hormones is enormous, as they take part in variousmetabolic processes and influence the tissue growthand differentiation [1, 2]. Iodine deficiency leads tomorphological and functional changes in the thyroidgland, to decreased thyroid hormones production and,as a result, to pathological conditions in humans andanimals [3, 4]. Iodine deficiency has remained a problemfor many countries, including Russia [5–7]. More thanhalf of the regions in the Russian Federation are iodinedeficient, and 60% of the population suffers from iodinedeficiency [8].A decisive role in iodine deficiency prevention isgiven to the production of iodine-enriched foods ofmass consumption (salt, milk, bread, meat products)[9–11]. One of the ways to produce iodized livestockproducts is addition of iodine to farm animal feed. Theiodine content in milk can reach 500 μg/kg and more, inchicken eggs – up to 60 μg/egg [15–18]. Considering theaccumulation of significant amounts of iodine in milkand eggs, the European Food Safety Agency (EFSA) hasset a maximum level of iodine in feed: for dairy cowsand small dairy ruminants – 2 mg/kg, for laying hens –3 mg/kg of feed dry matter [18].61Bolshakova L.S. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 60–66The iodine content in meat is directly related toits content in feed. However, even with significantconcentrations of the trace element in the diet its contentin muscle tissue of animals and poultry is much lowerthan in milk and eggs [19–21]. According to Flachowsky,the proportion of iodine absorbed from feed is 0.3%for pork and less than 1% for beef compared to 30–40% for milk and 10–20% for eggs [22]. Significantconcentrations of iodine in milk and eggs are explainedby the fact that not only the thyroid gland, but alsoexocrine glands, such as salivary, gastric, cervicaluterine, and lacteous glands, can uptake iodine from theblood [23, 24].The accumulation of iodine in the muscle tissueof animals and poultry when entering the body in aninorganic form is negligible. Iodine in the form of iodideion is almost completely absorbed in the gastrointestinaltract, most part of it is used by the thyroid gland, some iscaptured by exocrine glands and leukocytes, and the restis excreted from the body [25].Currently, there is a growing interest in the study oforganic forms of minerals. Iodine in organic moleculesis more stable during feed storage. A number ofstudies have found a positive effect of organic iodinecompounds on iodine accumulation in the body andthe productivity of farm animals [26, 27]. He et al.showed that Laminaria digitate alga in the diet of pigsled to a 45% increase of iodine content in muscle tissuecompared to the control group whose diet includediodine in the form of potassium iodide [28]. Promisingsources of organic iodine are iodized milk proteinscontaining iodotyrosines. Iodotyrosines of milk proteinsare analogues of natural compounds produced by thethyroid gland and involved in the metabolism of iodine.Iodine accumulation in organs and tissues entering aspart of organic compounds has not been sufficientlystudied.The purpose of the work was to study the mechanismof absorption and accumulation of iodotyrosines, whichare part of milk iodized protein, in the animal muscletissue.STUDY OBJECTS AND METHODSThe objects of the study were iodized milk protein(“Chemical Technologies”, Russia) and potassium iodide(“Iodobrom”, Russia). In iodized milk protein, the matrixfor iodization is whey proteins, unlike in “Iodcasein”,where the matrix is milk casein.Iodized milk protein is obtained by the Lublinskijet al. method [29], which involves mixing protein rawmaterials with an aqueous solution of inorganic iodineand enzyme treatment. A mixture of skimmed freshmilk and proteins of different natural origin is used asa protein raw material. In iodized milk protein, iodine ispresent in the form of mono- and diiodotyrosines, whichare full analogues of natural organic iodine compounds.The content of total iodine in iodized milk protein is 2%,monoiodotyrosine ‒ 1.32%, and diiodotyrosine ‒ 0.64%.The study included two