The Journal of Nutrition Vol. 129 No. 1 January 1999, pp. 174-180

Single and Multiple Selenium-Zinc-Iodine Deficiencies Affect Rat Thyroid Metabolism and Ultrastructure^1,2

Manuel Ruz³, Juana Codoceo, Jose Galgani, Luis Muñoz^*, Nuri Gras^*, Santiago Muzzo, Laura Leiva, and Cleofina Bosco^**

Center for Human Nutrition, Faculty of Medicine, University of Chile, Santiago, Chile; ^* Chilean Commission for Nuclear Energy, Santiago, Chile;  Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile; and ^** Program of Morphology, Faculty of Medicine, University of Chile, Santiago, Chile

	ABSTRACT

Abstract Introduction Methods Results Discussion References

This study was conducted to evaluate the effects of single and combined deficiencies of Se, Zn and I on thyroid function in rats. Rats were fed amino acid-based diets for 6 wk starting from weaning. The diets contained either low or adequate amounts of these minerals. In addition to the control and control pair-fed groups, seven experimental groups were formed: Se deficient (Se); I deficient (I); Zn deficient (Zn); Se and I deficient (SeI); Zn and I deficient (ZnI); Se and Zn deficient (SeZn); and Se, I and Zn deficient (SeIZn). Serum triiodothyronine (T₃) was significantly lower than in controls in Zn, SeZn and SeI groups. Serum total thyroxine (T₄) and free T₄ were significantly lower and thyroid-stimulating hormone (TSH) greater in all iodine-deficient groups, regardless of Se or Zn status. Thyroid glutathione peroxidase activity was significantly reduced in Se and SeZn groups. Nevertheless, in the groups with a concurrent I deficiency, the activity of this enzyme was significantly greater than in controls. Severe alterations of the follicle cellular architecture, including signs compatible with apoptosis, were observed in the Zn and SeZn groups. These alterations appeared to be less severe when iodine deficiency was simultaneously present. Single and multiple deficiencies of Se, Zn and I have distinct effects on thyroid metabolism and structure.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The thyroid gland synthesizes two major hormones, triiodothyronine (T₃),⁴which is the main biologically active thyroid hormone, and thyroxine (T4), which is the precursor of the former. Thyroid hormones promote cellular growth and development (Cavalieri 1997).

The thyroid gland is morphologically organized in follicles. These contain the colloid, surrounded by a single-layer epithelium. The shape of the epithelial cells is strongly affected by thyroid-stimulating hormone (TSH). The synthesis of T₄ and T₃ occurs within thyroglobulin at the cell-colloid interphase. Microvilli project from the surface of the follicle into the colloid, followed by endocytosis of thyroglobulin, which is in turn hydrolyzed to release the hormones and transferred through the basal membrane to the capillary (Greenspan 1994).

Several micronutrients are involved in thyroid hormone metabolism. Iodine is crucial for the formation of the hormones at the thyroid gland (Clugston and Hetzel 1994). Between 59 and 65% of total body iodine is contained in the thyroid hormones. Selenium and zinc also have important roles in thyroid metabolism. Selenium participates in the extrathyroidal deiodination of T₄ to the active form T₃ (Arthur et al. 1993). Zinc, in addition to its participation in protein synthesis, is involved in T₃ binding to its nuclear receptor (Miyamoto et al. 1991).

Iodine deficiency is among the three most common nutritional deficiencies worldwide (WHO/UNICEFF/ICCIDD, 1994). Zinc deficiency is thought to be a prevalent condition in less affluent societies (Gibson 1994, Ruz 1995). Selenium deficiency, although less prevalent than iodine and zinc deficiencies, has also been reported in some areas of the globe (Levander 1987). Current evidence indicates that the simultaneous occurrence of nutritional deficiencies of more than one of these micronutrients could be more common than previously considered (Diplock 1992, Vanderpas et al. 1993). These situations may have important implications regarding the form in which such deficiencies are expressed. For instance, combined deficiency of selenium and iodine has been suggested as a potential determining factor in the development of the myxedematous or nervous form of endemic cretinism (Goyens et al. 1987, Vanderpas et al. 1990).

