Effect of a glutathione-containing dinitrosyl iron complex on the oxidative metabolic state and crystallogenic properties of rat blood plasma: a preclinical experimental study

INTRODUCTION

The multifaceted regulatory role of nitric oxide (NO) in biological systems predetermines the importance of examining the possibilities of controlling the level of this compound in organs and tissues externally. Besides its most well-known biological effect, namely vasodilatory activity, the study also shows the participation of this compound in neurotransmission, modification of blood coagulation processes, intracellular killing during phagocytic respiratory burst, membranotropic activity, and so on [1–4]. Moreover, it is important to note the extremely short lifetime of a nitrogen monoxide molecule, averaging six seconds in the free state [5]. This imposes strict requirements on the metabolism regulation of the compound and highlights the need for the presence of substances that temporarily deposit NO or create conditions for its synthesis (if necessary) in the organism [7–8].

Currently, there are several fundamentally different ways of exogenous modulation of NO metabolism. These include administering the L-arginine substrate of NO synthase, using selective inhibitors of this enzyme which influence the release of the compound, as well as employing a wide range of pharmacological donors [9–11]. Dinitrosyl iron complexes (DNICs) with various ligands hold a special place among such donors since they are considered to be a natural deposited form of nitric oxide [12–15]. At the same time, the biological effects of exogenous DNICs are not sufficiently elaborated yet [16]. Experimental data obtained by in vivo studies suggest that DNICs have marked antioxidant properties [17][18]. This hypothesis was confirmed by modeling oxidative stress in vitro (by introducing highly concentrated ozonized saline solution into bodily fluid samples) and in vivo (by modeling thermal injury in rats) [19]. On the other hand, the abovementioned data need to be confirmed in vivo and in healthy animals.

The research aims to study the effect of a glutathione containing dinitrosyl iron complex on the oxidative metabolism parameters and crystallogenic activity of rat blood.

METHODS

Experimental animals

The experiment was conducted on 60 sexually mature male Wistar rats weighing about 250 g. The animals were obtained from the Stolbovaya breeding nursery, a branch of the Scientific Center for Biomedical Technologies of the Federal Medical-Biological Agency, in the autumn-winter period.

Housing and welfare

The animals were kept in the vivarium of the University Experimental Biological Clinic at Privolzhsky Research Medical University of the Ministry of Health of the Russian Federation (hereinafter referred to as the vivarium) in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and the rules for working with animals based on the provisions of the Declaration of Helsinki, the recommendations contained in EC Directive 86/609/ECC, and the European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes. The animals were fed the standard water and food diet with free access to food and water.

Study design

The study was randomized. Drug administration and biological material collection were conducted in the vivarium. The laboratory stage of the research was performed at the Medical Biophysics Laboratory of the University Clinic at Privolzhsky Research Medical University of the Ministry of Health of Russia. Fig. 1 shows the block diagram of the study design.

Sample size

The animals were divided into 6 groups with 10 individuals in each group using the envelope method. Group 1 included intact (without any procedures) individuals. In group 2, the rats were administered daily intraperitoneal injections of 1 ml. of 0.9% sodium chloride solution for 10 days. The rats included in the other four groups received daily intraperitoneal injections of 1 ml of dinitrosyl iron complexes with glutathione ligands in saline with different agent concentrations: 0.15 mM for group 3; 0.30 mM for group 4; 0.45 mM for group 5; 0.60 mM for group 6. The prespecified analysis of the normal distributions for the age and weight variables of the rats in the groups using the Shapiro-Wilk test showed that there is no normal distribution (Gaussian) law in three age groups (p < 0.05) and in one weight group (p < 0.05). To prove that the age and weight of the rats were uniform, a nonparametric comparison method, namely the Kruskal-Wallis test, was employed. Table 1 presents the central tendency data in the form of the median and quartiles (Q1 — the first quartile or the 25^th percentile and Q3 — the third quartile or the 75^th percentile).

For age and weight, the differences in the median values for different groups of rats were not statistically significant with p = 0.253 and p = 0.778, respectively.

Eligibility Criteria

Inclusion criteria

Two-month-old male Wistar rats weighing about 250 g, without visible physical development abnormalities and injuries were included in the study.

Exclusion criteria

Animals weighing more than 250 ± 1 g, aged less than 56 and more than 64 days, female individuals, as well as animals with visualized developmental abnormalities and injuries were not included in the study.

Randomization

60 animals were selected according to the inclusion and exclusion criteria. The animals were allocated to groups randomly, namely by envelope method. Each animal was assigned one of the six group numbers extracted from an opaque envelope containing 60 pieces of paper with the group numbers. Depending on the group number indicated in the envelope, all animals were divided into six groups of 10 animals each.

