Human Nutrition and Metabolism Research Communication A Single 60-mg Iron Dose Decreases Zinc Absorption in Lactating Women1,2
are greatest. Lactating mothers need zinc for milk production, immune function, and cell replication and growth (1). One maternal adaptation to meet the increased demand for zinc during lactation is an increase in intestinal absorption (2,3). Iron supplementation has been shown to prevent the increase in zinc absorption that is typically seen during early lactation (2). In a longitudinal study of changes in zinc absorption during the reproductive cycle, decreased zinc absorption was reported during lactation in four women prescribed iron supplements throughout pregnancy (120 mg/d) and lactation (76 mg/d). This finding merits further investigation and a study designed a priori to determine the effect of iron supplementation on zinc absorption. Data from the third National Health and Nutrition Examination Survey suggest that women often continue taking prenatal supplements postpartum (4) despite evidence that consumption of excess iron by healthy lactating women is unnecessary (5). Based on Institute of Medicine and WHO recommendations of 30 – 60 mg of iron per day (6), prenatal supplements contain 30 –75 mg of iron. These amounts of iron far exceed the recommended dietary allowance of 9 mg for lactation (5). Consumption of prenatal supplements high in iron may affect the enhanced intestinal absorption of zinc, the primary mechanism by which the high demand for zinc is met during lactation (2,3). Decreased zinc absorption at this time may impair maternal zinc status and possibly jeopardize maternal health. This study was designed to examine the effect of a single iron supplement on zinc absorption during lactation.
(Manuscript received 5 January 2002. Initial review completed 15 February 2002. Revision accepted 21 March 2002.) Carolyn S. Chung, David A. Nagey,* Claude Veillon,† Kristine Y. Patterson,† Robert T. Jackson and Phylis B. Moser-Veillon3 Department of Nutrition and Food Science, University of Maryland, College Park, MD; *Department of Gynecology and Obstetrics, The Johns Hopkins University, Baltimore, MD; † Beltsville Human Nutrition Research Center, U.S. Department of Agriculture, Beltsville, MD
ABSTRACT This study determined whether a single 60-mg dose of ferrous sulfate interferes with fractional zinc absorption (FZA) at 7–9 wk of lactation. In a crossover design, 5 exclusively breast-feeding women were given either a single 60-mg iron supplement or no supplement. FZA was measured by analyzing zinc stable isotope tracers (70Zn and 67Zn) in urine samples collected for 7 d after isotope dosing. A 0.7-mol intravenous (IV) infusion of 70Zn as ZnCl2 in saline was followed by a 0.03-mmol oral dose of 67 Zn as ZnCl2 given with a standardized meal. After a 7-d wash-out period, the supplement given was reversed and a second FZA measurement was taken. FZA was calculated from isotopic enrichments in urine measured by inductively coupled plasma mass spectrometry. Hemoglobin, plasma ferritin and transferrin receptor, and plasma 5ⴕ-nucleotidase, plasma zinc and erythrocyte zinc did not differ before the two measurements of zinc absorption. When women were given a single iron supplement, FZA was significantly lower, 21.7 ⴞ 1.7% compared with 26.9 ⴞ 2.6% when no supplement was given (P ⴝ 0.032). A single 60-mg iron dose significantly decreases FZA during early lactation. J. Nutr. 132: 1903–1905, 2002. KEY WORDS: ● zinc absorption ● lactation ● stable isotopes
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SUBJECTS AND METHODS Subjects and study design. The protocol for this study was approved by the Institutional Review Board of the University of Maryland and the Joint Committee for Clinical Investigation of the Johns Hopkins Hospital and Bayview Medical Center. All participants gave informed written consent. Women in their third trimester of pregnancy and early lactation were recruited through flyers as well as advertisement in the Johns Hopkins Hospital newsletter. An initial telephone screening was conducted to identify healthy women who met the following criteria: ⱖ21 y old, planned to breast-feed exclusively for at least 3 mo, nonsmoking, nonabusers of drugs or alcohol, not receiving iron therapy and with no previous obstetric or gynecological complications. Five Caucasian women completed the study and breast-fed exclusively for the study duration. Participants entered the study between 7 and 9 wk postpartum. Upon entry into the study, women were asked to take a daily multivitamin supplement containing 18 mg elemental Fe as ferrous fumarate and no zinc (CVS daily multivitamin, CVS Pharmacy, College Park, MD) for the duration of the study. Women were randomly assigned to either iron treatment (60 mg iron as ferrous sulfate, CVS pharmacy brand, College Park, MD) or no iron at the first visit. Baseline spot morning urine and fasting blood samples were collected upon arrival at the Johns Hopkins Hospital. After collection, ⬃0.7 mol (50 g) of 70Zn was infused into each subject. Subjects also received a 0.03 mmol (2 mg) oral dose of 67Zn as 67 ZnCl2 in a Crystal Lite (Kraft Foods, Glenview, IL) lemonade beverage with either the randomized iron supplement or no supplement. A stan-
iron supplementation
Supplemental iron taken during lactation may impair zinc absorption at a time when physiologic needs for maternal zinc 1 Presented at Experimental Biology 2000, American Society for Clinical Nutrition Young Investigator Award Competition, March/April 2000, Orlando, FL [C. S. Chung, D. A. Nagey, C. Veillon, K. Y. Patterson, R. T. Jackson, P. B. Moser-Veillon. (2000)]. A single 60-mg iron dose decreases zinc absorption during early lactation [FASEB J. 14(4): A476]. 2 Support was provided by the UM/FDA Joint Institute for Food Safety and Applied Nutrition (JIFSAN), College Park, MD. 3 To whom correspondence should be addressed. E-mail:
[email protected].
