Calcium Inhibition of Inorganic Iron Absorption in Rats

GASTROENTEROLOGY 1983;84:90-101

Calcium Inhibition of Inorganic Iron Absorption in Rats JAMES C. BARTON, MARCEL E. CONRAD, and RICHARD T. PARMLEY Division of Hematology/Oncology, Institute of Dental Research, and Department of Ped iatrics and Pathology, the University of Alabama in Birmingham, Birmingham, Alabama'

Calcium significantly diminishes the absorption of ferrous and ferric iron in a dose-related manner, whether the test doses of calcium and radioiron are administered ornily or introduced into isolated intestinal segments; the effect is maximal in the duodenum and jejunum. Electron microscopic observations and quantitative studies of the mucosal uptake and transfer of iron show that calcium decreases the entry of iron into the microvilli of intestinal epithelial cells. Animals fed a high-calcium, iron-replete diet developed iron depletion ; animals consuming high-culcium food of marginal iron content developed mild iron deficiency anemia. Further, more radioiron was absorbed from human milk than either bovine milk or human milk supplemented with calcium . These data suggest that individuals consuming a high-calcium diet containing marginal amounts of iron could develop iron deficiency anemia and may explain why infants who are fed cow's milk have a greater incidence of iron deficiency anemia than those fed human milk. A relationship between dietary calcium content and iron absorption has been postulated for many years. Reconstitution of hemoglobin and tissue iron levels occurred less rapidly in iron-deficient rats fed 0.5 mg iron daily in a food that contained either 1% or 3% CaC0 3 than in rats fed normal-calcium control diets; the addition of comparable quantities of calcium as either lactate, chloride, or diphosphate to control diets had a similar effect (1). Likewise, excess dietary CaC0 3 delayed the restoration of blood and tissue iron Received April 13, 1982. Accept ed Augu st 6, 1982. Address reprint requests to: James C. Barton , M.D., Uni versit y of Alabama in Birmingham, Division of Hematology/Oncology , Room 42 3, Lyons-Harrison Research Building, Birm ingham, Alabama 35294 . This work was supported by Grants AM 21416, 1K AM 00752 , HD 14134 , and DE 02670 from th e National Institutes of Health and by the Veterans Administration. © 1983 by the American Gastro ent erologi cal Association. 0016-5085/83/010090-1 2$03 .00

levels in iron-deficient rats fed diets containing 39 mg iron/kg (2), and oral calcium carbonate administration diminished the whole-body retention of dietary iron (3). Weanling rats fed 0.20 mg iron daily developed anemia when dietary calcium levels were four times optimal (4). Anemia was more prominent and heart size was increased among female mice and their litters when the dams were fed excessive CaC0 3 (5); the anemia could be corrected by the addition of greater quantities of iron to the diet (6). Among irondeficient rats fed semisynthetic diets containing marginal quantities of iron (23 -30 mg iron/kg, a fivefold increase in the amount of bone meal used was associated with a subnormal rate of hemoglobin regeneration corrected by additional dietary iron intake (7) . Mice that consumed diets containing 2129 mg iron/kg fortified with either calcium carbonate, lactate, or chloride had subnormal liver stores , reduced hemoglobin regeneration, and increased heart size (8). Similarly, rats fed diets containing 2425 mg iron/kg with either 2% csci, or 2% CaC0 3 added had decreased liv er, spleen, kidney, and lung iron levels after 12 wk (9). Oral CaC0 3 administration reduced the quantity of oral radioiron incorporated into hemoglobin in iron-depleted rats (10). These experiments in rodents clearly suggest that supranormal dietary calcium levels or Ca/Fe ratios, or both, are associated with diminished inorganic iron absorption. It is difficult to determine the effects of calcium upon iron absorption from these animal studies. Excessive and unphysiologic dosages of calcium were used in many of them, and others used carbonate or phosphate salts, which precipitate iron in the intestinal lumen. Lastly, none of these experiments measure iron absorption directly and depend upon body retention or some effect related to it. Two pertinent studies have been reported in humans; in both, the levels of food calcium used did not exceed normal. Four men were fed various natural diets representing 15 .6-17 mg iron and constant quantities of phosphorus daily; three levels of

January 1983

IRON ABSORPTION AND CALCIUM

91

Table 1. Ingredi ent s of Norm al Iron Experim ental Diets Ingred ients (g/kg)

High ca lci um

Normal ca lci um

Low ca lci u m

Casein" Cornstarch" Cottonseed oil " Vitamin mix , AOAca Calcium ch loride" Calcium cit rate" Calcium dih ydrogen phos phat e" Ferric am mo ni um citra te" Potassium h ydro gen phosphat e" Other mineral s"

260 .06 577.2 5 80.0 2.0 33.68 12.33 4.51 0.61 18.51 11.05

260.06 604. 60 80.0 2.0 6.32 12.33 4.51 0.61 18. 51 11.05

260 .06 609. 20 80 .0 2.0

16.86 4.50

5.66 4.50

2.66 4.50

Total calcium Total phosphorus a Supplied by Teklad Mills, Madison, Wis e.

b

o

12.33 4.5 1 0.61 24. 75 11.0 5

Various commer cial sour ces; all of reagent grad e.

stant quantities of phosphorus daily ; three levels of dietary calcium were associated with decreasing recovery of stool iron, It was concluded that either increasing quantities of calci u m within a normal range might enhance iron absorption directly by an unknown m echanism, or that calcium might bind phytate and phosphate to permit greater iron absorption (11). Numerous inorganic radioiron absorption tests were performed in 34 volunteers who consumed semisynthetic food labeled with an extrinsic radioiron ta g (12). M eals that contained calcium or phosphate , or both, in normal quantities (added as CaCl z, CaHP0 4 , or K zHP0 4 ) did not alter inorganic radioiron absorption by com paris o n with control meals. The combination of CaHP0 4 an d KzHP0 4 did significantly diminish iron absorption , suggesting that a poorly absorbable ca lci u m -p h os p h ate-iron complex may have been formed under these circumstances (12) , The current studies were performed to delineate the effects of physiological concentrations of ca lcium upon inorganic iro n absorption , to investigate the mechanism of action of ca lc iu m o n iron absorption, and to d et ermine the p ossible physiologic significance of these findings.

