The effect of iodosteviol glycoside rebaudioside «A» on the pro- and antioxidant systems of tissues in experimental hypothyroidism

L.F. Rakhmatullina¹, V.N. Kozlov², G.A. Bayburina¹,

D.E. Bayburina¹, F.Kh. Kamilov¹

¹ FSBEI HE Bashkir State Medical University of the Ministry of Health of Russia,

Ufa, Russian Federation;

² FSBEI HE «K.G. Razumovsky Moscow State University of Technologies and Management (FCU)», Bashkir Institute of Technologies and Management, Meleuz, Russian Federation

Abstract. Purpose of the study. To assess the severity of free radical oxidation processes, the activity of the components of the enzymatic link and the antioxidant system of tissues in conditions of iodine deficiency in experimental hypothyroidism and to study the effectiveness of the subsequent action of the new iodosaccharide complex based on rebaudioside «A». Methods. The state of the prooxidant system was studied by the method of iron-induced chemiluminescence and by the content of products in tissues that react with 2-thiobarbituric acid (TBA). The activity of superoxide dismutase (SOD), glutathione peroxidase (GPO) and catalase was determined. Iodine deficiency hypothyroidism was simulated in sexually mature male white outbred rats by intragastric administration of thiamozole (mercazolil) for 21 days at a rate of 2.5 μg/100g of body weight. The development of hypothyroidism was controlled by determining the content of thyroid-stimulating hormone (TSH) and free thyrxine (fT4) in blood plasma. Results. The development of hypothyroidism was accompanied by an increase in the tissues (brain, liver, kidneys) of indicators of the basal iron-induced activity of radical formation, a statistically significant increase in the level of TBA-active compounds, characterizing the development of oxidative stress. At the same time, a pronounced decrease in the activity of SOD, GPO and catalase was observed. The usage in the recovery period after the cessation of intoxication with thyreostatic iodosteviol glycoside rebaudioside «A» at a dose of 2-3 μg of iodine/100 g body weight of rats daily for 30 days restored the functional state of the thyroid gland, promoted an increase in the activity of antioxidant defense enzymes and a decrease in the course of free radical oxidation processes.

Keywords: experimental hypothyroidism, free radical oxidation, superoxide dismutase, glutathione peroxidase, catalase

There is no conflict of interest.

Contact details of the corresponding author:

Gulnar A. Bayburina

gulnar.2014@mail.ru

Received 23.11.2020

For citation:

Rakhmatullina L.F., Kozlov V.N., Bayburina G.A., Bayburina D.E., Kamilov F.Kh. The effect of iodosteviol glycoside rebaudioside «A» on the pro- and antioxidant systems of tissues in experimental hypothyroidism. http://vestnikural.ru/article/1152 [Online] Vestn. Ural. Med. Akad. Nauki. = Journal of Ural Medical Academic Science. 2020, Vol. 17, no. 4, pp. 299–312. DOI: 10.22138/2500-0918-2020-17-4-299-312 (In Russ)

More than two thirds of the administrative territories of Russia belong to the zones of iodine deficiency biogeochemical provinces. The relevance of the problem of iodine deficiency is also determined by the low efficiency of mass prevention: the median of ioduria among Russians averages 82.2 μg/l (the norm is 150-200 μg/l) [1]. In hypothyroidism caused by low iodine consumption and other reasons, most organs and systems are affected, the formation of reactive oxygen species, the level of lipid peroxidation products increase, oxidative stress develops, and the effectiveness of enzymatic and non-enzymatic links of the antioxidant system decreases [2, 3, 4].

Mass prevention of iodine deficiency, recommended by WHO experts, is the most effective and economical method of replenishing insufficient iodine intake in the population, which is achieved by introducing iodine (iodide or potassium iodate) into table salt used in households or the food industry [5]. More than 120 countries around the world have chosen salt iodization as a national strategy for overcoming iodine deficiency and have adopted coordinating legislation. The measures taken and the results of the implementation of this strategy allowed the International Committee for the Control of Iodine Deficiency Diseases and the Global Iodine Network to include most countries in the list of target levels of iodine nutrition [6]. At the same time, there are more and more reports of the backside effects of iodized salt use (development of hyperthyroidism induced by iodine, autoimmune thyroiditis, goiter) [7, 8, 9]. Hyperiodization develops as a result of the rapid absorption of iodine, which is excessively supplied with table salt. At the same time, iodide alters metabolic processes in the cells of the thyroid follicle, influencing the cascade of signal transmissions of cAMP and Ca²⁺-inositol triphosphate pathways [7]. Persons who were early in the iodine deficiency state are more sensitive to an increase in iodine content, they are more likely to have thyroid dysfunction with an increase in the intake of a trace element even to a physiological level [10].