0022-3166/02 $3.00 © 2002 American Society for Nutritional Sciences.
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CHUNG ET AL.
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dardized snack of a blueberry muffin was provided for women after the labeled beverage was consumed. Spot morning urine samples were collected for 7 d after administration of stable isotope doses. After a 7-d wash-out period, the protocol was repeated at the second visit. Women who had previously been assigned the iron supplement at the first visit received no supplement at the second visit and vice versa. Sample collection and analyses. Women were instructed to fast for 3 h before they arrived at The Johns Hopkins Hospital. Spot morning urine samples collected at home were collected upon rising. All urine samples were collected in polypropylene containers and the subject’s height and weight were measured. Fasting plasma samples were collected in plastic syringes (Sarstedt, Newton, NC) containing a zinc-free sodium heparin solution (Bovine Lung, Sigma, St. Louis, MO). Hemoglobin was measured at each visit using a HemoCue (HemoCue AB, Angelholm, Sweden). Blood samples were kept on ice for a maximum of 2 h then centrifuged at 2400 ⫻ g for 10 min at room temperature. Plasma was separated from the RBC and then samples were stored in polypropylene vials and frozen at ⫺70°C for later analyses. Urine was acidified to pH 2.0 with concentrated HCl (Seastar Chemicals, Seattle, WA) and stored at ⫺70°C for fractional zinc absorption (FZA)4 determination by a dual stable isotope method (7,8). Isotope preparation. Stable isotopes of zinc were obtained as the oxide (Oak Ridge National Laboratory, Oak Ridge, TN). The intravenous (IV) isotope, 70Zn, was prepared by dissolving the labeled zinc oxide into a few drops of 1 mol/L HCl (Seastar Chemicals) and adjusting the concentration to 20 mg 70Zn/L with sterile saline. The solution was tested for sterility and pyrogenicity (National Institutes of Health Pharmacy, Bethesda, MD) divided into 6.0-mL aliquots and sealed in sterile vials. Each IV dose was ⬃2.5 mL (2 doses/vial) or ⬃0.7 mol (50 g) 70Zn/IV dose. The oral isotope, 67Zn, was prepared by dissolving the labeled zinc oxide in a few drops of 1 mol/L HCl (Seastar Chemicals) and adjusting the concentration to 2.0 mg 67 Zn in 0.5 mL deionized water. For each subject, 0.5 g of 67Zn solution was weighed out into a polypropylene test tube and equilibrated for 24 h with ⬃10 mL of Crystal Lite solution. Zinc absorption method. FZA was estimated from isotopic enrichments of urine samples on days 4 – 6 (7). Ultrapure acids and bases (Seastar Chemicals) were used to dilute samples for inductively coupled plasma mass spectrometry (ICP-MS) analysis (8,9). Urine samples were digested using Ultrex nitric acid and hydrogen peroxide (9). The samples were dissolved in diluted nitric acid and ammonium acetate buffer (0.9 mol/L) added to adjust the pH to 5.0. Zinc was then extracted using trifluoroacetylacetone, pyridine, and HPLCgrade hexane and then back extracted from hexane into 0.1 mol/L nitric acid. Extracted samples were diluted to a natural zinc concentration of 75 g/L (75 ppb) for ICP-MS analysis. Instrument sensitivity was adjusted so that 75 g/L (75 ppb) of natural zinc gave a counting rate of ⬃300,000 counts per second at m/z of 66Zn. Calculations. The amounts of tracers were determined by using the following equation, based on the equations of Turnlund et al. (10) mt ⫽
the equation below (7). The average values of isotope incorporation into urine from d 4 to 6 were used to determine FZA at each time point according to the following equation: 70
FZA共%兲 ⫽ 67