Thus, there is a need to explore the effects of nutritional deficiencies of iodine, selenium and zinc, either alone or in combination. In this study, the effects of the interaction among zinc, selenium and iodine on thyroid metabolism and structure were evaluated in rats.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Wistar rats of both sexes, obtained from the Center for Human Nutrition animal unit, Faculty of Medicine, University of Chile, were fed amino acid-based diets for 6 wk starting from weaning (21 d). Diets met AIN guidelines for preparing diets for experimental animals (AIN 1993). To achieve a low level of contamination by the protein source, modified L-amino acid-based diets were used. The diets were purchased from Dyets (Bethlehem, PA). All nutrient levels were kept constant except those of Zn, Se and I as follows: low Zn, 3.5 mg/kg; adequate Zn, 38 mg/kg; low Se, <0.05 mg/kg; adequate Se, 0.18 mg/kg; low I, <0.05 mg/kg; adequate I, 0.20 mg/kg. Thus, seven groups were formed with all possible combinations: selenium deficient (Se); iodine deficient (I); zinc deficient (Zn); selenium and iodine deficient (SeI); zinc and iodine deficient (ZnI); selenium and zinc deficient (SeZn); selenium and iodine and zinc deficient (SeIZn). In addition, two groups receiving adequate amounts of Zn, I and Se in their diets were used as controls. One had free access to food (control) and the other was pair-fed according to the food consumption observed in the Zn group (control pair-fed). The rats were individually housed in stainless steel cages and had free access to deionized drinking water. The animal unit had a controlled environmental temperature and a 12h light:dark cycle. The entire experiment was conducted in three sequential batches. To avoid variations due to this procedure, each batch had a similar number of rats from all experimental groups. There were no significant differences among batches in selected variables of the same experimental groups, such as body weight gain, thyroid weight and serum thyroid hormones. This was interpreted as an indicator of homogeneity of batches.

After 6 wk of consuming the experimental diets, the rats were deprived of food and anesthetized with ether. Blood was drawn with a syringe and needle, free of the trace elements under investigation, by cardiac puncture before the rats were killed. Blood was transferred to a test tube with no anticoagulant to obtain serum and to a container containing heparin as anticoagulant to obtain plasma. Both serum and plasma were immediately frozen at 20°C until analysis.

Serum T₃ and total and free T₄ concentrations were determined according to the RIA method suggested by Larsen (1976). TSH concentration was determined in serum by using the Rat Thyroid Stimulating Hormone Radioimmunoassay kit (rTSH RIA) provided by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, Bethesda, MD). Plasma Zn and Se concentrations were determined by instrumental neutron activation. The samples were irradiated for 24 h at a thermal neutron flux of 10¹³ neutron/(cm²·s) in the RECH-1 nuclear reactor at La Reina Nuclear Center, Santiago. After 15 d of decay, the -radiation from the long-lived radionuclides generated was measured in a high resolution -spectrometer (Gras et al. 1992 and 1993). The equations for quantitation of the minerals, i.e., ⁶⁴Zn(n,) ⁶⁵Zn and ⁷⁴Se(n,) ⁷⁵Se, have been described in detail elsewhere (Travesi 1975). Accuracy and precision of the determinations were assessed by repeated determinations of the reference material IAEA-A-13 animal blood (International Atomic Energy Agency, Vienna, Austria). Certified values were as follows (means ± sd): Zn, 13 ± 1 µg/g and Se, 0.24 ± 0.07 µg/g. Values determined by analysis were: Zn, 14 ± 1 µg/g and Se, 0.22 ± 0.04 µg/g and were not significantly different from the corresponding certified values. Precision was 10% for Zn and 7% for Se. During the entire process of handling and analysis of samples, special care was taken to avoid contamination.

Plasma alkaline phosphatase (EC 3.1.3.1 ) activity was measured by using p-nitrophenyl phosphate as substrate (Sigma Chemical, St Louis, MO). Plasma and erythrocyte glutathione peroxidase (EC 1.11.1.9) activities were conducted according to the method of Paglia and Valentine (1967).

The thyroid gland was removed from the rats and stored under three different conditions as follows: 1) with no additives and frozen at 20^o C to determine the glutathione peroxidase activity as described above; 2) fixed in 10% neutral formalin for light microscopy studies; and 3) fixed in 3.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.3, at 4^oC for 3 h, for transmission electron microscopy studies.

