Effects of Succinate-Based Antioxidant on in vitro Conversion of Methemoglobin in Oxyhemoglobin

Цель исследования — показать возможность использования антиоксиданта на основе янтарной кислоты для восстановления метгемоглобина до оксигемоглобина в крови in vitro. Материалы и методы. Забор крови в микроветты, содержащие ЭДТА, производили у пяти здоровых доноров при профилактических осмотрах. В кровь in vitro добавляли раствор NaNO2 для получения метгемоглобина (MetHb). В качестве антиоксиданта использовали комплексный препарат, состоящий из следующих активных компонентов: янтарная кислота, инозин, рибофлавин и никотинамид. Измеряли спектр поглощения суспензий эритроцитов с различным содержанием препарата Dι(λι)exper с шагом 1 нм. Методом нелинейной регрессии рассчитывали концентрации производных гемоглобина в суспензиях. Результаты. В экспериментах, когда метгемоглобин взаимодействовал с препаратом, оптическая плотность пиков, характерных для оксигемоглобина, увеличивалась, а спектральный пик метгемоглобина снижался. Чем больше были концентрация препарата и время инкубации, тем эффективнее был процесс восстановления метгемоглобина до оксигемоглобина. Заключение. Экспериментально доказали, что при начальной концентрации метгемоглобина в крови 91—93% добавление препарата снижает его концентрацию до 25—27%. В то же время, благодаря автовосстановлению, концентрация метгемоглобина уменьшается только до 84%. Показанный эффект может иметь практическое применение при критических состояниях, при хранении донорской крови, при проведении гемотрансфузии, при воздействии физико-химических факторов на кровь.


Introduction
Endogenous and exogenous intoxications of various causes may lead to the formation of active forms of oxygen (ROS) in the blood and modify hemoglobin molecules in erythrocytes.At the same time, the oxidation processes Fe 2+ Fe 3+ occurs, and molecules of deoxyhemoglobin (Hb) and oxyhemoglobin ( HbO 2 ) are converted to methemoglobin (MetHb) [1][2][3].In its normal state, the body maintains a biochemical balance, and methemoglobin is always present in small amounts (0.5-1.5%) in human blood.
The imbalance between the amount of ROS formed and the antioxidant defense mechanisms may occur in cells under the influence of various endogenous and exogenous factors: bacteria, viruses, chemical agents, a number of pharmaceuticals, environmental pollution, ionizing radiations, traumas, massive bleeding, etc. [4][5][6].In addition, methemoglobinemia can be caused by an overdose of some drugs (lidocaine, novocaine, antimalarials, etc.) and poisoning with chemicals (aniline dyes, nitrates, chlorobenzene) [7].With increasing of oxidative processes, own antioxidant system can no longer deal with an increased amount of active forms of oxygen and nitrogen and MetHb formation.If MetHb level is higher than the physiological level, the disturbance of gas exchange occurs, because the oxidized heme is not capable to deliver the oxygen to tissues.At the level of MetHb of more than 30%, irreversible changes in vital organs can develop [4].It is known that methylene blue is used to treat methemoglobinemia.However, it is not always effective and has a number of toxic side effects [4,5].
Therefore, the search for drugs that can enhance the effectiveness of the antioxidant system of erythrocytes and prevent the formation of ROS and MetHb is always urgent.
Substances containing natural ingredients are of a special interest.One of such drugs is cytoflavin (STPF «POLISAN», Russian Federation).Cytoflavin as a therapeutic category relates to «a metabolic regulator», pharmacological effects of which is regulated by a combined action of the formulation' ingredients: succinic acid, inosine, nicotinamide, riboflavin mononucleotide sodium [8].Cytoflavin has been shown to serve as a pharmacological agent capabale to aid in enchancing the energy formation, decreasing reactive oxygen species (ROS), activating metabolic processes after ischemia and reoxygenation [9,10].
The aim of the work is to show the feasibility of using of cytoflavin for in vitro reduction of excessive methemoglobin to oxyhemoglobin in blood.

