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Scientific research report
EFFECT OF SYNTHESIT IRON CITRATE ON THE ACTIVITY OF
ENZYMES OF PURINE METABOLISM IN INDIVIDUALS
OF DIFFERENT AGE GROUPS
Report prepared by:
Sergey Zuykov,
PhD Candidate in Biological Sciences
Dmitry Kaplun,
Jr. Scientist, Biology
Introduction
One of the central problems of modern biology is the problem of differentiation,
aging, death and replacement of damaged cells. This problem is related to the study of
ontogenesis of entire body, and it is considered one of the major aspects in modern
biological theories of aging in humans [1]. Recently, scientists of different specialties
have reached a conclusion that the basis of many pathological processes in the body,
leading to various diseases, as well as premature aging, is the same phenomenon. This is
the damage of cell membranes and other structures inside the cell by oxygen free radicals
(OFR) [2;3].
Among numerous theories of aging, the free radical theory of aging (FRTA) is the
key theory. The central tenets of the theory were defined more than 50 years ago. This
theory explains not only the mechanism of aging but a wide range of related processes,
such as cardiovascular diseases (CVD), age-related immunosuppression and brain
dysfunction, cataracts, cancer and other diseases, but also adaptive processes aimed at
correcting age-related metabolic disorders. Currently, this theory is developing
particularly fruitfully and is the leading fundamental theory in gerontology [4].
Age-related changes in any organ of the body are the result of internal processes
and the work of other organs. As the body ages, the number of FR increases, and there is
a higher risk of various age-related diseases, such as atherosclerosis, diabetes, obesity,
CVD, cancer, and etc. With age, incidence rates of these pathologies increase sharply,
most likely due to a decrease in the effectiveness of mechanisms of cell renewal,
metabolic disorders, failure of the regulatory system, the formation of oxidative stress
(OS) caused by stimulation of the formation of FR, as well as a decreased activity
of antioxidant systems. Therefore, aging and the accompanying increase in the level of
FR is one of the fundamental risk factors for the development of most pathologies.
In recent years, nucleotide exchange and FR are frequently mentioned together.
One of the reasons is the connection of nucleotide metabolic enzymes with the formation
of FR. Despite the fact that mitochondria are considered the main system of FR generation
in the cell, and their oxidative damage is recognized as one of the contributing factors
leading to aging and associated diseases [5;6], nevertheless, along with mitochondria,
high FR production is generated by many enzymatic and non-enzymatic reactions with
metalloproteins, one of which includes purine metabolic enzymes [7;8]. Moreover, in
cells devoid of mitochondria, the degradation of purine nucleotides is considered one of
the key processes that generate FR [9;10].
The enzymes, Adenosine deaminase (ADA, EC 3.5.4.4.) and Xanthine oxidase
(XO, EC 1.17.3.2) that participate in the regulation of purine nucleotide catabolic process
may be the markers of cell differentiation, proliferation, growth factors, and also play the
role of enzymatic sources for radical formation [11-13] (fig. 1).
ADA catalyzes the reaction of converting adenosine to inosine, while toxic
ammonia (NH3) is also formed, then inosine is converted into hypoxanthine (xanthine) –
a substrate for the enzyme XO – which is considered a generally recognized generator of
the superoxide anion radical (O2•−) and hydrogen peroxide (H2O2), catalyzing the final
stage of purine breakdown to uric acid.
For biochemical diagnostic methods, the most accessible and significant material
is blood plasma. Previously, it was assumed that the activity of enzymes of purine
metabolism in plasma may reflect their activity in body tissues [14; 15]. However, the
level of enzymatic activity in plasma does not certainly consist only of plasma and tissue
dissociation enzymes. The enzymes of the formed elements of blood are also involved,
and one of which are red blood cells capable of relatively rapid formation. An important
role of erythrocyte dysfunction in the development of hypoxia and increased FR
production should be considered [16].
Fig. 1. Catabolism of Purine Nucleotides
Thus, with aging, the catabolism of purine nucleotides increases followed by an
increased activity of ADA and XO. This is the trigger mechanism for the oxidative
destruction of various protein molecules, playing a key role in the molecular mechanisms
of the development of the OS.
