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,, and, 5
* Department of Foods and Nutrition, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada, and Nutrition Research Division, Food Directorate, Health Protection Branch, Health Canada 2203C, Ottawa, Ontario K1A 0L2, Canada
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENT
FOOTNOTES
LITERATURE CITED
ABSTRACT 
The interaction of dietary selenium and iodine on the activities of the selenoenzymes, selenium-dependent glutathione peroxidase (GSH-Px), and type I deiodinase (DI-I), and the thyroid hormones thyroxine (T4) and triiodothyronine (T3) were studied. Male weanling Sprague-Dawley rats were fed an AIN-93G diet for 6 wk with modified selenium and iodine concentration as follows: three levels each of iodine and selenium (0.03, 0.2 added and 1.0 added mg iodine/kg diet, and 0.05, 0.18 added and 1.0 added mg selenium/kg diet) were used in a 3 × 3 factorial design. Renal, but not hepatic, DI-I activity was lower in rats with low selenium intake than in controls. Circulating T3 concentration was not affected by the dietary levels of iodine or selenium. Unlike in liver, kidney and erythrocytes, thyroidal GSH-Px activity was not lower than in controls in rats with low selenium intake, but was significantly higher when iodine intake was low. Significant interactions of iodine and selenium on serum T4 and thyroidal GSH-Px activity were observed. Serum T4 was maintained at control levels when both dietary iodine and selenium were low, but not when iodine alone, or selenium alone, was low. Activity of thyroidal GSH-Px was lowest in rats fed a diet containing high iodine and low selenium. The results suggest that high iodine intake, when selenium is deficient, may permit thyroid tissue damage as a result of low thyroidal GSH-Px activity during thyroid stimulation. A moderately low selenium intake normalized circulating T4 concentration in the presence of iodine deficiency.
KEY WORDS: iodine · selenium · selenoenzymes · thyroid hormones · ratsINTRODUCTION
Adequate amounts of both iodine (I) and selenium (Se) are required for optimal thyroid hormone (TH)6 metabolism. As a structural component of TH, I is a primary requirement for TH synthesis. Thyroxine (T4) is produced solely by the thyroid and, although it is the most abundant TH form, it is relatively biologically inactive. Triiodothyronine (T3), the biologically active TH form, is present in comparatively small concentrations; most T3 is produced by the deiodination of T4 in peripheral tissues. Low availability of I to the thyroid decreases TH synthesis dramatically. TH production is controlled by thyroid stimulating hormone (TSH), secreted by the pituitary gland in response to circulating levels of TH and by thyroidal autoregulatory mechanisms in response to I availability.
Selenium also plays a role in the control of TH metabolism. The deiodinating enzyme, which produces most of the circulating T3, type I iodothyronine 5
-deiodinase (DI-I) (E.C. 1.11.1.8), is a selenoenzyme with most of the activity occurring in liver, kidney and thyroid. Deiodination of T4 is also catalyzed by type II 5-deiodinase (DI-II), which produces T3 primarily for local use, and occurs in the central nervous system, pituitary gland and brown adipose tissue. The deiodinase enzymes and their activities have been reviewed in detail (Köhrle 1994
). Selenium may also play an indirect role in the control of TH synthesis because it is required by another selenoenzyme, Se-dependent glutathione peroxidase (GSH-Px) (E.C. 1.11.1.9). In the thyroid, GSH-Px is thought to be the main antioxidant system for neutralizing cytotoxic H2O2 and its oxidative by-products (Combs et al. 1975). Hydrogen peroxide is produced by the thyroid as a cofactor in TH synthesis (Dumont 1971).
The complex relationship between I and Se in TH metabolism has raised questions concerning the interaction between varying levels of dietary I and Se. For example, it has been suggested that Se deficiency can further compound the adverse effects of I deficiency; the increased plasma TSH and thyroid weights seen with I deficiency were further increased by concurrent Se deficiency (Arthur et al. 1992). On the other hand, there is evidence suggesting that Se deficiency has a moderating effect on the clinical variables associated with low I availability, in which the decreased serum T4 and T3 and increased TSH and thyroid weight seen in hypothyroid rats were reversed by Se deficiency (Golstein et al. 1988).
