Orel, Орловская область, Россия
Moscow, г. Москва и Московская область, Россия
Introduction. One of the ways to the solve iodine deficiency problem is the addition of iodine to farm animal feed. It allows producing iodized livestock products. Promising sources of organic iodine are iodotyrosine-containing iodized milk proteins. Organic iodine accumulation in organs and tissues has not been sufficiently studied. Study objects and methods. We determined iodotyrosine content in rat blood plasma and in pig muscle tissue. For this purpose, high performance liquid chromatography with mass spectrometric detection and cathodic stripping voltammetry were used. Results and discussion. At the first stage of the study, we examined iodotyrosines in rat blood plasma after a single administration of iodized milk protein or potassium iodide (30 μg I/kg weight) at specific time intervals. A significant increase in the concentration of monoiodotyrosine and diiodotyrosine was recorded 4 and 24 h after the administration. At the second stage, we studied the accumulation of iodotyrosines in the muscle tissue of pigs during their fattening period (104 days). The diet of the control animal group included potassium iodide (0.6 mg I/kg of feed). The experimental groups A and B got iodized milk protein (0.3 and 0.6 mg I/kg of feed, respectively). Monoiodotyrosin content in the muscle tissue of pigs of the experimental groups was 3.0 and 5.2 times higher than that in the control group. Diiodotyrosine content was 4.9 and 8.2 times higher. In the experimental group A, iodine content in muscle tissues was 26% higher than that in the control group, in the experimental group B it was 72% higher. Calculations of iodine intake balance and its accumulation in muscle tissues showed that in animals whose diet included iodized milk protein, the iodine assimilation was much higher (0.70 and 0.53%) than in the control group (0.21%). Conclusion. Iodotyrosines from iodized milk protein are absorbed by the gastrointestinal tract in an unchanged form and accumulate in muscle tissues. The findings give more clear understanding of physiological and biochemical mechanisms of organic iodine absorption in animals.
Iodine, iodotyrosines, plasma, muscle tissue, iodized milk protein, absorption
hormones is enormous, as they take part in various
metabolic processes and influence the tissue growth
and differentiation [1, 2]. Iodine deficiency leads to
morphological and functional changes in the thyroid
gland, to decreased thyroid hormones production and,
as a result, to pathological conditions in humans and
animals [3, 4]. Iodine deficiency has remained a problem
for many countries, including Russia [5–7]. More than
half of the regions in the Russian Federation are iodine
deficient, and 60% of the population suffers from iodine
deficiency [8].
A decisive role in iodine deficiency prevention is
given to the production of iodine-enriched foods of
mass consumption (salt, milk, bread, meat products)
[9–11]. One of the ways to produce iodized livestock
products is addition of iodine to farm animal feed. The
iodine content in milk can reach 500 μg/kg and more, in
chicken eggs – up to 60 μg/egg [15–18]. Considering the
accumulation of significant amounts of iodine in milk
and eggs, the European Food Safety Agency (EFSA) has
set a maximum level of iodine in feed: for dairy cows
and small dairy ruminants – 2 mg/kg, for laying hens –
3 mg/kg of feed dry matter [18].
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The iodine content in meat is directly related to
its content in feed. However, even with significant
concentrations of the trace element in the diet its content
in muscle tissue of animals and poultry is much lower
than 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]. Significant
concentrations of iodine in milk and eggs are explained
by the fact that not only the thyroid gland, but also
exocrine glands, such as salivary, gastric, cervical
uterine, and lacteous glands, can uptake iodine from the
blood [23, 24].
The accumulation of iodine in the muscle tissue
of animals and poultry when entering the body in an
inorganic form is negligible. Iodine in the form of iodide
ion is almost completely absorbed in the gastrointestinal
tract, most part of it is used by the thyroid gland, some is
captured by exocrine glands and leukocytes, and the rest
is excreted from the body [25].