A positive effect on the state of the thyroid gland was noted with the consumption of iodized tea, milk and dairy products, bread and bakery products, eggs and other everyday products, as well as seafood and seaweed [7, 8, 11, 12, 13]. Unlike iodized salt, iodine in them is stabilized by an organic matrix, which ensures the stability of the microelement in the upper gastrointestinal tract, contributes to its more uniform absorption over time, preventing the development of the Wolff-Chaikov effect [3, 7].

To expand the range of iodine-containing fortified products, particular attention is drawn to carbohydrate structures (pectins, insulins) used in the food industry, which have adjuvant properties relate to iodine [12, 13].

The aim of the study was to assess the severity of free radical oxidation processes, the activity of the enzymatic link of the antioxidant system of tissues in conditions of iodine deficiency in experimental hypothyroidism and the effectiveness of the subsequent action of a new iodosaccharide complex based on steviol glycoside rebaudioside «A».

Materials and methods

The studies were carried out on nonlinear white male rats of 190-230 g kept in a vivarium on a balanced diet (compound feed for laboratory animals, CJSC «Assortment-Agro», Russia) with free access to water. The experiments were carried out in compliance with ethical standards and recommendations for the humane treatment of laboratory animals (order of the Ministry of Health of Russia No. 199n dated 01.04.2016 «On the approval of the Rules of Good Laboratory Practice»). The animals were slaughtered under light ether anesthesia.

The animals were divided into 4 groups: the first (control) were intact. Iodine deficiency in rats of the second, third and fourth groups was simulated by intragastric administration of thiamazole for 21 days at a dose of 2.5 mg/100 g of body weight daily. The animals (second) of the experimental group were killed on the 22nd day, and the rats of the third (comparison) and fourth (main) groups — 30 days after the last administration of the thyreostatic. In the recovery period, the rats of the third group were on vivar food, and the fourth group received an additional oral solution of a new iodosaccharide complex at the rate of 2-3 μg of iodine per 100 g of body weight daily [14]. Rebaudioside «A» is isolated from the plant Stevia rebaudiana Bertani and is used in the food industry as a safe sweetener replacing sucrose. It is stable during storage, compatible with food technologies, biodegradable, has hypotensive and hypolipidemic effects, and is used in the treatment of obesity and reduction of excess weight [15].

In the blood plasma of animals, the content of TSH and cT4 was determined by the enzyme-linked immunosorbent assay on a StatFox-2100 analyzer (USA) using reagent kits DRG Diagnosties GmbH (Germany) and T4 free — IFA-Best (ZAO «Vector Best», Russia). In tissue homogenates (brain, liver, kidneys), the processes of free radical oxidation were assessed by the chemiluminescence method (chemiluminometer KhL-03, Russia) with automatic processing of the results. Free radical oxidation was induced with a solution of ferrous iron salt. In tissue homogenates, the content of compounds reacting with 2-thiobarbituric acid (reagents of GLL Agat-Med), the activity of catalase [16], superoxide dismutase, and glutathione peroxidase (reagents of Radox Laboratories Ltd, Denmark) were also determined.

Statistical processing of the results was carried out using the Statistica 8.0 software with the calculation of mean values and standard deviations (M±σ), the data were processed using one-way ANOVA. Post-hoc analysis and Bonferroni test were used for post hoc comparisons.

Results and discussion

The introduction of thiamazole, which inhibits the processes of iodination of tyrosine radicals in thyreglobulin, was accompanied in rats of the experimental group by a decrease in the content of free thyroxine in the blood plasma and an increase in thyrotropin, which is characterized by hypothyroidism (Table 1). In animals of the comparison group, which were on vivar nutrition during the recovery period for 30 days, after the termination of the administration of the thyreostatic, the hypofunction of the thyroid gland continued to persist. In the fourth main group of rats receiving iodosteviol glycoside rebaudioside «A» in the recovery period, there was a decrease in TSH and an increase in fT4 to the level of values in the control group, which indicates the normalization of the pituitary-thyroid status.