Plus, an increase in the activity of ADA and XO is observed not only with aging,
but also with a number of inflammatory diseases, CVD, atherosclerosis, rheumatism, viral
hepatitis, cirrhosis, cancer.
Research aim
This research aimed to analyse the effect of Synthesit iron citrate on the activity of
key enzymes of purine nucleotides in blood plasma and red blood cells in healthy
individuals of different age groups.
Materials and methods
The present research was conducted in vitro in blood plasma and hemolysate of red
blood cells obtained through double freezing of washed red blood cells. We examined 21
relatively healthy volunteers aged 40 to 80 years (including 15 men and 6 women) who
do not have any oncological pathologies, diabetes mellitus or other serious systemic
pathologies.
The distribution of the volunteers by age groups and statistical processing of the
distribution data for normality is shown in the figure below (fig. 2).
Fig. 2. Age groups of volunteers
As a result of the experiment, 2 individuals (men aged 61 and 62 years) from a 21-
volunteer group did not show any changes in the activity of the studied enzymes under
the impact of Synthesit iron citrate, therefore, for further analysis of statistically
significant results, we analysed a group of 19 people (13 men and 6 women).
To observe the impact of age on the activity of purine metabolic enzymes, as well
as to identify age-related characteristics of changes in the activities of the studied enzymes
under the impact of Synthesit iron citrate, all individuals were divided into two groups
according to their age: the first age group (middle age) was of 9 people aged from 40 to
59 years, and the second group (elderly) of 10 people aged from 60 to 79 years.
Age
Num
ber o
f volu
nteers
Blood plasma extraction method. Whole blood was collected from the ulnar vein
into a test tube with a 3.8% sodium citrate solution (the blood and citrate ratio is 5:1),
centrifuged at 4000 rpm for 15 minutes. Plasma was extracted. The completeness of the
deposition of blood cells was monitored microscopically.
Red blood cell (RBC, erythrocyte) hemolysis [17]. 5 ml of whole blood was
collected from the ulnar vein into a test tube with a 3.8% sodium citrate solution (the
blood and citrate ratio is 5:1), centrifuged at 3000 rpm for 15 minutes (OPN-3 centrifuge,
g=1200). Supernatant fluid was removed, the red blood cells were washed twice with
physiological saline: 1.5 ml of red blood cells were brought to a volume of 4.5 ml with
physiological saline solution three times and centrifuged for 10 minutes at 3000 rpm
(OPN-3 centrifuge, g=1200). The red blood cells washed using this method were
hemolysed with distilled water in a ratio of 1:150. 1 ml of the hemolysate contains 0.0066
million RBCs.
Lowry protein assay. The protein was determined in accordance with the
procedure described by O. H. Lowry [18]. Principle: Lowry’s method is based on
measuring the color intensity of a solution in which a color reaction to a protein (Folin
reaction) with tyrosine and cysteine radicals of a protein molecule is carried out, which
includes the reduction of phosphoric-molybdenum and phosphoric-tungsten acids (Folin-
Ciocalteu reagent) with the formation of a complex compound of blue color.
Reagents:
1. 2% sodium carbonate (Na2CO3) solution in 0.1 n. sodium hydroxide solution;
2. 0.5% solution of copper sulfate in 1% solution of potassium tartrate or sodium;
3. Alkaline copper solution: 50 ml of reagent #1 and 1 ml of reagent #2
(stable for 2 days);
(годен в течение двух суток);
4. Folin-Ciocalteu reagent;
5. Physiological saline solution.
Research methodology
0.98 ml of NaCl saline solution, 0.02 ml of 10-fold diluted blood plasma (or 50-
fold diluted erythrocyte hemolysite) and 2 ml of solution #3 were poured into a test tube.
Then everything was mixed and incubated for 10 minutes in a thermostat at 37°C.
Meanwhile, a control tube was prepared that contained 1 ml of NaCl physiological saline
solution and 2 ml of solution #3. 0.2 ml of Folin-Ciocalteu reagent was added to both test
tubes after incubation, then it was mixed and incubated for 30 minutes at 37°C.
