedekind and Baker (1990a) have shown that phosphorus per se is the Mnantagonizing factor in commercial phosphorus supplements.
152
BAKER AND HALPIN REFERENCES
Anonymous, 1985. Interactions of dietary iron and zinc in the chick. Nutr. Rev. 43:121-122. Bafundo, K. W., D. H. Baker, and P. R. Fitzgerald, 1984. The iron-zinc interrelationship in the chick as influenced by Eimeria acervulina infection. J. Nutr. 114:1306-1312. Borg, D. C, and G. C. Cotzias, 1958. Incorporation of manganese into erythrocytes as evidence for a manganese porphyrin in man. Nature 182: 1677-1678. Cohen, R. R., 1967. Anticoagulation, centrifugation time and sample replicate number in the microhematocrit method for avian blood. Poultry Sci. 46:214-218. Crosby, W. H., J. I. Munn, and F. W. Furth, 1954. Standardizing a method for clinical hemoglobinometry. US Armed Forces Med. J. 5:693-703. Czarnecki-Maulden, G. L., 1990. Iron bioavailability. Pages 73-84 in: Proceedings of the Guelph Nutrition Conference, Guelph, Ontario, Canada. Davis, C. D., D. M. Ney, and J. L. Greger, 1990. Manganese, iron and lipid interactions in rats. J. Nutr. 120:507-513. Deobald, H. J., and C. A. Elvehjem, 1935. The effect of feeding high amounts of soluble iron and aluminum salts. Am. J. Physiol. 111:118-123. Diez-Ewald, M., L. R. Weintraub, and W. H. Crosby, 1968. Interrelationship of iron and manganese metabolism. Proc. Soc. Exp. Biol. Med. 129: 448-451. Farrow, G., A. S. Glassman, P. Vohra, and F. H. Kratzer, 1982. Effect of high fat and iron levels on the growth and mortality of chickens. Poultry Sci. 62:85-90. Flanagan, P. R., J. Haist, and L. S. Valberg, 1980. Comparative effects of iron deficiency induced by bleeding and a low iron diet on the intestinal absorptive interactions of iron, cobalt, manganese, zinc, lead and cadmium. J. Nutr. 110:1754-1763. Fly, A. D., O. A. Izquierdo, K. R. Lowry, and D. H. Baker, 1989. Manganese bioavailability in a Mnmethionine chelate. Nutr. Res. 9:901-910. Freedland-Graves, J., Y. X. Li, X. H. Wir, R. Tesar, and R. Pobocik, 1990. Manganese status of osteoporotics and age-matched healthy women. Fed. Am. Soc. Exp. Biol. J. 4:A777. (Abstr.) Halpin, K. M, and D. H. Baker, 1986. Long-term effects of corn, soybean meal, wheat bran, and fish meal on manganese utilization in the chick. Poultry Sci. 65: 1371-1374. Halpin, K. M., D. G. Chausow, and D. H. Baker, 1986. Efficiency of manganese absorption in chicks fed corn-soy and casein diets. J. Nutr. 116:1747-1751. Hartman, R. H., G. Mattone, and G. H. Wise, 1955. Effect of high dietary manganese on hemoglobin formation. J. Nutr. 55:429-439. Ho, S. Y., W. J. Miller, R. P. Gentry, M. W. Neathery, and D. M. Blackmail, 1984. Effects of high but nontoxic dietary manganese and iron on their metabolism by calves. J. Dairy Sci. 67:1489-1495. Hurley, L. S., C. L. Keen, and B. Lonnerdal, 1983. Aspects of trace element metabolism during development. Fed. Proc. 42:1735-1739. Maines, M. D., 1980. Regional distribution of the enzymes of haem biosynthesis and the inhibition of 5-aminolevulinate synthase by manganese in the rat brain. Biochem. J. 190:315-321.
Matrone, G., R. H. Hartman, and A. J. Clawson, 1959. Studies of a manganese-iron antagonism in the nutrition of rabbits and baby pigs. J. Nutr. 67: 309-317. McGhee, F., C. R. Creger, and J. R. Couch, 1965. Copper and iron toxicity. Poultry Sci. 44:310-312. National Research Council 1984. Page 12 in: Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. O'Dell, B. L., P. G. Reeves, and R. F. Morgan, 1976. Interrelationships of tissue copper and zinc concentrations in rats nutritionally deficient in one or the other of these elements. Pages 411-421 in: Trace Substances in Environmental Health. University of Missouri, Columbia, MO. Parsons, C. M., D. H. Baker, and C. C. Welch, 1989. Effect of excess zinc on iron utilization by chicks fed a diet devoid of phytate and fiber. Nutr. Res. 9: 227-231. Smith, K. J., 1970. Practical significance of gossypol in feed formulation. J. Am. Oil Chem. Soc. 47: 448-450. Southern, L. L., and D. H. Baker, 1982. Iron status of the chick as affected by Eimeria acervulina infection and by variable iron ingestion. J. Nutr. 112: 2353-2362. Southern, L. L., and D. H. Baker, 1983a. Eimeria acervulina infection in chicks fed deficient or excess levels of manganese. J. Nutr. 113:172-177. Southern, L. L., and D. H. Baker, 1983b. Excess manganese ingestion in the chick. Poultry Sci. 62: 642-646. Southern, L. L., and D. H. Baker, 1983c. Eimeria acervulina infection and the zinc-copper interrelationship in the chick. Poultry Sci. 62:401-404. Southern, L. L., and D. H. Baker, 1983d. Zinc toxicity, zinc deficiency and the zinc-copper interrelationship in Eimeria acervulina-infected chicks. J. Nutr. 113: 688-696. Steel, R.GX>., and J. H. Torrie, 1980. Pages 137-171 in: Principles and Procedures of Statistics. A Biometrical Approach. 2nd ed. McGraw-Hill Book Co., New York, NY. Strause, L., and P. Saltman, 1987. Role of manganese in bone metabolism. Page 146 in: Nutritional Bioavailability of Manganese. C. Kies, ed. American Chemistry Society Symposium Series 354. American Chemistry Society Publishing Co., Washington, DC. Tanksley, T. D., Jr., 1970. Use of cottonseed meal in swine rations. Feedstuffs 42:22. Thomas, J. W., 1970. Metabolism of iron and manganese. Jf. Dairy Sci. 53:1107-1123. Thomson, A S * . , D. Olatunbosun, and L. S. Valberg, 1971. Interrelation of intestinal transport system for manganese and iron. J. Lab. Clin. Med. 78:642-655. Thomson, A3JR., and L. S. Valberg, 1972. Intestinal uptake of iron, cobalt, and manganese in the irondeficient rat Am. J. Physiol. 223:1327-1329. Wedekind, K. J., and D. H. Baker, 1990a. Manganese utilization in chicks as affected by excess calcium and phosphorus ingestioa Poultry Sci. 69:977-984. Wedekind, K. J., and D. H. Baker, 1990b. Effect of varying calcium and phosphorus level on manganese utilization. Poultry Sci. 69:1156-1164. Wedekind, K. J., M R. Murphy, and D. H. Baker, 1989. Influence of manganese status and excess dietary phosphorus on manganese turnover in chicks. Poultry Sci. 68(Suppl. 1):157. (Abstr.)