Table 1

Changes in the pituitary-thyroid status during intoxication of rats with thiamazole and subsequent rehabilitation by the iodosaccharide complex, M±δ

Notes: in this and the following tables, the statistical significance of the differences p — with the control group, p₁ — with the experimental group and p₂ — with the comparison group. ANOVA, Bonferroni test.

Table 2

Intensity of chemiluminescence of rat tissue homogenates during experimental hypothyroidism and subsequent correction of iodine deficiency with the iodosaccharide complex, M±δ

The hypofunction of the thyroid gland in iodine deficiency was accompanied by changes in the pro- and antioxidant systems. In the rats of the experimental group, the spontaneous luminosity, which characterizes the basal activity of radical formation, increased statistically significantly in the tissues (Table 2). In brain homogenates, the spontaneous luminosity in hypothyroidism was higher than in intact rats by 51.2% (p=0.0007), in the liver by 82.6% (p=0.0001), in the kidneys by 45.6%. (p=0.0008). The amplitude of the rapid flash, which reflects the severity of radical formation after the induction of the process by ferrous ions, also increased, but reached statistically significant differences only in the liver. The maximum luminosity and light sum are indicators that indicate the ability of free radical processes in the biosubstrate when initiated by metal ions of variable valence. In animals with iodine-deficient hypothyroidism, these parameters in tissues were also significantly higher. In animals of the comparison group, the markers of free radical oxidation decreased, however, the spontaneous luminosity in all studied tissues remained elevated, characterizing the continuing intensification of radical formation. In the brain of this group of rats, at a higher level than in the animals of the control group, the maximum luminosity was preserved, in the liver and kidneys — the amplitude of the rapid flash and the light sum (p<0.02).

In the rats of the basic group, which received the iodosaccharide complex during the recovery period, the chemiluminescence markers decreased and did not differ statistically significantly from the data in the control group.

The results of studying the content of substances in rat tissues that react with thiobarbituric acid, which are mainly secondary products of lipid peroxidation (malondialdehyde, 4-hydroxy-2-nonenal and other aldehydes and ketones), do not contradict the data of the study of free radical oxidation processes by chemiluminescence (Table 3). In rats of the experimental group, the level of lipid peroxidation products was increased, in animals of the main group, their content sharply decreased, but did not reach the control level, although in the tissues of the brain and liver it was significantly lower than in the comparison group.

Table 3

Intensity of lipid peroxidation processes according to the level of accumulation of products that react with thiobarbituric acid in homogenates of rat tissues with experimental hypothyroidism and subsequent correction of iodine deficiency by iodosaccharide complex, M±δ

Thus, the results of studying the processes of free radical oxidation in tissues show that with the development of iodine deficiency hypothyroidism, there is an increase in oxidative processes with the development of oxidative stress, expressed in different organs with different intensities. The subsequent use of a new iodine-containing complex based on rebaudioside «A» in the recovery period leads to a decrease and normalization of free radical oxidation processes against the background of improving the functional state of the thyroid gland and reaching the physiological level of thyrotropin secretion.

However, until now there is no consensus on the mechanisms of the effect of iodinated thyroid hormones on the processes of free radical oxidation. When discussing the mechanisms of development of oxidative stress in thyroid dysfunction, the ability of thyroid hormones to modulate mitochondrial function (the mass of mitochondria, the course of oxidative phosphorylation, the intensity of biosynthesis of mitochondrial proteins, phospholipids, DNA) and, as a consequence, changes in the level and activity of prooxidants, the development and severity of free radical processes are discussed [4, 16], as well as shifts in the degree of saturation of fatty acids as the main objects of lipid peroxidation of biological membranes, changes in the level of receptors, increased sensitivity to catecholamines, and other mechanisms [2, 3, 17, 18]. The imbalance in the system of pro- and antioxidants during hypothyroidism may be associated with a decrease in the activity of the antioxidant system, which develops against the background of intensification of free radical oxidation processes [2, 3, 19]. However, according to other authors [20], in experimental hypothyroidism caused by the administration of a thyrostatic, the intensification of lipid peroxidation is accompanied by an increase in the activity of antioxidant defense enzymes.