All samples were thoroughly mixed, avoiding the formation of foam, using
photoelectric colorimetry at a wavelength of 670 nm in a 0.5 cm thick cuvette. The protein
content in the sample is determined according to the calibration schedule.
Determination of the activity of the enzyme of purine nucleotide metabolism -
adenosine deaminase (ADA) [19; 20]. ADA is a key catabolic enzyme of adenosine
(deoxyadenosine) metabolism that catalyzes its hydrolytic deamination into inosine
(deoxyinosine) and ammonia.
Adenosine + Н2О → inosine + NH3
Principle: the assay is based on a change in the optical density of the reaction
mixture at a wavelength of 265 nm due to the accumulation the product of adenosine
deamination – inosine that was recorded on a Specord-200 spectrophotometer (hydrogen
lamp).
Reagents:
1) 0,1 М Na- phosphate buffer, рН 7,0;
2) 0,36x10-4 М adenosine dissolved in 0,1 М Na- phosphate buffer.
Determination: the incubation medium contains following components:
1. Adenosine dissolved in Na- phosphate buffer - 0,3 ml;
2. Na- phosphate buffer - 2,7 ml.
The incubation medium was heated in a thermostat at 37°. After that, blood plasma
or erythrocyte hemolysate was added (dilution by 10 times or 50) in the amount of - 0,02
ml.
An incubation medium consisting of an adenosine solution and a buffer was poured
into a spectrophotometer cuvette (1 cm), then an enzyme was added. The initial value of
the optical density was measured at a wavelength of 265 nm using a comparative solution
that contained adenosine, a buffer and a physiological solution of NaCl. Then the sample
was incubated at 37°C for 30 minutes and the optical density was measured a second time.
ADA activity (nmol/(min×mg)) according to the formula:
A = ∆D × 109 / (C × 1000 × t × E)
where ∆D – the difference between the optical density at the 10th minute of
measurement and at zero minutes of measurement, at a wavelength of 265;
С – protein concentration, mg/ml (Lowry protein assay), multiplied by 1000 to
convert into mg/l;
t – incubation time (30 min);
109 – conversion rate from mol/(min×mg) to nmol/(min×mg);
E - molar extinction coefficient (inosine = 12300 L/(mol×cm)).
Determination of the activity of Xanthine oxidase (XO) [21]. XO is a key
catabolic enzyme of purine nucleotide metabolism that catalyzes two consecutive and
final stages: the reaction of oxidation of hypoxanthine to xanthine and then to uric acid:
hypoxanthine + 2О2 + Н2О → xanthine + 2O2∙- + 2Н+
xanthine + 2О2 + Н2О → uric acid + 2O2∙- + 2Н+
Principle: the assay is based on the ability of the enzyme to generate O2•− when
converting hypoxanthine (xanthine) into uric acid, the content of which can be judged by
the rate of reduction of nitroblue tetrazolium into a colored product – formazan that has
a maximum light absorption at a wavelength of 540 nm.
Reagents:
1. 0,05 М Na- phosphate buffer, pH 7,8 with 1 mM EDTA: 6 g sodium dihydrogen
phosphate (NaH2PO4) + 292 mg EDTA dissolve in 500 ml of distilled water
and 0.1 M sodium hydroxide (NaOH) solution bring the pH to 7.8, then dilute
the solution with water to 1L;
2. Substrate mixture: 100 ml of 0.05 M sodium-phosphate buffer + 680 mcg (50
microns, μm) hypoxanthine + 460 mcg (15 microns, μm) of phenazine
methosulfate + 5.71 mg (420 microns, μm) of nitroblue tetrazolium + 140 mg
gelatin. The substrate mixture is prepared before the research or stored frozen.
Determination: 3 ml of the substrate mixture is poured into the cuvette of the
spectrophotometer (the optical path length is 10 mm) and heated for 5 minutes at 37°C.