Materials and Methods
The blood samples (150 μl) were withdrawn from five donors into microvettes with EDTA (Sarstedt AG and Co., Germany) during prophylactic screenings.
The study was performed in accordance with the principles of the Declaration of Helsinki and was approved by Ethics Committee of Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology, V. A. Negovsky Scientific Research Institute of General Reanimatology.An informed consent was obtained from each participant prior to the study.
For in vitro oxidation of hemoglobin in blood, we used sodium nitrite NaNO 2 solution.To make the sodium nitrite stock solution, 1.4 g of sodium nitrite NaNO 2 (Sigma-Aldrich Co. LLC) was diluted in 10 ml of phosphate buffer (PBS) pH=7.4 (MP Biomedicals, USA) to form the «solution L».The latter was diluted 10 times to obtain the solution S. Сytoflavin (STPF «POLISAN», Russian Federation) is a complex drug containing the following active ingredients: succinic acid 100 g, inosine 20 g, nicotinamide 10 g, riboflavin sodium phosphate (riboflavin) 2 g, and the following excipients: meglumine 165 g, sodium hydroxide 34 g, water for injections qs up to 1 liter [8].
The absorbancy of solutions was measured by a digital spectrophotometer Unico 2800 (USA).The absorption spectra D l (λ l ) exp of erythrocyte suspension in buffer (PBS pH 7.4) was measured at a wavelength range 500-700 nm with 0.5-1 nm increments.Here λ l stands for the wavelengths of light for which the absorbancy were measured, and l is the number of wavelength.
The Nonlinear Fitting method was used to find the concentrations of hemoglobin derivatives [7,[11][12][13].The empirical spectrum D l (λ l ) exp was approximated by the theoretical curve D l (λ l ) theor which fits the experimental curve in the best way.Under approximation, the light absorption by different hemoglobin derivatives was considered.The effects of Rayleigh light scattering on structures with size D λ (coefficient S) and light scattering on particles with size D λ (coefficient K) were taken into account: Here, the index h stands for hemoglobin derivatives: HbO 2 (oxygenated hemoglobin), Hb (deoxygenated hemoglobin), MetHb (methemoglobin), HbNO (ferrous nitrosyl hemoglobin), MetHbNO 2 , (nitrite bound methemoglobin, MetHbNO (ferric nitrsoyl hemoglobin).L stands for the thickness of a solution layer.
To calculate the concentration of hemoglobin derivatives, the method of curve approximation was used by means of the Origin software (OriginLab, Northampton, MA).By means of Function Builder, the function D l (λ l ) theor (equation ( 1)) was created.In this case, the status of dependent variable was assigned to D l (λ l ) exp .Parameters of individual absorption at different wavelengths ε lh (λ l ) were independent variables [10].Independent concentrations of CHbO2, CHb, CMetHb, CHbNO, CMetHbNO2 , CMetHbNO, and scattering coefficients K and S were considered model parameters.These model parameters have been chosen in a manner where the theoretical curve D l (λ l ) theor describes the experimental data D l (λ l ) exp the best.In the approximation, the condition that C i 0 must be satisfied.был присвоен D l (λ l ) exp .Индивидуальная поглощательная способность при различных длинах волн ε lh (λ l ) являлись независимыми переменными [10]

Экспериментальные исследования
We carried out experiments in vitro according the following scheme (Fig. 1).
Microvettes with fresh whole blood (150 μl) were prepared (stage 1). 10 μl of NaNO 2 solutions with the concentration С= 0.14 g / ml were added to each microvette with whole blood (stage 2).Therefore, in microvettes, the concentration of NaNO 2 was 100 mM.The suspensions with this concentration we called NaNO 2 (L).In addition, we studied the effects of NaNO 2 with a concentration of 10 mM (NaNO 2 (S)).Within 10 min of incubation, MetHb was formed, and blood turned brown.NaNO 2 solution was not added to the reference microvettes (reference sample).In the 3d stage, the suspensions were washed from NaNO 2 .For this, suspension with red blood cells (RBC) from each microvette was added into a corresponding Eppendorf tube with 1 ml of PBS.The suspensions in Eppendorf tubes were centrifuged (1500 min -1 , 5 min), then the supernatant was removed and 1 ml of buffer was added again to each Eppendorf tube, and they were centrifuged again.Thus the concentration of NaNO 2 substantially diminished after the third washing and was close to 0. After the third washing, the total volume of erythrocyte suspension was 150 μl in each Eppendorf tube, which is equal to the baseline volume of blood.
