The effect of salt supplements on thyroid hormones and quality of pregnancy in female hypothyroid rats

Abstract

Background.

The use of nutrient supplements along with medication to optimize the treatment of diseases yields desirable outcomes. Hypothyroidism causes abnormalities in cells, and organs, and induces gene expression changes. The use of salt supplements and vitamins considerably helps to treat hypothyroidism.

Objectives.

To evaluate the effect of a food supplement containing iron, iodine, and folic acid on thyroid hormones changes as well as the quality and quantity of hypothyroid female rat’s offspring.

Materials and Methods.

In the current experimental study, 40 female rats were divided into six experimental and two control groups. The study was conducted in three phases. In the first phase, the role of a combinatory supplement along with levothyroxine to treat hypothyroidism by assessing T3, T4, and TSH hormones was investigated. In the second phase, the dose-depended effects of a combinatory supplement were investigated. Additionally, in the third phase, the quality and quantity of the next generation were measured in the hypothyroid female rats receiving the salt supplement.

Results.

The plasma level of T3, T4 and TSH in hypothyroid rats receiving nutrient supplements indicated that the use of combinatory supplements along with levothyroxine could have desirable effects on the treatment of hypothyroidism to such an extent that the level of T3 and T4 hormones in the intervention group was significantly higher than that of the control group (P≤0.01). The second phase demonstrated that the desired effects of combinatory supplements on the serum levels of T3, T4, and TSH hormones were dose-dependent so that by increasing the dosage of supplementation, a significant decrease in the TSH level was observed (P <0.05), while T3 and T4 levels increased (P <0.01).

The results of the third phase demonstrated that salt supplements could be effective in reducing the number of dead or preterm pups, and the use of mineral salts along with levothyroxine could promote a healthy birth.

Conclusion.

Salt supplements have considerable effects on the health status of the offspring of hypothyroid rats, resulting in the birth of more healthy pups and reducing the rate of abortion or preterm births.

Introduction

Thyroid hormones stimulate the proliferation of erythrocyte precursors both directly and via erythropoietin production enhancement, while iron-deficient anemia negatively influences thyroid hormone status (1-3). Thyroid diseases are among the most common endocrine disorders. Animal and human data indicate that iron deficiency disrupts thyroid metabolism. On the other hand, iron therapy can improve thyroid function and even decrease the dose of levothyroxine (4, 5).

Thyroid disease is the most common problem during pregnancy after diabetes (6). Thyroid function changes during pregnancy, and may result in thyroid failure due to physiological changes (6-8). It is shown that thyroid failure exerts destructive impacts on maternal metabolism and embryonic growth, including less weight at birth and congenital hypothyroidism (9).

Thyroid needs adequate amounts of iodine to synthesize hormones during the embryonic period, and the embryo’s thyroid gland competes with the mother’s thyroid gland in iodine intake (10). Thyroid hormones play a crucial role in developing the nervous system and the human brain, especially in the first trimester of pregnancy (11). Thyroid hormones induce the expression of some genes in the neural tissue. T3 and T4, but not TSH, pass through the placenta. In premature pups, the serum level of T4 is lower than that in mature pups, but the TSH level is usually the same in both premature and normal pups (12).

Hypothyroidism is caused by decreased secretion of thyroid hormones, and causes reduction in the basal metabolic rate (BMR) (13). Under such conditions, the thyroid gland does not adequately synthetize hormones necessary for normal tissue functions (14).

There are various conditions contributing to the development of hypothyroidism, including reduced iodine levels, hypoactive thyroid gland, and autoimmune diseases (15). Generally, hypothyroid patients complain of weight gain (16, 17). For the successful treatment of hypothyroidism, the level of thyroid hormones should be within the normal range in the peripheral tissue (18). Levothyroxine is used to treat hypothyroidism, but it has a different side effect (19).

The current study aimed to evaluate whether orally administration of combined iron salt, folic acid, and iodine could affect thyroid hormones and the offspring of hypothyroid rats.

Materials and methods

In the current study, forty Wistar female rats (twenty days old) weighing 50 g (in average) were obtained from the breeding colony of the Semnan University of Medical Sciences (SUMS), Semnan, Iran. The rats were divided into six experimental and two control groups. In the experimental group, to induce hypothyroidism, 10 mg/L methimazole was added to the water for 20 days, and the control group received pure water. Afterwards, blood samples were taken from the tail vein to confirm hypothyroidism induction. After confirmation of hypothyroidism in the experimental groups, salt supplementation plus medication was started.