In this regard, the main enzymes of the antioxidant system were determined in the tissues — superoxide dismutase (SOD), glutathione peroxidase (GPO) and catalase (Table 4).

Table 4

The activity of antioxidant enzymes in the tissues of rats with experimental hypothyroidism and with its subsequent correction by iodosaccharide complex, M±δ

The development of hypothyroidism in animals of the experimental group was accompanied by a statistically significant decrease in the activity of antioxidant enzymes in rats of the comparison group: after a 30-day recovery period, the activity of enzymes was partially restored, but the activity of SOD remained significantly reduced in all tissues, the activity of GPO and catalase was significantly lower than in the control group in the liver and kidneys. In animals fed iodine-enriched diet (basic group), by the end of the recovery period, in contrast to rats in the comparison group, a statistically higher level of activity of antioxidant enzymes in tissues was observed. These changes in the indicators of the enzymatic link of the antioxidant system occurred against the background of normalization of the content of thyrotropin and free thyroxine in the blood.

The results obtained underline that metabolic shifts of pro- and antioxidant systems in tissues established in animals are associated with changes in the functional state of the thyroid gland, and indicate the effectiveness of the effect of a new iodosaccharide complex based on steviol glycoside rebaudioside «A» on these processes.

Conclusion

Experimental hypothyroidism caused by daily administration of thiamazole (mercazolil) at a dose of 2.5 mg/100 g of animal weight during three weeks leads to the development of oxidative stress with increasing of the intensity of chemiluminescence and the level of lipid peroxidation products in the tissues of the brain, liver and kidneys and decreasing of activities of the main antioxidant enzymes — superoxide dismutase, glutathione peroxidase and catalase.

Iodine enrichment of the nutrition of animals with hypothyroidism by daily addition to the feed of an iodosaccharide complex based on steviol glycoside rebaudioside «A» at the rate of 2.5 μg of iodine per 100 g of body weight during a monthly recovery period helps to restore the functional state of the thyroid gland, indicators of pro and antioxidant systems.