The reaction is started by adding 0.1 ml of blood plasma (tissue homogenate, erythrocyte
hemolysate, dilution by 10 times or 50 - for RBCs). After that, the growth rate of the
optical density of the sample is recorded for 30 minutes at a wavelength of 540 nm against
an incubation medium of equal volume, where distilled water is added instead of blood
plasma (tissue homogenate, erythrocyte hemolysate).
XO activity (micromole (µmol) /(min×mg)) according to the formula:
A = (∆Е × V.r.m × 106) × r / (C × V.s × 1 × ε × t)
where ∆E – differential sample extinctions before and after incubation;
V.r.m. – reaction mixture volume (3,1 ml);
V.s – sample volume (0,1 ml);
106 – conversion factor from mol to mmol;
r – delusion rate (10 – for blood plasma, 50 – for erythrocyte hemolysate);
1 – optical path length (1 cm);
ε – molar absorptivity of formazan (7200 М-1×cm-1)
t - incubation time (30 min);
С - protein concentration, mg/ml (Lowry protein assay).
To analyse the effect of Synthesit iron citrate on the activity of enzymes of purine
nucleotide metabolism the product was used in powdered form preliminarily diluted in
0.1 M Na-phosphate buffer, pH 7.0 (to determine the activity of ADA) or in 0.05 M
sodium-phosphate buffer, pH 7.8 with 1 mM EDTA (to determine the activity of CO), at
a concentration of 1.5 mg per 10 ml of buffer. For the further research process we used
Synthesit iron citrate at a concentration of 0.0025 mg/ml or 2.5 mcg/ml.
Thus, to study the effect of Synthesit iron citrate effect on the change of ADA
activity, the incubation medium contained the following components: Adenosine
dissolved in Na-phosphate buffer - 0.3 ml; Na-phosphate buffer - 2.65 ml; Dissolved
Synthesit iron citrate in Na-phosphate buffer - 0.05 ml.
To study the effect of Synthesit iron citrate on the change XO activity, the
incubation medium contained the following components: Substrate mixture - 2.95 ml;
Dissolved Synthesit iron citrate in Na-phosphate buffer - 0.05 ml.
The activity of all enzymes was determined by spectrophotometric method.
Statistical data analysis was carried out using the program Statistica 10.0, Statsoft, USA.
To check the distribution for normality, the Shapiro-Wilk W test was performed that
allows to conduct an accurate check even with small sample sizes [22; 23]. The average
values of two samples were compared in the research. To compare independent samples,
in case of a normal distribution law, the Student's t criterion was used, and in case of a
distribution different from the normal law, the Wilcoxon W-criterion was used. The data
is shown in tables, figures and presented in the text in the form of average values (M) and
their standard deviations (σ). When testing statistical hypotheses, the choice of an
adequate comparison criterion was carried out in accordance with the recommendations
of the GCP, ICH Statistical Principles for Clinical Trials, critical values were calculated
at the significance level of p<0.05 [24].
The research was conducted with the consent of volunteers who were previously
acquainted in detail with the research objectives and gave their written, informed consent
to sampling carried out under the direct supervision of a doctor. The study complies with
the ethical principles of clinical trials and the statements of the World Medical
Association Declaration of Helsinki, does not violate the interests of the patient and does
not harm his health.
Research results and discussion
After analysing the activity of ADA and XO data in the group of 40-59 y.o. and
60-79 y.o. volunteers, we observed that the activity of the enzymes of purine metabolism
in the blood plasma of elderly people (60-79 years) was significantly higher than in
middle-aged people (40-59 y.o.) (fig. 2).
Change in ADA activity Change in XO activity
Fig. 2. Change in activity of enzymes of purine metabolism in blood plasma with aging (М±σ).
A similar dynamics was found in red blood cells – in the elderly, the activity of the
studied enzymes was also significantly higher than in middle-aged individuals (fig. 3).
Change in ADA activity Change in XO activity
Fig. 3. Change in activity of enzymes of purine metabolism in RBCs with aging (М±σ).
This corresponds to the results obtained earlier, and also does not contradict the
research reference database regarding the increase of these enzymes in the blood plasma
with aging, as well as diseases associated with aging, such as CVD, atherosclerosis,
neurodegenerative, rheumatoid, oncological diseases and many others [25-27].
