Increased Morbidity and Its Possible Link to Impaired Selenium Status

Abstract

This chapter summarizes the latest information on the main differences in the chemical properties of selenium proteins and their sulfur analogues, Se proteins and their functions, Se-accumulating proteins, the relationship between Se and hemoglobin, Selenium in gerontology, Selenium and iodine deficiency conditions, Se and immunity, Selenium as an antioxidant in nitrite poisoning. Also discussed are some of the results of the first studies on protein enrichment with selenium carried out in the seventies of the last century. This native protein was natural silk fibroin. Fibroin has since become an important tool for human health and healing. It was discovered that when selenium-containing inorganic compounds were added to mulberry silkworm feed, selenium atoms formed additional sulfur-like bonds in fibroin macromolecules. This resulted in additional branching of protein macromolecule. Selenium atoms in the fibroin structure have a sufficiently high electron affinity, act as small traps and capture migrating electrons. This leads to a reduction of free radicals, which are generated by external influences such as mechanical, thermal, electrical and radiation.

1. Introduction

One of the trace elements, the lack of which has a significant impact on human health, is selenium (Se). It is a part of many proteins and key antioxidant enzymes involved in many metabolic processes and has antioxidant and immunoregulatory properties. Its deficiency leads, first, to the weakening of the antioxidant defense system and immunity, which causes the development of several diseases. The content of selenium in the human body depends on the level of its dietary intake, which is closely related to the distribution of the element in the biosphere of the region of residence. At status of selenium in Azerbaijan, as well as in many other countries, is close to deficiency, and its decrease is connected with the worsening of the ecological situation. The problem of selenium supply to the population of Azerbaijan at the present time is urgent and requires the adoption of appropriate measures to solve it.

Selenium is an essential, absolutely essential element for the life activity of many organisms (from viruses to mammals) and, mainly, humans. Despite the fact that its gross content in a 70 kg human body is only 14–15 mg, it is directly involved in many vital regulatory processes [1, 2, 3]. Its distribution in the Earth’s crust is insignificant, the so-called clark makes only 10 5%, and, thus, it is distributed very unevenly. It is accepted to consider soils that a content of less than 10 5% of selenium as poor and more than 10 5% as rich soils [4]. Proceeding from this the content of selenium in products depends on its regional provisioning and, consequently, provisioning of selenium (selenium status) in human organisms can vary greatly even within one country. At the same time, it was found that different organisms absorb selenium unevenly. Some plants belonging to cereals and astragals can serve as indicators of soil selenium supply.

Despite the fact that the selenium content in the ocean is very low, some species of aquatic organisms, including various algae (e.g., spirulina) have the ability to accumulate it in their tissues [4]. In addition to species specificity, there is also organ specificity. In the liver, kidneys, retina, thyroid gland, adrenal glands, testes, blood cells (lymphocytes, platelets, red blood cells), and nerve cells the selenium content is high, which indicates its importance in their functioning [3, 5].

However, despite the tremendous progress in the understanding of the biological role of selenium achieved over the last 50 years, its true potential as a biologically active substance is far from being disclosed. The history of research on the biological properties of selenium covers characteristic stages since 1817, from the moment of its discovery by I. Berzelius as a chemical element [6, 7]. In 1957 the American scientist K. Schwarz proved its anti-necrotic value in a number of animals, the so-called anti-necrotic factor - 3 [8]. Since then, the attitude towards selenium as a purely toxic element shifted to the desire to study its useful biological functions [9, 10]. Thus, in 1973 it was found that the previously well-known anti-peroxide, hemoglobin-protective enzyme glutathione peroxidase (GPX) [11] is a selenium-dependent protein, and its functions as an antioxidant are much broader than had been commonly thought [12, 13]. In 1970–1980 the existence of other selenoproteins was established, and that selenium is localized actually in all cells of the organism [14, 15, 16, 17].

In the 1990s, three selenium-containing enzymes at different levels involved in regulating iodine metabolism were identified [18]. These discoveries stimulated even greater interest in its intracellular regulatory functions. Over 30 selenium-containing proteins have been identified in cells of various organs and tissues encoded by about 25 genes. Specific physiological functions were established in some of these proteins, while many of them had antioxidant properties [19].

At the same time, a unique mechanism of selenoprotein synthesis was discovered with the use of the so-called SESIS mechanism. It contains the 21 obligatory amino acid Se-cysteine (Sec), encoded by the UGA stop codon in the mRNA structure. Selenium is incorporated into selenoproteins via Se cysteinyl tRNA, which in turn is synthesized by transferring the selenium group into selenium-tRNA from selenophosphate. This mechanism is unique in that it is co-translational in that protein synthesis on ribosomes occurs simultaneously with the synthesis of the 21st amino acid (i.e., conversion of serine to Se cysteine) [19, 20, 21, 22, 23, 24].

Farhan Saeed et al. show that there is great potential for selenium to affect the immune system, for example, the antioxidant peroxidase GSH probably protects neutrophils from oxygen radicals that are produced to destroy ingested foreign organisms [25]. Selenium affects both the innate, “maladaptive” and the acquired, “adaptive” immune system. Selenium-deficient lymphocytes are less able to proliferate in response to mitogen, and in macrophages, its deficiency impairs the synthesis of leukotriene B4, which is essential for neutrophil chemotaxis. The humoral system is also affected by selenium deficiency; for example, IgM, IgG, and IgA titers are reduced in rats, and IgG and IgM titers are reduced in humans [2].

Linda Johansson et al. showed that selenocysteine (Sec), the 21st amino acid, exists in nature in all kingdoms of life as the defining element of selenoproteins. Sec is an analog of cysteine (Cys) with a selenium-containing selenium group instead of the sulfur-containing thiol group in Cys. The selenium atom gives Sec completely different properties than Cys. The most obvious difference is the lower pKa of Sec and the fact that Sec is a stronger nucleophile than Cys. Proteins containing Sec are often enzymes that utilize the reactivity of the Sec residue in the catalytic cycle. Therefore, Sec is usually necessary for their catalytic efficiency [26].

Moghadaszadeh B. and Beggs A.H. in their article show an overview of human selenoprotein expression and function and schematically depict the process of Sec codon recognition and Sec insertion requiring several trans-acting factors including tRNA^Sec, Sec-specific elongation factor and SECIS-binding proteins. It has been observed that targeted deletion of the tRNA^Sec Trsp gene leads to an embryonic lethal phenotype in mice [27]. To illustrate the scheme, let us show the above-described processes in Figure 1.

Figure 1.

Selenoproteins and their impact on human health through diverse physiological pathways [27].

