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Department of Nutrition and Food Science, Auburn University, Alabama
Address reprint requests to: Sareen S. Gropper, Ph.D., R.D., Department of Nutrition and Food Science, 328 Spidle Hall, Auburn University, AL 36849. E-mail: sgropper{at}auburn.edu
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ABSTRACT |
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 TOPFOOTNOTES ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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Methods: Serum copper and ceruloplasmin concentrations, erythrocyte superoxide dismutase activity, serum ferritin, transferrin receptors, hemoglobin and hematocrit were studied in 15 females with non-anemic iron depletion before and after five weeks of iron supplementation and in 12 healthy iron-adequate females aged 19 to 28 years.
Results: Mean hemoglobin, hematocrit and ferritin concentrations of the control group (144 ± 11 g/L, 43 ± 3% and 38 ± 15 µg/L, respectively) were significantly higher than those of the iron depleted group prior to supplementation (134 ± 9 g/L, 39 ± 2% and 11 ± 6 µg/L, respectively). The serum transferrin receptor to serum ferritin ratio was significantly greater for the iron depleted group prior to supplementation (890 ± 753) versus the control group (151 ± 61). Mean serum copper and ceruloplasmin concentrations and erythrocyte superoxide dismutase activity of the iron-adequate control group (20.0 ± 5.7 µmol/L, 463 ± 142 mg/L and 527 ± 124 U/mL, respectively) were significantly higher than those of the iron depleted group (12.4 ± 3.8 µmol/L, 350 ± 108 mg/L and 353 ± 186 U/mL, respectively) prior to supplementation. Following iron supplementation, hematocrit and ferritin concentrations of the iron depleted group significantly increased to 42 ± 3% and 26 ± 8 µg/L, respectively. Mean serum transferrin receptor concentrations and the serum transferrin receptor to ferritin ratios significantly decreased in the iron depleted group following supplementation (6.1 ± 1.6 mg/L to 4.6 ± 1.5 mg/L and 890 ± 753 to 198 ± 114, respectively). Iron supplementation also significantly increased the mean serum copper concentration to 14.2 ± 5.4 µmol/L and, in subjects with serum ferritin concentrations 
12 µg/L, the mean serum ceruloplasmin concentration.
Conclusions: Non-anemic iron depletion characterized by low iron stores is associated with negative impacts on copper status. Iron supplements improved indices of iron status and serum copper and ceruloplasmin concentrations. Whether the diminished serum copper and ceruloplasmin concentrations and superoxide dismutase activity are associated with free radical damage to iron depleted cells requires further investigation.
Key words: non-anemic iron depletion, copper, ceruloplasmin, superoxide dismutase, females
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INTRODUCTION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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Antioxidants are a group of compounds that protect the body by scavenging or preventing formation of potentially harmful free radicals or reactive species. Free radicals and reactive species are formed in the body during normal physiological processes such as electron transport as well as following exposure to environmental factors such as ultraviolet radiation. While the antioxidant roles of several vitamins and minerals are well established, the effects of nutrient deficiencies on the body’s oxidant defense system are less studied.
One of the most well-studied nutrient deficiencies is iron deficiency anemia, which has been shown to diminish three mineral-dependent antioxidant enzymes. Studies have demonstrated that iron deficiency anemia significantly decreases glutathione peroxidase activity in red blood cells of rats [5,6] and humans [4,8] and also in the liver, kidney, heart, and brain of rats [6]. Iron deficiency anemia also has been shown to significantly decrease catalase activity in red blood cells of humans [4,9] and rats [5] and in the gastrointestinal mucosa of rats [7]. Both glutathione peroxidase and catalase are important in the removal of the destructive reactive oxygen species hydrogen peroxide from cells. Decreased erythrocyte superoxide dismutase activity of humans also has been demonstrated in the presence of iron deficiency anemia [4,9]. Superoxide dismutase disposes of superoxide radicals generated in cells. Because studies have focused on the effects that iron deficiency with anemia has on these mineral-dependent antioxidant enzymes, it is unknown if these enzymes are affected prior to the anemia stage and, if so, whether iron supplementation will improve the antioxidant enzyme status.