Salt supplements containing KI: 415 mg/L, FeSO₄: 25 mg/L, folic acid: 500 mg/L and the concentrations were totally considered one unit of supplement. For the second phase of the study, to investigate the dependence of rats on supplements dosage, supplements at 0.5, 1 and 2 units were also given. To treat the control and experimental groups, Levothyroxine (25 μg/kg/day) and saline were intraperitoneally injected.

There were six groups in the first phase of the study (Table 1). In this phase, the study objective was to investigate the effect of nutrient supplementation, compared to levothyroxine, on healthy and hypothyroid rats (Table 1).

Table 1.

Intervention groups to investigate the first phase of the study. G1, the negative control, and G2, the positive control groups for hypothyroidism, and G3-G5 as the intervention groups for hypothyroidism. Rats in Group G6 were the healthy recipients

Compounds	Groups
	G1	G2	G3	G4	G5	G6
Methimazole	-	+	+	+	+	-
Drug	Normal saline	Normal saline	-	Levothyroxine	Levothyroxine	-
Salt Supplement	-	-	(1 unit)+	+(1 unit)	-	(1 unit) +

In the second phase of the study, five groups were investigated: groups I and II were respectively considered as negative and positive hypothyroid controls same as the ones used in the first phase (G1 and G2), and groups III, IV, and V receiving the supplement at different dosages (Table 2).

Table 2.

Intervention groups in the second phase of study; Group I, the negative control and Group II the positive control for hypothyroidism; groups III, IV, and V received different dosages of combinatory supplements

Compounds	Groups
	I	II	III	IV	V
Methimazole	-	+	+	+	+
Supplement	Normal saline	Normal saline	0.5 unit	1unit	2 unit

Male rats were then added to the groups for mating. During pregnancy, the treatment was continued according to the previous procedure. To measure thyroid hormones, blood samples were also taken daily during the treatment.

In the phase three (at the end of pregnancy), healthy, dead, and preterm pups were assessed. Blood samples were taken from mothers to measure the serum levels of TSH, T3, and T4 by ELISA.

The study protocol and the experiments were in accordance with international rules and regulations and the recommendations of the Ethics Committee for laboratory animals. The collected data were analyzed via the SPSS software using the analysis of variance and Tukey’s post hoc test. All data were expressed as mean ± SD of the detected variables, and P<0.05 was considered significant.

Results

In Phase I, Figure 1 shows the results of the serum levels of TSH, T4, and T3 in negative and positive control groups as well as the intervention groups (supplements plus levothyroxine). According to the significant increase in T4 and T3 (P <0.01) and significant reduction in TSH (P <0.001) in the intervention groups, compared to control groups, it can be concluded that supplements containing iron, iodine, and folic acid along with levothyroxine had better effects on the level of thyroid hormones against thyroid hormones alone.

Figure 1.

Changes in the serum levels of T3, T4, and TSH in hypothyroid rats compared to the control groups (A, B and C) and hypothyroid rat’s serum treated with different supplements doses (D, E and F). The results of three different runs were expressed as mean and standard deviation and finally were compared to the control groups based on the considered P-values (*, P≤0.05, ** P≤0.01, and *** P≤0.001) to indicate the statistically significant changes of the mentioned hormones.

Methimazole significantly reduced the levels of T3 and T4 (P<0.05) and increased TSH levels (P <0.01) compared to the negative control group (normal rats), and treatment of hypothyroid rats with combinatory supplements had desirable outcomes. As Figure 1 shows, in the G3 hypothyroid group, T3 level decreased significantly (P<0.05) compared to the negative control group (G1), but in the same group, T4 levels increased significantly (P<0.01). The TSH level decrease in the G3 group was very significant.

In the G4 group receiving levothyroxine plus supplement to treat hypothyroidism a significant increase in T3 and T4 levels was observed compared with the G1 control group; it is noteworthy that the levels of these hormones in the G4 group were even significantly higher than in the G5 group. The level of TSH in the group G6 was significantly lower than in the G5 group (P <0.001). However, the highest levels of T3 and T4 were observed in normal rats (the G6 group) that only received salt supplements and the TSH level in these rats was similar to the healthy controls (G1 group). The results indicated that nutrient supplementation in rats could improve thyroid function and subsequently increased the levels of T3 and T4 without remaining a significant impact on TSH level (Fig. 1).