Methods Animal and Diet Preparation Male albino rats of a pathogen-free Wistar strain (Southern Animal Farm s, Inc., Pratt vill e, Ala.) were used in all experiments. Th e pri nciples of laborator y animal care, as pro mulgated by the National Research Coun cil , were observed . Except as specified, all animals were housed in polypropylene cages floored with stainless stee l grids to pr event pica for feces and absorbent bedding in a room provided with automatically contro lled temp erature (22 ± OS C) and lighting (light 7:00 A M to 6:00 PM) . After weaning, th e rats were given a standard pelleted food (LabBlox; Allied Mills, Inc., Chicago, Ill.) and free access to tap

water. At th e start of experimental man ipulation, rats had mean weights of 200-225 g; one experime nt used juvenile rats whose weights were 65- 80 g. All statistical comp arisons were mad e amon g groups of rats of similar weights. Mild to mod erat e iron deficiency was produced by a single bleed ing of 3 ml from th e retroorbit aI venous sinus with a heparini zed microhematocrit tub e after int raperitoneal pentobarbital anes thes ia (4 mg/l00 g). Iron load ing was accomplished by intramuscular injection of 1 ml of iron-d extran (50 mg Fe) (Imferon ; Merrell-National Laboratories, Inc., Cincinnati , Oh .). Iron-lo ad ed and normal con trol anim als were th en fed norm al calcium, norm al iron content control d iets . Bled animals were given normal calcium, severely iron-deficient diets described below. Rats were fed their resp ective diets 2 wk before th e performance of isol ated gut loop experime nts. For experimental preparati on, th e rats received demineraliz ed , deionized water, and powdered diets , exclu sively. Experim ental diets were modifi cations of a previously described semisynthe tic normal protein test diet (13). Calcium chloride was substituted for calcium carbonate as a major source of calcium. Low-calcium, normal control, and high-calcium diets were prepared , all containing - 85 mg Fe/kg (Table 1). Sim ilar changes in the calcium content of diets have been shown prev iou sly to alter calcium metabolism in rats after at least 2 wk of feeding as measured by femur weight but do not affect serum che mistry values (tota l prot ein . albumin, calcium, phosphorus, alkaline phosphatase, and creatini ne) (14). Mildl y irondeficient and severely iron-deficient diets of high, normal , and low calcium content were compounded by the omission of various quantities of ferric ammonium citrate. Diet samples were digested in nitric and perchloric acids, and their calcium and iron conten ts were determined by atomic absorption spectrometry (Perkin-Elmer Model 303; Perkin-Elmer Corp ., Instrument Div., Norw alk, Conn.). Calcu lated and measured values were similar for all diets (Table 2). Except as specified for iron and calcium cont ent s, all diets met th e nutrient requ irements of the rat specified by the National Acade my of Scie nces (15). Before most experim ents, ani mals were fed experimental diets ad libitum for 4 wk. For retention studies, the designated diet s were continued throughout th e duration of the studies.

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BARTON ET AL.

Table 2. Iron and Calcium Contents of Experimental Diets Fe determined

Ca determined

(m glkg)

(mglkg)

High Ca, normal Fe Normal Ca, normal Fe Low Ca, normal Fe

83 .2 87 .2 89 .6

17.5 6 .8 3.2

High Ca, mildly Fe-deficient Normal Ca, mildly Fe-deficient Low Ca, mildly Fe-deficient

20.4 21.6 20 .0

15 .5 6.3 3.0

2.0 2.0 2.0

15.9 6.1 3.6

Diet

High Ca. severely Fe-deficient Normal Ca, severely Fe-deficient Low Ca, severely Fe-deficient

Absorption Measurements Using Isolated Gastrointestinal Loops Iron absorption studies were performed by measurement of total-body radioactivity in a small animal whole-body scintillation detector (Packard-ARMAC; Packard Instrument Co., Inc., Downers Grove, Ill.). Iron-59 as ferrous chloride, sp act 21.9 Ci/g (New England Nuclear, Boston, Mass.) , was used. Intestinal loop experiments were performed in rats fasted overnight from food but not water. Under intraperitoneal pentobarbital anesthesia (4 mg/l00 g), the urethra was tied with silk suture to prevent urinary loss of absorbed iron. A laparotomy was performed, the intestinal segment was isolated proximally and distally with umbilical tape, and the bile duct was ligated with silk suture. Although the presence of bile in the small intestine enhances iron absorption (16), the bile duct was ligated to prevent enterohepatic circulation of iron during the experiments. For most studies, absorption from the entire small intestine was quantified, but in other experiments, stomach, duodenum, jejunum, ileum, or colon were used. A standard test dose of 56 /-tg of radiolabeled iron was injected into the isolated intestinal segment. For entire small intestinal loops, a volume of 1 ml was used (1.0 ml of 1 mM FeCl 2 + 1 i-tCi 59Fe, pH 4.0). For injections of the stomach, duodenum, jejunum, ileum, and colon, 0.5 ml was given (0.5 ml of 2 mM FeCh + 1 i-tCi 59Fe, pH 4.0). This reduced volume of test solution (0.5 ml] by comparison with the 1.0 ml used in entire small intestinal loops was required for optimal loop filling , requiring a twofold increase in iron concentration to maintain a constant test dose of iron. To assess the effects of intraluminal calcium on iron absorption, a standard iron dose was compounded in nonradiolabeled CaCl2 carrier solutions ranging from 1 to 200 mM calcium concentration. The effects of chloride ion concentration were assessed using a standard iron dose compounded in nonradiolabeled NaCI carrier solutions. Chloride ion concentrations selected were equivalent to those in experiments using CaCh solutions as carriers. In a single study, iron and iron/calcium solutions were reduced to 1000th of the concentrations given above.

The solutions were injected by entering the gut lumen proximal to the proximal ligature with a 21-gauge hypodermic needle, passing it intraluminally through the ligature loop, tightening the ligature, and then injecting the test dose in the isolated segment, with subsequent withdrawal of the needle and tying of the ligature. Gastric injections were performed with an olive-tipped oroesophageal needle. The abdomen was then closed with stainless steel clips, and the rats were placed in 1-qt vented cardboard ice cream containers. Total body radioactivity was measured in a whole-body detector and compared with a 250-ml water-filled plastic bottle containing a test dose equal to that injected into the animals. Four hours after administration of the test dose, each animal was killed by cervical dislocation. Isolated intestinal segments were excised from the carcass, and whole-body radioactivity was again measured and compared with the original whole-body radioactivity.