Parameters used for the diagnosis of iron deficiency anemia include measurement of packed cell volume (hematocrit) and/or hemoglobin which when below the lower normal limit are widely accepted as diagnostic of iron deficiency anemia [10,11]. For adult females, iron deficiency anemia is diagnosed with a hemoglobin <120 g/L and a hematocrit <36%. To distinguish iron deficiency anemia from other non-iron deficiency anemias, serum ferritin, which reflects iron stores, and at least one additional assay, such as serum iron, transferrin saturation, iron binding capacity or more recently serum transferrin receptors, are measured [10]. However, unlike hemoglobin and hematocrit for which cut-off values are fairly standard, the stated normal range for serum ferritin is controversial [10]. The cut-off values considered to be below normal for serum ferritin reported in the literature are 12, 15 and 20 µg/L [10,11].
The purposes of this study were first to examine copper status, specifically two copper dependent antioxidant enzymes, ceruloplasmin and superoxide dismutase, and serum copper concentrations in non-anemic college-aged females with varying degrees of iron depletion and, secondly, to examine the effects of iron supplementation on these same parameters.
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SUBJECTS AND METHODS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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20 µg/L. The term "iron depletion" will be used. Fifteen subjects with non-anemic iron depletion and 12 iron-adequate controls participated in the study. To further examine potential associations between iron depletion and copper status, subjects with iron depletion were subdivided into two groups: those with serum ferritin concentrations 12 µg/L and those with serum ferritin concentrations between 13 and 20 µg/L. No subjects reported use of oral contraceptive agents. A letter of informed consent was signed by each participating subject. Approval for this study was received from the Institutional Review Board for the Use of Human Subjects in Research at Auburn University.
Biochemical Assays
Subjects reported to the laboratory between 7:30 and 9:00 a.m. after at least a seven-hour fast. Blood samples were drawn from an antecubital vein into unheparinized Vacutainer® tubes using 20 gauge Vacutainer® multiple-sample needles by a certified phlebotomist. Aliquots of blood taken from the tubes were transferred to heparinized microhematocrit capillary tubes and Microtainer Brand® tubes for analysis of hematocrit and hemoglobin, respectively. Hematocrit concentrations were measured using a microhematocrit centrifuge (Damon/IEC Division, Needham Hts., MA). Hemoglobin concentrations were measured using a spectrophotometric assay (Sigma Diagnostics, St. Louis, MO). Remaining blood in tubes was centrifuged at 2200 rpm for 20 minutes at 4°C (Beckman Model J-6B centrifuge, Beckman Instruments, Inc., Palo Alto, CA). Aliquots of serum were analyzed for ferritin and transferrin receptors using enzyme-linked immunosorbent assays (Ramco Laboratories, Houston, TX). Additional aliquots of serum were stored in acid-washed microcentrifuge tubes at -80°C until further analysis of serum for copper and ceruloplasmin concentrations. In addition, serum was analyzed for glutathione peroxidase and selenium concentrations; the results of these assays have been published elsewhere [12]. Red blood cells were resuspended in phosphate-buffered saline (1:4 dilution) and frozen at -80°C for determination of superoxide dismutase activity. Serum copper concentrations were determined using flame atomic absorption spectrophotometry (Perkin-Elmer Model 5100, Norwalk, CT). Bovine serum (SRM 1598) certified for copper by the National Institute of Standards and Technology (Gaithersburg, MD) served as the standard reference material. Serum copper concentrations of the standard were quantified at 11.3 ± 0.3 µmol/L with values certified at 11.6 ± 0.6 µmol/L. Ceruloplasmin concentrations were analyzed using radial immunodiffusion with concentrations of samples determined from calibration curves of controls (The Binding Site Ltd., Birmingham, UK). Erythrocyte superoxide dismutase activity was analyzed using a spectrophotometric assay (OXIS Health Products, Inc., Portland, OR). Bovine superoxide dismutase served as a standard. Analyses of the SOD standard agreed within 5.7% of the assayed value. All assays were done in at least duplicate to ensure a variation of <5% to 7%.