Hence, the acceleration of purine metabolism after 60 that is manifested by an
increase in the activity of ADA and XO in blood plasma and red blood cells, contribute
to the increased production of FR, which, according to the feedback mechanism, further
stimulate this metabolism and disrupt the work of various proteins, including those that
are involved in the system of protection against FR leading to their oxidation. Such
proteins include enzymes of antioxidant protection and transfer proteins - lipoproteins,
albumins, globulins, hemoglobin and others [28-31].
After that, we conducted a comparative analysis of the effect of Synthesit iron
citrate on the activity of purine nucleotide catabolism enzymes. The obtained results show
that the addition of Synthesit iron citrate solution with a concentration of 2.5 mcg/ml
affects the work of the key enzymes of the breakdown of purine nucleotides - ADA and
XO, both in the elderly and in middle-aged individuals leading to a decrease in their
activity (Table 1).
Table 1.
Change in the activity of enzymes of purine metabolism in blood plasma and red
blood cells with Synthesit iron citrate of two age groups (М±σ).
Age groups
Research material
Blood
plasma
Blood plasma
+ Synthesit
iron citrate
solution
Erythrocyte
hemolysate
Erythrocyte
hemolysate +
Synthesit iron
citrate solution
ADA, (nmol/min×mg)
40-59 (n=9) 2,13±0,25 1,20±0,21* 9,08±1,90 6,52±2,37*
60-79 (n=10) 2,86±0,26 1,99±0,51* 13,7±0,93 11,3±0,75*
XO, (µmol/min×mg)
40-59 (n=9) 0,220±0,053 0,088±0,023* 5,31±0,51 4,18±0,30*
60-79 (n=10) 0,376±0,062 0,196±0,067* 6,49±0,55 4,91±0,45*
Note: * – values at the significance level of p<0.05
Thus, in the blood plasma of middle-aged people, when Synthesit iron citrate is
added, there is a significant decrease in the activity of ADA by 1.8 times and XO – by
2.5 times. At the same time, in elderly people, the activity of the studied enzymes also
significantly decreases with the presence of a solution of Synthesit iron citrate - by 1.4
times for ADA and by 1.9 times for XO, accordingly (Fig. 4). Consequently, it is clear
from the obtained results that a more pronounced inhibitory effect of Synthesit iron citrate
on the activity of the studied enzymes in blood plasma is manifested in middle-aged
people (40-59 years).
Change in ADA activity Change in XO activity
Fig. 4. Change in the activity of enzymes of purine metabolism in blood plasma with Synthesit
iron citrate in individuals of different age groups (М±σ).
A similar dynamics of change in the activity of purine catabolic enzymes was also
found in red blood cells. In the 40-59 years age group the activity of ADA under the effect
of Synthesit iron citrate decreases by 1.4 times, and XO – by 1.3 times. In the red blood
cells of people aged 60-79 years, the activity of ADA with the presence of Synthesit
solution was 1.2 times lower, and 1.3 times lower for XO (Fig. 5).
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
40-59 лет 60-79 лет
Активность АДА без «Синтезита»Активность АДА при добавлении «Синтезита»
0,000
0,050
0,100
0,150
0,200
0,250
0,300
0,350
0,400
0,450
0,500
40-59 лет 60-79 лет
Активность КО без «Синтезита»Активность КО при добавлении «Синтезита»
0,0
2,0
4,0
6,0
8,0
10,0
12,0
14,0
16,0
18,0
40-59 лет 60-79 лет
Активность АДА без «Синтезита»Активность АДА при добавлении «Синтезита»
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
40-59 лет 60-79 лет
Активность КО без «Синтезита»Активность КО при добавлении «Синтезита»
40-59 years 60-79 years 40-59 years 60-79 years
40-59 years 60-79 years 40-59 years 60-79 years
Change in ADA activity Change in XO activity
Fig. 5. Change in the activity of enzymes of purine metabolism in RBCs with Synthesit iron
citrate in individuals of different age groups (М±σ).