Physicochemical State of Iron Because iron solubility in vitro is affected by other dietary constituents and has been correlated with absorption (17), titration experiments were performed to determine if calcium might physically alter the state of iron. A 100-ml amount of each of the following solutions was prepared at pH 2.0: 1 mM FeCh; 1 mM FeCh in 1 mM CaCh ; 1 mM FeCh in 10 mM CaCI2 ; and 1 mM FeCh in 100 mM CaCho One microcurie of iron-59 was added to each 100-ml solution. After mixing and centrifugation at 3000 rpm for 30 min (which removed <1 % of the total initial radioactivity) , 1 ml of each solution was removed as a standard. Each solution was titrated against 0.1 N NaOH to pH 10.0, and z-ml samples were removed at multiple pH values. Aliquots taken from each titration were recentrifuged, and 1 ml of supernatant was removed from each for counting in a Packard Auto-Gamma spectrometer (Packard Instrument Co., Inc.). Solubility of iron in water and calcium chloride solutions was expressed as the percentage of standard as a function of pH. Supernatants from control and calcium-containing solutions similarly prepared and tirated were applied to 1.5 x 25-cm Sephadex G25 columns equilibrated to the appropriate pH value. Column elution fractions of 0.1 ml were counted for radioiron content to detect peaks of radioactivity, which would indicate the presence of soluble iron-containing polymers or macromolecules. Ferric ion concentrations in iron and iron/calcium solutions were determined using the thiocyanate color reaction (18). The lower limit of sensitivity in this assay was 0.01 /-tg Fe 3 +/ml. The following solutions were prepared at pH 4.0: 1 mM FeCh; 1 mM FeCh in 1 mM CaCI2 ; 1 mM FeCl 2 in 10 mM CaCI2 ; 1 mM FeCl 2 in 100 mM CaCI2 ; and 1 mM FeCla. Two milliliters of each solution was incubated for 15 min at 37°C, and the concentration of ferric iron was determined. Similarly, 0.5 ml of each of the above solutions was injected into isolated rat duodenal loops as previously described. The loops were aspirated with a 21gauge needle after 15 min, the aspirate volume was measured, and its ferric iron concentration was quantified.

January 1983

Localization of the Calcium Effect Seventy-two rats reared on a standard pelleted diet were fasted from food but given continued access to water overnight. After the administration of intraperitoneal pentobarbital anesthesia, duodenal loops were isolated and injected with a standard iron dose. At intervals of 2 min-4 h, groups of 8 rats were killed, and the duodenal loop was excised and immediately flushed from the pylorus with 10 ml of cold 0.9% NaCl, using an olive-tipped needle. Absorbed radioiron in the carcass as well as that radioiron retained in the washed duodenal segment was quantified using the whole-body counter. Radioiron remaining in the washed duodenal segment was defined as mucosal retention; mucosal uptake was defined as the sum of absorbed radioiron and mucosal retention. Three additional groups of 72 rats each were similarly studied. Duodenal segments were injected with 0.5 ml of 2 mM FeClz + 1 IJ-Ci 59Fe in 2, 20, and 200 mM CaClz, pH 4.0. respectively.

Electron Microscopy For ultrastructural studies, groups of 2 animals each were given 0.5 ml of 0.9% NaCl, 0.5 ml of 2 mM FeCl z, or 0.5 ml of 2 mM FeClz/20 mM CaCl z into isolated duodenal loops. After 15 min, the midduodenum was excised and finely minced in 3% glutaraldehyde/0.1 M cacodylate buffer, pH 7.35, and allowed to fix for 1 h at 22°C.The specimens were then rinsed in 0.1 M cacodylate/ 7% sucrose buffer and incubated for 30 min at room temperature in a fresh 1% acid ferrocyanide solution for demonstration offerric iron (19.20). Some specimens were similarly incubated in an acid ferricyanide solution for the demonstration of ferrous iron. The tissues were then rinsed three times in cacodylate-sucrose buffer, postfixed for 1 h in 1% Os04/0.1 M cacodylate buffer, routinely dehydrated in graded alcohols and propylene oxide, and embedded in Spurr low-viscosity medium. Thin sections (70 nm) were examined without counterstaining in a Philips 300 electron microscope (Phillips Electronic Instruments, Inc. Mahwah, N.J.) at an accelerating voltage of 60 kV.

Absorption, Gastrointestinal Transit Time, and Retention Measurements in Intact Rats Measurements of iron absorption and gastrointestinal transit time in intact rats were performed after dosing with radioiron using an olive-tipped oroesophageal needle under light ether anesthesia. After dosing, the animals were placed in individual metabolic cages (Econo-Metabolism Unit; Maryland Plastics, Inc .. bioMedic Corp., Federalsburg, Md.) and allowed to resume feeding ad libitum. The radioiron content of stool, collected separately from urine, was determined at intervals. After 7 days, the rats were killed by cervical dislocation, the entire gastrointestinal tract was excised, and radioiron remaining in the carcass was quantified. Radioiron in the stool and in the gastrointestinal tract was defined as unabsorbed iron. Gastrointestinal transit time was expressed as cumulative stool radioiron over time as a fraction of the unabsorbed portion of the administered dose of radioiron. A similar

IRON ABSORPTION AND CALCIUM

93

technique for quantification of gastrointestinal transit time was concurrently performed in additional animals using chromic chloride, a poorly absorbed salt (21). Each rat was given 1 IJ-Ci of 51CrCb. sp act 50-500 Ci/g Cr (New England Nuclear, Boston, Mass.], Using a breast pump, 15 ml of human milk was obtained from each of 8 volunteers. and samples were pooled. Fresh, raw bovine milk pooled from ~500 Holstein cattle was a gift of Sealtest Dairy Products, Birmingham. Ala. Protein, fat. and lactose contents of the milks were quantified using standard dairy methods (22-24). Calcium, phosphorus, and iron contents were determined using atomic absorption spectrometry; phosphorus determinations required a special ashing procedure (25). All analyses for unaltered human and cow's milk agreed closely with published values (26). The absorption of radioiron from human and bovine milk was quantified in rats as described above 1 wk after an oral dose of 1 ml of milk containing 56 IJ-g of Fe as FeClz and 1 IJ-Ci of 59Fe per animal. Sufficient CaClz was added to the human milk given some rats to elevate its calcium content to that of cow's milk; extrinsic radioiron absorption from this milk was also quantified.