where 70ZnIV is the IV dose, 67Znoral is the oral dose, and 67Zn and 70 Zn are the amounts of the two tracers in the biological sample, all expressed in mass terms. Plasma and erythrocyte analyses. Plasma was analyzed to determine iron and zinc concentrations. Plasma ferritin and transferrin receptor were analyzed using an ELISA kit (RAMCO, Houston, TX). Plasma 5⬘-nucleotidase was analyzed using a spectrophotometric kinetic assay by Dr. Robert DiSilvestro, Ohio State University (11). Plasma was acidified with mineral-free HCL (Ultrex, Baker Chemical, Bricktown, NJ) for zinc analysis by atomic absorption spectrometry (AAS) (model 5000, Perkin Elmer, Norwalk, CT) (12). Erythrocyte zinc concentrations were also determined by AAS after a wet digestion with concentrated nitric acid and 8.8 mol/L (30%) hydrogen peroxide (13). Dietary iron and zinc intakes were not determined. Subjects served as their own controls in this study; therefore, habitual dietary intakes were assumed to be constant within subjects. Typical zinc intakes for similar groups of lactating women in Maryland are 10 –12 mg/d (13–15). Statistical analysis. Means and SEM were calculated. Data were analyzed using a one-tailed, paired t test with differences considered significant at P ⬍ 0.05 (GPInstat version 2.0, San Diego, CA). Values are presented as means ⫾ SEM.
RESULTS The age of subjects was 33.4 ⫾ 4.1 y; the body mass index of the women was 22.8 ⫾ 1.8 kg/m2. Hemoglobin, plasma ferritin, and plasma transferrin receptor measurements taken before each measurement of FZA were within normal limits and did not differ between the two visits (Table 1). One subject had hemoglobin concentrations below 120 g/L and was counseled to see her physician for follow-up. Plasma and erythrocyte zinc and plasma 5⬘-nucleotidase measurements taken before each measurement of FZA did not differ between the two visits. Urinary FZA after an iron supplement was given was significantly lower than when no supplement was given (P ⫽ 0.032). DISCUSSION Results of this study demonstrate that a single 60-mg iron supplement decreases zinc absorption during early lactation. A previous report of decreased zinc absorption in lactating
T 䡠 Wt 䡠 关Ain ⫺ 共Ri/x 䡠 Axn兲兴 Wn 䡠 关共Ri/x 䡠 Axt兲 ⫺ Ait兴 ⫹ Wt 䡠 关Ain ⫺ 共Ri/x 䡠 Axn兲兴
where T is the total amount of zinc in the sample; mt is the amount of enriched zinc tracer; Wn is the atomic weight of natural or unenriched zinc tracer; Wt is the atomic weight of the enriched zinc tracer; A is used to designate atomic abundance with the subscripts indicating the isotope and the source of the isotopes; i is the tracer (67Zn or 70Zn) isotope; x is the reference (66Zn) isotope; n is the natural element; t is the enriched stable isotope tracer; and Ri/x is the ratio of reference to tracer isotope. Calculation of fractional zinc absorption. FZA of the oral zinc dose was determined by the relative amounts of the two (67Zn and 70 Zn) tracers in the IV and oral doses, and in the urine samples using
4 Abbreviations used: AAS, atomic absorption spectrometry; FZA, fractional zinc absorption; ICP-MS, inductively coupled plasma mass spectrometry; IV, intravenous.
67 Zn IV Zn ⫻ 70 ⫻ 100 Zn oral Zn
TABLE 1 Iron and zinc status and fractional zinc absorption (FZA) of lactating women with and without an iron supplement 1 Measurement Hemoglobin, g/L Plasma ferritin, ug/L Plasma transferrin receptor, mg/L Plasma zinc, umol/L Erythrocyte zinc, umol/g Plasma 5⬘ nucleotidase, U/L FZA,3 %
Iron2
No iron
120.7 ⫾ 8.0 30.9 ⫾ 11.5 5.0 ⫾ 1.2 13.0 ⫾ 2.9 0.189 ⫾ 0.006 3.1 ⫾ 0.1 21.7 ⫾ 1.7*
120.7 ⫾ 9.0 27.8 ⫾ 12.3 3.3 ⫾ 0.9 11.5 ⫾ 1.0 0.178 ⫾ 0.003 3.0 ⫾ 0.4 26.9 ⫾ 2.6
1 Values are means ⫾ SEM, n ⫽ 5. *Different from no iron, P ⬍ 0.032. 2 Iron supplement given as a single 60 mg dose of ferrous sulfate. 3 Determined using urine.