ADA is an enzyme widely distributed in human and animal tissues. At present, the
enzyme from human red blood cells has been most well characterized, its physico-
chemical and kinetic parameters, isoenzyme composition, and amino acid sequence have
been studied. It plays a major role in the development and function of blood cells;
deviations in its activity in blood cells are associated with the development of certain
diseases.
The level of activity of ADA determines the ratio of the concentration of adenosine
and inosine in a cell - an increase in adenosine levels occurs with a decrease in the activity
of ADA.
Adenosine is a regulatory molecule that controls the function of cells of the immune
system, and also the cell function of neuromuscular, secretory and other systems, like
cyclic nucleotides and calcium ions (Са2+) [32-34].
Consequently, the suppression of the activity of ADA caused by the action of
Synthesit iron citrate preserves adenosine for its subsequent effects.
It is known that under normal conditions, Xanthine oxidase (XO) and xanthine
dehydrogenase (XDH) are two interconvertible forms. Xanthine dehydrogenase (XDH)
can reversibly or irreversibly transfer to XO, as a result of the formation of disulfide bonds
of cysteine residues (Cys535 and Cys992) as well as with the participation of sulfhydryl
oxidases [35], or limited proteolysis [36] involving Ca2+ dependent proteases.
The peculiarity of the action of this enzyme is that it functions as a complex in two
ways: the enzyme can work as an oxidase, and as a dehydrogenase. According to the ratio
of the activity of XO and XDH, it is possible to assess the intensity of oxidant and
antioxidant processes.
In the blood plasma, basically the entire enzyme is presented in the oxidase form
as a result of the action of serum proteases [37]. It was found that the depletion of ATP
reserves occurs under hypoxic conditions that leads to a change in the membrane gradient
of Ca2+. An increase in the level of Ca2+ activates Ca2+ - dependent proteases, which
participate in converting XDH into XO, stimulating an increase in the production of FR,
which, according to the feedback principle, further stimulate the activity of the enzyme,
inducing OS [38].
It is an interesting fact that modern scientific data considers the enzymes of purine
catabolism as angiogenic [39] and growth factors [40].
Thus, by inhibiting the XO with Synthesit iron citrate, the level of FR decreases,
normalizing the oxidative potential of blood plasma and formed elements.
Also, we would like to draw attention to the fact that initially, for the research, the
blood plasma and erythrocyte hemolysate of 21 relatively healthy volunteers were used
(see materials and methods). However, as a result of the research, no changes in the
activity of purine metabolic enzymes under the effect of Synthesit iron citrate were
observed in both blood plasma and red blood cells of 2 male volunteers in the group aged
60-79 years. At the same time, the activity of the studied enzymes was significantly higher
in those individuals than in volunteers aged 40-59 years.
In this case, the absence of any effect of Synthesit iron citrate on the activity
indicators of purine metabolic enzymes in these volunteers, whose blood was used during
the research may be related to their individual characteristics of the body. This aspect
requires additional research.
Conclusion
1. In vitro experiment showed that Synthesit iron citrate promotes the reduction in
the activity of key enzymes of the breakdown of purine nucleotides in blood
plasma and in red blood cells. The effect was observed in most of the peripheral
blood samples (90%).
2. More pronounced inhibitory effect of Synthesit iron citrate solution was
observed in a group of middle-aged people (40-59 years). Since catabolic
processes increase with aging, in this case, the breakdown of purine nucleotides
(due to the stimulation of ADA and XO activities), which contributes to an
increase in the level of FR in the body, followed by the further OS – one of the
key causes of aging and age-associated diseases, including CVD, metabolic
(obesity, type 2 diabetes mellitus) and oncological disorders. Therefore, the use
of Synthesit iron citrate can be recommended as a geroprotector, especially for
people who are at risk for age-related pathologies.
3. The decrease in the activity of ADA with Synthesit iron citrate contributes to an
increase in the intracellular and extracellular levels of adenosine. It is known
that cells with a high level of adenosine, under certain conditions, are more
resistant to the oxidative action of FR, contributing to the stimulation of
enzymes and low-molecular-weight antioxidants, such as superoxide dismutase
(SOD), catalase, glutathione peroxidase (GPx) , glutathione reductase (GR) and
glutathione, thereby protecting the cell from OS [41-44].