Thus, according to the author [27], all animal specific (acting) Se proteins are Se cysteine-containing natural compounds in the active center. In the organic world, selenium is usually in the form of the amino acids selenocysteine (Sec) and selenomethionine (SeMet), which differ in the presence of selenium instead of sulfur. This substitution is predictably related to the fact that selenium is closer to serine than to other chalcogenes in its physical and chemical properties: atomic radius value, electronegativity value and polarizability of the oxidation degree. All these parameters determine the increased nucleophilicity, which provides higher catalytic activity of Se-proteins in relation to their sulfur-containing counterparts. However, despite the obvious advances in this field, there is still no clear understanding of all sides of this mechanism.

The main differences in the chemical properties of selenoproteins and their sulfur analogues are due to a significant difference in the values of the dissociation constants (pKa), which for Sec is 5.1, and for Cys 8.3 [28, 29]. This circumstance makes thiolates (ionized form) less reactive than selenolates.

Kohrle J. reports that in experimental animal models prolonged and severe selenium deficiency leads to necrosis and fibrosis after high iodide loads. Combined iodide and selenium deficiency, such as in central Zaire, is thought to cause a myxedematous form of endemic cretinism. Insufficient selenium intake and diagnostically low serum selenium levels correlate significantly with the development of thyroid carcinoma and other tumors. Although selenium intake controls the expression and translation of selenocysteine-containing proteins, no direct correlation has been found between tissue selenium content and the expression of various thyroid selenoproteins, suggesting that other regulatory factors contribute to or override selenium-dependent expression control, such as in adenoma, carcinoma or autoimmune thyroid disease. Because both micronutrients, iodine and selenium, were leached from the topsoil during and after the ice age in many regions of the world, an adequate supply of these essential compounds must be provided by either a balanced diet or supplements [30].

Gustin C. et al. state that jodine (J) and selenium (Se) are necessary for the synthesis of thyroid hormones. Iodine and selenium interact. Pregnancy increases the mother’s need for iodine [31]. And Mayunga K.C. et al. reported inadequate iodine levels in pregnant Dutch women [32]. Because as there is no enough information about their selenium intake, we examined iodine status and selenium intake in relation to iodine and selenium supplementation during pregnancy. The authors concluded that research on the 21st amino acid, selenocysteine, has progressed over the past 30 years from the intriguing discovery of Sec in a few select proteins to the recognition of Sec as an important component of many living organisms, associated with human disease and translated into an extension of the genetic code. The field of study of proteins naturally containing selenocysteine is growing rapidly, with new selenoproteins being discovered that have yet to be characterized. The ability to produce synthetic selenoproteins should facilitate such research, as well as open up new possibilities for biotechnological techniques based on the unique properties of selenocysteine. They are confident that the biochemistry of selenium-based proteins will form the basis for several future technologies of both fundamental and medical importance.

In experimental animal models, long-term and strong selenium deficiency leads to necrosis and fibrosis after high iodide loads. Combined iodide and selenium deficiency, such as in central Zaire, is thought to cause the myxedematous form of endemic cretinism [33]. The trace element selenium is of essential importance for the synthesis of a set of redox active proteins. Kamil Demircan et al. [34], studied three additional biomarkers of serum selenium status in relation to overall survival and recurrence after diagnosis of primary invasive breast cancer in a large prospective cohort study. They concluded that the prediction of mortality based on all three biomarkers was superior to established tumor characteristics such as histologic grade, number of lymph nodes involved, or tumor size. Se-status assessment at breast cancer diagnosis identifies patients at exceptionally high risk for poor prognosis.

2. The experimental part of the study

2.1 The main differences in the chemical properties of selenium proteins and their sulfur analogues

The main differences in the chemical properties of selenoproteins and their sulfur analogues are caused by a significant difference in the dissociation constants (pKa) values, which is 5.1 for selenocysteine (Sec), and 8.3 for cysteine (Cys) [28, 29]. This circumstance makes thiolates (in ionized form) less reactive than selenolates.

2.1.1 Se-proteins and their functions

Animal (mammalian) Se proteins are commonly divided into 3 categories: [3, 18].

1. True selenium-specific selenoproteins that include Se cysteine in the active center;

2. Proteins that do not include specific selenium; Selenium-binding proteins, such as (SBP 1)

Among the identified 30 specific Se proteins encoded by 25 genes, only a small fraction of them has specific physiological functions [19]. A hierarchy of “sensitivity” of Se protein synthesis to dietary intake of selenium has now been discovered and it is postulated that the hierarchy of mRNA expression is closely related (deterministic) to the importance of this or that selenoprotein in cellular hemostasis [35]. The organ-tissue specificity of selenoprotein distribution, i.e., their localization by tissue principle, exemplified by the glutathione peroxidase family, has also been established. While GPX is present in many cell types, GPX is expressed only in the gastrointestinal tract, GPX in intercellular medium and blood plasma, GPX in the nasopharyngeal epithelium, TRXR3 is localized in testes, iodothyronine deiodinases in thyroid tissues, etc. [36].

The high antioxidant properties of selenium were first established back in the 60-the 70s of the last century. And since the previously well known antioxidant enzyme (GPX) turned out to be a selenium protein, the AO properties of many newly discovered Se proteins were discovered. However, Se proteins were found to have many other important biological properties in addition to their antioxidant properties, such as regulation of thyroid hormone activity, participation in the regulation of non-specific immune response, inhibition of inflammatory, chemotactic, and phagocytic reactions, influence on reproductive functions (male infertility), participation in redox reactions. The authors [36] briefly describe both the function of these selenoproteins and the regulation of their expression depending on Se status and tabulate data for 40 proteins important for understanding the function and significance, effects of dietary selenium, and subcellular localization.

2.1.2 Se accumulating proteins

It turned out that UGA serves as a stop-signal and selenocysteine codon in the genetic code, but there are no computational methods to determine its coding function, which means that most selenoprotein genes are wrong. Gregory V. Krukov et al. identified selenoprotein genes in sequenced mammalian genomes using methods based on determining structures of selenocysteine RNA insertions by coding for UGA codon potential and presence of cysteine-containing homologs. They found that the human selenoproteome consists of 25 selenoproteins [37].

Based on the SECIS method applied to mammalian genomes, the authors identified SECIS candidate elements in the human genome using the SE CIS2.0 program [37]. Structural and thermodynamic features of SECIS elements were analyzed using this program. The candidate elements were about 10 times more selective (for the same specificity) than the original SECISearch version [38]. They then identified human/mouse and human/rat SECIS pairs using the SECISblastn program, which analyzes the evolutionary conservation of mammalian SECIS elements. In addition, they analyzed genomic sequences upstream of SECIS candidate elements using geneid [39], a gene prediction program that identifies open reading frames (ORFs) with high coding potential and containing infra-labeled TGA codons.

