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Laboratory for Human Nutrition, Institute of Food Science, Swiss Federal Institute of Technology, Zürich, Switzerland and * Physiology and Animal Husbandry, Institute of Animal Sciences, Swiss Federal Institute of Technology, Zürich, Switzerland
2To whom correspondence should be addressed. E-mail: sonja.hess{at}ilw.agrl.ethz.ch.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • anemia • iron deficiency • thyroid peroxidase • food restriction • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–8
). In regions of endemic goiter, the thyroid response to iodized oil is impaired in children with IDA compared with Fe-sufficient children (9
). In addition, Fe supplementation of goitrous children with IDA improves the efficacy of iodized oil and iodized salt (10
,11
).
The mechanism by which Fe status influences thyroid and iodine metabolism is unclear. IDA could impair thyroid metabolism through anemia and lowered oxygen transport (12
,13
). IDA may also alter central nervous system control of thyroid metabolism (14
) and nuclear T3 binding (15
). Another potential mechanism is impairment of thyroid peroxidase (TPO) activity. TPO is a 103-kDa Fe-dependent enzyme located at the apical membrane of the thyrocyte (16
). TPO catalyzes the first two steps of thyroid hormone synthesis, iodination of thyroglobulin and coupling of the iodotyrosine residues (17
). TPO activity requires a heme protein attached to ferriprotoporphyrin IX or a closely related porphyrin (18
,19
). IDA lowers the activities of other heme-containing enzymes, i.e., cytochrome oxidase, myeloperoxidase and succinate-ubiquinone oxidoreductases all are sensitive to depletion during Fe deficiency (20
). Similarly, IDA could lower TPO activity and thereby interfere with iodine metabolism in the thyroid. Therefore, we investigated whether TPO activity is decreased in Fe-deficient anemic rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The Veterinary Department of the Canton of Zurich gave ethical approval for the study. Male weanling Sprague-Dawley rats (ZUR:SD, Institut für Labortierkunde, University Zurich) were randomly assigned to 7 groups (n = 12) at 21 d of age. Three groups were assigned to receive low Fe (ID) diets of 3, 7 and 11 µg Fe/g, respectively, and three groups were pair-fed (PF) the control, Fe-sufficient diet (35 µg Fe/g) to each of these Fe-deficient groups. All 6 groups are referred to as food restricted in this paper although the reduced food intake in the IDA groups was due to anorexia. One group consumed the normal Fe diet ad libitum (CN). The low Fe and normal Fe diets were prepared by Dyets (Bethlehem, PA). Other than their Fe content, the diets were equivalent and conformed to the recommendations for AIN-93 purified diets (21
). The Fe content of all diets at baseline was confirmed by atomic absorption spectroscopy (SpecrAA-300/400 with GTA-96 Graphite Tube Atomizer, Varian Techtron, Mulgrave, Victoria, Australia). Each pair of Fe-deficient and pair-fed rats was matched by body weight. At the beginning of the study, all rats consumed food ad libitum for 4 d to adapt to the new conditions. For the remainder of the 4-wk feeding period, the rats were fed daily. The amount of diet provided to a PF control rat was equal to the consumption of its Fe-deficient partner on the previous day. Rats were individually housed in plastic cages with grated stainless steel floors in random order. The rats were kept under controlled conditions at 21°C temperature and 55% humidity with a daily 12-h light:dark cycle. Rats consumed Millipore water (Milli-Q UF Plus, Millipore, Bedford, MA) ad libitum. To prepare the rats and reduce the stress response at the time of anesthesia, all were picked up and handled daily. After a feeding period of 29 d, pentobarbital anesthesia (0.16 mg/g body) was induced intraperitoneally by injection. Blood was collected by cardiac puncture into EDTA-coated tubes and the rats killed by exsanguination. Thyroids were immediately dissected and removed. To ensure complete dissection of the thyroids, the procedure was done using a dissecting microscope with an animal technician experienced in rat neck dissection. The dissected thyroids were wrapped in aluminum foil, shock frozen in liquid nitrogen and stored at -60°C.
Laboratory analysis.