Assessment of Body Iron Stores Microhematocrits of blood removed from the retroorbital venous sinus were determined. Serum iron concentration and total iron-binding capacity were performed by standard methods (27) using reagents and standards obtained from G. Frederick Smith Chemical Co.• Columbus, Oh. Bone marrow iron stores were rated using proximal femoral marrow from each rat stained with Prussian blue (28). The number of Prussian blue-positive macrophages observed in 100 microscopic fields (x 1000) of uniform one-cell thickness per rat was defined as a quantification of storage iron. In animals used for absorption measurements, a microhematocrit only requiring ~50 IJ-l blood was performed in each rat at the beginning of absorption measurement.

Calculations and Statistics In all animal experiments except for ultrastructural studies, groups of 8 rats matched for weight upon initiation of the experiment were used for each data point. All measurements of radioactivity were corrected for radiodecay and resolving time delay by comparison with appropriate standards. Absorption. retention. mucosal retention, mucosal uptake, and ultrastructural data are expressed as the mean ± 1 SEM. Single comparisons were made using Student's t-test for unpaired data. A modification of Tukey's test of all comparisons among means was used for the statistical evaluation of experiments involving three or more treatment groups (29). A level of probability ~0.05 was defined as statistically significant.

Results Gut Loop Studies Using entire small intestinal loops, radioiron absorption from a standard dose of iron (56 JLg) as

94

BARTON ET AL.

GASTROENTEROLOGY Vol. 84, No. 1

Table 3. Eff ect of Carrier Solutions on Radioiron Absorption from Entire Small Int estine CaCl 2 carrier , co ncen tra tion FeCl 2 test do se , concen tration Absorption, %

Cont rol 1 mM 34.7 ± 1.8

1 mM 1 mM 37.5 ± 2.2

lOmM 1 mM 26.4 ± 1.8"

100 mM 1 mM 21.8 ± 0.3"

NaCl carrier, concentration FeCl 2 test dose , concentration Absorption. %

Control 1 mM 31.5 ± 1.6

2mM lmM 29.8 ± 1.9

20 mM lmM 29.9 ± 2.4

200 mM lmM 30.3 ± 2.1

CaCl 2 carri er. con centration FeCI, test dose. concen tration Absorption , %

Control 1 fLM 50.8 ± 2.8

1 fLM 1 fLM 54.6 ± 2.1

10 fLM 1 fLM 54.4 ± 4.5

100 fLM 1 fLM 56.4 ± 3.3

CaCl 2 carrie r , concentrati on FeCl 3 test dose, concentration Absorption, %

Cont rol 1 mM 9.0 ± 1.2

1 mM 1 mM 6.9 ± 1.2

10 mM lmM 4.4 ± 0.60

100 mM 1 mM 1.6 ± 0.30

a Signifi cant at p

::5

0.02.

ferrous chloride was not affected when a carrier solution of 1 mM CaCh was used. Significant decreases in iron absorption were obs erved with CaClz concentrations of 10 mM and 100 mM (Table 3). A similar experiment was performed in which the standard iron dose was administered in NaCI solutions. No significant change in radioiron absorption was noted by comparison to controls (Table 3). To investigate the relationship of the Ca/Fe ratio on iron absorption, an iron dose of 56 ng was tested using a control solution (without CaCl z) and carrier solutions of 1 pM, 10 pM , and 100 pM CaCl z concentrations. Radioiron absorption in animals receiving iron/calcium solutions was not significantly altered by comparison with controls (Table 3). A similar experiment was performed using ferric chloride with and without the addition of 1 mM , 10 mM , and 100 mM concentrations of CaCl z (Table 3). Significantly less radioiron was absorbed from the solution of ferric chloride than ferrous chloride. Calcium significantly diminished the absorption of radioiron from test doses containing ferric iron. Iron abso rption was then quantified in various gastrointestinal segments (Table 4). Th e addition of 200 mM CaClz significantly reduced iron absorption in the duodenum (10.4 ± 1.9% vs. 33 .6 ± 1.9%

Table 4. Per centage of Radi oiron Abs or pt ion from Isolated Gastrointe stinal Segm ents Test Dose Stomac h" Duodenum" [ejunum? Ileum" Colona

0.5 ml of 2 mM FeCl 2 2.1 33.6 13.9 0.4 0.4

± 0.7 ± 1.9

± 2.2 ± 0.1 ± 0.1

0.5 ml of 2 mM FeCI2 / 200 mM CaCl 2 2.1 10.4 4.6 0.3 0.4

± 0.1

± 1.9 b ± LO b

± 0.1 ± 0.1

a Stomach = di stal eso phagus to py loru s; du odenum = pyloru s to ligam ent of Treitz, - 4 cm; ileum = 4-cm segment prox imal to ileocecal valve; colon = all int estine distal to ileocecal valve. b Signifi cant at p ::5 0.05.

control) and in the jejunum (4.6 ± 1.0% vs. 13 .9 ± 2.2% control). Decreased iron absorption from the ileum was observed in the presence of calcium (0.3 ± 0.1 % vs. 0.4 ± 0 .1 % control), but the difference was not significant. No differences between experimental and control rats were observed when the stomach or colon was used as the absorptive site. Iron absorption in iron-deficient animals was significantly decreased in the presence of calcium (29 .7 ± 3.0% vs . 51.9 ± 2.4% iron-deficient controls) as shown in Table 5; a similar effect wa s also observed in iron-loaded rats (0 .6 ± 0.1% vs . 2.1 ± 0.5% ironloaded controls) . When either 100 mM CaClz, nonradiolabeled 100 mM FeCl z, or 200 mM NaCI were given 15 min after a standard radiolabeled iron dose into isolated entire small intestinal loops, no significant alteration in iron absorption was observed (data not shown).