IRON SUPPLEMENTATION AND ZINC ABSORPTION
women supplemented with iron supports our finding (2). The previous study found that the FZA of an iron-supplemented group was 13% compared with 31% in a nonsupplemented group. Our study showed that women taking a single iron supplement had a FZA of 22% compared with 27% when the same individuals took no supplement. The difference in the percentage of zinc absorbed between the two studies may be attributable to different doses and lengths of iron supplementation used. Women in the previous study were prescribed iron supplements as ferrous sulfate from pregnancy to 7–9 wk postpartum. Doses prescribed ranged from 50 to 130 mg of elemental iron from as early as 8 –10 wk gestation to 34 –36 wk gestation and 50 – 65 mg of elemental iron from 34 to 36 wk gestation through 7–9 wk postpartum. Our study used a single 60-mg dose of elemental iron taken at 7–9 wk postpartum yet still produced a significant effect of decreased zinc absorption. Our data provide additional evidence that an interaction between iron and zinc occurs during lactation. The interaction between iron and zinc has been explained by an increase in the relative abundance of ions that compete for the same absorptive pathway in the intestine (16) and may result in inhibition of zinc absorption. Supplemental iron taken during early lactation may saturate a limited number of intestinal carrier sites, such as a possibly shared receptor recently identified as NRAMP2 (17), and depress zinc absorption. NRAMP2 is likely to be the membrane transporter that functions in controlling iron entry across the apical membrane and in the export of iron out of endosomal vesicles (18). The role of NRAMP2 in zinc transport, however, is less clear. The interaction between iron and zinc could be explained in part on the basis of a multi-ion function for this transporter (17). A recent study in Chinese women indicated that conservation of endogenous zinc occurs during lactation in subjects with a low zinc intake (19). Endogenous excretion of zinc may also be influenced by iron supplementation; unfortunately, we were unable to collect fecal samples to test this hypothesis. Hemoglobin, plasma ferritin and transferrin receptor analyses showed iron status was replete before the two measurements of zinc absorption. In one individual, subject 405, hemoglobin and ferritin concentrations were below the WHO standard cut-off values (20). Although iron status for subject 405 was the lowest in the group, it is interesting that when iron was not given, her FZA value was also the highest in the group. One possible explanation for this could be that low iron status may increase NRAMP2 transporters, which have also been shown to transport zinc (21), thereby increasing the opportunity for zinc absorption. Zinc status before the two measurements of zinc absorption did not differ. Plasma and erythrocyte zinc concentrations were within normal ranges for lactation (11–18 mol/L and 0.158 – 0.197 mol/g) (13) and were not significantly different before an iron supplement was given. Plasma zinc measured before an iron supplement for subject 406 was high at 27.9 mol/L compared with 12.7 mol/L measured before no supplement. Because 27.9 mol/L falls above the normal range, it is possible that the sample was contaminated. Plasma 5⬘nucleotidase activities did not differ before the two measurements of zinc absorption. Plasma 5⬘-nucleotidase activities of women in our study were low compared with the normal range for young adult men and women (4.0 ⫾ 0.7 U/L) (Dr. Robert DiSilvestro, Ohio State University, personal communication). Plasma 5⬘-nucleotidase enzyme activity may be useful in detecting marginal zinc deficiency because it is responsive to acute changes in zinc intake in adults (22). However, more data are required to interpret our results in the context of lactation.