4. Aging, like most pathologies, is characterized by the presence of hypoxic states,
which is accompanied by an increase in the activity of ADA [45]. Therefore,
the suppression of the activity of this enzyme with Synthesit iron citrate
stimulates the release of adenosine under hypoxic conditions, dilating blood
vessels and promoting better flow, as well as contributing to an increase in the
level of nitrogen monoxide (NO), a strong vasodilator, normalizing vascular
tone, blood circulation, as well as oxygen transport to cells [46-49]. Many cells
involved into the production of adenosine have adenosine receptors embedded
in the plasma membrane. In the cardiovascular system, they are found on the
surface of atrial cardiomyocytes, ventricles and the conducting system of the
heart, in the endothelium and smooth muscle cells of the vessel walls. Thus,
acting through stimulation of the release of adenosine and NO, Synthesit iron
citrate can have antihypoxic and antiadrenergic properties, having a hypotensive
effect, thereby acting as a kind of cardioprotector.
5. Adenosine is able to stimulate an increase in the level of ATP in cells [49; 50],
and an increase in the activity of ADA leads to a decrease in the level of
adenosine. A suppression of ADA activity using Synthesit iron citrate leads to
the regulation of cell bioenergetics, performing control in the need and
consumption of energy.
6. Also, the maintenance of vascular tone, microcirculation and normal oxygen
delivery to cells can be carried out by suppressing the activity of XO with the
use of Synthesit iron citrate. It was found that with aging, XO participates in the
formation of vascular OS, which leads to a decrease in endothelium-dependent
dilatation, by reducing NO [51]. FR generated by XO are involved in the
oxidation of low-density lipoproteins and other proteins, contributing to the
early risk of atherosclerosis, hypertension, heart failure, coronary heart disease,
diabetes, as well as the formation of microthrombosis [52]. At the same time,
FR generated by XO contribute to the disturbance of Ca2+ - ATPase of the
sarcoplasmic reticulum of smooth muscle cells, thereby inhibiting the transport
of Ca2+ that leads to vascular damage in various pathological situations.
7. In erythrocytes, FR generated by purine metabolism enzymes contribute to the
oxidation of cysteine residues of hemoglobin with the formation of cross-
disulfide bonds and aggregation of hemoglobin protomers with the formation of
Heinz bodies. The presence of Heinz bodies affects the plasticity of the
erythrocyte membrane, it loses its ability to deform when red blood cells pass
through the capillaries. This causes a disturbance of the integrity of the
membrane, which leads to hemolysis of red blood cells. Thus, inhibiting the
enzymes of purine metabolism with Synthesit iron citrate, there is a decrease in
the level of FR, as well as, possibly, extracellular accumulation of adenosine,
which is one of the first steps in the protective auto- and paracrine cascade signal
aimed at limiting cell damage in response to adverse conditions [53],
contributing to the preservation of the integrity of blood cells and preventing
the oxidation of hemoglobin in red blood cells and, as a result, increasing
oxygen delivery to cells.
8. Moreover, O2•− generated by XO acts as a precursor for other forms of FR,
which have a more pronounced cytotoxic effect, disrupting the mechanisms of
oxidation and phosphorylation during tissue respiration, the main function of
which is to maintain thermoregulation, metabolic and energy balance in the cell
[54]. A suppression of XO activity using Synthesit iron citrate stimulates tissue
respiration and oxidative phosphorylation, thereby contributing to the
normalization of biological oxidation processes and ATP synthesis.
List of abbreviations
Ca2+ – Calcium ions
H2O2 – Hydrogen peroxide
NO – Nitric oxide
NH3 – Ammonia
O2•− – Superoxide anion radical
ADA – Adenosine deaminase
ATP – Adenosine triphosphate
XDH – Xanthine dehydrogenase
XO – Xanthine oxidase
OS – Oxidative stress
FR – Free radicals
OFR – Oxygen free radicals
CVD – Cardiovascular disease
FRO – Free radical oxidation
RBCs – Red blood cells
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