Blinding

The head of the study, A. K. Martusevich, had information about the allocation of animals to groups. The author team assessed the results and analyzed the obtained data without introducing additional persons.

Outcome measures

The study outcome was the assessment of the crystallogenic properties and oxidative potential of blood under conditions of injecting various DNIC doses.

Experimental procedures

The crystallogenic properties and oxidative potential of blood were assessed in bodily fluid (blood). In the animals of all groups, blood samples were obtained from the sublingual vein. In the rats of the first (intact) group, blood samples were collected once, whereas in the representatives of the other groups — twice (before and immediately after the dosing cycle). 1 ml of the studied solutions was administered daily for 10 days.

DNICs with glutathione ligands were synthesized according to the method of A. F. Vanin [20]. The compound concentration in the saline solution was determined spectrophotometrically at wavelengths of 310 and 360 nm by known extinction coefficient and amounted to 3.1 mmol/L.

The activity of pro- and antioxidant systems was studied in rat blood plasma using Fe²⁺-induced biochemiluminescence (BChL-06, Russia). The following evaluation parameters were used: biochemiluminescence light sum for 30 s, which is usually considered as an indicator of lipid peroxidation (LPO) intensity (arbitrary units). The other parameters included the total antioxidant systems activity (AOA, arb. units) considered as the intensity criterion, as well as the slope of the chemiluminescence kinetic curve tg 2б, and malondialdehyde (MDA) concentration in blood plasma (mmol/L).

The crystallogenic properties of blood serum were studied by classical crystalloscopy. The results of the bodily fluid intrinsic structure were criterially evaluated. We used a specialized system of parameters [21] including crystallizability (CR, points), i. e. the density of the crystalline elements in the specimens mounted on microscope slides; structural index (SI, points), which characterizes the complexity of emerging structures (from amorphous solids to highly branched dendrites); the degree of facies destruction (DFD, points), which indicates the destruction level of the elements of the specimens on microscope slide; the marginal zone pronouncedness (M_z, points).

Animal care and monitoring

The animals were kept under standard vivarium conditions with free access to food and water. At the end of the research, the animals were removed from the experiment under general anesthesia with tiletamine hydrochloride (60 mg/kg) and xylazine hydrochloride (6 mg/kg) administered intramuscularly.

Statistical procedures

Principles of sample size determination

No preliminary sample size calculation was made.

Statistical methods

The numerical samples were tested for compliance with the normal (Gaussian) distribution law using the Kolmogorov-Smirnov or Shapiro-Wilk tests. In case of departure from normality, descriptive statistics were presented as the median and the first and third quartiles of Me (Q1–Q3). In case of normality, descriptive statistics were presented as mean and standard deviation (M ± SD). When analyzing the influence of a factor on all groups, one-way analysis of variance (ANOVA) was used according to the F-test for normally distributed samples. Pairwise comparisons were made using Student’s t-test for independent samples. When analyzing the influence of a factor on all groups, one-way ANOVA was employed using the Kruskal-Wallis test for samples with a distribution departing from normality. Pairwise comparisons were made using the Mann-Whitney U test for independent samples. The statistical significance level was p ≤ 0.05. Calculations were made using software packages MS Office 2013 (Microsoft Corporation, USA) and Statistica 10 (StatSoft, USA). The values were normalized. The average indicator values for the group of untreated animals are taken as 100%. The data are presented as histograms.

RESULTS

It was found that infusions of saline solution not containing the studied substance had no significant effect on both the intensity of lipid peroxidation in rat blood plasma and on its total antioxidant activity. Conversely, employing a physiological nitric oxide donor in all used dosages changed the values of the abovementioned parameters (Table 2). In particular, the intensity of lipid peroxidation demonstrated a marked statistically significant dependence on the administered DNIC concentration (according to the F-test for one-way ANOVA with p = 0.049, where the concentration of the DNIC solution acts as a factor).

Thus, when the animals were administered a minimal dose of the compound (1 ml of 0.15 mM solution), no significant indicator deviations from the indicator for the intact animal group were observed (p = 0.940). When the concentration of the compound in the solution (0.3 mM and above) was increased, a decrease in the intensity of lipid peroxidation processes was observed, reaching a minimum during a course of infusions of a 0.45 mM DNIC solution, i. e. in the animals of group 5 (p < 0.05 by Student’s t-test for independent samples between the indicator pairs for group 5 and the indicators for the first, second, and third groups of animals, except for groups 4 and 6). A further increase in the dose of the administered NO donor had a less marked effect on the parameter level, which may be caused by the formation of an excess of the substance due to the partial destruction of complexes with nitric oxide release and the transformation of the latter into peroxynitrite, one of the most powerful oxidizing bioradicals [19, 21].