Next, an appropriate amount of cytoflavin was added to each Eppendorf tube (step 4).Before adding cytoflavin
The preparation of suspensions of RBC with cytoflavin was carried out according to scheme 2-5 and Table 1.The total volume of the suspension was always kept at 150 μl (L/S) means the NaNO 2 solution at a concentration equal L or S.
Reference solutions (NaNO 2 solution was not added, but cytoflavin was added) were carried out according to the equation:

RBC+(75-CYT i )PBS+CYT i (μl) (2)
Such suspensions were designated as The investigated samples (NaNO 2 solution was added followed bycytoflavin was also added after washing) were carried out according to the equation

RBC NaNO 2 (L/S) CYT i (5)
In our experiments the CYT i values were as follows: CYT 0 = 0 μl CYT, CYT 2.5 = 2.5 μl CYT, CYT 5 = 5 μl CYT, CYT 10 = 10 μl CYT In this case, CYT i is not a concentration, but the volume of cytoflavin in 150 μl of suspension.To calculate the volume concentration of cytoflavin in each Eppendorf tube, we can use the formula: CYT i /150 (μl/ml).
Hematocrit in Eppendorf tubes nearly almost coincided with the baseline for each series of experiments at an accuracy of 10%.The RBC suspensions were incubated with CYT i from 1 hour to 24 hours; then, the absorption spectra were measured (stage 5).For measuring of absorption spectra, V susp =20 μl of suspension from Eppendorf tube were added to a quartz cuvette with 2.4 ml of PBS and optical density was measured.This process was repeated for the suspension in each Eppendorf tube.
The experiments for blood taken from each donor were carried out thrice.For each Eppendorf tube, the spectrum was measured thrice.Thus, 45 series of experiments were carried out according to the scheme (Fig. 1).In each series, the spectra of reference solutions as well as of solutions with different amount of cytoflavin were measured.The spectra shown further in Figs. 2 and  The statistical analysis of experimental data was performed using the Origin software (OriginLab, Northampton, MA).To determine unknown concentrations of hemoglobin derivatives the fitting curve was constructed according the scheme: Analysis -Fitting -Nonlinear Curve Fit -the Function (1).In resulting approximation, the R-Square parameter, the quantitative representatve of the fitting level, was determined.The R-Square parameter should be greater than 0.98.Data are shown as the mean±standard deviation.We used One-Way ANOVA for determining statistical significance of effect of cytoflavin in different concentrations on reduction of MetHb in comparison with autoreduction values (RBCNaNO 2 (L/S)CYT 0 ).Significant differences were reported at P 0.05.

Results and Discussion
The experiments demonstrated that cytoflavin administered into a suspension of RBC contributed to the reduction of methemoglobin to oxyhemoglobin.After addition of NaNO 2 , almost all hemoglobin molecules were transformed to methemoglobin, for which certain peaks in the absorption spectrum were specific.The peak at the wavelength λ=630 nm was most spe-  The addition of different amount of cytoflavin (CYT i ) resulted in changing the absorption spectra, and the concentration dependence was observed.
For suspensions L after 1 hour of incubation with cytoflavin, spectra typical for MetHb were observed for all samples (Fig. 2, a 1 ).It can be concluded that CYT 0,2.5,5,10 almost did not affect the reduction of MetHb.
After 24 hours of incubation, for the sample RBCNaNO 2 (L)CYT 0 , in which cytoflavin was not added, a spectrum typical for MetHb (Fig. 2, b 1 brown curve) was observed.The process of autoreduction occurred [14], however it was insignificant.