TPO preparation and analysis was done using a modified mini-assay method of Hosoya et al. (22
). The thyroids were thawed, washed three times with cold saline, blotted on filter paper and weighed. They were then repeatedly manually homogenized in a 1.5 mL Eppendorf tube with a glass pestle in 30 µg/L buffer A (0.25 mol/L sucrose, 20 nmol/L Tris-HCl, pH 7.4, 100 mmol/L KCl, 40 mmol/L NaCl, 10 mmol/L MgCl2)/mg original tissue and cooled in ice between each repetition. After centrifugation at 9000 x g for 1 min, the pellet was again homogenized in buffer A and centrifuged. The combined supernatants were ultracentrifuged at 25,900 x g and 4°C for 4 h. The pellet was suspended in 30 µg/L buffer A/mg original tissue and solubilized in an ice-cold ultrasound bath. This procedure resulted in a higher enzyme activity than the recommended treatment with desoxycholate and/or trypsin (22
–24
). Therefore, the reaction mixture was not treated with desoxycholate. TPO activity was measured by the method using guaiacol and iodide as the second substrate (22
). After ultracentrifugation, supernatants of ID groups and CN group (n = 2) had no measurable TPO activity by the guaiacol and iodine assay. For the guaiacol assay, the reaction mixture had a total volume of 450 µL and contained 33 mmol/L guaiacol, 0.27 mmol/L H2O2, 33 mmol/L sodium phosphate buffer (pH 7.4) and a total of 50 µL of enzyme mixture. The reaction was started by the addition of 10 µL H2O2 and followed spectrophotometrically at 470 nm and 25°C. For the iodide assay, the reaction mixture contained 0.135 mmol/L H2O2, 4.95 mmol/L potassium iodide, 33 mmol/L sodium phosphate buffer (pH 7.0), 50 µL of enzyme mixture and had a total volume of 400 µL. After the reaction was started with 10 µL of H2O2, it was followed spectrophotometrically at 350 nm and 25°C. For both assays, the total of 50 µL of enzyme mixture contained three different volumes of sample solution (20, 30, 40 µL). One guaiacol unit (GU) and iodide unit (IU), respectively, represent the amount of enzyme that produced a change of 1.0 optical density unit/s. Protein concentration was determined using the advanced protein assay (Cytoskeleton, Denver, CO). Hemoglobin (Hb) concentration was measured in triplicate in whole blood using the cyanmethemoglobin method (Sigma Diagnostics, St. Louis, MO). Whole-blood samples were centrifuged at 1700 x g for 15 min, and plasma samples were stored at -20°C. Total T4 and total T3 plasma concentrations were determined by RIA kits for veterinary use (Immunotech S.A., Marseille, France).
Statistical analysis.
Data processing and statistics were done using SPSS 10.0 (SPSS Chicago, IL). Data were analyzed by a one-way ANOVA with post-hoc comparisons performed by Tukey’s test. Multiple regression was used to compare effects of IDA and food restriction using Hb and mean daily food intake, respectively, as indicators. Differences with P-values < 0.05 were considered significant. Values are means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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or relative thyroid weights (4.8 ± 0.9 g/100 g body). Thyroid protein content was significantly lower in the ID-3 group than in the CN group (Table 1)
. By multiple regression, decreased food intake in both the PF and ID groups significantly reduced thyroid protein concentration (P < 0.05; data not shown).
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.
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. Multiple regression showed a significant independent effect of IDA on TPO activity expressed as GU/thyroid (P < 0.001). Compared with TPO activity (GU/thyroid) in the CN group, TPO activity in the ID-3, ID-7 and ID-11 groups was reduced 56, 45 and 33%, respectively (Fig. 1
). IDA also reduced TPO activity expressed as GU/mg thyroid (P < 0.01), IU/total thyroid (P < 0.005) and IU/mg thyroid (P < 0.05). IDA did not affect TPO activity/mg protein (data not shown). Food restriction also significantly affected TPO activity. As modeled by multiple regression, TPO activity was decreased by food restriction whether expressed as GU/total thyroid (P < 0.0005), GU/mg thyroid tissue (P < 0.05), IU/total thyroid (P < 0.0005), IU/mg thyroid (P < 0.0005) or IU/mg protein (P < 0.05). There was a strong association between Hb, daily food intake and TPO activity expressed as GU/thyroid (R2 = 0.40) and as IU/thyroid (R2 = 0.55) (Table 4)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) reported 10% lower T3 levels in both moderate-to-severe IDA (Hb, 75 g/L) and Fe deficiency without anemia. Beard et al. (7
) compared women with mild IDA (Hb, 110 g/L) with Fe-sufficient controls, and reported that the anemic group had significantly lower serum T3 and T4 and significantly greater TSH. In Fe-deficient women without anemia, serum T3 was significantly lower and TSH significantly greater than in Fe-sufficient controls. Fe treatment of the IDA women significantly increased serum T3 concentrations (7
). Plasma T4 and T3 concentrations were also lower in Fe-deficient rats (2
–4
). Rats with Fe deficiency and moderate IDA (Hb, 85 g/L) have reduced conversion of T4 to T3 (1
), and lower serum T4 and T3 concentrations than controls (5
). Fe-deficient rats have significantly lower hepatic thyroxine-5'-deiodinase activity, with hepatic production of T3 only 46% that of controls (7
). Weanling rats fed Fe-deficient diets had significantly blunted TSH responses to exogenous thyrotropin-releasing hormone, reduced turnover of serum T3 (
50% lower than controls) and lower hepatic thyroxine-5'-deiodinase activity (6
). [125I]T3 binding in hepatic nuclei was lower in Fe-deficient than in control rats (15
).