Currently, there is a growing interest in the study of
organic forms of minerals. Iodine in organic molecules
is more stable during feed storage. A number of
studies have found a positive effect of organic iodine
compounds on iodine accumulation in the body and
the productivity of farm animals [26, 27]. He et al.
showed that Laminaria digitate alga in the diet of pigs
led to a 45% increase of iodine content in muscle tissue
compared to the control group whose diet included
iodine in the form of potassium iodide [28]. Promising
sources of organic iodine are iodized milk proteins
containing iodotyrosines. Iodotyrosines of milk proteins
are analogues of natural compounds produced by the
thyroid gland and involved in the metabolism of iodine.
Iodine accumulation in organs and tissues entering as
part of organic compounds has not been sufficiently
studied.
The purpose of the work was to study the mechanism
of absorption and accumulation of iodotyrosines, which
are part of milk iodized protein, in the animal muscle
tissue.
STUDY OBJECTS AND METHODS
The objects of the study were iodized milk protein
(“Chemical Technologies”, Russia) and potassium iodide
(“Iodobrom”, Russia). In iodized milk protein, the matrix
for iodization is whey proteins, unlike in “Iodcasein”,
where the matrix is milk casein.
Iodized milk protein is obtained by the Lublinskij
et al. method [29], which involves mixing protein raw
materials with an aqueous solution of inorganic iodine
and enzyme treatment. A mixture of skimmed fresh
milk and proteins of different natural origin is used as
a protein raw material. In iodized milk protein, iodine is
present in the form of mono- and diiodotyrosines, which
are 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 blood
plasma was studied after a single injection of iodized
milk protein or iodide potassium. The study used Wistar
rats aged 8–10 weeks obtained from a licensed source
(Andreevka branch of Scientific Centre of Biomedical
Technologies, Moscow, Russia). The experiments
were performed in the vivarium of Gorbatov Federal
Research Center of Food Systems of Russian Academy
of Sciences, Moscow. All manipulations with the rodents
were carried out in strict accordance with the protocol of
research and the current regulatory documentationIII [30].
After five-day adaptation the animals were divided into
groups (six animal units in each) and placed in plastic
cages (“Tecniplast”, type IV S) on a fine wood chips
litter. The rats had unlimited access to tap water and
food. We used complete feed (by “Laboratorkorm”).
After the adaptation the animals randomly were
divided into three groups. The scheme of the experiment
is presented in Table 1.
The drug aqueous solutions were injected once
with an intragastric probe. Dosages of drugs were
30 μg iodine/kg of body weight. After the injections, the
animals were subjected to food deprivation for no more
than 24 h.
Four animals from each group were subjected to
euthanasia in a CO2 chamber (VetTech, UK) 1 h, 4 h and
24 h after administration. Blood plasma was taken and
stored at –30°C for future experiments.
Mono- and diiodotyrosine content in rats’ blood
plasma was determined by liquid chromatography
with mass spectrometry detection [31]. Samples
were dried, degreased, and subjected to enzymatic
hydrolysis using Streptomyces griseus proteases. The
extraction and purification of iodotyrosines from the
samples was performed by solid-phase extraction,
followed by derivatization of the extract. Identification
of the analytes was carried out according to the
absolute retention time of chromatographic peaks of
iodthyrosine recorded in the mode of monitoring of
multiple reactions. The iodotyrosine concentration was
determined 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 Number
of animals
Diet
Control 20 Balanced common diet (CD)
Experimental
group A
16 CD + iodized milk protein
(1500 μg/kg body weight)
Experimental
group B
16 CD + potassium iodide
(39 μg/kg body weight)
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The results of the research were processed by
parametric methods of variational statistics using the
Student t-criterion for unrelated groups (P < 0.05) [32].
Arithmetic mean M, mean square deviation m, and the
mean error of arithmetic mean σ were determined to
calculate the reliability of the differences between the
two samples.
At the second stage of the research we studied
iodotyrosines accumulation in muscle tissue of large
white pigs at the age of 4 months. Three groups of
20 animals each were formed. The selection of groups
was carried out on the analogue principle. The duration
of the experiment was 104 days. All the animals were
kept on a balanced diet, fed twice a day. The diet
included wheat, barley, corn, soybeans, peas, wheat
meal, fish forage flour, yeast, as well as minerals and
vitamins. The chemical composition of the feed was
balanced by the main nutrients and depended on the
fattening period. Access to water was free.