Iron Supplementation
Subjects with iron depletion (serum ferritin 20 µg/L) were given a five week supply of ferrous sulfate capsules (Slow Fe, Ciba Inc., Woodbridge, NJ), each providing 50 mg elemental iron (160 mg ferrous sulfate). Subjects were instructed to take one capsule daily with a meal for five weeks and not to change dietary habits or exercise routines. At the end of five weeks, subjects returned to the laboratory for blood withdrawal and reassessment of all blood parameters. Compliance was monitored by follow-up phone calls made weekly throughout the study. A longer period of iron supplementation was not feasible due to constraints imposed by the academic calendar; many of the subjects in the study were graduating seniors and leaving Auburn.
Anthropometric and Dietary Information
Height and weight of all subjects were measured using standard techniques. While determination of the cause(s) for the low iron stores was not a focus of this study, a 24-hour diet recall was completed at the start and end of the study by each subject with the assistance of a registered dietitian to acquire information about usual iron intake by college-age females. Diet recalls were analyzed for energy, protein, meat, iron, copper and zinc intakes using diet analysis software (Food Processor, ESHA, Salem, OR) and compared to recommended intakes based on the Dietary Reference Intakes for iron, copper, and zinc [13].
Statistical Analysis
Statistical analyses were conducted using InStat (GraphPad Software, San Diego, CA) and StatView (SAS Institute, Inc., Cary, NC). Analysis of variance (ANOVA) and repeated measures ANOVA were used to analyze mean differences for all measured parameters between the control group and iron depleted group both before and after supplementation, and between the control group (serum ferritin >20 µg/L), subjects with iron depletion with serum ferritin concentrations 12 µg/L pre- and post-supplementation, and subjects with iron depletion with serum ferritin concentrations between 13 and 20 µg/L pre- and post-supplementation. When ANOVA results were significant, p < 0.05, identification of significant differences between groups was based on Tukeys multiple comparisons test. A Dunnett’s test also was employed to determine group differences from the controls. Transferrin receptor and ferritin data were transformed to a log scale to obtain normal distributions. Pearson correlations were used to assess the relationships between and within indices of copper and iron status.
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RESULTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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20 µg/L; of these 15 subjects, nine were iron deficient with a serum ferritin concentration 12 µg/L, and six subjects had serum ferritin concentrations ranging from 13 to 20 µg/L. Twelve subjects were iron-adequate with serum ferritin levels >20 µg/L and served as the control group. Mean ± standard deviation (SD) height, weight and age did not differ significantly between the iron depleted group (n = 15, 162.7 ± 6.1 cm, 57.7 ± 7.2 kg and 21.7 ± 2.0 years, respectively) and the iron-adequate control group (n = 12, 163.4 ± 6.1 cm, 60.0 ± 12.3 kg and 23.2 ± 1.9 years, respectively). While determination of cause(s) of iron depletion was not a purpose, dietary intake of subjects was assessed. Dietary total iron intake and heme iron intake did not significantly differ between the iron depleted group (14.5 ± 8.5 mg/day and 0.7 ± 0.6 mg/day, respectively) and the iron-adequate control group (24.2 ± 23.5 mg/day and 0.5 ± 0.6 mg/day, respectively). Similarly, no significant differences were found in copper and zinc intakes between the iron depleted (1.0 ± 0.6 mg/day and 6.8 ± 4.0 mg/day, respectively) and the control (0.9 ± 0.7 mg/day and 10.0 ± 4.5 mg/day, respectively) groups. Based on the 2001 RDA for iron for adult females of 18 mg/day, the iron deficient group consumed a mean iron intake of 81% of the RDA with 53% of subjects ingesting <2/3 the RDA for iron. The control group consumed a mean iron intake that was 134% of the RDA with 45% of subjects ingesting a dietary iron intake of <2/3 the RDA for iron. Based on the 2001 RDA for copper of 0.9 mg/day for adults, the mean dietary copper intakes by the iron deficient group and control group were 111% and 100%, respectively, of the RDA. Mean zinc intakes were 85% of the 8 mg/day RDA for the iron deficient group and 125% of the RDA for the control group. No significant differences were found in dietary energy, protein or meat intakes between the iron depleted group and the iron-adequate group (data not shown).