stages. At the first stage,we studied the mechanism of iodotyrosine absorption.For that, mono- and diiodotyrosine content in rat bloodplasma was studied after a single injection of iodizedmilk protein or iodide potassium. The study used Wistarrats aged 8–10 weeks obtained from a licensed source(Andreevka branch of Scientific Centre of BiomedicalTechnologies, Moscow, Russia). The experimentswere performed in the vivarium of Gorbatov FederalResearch Center of Food Systems of Russian Academyof Sciences, Moscow. All manipulations with the rodentswere carried out in strict accordance with the protocol ofresearch and the current regulatory documentationIII [30].After five-day adaptation the animals were divided intogroups (six animal units in each) and placed in plasticcages (“Tecniplast”, type IV S) on a fine wood chipslitter. The rats had unlimited access to tap water andfood. We used complete feed (by “Laboratorkorm”).After the adaptation the animals randomly weredivided into three groups. The scheme of the experimentis presented in Table 1.The drug aqueous solutions were injected oncewith an intragastric probe. Dosages of drugs were30 μg iodine/kg of body weight. After the injections, theanimals were subjected to food deprivation for no morethan 24 h.Four animals from each group were subjected toeuthanasia in a CO2 chamber (VetTech, UK) 1 h, 4 h and24 h after administration. Blood plasma was taken andstored at –30°C for future experiments.Mono- and diiodotyrosine content in rats’ bloodplasma was determined by liquid chromatographywith mass spectrometry detection [31]. Sampleswere dried, degreased, and subjected to enzymatichydrolysis using Streptomyces griseus proteases. Theextraction and purification of iodotyrosines from thesamples was performed by solid-phase extraction,followed by derivatization of the extract. Identificationof the analytes was carried out according to theabsolute retention time of chromatographic peaks ofiodthyrosine recorded in the mode of monitoring ofmultiple reactions. The iodotyrosine concentration wasdetermined based on the area of chromatographic peaks.I State Standard 31886-2012. Principles of Good Laboratory Practice(GLP). Application of the GLP principles to short term studies.Moscow: Standartinform; 2013. 10 р.II State Standard 33044-2014. Principles of good laboratory practice.Moscow: Standartinform; 2015. 12 р.Table 1 Experiment scheme (stage I)Group Numberof animalsDietControl 20 Balanced common diet (CD)Experimentalgroup A16 CD + iodized milk protein(1500 μg/kg body weight)Experimentalgroup B16 CD + potassium iodide(39 μg/kg body weight)62Bolshakova L.S. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 60–66The results of the research were processed byparametric methods of variational statistics using theStudent t-criterion for unrelated groups (P < 0.05) [32].Arithmetic mean M, mean square deviation m, and themean error of arithmetic mean σ were determined tocalculate the reliability of the differences between thetwo samples.At the second stage of the research we studiediodotyrosines accumulation in muscle tissue of largewhite pigs at the age of 4 months. Three groups of20 animals each were formed. The selection of groupswas carried out on the analogue principle. The durationof the experiment was 104 days. All the animals werekept on a balanced diet, fed twice a day. The dietincluded wheat, barley, corn, soybeans, peas, wheatmeal, fish forage flour, yeast, as well as minerals andvitamins. The chemical composition of the feed wasbalanced by the main nutrients and depended on thefattening period. Access to water was free.The control group received potassium iodide in theamount of 0.6 mg I/kg of feed, while the experimentalgroups A and B received iodized milk protein in theamounts of 0.3 and 0.6 mg I/kg of feed, respectively. Thescheme of the experiment is presented in Table 2.During the experiment we recorded average dailyfeed consumption, as well as initial and final weight ofanimals. At the end of the study, three pigs from eachgroup were slaughtered and butchered. We