By analyzing predicted human selenoprotein genes using MSGS (mammalian selenoprotein gene signature) criteria [37, 40], which test selenoprotein homologs for the presence and conservation of ORFs intraframe TGA codons and SECIS elements, the authors concluded that SelH, SelI, SelO, SelS and SelK mRNA are present in various tissues and cell types. However, GPx6mRNA was found only in embryos and olfactory epithelium, and SelV mRNA expression was limited to the testes, where it was present in the seminal tubules. The authors’ predictions regarding the secondary structure and organization of the protein showed that, like all previously described mammalian selenoproteins, GPx6, SelH, SelO, and SelV are globular proteins. However, SelK and SelS were predicted membrane proteins. They expressed SelK and SelS fusions containing the C terminal tag of green fluorescent protein (GFP) in CV 1 cells and found that the fusion products were indeed on the plasma membrane. Thus, SelK and SelS appeared to be the first known selenoproteins of the plasma membrane.

SBP selenium-binding proteins can be said to be included proteins in which the form of selenium is unknown. Although Se is stably bound, probably through the selenosulfide bond. One of them, SeBP 1 (Se Binding protein), has been intensively studied recently due to its prominent role in tumor growth [41, 42].

2.1.3 Relationship of Se and hemoglobin

The comparative distribution of Se over the two major erythrocyte proteins, HA and Hb, in humans and animals with different selenium metabolism (different sensitivity to Se deficiency) was studied in detail in 80 90 years by such researchers as M.A. Belstein, J.A. Butler, K.D. Thomson, P.D. Wanger and others [43]. They showed the predominance of Se inclusion in human and some primate hemoglobin (90% of all Se in erythrocytes) versus low Se HPC coverage (10%). At the same time, in the erythrocytes of animals sensitive to Se deficiency, such as sheep, rats, hamsters, etc., the proportion of Se included in the HPC is significantly higher than in humans, some primates, etc. These objects in conditions of selenium deficiency signs of sensitivity of selenium deficiency pathologies (liver and kidney necrosis, white muscle disease, exudative diathesis) and have rather high levels of GPX activity in organs and in erythrocytes, and their hemoglobin has a low capacity (0.1 0.2) to absorb selenium. Organisms (guinea pig, human, some primates) sensitivity dependence on selenium deficiency usually also have reduced GPX in the organ activity, and most of the intraerythrocyte selenium is included in the hemoglobin fraction.

Using the example of the inhabitants of Azerbaijan (Baku), we have shown that 3/4 of erythrocyte Se enters the hemoglobin fraction at a ratio of 1 Se atom per 300–1000 Hb molecules. Selenium is incorporated into hemoglobin by sulfur substitution predominantly in cysteine residues at the βCys93 position. Considering that it will affect the electronic environment of proximal histidine, which is in close proximity to heme, one can assume that it will enhance its antioxidant protection [43, 44, 45].

We examined the effects of sodium nitrite and sodium selenite in their joint and single action on the processes of oxidation of hemoglobin (Hb), lipid peroxidation (LPO), the activity of antioxidant (AO) enzymes glutathione peroxidase (GP) and catalase in human red blood cells in-vitro. Nitrite was found to have a significant effect on the oxidative processes in erythrocytes and Hb, while sodium selenite attenuated the development of the nitrite-induced oxidative process in erythrocytes and reduced the formation of methemoglobin (MetHb) by 25–40%. Having a significant effect on the oxidative process in erythrocytes, nitrite does not lead to a marked increase in lipid peroxidation rates in erythrocytes. Under the influence of nitrite, there is a slight change in the activity of AO enzyme GP (up to 20–30%), and the activity of catalase in all cases drops significantly (1.5–2 times). Nitrite in the incubation medium increases the concentrations of membrane oxyhemoglobin and MetHb, while sodium selenite has an inhibitory effect on this process [46, 47, 48].

Based on the fact that in the human body de novo synthesis occurs for a long time (up to 48–72 hours) in the liver and in the ready form comes with the blood stream to the erythrocytes, experiments were conducted to study the oxidative resistance of erythrocytes and hemoglobin to the damaging effects of such environmental factors as high pressure electric field, ozone, UV-radiation [43]. Here it was found that selenium incorporated into hemoglobin during the first 2 hours increases resistance to them without additional contribution of AO selenium-induced synthesis of GPC enzyme. On the other hand, it was shown that under conditions of selenium deficiency (blood of pregnant women, as a natural model of selenium deficiency) hemoglobin is impoverished in selenium, as are red blood cells, which is accompanied by a decrease in the antioxidant properties of Hb and red blood cells.

At the same time, the Hb activity in erythrocytes is weakly altered even in the third trimester of pregnancy. This is further evidence that Hb enzyme activity does not always adequately reflect selenium status [43, 44]. Regarding the effect of selenium on the health of pregnant women, it can be noted that pregnancy pathologies such as threatened termination, intrauterine fetal delay are accompanied by a decrease in selenium levels and Hb activity in serum, erythrocytes with an increase in lipid peroxidation (LPO) of erythrocytes [43, 44]. Selenium deficiency has been found to impair the regulation of nutrient transport through the placenta [49, 50]. In addition, serum selenium levels may serve as a risk marker for hypertension in pregnancy [51]. In addition, we can add that selenium deficiency can affect many health parameters, including the cognitive functions of children in the first few years of life, and also significantly increases the risk of adverse pregnancy development in various infections [52, 53]. The effect of sodium selenite on the development of lipid peroxidation (LPO) was studied. We also studied the accumulation of methemoglobin (MetHb) by selenium, the state of reduced glutathione (GSH) and glutathione peroxidase (GP) activity in isolated erythrocytes in incubation medium containing different final concentrations of sodium selenite (Na₂SeO₃). Low (1 M, 5 M) concentrations of sodium selenite were found to have little effect on glutathione, while at high (50 M and 100 M) concentrations there was a marked depletion of glutathione, and the activity of glutathione, which has glutathione as the main oxidation substrate, was also significantly reduced. Characteristically, high-end concentrations of lead to increased oxidative processes in both hemoglobin and erythrocytes. Conversely, low sodium selenite concentrations lead to a decrease in the accumulation of active thiobarbituric acid (TBA) and MetHb products. It has been suggested that the stimulation of oxidative processes by high concentrations of sodium selenite is associated with the inhibition of the key antioxidant enzyme GP, which is due to the formation of Se [48].

2.1.4 Selenium in gerontology

Aging can be represented as a process of continuous destruction inherent in all objects of animate and inanimate nature, a consequence of the second principle of thermodynamics, and an organism as an open thermodynamic system that dissipates its heat and simultaneously consumes free energy of high-potential light or chemical from outside. The existence and maintenance of complex dissipative structures of living organisms is possible due to the constant flow of energy, as well as the continuous reproduction of genetic information and structures in the process of cell division. Agerelated changes in somatic cells of multicellular organisms are caused by a decrease in proliferative potential and free radical reactions, the main source of which is oxygen reduction performed by mitochondria, microsomes, and NADPH oxidant systems of phagocytes and other specialized cells.