In Phase II, salt supplements in 0.5, 1, and 2 units were used to assess the effects of dose-dependence and dose changes on T3, T4, and TSH serum levels to treat hypothyroid rats. Levothyroxine was not given to rats in these three groups since the study only aimed at evaluating the effects of salt supplements on hypothyroidism.

As shown in Figure 1, in a dose-dependent manner (graph D, E and F), increase in the dose of salt supplements increased T3 and T4 levels in hypothyroid rats (II) compared to the positive control group. The dose-dependent increase in the T4 level was more than that in the control group (healthy rats(GI), but the T3 level in doses of 0.5 and 1 unit was lower than that of the negative control group, and in the dose of 2 unit was higher than that of the negative control group (P<0.05). The TSH hormone also reduced in a dose-dependence manner by increasing the dose of supplements in III, IV, and V groups (P <0.05), and the lowest TSH level was observed in Group V, but the TSH level in Group V was higher than that of Group I (negative control).

In phase III, the quantity and quality (healthy, premature) of pups were investigated in female hypothyroid rats. The quality of pups was evaluated through direct observation. The results of the current study indicated that the offspring of the hypothyroid mothers who received no treatment (G2) was dead, and vice versa, all pups of healthy mothers who received salt supplementation (G6) were born with healthy appearance, but 10% lost was observed in the offspring of the healthy rats who did not receive the supplement (G1). These results demonstrated that the salt supplementation could be effective in the birth of healthy pups, even in mothers with normal thyroid function.

In hypothyroid groups, along with levothyroxine, receiving salt supplements had desirable effects on reduction of premature or dead births (Table 3). In addition, in Table 4, it was found that by increasing the salt supplementation dosage, the mortality rate in the embryos of hypothyroid mothers decreased dramatically.

Table 3.

Number of Pups born in intervention and control groups

Treatment	Groups
	G1	G2	G3	G4	G5	G6
Pups number	10	13	22	24	14	13
Dead embryo	1(10%)	12(92%)	11(50%)	1(4%)	2(14%)	0(0%)
Premature pups	0(0%)	1(7%)	8(36%)	1(4%)	3(21%)	0(0%)
Healthy pups	9(90%)	0(0%)	2(0%)	22(91%)	9(64%)	13(100%)

The level of TSH also reduced in a dose-dependent manner (P <0.05), but this reduction even in the dose of 2 unit was not comparable to the negative control group; nevertheless, the TSH level decreased by 50% in the dose of 1 unit compared to the positive control group (Chart F of Fig. 1).

Table 3 presents the results of pregnancy in hypothyroid rats based on the total number of pups as well as the number of dead, premature, and healthy born pups in each group, compared with the control groups.

All G6 pups born from mothers with normal thyroid function receiving salt supplements were in a healthy condition (Table 3); the finding was more valuable when compared to the results of Group G1 (mothers of normal thyroid who did not receive any supplementation). Therefore, mortality in these pups was 10% in Group G1 that did not receive salt supplementation, while the rate dropped to 0% in Group G6 that received salt supplements. In addition, comparison of the quality and quantity of births in hypothyroid mothers of G3, G4, and G5 groups showed that the co-administration of levothyroxine and salt supplements (G4 group) had desirable effects on the birth of healthy pups.

Table 4 presents the results of pregnancy in hypothyroid rats that received 0.5, 1 and 2 unit of salt supplements based on the total number of pups as well as the number of dead, premature, and healthy born pups in each group, compared to the control groups.

Table 4.

Evaluation of the quality and quantity of Pups were born from mothers with hypothyroidism (treated with supplements) compared to the control group

Treatment	Groups
	I	II	III	IV	V
Pups number	10	13	23	24	21
Dead embryo	1(10%)	12(92%)	14(60%)	1(4%)	8(38%)
Premature pups	0(0%)	1(7%)	6(26%)	1(4%)	5(23%)
Healthy pups	9(90%)	0(0%)	3(13%)	22(91%)	9(42%)

The results demonstrated that the administration of different doses of salt supplements was effective in the process of healthy pups’ birth in hypothyroid female rats to such an extent that by increasing salt supplementation dosage, the pups’ mortality showed a decreasing trend (Table 4).

Discussion

The study was conducted to determine the effect of salt supplementation on the plasma level of thyroid hormones and the survival rate of pups born from hypothyroid rats as well as to compare the results to levothyroxine alone recipient groups.