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Our data demonstrate the interaction between a single dose iron supplement and zinc absorption during early lactation. Another study in healthy individuals showed decreased iron absorption in response to a calcium supplement taken with a meal but no effect of long-term calcium supplementation on iron status (23). The relationship between supplemental iron and zinc absorption during lactation may be similar. Longerterm iron supplementation may not affect zinc absorption. In summary, a single 60-mg dose of iron decreases zinc absorption during lactation. These data provide evidence for an iron and zinc interaction during the unique physiologic state of lactation. The effect of long-term iron supplementation on zinc absorption during lactation warrants further study. LITERATURE CITED 1. Food and Nutrition Board & Institute of Medicine (1991) Nutrition During Lactation. Subcommittee on Nutrition During Lactation, Committee on Nutritional Status During Pregnancy and Lactation. National Academy Press, Washington, DC. 2. Fung, E., Ritchie, L. D., Woodhouse, L. R., Roehl, R. & King, J. C. (1997) Zinc absorption during pregnancy and lactation: a longitudinal study. Am. J. Clin. Nutr. 66: 80 – 88. 3. Moser-Veillon, P. B., Patterson, K. P. & Veillon, C. (1995) Zinc absorption is enhanced during lactation. FASEB J. 9: A729. 4. Heimbach, J. Profile of dietary supplement use. In press: Bioavailability of Dietary Supplements conference: Key Issues in Defining the Research Agenda. NIH Office of Dietary Supplements, Bethesda, MD. 5. Food and Nutrition Board & Institute of Medicine. (2001) Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. National Academy Press, Washington, DC. 6. WHO/UNICEF/UNU. (1996) Indicators for assessing Fe deficiency and strategies for its prevention. WHO, Geneva, Switzerland. 7. Friel, J. K., Naake, V. L., Miller, L. V., Fennessey, P. V. & Hambidge, K. M. (1992) The analysis of stable isotopes in urine to determine the fractional absorption of zinc. Am. J. Clin. Nutr. 55: 473– 477. 8. Patterson, K., Veillon, C., Moser-Veillon, P. & Wallace, G. (1992) The determination of zinc stable isotopes in biological materials using isotope dilution ICP-MS. Anal. Chim. Acta 258: 317–324. 9. Veillon, C., Patterson, K. & Moser-Veillon, P. (1996) Digestion and extractions of biological materials for zinc stable isotope determination by inductively coupled plasma mass spectrometry. J Anal. At. Spectrom. 11: 727–730. 10. Turnlund, J. R., Michel, M. C., Keyes, W. R., King, J. C. & Margen, S. (1982) Use of enriched stable isotopes to determine zinc and iron absorption in elderly men. Am. J. Clin. Nutr. 35: 1033–1040. 11. Bertrand, A. & Buret, J. A. (1982) One step determination of serum 5⬘nucleotidase using a centrifugal analyzer. Clin. Chim. Acta 119: 275–284. 12. Smith, J. C., Butrimovitz, G. P. & Purdy, W. C. (1979) Direct measurement of zinc in plasma by atomic absorption spectroscopy. Clin. Chem. 25: 1487–1491. 13. Moser, P. B. & Reynolds, R. D. (1983) Dietary zinc intake and zinc concentrations of plasma, erythrocytes, and breast milk in antepartum and postpartum lactating and nonlactating women: a longitudinal study. Am. J. Clin. Nutr. 38: 101–108. 14. Klein, C. J., Moser-Veillon, P. B., Douglass, L. W., Ruben, K. A. & Trocki, O. (1995) A longitudinal study of urinary calcium, magnesium, and zinc excretion in lactating and nonlactating postpartum women. Am. J. Clin. Nutr. 1995: 779 –786. 15. Moser-Veillon, P. B. & Reynolds, R. D. (1990) A longitudinal study of pyridoxine and zinc supplementation of lactating women. Am. J. Clin. Nutr. 52: 135–141. 16. Solomons, N. W. (1986) Competitive interaction of iron and zinc in the diet: consequences for human nutrition. J. Nutr. 116: 927–935. 17. McMahon, R. J. & Cousins, R. J. (1998) Mammalian zinc transporters. J. Nutr. 128: 667– 670. 18. Wood, R. J. & Han, O. (1998) Recently identified molecular aspects of intestinal iron absorption. J. Nutr. 128: 1841–1844. 19. Sian, L., Krebs, N. F., Westcott, J. E., Fengliang, L., Tong, L., Miller, L. V., Sonko, B. & Hambidge, M. (2002) Zinc homeostasis during lactation in a population with a low zinc intake. Am. J. Clin. Nutr. 75: 99 –103. 20. WHO/UNICEF/UNU. (1996) Indicators for Assessing Iron Deficiency and Strategies for Its Prevention. WHO, Geneva, Switzerland. 21. McMahon, R. J. & Cousins, R. J. (1998) Regulation of the zinc transporter ZnT-1 by dietary zinc. Proc. Natl. Acad. Sci. USA 95: 4841– 4846. 22. Bales, C. W., DiSilvestro, R., Currie, K., Plaisted, C., Joung, H., Galanos, A. & Lin, P. H. (1994) Marginal zinc deficiency in older adults: responsiveness of zinc status indicators. J. Am. Coll. Nutr. 13: 455– 462. 23. Minihane, A. M. & Fairweather, S. J. (1998) Effect of calcium supplementation on daily non-heme iron absorption and long-term iron status. Am. J. Clin. Nutr. 68: 96 –102.