According to V.A. Gusev, the magnitude of the flux of reactive oxygen species is related to the intensity of the basic metabolism. The accumulation of damage in cells and the rate of aging depend on the ratio of reactive oxygen species formation and their deactivation by the enzymatic antioxidant defense system. The reason for the inevitable occurrence, leakage and dissipation of reactive oxygen species during energy conversion in mitochondria is the second law of thermodynamics, which excludes 100% efficiency of such processes. Comparison of specific superoxide dismutase activity in human granulocytes, platelets, erythrocytes and lymphocytes with the ability of these cells to exogenously generate superoxide radicals allowed to trace the relationship of these factors to the lifetime of cells in blood, which varies from 12 hours to several years [54].

A physiological process, similar to pregnancy, associated with the weakening of AR status and activation of free-radical processes is old age. Currently, there are two main hypotheses of the development of old age, one of which is genetic, i.e. programmed, and the second one is based on the acceleration of free-radical processes leading to AR depletion in the organism [35]. This hypothesis was first proposed by Harman D. and is still a priority [55]. Although there is no clear link between these hypotheses, there is strong evidence that free-radical reactions accelerate with age, having a negative impact on physiological processes related to age [56]. AO minerals such as selenium and zinc have been found to be involved in maintaining metabolic homeostasis in older adults.

Their deficiency increases with age, which is probably a significant cause of premature aging [35]. H. Steinbrenner and S. Helmut [57], believe that antioxidant selenium enzymes as well as pro-oxidant effects of selenium compounds on tumor cells are involved in the anticancer effects of selenium. Brigelius-R. Floh́e and M. Matilde [58] argue that collectively, selenium-containing GPx (GPx1, x4&x6) as well as their non-selenium congeners (GPx5, x7&x8) have become key players in important biological contexts far beyond hydroperoxide detoxification. In the pathogenetic mechanisms of aging, LPO activation plays an important role against the background of decreased AR status of the organism, which can be corrected by the use of Se drugs.

Using the example of the inhabitants of Azerbaijan (Baku), we have shown that 3/4 of erythrocyte Se enters the hemoglobin fraction at a ratio of 1 Se atom per 300–1000 Hb molecules. Selenium is incorporated into hemoglobin by sulfur substitution predominantly in cysteine residues at the βCys93 position. Considering that it will affect the electronic environment of proximal histidine, which is in close proximity to heme, one can assume that it will enhance its antioxidant protection.

2.1.5 Selenium and iodine deficiency conditions

In the development of iodine deficiency states, in addition to iodine itself, as it has been discovered relatively recently, in the last 20–25 years, the provision of the trace element selenium to the body is of great importance. This is the main molecular synergist that has key regulatory significance in thyroid gland (TG) functioning. Characteristically, iodine and selenium act at the cellular level in all organs of the body, with amounts and requirements of the same order (14 mg (Se) and 20–35 mg (J)), and daily intake is (60–120 mg Se and 150–250 mg J [59, 60]). It turned out that many patients have a clear selenium deficiency along with iodine deficiency, indicating that iodine deficiency conditions (including goiter) cannot be cured by iodine supplementation alone. It has been experimentally proven that even under conditions of normal iodine intake, selenium deficiency leads to necrosis and thyroid fibrosis [61]. The importance of not only iodine, but also selenium in the treatment and prevention of thyroid diseases is recognized by all leading specialists, and the study of this problem is urgent [62].

It is now established that selenium is involved in the metabolism of thyroid hormones because it is a component of deiodinases, a family of selenoenzymes including selenocysteine and 5′-iodothyronine involved in the transformation (conversion) of T 4 to TK, performing deiodination of the outer ring of T 4. Deiodinases belong to the family of selenoenzymes that include selenocysteine. One of the important enzymes responsible for the conversion of thyroxine to 3, 5, 3^′triiodothyronine, 5^′ iodothyronine deiodinase type 1 (D1) [18, 63], was first shown to be a selenoenzyme in 1990–1991. The findings explained why the conversion of T 4 to TK was reduced in the selenium-deficient experiment, leading to the development of hypothyroidism symptoms. Many studies have focused on deiodinase type 2 (D2). In humans, plasma T 3 is formed in the thyroid gland (20%) and by peripheral deiodination (80%).

Accordingly, the role of D1 and D2 in the formation of circulating T 3 remains unknown, but there is speculation that D2 may play a greater role in this process. Deiodinase type 3 (D3) catalyzes the conversion of T 4 and T 3 to inactive metabolites [64]. It is expressed in high concentration in the placenta and regulates the concentration of circulating fetal thyroid hormones throughout gestation. The action of selenium-dependent deiodinases in tissues is under the control of the selenium diet and is realized with the participation of thyrotropic hormone [65, 66]. The effect of both isolated selenium deficiency and selenium deficiency combined with iodine deficiency on the human body is of interest to researchers, since pronounced combined deficiency of these elements is a problem in many regions of Central Africa (Congo, Zaire, Sudan), Tibet and some European countries [62].

Of particular interest is the fact that during pregnancy iodine deficiency often leads to the development of thyroid diseases, mainly due to the doubled need for iodine and other important elements, primarily selenium, the lack of which in addition to its direct effect on iodine metabolism and thyroid hormones contributes to other dangerous pathologies, including infant mortality syndrome [67]. It should be added that all over the world due to deteriorating environmental conditions (heavy metals, acid rain, intensive chemicalization of agriculture, etc.) the content of mobile forms of selenium in soils is constantly decreasing, which is reflected in the selenium status in the human body. The role of selenium in the development of iodine deficiency states is not fully understood, and data on the relationship between selenium deficiency in food and preservation of thyroid function require further study [62].

Taking into consideration that deficiency of iodine and selenium in living organisms increases the risk of thyroid gland diseases, malignant neoplasms, cardiovascular pathology, and other serious diseases, the issue of provision of an organism with these microelements is actually all over the world, including CIS countries. This problem is also extremely important for Azerbaijan. It is noted that the microelement selenium is closely connected with iodine metabolism in organisms that is of key importance for thyroid gland functioning. The importance of not only iodine but also selenium in the treatment and prevention of thyroid diseases is recognized by all leading world experts studying this problem. In this connection it is necessary to further study in detail the joint functioning of these elements in organisms, consider the development of a new state strategy for the liquidation of iodine deficiency in Azerbaijan, and possible revision of current salt iodization program in favor of the medicinal prophylaxis with iodine-containing oil capsules with additional use of selenium preparations and continuous monitoring of iodine supply, use the existing positive experience of the international organization “World Doctors” (1998–2004) [62].