Statistical analyses were conducted using the Stata software (Stata Corporation, College Station, TX). Intergroup comparisons were performed by one-way ANOVA, followed by the Bonferroni multiple comparisons test. When necessary, variables were log-transformed to achieve normality before proceeding with the statistical procedures. Variables transformed included serum T₃, total and free T₄, and TSH. A probability value 0.05 was considered to be significant (Snedecor and Cochran 1980).

	RESULTS

Abstract Introduction Methods Results Discussion References

Body weight was significantly lower in all groups with Zn deficiency (Table 1). Plasma zinc was lower in all groups fed the low Zn diets, regardless of the presence of selenium and iodine deficiencies. Plasma alkaline phosphatase did not differ among groups. Plasma selenium concentration, and plasma and erythrocyte glutathione peroxidase activities were dramatically reduced in the groups fed the low Se diets.

Thyroid weight was markedly affected by the presence of iodine deficiency (Table 2). The I and SeI groups had the highest weights, which were significantly greater than all other groups. The ZnI group had lower thyroid weight than the I group, but greater than controls. Serum TSH concentrations correlated with thyroid weight (r = 0.68, P < 0.01).

Serum T₃ concentrations were lower in SeI, SeZn and Zn groups than in the controls. Serum total T₄ and free T₄ were significantly lower and TSH greater in all I groups, regardless of Se or Zn status. The Zn group had significantly lower T₄ concentrations than controls. This difference, however, was of a smaller magnitude than the reduction noted in the I groups. Lower activities of thyroid glutathione peroxidase were observed in selenium-deficient rats with and without concomitant Zn deficiency. Nevertheless, the activity of this enzyme was significantly greater than in controls in rats with concurrent I deficiency.

Histological examination of the thyroid gland conducted by light microscopy (not shown), revealed that those groups with iodine deficiency, regardless of the simultaneous presence of Se and/or Zn deficiency, showed a series of morphologic alterations, i.e., cylindric epithelial cells, diminution or absence of colloid and dilatation of blood capillaries. In Se-deficient rats, no major morphologic alterations of the thyroid were apparent. Thyroids of Zn-deficient rats had flattened epithelial cells and colloid accumulation.

Transmission electron microscopy studies of the rats' thyroid glands permitted the observation of morphologic details important for understanding some of the micronutrient interactions (Figs. 1 and 2).

View larger version (147K): [in this window] [in a new window]   Fig 1. Thyroid ultrastructure in control rats, and in those with single selenium, iodine and zinc deficiencies. Panel A (control): the micrograph shows two epithelial cells of the follicle with regular nucleus and mitochondria. Supranuclear Golgi apparatus, dense bodies resembling lysosomes, and smooth and granular endoplasmic reticulum are present. Colloid at the upper right is denoted by an asterisk and capillary at the lower left by a star; magnification was X14,000. Panel B (Se group): the epithelial cells present large apical microvilli to the colloid (asterisk) region, supranuclear intracytoplasmic colloid vesicles and a large canalicular system. Active fibroblasts are present in the interstitium (f) with bundles of collagen fibrils (arrow-heads), some being cut in cross sections and others in longitudinal sections; capillaries are indicated by stars; magnification was X7000. Panel C (I group): there is an abundance of mitochondria, supranuclear colloid vesicles and a dilated smooth endoplasmic reticulum. The nucleous of the basal epithelial cell evidences hyperplasia. Colloid at the upper right is noted by an asterisk and part of a capillary at the lower left by a star. Normal collagen fibrils are indicated by the arrow-head. A fibrocyte (f) is present in the interstitium; magnification was X5000. Panel D (Zn group): debris of degenerating epithelial cells (arrows) at the colloid surface (asterisk) is observed upon a second cellular layer. Below the basement membrane, a fibroblast (f) and some collagen fibrils (arrow-head) in the interstitium are seen. Close to the fibroblast, there is a macrophage (m) with cytoplasmic extensions and pinocytotic vesicles. Capillary indicated by a star; magnification was ×8000.