Indicators of iron and copper status for the 12 iron-adequate controls and the 15 females with iron depletion are presented in Table 1. The iron depleted group had significantly lower mean hematocrit, hemoglobin and ferritin concentrations before iron supplementation compared to the control group. After five weeks of iron supplementation of the iron depleted group, mean hematocrit and ferritin concentrations significantly increased from pre-supplementation values, and serum ferritin concentrations rose to within normal ranges for healthy adult females. However, the mean post supplementation serum ferritin concentration for the iron depleted group remained significantly lower than that exhibited by the controls at the start of the study.
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Analysis of diet at the end of the iron supplementation period revealed significantly higher intakes of iron including that from supplements post treatment (65.4 ± 11.4 mg iron/day) versus pre-supplementation (14.5 ± 8.5 mg/day). Dietary intakes of energy, protein, meat, heme iron, copper and zinc (data not shown) did not differ significantly between the pre- and post-supplementation period in the iron depleted group.
Subdivision of the 15 iron depleted subjects into two groups, those with serum ferritin concentrations 12 µg/L and those with concentrations 13–20 µg/L (shown on Table 2), and comparisons among these groups with the control group (data reported in Table 1) reveal statistically significant differences in several indices of iron and copper status. Mean serum ferritin and hemocrit were significantly greater in the control group versus those with serum ferritin 12 µg/L and versus those with serum ferritin 13–20 µg/L. The mean serum transferrin receptor concentration for the serum ferritin 12 µg/L group was significantly higher than that of both the control group and the serum ferritin 13–20 µg/L group. The mean blood hemoglobin concentration of the control group was significantly greater than that of the serum ferritin 12 µg/L group.
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12 µg/L group and serum ferritin concentrations in both groups significantly increased from respective pre-supplementation values (Table 2). The mean serum transferrin receptor concentration decreased significantly in the serum ferritin 12 µg/L group, and the transferrin receptor to ferritin ratio decreased significantly in both groups following iron supplementation (Table 2). Indices of copper status are reported in Tables 1 and 2, which show comparisons between the control group and the entire iron depleted group and further comparisons between the subdivided iron depleted group, respectively. Mean serum copper concentrations were significantly lower in the iron depleted group (n = 15, before supplementation) than in the control group (Table 1). All (100%) of the control subjects had serum copper concentrations within the normal range, 12.6–24.4 µmol/L. In contrast, 11 of 15 (73%) subjects in the iron depleted group exhibited serum copper concentrations below the lower limit of normal. After iron supplementation, the mean serum copper concentration significantly increased in the iron depleted group (Table 1). The mean serum copper concentration after supplementation, however, was still significantly lower than that of the healthy controls measured at the start of the study.
Analysis of serum copper concentrations of controls, subjects with serum ferritin concentrations 12 µg/L, versus those with serum ferritin concentrations 13–20 µg/L found significant differences between the control group and subjects with serum ferritin 12 µg/L, and between the control group and those with serum ferritin 13–20 µg/L prior to supplementation (Table 2). Serum copper concentrations of iron deficient subjects with serum ferritin concentrations 12 µg/L versus those with concentrations 13–20 µg/L did not significantly differ prior to supplementation. After five weeks of iron supplementation, the mean serum copper concentrations of both iron depleted groups rose with that of the serum ferritin 12 µg/L group approaching statistical significance (p = 0.087).
The mean serum ceruloplasmin concentration was significantly lower in the iron depleted group (n = 15) before supplementation than in the control group (Table 1). However, all subjects both in the control and iron depleted groups had serum ceruloplasmin concentrations above the lower limit of normal, 180 mg/L. After iron supplementation, mean serum ceruloplasmin concentration increased in the iron depleted group, but the increase was not statistically significant. When compared to the control group at the start of the study, however, no statistically significant difference was found in serum ceruloplasmin between the control group and the iron depleted group post supplementation (Table 1).