2.1.6 Se and immunity

A number of micronutrients, including Se, are known to be important in maintaining a “proper” immune response. Selenium is essential for the efficient formation and functioning of virtually all components of the immune system, including the major immune cells: neutrophils and macrophages, NK (natural cell) killers, T lymphocytes and B lymphocytes [68, 69]. In particular, it is well known that high Se levels in the body stimulate the proliferation and differentiation of CD4 + T helper cells (Th) [70]. Selenium is also important for the cytotoxicity function of CD8 + T cells and NK cells Se levels have a significant impact on innate immunity function, in particular macrophage activity depends on selenium levels for their signaling and antigenic abilities [69]. Added to this is the fact that selenium is actively involved in regulating the activity of such interleukins as IL 1, IL 6, IL 10, TNF through the coordination of the nuclear transcription factor NF kB, which is inhibited by selenium. At the same time, the expression of such inflammatory cytokines as IL 2, IL 8, and IL 18 is stimulated [71].

T cells have an increased sensitivity to oxidative stress, and when deficient in selenium proteins, T cells cannot proliferate in response to stimulation of T cell receptors due to loss of generation of reactive oxygen species and nitrogen [69, 70].

To date, the Se proteins involved in the formation of the immune response have been most fully characterized: the GPX, TXNRD, and DIO families and proteins such as MSRBI, SPS2 [69].

Analysis of the available data suggests the effect of selenium deficiency on innate and adaptive immunity. However, selenium supplementation does not always produce positive results. This is particularly evident in the case of tumor growth, where there are no clear positive results on the use of selenium supplementation for cancer control [72].

We will not address this topic in detail in our review, but we will note the main points. Back in the 60’s and 70’s a Canadian researcher R. Schamberger noted that in biogeochemical provinces rich in selenium the incidence of cancer was much lower than in selenium-poor regions [73]. This work initiated a broad study of the role of selenium in tumor growth. In the 70s on the initiative of Prof. G.B.Abdullaev our laboratory staff began to study migration of endogenous and exogenous selenium in the rat organism - Giren carcinoma, Wakor carcisarcoma, M1 sarcoma. A sitadic character of selenium accumulation in these tumors was shown (exchange of selenium between the tumor and rat organs and tissues), i.e. affinity of selenium accumulation in malignant tumors was established, which suggests that tumors need selenium as an antioxidant for their development [74]. Established on experimental animals inhibition of tumor growth by a number of selenium compounds stimulated their use as adjuvants in oncology. However, conflicting results were obtained here [72].

We studied the sitadic nature of selenium accumulation in these tumors (exchange of selenium between tumor and rat tissue organs). We found that selenium atoms accumulate affinely in malignant tumors. This suggests that tumors need selenium - as an antioxidant - for their development. And a high dose destroys them. This was reported at the 1st Scientific Conference “Selenium in Biology” in Baku, 1974 [74].

In this regard, some researchers have tried to use already toxic doses of selenium compounds to apply them as proxidants, which can penetrate into tumors as toxicants and thus inhibit tumor development. In some cases, positive results are achieved on esperiments, but this is not universal. Therefore, manipulations of individual selenoproteins at sub-toxic doses may be useful to study the immune system and to identify the molecular mechanisms of selenoprotein regulation of immunity. These mechanisms should include pro-oxidative and proteomic activities that provide suppression of cancer development (apoptosis, necrosis, paranthosis) [72].

2.1.7 Selenium as an antitoxicant in nitrite poisoning

One of the main targets of the toxic effects of nitrites is hemoglobin, which has an increased oxidative affinity (formation of methemoglobin and other oxidative derivatives) for nitrites [75]. There is extensive data on the use of antioxidants of different nature to attenuate nitrite toxicity, including through the break-down of nitrite metabolites (peroxynitrite, etc.). In particular, there is data on the AO action of selenium-containing substances: Se-proteins and Se-amino acids or other selenium compounds (usually acting similarly to SH-containing compounds, but with a greater efficiency) [76, 77]. There is evidence that some selenoproteins can catalyze the breakdown of ONOO (an aggressive radical capable of oxidizing cellular structures) with a high 2nd order final reaction rate. It has been suggested that hP x acts as a peroxynitrireductase, reducing ONOO and protecting hemoglobin from oxidation and nitrification [78].

There are several indications in the literature that sodium selenite is readily incorporated into erythrocytes (selenium pump), where it undergoes complex metabolism, interacting with hemoglobin, affecting its properties, with subsequent release from erythrocytes into plasma as part of various albumin [79, 80]. Thus, selenium incorporated into erythrocytes as an active intermediate can affect oxidative processes induced by nitrites or their metabolites. The transfer of selenium from erythrocytes into plasma is carried out through the membrane anion exchanger AE1 through a complex interaction of membrane SH-proteins including transported selenium, interaction with plasma albumin. NO in vitro/in vivo is formed through the inherent nitrite reductase activity of hemoglobin according to the scheme: Hb + NO²⁻MetHb + NO + H₂O [81].

On the other hand, NO, as the main metabolite of the NO²⁻ ion in vitro and in vivo, interacts with hemoglobin in the same complex way, binding directly to heme (nitrosyl hemoglobin HbNO) or including in the SH group of α or β peptide chains (nitrosohemoglobin SNHb) as NO⁺ nitrosonium cations [48, 75]. Of particular interest is the incorporation of NO into the β chain of hemoglobin at the βCys93 position, which has important physiological significance for its vasodilator function. This circumstance is also interesting because selenium from sodium selenite, i.e., selenium replacing sulfur in the β − chain of cysteine, is also included in this position. In other words, selenium, along with NO, is included in the same site of the hemoglobin β chain (βCys93) [79, 80].

At the same time, the frequency of selenium presence in Hb for humans in norm according to one data is 1: 225 [80], according to other data Se: Hb1: 300 [43, 44]. Normally, the frequency of NO inclusion in Hb is NO: Hb 1: 1000 (but in extreme cases may reach 1: 100), i.e., the number of inclusions in β chain is normally higher for selenium than for NO, and the inclusion of NO directly in β chain is even lower (40%) [82].

When dietary conditions change (nitrite poisoning or nitrogen deficiency) of both nitrite and selenium (excess or deficiency in the diet), the NO: Hb1: 1000 and Se: Hb 1: 300 ratio may change significantly, especially for nitric oxide due to the extensive use of nitrate/nitrite in agricultural production and food industries. In this case, excess NO can stimulate oxidative stress as one manifestation of nitrite toxicity. Thus, inclusion of selenium at the same site (βCys93) may create competition for NO and thereby reduce the oxidative burden on hemoglobin, in addition to the action of GPx as a natural defender against oxidation.