View larger version (149K): [in this window] [in a new window]   Fig 2. Thyroid ultrastructure in rats with multiple deficiencies of selenium, iodine and zinc. Panel A (SeI group): there is an abundance of large and irregular mitochondria with a dilated smooth endoplasmic reticulum. Colloid at the upper left is noted by an asterisk and capillaries at the lower right by stars; magnification was ×8500. Panel B (ZnI group): an abundance of mitochondria at the basal region of the epithelial cell of the follicle is seen. Colloid at the upper left is denoted by an asterisk and capillary at the lower right by a star; magnification was X8550 Panel C (SeZn group): debris of degenerating epithelial cells (arrow) is observed at the colloid surface (asterisk). A second layer of degenerating epithelial cells with pycnotic dark nucleous and dilated cytoplasmic spaces is seen. The interstitium (i) has accumulated amorphous extracellular matrix; magnification was X10,000. Panel D (SeIZn group): there is an abundance of mitochondria in the upper and basal region of the epithelial cells of the hyperplastic follicle. Dilated smooth endoplasmic reticulum and canalicular systems are observed. Colloid at the upper right is denoted by an asterisk and capillary at the lower left by a star; magnification was X7200.

The control pair-fed group (not shown) did not differ from the freely fed control group. The Zn and SeZn groups had the most dramatically altered follicle cellular morphology (Figs. 1D and 2C, respectively). In those groups, signs compatible with apoptosis were observed, i.e., nucleus deformation, nuclear chromatin condensation, compaction of cytoplasmic organelles and dilatation of the smooth endoplasmic reticulum. Nevertheless, in the SeIZn rats (Fig. 2D), the alterations were less severe.

With the exception of fibrosis at the interstitium, selenium deficiency caused minor alterations of the follicular cells (Fig. 1B); the presence of long apical microvilli and the enlargement of the canalicular system, with a concomitant increase of intracytoplasmic colloid-containing vesicles were noted

Iodine deficiency alone (Fig. 1C) was characterized by hyperplasia as shown by the presence of more than a single cellular layer and an increase in mitochondria and in the smooth endoplasmic reticulum at the basal cellular region. In addition, a decreased number of apical microvilli were noted. In rats with multiple micronutrient deficiencies, including iodine deficiency, the morphologic changes were closely related to those resulting from the lack of iodine alone.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The importance of adequate nutrition for thyroid function is undisputed. Energy intake is a strong determinant of circulating levels of thyroid hormones. Overfeeding increases T₃ production, whereas energy restriction decreases serum T₃ concentrations. The latter is mediated by a reduction in the extrathyroidal conversion of T₄ (Katzeff et al. 1990).

Micronutrients such as iodine, zinc and selenium are also important in thyroid metabolism. Clugston and Hetzel (1994) reviewed extensively the role of iodine in thyroid metabolism. Severe iodine deficiency causes goiter and cretinism. Milder degrees of deficiency have detrimental effects on energy metabolism, cardiovascular function, bone turnover and neuromuscular alterations, among others.

Arthur (1997) and Greenspan (1994) summarized the most relevant aspects of selenium involvement in thyroid function. Selenium is a component of deiodinase type I, which transforms T₄ into T₃ in liver, kidney, muscle and thyroid. Recent evidence suggests that deiodinases type II and type III may also be selenium dependent (Arthur 1997). Deiodinase type II plays an important role in providing intracellular T₃ to the brain and pituitary. Selenium also plays a role in oxidative stress control at the thyroid as a component of the enzyme glutathione peroxidase.

In terms of zinc and thyroid function, Morley et al. (1980) and Kralik et al. (1996) reported that zinc deficiency significantly reduced serum T₃ concentrations in rats. In a zinc depletion-repletion study conducted in humans, Wada and King (1986) observed that circulating TSH, total T₄ and free T₄ tended to decrease during the depletion phase, returning to control levels after zinc repletion. Only the decrease in free T₄ was significant, however. Lukaski et al. (1992) found that Zn-deficient rats showed decreased adaptation to stress induced by cold exposure. These authors attributed the effects noted to reduced circulating T₄ and T₃. In our study we observed similar trends, although only the lower value of T₃ was significant.