The control group received potassium iodide in the
amount of 0.6 mg I/kg of feed, while the experimental
groups A and B received iodized milk protein in the
amounts of 0.3 and 0.6 mg I/kg of feed, respectively. The
scheme of the experiment is presented in Table 2.
During the experiment we recorded average daily
feed consumption, as well as initial and final weight of
animals. At the end of the study, three pigs from each
group were slaughtered and butchered. We calculated
weight before the slaughter, kg; weight of hot carcass,
i.e. weight after slaughter and visceration, kg; carcass
yield, i.e. the ratio of the hot carcass weight to the weight
before slaughter, %; chilled carcass weight (after 24 h
storage 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 determined
by high performance liquid chromatography with mass
spectrometric detection according to [31].
Iodine content was assessed on a TA-Lab
voltammetric analyzer (“Tomanalit”, Russia). The
method includes mineralization of samples and
subsequent analysis of their aqueous solutions with
the help of cathodic stripping voltammetry. During
the mineralization process and subsequent ultraviolet
irradiation of the solution of the mineralized sample
solution all forms of iodine are transformed into iodide
ions. Iodide ions are concentrated on silver modified
or mercury-film electrodes in the form of low-soluble
sludge followed by cathodic reduction of the sludge with
linear change of potential. The resulting cathodic peak at
the potential minus (0.4 ± 0.05) B for the modified silver
electrode and minus (0.3 ± 0.05) B for the mercury film
electrode is an analytical signal. The content of iodide
ions in the solution of the prepared sample is determined
by the method of standard additives of the certified
mixture of iodide ions.
The amount of iodine absorbed from feed (%) was
calculated as a ratio of iodine accumulated in muscles
during fattening to an amount of iodine consumed with
feed. The amount of accumulated iodine in muscle
tissue was determined by subtracting the amount of
iodine in muscles at the beginning of the experiment
from the amount of this trace element at the end.
The initial amount of iodine was estimated based on
the iodine content established at the consumption of
inorganic iodine, taking into consideration the initial
mass of muscle tissue. The latter was calculated based
on the muscle tissue yield determined at the end of the
experiment.
Statistical processing of results was carried out using
the method of dispersion analysis (P < 0.05) [32]. The
data are presented as arithmetic mean M and standard
square deviation m.
RESULTS AND DISCUSSION
The mechanism of inorganic iodine compounds
absorption is studied quite well. Iodine in the form
of iodide ion is absorbed in the stomach and upper
intestine for 30 min. The thyroid gland takes from 5
to 30% of iodine, some part is used by leukocytes and
exocrine glands [25]. Organic iodide is believed to
detach from the organic molecule in the liver and enters
the blood in the form of iodide ion [33]. However, in the
process of presystemic metabolism of iodized amino
acids, in particular iodotyrosines, iodine detachment
may not occur, and they enter the systemic blood flow
unchanged. Absorption of organic selenium in the form
of selenomethionine carries in a similar manner [34, 35].
To define the features of iodotyrosine metabolism
in animals, we determined the concentration of
monoidotyrosine and diiodotyrosine in rats’ blood
plasma. We tested control sample (with no iodide),
experimental sample A (with iodized milk protein), and
experimental sample B (with potassium iodide). The
results of the study are presented in Table 3.
Table 3 shows that the monoiodotyrosine
concentration in the blood plasma of intact animals
(control group) did not change throughout the
experiment. After administration of iodized milk
protein, the concentration of monoiodotyrosine did
not differ from the control group in 1 h, but was
Table 2 Experiment scheme (stage II)
Groups Diet Monoidotyrosine
content,
mg/kg feed
Diiodtyrosine
content,
mg/kg feed
Control CD + potassium
iodide (0.6 mg I/kg)
– –
Experimental
group A
CD + iodized milk
protein (0.3 mg I/kg)
0.2 0.097
Experimental
group B
CD + iodized milk
protein (0.6 mg I/kg)
0.4 0.194
CD is balanced common diet
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significantly higher 4 and 24 h later (P < 0.05). In 4 h
and 24 h monoiodotyrosine content in the sample A
exceeded that in the control sample by 8 and 3.6 times,
respectively.