Moreover, relatively recently, it was shown using transgenic mice that the amino acid residue β 93sus itself confers certain AR properties on erythrocytes during hydrogen peroxide stimulation of the ferric forms of hemoglobin [83]. Earlier, a similar idea was put forward by Mansouri [84] when studying the sodium-dependent oxidation of hemoglobin, that βCys93 has a protective AR function for hemoglobin. As for selenium, we previously showed that a 2-hour incubation of human erythrocytes with sodium selenite (Na₂SO₃) leads to a doubling of the selenium content in the hemoglobin fraction, increasing the AR properties of both hemoglobin and erythrocytes (LPO reduction). The authors explain this by the lower electronegativity of selenium atoms in relation to the sulfur atoms they replace [48].

The question of how such low NO inclusions in hemoglobin can exert significant physiological effects remains to be fully elucidated, despite impressive achievements in this field (recognition of NO as a gas molecule, etc.). To a certain extent, this also applies to selenium, whose content in hemoglobin is comparable to NO, but its physiological role, in addition to that of AR, has not been elucidated. And the fact that an essential part of NO in hemoglobin is at the same site together with selenium suggests a close interaction of these two ligands in comparable proportions. Which makes it interesting to study this issue.

2.1.8 Selenium regulation of oxidative processes in blood of rats induced by sodium nitrite

The role of selenium in moderate doses of sodium nitrite on rat erythrocytes was studied in vivo. Rats were exposed to single and combined Na₂SeO₃ [0.5 mg/kg] and NaNO₂ [30 mg/kg] by intraperitoneal injections and subsequent exposures with periods of 1, 2, 3, and 12, 48 h. Administration of sodium nitrite with exposures at 1 and 3 h in rats resulted in a marked accumulation of MetHb and already by 1 h reached 30%, which during the following 2–3 h monotonically decreased to 30% of the maximum level reached. By 12 and 48 h of exposure, the level of MetHb was little or no different from the control, respectively. Under the action of nitrite in the erythrocyte suspension was found to decrease (by 30% of control) the content of products reacting with thiobarbituric acid (TBA). A single injection of sodium selenite did not lead to changes in MetHb and lipid peroxidation (LPO). At short-term exposure (1–3 h), combined administration of selenite and sodium nitrite resulted in a decrease in nitrite-induced accumulation of MetHb by 35% and an increase in accumulation of LPO products compared with the single nitrite action. At the same time, the order of administration had no effect on the final result.

At prolonged exposure, preinjected selenite at 48 h followed by nitrite [with 1 h incubation] led to a decrease in nitrite-induced MetHb accumulation by 16 and 41% of LPO values, whereas selenite injected 1 h after nitrite [48 h exposure] had no effect on MetHb accumulation and slightly (10%) reduced LPO values. Changes in the activity of antioxidant enzymes, glutathione peroxidase, and catalase, were examined. The activity of catalase decreased in all variants of exposure to sodium nitrite. Selenite did not lead to a significant increase in the activity of GPX under short-term exposure, while nitrite led to its inhibition. Exposure to selenite combined with nitrite had little effect on the NaNO₂-induced decrease in GP activity. The decrease in nitrite-induced accumulation of MetHb, when sodium selenite is administered during the first 1–3 h, is probably more related to the very fact of selenium inclusion in the Hb molecule than to the effect of additional contribution of GP, whose activity is not significantly increased during this period of exposure. Based on the position of the spectral maxima for HbO₂ and doxHb, we note that NaNO₂ increases MetHb by reducing HbO₂, and selenite inhibits this effect [47].

2.1.9 Se and Covi̇d-19

The discovery of a significant role of selenium deficiency in COVID-19 development has led to increased interest in the question of selenium-virus interactions. To date, there are many studies on this topic, a huge amount of clinical material has been accumulated, but a number of unresolved questions remain.

Here we will touch upon only some of the issues in the interaction of selenium with viruses in humans [85, 86, 87]. The mechanism of selenium antiviral action is multifaceted and covers a number of stages of viral infection, from virus invasion into healthy cells to fighting its consequences. Below is a brief list of the beneficial properties of selenium sodium selenite (the main inorganic selenium compound used in biology and medicine) in the treatment of viral infections, using HIV and Ebola as examples [85, 86, 87]. Sodium selenite (Na₂SO₃) can act as a contact interrupter between virions (SARS CoV 1, SARS CoV 2) and the membrane apparatus of healthy cells (host). Specifically, the SARS CoV 2 virion itself consists of a hydrophobic envelope with protein spikes on the outside and a carrier of its genome, mRNA, on the inside.

The proteins of these spikes interact with the membrane apparatus of the “host” cells, i.e. the organism attacked by the virus, mainly through the membrane integral cell protein, the angiotensin-converting enzyme ACE2 (angiotensin) and with the subsequent disruption of membrane integrity, facilitating the penetration of the virus genetic material into healthy cells. Subsequently, this mRNA is incorporated into the host cell genome, modifying it, after which the virus replicates at the expense of the host cell resources [88, 89]. Thus, interrupting the contact of virus spikes with the membranes of healthy cells by changing the structure of any spike proteins is a preventive measure to suppress the development of infection [90]. This hypothesis is presented in detail in the work of M. Kieliszek and B. Lipinski [91].

Sodium selenite (Na₂SO₃), being a small and non-polar molecule, easily passes through cell membranes by passive transport, has an active intracellular metabolism of selenium, which is accompanied by oxidation of intracellular sulfur-containing proteins with simultaneous reduction of selenite (+4) to selenide (2). Taking into consideration that selenium and sulfur are quite similar in their chemical properties, it can be supposed that when entering the body as a chemically more active element, selenium will replace sulfur in sulfur-containing cysteine (2 amino 3 mercaptopropanoicacid) or when interacting with SH-groups of proteins it takes away the hydrogen atom from thiols, thereby oxidizing them, forming R S S R and R S Se S R type bonds [92, 93]. In the case of viral infection, sodium selenite will also interact with viral sulfur-containing proteins, including disulfidisomerase (PDI) located in Covid − 19 spikes, deactivating it as an enzyme according to the scheme:

$$PDI-{\left(SH\right)}_2+S{e}^{4+}\to PDI-S-S-PDI+S{e}^{2+}$$E1

This means that sodium selenite can contribute to the disruption of contact viral entry into healthy cells [90, 91]. As mentioned above, genomic antisense interactions lead to selenium deficiency, which leads to a decrease in selenium enzyme resources, primarily thyroredoxin reductase, a supplier of protons for the needs of DNA synthesis in healthy cells. This leads to increased consumption of selenium by the body, which is necessary for the synthesis of selenoproteins, both own and “viral”. As a consequence, a selenium deficiency condition occurs, leading to the formation of reactive oxygen species [94], weakened immunity against the background of oxidative stress and decreased antioxidant protection of the body. Sodium selenite is a successful form of selenium in this respect, promoting its rapid penetration into cellular structures and overcoming the blood–brain barrier [95]. This property allows the body to use selenium from sodium selenite to maintain vital selenoprotein levels, protecting it from oxidative stress.