calculatedweight before the slaughter, kg; weight of hot carcass,i.e. weight after slaughter and visceration, kg; carcassyield, i.e. the ratio of the hot carcass weight to the weightbefore slaughter, %; chilled carcass weight (after 24 hstorage at 4 ± 2°C), kg; and mass of muscle tissue, kg.M. longissimus dorsi was sampled for chemical analysis.Iodotyrosine content in muscle tissue was determinedby high performance liquid chromatography with massspectrometric detection according to [31].Iodine content was assessed on a TA-Labvoltammetric analyzer (“Tomanalit”, Russia). Themethod includes mineralization of samples andsubsequent analysis of their aqueous solutions withthe help of cathodic stripping voltammetry. Duringthe mineralization process and subsequent ultravioletirradiation of the solution of the mineralized samplesolution all forms of iodine are transformed into iodideions. Iodide ions are concentrated on silver modifiedor mercury-film electrodes in the form of low-solublesludge followed by cathodic reduction of the sludge withlinear change of potential. The resulting cathodic peak atthe potential minus (0.4 ± 0.05) B for the modified silverelectrode and minus (0.3 ± 0.05) B for the mercury filmelectrode is an analytical signal. The content of iodideions in the solution of the prepared sample is determinedby the method of standard additives of the certifiedmixture of iodide ions.The amount of iodine absorbed from feed (%) wascalculated as a ratio of iodine accumulated in musclesduring fattening to an amount of iodine consumed withfeed. The amount of accumulated iodine in muscletissue was determined by subtracting the amount ofiodine in muscles at the beginning of the experimentfrom the amount of this trace element at the end.The initial amount of iodine was estimated based onthe iodine content established at the consumption ofinorganic iodine, taking into consideration the initialmass of muscle tissue. The latter was calculated basedon the muscle tissue yield determined at the end of theexperiment.Statistical processing of results was carried out usingthe method of dispersion analysis (P < 0.05) [32]. Thedata are presented as arithmetic mean M and standardsquare deviation m.RESULTS AND DISCUSSIONThe mechanism of inorganic iodine compoundsabsorption is studied quite well. Iodine in the formof iodide ion is absorbed in the stomach and upperintestine for 30 min. The thyroid gland takes from 5to 30% of iodine, some part is used by leukocytes andexocrine glands [25]. Organic iodide is believed todetach from the organic molecule in the liver and entersthe blood in the form of iodide ion [33]. However, in theprocess of presystemic metabolism of iodized aminoacids, in particular iodotyrosines, iodine detachmentmay not occur, and they enter the systemic blood flowunchanged. Absorption of organic selenium in the formof selenomethionine carries in a similar manner [34, 35].To define the features of iodotyrosine metabolismin animals, we determined the concentration ofmonoidotyrosine and diiodotyrosine in rats’ bloodplasma. We tested control sample (with no iodide),experimental sample A (with iodized milk protein), andexperimental sample B (with potassium iodide). Theresults of the study are presented in Table 3.Table 3 shows that the monoiodotyrosineconcentration in the blood plasma of intact animals(control group) did not change throughout theexperiment. After administration of iodized milkprotein, the concentration of monoiodotyrosine didnot differ from the control group in 1 h, but wasTable 2 Experiment scheme (stage II)Groups Diet Monoidotyrosinecontent,mg/kg feedDiiodtyrosinecontent,mg/kg feedControl CD + potassiumiodide (0.6 mg I/kg)– –Experimentalgroup ACD + iodized milkprotein (0.3 mg I/kg)0.2 0.097Experimentalgroup BCD + iodized milkprotein (0.6 mg I/kg)0.4 0.194CD is balanced common diet63Bolshakova L.S. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 60–66significantly higher 4 and 24 h later (P < 0.05). In 4 hand 24 h monoiodotyrosine content in the sample Aexceeded that in the control sample by 8 and 3.6 times,respectively.In the sample B, a 35% increase in monoiodotyrosineconcentration was recorded only after 24 h (P < 0.05).This can be explained by a more active synthesis ofthyroid hormones after an increased iodine intake intothe thyroid gland. Iodized tyrosines are formed in thegland during thyroglobulin proteolysis and can flow intothe bloodstream along with hormones.The content of diiodotyrosine and monoiodotyrosinein the control sample was at the same level during theexperiment (Table 3). 4 and 24 h after administration ofiodized milk protein, the concentration of diiodotyrosinein rats of the experimental group A exceeded thatibn rats of the control group by 6.8 and 3.9 times,respectively (P < 0.05). Introduction of potassium iodidedid not cause an increase in diiodotyrosine content inrats of the experimental group B.According to the finding, iodized milk proteinincreased significantly iodotyrosine content in rats’blood plasma. Concentrations of monoiodotyrosine anddiiodotyrosine were maximal 4 h after administrationof iodized milk protein. This is probably due to the factthat milk whey protein digestion takes 2–3 h. In thelater period, the concentration of amino acids in theblood reaches maximum, and then it decreases, which isconfirmed by our findings. It is also should be noticedthat the content of monoiodotyrosine in blood was twiceas much as that of diiodotyrosine. Such ratio of iodizedamino acids consists with their content in iodizedmilk protein.Thus, an increased concentration of iodized aminoacids in rat blood plasma after taking iodized milkprotein may indicate that monoiodotyrosine anddiiodotyrosine are able to enter into systemic bloodstream unchanged, without being deodized in the liverduring presystemic metabolism.Getting into the systemic blood stream, amino acidsbegin to be distributed to various organs and tissues ofthe body. At the next stage, we studied the accumulationof iodtyrosines and the degree of iodine absorption inthe muscular tissue of the pigs.We did not find statistically significant differencesbetween the control and experimental groups (Table 4).The results of the research showed that iodine inorganic form in the diet of pigs did not have a significantimpact on the slaughter parameters of the animals(Table 5).The yield of carcasses was the same in pigs ofthe control and experimental groups and amountedto 70%. The content of muscle tissue in pig carcagesvaried according to the iodine-containing supplementconsumed. Muscle tissue yield of the animals in theexperimental groups exceeded that in the control by0.45% (P < 0.05), which indicates the positive effect ofiodized milk protein on this parameter.Table 3 Iodotyrosine concentration in rat blood plasma,ng/mL (n = 4)Time, hGroupsControl Experimentalgroup A (iodizedmilk protein)Experimentalgroup B(potassium iodide)M m σ M m σ M m σMonoiodthyrosine concentration0 0.093 0.01 0.01 – –1 0.098 0.01 0.01 0.11 0.01 0 0.078 0.01 0.014 0.08 0.02 0.01 0.72* 0.07 0.04 0.1 0.02 0.0124 0.085 0.01 0.01 0.31* 0.03 0.02 0.115* 0.01 0.01Diiodotyrosine concentration0 0.05 0.01 0 – –1 0.048 0.01 0.01 0.043 0.01 0.01 0.035 0.01 0.014 0.04 0.02 0.01 0.27* 0.03 0.02 0.04 0.01 0.0124 0.04 0.01 0 0.155* 0.01 0.01 0.05 0.01 0* statistically significant differences (P < 0.05) from the indicatorof the animals in the control groupTable 4 Performance parameters of test pigs (M ± m, n = 20)Indicator GroupsControl Experimentalgroup AExperimentalgroup BInitial bodyweight, kg47.55 ± 7.52 49.65 ± 7.77 48.95 ± 8.98Final bodyweight, kg113.25 ± 6.63 118.25 ± 7.41 115.59 ± 8.07Averagedaily weightgain, g/day631.69 ± 19.96 659.60 ± 18.27 640.81 ± 35.37Averagedaily feedintake,kg/day2.3 ± 0.19 2.3 ± 0.17 2.3 ± 0.12Table 5 Slaughter parameters and muscle yield of test pigs(M ± m, n = 3)Parameter GroupsControl Experimentalgroup AExperimentalgroup BPre-slaughterweight, kg110.09 ± 3.10 111.68 ± 2.79 110.21 ± 3.52Hot carcassweight, kg77.09 ± 2.07 78.18 ± 2.00 77.12 ± 2.50Chilled carcassweight, kg75.76 ± 2.07 76.87 ± 2.00 75.79 ± 2.49Muscle tissueweight, kg63.49 ± 1.82 64.76 ± 1.62 