In the sample B, a 35% increase in monoiodotyrosine
concentration was recorded only after 24 h (P < 0.05).
This can be explained by a more active synthesis of
thyroid hormones after an increased iodine intake into
the thyroid gland. Iodized tyrosines are formed in the
gland during thyroglobulin proteolysis and can flow into
the bloodstream along with hormones.
The content of diiodotyrosine and monoiodotyrosine
in the control sample was at the same level during the
experiment (Table 3). 4 and 24 h after administration of
iodized milk protein, the concentration of diiodotyrosine
in rats of the experimental group A exceeded that
ibn rats of the control group by 6.8 and 3.9 times,
respectively (P < 0.05). Introduction of potassium iodide
did not cause an increase in diiodotyrosine content in
rats of the experimental group B.
According to the finding, iodized milk protein
increased significantly iodotyrosine content in rats’
blood plasma. Concentrations of monoiodotyrosine and
diiodotyrosine were maximal 4 h after administration
of iodized milk protein. This is probably due to the fact
that milk whey protein digestion takes 2–3 h. In the
later period, the concentration of amino acids in the
blood reaches maximum, and then it decreases, which is
confirmed by our findings. It is also should be noticed
that the content of monoiodotyrosine in blood was twice
as much as that of diiodotyrosine. Such ratio of iodized
amino acids consists with their content in iodized
milk protein.
Thus, an increased concentration of iodized amino
acids in rat blood plasma after taking iodized milk
protein may indicate that monoiodotyrosine and
diiodotyrosine are able to enter into systemic blood
stream unchanged, without being deodized in the liver
during presystemic metabolism.
Getting into the systemic blood stream, amino acids
begin to be distributed to various organs and tissues of
the body. At the next stage, we studied the accumulation
of iodtyrosines and the degree of iodine absorption in
the muscular tissue of the pigs.
We did not find statistically significant differences
between the control and experimental groups (Table 4).
The results of the research showed that iodine in
organic form in the diet of pigs did not have a significant
impact on the slaughter parameters of the animals
(Table 5).
The yield of carcasses was the same in pigs of
the control and experimental groups and amounted
to 70%. The content of muscle tissue in pig carcages
varied according to the iodine-containing supplement
consumed. Muscle tissue yield of the animals in the
experimental groups exceeded that in the control by
0.45% (P < 0.05), which indicates the positive effect of
iodized milk protein on this parameter.
Table 3 Iodotyrosine concentration in rat blood plasma,
ng/mL (n = 4)
Time, h
Groups
Control Experimental
group A (iodized
milk protein)
Experimental
group B
(potassium iodide)
M m σ M m σ M m σ
Monoiodthyrosine concentration
0 0.093 0.01 0.01 – –
1 0.098 0.01 0.01 0.11 0.01 0 0.078 0.01 0.01
4 0.08 0.02 0.01 0.72* 0.07 0.04 0.1 0.02 0.01
24 0.085 0.01 0.01 0.31* 0.03 0.02 0.115* 0.01 0.01
Diiodotyrosine concentration
0 0.05 0.01 0 – –
1 0.048 0.01 0.01 0.043 0.01 0.01 0.035 0.01 0.01
4 0.04 0.02 0.01 0.27* 0.03 0.02 0.04 0.01 0.01
24 0.04 0.01 0 0.155* 0.01 0.01 0.05 0.01 0
* statistically significant differences (P < 0.05) from the indicator
of the animals in the control group
Table 4 Performance parameters of test pigs (M ± m, n = 20)
Indicator Groups
Control Experimental
group A
Experimental
group B
Initial body
weight, kg
47.55 ± 7.52 49.65 ± 7.77 48.95 ± 8.98
Final body
weight, kg
113.25 ± 6.63 118.25 ± 7.41 115.59 ± 8.07
Average
daily weight
gain, g/day
631.69 ± 19.96 659.60 ± 18.27 640.81 ± 35.37
Average
daily feed
intake,
kg/day
2.3 ± 0.19 2.3 ± 0.17 2.3 ± 0.12
Table 5 Slaughter parameters and muscle yield of test pigs
(M ± m, n = 3)
Parameter Groups
Control Experimental
group A
Experimental
group B
Pre-slaughter
weight, kg
110.09 ± 3.10 111.68 ± 2.79 110.21 ± 3.52
Hot carcass
weight, kg
77.09 ± 2.07 78.18 ± 2.00 77.12 ± 2.50
Chilled carcass
weight, kg
75.76 ± 2.07 76.87 ± 2.00 75.79 ± 2.49
Muscle tissue