The main arguments for using sodium selenite in adjuvant treatment are as follows: 1. In model experiments, selenium inhibited RNA and DNA polymerase reactions; 2. Inhibited nuclear factor NF kB activity; 3. Regulates immune response, including inflammatory process; 4. it has an anti-aggregation effect by inhibiting the formation of thromboxane [86].

2.2 Preliminary research towards selenium-enriched protein - natural silk fibroin

Bioactive peptides are known for their high tissue affinity, specificity and effectiveness in health promotion. In this sense, fibroin and sericin of natural silk have a special place. Natural silk is a valuable textile raw material of animal origin. It is a product of excretion of silk-producing glands of animals, mainly silkworms (type of arthropods, class of insects). Among them, the most industrially important is the domesticated mulberry silkworm (Bombyx mori L., a mulberry type silkworm), which feeds on mulberry leaves. By the end of V age, the caterpillars reach maturity and curl up into a cocoon that protects the pupa from adverse environmental conditions and silkworm enemies. Maturity occurs when a dense mass of silk, namely the protein fibroin (pure silk thread) and the protein sericin (sticky mass), is formed in the caterpillar silk gland.

If we consider the consumption of silk proteins, fibroin and sericin, from cocoons as bioactive peptides and hydrolysates of food proteins, which are known to be beneficial for human health, then modern silk production should contribute to food production and therefore equally to clothing, food and housing.

Enzymatic hydrolysis is a powerful tool for producing bioactive peptides and hydrolysates from fibroin and sericin. Motoyuki Sumida and Vallaya Sutthikhum [96], based on their experience of studying silk digestion enzyme for over 20 years, summarize current knowledge on bioactive peptides and hydrolysates produced from B. mori L. and wild silkworm fibroin and sericin using proteases, and their potential for human health promotion. They encourage researchers associated with silk proteins - fibroin and sericin - to conduct further comprehensive research on bioactive peptides and hydrolysates of fibroin and sericin derived from domesticated and wild silkworms. As such, these ingredients are expected to become fruitful resources for the well-being of mankind. In keeping with this principle, our results on fibroin enrichment with selenium are also becoming important in this field.

Furthermore, the aqueous solution of silk fibroin is suitable for preparing various silk fibroin films, hydrogels, porous materials, microspheres and the like used in cosmetics, skin care products, tanning lotions, tissue-engineered materials, drug carriers, artificial skin and the like. Since stable aqueous silk fibroin solution can be stored for a long time [97], it is obvious that enriching fibroin with selenium simultaneously increases the intelligence and innovativeness of aqueous fibroin solution.

Ch. Wen et al. [98] note that conventional inorganic Se supplements have drawbacks such as toxicity and low bioavailability. Enriched Se proteins and their hydrolysates show good bioactive properties, mainly including antioxidant activity, immune regulation, neuroprotective activity and inhibition of hyperglycaemia, among others. The authors advise that future studies should focus on the relationship between the metabolism of Se-enriched proteins and the metabolic pathways of selenoregulatory proteins using multiomics technology. In addition, in their opinion, the structure–activity relationship of Se-enriched proteins/hydrolysates from different sources should be comprehensively studied to further elucidate their bioactivity mechanism and test their beneficial properties in vivo. Considering this, as well as the findings of M. Puccinelli et al. [99] that increasing the amount of selenium in plant foods is a good way to increase Se intake in animals and humans, and the advice of the authors [96] above, our results on fibroin selenium enrichment may become important in this field.

2.2.1 Introduction of selenium into the fibroin structure

Selenium was introduced into the fibroin structure using our developed method [100]. Two batches of “Sheki-2” silkworm caterpillars were selected for this purpose. Starting from the fourth instar, the experimental batch was fed a preparation of sodium selenite (Na₂SeO₃); fresh mulberry leaves before feeding were sprayed with 0.1% solution of sodium selenite in distilled water, carefully dried, then caterpillars were fed every 48 hours. The dose of sodium selenite was taken at the rate of 4 mg per kg of live weight of the caterpillars. A control batch of caterpillars was fed with normal mulberry leaves. The temperature, humidity, light and feeding frequency of both batches were the same.

2.2.2 Preparation of pure fibroin

To purify fibroin obtained from silkworm (B. mori) cocoon filaments, we used the well-known sericin dissolution method [101]. Equal volumes of 0.05 M solutions of sodium carbonate Na₂CO₃ and sodium hydrogen carbonate NaHCO₃ were taken and the cocoons freed from their shells were boiled in them for 30 min. This allows fibroin to be separated from sericin. After washing fibroin five times in warm distilled water, the residual sericin in the sample was tested using a biurette reaction as follows: 2 ml of water remaining after the third wash of silk fibroin was added to a double volume of 30% CuSO₄ solution and the mixture was stirred again thoroughly. If sericin is present in the sample, it turns red-purple. Washing was continued until the sericin was completely absent.

The obtained fibroin was dried in a desiccator at 340 K, in glass cups, until constant weight. Fibroin was then extracted for 12 h with ethyl alcohol (20 g fibroin/500 ml 95% ethyl alcohol) to remove the waxes and for 12 h with petroleum ether (20 g fibroin/500 ml petroleum ether) in a Soscelet apparatus (extractor) to remove the fats.

2.2.3 Determination of selenium content in fibroin

The photometric method of selenium determination is one of the most convenient and up to now widely used in analyses of this element. This is primarily due to the availability of analytical equipment and the convenience and simplicity of the method [102]. To determine selenium content in fibroin, we used fluorimetric method adapted for biological samples [103]. Based on the ability of selenium to form in dilute solutions with 2, 3 − diaminonaphthalene a fluorescent complex - diazoselenols with a wide area (λ_max = 520 nm), when excited by UV light with λ_max = 366 nm (Figure 2).

Figure 2.

In dilute solutions with 2,3-diaminonaphthalene, selenium forms fluorescent complexes, diazoselenols, with a wide spectral range, when excited by ultraviolet light.

The sensitivity of the method is 0.002 μg selenium per 1 ml of extract. Selenium content was determined in fibroin, its crystalline part and raw silk. Therefore, mineralization of the samples was carried out first. For this purpose a mass of dry sample (100 mg) was poured with concentrated nitric acid (5–7 ml), incubated for 24 hours in the dark, then 3–4 ml of 30% chloric acid was added. Using a reflux condenser the resulting mixture was heated first on a weak flame for 30 min and then on a strong flame.