Questions pertaining to the complex relationship between I and Se have also been raised during supplementation trials. Supplementation of humans with Se when both I and Se are deficient, may cause a rapid increase in thyroidal GSH-Px, neutralizing the H2O2 produced and thus decreasing TH production to dangerously low levels (Corvilain et al. 1993). The restoration of DI-I activity after Se supplementation would increase the deiodination of T4 to T3 and T3 to diiodothyronine, and the increased catabolism of TH could result in loss of I from the system, thus exacerbating the hypothyroid condition of rats (Golstein et al. 1988).
Contempré et al. (1993) have also demonstrated that a high iodine dose given to rats deficient in both I and Se produced greater thyroid tissue damage that was later shown to be irreversible (Contempré et al. 1995), than when a high I dose was given to rats that were previously only I deficient.
Thus, there is a demonstrated need to clarify the effects of both combined I and Se deficiency and of imbalanced intakes of the two elements on TH metabolism, which may result when supplementation with one mineral is provided when both were previously deficient. The hypotheses of the present study were to determine whether combined I and Se deficiencies produce a more severe hypothyroid condition than with I deficiency alone and whether unbalanced intakes of I and Se produce any subclinical symptoms of perturbed TH metabolism.
MATERIALS AND METHODS
) with modified Se and I concentrations in a 3 × 3 factorial design with diets containing a combination of low, normal (as recommended by the AIN-93G diet formula) (Reeves et al. 1993) or high concentrations of I and Se. The basal (low) diet contained 0.03 mg I/kg diet (by analysis), and 0.2 and 1.0 mg I/kg diet were added in the form of potassium iodate to prepare the normal and high I diets, respectively. The basal diet also contained 0.05 mg Se/kg diet (by analysis), and 0.18 and 1.0 mg Se/kg diet were added in the form of sodium selenate to prepare the normal and high Se diets, respectively. Basal diets containing moderately low, as opposed to severely deficient, concentrations of I and Se compared with control concentrations, were chosen so that any interactions between these micronutrients could be detected. The control group were those rats fed the normal I and normal Se diet. Rats were housed individually in stainless steel cages and given free access to food and distilled water for 6 wks. The diet groups consuming the normal Se diet contained six rats and all other diet groups contained seven rats. Food consumption and body weights were recorded weekly. Food source was removed the afternoon prior to the day of necropsy. Animals were killed via exsanguination from the abdominal aorta following anesthetization with 5% isoflurane in O2 (500 mL/min). Liver, kidney and thyroid were removed and stored at 
80°C until the time of analysis. Whole blood was removed from the abdominal aorta. Serum was separated for use in the TH analyses and stored at 20°C. For the GSH-Px assay, erythrocytes were separated by centrifuging a hematocrit tube containing whole blood at 13,700 × g at 4°C. Erythrocytes were released into 1.0 mL saline solution and stored at 80°C until the time of analysis.
Iodine analysis.
Diet and liver I concentrations were determined using acid digestion followed by colorimetric analysis according to a modified method of Fischer et al. (1986). Concentrated nitric acid (10 mL) was added to Kjeldahl flasks containing sample (~1.0 g) and left overnight at room temperature. Sulfuric (5.5 mL) and perchloric (20.0 mL) concentrated acids were added, and flasks were then heated at medium temperature for 30 min, with cold-fingers inserted to control the evaporation rate. Nitric and perchloric acids were then distilled off at high heat and the cooled digests were made up to 100 g with distilled, demineralized water. Test tubes containing 1.0 mL digest and 300 µL each of 100 g/L NaCl, arsenic reagent (6 g arsenic trioxide, 400 mL 1.25 N NaOH, 112 mL concentrated sulfuric acid, diluted to 2 L with H2O), and ceric reagent (20 g ceric ammonium sulfate, 112 mL sulfuric acid, diluted to 2 L with H2O) were incubated at 37°C for 30 min. Iodine was determined on a Hewlett-Packard diode array spectrophotometer (8452A, Mandel Scientific Company, Guelph, ON, Canada) at 410 nm. Standards were prepared from a stock solution of 1.0 g/L iodic acid (0.1-0.6 mg/L). A standard reference material (nonfat skim milk powder, #1549, National Bureau of Standards, Washington, DC) was used for quality control.
GSH-Px activity analysis.