weight, kg
63.49 ± 1.82 64.76 ± 1.62 63.85 ± 1.95
Muscle tissue
yield, %
83.80 ± 0.11 84.25 ± 0.12* 84.25 ± 0.21*
* statistically significant differences (P < 0.05) from the indicator
of the animals in the control group
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When determining iodotyrosines in the muscle
tissue of pigs (Fig. 1), it was found that contents of
monoidotyrosine and diiodotyrosine in the animals
from the experimental groups were significantly higher
than those from the control group. The content of
monoiodotyrosine in the muscle tissue of pigs from the
experimental groups A and B was by 3.0 and 5.2 times,
and diiodotyrosine – by 4.9 and 8.2 times higher than
in the control group, respectively. In the experimental
groups, with the increase in the content of iodtyrosines
in feed, their concentration in meat also increased, but
not directly proportional to the increase in the amount
of iodized amino acids consumed. The results recorded
during this stage were obtained for the first time. For
the last five years there have been no available data
confirming or refuting our findings.
The content of total iodine in animals of the
experimental groups was higher compared to that in pigs
from 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 in
the control group, the concentration of iodine in muscles
was 26% higher. In the experimental group B, with the
equal content of iodine in the feed, the concentration
of iodine in muscle tissue was 72% higher than in the
control group.
Calculations of iodine intake balance and its
accumulation in muscle tissue showed that in animals
receiving iodine in the form of iodized milk protein, the
degree of iodine absorption was much higher than that in
the control group. According to Franke et al., inorganic
iodine absorption in the muscle/fat fraction is not more
than 0.24%, which corresponds to our result recorded
in the control group [36]. Besides, the researchers
found a tendency of iodine absorption decreasing with
an increase in its content in feed. This pattern was
also found in our study. In the experimental group A,
where iodine content in the diet was twice less, iodine
absorption was higher than that in the experimental
group B.
The data obtained confirm the assumptions of some
authors about better absorption and more intensive
accumulation of organic iodine compounds in animals.
For example, Banoch et al. observed a similar effect
when adding iodine-rich algae Chlorella spp. compared
to potassium iodide. A noticeable effect of iodine
introduced into the feed on the pork quality was not
established [37].
CONCLUSION
The results of the study showed that iodotyrosines
entering the body of animals in the form of iodized milk
protein can be absorbed in the gastrointestinal tract
in an unchanged form without iodine detachment and
can accumulate in the muscle tissue. At the same time,
there was a significant increase in the concentration
of monoiodthyrosine and diiodotyrosine in the blood
plasma of experimental animals. The content of iodized
tyrosines in the muscle tissue of animals whose diet
included iodine in the form of iodized milk protein
significantly exceeded that in animals whose diet
included inorganic iodine. In addition, it should be noted
that the proportion of absorbed iodine from organic
compounds is much higher than the absorption degree of
inorganic iodine.
The findings can provide more clear understanding
about physiological and biochemical mechanisms
of organic iodine absorption in animals.
CONTRIBUTION
Concept and research design, statistical processing,
and editing – L.S. Bolshakova. Collection and material
processing, text writing – L.S. Bolshakova, D.E. Lukin.
CONFLICT OF INTEREST
The authors state that there is no conflict of interest.
ACKNOWLEDGEMENTS
The authors express their gratitude to A.B. Lisitsyn,
Academician of the Russian Academy of Sciences,
Doctor of Technical Sciences, Professor, Laureate
of the State Prize of the Russian Federation, and
I.M. Chernukha, Doctor of Technical Sciences,
Professor, for their methodical assistance with the
research.
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