63.85 ± 1.95Muscle tissueyield, %83.80 ± 0.11 84.25 ± 0.12* 84.25 ± 0.21** statistically significant differences (P < 0.05) from the indicatorof the animals in the control group64Bolshakova L.S. et al. Foods and Raw Materials, 2020, vol. 8, no. 1, pp. 60–66When determining iodotyrosines in the muscletissue of pigs (Fig. 1), it was found that contents ofmonoidotyrosine and diiodotyrosine in the animalsfrom the experimental groups were significantly higherthan those from the control group. The content ofmonoiodotyrosine in the muscle tissue of pigs from theexperimental groups A and B was by 3.0 and 5.2 times,and diiodotyrosine – by 4.9 and 8.2 times higher thanin the control group, respectively. In the experimentalgroups, with the increase in the content of iodtyrosinesin feed, their concentration in meat also increased, butnot directly proportional to the increase in the amountof iodized amino acids consumed. The results recordedduring this stage were obtained for the first time. Forthe last five years there have been no available dataconfirming or refuting our findings.The content of total iodine in animals of theexperimental groups was higher compared to that in pigsfrom the control group (Table 6).At the same time, even in the experimental group A,where iodine content in the diet was twice less than inthe control group, the concentration of iodine in muscleswas 26% higher. In the experimental group B, with theequal content of iodine in the feed, the concentrationof iodine in muscle tissue was 72% higher than in thecontrol group.Calculations of iodine intake balance and itsaccumulation in muscle tissue showed that in animalsreceiving iodine in the form of iodized milk protein, thedegree of iodine absorption was much higher than that inthe control group. According to Franke et al., inorganiciodine absorption in the muscle/fat fraction is not morethan 0.24%, which corresponds to our result recordedin the control group [36]. Besides, the researchersfound a tendency of iodine absorption decreasing withan increase in its content in feed. This pattern wasalso found in our study. In the experimental group A,where iodine content in the diet was twice less, iodineabsorption was higher than that in the experimentalgroup B.The data obtained confirm the assumptions of someauthors about better absorption and more intensiveaccumulation of organic iodine compounds in animals.For example, Banoch et al. observed a similar effectwhen adding iodine-rich algae Chlorella spp. comparedto potassium iodide. A noticeable effect of iodineintroduced into the feed on the pork quality was notestablished [37].CONCLUSIONThe results of the study showed that iodotyrosinesentering the body of animals in the form of iodized milkprotein can be absorbed in the gastrointestinal tractin an unchanged form without iodine detachment andcan accumulate in the muscle tissue. At the same time,there was a significant increase in the concentrationof monoiodthyrosine and diiodotyrosine in the bloodplasma of experimental animals. The content of iodizedtyrosines in the muscle tissue of animals whose dietincluded iodine in the form of iodized milk proteinsignificantly exceeded that in animals whose dietincluded inorganic iodine. In addition, it should be notedthat the proportion of absorbed iodine from organiccompounds is much higher than the absorption degree ofinorganic iodine.The findings can provide more clear understandingabout physiological and biochemical mechanismsof organic iodine absorption in animals.CONTRIBUTIONConcept and research design, statistical processing,and editing – L.S. Bolshakova. Collection and materialprocessing, text writing – L.S. Bolshakova, D.E. Lukin.CONFLICT OF INTERESTThe authors state that there is no conflict of interest.ACKNOWLEDGEMENTSThe authors express their gratitude to A.B. Lisitsyn,Academician of the Russian Academy of Sciences,Doctor of Technical Sciences, Professor, Laureateof the State Prize of the Russian Federation, andI.M. Chernukha, Doctor of Technical Sciences,Professor, for their methodical assistance with theresearch.