The peaks at λ=542 nm and λ=577 nm characteristic for HbO 2 (Fig. 2, b 1 , green curve) began to appear for a sample with cytoflavin RBCNaNO 2 (L)CYT 2.5 .However, the reduction of MetHb to HbO 2 and Hb was incomplete.During this time of incubation, only 59 % of methemoglobin was reduced.
An increase in concentration of cytoflavin to CYT 10 significantly enhanced the effect of reduction of methemoglobin in samples RBCNaNO 2 (L)CYT 5 (Fig. 2, b 1 , cyan curve) and RBCNaNO 2 (L)CYT 10 (Fig. 2, b 1 , blue curve).The peaks characteristic for oxyhemoglobin almost reached the reference values observed in sample RBCCYT 0 without the impact of NaNO 2 (Fig. 2, b 1 red curve).In this case methemoglobin was already reduced to about 25-27%.
As for suspensions S after 1 hour of incubation, spectra characteristics for a combination of different hemoglobin derivatives (Fig. 2, a 2 ) were observed for all samples.For all concentrations of CYT 0,2.5,5,10 , spectra were approximately the same with a small but obvious peak at λ=630 nm.This peak corresponded to 30-40% of MetHb in suspensions.After 24 hours of incubation, for the sample RBCNaNO 2 (S)CYT 0 in which cytoflavin was not added, this peak remained the same (Fig. 2, b 2 , brown curve).
However, after incubation for t 24 =24 hours, the suspension with cytoflavin RBCNaNO 2 (L)CYT 10 turned red (Fig. 3, b) that clearly demonstrated the process of reduction of methemoglobin to oxyhemoglobin in the suspension.The suspension RBCNaNO 2 (L)CYT 0 without cytoflavin remained brown, i. e. the process of autoreduction was almost absent.Reference suspension RBCC 0 without addition of NaNO 2 and cytoflavin was red at a baseline and in 24 hours.
These images qualitatively illustrate the effect of changes in the concentrations of hemoglobin derivatives that is in good agreement with the quantitative results of experiments.
During reactions, the formation of the following hemoglobin species are possible: HbO 2 , Hb, MetHb, HbNO, MetHbNO 2 , MetHbNO.Curve fitting method was used.Experimental relations D l (λ l ) exp and fitted curves D l (λ l ) teor are presented in Fig. 4 Concentrations of six hemoglobin derivatives were identified by best approximation of the experimentally measured spectra by theoretical curves.
Fig. 4, a and Fig. 4, b show good approximation of experimental spectra by theoretical curves for calculated concentrations of hemoglobin derivatives.
The kinetics of reduction of hemoglobin derivatives in the presence of cytoflavin was studied in the experiments (Fig. 5) once every 3 hours during 24 hours of incubation (suspension L).The dependences of changes in hemoglobin derivatives concentrations with с Fe 3+ -MetHb+MetHbNO 2 +MetHbNO are shown in Fig. 5, a.The dependences of changes in hemoglobin derivatives concentrations with Fe 2+ -HbO 2 +Hb+HbNO are shown in Fig. 5, b.
Cytoflavin increased the concentration of Fe 2+ hemoglobin derivatives and decreased concentration Fe 3+ Hb.
Importantly, the action of cytoflavin developed gradually over time.This was demonstrated by the kinetics of the level of significance.For each incubation timepoint, data for each concentration of cytoflavin CYT 2.5 , CYT 5 , CYT 10 were compaired to corresponding data of CYT 0 (autoreduction).At timepoints t 1 =1 hour and t 3 =3 hours, there were no significant differences in a drug action at all concentrations of cytoflavin.