In addition to its direct effect on thyroid function, zinc deficiency can indirectly affect thyroid hormone status by decreasing energy intakes. To control for this factor, we used a control pair-fed group. This group was fed the control diet, which had adequate contents of all nutrients, but supplied in amounts similar to those consumed by rats in the Zn group. On average, the amount of diet consumed by these groups was 80% of that of the control group.

Much of what is known about the effects of these micronutrients on thyroid metabolism has been learned from studies involving single nutrient deficiencies. Nevertheless, two- and three-way nutrient interactions may have distinct manifestations in thyroid function. With the notable exception of the iodine-selenium interaction, which has received much attention in the last decade, few studies of two nutrient deficiencies have been reported. To the best of our knowledge, this is the first study designed to evaluate the effects of combined Zn, I and Se deficiencies on thyroid gland function and structure.

The effects of combined selenium and iodine deficiencies have been evaluated in different fluids and tissues, such as plasma (Beckett et al. 1993), central nervous system (Campos-Barros et al. 1997, Mitchell et al. 1996), brown adipose tissue (Mitchell et al. 1996), liver (Mitchell et al. 1997) and thyroid (Hotz et al. 1997, Mitchell et al. 1997). Beckett et al. (1993) reported that the combined Se and I deficit significantly lowered thyroidal T₃ and thyroidal and plasma T₄ compared with rats with single iodine deficiency. Also, plasma TSH concentrations and thyroid weight were significantly greater in the former. Our results support some of those observations. We noted similar trends as described above regarding T₄ and TSH, although the differences in our study were not significant. In terms of plasma or serum T₃ concentrations, both studies found a slight decrease in the rats with combined I and Se deficiencies compared with I-deficient rats. In contrast, Hotz et al. (1997) found that combined Se and I deficiency in rats did not further antagonize thyroid hormone metabolism beyond what was observed with I deficiency alone. Differences in severity of selenium deficiency in these studies may help to explain part of the discrepancies in the results. For instance, the study of Beckett et al. (1993) used diets containing <0.005 mg Se/kg, whereas the study of Hotz et al. (1997) used 0.05 mg Se/kg. Our study can be placed at an intermediate point between these two.

Selenium deficiency lowered glutathione peroxidase activity in the thyroid gland. Interestingly, in the rats with combined selenium and iodine deficiencies, the activity was markedly greater, suggesting some degree of selenium redistribution under these conditions. A similar observation was reported by Hotz and colleagues (1997).

Histologic evaluations conducted by Contempré et al. (1996) in thyroid glands of selenium-deficient animals showed an active fibrotic process in which the inflammatory reaction and excess of transforming growth factor- play a key role (Contempré et al. 1996). Our observations were compatible with the presence of fibrosis in selenium-deficient rats. As a result of the selenium-iodine interaction, potential consequences for human health have been suggested (Contempré et al. 1991, Goyens et al. 1987, Vanderpas et al. 1990 and 1993).

In terms of combined Zn and I deficiencies, the available information is scarce. Smit et al. (1993) concluded that Zn deficiency induced in iodine-deficient rats did not cause additional biochemical alterations. In general terms, our observations agreed with this conclusion. We did not observe significant differences between the ZnI and I groups in serum TSH, total T₄ and free T₄, but we did with respect to T₃. Serum T₃ concentrations were lower in the I group than in the ZnI group.

One of the most intriguing observations in our study was the overriding effects of superimposed iodine deficiency on rats deficient in Zn, or Zn and Se. The most severe morphologic alterations of the thyroid cell were caused by Zn deficiency alone or in combination with the lack of Se. However, when I deficiency was simultaneously present, cellular changes were not as severe as seen in conditions of adequate I.

Two main mechanisms by which zinc deficiency may affect cell structure can be postulated. The first is related to the role of zinc in a number of enzymes involved in nucleic acid metabolism and protein synthesis (Valle 1990). The second mechanism is related to the critical role of zinc as a regulatory agent in apoptosis. In apoptosis, activation of an endonuclease is responsible for chromatin cleavage and major nuclear morphologic changes, as typically observed during this process (Arends et al. 1990). An intracellular pool of zinc atoms is crucial for modulating the activity of this endonuclease (Sunderman 1995, Treves et al. 1994). More recently, Perry et al. (1997), found that intracellular zinc atoms can prevent apoptosis not only by acting at the endonuclease level, which is a rather late stage in the apoptotic process, but also in an early stage by inhibiting proteolysis of the so called "death substrate" poly(ADP-ribose)polymerase (PARP).