In the present study, T3 and T4 levels were significantly decreased in IDA rats, in agreement with previous studies (1
,2
). The T3 levels in IDA rats were only 65–68% of those in the CN group. However, T3 did not decrease in a dose-response fashion with increasing severity of IDA, consistent with the data of Brigham and Beard (8
). Increasing severity of IDA did produce a significant step-wise decrease in mean T4 (Table 2)
. Fe deficiency may impair activity of hepatic 5'-deiodinase, which catalyzes the conversion of T4 to T3 (8
,26
,27
). The decreased T3 levels in IDA rats in the present study may be related to decreased deiodinase activity and reduced peripheral formation of T3 (6
).
IDA may affect thyroid metabolism by several mechanisms. Using an in vitro incubation method, Kaplan and Utiger (28
) found that outer ring deiodinase activity is not affected by either ferric or ferrous iron. Thyroid metabolism could be impaired by Fe deficiency through anemia and lowered oxygen transport, similar to the thyroid impairment of hypoxia (12
,13
). Fe deficiency may influence iodine deficiency disorders through alterations of the central nervous system control of thyroid metabolism (14
) or through modification of nuclear T3 binding (15
). Our findings are the first to suggest an alternate contributory mechanism. TPO activity in the thyroid, measured by both the guaiacol and iodide assays, was clearly sensitive to body Fe depletion. These data suggest that impairment of TPO activity contributes to the adverse effects of IDA on thyroid and iodine metabolism.
Of interest is the discrepancy between TPO activity expressed per total thyroid and per mg thyroid compared with TPO activity expressed per mg protein. There was no independent significant effect of IDA on TPO activity/mg protein. Because there was no independent reduction in protein concentration/mg thyroid by IDA, this cannot explain the discrepancy. The precision of the protein assay (CV = 4.7%) was adequate and is unlikely to have obscured a potential difference. We are therefore unable to explain the lack of an independent effect of IDA on TPO activity expressed per mg protein.
We included pair-fed controls to distinguish the effects of reduced food intake associated with IDA from IDA per se as a cause of lowered thyroid hormone levels and TPO activity. This is important because reduced food intake in rats predictably lowers serum concentrations of thyroid hormones (29
–33
). During the final 10 d of the present study, PF-3, PF-7 and PF-11 rats were food restricted by 41, 26 and 23%, respectively, relative to the CN group. Consistent with previous reports, food restriction in our PF groups significantly reduced T4 compared with the CN group. Lower food intake also reduced thyroid protein concentration. In addition, food restriction was an independent predictor of reduced TPO activity. However, comparing the IDA rats to their PF controls showed that IDA per se had a clear effect on T4 and T3 levels and TPO activity (Tables 2
and 3)
.
These data provide a possible explanation for the observed impairment in thyroid response to iodine repletion in goitrous children with IDA (9
). By reducing TPO activity, Fe deficiency may decrease iodine incorporation into thyroglobulin and subsequent coupling of iodotyrosines to form thyroid hormone. These data also provide a potential mechanism to explain findings in our previous studies showing that Fe supplementation in goitrous children with IDA improves the thyroid response to both iodized oil and iodized salt (10
,11
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CN, control; GU, guaiacol unit; Hb, hemoglobin; ID, iron deficient; IDA, iron deficiency anemia; IU, iodine unit; PF, pair-fed; T3, triiodothyronine; T4, thyroxine; TPO, thyroid peroxidase; TSH, thyrotropin.
Manuscript received 27 November 2001. Initial review completed 8 January 2002. Revision accepted 27 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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