Fig. 2 represents histograms of the MetHb percentage in suspensions of the L series (Fig. 2, a) and S series (Fig. 2, b) depending on different concentrations of NaNO 2 and corresponding different baseline levels of methemoglobin.At t 1 =1 hour, there were no significant differences between MetHb levels for all concentrations of cytoflavin ( CYT 2.5 , CYT 5 and CYT 10 ) compared to CYT 0 (Fig. 2).After t 24 =24 hours, for both series, there were significant differences between group with cytoflavin and control suspensions with no drug.Moreover, significance level was different for different concentrations of cytoflavin: (a) for CYT 2.5 * -P<0.05(for S) and ** -P<0.01 (for L); (b) for CYT 5 and CYT 10 , ** -P<0.01 (for S) and *** -P<0.001(for L).
There were no statistical differences between values of MetHb (%) due to autoreduction in samples after 24 hours and 1 hour, P=0. 4.
In a separate experiment, it was shown that under the action of cytoflavin, the excessive methemoglobin is converted to oxyhemoglobin and not to carboxyhemoglobin.The standard methods based on the use of sodium dithionite were employed in this experiment.After addition of dithionite, the spectral parameters specific for oxyhemoglobin disappeared, and the spectrum appeared to exhibit only one maximum typical for deoxyhemoglobin at λ=554 nm.
In our experiments, hemoglobin was converted into methemoglobin when erythrocyte suspension was exposed to NaNO 2 , and as a result, there was a conversion Fe 2+ Fe 3+ .Under such conditions, the formation of following hemoglobin derivatives becomes possible: with iron Fe 3+ (et MetHb+MetHbNO 2 +MetHbNO) or Fe 2+ (HbO 2 +Hb+HbNO) in the solution [16].Only HbO 2 of these 6 components is capable to deliver the oxygen to the tissues whereas Hb is potentially capable to attach oxygen.Oxidative processes in the blood lead to tissue hypoxia, to shape changes of RBC and to distortion of the membrane nanostructure [17][18][19].Exact manner in which all these derivatives of hemoglobin interact with one another is not clear enough.For example, possible reduction reactions were shown in work [20].The change in the forms of hemoglobin was investigated not only in vitro, but also in vivo [2,3,14].Reactions of conversion Fe 3+ Fe 2+ and Fe 2+ Fe 3+ are associated with redox processes in the RBC.Therefore, the addition of pharmaceuticals to the RBC suspension, which affects the redox processes, may also affect the interactions of hemoglobin derivatives.As a result, the ratio of the concentrations of hemoglobin derivatives changes significantly.Earlier we have shown that perfluorocarbon com- Высокое содержание MetHb в крови может возникать при отравлении анилином и его производными, некоторыми местными анестетиками, нитритами и нитратами [1,3,6].В связи с развитием фармацевтической и химической промышленности метгемоглобинемия экзогенной обусловленности встречается очень часто [15].
It could be particularly useful, however, to study biomimetic pharmaceuticals, which are similar to natural, functionally significant components of the body.The positive effect of cytoflavin on the human body is known since 2004 due to clinical use in cardiosurgeries, postsurgery and after cerebral infarctions and other cerebrovascular diseases.Cytoflavin provided a cytoprotective effect.It is a metabolic regulator.This drug has an antioxidant and antihypoxic action, exerting a positive effect on energy production in cells reducing the production of free radicals and restoring antioxidant enzyme activity.Cytoflavin has an anti-ischemic effect, improves coronary and cerebral blood flow limiting the zone of necrosis [9,10,[21][22][23].
Clinical results of the studies of the antioxidant effect of cytoflavin in the human body are presented in a publication [10].It was of interest to investigate the effect of cytoflavin on hemoglobin in a direct experiment in vitro.Further, these studies may reveal the mechanisms of antioxidant action of this drug.
Pharmacological effects of cytoflavin are regulated by the combined action of several components of the formulation as described [8,9].The complex formulation is composed of two metabolites (succinic acid and inosine) and two coenzymes (riboflavin mononucleotide and nicotinamide).The pharmacological effects of each component are well-known.Each component contributes to the redox processes in the RBC suspension.