How a cell severely affected by zinc deficiency can have the damage partially ameliorated when an additional nutrient deficiency occurs, specifically iodine, is not known. Nevertheless, to provide possible explanations for these results, it is useful to consider the Type I and Type II nutrient deficiencies discussed by Golden (1989). Briefly, a Type I nutrient deficiency is characterized by a reduction of tissue concentration of the nutrient accompanied by defects in one or more metabolic pathways with a resultant loss of function and selected clinical signs. There is no primary effect on growth. The most prominent example of Type I nutrient deficiency is iron deficiency. Also included in this category are iodine and selenium deficits, among others.

Zinc deficiency belongs to the Type II nutrient deficiencies. These present a primary growth diminution, possibly as a way of reducing the demand for Zn by the most metabolically active tissues. No major reductions of the nutrient concentration in tissues and fluids are seen. Clinical signs are relatively nonspecific and are the result of generalized disfunction. In the specific case of Zn, when deficiency occurs, a series of homeostatic changes take place such as reduction of food intake, decreased growth rate, increased zinc absorption and reduced Zn excretion. There is a high capacity to redistribute Zn to those tissues most urgently needing it. Zinc deficiency induces muscle catabolism (Clegg et al. 1989) to free Zn to support the synthesis of essential proteins. In iodine sufficiency, however, the thyroid gland does not seem to be among the recipient tissues of high priority. This situation apparently changes when iodine deficiency is superimposed on the existing Zn deficit. If this assumption is correct, it may explain the less severe cellular alterations observed under these circumstances. The presence of more Zn atoms at the thyrocyte would lower the endonuclease activity and presumably reduced PARP proteolysis, causing in turn less apoptotic activity as evidenced through the ultrastructural evaluation conducted in this study.

Production and release of TSH are stimulated by the lowering mainly of T₃, but also T₄ concentration. In all I-deficient groups, TSH levels were significantly greater than in controls. Nevertheless, it was noteworthy that in our study, despite the reductions of T₃ in Zn and ZnSe groups, this was not accompanied by increased serum TSH concentrations. Interestingly, this hormonal pattern is similar to that seen in the "low T₃ syndrome," an apparent adaptative mechanism to severe illness that reduces oxygen and other metabolic demands (Greenspan 1994). A possible explanation for these observations may lie in the selected effects of zinc on thyroid hormone metabolism. The nuclear receptor for T₃ is partially Zn dependent. There are two cysteine-zinc fingers at the central DNA binding domain (Greenspan 1994, Miyamoto et al. 1991). Thus, under Zn deficiency conditions some degree of resistance may exist at the hypophysis, which in turn, may interfere with the adequate reception and interpretation of signals. Also, zinc participates in the formation and mechanism of action of thyrotropin-releasing hormone (TRH). Pekary and colleagues (1991) reported that the processing of prepro-TRH to form TRH is zinc dependent via post-translational processing enzymes such as carboxypeptidase H. The mechanism of action of TRH at the hypophysis is bimodal. First, it stimulates the release of stored hormone; second, it stimulates transcription. The intimate mechanism involves an increase in intracellular Ca and an activation of protein C kinase. Protein C kinase is a Zn-metalloenzyme; specifically, the regulatory domain coordinates four atoms of zinc (Quest et al. 1992).

Single or combined Zn, Se and I deficiencies have distinct effects on thyroid function and structure. Although some combinations may worsen the condition existing when only one nutrient is deficient, others seem to reduce the severity of the damage. The potential occurrence of simultaneous deficiencies of two or three of these essential minerals in human populations deserves further studies designed to clarify the nature and effect of these interactions.

	FOOTNOTES

Manuscript received 14 May 1998. Initial reviews completed 11 June 1998. Revision accepted 1 October 1998.

	ACKNOWLEDGMENTS

The authors express their gratitude to Nancy Linero, Antonio Negrete, Augusto Aldunce and Ximena Pino, from the Center for Human Nutrition of the Faculty of Medicine, University of Chile, for their continuous support throughout the study. Their collaboration is very much appreciated.

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