A solution of HClO₄ was added from time to time and waited for the appearance of white vapors of perchloric acid until the solution was completely discolored. After cooling down, 10 mL distilled water was added to the mixture and heated again until the perchloric acid vapor appeared. Then the mixture was cooled down again and 2 mL of a 2% Determination of selenium content in fibroin Ethylenediaminetetraaceticacid (EDTA) solution was added. The pH of the solution was then adjusted to 1.0 using 10.0% concentrated hydrochloric acid and 25% ammonia solution. The mixture was stirred and 5 ml of 0.05% solution of 2, 3 diaminonaphthalene (in 0.1 N HCl) was added. The solutions were put on a boiling water bath for 5 min, cooled in the dark for 30–40 min. Then they were poured into a separating funnel with 5 ml of freshly distilled cyclohexane (or hexane) and extracted for 1 min. After separation of the phases the aqueous solution was discarded and the organic phase was poured into a cuvette for measurement. Fluorescence was measured on a sensitive FAS-1 fluorimeter. In each batch of determination a blank test was run through the whole assay cycle and an appropriate correction was introduced into the calculation of the selenium content of the samples. The selenium content of the test samples was calculated by plotting calibration curves.

In each batch of determinations a blank test was carried out throughout the analysis and an appropriate correction was entered into the calculation of the selenium content of the samples. By constructing calibration graphs, the selenium content of the test samples was calculated.

Daily measurements of caterpillar weight have shown that from the age IV, with the exception of the molting period, the weight of each caterpillar increases from 0.2 to 6.0 g. Already from the end of age IV, a difference in the weight of experimental (b) and control (a) caterpillars can be detected (Figure 3), with the former starting to curl one day earlier. This indicates that feeding the caterpillars with sodium selenite increases cellular metabolism and accelerates growth and development [104].

Figure 3.

Changes in mulberry silkworm caterpillar weight as a function of feeding time: A - for control batches; b - for test batches.

The effect of selenium on the growth, development, and productivity of mulberry silkworm has been studied. It is established that the yield of raw silk in experimental cocoons is 2.0–2.5% higher than in control cocoons, the metric number of yarn is better. Thus, to increase cocoon yield and improve the quality of raw silk one may recommend feeding silkworm caterpillars with sodium selenite every 48 hours at the rate of 4 micrograms of sodium selenite per gram of live weight of caterpillars from the 4th instar.

Figure 4 shows the change in selenium content in fibroin depending on the dose of sodium selenite sprayed on mulberry leaves during caterpillar feeding. The figure shows that when the dose of sodium selenite in the feed is increased to 50 μg per caterpillar, the selenium content increases from 0.04 to 0.27 mg per 1 kg of fibroin. Further increases in feed dose do not change the amount of selenium in fibroin. Consequently, Se has a negligible enrichment in fibroin. This indicates that not all the selenium from the feed is transferred to fibroin.

Figure 4.

Dependence of selenium content in fibroin on the dose of sodium selenite received by the silkworm caterpillar in the feed.

When the single dose of sodium selenite is increased above 4 mg per 1 kg live weight, caterpillar poisoning has been observed.

2.2.4 Effect of selenium on some fibroin properties

We found that when selenium is introduced into the structure of fibroin, it either replaces sulfur in the bridges between the subunits of macromolecules or forms additional lateral branching, which leads to a decrease in the rate constant of free radical formation in the matrix under the influence of UV-irradiation. In this case selenium atoms, replacing sulfur in macromolecules or forming additional branching like sulfur, lead to the capture of a great number of migrating electrons, thus reducing the rate of registered free radicals. This seems to explain the resistance of silk to radiation damage [105].

We investigated the effect of selenium on the time and temperature dependence of the strength of a cocoon yarn. It was found that at a constant tensile stress applied to the yarn, the value of the breaking time for the control samples was significantly lower, i.e. the strength of the control samples at a constant mechanical stress was lower than for the experimental samples. Similarly, with the same tensile time for the control specimens, the mechanical stress value is significantly higher, i.e. the control specimens withstand a higher load at a given temperature.

On the basis of the literature (S.B. Ratner, 1990) and the above experimental data on the study of the time and temperature dependence of the cocoon thread strength, as well as the nature of the material studied, it can be concluded that Se entering the fibroin structure changes its molecular and supramolecular structure. This, in turn, leads to a more uniform distribution of mechanical stress along the macromolecular chains, which is reflected in a reduction of the structure-sensitive parameter γ. Ultimately, the strength properties of the cocoon yarn are improved [106].

It is known that branching creates an obstacle for the proper stacking of macromolecules during their crystallization. Therefore, a change in the macro-molecular structure of fibroin when selenium is introduced should also be reflected in its supramolecular structure. Our data show that selenium introduction into fibroin structure decreases the degree of its crystallinity. This can be explained by the fact that Se getting into the fibroin structure forms additional branching of fibroin macromolecules. As a result, the mobility of branched macromolecules and their segments decreases during formation of the crystalline phase. Due to this slowing down, there is not enough time for the folding of the branched macromolecules and the amorphous part of the fibroin microfibrils increases [107].

To determine the nature of the change in fibroin structure following the introduction of selenium, we investigated the thermomechanical [108], deformation characteristics of fibroin [109]. In order to adequately determine the dependence of the number of amorphous sites on the concentration of selenium introduced into the fibroin, we used spin probe method, infrared spectroscopy, X-ray structure and derivatogravimetric analysis. The results are well explained by assuming that the mechanical stresses are unevenly distributed along the macromolecule chains. Selenium atoms, playing the role of a prophylactic antioxidant in fibroin, increase the resistance of the material to the effects of spark discharge. The study of these characteristics of fibroin provides qualitative information about the action of selenium, i.e. it is only indirectly possible to trace changes in the state of the amorphous sites.

It was found for the first time that during twisting of mulberry silkworm cocoon under the influence of jet stretching, caterpillar pressure, peculiarities of silk-screen structure and speed gradient crystallization of fibroin (orientation process) accompanied by formation of two modifications - CEC (crystals with elongated chains) and CFC (crystals with folded chains) occurs. Upon increasing the temperature in the derivatogravimetric chamber, crystallites with elongated fibroin chains begin to break up first, followed by crystallites with folded chains. The depth and width of DTGA minimum in low temperature region corresponding to the destruction (disordering) of EWC is much larger than EWC minimum in high temperature region. In the case of selenium-enriched fibroin, the minimum corresponding to EWC almost disappears. Thus, the introduction of selenium into the fibroin structure decreases the number of SSCs and leads to a preferential increase in the amorphous part of the polymer [110]. Fibroin is known to consist of hydrophobic and hydrophilic amino acid residues and is highly hygroscopic. It therefore quickly absorbs moisture available in the atmosphere and an equilibrium between air humidity and fibroin is established. Moisture ingress into fibroin quickly changes its electrical resistivity ϱ, polarization ε and dielectric constant tgδ, which makes it possible to determine air humidity by measuring R, C and tgδ. Based on these properties of fibroin, we created and patented a humidity sensor based on the selenium-enriched crystalline part of fibroin, which has a fast response and high sensitivity (M.Y. Bakirov et al. [111]). Due to the selenium content, this sensor is more resistant to aggressive environments than other materials and has a low temperature coefficient.