The mean values of the total blood plasma antioxidant activity were found to be dependent on the DNIC solution concentration. In particular, no significant differences in the indicator were observed in the rats receiving only saline infusions (according to Student’s t-test, p = 0.915). When DNIC was added to it in any of the studied concentrations, an increase in this parameter value was observed (according to the F-test for one-way ANOVA, p = 0.005, where the DNIC solution concentration acts as a factor) (Table 2). This tendency was least pronounced for the minimum compound dose (0.15 mM). Thus, in the range of 0.15–0.45 mM DNIC, an increase in the total antioxidant activity of plasma was recorded: for the 0.15, 0.30, and 0.45 mM concentrations it was 1.07, 1.24, and 1.31 times, respectively, relative to the indicator values for the group of the untreated animals. For group 6 the increase amounted to 1.13 times. According to Student’s t-test, the differences are statistically significant (p < 0.05) for groups 4, 5, 6. A further increase in the amount of the administered compound (up to 0.6 mM) produced the opposite effect. In this case, the total antioxidant activity only increased by 13% compared to the healthy animals (p < 0.05). We believe that the mechanism of these shifts is similar to the one presented above with regard to the dynamics of lipid peroxidation processes with the considered nitric oxide donor.

The biochemiluminescent analysis results characterizing the oxidative metabolism components were additionally verified by assessing the concentration of a stable lipid peroxidation product, namely malondialdehyde (MDA), in the blood plasma of the animals in the formed groups (Table 2). In particular, we detected no significant dynamics of this parameter in the rats that only received saline injections (p = 0.655). Moreover, minor changes in the indicator values were recorded in the group of the animals that were administered the minimal DNIC concentration (p = 0.533). At the same time, a twofold increase in the effective concentration of the compound (up to 0.3 mM) significantly enhanced the malondialdehyde level reduction in blood plasma (-24%; p = 0.049 compared with the healthy individuals). Similar behavior was observed when using a concentration of 0.45 mM (-14%; but p equaled 0.247 and was not statistically significant). What is more, a further increase in the DNIC dose (up to 4 times the minimal dose) contributed to a less marked decrease in the level of the studied metabolite of lipid peroxidation (-5%; p equaled 0.717 and was also statistically not significant). The change in the malondialdehyde value according to one-way ANOVA, where the factor is the DNIC solution concentration, is statistically significant (p = 0.050).

Since the values of oxidative metabolism indicators in absolute units differed from each other by several orders of magnitude, all values were normalized. The average values of the indicators for the group of untreated animals were taken as 100%. We obtained a histogram (Fig. 2–4) for all oxidative metabolism indicators. The most statistically significant changes (p = 0.005) were for the AOA indicator (Fig. 3).

It is found that administering saline solution containing no natural nitric oxide donor to animals had no significant effect on the intrinsic crystallization parameters of the bodily fluid. Thus, the differences in the median values for the indicators between groups one and two were not statistically significant. According to the Mann-Whitney U test, p equaled 0.650 for the SI indicator, 0.705 for the CR indicator, 0.706 for the DFD indicator, and 0.571 for the M_z indicator (Table 3).

At the same time, the use of DNIC solutions changed the values of these indicators in comparison with the intact animals. However, the influence of different solution concentrations as a factor was not statistically significant for all indicators.

For instance, the median value of the serum facies structural index, or the SI index, differed in the compared groups with respect to the Kruskal-Wallis test, but p = 0.306 did not show statistical significance. This parameter reflects the structure complexity of facies elements. The range from 1 to 2 arb. units is characterized by the presence of both single-crystalline and dendritic elements in the specimen on the microscopic slide. Moreover, the increase in the indicator value testifies to an increase in the proportion of the latter in the crystallogram. The maximum median value of the structural index was determined when rats were administered a saline solution containing 0.3 mM of DNIC (Table 3 and Fig. 5). In this case, the median value of the parameter exceeded the median for the group of animals with physiological values by 2.0 times (p = 0.029), which is statistically significant. Besides, the indicator value achieved at an agent concentration of 0.15 mM was also 2.0 times higher, but not statistically significant (p = 0.098). It should be noted that at a DNIC concentration of 0.6 mM, this indicator value, on the one hand, was higher than the median value characteristic of the intact rats. On the other hand, it was lower than the median value for the group of animals treated with a 0.3 mM DNIC solution.

We also recorded changes in relation to the crystallizability of blood serum facies, or the CR index, which is the main quantitative criterion for assessing the intrinsic crystallization of blood serum (Table 3 and Fig. 6). For this indicator, changes in accordance with one-way ANOVA using the Kruskal-Wallis test showed greater statistical significance of the influence of the DNIC solution concentration factor (p < 0.001).