GSH-Px activity was determined in erythrocytes, liver, kidney and thyroid. The method has previously been described in detail (L'Abbé et al. 1991). Homogenized tissues (1:10 wt/v) were diluted with glutathione reagent (2 mmol/L glutathione in 10 mmol/L phosphate buffer, pH 7.0) as follows: liver and kidney 1:10, thyroid 1:6, wt/v. The assay mixture contained 5 mmol/L EDTA, 0.5 mmol/L sodium azide, 2 mmol/L reduced glutathione, 0.24 mmol/L NADPH and 1 × 103 U/L glutathione reductase and 0.3 mmol/L tert-butyl hydroperoxide as substrate and was performed at 37°C. Hemoglobin was analyzed using Drabkins reagent (#525-2 Sigma Diagnostics, St. Louis, MO) in an automated assay at 30°C with a 415/450-nm filter. The assay was standardized with human lyophilized hemoglobin, using 0.36 and 1.09 µg/L as low and high standards, respectively. Total protein content of tissues was determined using an Abbott QuickStart Total Protein kit (#LN 5A13-22, Abbott Laboratories, Mississauga, ON, Canada) following manufacturer's instructions. Low and high total protein standards of 2 and 8 g/L, respectively, were used (Agent, Abbott Laboratories). Homogenates from the GSH-Px analysis, after further dilution with glutathione reagent (liver, 1:4; kidney, 1:5; thyroid, 1:6), were used for protein analysis. Results were expressed as milliunits GSH-Px per milligram hemoglobin or per milligram protein, where one unit of activity catalyzed the oxidation of 1.0 mmol of reduced NADPH/min.
DI-I activity analysis.
DI-I activity was determined in liver and kidney. Full details of the procedure have been described elsewhere (Hotz et al. 1996). Tissue homogenates were incubated in a 125I-reverse T3 substrate solution (0.005 125I-reverse T3) at 37°C. Released 125I was separated using centrifuge filter units containing 1 g Dowex-50W resin. Radiolabel in the total filtrate was measured on an Auto-Gamma 5000 series gamma counter system (#5530, Packard Instrument, Downers Grove, IL). Total protein was determined on the same homogenate used for the DI-I assay by the procedure described for GSH-Px activity analysis. Results were expressed as picomoles I released per minute per milligram protein.
Thyroid hormone analysis.
Serum T4 was determined using a commercial T4 reagent kit (#445995, Beckman Instruments, Brea, CA) on a Beckman SYNCHRON CX System. T4 in serum competes with a T4 conjugate for T4-specific antibody in which the amount of T4 conjugate not binding with antibody determines the rate of substrate hydrolysis (O-nitrophenyl -
-D-galactopyranoside to O-nitrophenol), as determined by absorbance change at 410 nm. Serum T3 was determined using a commercial T-Uptake reagent kit (#445999, Beckman Instruments) using the same system as for T4 analysis. T4 conjugate binds to unoccupied sites of thyroxine-binding protein in the sample serum. Thyroxine-binding protein is also a carrier for T3. The amount of conjugate binding to the protein reduces the rate of substrate hydrolysis as determined by the same reaction mechanism described for T4 analysis. Equivalency was assessed by correlation analysis of serum samples to RIA by the manufacturer.
Statistical analysis.
The significant effects due to diet treatments were determined by two-way ANOVA, and significant differences between group means were determined by Least Squared Differences (P 
0.05) (Winer 1971). For the analysis of the effect of dietary I, in which dietary Se had no significant effect on the response of a variable, the data for all Se groups were pooled. When dietary Se level had a significant effect on the response of a variable, Least Squared Differences were determined between the group means from the normal Se intake groups only. The analysis for the effects of dietary Se is presented in a manner similar to that for the effects of dietary I. In the case of a significant interaction between I and Se, data were reported in figures for all treatment combinations. Statistical analysis was performed using the software package CSS: Statistica (StatSoft, Tulsa, OK).
RESULTS
Table 1.
Effects of dietary iodine concentration on tissue iodine, thyroid hormones and glutathione peroxidase activity in rats1,2
Table 2.
Effects of dietary selenium concentration on type I deiodinase, glutathione peroxidase and thyroid hormones in rats1,2
DISCUSSION ACKNOWLEDGMENT 
Fig. 1.
Serum thyroxine (T4) concentrations in rats fed various levels of dietary iodine (I) and selenium (Se). Bars represent means ± SEM; for low and high Se groups, n = 7 and for normal Se groups, n = 6. The low, normal and high diets contained I: 0.03 mg I/kg diet (basal), basal + 0.2 mg I/kg diet, and basal + 1.0 mg I/kg diet, respectively, and Se: 0.05 mg Se/kg diet (basal), basal + 0.18 mg Se/kg diet, and basal + 1.0 mg Se/kg diet, respectively. Significant differences between means were determined by a Least Squared Differences test (P 0.05) and are indicated by the lowercase letters.