Physicochemical Studies The physicochemical state of iron in calcium solutions was first studied using titration methods. As shown in Figure 1, iron is more soluble in acid solutions and increasing amounts are precipitated as pH increases, particularly from pH 4 to pH 6. Iron in 100 mM CaClz was somewhat less soluble at pH 4 than in the presence of less calcium, but was slightly more soluble at pH 5-6 (Figure 1). Altogether, however, reduced iron solubility in the presence of calcium (particularly 10 mM CaCl z) could not explain the observed differences in iron absorption (Figure 1). Supernatants obtained in these experiments were chromatographed on Sephadex G-25 columns ; this demonstrated no evidence of soluble iron-containing polymers or macromolecules. Recovery of radioiron from the columns was > 9 5%. Ferric ion concentrations were quantified in various FeClz/CaCl z solutions. Ferrous chloride and ferr ic chloride solutions, without CaCl z, were us ed as controls. Ferric ion concentrations were not signifi-

IRON ABSORPTION AND CALCIUM

January 1983

95

Table 5. Radioiron Absorption from Small Intestinal Loops

Dose Absorption, % Microh ematocrit, % Starting weight. g Finish weight, g o

Significant at p

Fe + 29.7 ± 36.4 ± 207 ± 230 ±

Fe 51.9 36.5 206 233

± ± ± ±

2.4 1.3 3 6

Ca 3.0 0 1.2 2 8

Fe 31.5 46.4 204 249

of Calcium

Effect

Using isolated duodenal loops, further studies were performed to quantify the temporal relationships of iron absor ption, mucosal iron retention, and mucosal uptake of iron. Iron absorption proceeded more slowly in rats receiving calcium (Figure 2B) by comparison with control rats (Figure 2A) . In the absence of calcium, the mucosal retention of iron reached a maximum value after 5 min and fell rapidly; a slower phase of declining mucosal radioiron retention occurred from 1-4 h after dosing

100

c .S!

/m M FeC/2

80

60

60

40

40

20

20

]

0

4

..

Ol~

on.. /00

'" c

. .

8

45

pH

.S

I m M FeC/2 /

/ m M FeC/2/ImM CoC/2

/0 0

80

III

0 '0

/O mM CO CI2

2

4

100

eo

80

60

60

40

4

20

20

4

± 3

± 8

Fe + Ca 20.6 ± 2.1 0 46.4 ± 0.3 205 ± 3 25 7 ± 7

Fe 2.1 45 .6 203 243

± 0.5 ± 0.7 ± 3 ± 5

Fe 0.6 45.4 208 253

+ Ca ± 0.1 0

± 0.5 ± 3

± 7

6

8

/0

45

pH

8

/0

(Figure 2A). When a standard iron dose was administered in 2 mM CaClz, peak mucosal radioiron retention occur red at 20 min after dosing, and at 30 min when a 20 mM CaClz carrier solution was used. With a 200 mM CaCl z carrier dose, pe ak mucosal retention levels were observed 1 h after dosing (Figure 2B). Percentage of mucosal uptake of radioiron during the first 10 min after injection of the duodenal segment in these experiments was inversely related to the concentration of CaCl z in the carrier solution (Figure 3). Electron Microscopy

Specimens from animals given iron demonstrated considerably more acid ferrocyanide staining in microvilli and apical cytoplasm than did specimens from rats given iron/calcium or saline solutions (Figure 4A , B). In animals given the iron/ calcium solution, numerous plasmalemmal stain deposits were observed on microvilli (Figure 4B) . Midduodenal microvilli exposed to iron alone had 395 ± 110 (SEM) intravillous stain deposits per 10 0 0 microvilli, while th ose exposed to iron/calcium solutions had 78 ± 19 intravillous deposits per 1000 microvilli (p < 0 .0 1 ). In rats receiving saline alone, even fewer stain deposits were observed (36 ± 12, P < 0.05). Stain deposits on lateral membranes of animals given iron or iron/calcium appeared similarly increased when compared with saline controls as described previously for solutions of iron alone (30).

pH ImM Fe C/2//00mMC oCI2

2

~ ll.

± 2.5 ± 1.6

0.01.

:5

cantly increased in FeClz/CaClz solutions. Similar solutions were the n injected into isolated duodenal loop s, asp irate d after 15 min, their volume s were measured, an d the ferric ion concentration was qu antified. Again no in crease in ferric ion concentrations was observed . Site

Iron loading

Normal control

Iron deficiency

Body iron status

4

6

8

10

pH

Figure 1. Effect of pH on the solubility of radioiron in calcium chloride solu tions.

Intestinal Transit Time and Body Iron Retention

Because the preceding results suggested that increased dietary calcium content might decrease the absorption of dietary iron and subsequently produce iron deficiency, three groups of adult rats were fed diets of high, normal, and low calcium contents for 4 wk. All diets contained normal quantities of iron (-85 mg/kg). Iron absorption was quantified in isolated small intestinal loops after the animals were faste d overnight from food but not water. Rats previously fed high-calcium diets had signifi -

96

GASTROENTEROLOGY Vol. 84, No. 1

BARTON ET AL.

35

~30 frTl'7\

~25! ::z

!) I

C>

~ 20

C>

1 \,

y-------------r---------------------------1

I

!!:

Absorption --.. Mucosal Retention o----{)

~ 10

!i.... ........~

5

A 60

....

120

TIME. MINUTES

180

240

35

g 30

...

~ 25 ':Ii C>

~ 20 a::

...

C>

en

""" :z: 15 C>

a::

~

.... """ e

,

Absorption ....Mucosal Retention o----{)

10 ~

""' a:: .... ....

B 60

120

TIME. MINUTES

180

240

Figure 2. Absorption and mucosal retention of radioiron from isolated duodenal loops of control rats (A) and those given radioiron in ZOO mM GaGI, (B). Each point represents the mean ± 1 SEM, n = 8.