Differences in serum ceruloplasmin concentrations of controls, iron deficient subjects with serum ferritin concentrations 12 µg/L and those with serum ferritin concentrations 13–20 µg/L approached statistical significance (p = 0.0694). After five weeks of iron supplementation, the mean serum ceruloplasmin concentration of the serum ferritin 12 µg/L group rose significantly while that of the serum ferritin 13–20 µg/L group did not significantly differ from pre-supplementation.
Mean erythrocyte superoxide dismutase activity was significantly lower in the iron depleted group prior to iron therapy compared to the control group (Table 1). Following iron supplementation, the mean erythrocyte superoxide dismutase activity of the iron depleted group increased; however, this increase was not significant when compared to values prior to supplementation (Table 1). Post supplementation, the mean erythrocyte superoxide dismutase activity of the iron depleted group did not significantly differ from that of the healthy controls.
Comparisons of superoxide dismutase activity among iron deficient subjects with serum ferritin concentrations 12 µg/L, those with serum ferritin 13–20 µg/L, and the control group approach statistical significance (p = 0.051). After five weeks of iron supplementation, the mean superoxide dismutase activity of the serum ferritin 12 µg/L group rose approaching statistical significance (p = 0.088) while that of the serum ferritin 13–20 µg/L group did not significantly differ from pre-supplementation.
Significant positive correlations were found between serum copper and ceruloplasmin in the control group (r = 0.8313, p < 0.001), iron depleted group before supplementation (r = 0.9322, p < 0.0001) and iron depleted group after supplementation (r = 0.8966, p < 0.0001). No correlation was found between hemoglobin, hematocrit or ferritin and serum copper, ceruloplasmin, or superoxide dismutase activity in the iron depleted prior to supplementation or control groups.
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DISCUSSION |
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Iron deficiency anemia has been shown to affect several enzymes involved in the body’s oxidant defense system. Catalase, an iron dependent antioxidant enzyme, as well as nonferrous antioxidant enzymes such as glutathione peroxidase and superoxide dismutase have been shown to be diminished with iron deficiency anemia [4–9]. The results from this study support and extend these findings. In this study, serum ceruloplasmin concentrations and erythrocyte copper zinc superoxide dismutase activity were significantly lower in college-aged females with non-anemic iron depletion when compared to iron-adequate controls. Owen [15] reported increased hepatic copper and reduced release of ceruloplasmin from the liver of iron deficient rats. Kumerova et al. [9] reported significantly lower erythrocyte superoxide dismutase activity in 12 adults with iron deficiency anemia versus 50 healthy controls. Given that erythrocytes with low superoxide dismutase concentrations have been shown to be more susceptible to lipid peroxidation and hemolysis in vitro, states of iron deficiency without anemia may predispose individuals to increased free radical induced cell damage [16].
Concentrations of minerals, such as selenium and copper which are needed as cofactors for several enzymes, have been shown to be diminished with iron deficiency anemia [6,17–26]. In this study, subjects with iron depletion prior to iron supplementation had significantly lower serum copper concentrations than the controls. Moreover, 73% of the iron depleted subjects prior to iron therapy exhibited serum copper concentrations below the lower limit of normal. Results of other studies examining the effects of iron deficiency anemia on tissue copper concentrations vary. Iron deficiency in rats has been associated with significantly higher copper concentrations in the liver, spleen, muscle, bone and blood than found in healthy rats [16,21,24]. Similarly, one study in children also reported significantly higher serum copper concentrations in 60 children with iron deficiency anemia versus 64 healthy controls [27]. Two additional studies in rats, however, reported no significant changes in serum copper concentrations during iron deficiency [21,28]. Sherman and Tissue [29] found that the pups of rats fed an iron deficient diet exhibited increased copper concentrations in liver and spleen versus pups of rats fed a control diet. The authors suggested that copper is sequestered in tissues with iron deficiency and that with iron deficiency, less copper in the form of ceruloplasmin is needed in the blood to carry out ferroxidase roles. Shukla et al. [19] suggested that with iron deficiency, the reduction of iron allows for enhanced transport of nonferrous minerals into tissues and corresponding reductions in plasma.
While increased storage of copper in tissues in this study could account for the significantly lower serum copper and ceruloplasmin concentrations in the iron depleted subjects compared with those of the controls, decreased copper absorption and/or increased copper excretion also could have occurred. However, all subjects were healthy and did not report symptoms typically associated with malabsorption.