[View Larger Version of this Image (43K GIF file)]
Fig. 2.
Thyroidal glutathione peroxidase (GSH-Px) activity in rats fed various levels of dietary iodine (I) and selenium (Se). Bars represent means ± SEM; for low and high Se groups, n = 7 and for the normal Se groups, n = 6. The low, normal and high diets contained I: 0.03 mg I/kg diet (basal), basal + 0.2 mg I/kg diet, and basal + 1.0 mg I/kg diet, respectively, and Se: 0.05 mg Se/kg diet (basal), basal + 0.18 mg Se/kg diet, and basal + 1.0 mg Se/kg diet, respectively. Significant differences between means were determined by a Least Squared Differences test (P 0.05) and are indicated by the lowercase letters.
[View Larger Version of this Image (34K GIF file)]
, Beckett et al. 1993, and Meinhold et al. 1993), or even slightly higher (Meinhold et al. 1992). In the present study, serum T3 in the low I intake groups did not differ from controls and is attributed to greater direct synthesis of T3 from mono- and di-iodotyrosines (Abrams and Larsen 1973) and increased thyroidal DI-I activity (Erickson et al. 1982).
, Beckett et al. 1989 and 1992, Beech et al. 1995), the moderately Se-deficient diet used in this study (0.05 mg Se/kg diet) produced only minor effects on DI-I activity. Hepatic DI-I activity was unaffected by the Se-deficient diet in this study and renal activity was 81% of that of controls. Reduced DI-I activity in peripheral tissues is typically associated with elevated levels of circulating T4 (Arthur et al. 1990, Beckett et al. 1989 and 1992) because of lower rate of deiodination of T4 to T3 via the activity of DI-I.
and 1993, Meinhold et al. 1992). In the latter cases, the response mechanism that maintains T4 levels in this moderately high range has not yet been described.
).
, Golstein et al. 1988, Meinhold et al. 1992) in which Se deficiency either alleviated or had no effect on the characteristics of hypothyroidism, and counter those found by others (Arthur et al. 1992, Beckett et al. 1993) in which Se deficiency produced higher TSH concentrations and thyroid weights than those found with I deficiency alone. This inconsistency may also result from differences in the study designs used; whether concurrent Se deficiency aggravates I deficiency is dependent on the acuteness of the Se deficiency and therefore may not occur when Se deficiency is only moderate.
) and to be less susceptible to loss of GSH-Px activity (Beech et al. 1995, Behne and Kyriakopoulos 1993, Vadhanavikit and Ganther 1993) in Se deficiency than tissues such as liver and kidney. The need for maintenance of thyroidal GSH-Px activity may be indicative of its important function in the thyroid to neutralize H2O2 and prevent cytotoxicity.
). It was proposed that excess I in the thyroid, through a series of molecular reactions, would react with H2O2 to form free radicals and cause tissue damage. It was suggested that the greater potential for thyroid tissue damage during combined I and Se deficiency resulted from an impaired Se-dependent antioxidant system such as the GSH-Px system. The results in the present study support this theory because high I intake, when coupled with low Se intake, produced significantly lower GSH-Px activity than that in the control group (Fig. 2), thus increasing the potential for tissue damage. Furthermore, the stimulation of thyroidal GSH-Px activity by low I intake appeared to be limited by the amount of dietary Se because activity was greater with higher availability of Se (Fig. 2). If submaximal levels of GSH-Px activity occur while thyroidal activity is being stimulated, with a concomitant increase in H2O2 production, tissue necrosis may result. A study combining the biochemical and pathological aspects of this hypothesis would be required for confirmation.
FOOTNOTES
-deiodinase; DI-II, type II iodothyronine 5-deiodinase; GSH-Px, glutathione peroxidase; T3, triiodothyronine; T4, thyroxine; TH, thyroid hormone; TSH, thyroid stimulating hormone.
Manuscript received 12 September 1993. Initial reviews completed 13 November 1996. Revision accepted 19 February 1997.
LITERATURE CITED
-deiodinase and other selenoproteins.
Am. J. Nutr. (Suppl.)
1993;
57:310S-312S-deiodinase of rat thyroid, but not that of liver, is dependent on thyrotropin.
Endocrinology
1982;
111:434-440
a regulatory role for the trace element selenium?
Exp. Clin. Endocrinol.
1994;
102:63-89
[Medline]
-deiodinase (type I) activities.
J. Nutr.
1993;
123:1124-1128
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