cantly increased radioiron absorption by comparison with normal control rats (47.4 ± 2.4% vs. 34.2 ± 2.8% controls). Rats fed low-calcium diets had iron absorption similar to that of controls (31.8 ± 2.9% vs. 34.2 ± 2.8% controls). These results indicated that animals fed diets of high calcium, normal iron content had mild iron depletion. This could result from (a) calcium-related inhibition of iron absorption at the enterocyte microvilli, (b) increased gastrointestinal transit with consequent decreased dietary iron absorption, or (c) enhanced elimination of iron from the body. To measure concurrently intestinal transit time and iron absorption, three groups of normal adult rats were fed high, normal, and low calcium content diets without added iron (2 mg Fe/kg diet). Iron requirements were supplied by intramuscular injection of iron-dextran on a daily basis. After 4 wk, the rats were given a standard dose of radiolabeled iron and placed in individual metabolic cages. Stool and urine were collected separately at intervals and their radioiron contents were measured. Daily iron-dex-

tran injections were continued for the duration of the study. After 7 days, the rats were killed, and absorption and gastrointestinal transit time were quantified. Rats fed high-calcium diets had slightly increased transit time by comparison with normal controls from the second to sixth day after dosing, but the differences were not significant (data not shown). Rats fed the high-calcium diets, however, absorbed significantly less radioiron than normal control rats (17.7 ± 3.0% vs. 27.3 ± 3.0%, p < 0.01); this result suggested that gastrointestinal motility was not a major factor in diminishing dietary iron absorption from high-calcium diets. Rats fed lowcalcium food did not differ significantly from normal controls with respect to either motility or iron absorption. Quantification of gastrointestinal transit time using 51CrCl3 also showed no significant effect of dietary calcium upon transit time (data not shown). To study the effects of the various diets on net iron elimination from the body, an additional three groups of adult rats were fed high, normal, and low calcium content diets without added iron; daily intramuscular injections of iron-dextran were given throughout the experiments in a dose calculated to maintain a normal body iron concentration of 60 mg/ kg (60 IJ-g/g of daily weight gain) as described above. After 4 wk, each rat was given an intravenous tracer dose of radioiron in the dorsal vein of the penis under anesthesia (1 IJ-Ci of 59Fe in 0.5 ml of 0.9% NaCl, pH 6.0). Four weeks after dosing, no signifi-

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Figure 3. Mucosal uptake (mucosal retention + absorption) of radioiron from isolated duodenal loops. Each point represents the mean ± 1 SEM. n = 8.

January 1983

IRON ABSORPTION AND CALCIUM

97

Figure 4. A. Rat duodenum. In this animal given ferrous iron for io min, acid ferroc yanide stains ferric iron in micro villi (enlarged in inset) . apical cytoplasm. and on lateral membranes. x 20.000; inset x 40.000 . B. Rat duodenum. Ten minutes after the intraluminal administration of a FeCI2 /CaCI2 solution, stain deposits localizing the ferric iron are concentrated on the surfa ce of microvilli (enlarged in inset) and on lateral membranes. Note the relative paucity of intravillous and cytoplasmic stain deposits (see A). x 20,000; inset x 40.000.

dosing, no significant differences in radioiron retention among rats fed the various diets were observed (89.9 ± 1.4% high calcium, 90.0 ± 1.5% normal control, 91.8 ± 0.7% low calcium).

those fed high-calcium diets had significantly lower concentrations of serum iron (139 ± 12 JLg/dl vs. 186 ± 12 JLg/dl normal calcium controls); marrow iron stores and weight gain were also diminished (Table 6).

Calcium-Induced Iron Deficiency in Juvenile Rats

Male rats were fed standard pelleted food for 2 wk following weaning. The animals were then fed diets of high, normal, and low calcium content, which were either iron-replete (85 mg Fe/kg) or mildly iron-deficient (21 mg Fe/kg). After 4 wk . parameters of body iron stores were determined. In rats fed iron-replete diets . a decrease in marrow iron stores was observed among those receiving highcalcium diets (Table 6). No significant changes in weight gain, microhematocrit, serum iron concentration, or total iron-binding capacity of serum were observed. Among rats fed mildly iron-deficient diets,

Absorption of Extrinsic Radioiron from Milk

Intact weanling rats orally dosed with 1 ml of fresh human milk containing 56 JLg of radiolabeled iron absorbed significantly more radioiron than did rats given the same iron dose in bovine milk (41.7 ± 4.9% human milk vs. 24.0 ± 4.4% bovine milk, p < 0.01; calcium concentrations 0.04 and 0.11 g/100 ml milk, respectively). When CaCl z was added to human milk such that its calcium level approximated that of bovine milk, the iron absorption from this modified human milk was similar to that of bovine milk (28.3 ± 5.0% human milk + CaClz vs. 24.0 ± 4.3% bovine milk, p > 0.10; calcium concentrations

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Table 6. Effect of Dietary Calcium Level on Parameters of Body Iron Stores Dietary Ca level (g/kg)

Serum Fe

srrac-

(f.Lg/lOO ml)

(f.Lg/lOO ml)

Macrophages"

Dietury iron level 85 mg/kg 16.86 5.66 2.66

193 ± 21 205 ± 12 186 ± 10

454 ± 10 457 ± 11 432 ± 18

30.9 ± 3.0 c 4~.3 ± 5.2 42.0 ± 5.3

43.4 ± 0.9 42.5 ± 0.4 42.6 ± 0.5

139 ± 12 c 186 ± 12 186 ± 16

487 ± 8 516 ± 10 492 ± 6

24.6 ± 1.7c 29.5 ± 0.9 29.3 ± 1.0

40.1 ± 0.5 41.1 ± 0.7 42.3 ± 0.9

Microhematocrit (%)

Dietary iron level 20 mg/kg 16.86 5.66 2.66 a

Serum total iron-binding capacity. b Number of Prussian blue-positive proximal femoral bone marrow macrophages observed in 100 consecutive microscopic fields [x 1000) of uniform one-cell thickness per rat. C Values significant at 0.01.

0.10 and 0.11 g/100 ml milk, respectively). Our analysis of unaltered human milk showed, per 100 ml, protein 1.7 g, fat 5.2 g, lactose 6.6 g, calcium 0.04 g, phosphorus 13 g, and iron <0.1 mg. For unaltered bovine milk, we found protein 34 g, fat 3.0 g, lactose 4.3 g, calcium 0.11 g, phosphorus 103 g, and iron <0.1 mg, all per 100 rnl.