Succinic acid is a key biochemical molecule.It is employed for energy metabolism in plants, human and animal tissue representi ng an intermediate in the Krebs cycle.Studies have shown, however, that the role of succinic acid goes far beyond energy processes.Antihypoxic and antioxidant effects of succinic acid has also been demonsrated [9,25].
Inosine is used in to improve the quality of the donor blood components during long-term storage [26].Nicotinamide has been included as a component of a composition for cryopreservation.Nicotinamide is employed for membrane stabilization and fluidity [27].Riboflavin mononucleotide sodium (FMN-Na, Flavin mononucleotide) plays an important role in oxidation-reduction reactions.Riboflavin is a water soluble vitamin, also known as Vitamin B2.In the body, riboflavin is primarily found as an integral component of the coenzyme, one of which is riboflavin mononucleotide sodium.There is a significantly impared relationship between plasma riboflavin mononucleotide sodium and riboflavin in patients with critical illness [28].Flavins act as a cofactor in many enzymes and catalyse a wide variety of biological reactions due to one of the most versatile in vivo redox centers in its structure.Organic redox active materials dissolved in electrolyte materials have received increasing interest DOI:10.15360/1813-9779-2018-2-46-59
All four components of cytoflavin are capable to contribute to redox properties of hemoglobin derivatives.Presumably, a combined effect of cytoflavin components might lead to the reduction of methemoglobin to oxyhemoglobin in the presence of cytoflavin.In this case, there are two possible ways of the influence of cytoflavin on the processes of iron conversion Fe 3+ Fe 2+ .
Direct chemical involvement of cytoflavin components in a redox processes is possible.This influence can be similar to that of methylene blue or ascorbic acid used in the treatment of methemoglobinemia [5].
Binding of cytoflavin components to active oxidation centers, thereby participating in the inhibition of oxidation processes.
To calculate the concentrations of hemoglobin derivatives, the Nonlinear Fitting method was used for the approximation of the experimental spectra.Good agreement between the theoretical data and the experimental data with the coefficient R-Square 0.99 was obtained.The theoretical data agreed not only in the description of the spectrum, but also in the estimation of the residual K level.
The antioxidant property of cytoflavin was demonstrated in our work for the first time in a direct biophysical in vitro experiment.For this purpose, blood from healthy donors was used as control exhibiting initially normal concentration of oxyhemoglobin and methemoglobin.The situation of using already initially poisoned blood containing a large amount of methemoglobin was excluded.The use of cytoflavin in this model experiment led to the reduction of excessive methemoglobin to oxyhemoglobin.In this case, the reduction effect of cytoflavin occured only if the methemoglobin was located inside the cell.The effect of NaNO 2 on free hemoglobin in distilled water led to the appearance of methemoglobin.However, in this case, the addition of cytoflavin did not result in a reduction of methemoglobin to oxyhemoglobin.This interesting phenomenon will be studied in details in future experiments.
Performed experiments might contribute to future studies validating the role of cytoflavin in blood recovery after acute poisoning.Clarifying the role of separate components of the drug would contribute to mechanistic studies of cytoflavin action.

Conclusion
The revealed effects may have a fundamental and practical application.Based on results with cytoflavin it would be possible to study and develop pharmacological methods for reduction Fe 3+  может быть аналогично действию метиленового синего или аскорбиновой кислоты, используемых при лечении метгемоглобинемии [5].
w w w .r e a n i m a t o l o g y .c o m G E N E R A L R E A N I M A T O L O G Y ,2 0 1 8 , 1 4 ; 2 DOI:10.15360/1813-9779-2018-2-46-59 49 w w w .r e a n i m a t o l o g y .c o m G E N E R A L R E A N I M A T O L O G Y , 2 0 1 8 , 1 4 ; 2 DOI:10.15360/1813-9779-2018-2-46-59
to Fe 2+ in blood.Cytoflavin can be used directly in treatment of methemoglobinemia in clinics.It would be GE N E R A L R E A N I M A T O L O G Y , 2 0 1 8 ,1 4 ; 2 DOI:10.15360/1813-9779-2018-2-46-59Experimental Studies