In this regard, it is significant that the structural index and crystallization changes manifested in an increase in both parameters during intraperitoneal administration of DNIC to animals are unidirectional and indicate activation of the bodily fluid crystallogenic properties. At the same time, if the highest median value of the structural index was observed when using DNIC at a concentration of 0.3 mM, then the highest median value of crystallizability was registered with the introduction of 0.45 mM of DNIC. Thus, the difference in the crystallizability median between group 5 and the intact animals is statistically significant, p = 0.002. It should be noted that when using other agent concentrations, this indicator changes are significant.

The effect of the concentrations of the physiological nitric oxide donor on the destruction degree of crystalloscopic facies, or the DFD indicator in the form of medians, appears to be not statistically significant according to the Kruskal-Wallis test as p = 0.102 for all comparison groups (Table 3 and Fig. 7). The values of this indicator became higher with increasing the DNIC dose, but they did not exceed the mean value of the indicator (0.7 arb. units) at all concentrations except 0.6 mM. Such a parameter level indicates a weak pronouncedness of destructive processes during the formation of crystalline facies elements, indirectly showing the absence of a significant toxic effect of the compound. Moderate destruction of the sample structures was observed only when the highest concentration of the used compound (0.6 mM) was administered to the rats.

We also revealed uniform pronouncedness of the marginal protein zone of the specimen on the microscope slide under the action of different DNIC concentrations (Table 3 and Fig. 8). Thus, at all doses of the compound used, a decrease in the median values of this indicator was recorded. However, the statistical significance of the influence of the factor in the form of DNIC concentrations on the marginal protein zone of the specimen, or the M_z indicator, was not revealed, because p equaled 0.258.

DISCUSSION

Interpretation / scientific significance

Despite the already established numerous biological effects of DNICs associated with their ability to release nitrogen monoxide [1][22–28], relatively little attention has been paid to the antioxidant effects of the compound. They were first discovered by us earlier in experiments performed on a thermal injury model [19, 21]. The present study conducted on healthy laboratory animals as a test bio-object, enabled us to visualize this effect. We believe that it includes two components, namely the direct antioxidant properties of dinitrosyl iron complexes themselves and the corresponding activity of glutathione ligands. Together, they can ensure the functioning of DNIC as a pharmacological agent with marked antioxidant potential.

Research limitations

Within the framework of the study, we considered the factor of injecting animals with saline solution without the studied agent (dinitrosyl iron complexes). Nevertheless, we chose a limited range of doses of the compound for testing (from 0.15 to 0.60 mM). Based on previous studies, it was assumed that this range corresponds to the most intense bioregulatory activity of the compound. Nevertheless, going beyond this range may provide additional information about the biological effects of DNICs over a wider dose range. Additionally, due to the need to minimize the number of animal groups for bioethical reasons, we only used 4 concentrations of the compound. By increasing the number of points within the considered range, additional data can be obtained to clarify the identified dose-effect relationships.

Extrapolation

It is known that DNICs, which are used as a natural nitric oxide donor, can stimulate antioxidant status correction through qualitative inhibition of free radical oxidation of lipids [18][19][21]. Our experiment has revealed that lipid peroxidation intensity values decreased dose-dependently in comparison with those in the intact animals as the DNIC agent concentration increased. However, the exception was the highest concentration used (0.6 mM), which affected the lipid peroxidation indicator in the blood plasma to a lesser extent than the previous DNIC dose (0.45 mM). Nevertheless, in this case there was also a decrease in the indicator values in comparison with those in the intact rats.

Blood plasma malondialdehyde tests also confirmed a decrease and weakening of LPO intensity with an increase in the administered DNIC concentration. A similar tendency was recorded in relation to the total antioxidant activity of blood plasma. The ability of DNIC to produce antioxidant effects is due to the ability of complexes to intercept free radicals in conjunction with the subsequent recovery of the oxoferryl form of myoglobin. Interception of O₂- formed during superoxide decomposition is characteristic of DNIC with thiol-containing ligands [29–30].

In regard to the crystallogenic properties of rat blood serum, we registered changes indicating a positive effect of administering DNIC at concentrations of 0.3 and 0.45 mM. This was manifested in an increase in the key parameters, namely the structural index and facies crystallizability. At the same time, the indicators of the possible toxic effects of the agent used (the degree of facies destruction and the pronouncedness of the marginal protein zone) showed moderate deviations in comparison with those in the intact animals.

CONCLUSION

In general, the conducted studies indicate the presence of an antioxidant effect in glutathione containing DNICs. The pronouncedness of these properties demonstrates a dependence on their dose with a possible optimum lying in the range of 0.3–0.45 mM (agent dose — 2.86–4.29 µg/g of animal weight). The research has established the activating effect of glutathione containing DNIC injections on the crystallogenic potential of blood serum in healthy rats. This effect manifested itself in an increase in the density of crystalline elements and their complexity, and, as for the metabolic indicators, the maximum pronouncedness of this tendency corresponded to concentrations of 0.3 and 0.45 mM.