In the present study, supplementation alone increased the level of T3 and T4 hormones (P <0.05) and significantly decreased plasma levels of TSH (P<0.05) in healthy rats. In rats that received methimazole to induce hypothyroidism, salt supplements and/or levothyroxine or a combination form was administered. The current study showed that levels of T3 and T4 hormones in combination therapy were higher than those in levothyroxine therapy alone. Therefore, it can be concluded that the salt supplements can perfectly stimulate the thyroid gland, resulting in the increase of thyroid hormones levels.

In one study by Campbell et al. (1992), FeSO₄ and thyroxine tablets were concomitantly administered for 12 weeks to 40 patients, and the results showed that the plasma levels of TSH increased from 1.6 ± 0.4 to 5.4 ± 2.8 mU/L, but the level of T3 had not significant changes. The combination of iron with thyroxine produced a purple-colored complex with weak solubility, indicating the binding of iron to thyroxin. The combination of iron and thyroxine reduced the thyroxine activity in patients, which can be attributed to iron binding to thyroxin (22). The current study results regarding the use of salt supplements containing copper sulfate were different from those of the study by Capbell et al., to such an extent that the supplement containing FeSO₄ in the current study led to an increase in plasma levels of thyroid hormones but a significant reduction in TSH.

In the study by Rabeh et al. (2016), the effect of supplements containing iron, zinc, and vitamins E and C on thyroid hormones levels was studied in hypothyroid rats. The hypothyroidism was induced on rats using propylthiouracil; then, to treat them, the rate was supplemented with 40 mg/kg FeSO₄, 200 mg/kg zinc carbonate, 250 mg/kg/BW alpha tocopherol acetate, and 250 mg/kg/BW ascorbic acid. This combinatory supplement significantly increased thyroid hormones levels and reduced the TSH level. The level of glutathione peroxidase increased, which can be explained by the antioxidant activity of administered combinatory supplements. They suggested that this supplement could be used to treat hypothyroid patients (23).

The results of our study are consistent with those of the study by Rabeh et al. Thus, the supplement used in our study containing FeSO₄ at a concentration of 25 mg/kg was able, along with other mineral compounds, to have beneficial effects on secretion of hormones.

In one study by Sreelatha et al. (2017), 100 pregnant mothers with hyperthyroidism, who were in the first trimester of their pregnancy, were evaluated. FT3, FT4, and TSH levels in these women were assessed, and finally mortality rate in their embryos was examined. The results showed 2.1% abortion rate in the hypothyroid mothers (24). Evidently, a percentage of the embryos are missed due to hypothyroidism in the mothers, and the current study also showed that none of the embryos survived in Group G2 that received no intervention, but in Group G4 that concomitantly received levothyroxine and salt supplements, the premature birth rate reduced to 4%.

Iodine is a rare element that is essential for the synthesis of thyroid hormones. Concentration of the thyroid hormones in blood under the influence of iodine intake and changes in thyroid hormones levels interact with reproductive system (23). Casey et al. (2006) reported that the increased risk of abortion, preterm delivery, and low birth weight in mothers with hypothyroidism were evident. They examined 25,756 pregnant females in terms of thyroid hormones levels, and concluded that the risk of preterm delivery occurring before the week 34 of gestation was approximately twice in females with hypothyroidism, as compared to healthy pregnant mothers (24). The current study also found that the number of premature pups significantly reduced in mothers with hypothyroidism who received salt supplements plus levothyroxine (Table 3). One of the limitations of this study was the premature pups eaten by the mother, and to resolve this problem, the pups had to be separated from their mothers and screened immediately after delivery, and then they were returned to their mother’s cage.

In conclusion, hypothyroidism is associated with poor mental health and increased fetal mortality during pregnancy. During gestation, the need for thyroid hormone increases in the mother. Although it is indicated that females with hypothyroidism should increase their levothyroxine during pregnancy, the risk of pregnancy during hypothyroidism still threatens them. Since there is a limited rat model’s study on hypothyroid pregnant mothers to assess the effect of nutrient supplements on pregnancy process, it is suggested to extensively examine the same intervention on more animals and in subsequent steps, owing to the safety of the salt administered in the current study, it is recommended that it should be applied on the human population. Eventually, it seems that owing to salt properties, salt supplementation compensates for increased hormone requirement during pregnancy; thus, hypothyroid mothers do not need an increased dosage of hormones.

Conflict of interest

The authors declare that they have no conflict of interest.