Discussion The results of these experiments demonstrate that calcium chloride solutions between 1 and 100 mM decrease ferrous iron absorption from the small intestinal loops in a dose-related manner. The absence of an effect of sodium chloride carrier solutions on radioiron absorption indicates that neither chloride ion concentration nor osmolality were significant factors contributing to the inhibitory effect of ionic calcium on ferrous iron absorption in this study. When both ferrous and calcium ion concentrations were reduced 1000-fold, a significant percent increase in radioiron absorption was observed regardless of calcium concentration by comparison with the higher iron dose rats. Within the lower iron test dose group, increasing calcium concentrations produced a slight, but nonsignificant, percentage of iron absorption increase. Thus, the inhibitory effect of calcium depends more upon the absolute quantity of calcium present in the gut lumen than upon the molar CafFe ratio. The inhibition occurred only in segments of the gastrointestinal tract in which the absorptive capacity is responsive to body iron needs (31). Iron absorption from small intestinal loops was diminished by calcium in iron-deficient and in ironloaded rats. Neither calcium, iron, nor NaCI significantly decreasedradioiron absorption when injected 15 min after radioiron administration. These calcium-related effects might be explained by changes in the physicochemical state of iron, which would render it less acceptable for mucosal uptake and absorption. Titration experiments were performed but indicated no significant decrease in

iron solubility in calcium solutions by comparison with non-calcium-containing control solutions over a wide range of pH. Chromatographic studies showed no evidence of polymer or macromolecule formation to account for the findings of the absorption experiments. 'Quantification of ferric iron concentrations in iron and iron/calcium test solutions incubated in vitro and in duodenal loops indicated that increased oxidation of ferrous iron to the ferric form did not account for the effect of calcium on ferrous iron absorption. There remained other possible explanations for the effect of the calcium: (a) calcium retards the entry of iron into the mucosal cell and/or competes for mucosal acceptor substances common to absorptive pathways for both metals, (b) calcium delays the movement of iron from the mucosal cell into the circulation, and (c) combinations of the above. Previ01,1s in vivo iron absorption studies using gut loops (32) and in vitro experiments using everted gut sacs (33) have demonstrated evidence for the existence of a two-step intramucosal absorptive mechanism for iron. The first step involves entry of iron into the mucosal cell, a process that includes the conversion of iron to a ferric form at the level of the microvillus membrane, and its appearance within the microvilli (30). Peak mucosal radioiron retention was delayed by calcium in a dose-related manner. Similarly, the rate of mucosal radioiron uptake early in the absorptive process was progressively retarded by increasing calcium concentrations in carrier solutions. Further, the increased quantities of intravillous stain deposits seen in duodenal specimens from rats given iron alone, and the accumulation of plasmalemma stain deposits in animals given iron/calcium solutions suggest that the absorptive process for iron is interrupted by calcium in part at or in the microvillus membrane. These data confirm and extend the findings or Greenberger et al. (34) who observed that 2M CaCtz significantly decreased the radioiron uptake of brush borders derived from proximal and distal rat intestinal mucosa. The rate-limiting proc-

January 1983

ess for the transfer of iron to the blood from the mucosal cell, however, involves the extrusion of iron from the cells rather than its uptake from the intestinallumen (32). Data in this study suggest that once peak mucosal iron levels are achieved, their rate of decrease in the presence of calcium is less than in the absence of calcium. Using everted gut sacs from proximal rat duodena, it has previously been demonstrated that calcium inhibits the net serosal transfer of inorganic iron at concentrations that do not inhibit mucosal iron uptake (33). Thus, our study confirms that calcium decreases the mucosal uptake of inorganic iron, and suggests that calcium may also inhibit its subsequent delivery into the circulation. These results provide support for the hypothesis that calcium decreases iron absorption at least in part by competing for iron acceptor substances in the intestinal mucosa or by blocking transmembrane movement of iron into and perhaps out of the enterocyte. While the mechanism(s) by which iron traverses the mucosal cell is unknown, its absorptive pathway is not an exclusive one. The absorption of a number of metals is increased in iron deficiency (35), and of these, cobalt, lead, and zinc in particular have been shown to compete with iron for absorption (36-38). Vitamin D treatment of rachitic chicks, associated with increased calcium absorption, has been shown to increase the absorption of other metals including cobalt, zinc, and iron (39). Pollack et al. (35), however, found no increase in 47Ca absorption in iron-deficient rats using a single calcium dose. Manis (33) showed that iron inhibited the mucosal uptake and net serosal transfer of calcium. An inhibitory effect of calcium on the absorption of metals that seem to be related to iron absorption has been documented only for lead (14). Calcium concentrations of ~l mM significantly decreased the absorption of small doses of lead from rat intestinal loops. The molecular basis for the probable competition is unknown at this time. However, vitamin Ddependent calcium-binding protein, important to the intestinal transport of calcium (41) , also binds other divalent cations including iron, lead, cobalt, and zinc in a competitive manner (14,41). While it is unlikely that calcium-binding protein is of great physiologic importance to iron absorption, competition of metals for this and other as yet unidentified absorptive proteins would explain the inhibitory. effect of calcium on iron absorption. The decreased absorption of radioiron by intact rats fed high calcium content diets suggested that, under certain circumstances, such food might sufficiently diminish dietary iron absorption so that iron deficiency would result. Quantification of iron absorption from gut loops in animals similarly prepared demonstrated increased iron absorption, a

IRON ABSORPTION AND CALCIUM

99

sensitive indicator of early iron depletion. Finally, groups of immature rats were fed high-, normal-, and low-calcium diets containing normal or mildly deficient amounts of iron for 4 wk; parameters of body iron stores were then quantified. Regardless of dietary iron levels, evidence of iron deficiency was most prominent among rats consuming high-calcium diets. When dietary iron content was normal, these rats had decreased marrow iron stores. Fed mildly iron-deficient diets, animals given high calcium content food gained less weight and had lower serum iron concentrations by comparison with control rats, and marrow iron stores were diminished. Thus , at least under sonie conditions, dietary calcium excess can induce or accelerate iron deficiency, or both, by diminishing the absorption of dietary iron. Although there is a paucity of data regarding the effect of dietary calcium on human iron homeostasis, this study suggests that it may be one of nutritional significance. The average American adult daily diet contains substantial quantities of calcium (800-1000 mg) and relatively small quantities of iron (10-15 mg) (42). In the absence of either blood loss or pregnancy, such diets have not been associated with clinical iron deficiency. In infancy and childhood, however, iron deficiency is commonplace due to iron requirements for growth, the consumption of milk (a food of high calcium, low iron content) , and in some cases to pathologic occult gastrointestinal bleeding (43). Among 13 selected infants with iron deficiency, Wilson (44) found evidence for accelerated gastrointestinal blood loss, gastrointestinal mucosal abnormalities, and a high incidence of serum precipitins to whole cow's milk. In a later study, however, only 17 of 34 selected infants with iron deficiency were shown to have cow 's milk-induced gastrointestinal bleeding measurable with 51Cr_la_ beled erythrocytes (45). Alternatively, Naiman (46) proposed that the functional and morphologic gastrointestinal abnormalities observed in most irondeficient children are primarily due to nutritional iron deficiency, not to cow's milk sensitization, because in most children, iron therapy alone corrected the observed abnormalities (47) . Human milk contains -0.03 g of calcium/l 00 ml (7.5 mmollL) and bovine milk 0.12 g/lOO ml (30 mmollL) (26). Our results indicate that the concentration of calcium in bovine milk is sufficient to diminish significantly the absorption of physiologic quantities of inorganic radioiron in rats. Iron absorption in normal adults and infants was significantly greater from human than bovine milk (48,49); in this experiment the results could not have been explained by gastrointestinal blood loss. This observation on extrinsic iron absorption from human and bovine milks was confirmed in our studies in rats. Further, the addition of