The reason(s) for the association between iron depletion and serum copper and ceruloplasmin concentrations and erythrocyte superoxide dismutase activity in the present study are not clear and were not a part of the investigation. However, the findings of the present study showing copper status to be negatively associated with iron depletion without anemia are similar to those of other researchers who showed that iron deficiency anemia negatively affected selenium status, including serum and tissue selenium concentrations and glutathione peroxidase activity [4–8,23]. Moriarty et al. [6] reported decreased expression of glutathione peroxidase mRNA in the livers of iron deficient rats. Such findings suggest that iron may be needed for the translation of glutathione peroxidase and perhaps for other enzymes including superoxide dismutase and ceruloplasmin or for the transcription and/or translation of copper trafficking proteins.
Iron supplementation of the iron depleted group for five weeks resulted in significant increases in hemoglobin, hematocrit and ferritin, as well as serum copper and ceruloplasmin concentrations. With supplementation, mean concentrations of ferritin increased from 11 ± 6 µg/L to 26 ± 8 µg/L, a level no longer suggesting iron deficiency but still significantly less than that of the controls. These findings suggest that the five week duration of supplementation providing 50 mg elemental iron was insufficient to achieve control values, however, was sufficient to correct the iron depletion.
The significant increase observed in serum copper concentrations following iron supplementation in the present study is similar to the findings of Yetgin et al. [8,23] with selenium. These researchers found that iron therapy effectively increased serum selenium concentrations and glutathione peroxidase activity in children with iron deficiency anemia [8,23]. In the present study, five weeks of iron supplementation resulted in significant increases in serum copper concentrations; however, serum copper concentrations following this supplementation remained significantly less than those of the controls. These findings also suggest the need for a longer duration of iron supplementation. While superoxide dismutase activity increased but not significantly following iron supplementation, the increase was sufficient such that there was no statistically significant difference between the enzyme activity of the controls and the post supplemented iron depleted subjects. These findings suggest that iron supplementation was sufficient to provide enough copper to achieve superoxide dismutase activity comparable to the controls’ enzyme activity.
Similar to superoxide dismutase activity, serum ceruloplasmin concentrations increased but not significantly over pre-supplementation concentrations when all subjects with serum ferritin concentrations <20 µg/L were considered. In contrast, when only iron deficient subjects with serum ferritin concentrations 12 µg/L were considered, iron supplementation resulted in significant increases in mean serum ceruloplasmin concentrations. Lee and Matrone [30] found that rats provided with a combination of iron and copper exhibited a significantly greater rise in ceruloplasmin activity than rats given iron or copper alone suggesting a role for iron in ceruloplasmin activity.
Serum ceruloplasmin and copper have been found to be positively correlated [31,32]. These findings are consistent with the results of the present study in which a significant positive correlation was found between serum copper and ceruloplasmin concentrations. It has been speculated that this correlation could be expected given that over 90% of copper in serum is bound to ceruloplasmin [31,33] and that ceruloplasmin biosynthesis has been shown to be under the control of copper with the rate of biosynthesis dependent upon copper metabolism and tissue copper concentrations [34–36].
It is clear from this study that non-anemic iron depletion is associated with a negative impact on indices of copper status in college-aged females. According to NHANES III, 7.8 million adolescent girls and women of child-bearing age have non-anemic iron deficiency and, thus, also may have sub-optimal copper status [1]. While iron status was improved with five weeks of iron supplementation, serum ferritin and copper concentrations remained significantly less than those of healthy controls. Further investigation of the mechanism(s) by which iron depletion impacts copper status and function is warranted. Additional studies also are needed to determine if the reductions in serum ceruloplasmin and erythrocyte superoxide dismutase, two enzymes with roles in the body’s oxidant defense system, are associated with free radical damage to body cells and tissues.
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ACKNOWLEDGMENTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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Dr. McAnulty is currently at Appalachian State University, Department of Family and Consumer Sciences, Boone, NC 28608.
Received September 25, 2001. Accepted July 5, 2002.
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