REFERENCES

1.      Platonova NM, Troshina EA. Iodine deficiency: solving the problem in the world and in Russia (25 years of experience). Consilium medicum. 2015; 17(4):44-50 (In Russian).
2.      Remenyakina EI, Pavlyuchenko II, Ohremenko OS, Panasenkova YUE. Comparative analysis of the state of pro / antioxidant defense in patients with thyroid dysfunction of various origins. Sov-remennye problemy nauki i obrazovaniya. 2014; 2. URL: http://www.science-education.ru/116-12596. (In Russian).
3.      Lygdenov DV, Serdonova EV, Zhamsaranova SD. Influence of organic forms of iodine and zinc on the ratio of prooxidant and antioxidant systems of the body in iodine deficiency. Izvestiya vuzov. Prikladnaya himiya i biotekhnologiya. 2017; 7(23, Suppl.4): 36-43. (In Russian).
4.      De Castro AL, Tavares AV, Campos C, Fernandes RO, Siquera R, Conzatti A. et al. Cardioprotective effects of thyroid hormones in a rat model of myocardial infarction are associated with oxidative stress reduction. Mol Cell Endocrinol. 2014; 391(1-2):22-9. doi: 10.1016/j.mce.2014.04.010.
5.      WHO. Guideline: fortification of food-grade salt with iodine for the prevention and control of iodine deficiency disorders. Geneva, 2015.
6.      Mohort TV, Kolomiec ND, Petrenko SV, Fedorenko EV, SHepel’kevich AP, Solnceva AV. Problem of the iodine deficiency in Belarus Republic. Mezhdunarodnyj endokrinologicheskij zhur-nal. 2016; 73(1):11-18. (In Russian).
7.      Choudhry H, Nasrullah M. Iodine consumption and cognitive performance: Confirmation of adequate consumption. Food Sci Nutr. 2018; 6(6):1341-1351. doi: 10.1002/fsn3.694.
8.      Santos JAR, Christoforou A, Trieu K, McKenzie BL, Downs S, Billot L. et al. Iodine fortifica-tion of foods and condiments, other than salt, for preventing iodine deficiency disorders. Cochrane Database Syst Rev. 2019; 2(2):CD010734. doi: 10.1002/14651858.CD010734.pub2.
9.      Bali S, Tomar A, Nayak PK, Belwal R. No Longer Prevalent and Urinary Iodine Excretion Is above Normal among School Going Children in Jabalpur, India: Is This Major Health Problem Al-ready Solved? J Trop Pediatr. 2019; 65(5):457-462. doi: 10.1093/tropej/fmy076.
10.    Farebrother J, Zimmermann MB, Andersson M. Excess iodine intake: sources, assessment, and effects on thyroid function. Ann N Y Acad Sci. 2019; 1446(1):44-65. doi: 10.1111/nyas.14041.
11.    Dani DT. Negative effects of iodine deficiency and its elimination by iodine supplementation. In: Braverman LI, ed. Diseases of the thyroid gland. Moscow: Medicina. 2000. P. 378-391. (In Russian).
12.    Danilenko AL, Kamilov FH, Mamcev AN, Kozlov VN, Ponomarev EE. The effectiveness of the implementation of the «School milk» program in the prevention of iodine deficiency. Voprosy pitaniya. 2015; 84(2):53-58. (In Russian).
13.    Kozlov VN, Ponomaryov EE, Ponomareva LF. Vitamin composition of iodine-fortified bread. Privolzhskij nauchnyj vestnik. 2012; 4(8): 22-25. (In Russian).
14.    Kamilov FH, Konkina IG, Murinov YUI, Ivanov SP, Bayburina GA, Kozlov VN et al. Iodine-containing biologically active food supplement. Patent RF, N 2716971; 2020. (In Russian).
15.    Jiewen Z. Kinetics of rebaudioside A degradation in buffer solutions as affected by uv light exposure: diss. Auburn, Alabama; 2016. 90 p.
16.    Korolyuk MA, Ivanova LI, Majorova IG, Tokareva VEMethod for determination of catalase activity. Laboratornoe delo. 1988; 1:16-19. (In Russian).
17.    Jabbar A, Pingitore A, Pearce SHS., Zaman A, Iervasi G, Razvi S. Thyroid hormones and car-diovascular disease. Nat. Rev. Cardiol. 2017; 14(1):39-55. doi: 10.1038/nrcardio.2016.174.
18.    von Hafe M, Neves JS, Vale C, Borges-Canha M, Leite-Moreira A. The impact of thyroid hormone dysfunction on ischemic heart disease. Endocr Connect. 2019; 8(5):R76-R90. doi: 10.1530/EC-19-0096.
19.    Konoplyanko VA, Klebanov RD. Pathophysiological processes associated with hypothyroidism in experiment. Zdorov’e i okruzhayushchaya sreda. 2015; 2(25):102-105. (In Russian).
20.    Sabanov VI, Dzhioev IG, Lolaeva AT. The activity of lipid peroxidation, antioxidant protec-tion and the state of the myocardium in experimental hyper- and hypothyroidism. Mezhdunarodnyj zhurnal prikladnyh i fundamental’nyh issledovanij.2017; 2(6):241-244. (In Russian).

Authors

Liliana F. Rahmatullina

Federal State Budgetary Educational Institution of Higher Education «Bashkir State Medical University»

Associate Professor at the Department of Pathophysiology

3 Lenin str., Ufa, Russian Federation, 450008

Valeriy N. Kozlov

FSBEI HE «K.G. Razumovsky Moscow State University of Technologies and Management (FCU)», Bashkir Institute of Technologies and Management

34, st. Smolenskaya, Meleuz, Republic of Bashkortostan, Russian Federation, 453850

Doctor of Sciences in Biology, Associate Professor, Professor at the Department Food production technologies

Gulnar A. Bayburina

Federal State Budgetary Educational Institution of Higher Education «Bashkir State Medical University»

Candidate of Sciences in Medicine, Associate Professor at the Department of Pathophysiology

3 Lenin str., Ufa, Russian Federation, 450008

gulnar.2014@mail.ru

Dina E. Bayburina

Federal State Budgetary Educational Institution of Higher Education «Bashkir State Medical University»

3rd year student of the pediatric faculty

3 Lenin str., Ufa, Russian Federation, 450008

Felix H. Kamilov

Federal State Budgetary Educational Institution of Higher Education «Bashkir State Medical University»

Professor at the Department of biological chemistry, M.D.

3 Lenin str., Ufa, Russian Federation, 450008