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calcium to human milk significantly reduced iron absorption by rats from the milk. Although iron deficiency in infancy and childhood is affected by many complex dietary and nondietary factors, the inhibitory effect of calcium on iron absorption may contribute to the development of this widespread health problem.

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D.C. 1960. Official methods of anal ysis , cited in Oser BL, ed. Hawk 's ph ysiological chemistry. New York: McGraw-Hill Book Co., 1965:391. Association of Official Agricultural Chemists, Washington, D.C. 1960. Official methods of analysis, cited in Oser BL, ed . Hawk 's physiological chemistry. New York: McGraw-Hili Book ce.. 1965:389-90. Association of Official Agricultural Chemists, Washington, D.C. 1960 . Official methods of analysis, cited in Oser BL, ed . Hawk 's physiological chemistry. New York: McGraw -Hill Book Co., 1965:392. Chen PS, Toribara TY, Warner H. Microdetermination of phosphorus. Anal Chern 1956;28:1756-8. Association of Official Agricultural Chemists, Wash ington, D.C. 1960 . Official methods of analysis, cited in Oser BL, ed. Hawk 's physiological chemistry. New York: McGraw-Hill Book Co., 1965:369. Izak G, Lewis SM. Proposed recommendations for measurement of serum iron in human blood. In : Izak G, Lewis SM, eds . Modern concepts in hematology. New York: Academic Press , Inc., 1972:125-i53. Beutler E. Peripheral blood, bone marrow, and urine iron stains . In: Williams WJ, Beutler E, Erslev AJ, Rundles RW, eds . Hematology, 2nd ed . New York: McGraw -Hili Book Co., 1977:1589-90. Snedecor GW, Cochran WG. Statistical methods. 6th ed. Ames. Iowa : Iowa State Uni vers ity Press , 1967:275. Parmley RT, Barton JC, Conrad ME, Austin RL. Ultrastructural cytochemistry of iron absorption. Am J Pathol 1978 ; 93:707-28. Manis JG, Schachter D. Active transport of iron by intestine: effects of oral iron and pregnancy. Am J PhysioI1962 ;203:816.

32. Wheby MS, Jones LG, Crosby WHoStudies on iron absorption. Intestinal regulatory mechanism. J Clin Invest 1964;43 :143342. 33. Manis JG, Schachter D. Acti ve transport of iron by intestine: features of the two-step mechanism. Am J Ph ysiol 1962; 203:73-80. 34. Greenb erger NJ, Balcerzak SP:Ackerman GA. Iron uptake by isolated intestinal brush borders: changes induced by alterations in iron stores. J Lab Clin Med 1969;73:711-21. 35. Pollack S, George IN, Reba RC, Kaufman RM, Crosby WHo The absorption of nonferrous metals in iron deficiency. J Clin Invest 1965;44:1470-3. 36. Becker G, Huebers H, Rummel W. Intestinal absorption of cobalt and iron: mode of interaction and sub cellular distr ibution . Blut 1979;38 :397-406. 37. Barton IC, Conrad ME, Nub y S, Harrison L. Effects of iron on the absorption and retention of lead. J Lab Clin Med 1978;92:536-47. 38. Hamilton DL, Bellam y JEC, Valberg JD, Valberg LS. Zin c, cadmium, and iron interactions during intestinal absorption in iron-deficient mice. Can J Physiol Pharmacol 1978;56 :3849.

39. Wasserman RH. Studies on vitamin D3 and the int estinal absorption of calcium and other ions in the rechitic chick. J Nutr 1962;77:69-80. 40. Wasserman RH, Taylor AN. Vitamin D-dependent calciumbinding protein. Response to some ph ysiological and nutritional variables. J Bioi Chern 1968 ;243:3987-93. 41. Bredderman PJ, Wasserman RH. Chemical composition, affinity for calcium, and some related properties of the vitamin Ddependent calci um-binding protein. Biochemistry 1974; 13:1687-94. 42. Recommended dietary allowances. Nat! Acad Sci, Washington, D.C., 9th ed., 1980.

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43 . Hoag MS, Wallerstein RO, Pollycove M. Occult blood loss in iron deficiency anemia of infancy. Pediatrics 1961 ;27 :199203 . 44. Wilson JF, Heiner DC, Lahey ME. Studies on iron metabolism.

I. Evidence of gastrointestinal dysfunction in infants with iron deficiency anemia: a preliminary report. J Pediatr 1962; 60:787-800. 45 . Wilson JF, Lahey ME, Heiner DC. Studies on iron metabolism.

V. Further observations on cow 's milk-induced gastrointestinal bleeding in infants with iron-deficiency anemia. J Pediatr 1974;84:335-44. 46 . Naiman JL, Oski FA, Diamond LK, Vawter GF, Shwachman H.

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The gastrointestinal effects of iron-deficiency anemia. Pediatrics 1964;33:83-99. 47 . Diamond LK, Naiman JL. More on iron deficiency anemia (lett). J Pediatr 1967;70:304-5. 48. McMillan JA, Landaw SA, Oski FA. Iron sufficiency in breastfed infants and the availability of iron from human milk . Pediatrics 1976;58:686-91. 49 . Saarinen UM, Siimes MA, Dallman PRo Iron absorption in infants: high bioavailability of breast milk iron as indicated by the extrinsic tag method of iron absorption and by the concentration of serum ferritin . J Pediatr 1977;91:36-9.