Lithium Salts of Krebs Cycle Substrates as Potential Normothymic Antioxidant Agents

Abstract

Objective:

Aim of the present work was to study the antioxidant properties of lithium salts of Krebs cycle substrates and their influence on immune cells. Lithium is a well-known and widely used mood stabilizer. These lithium-based substances have a lot of potential properties because of the anionic component of the Krebs cycle substrates, which take part in basic intracellular biochemical process.

Materials and Methods:

Lithium salts of fumarate, pyruvate, malate, succinate, and citrate (as reference drug) were investigated in this study as antioxidants and immunomodulators. The antioxidant properties were studied by the voltammetry method, which evaluates oxygen radical scavenging capacity of lithium substances. Influence of the lithium compounds on the immune cells of human blood was indicated by the reaction of blast transformation of lymphocytes.

Results:

All tested substances and their mixes possessed antioxidant properties, more expressed in maximal therapeutic concentration. Lithium compounds showed no toxic influence on human blood immune cells and caused no significant changes in both spontaneous and stimulated proliferation.

Conclusion:

The results allow considering lithium salts of Krebs cycle substrates as potential normothymic agents (mood stabilizer) with antioxidant properties and low toxicity.

INTRODUCTION

The Krebs cycle or tricarboxylic acid (TCA) cycle is a well-known basic biochemical process, which provides a major aerobic source of adenosine triphosphate energy. During this cycle, a lot of intermediate molecules are produced, which take part in a number of vital metabolic functions in the brain. Some of the TCA cycle metabolites can reduce death of neurons during ischemia.[1] Initial studies that suggest abnormalities of Krebs cycle in the brain of patients with schizophrenia are available.[2] However, in what way brain metabolism responds to injury or disease is not fully understood. Here, we present the antioxidant and immunomodulating effects of lithium salts of Krebs cycle substrates, including succinate, malate, fumarate, pyruvate, and citrate. These salts can be considered as perspective psychotropic substances (normothymic agents) because of the presence of lithium. Lithium salts are still considered as the standard or first-line treatment of bipolar affective disorder.[3] Recent studies confirm the protective effect of lithium in oxidative stress.[4] Lithium causes complex and various actions on the human nervous system, including neuroprotective and neurotropic effects. These effects are mostly realized through the influences on neuroprotective protein Bcl-2, fermentation of glycogen synthase kinase-3 beta (GSK-3β) in neuronal tissue,[5] and regulation of genes transcription.[6] Astrocytes are also considered as a potential target of lithium, explaining its neuroprotective action.[7] Information confirming that lithium improves and stabilizes cognitive impairment in patients with Alzheimer’s disease is available.[8] Despite many benefits, lithium has a narrow therapeutic index and can cause toxic effects. Therapeutic level of lithium in blood for normothymic effect is 0.6 mmol/L, but side effect can occur at levels equal to 1.2 mmol/L and more. Regarding the aim of this study, it should be noted that free radicals may play an important role in the pathogenesis of mental disorders[9] because of the high brain vulnerability to oxidative damage. The study of the antioxidant activity of human blood confirmed the depletion of antioxidant defense systems in mental disorders.[10] Therefore, the development and study of new psychotropic antioxidants with low toxicity is an actual challenge. Combinations of lithium and substrates of Krebs cycle look promising because of their wide-spectrum bioactivity, low toxicity, and mood-stabilizing effects. Lithium citrate (brand names Litarex and Demalit) is used as a mood stabilizer in psychiatry, and here, we considered it as a reference drug.

MATERIALS AND METHODS

The following lithium compounds have been used in this study [Figure 1]: lithium malate, lithium fumarate, lithium succinate, and lithium citrate (reference drug, commonly used lithium medication). Ascorbic acid powder (Sigma-Aldrich, Steinheim, Germany) was used as a standard antioxidant for testing the antioxidant activity.

Figure 1.

Formulas of investigated lithium salts (based on Krebs cycle acids) and standard antioxidant ascorbic acid

All lithium salts were prepared extempore in reaction of the calculated amount of lithium carbonate (Sigma-Aldrich) and corresponding acids (Sigma-Aldrich, Germany) in bidistilled water under moderate heating up to 40°C. The resulting clear solutions were filtered and cooled. Then, ethanol maintained at a 4°C temperature was added to the solution. The formed fine crystalline precipitate was filtered off. Then the substrates were washed using ethanol, dried in air, and used in experiments. Formulas of all tested substances are shown in Figure 1.

Antioxidant activity assessment techniques

The voltammetric analyzer “TA-2” (Tomanalyt, Tomsk, Russia) was used for the evaluation of antioxidant activity. All tested lithium salts were diluted in water phosphate buffer (0.025 mol/L, pH = 6.86). The procedure of antioxidant activity assessment by voltammetry was described in detail in previous works.[11,12] Briefly, experiment based on process of oxygen electroreduction (ER О₂) at mercury film electrode with the formation of the reactive oxygen species (ROS). These processes play a major role in basic oxidation mechanism in living cells. Measurements of oxygen current with and without the tested substances make it possible to evaluate the level of radical ROS scavenging by the substances. The coefficient of antioxidant activity was calculated as follows:

where, C_o (μmol/L) is the oxygen concentration in water solution, I the ER O₂ current with the tested lithium salt, I_o the ER O₂ current without the substance, and t (minutes) the time of the ROS–antioxidant interaction.

The most important test for biological application is comparison in molar concentrations equal to minimal therapeutic concentration of lithium (0.6 µmol/mL of Li⁺ ions). It is the common level of lithium ions manifesting mood-stabilizing action. Another dosage corresponded to the maximal therapeutic concentration of lithium (1.2 µmol/mL of Li⁺). Lithium causes a lot of side effects in higher concentrations, which is why Li⁺ blood level is strictly monitored in patients undergoing treatment. Test of antioxidant activity in equal mass concentration was conducted for the comparison of different salts, including investigation of possible synergetic and antagonistic effects in mixes. All mixes of salts were prepared in 1:1 weight ratio with a final concentration of 0.1%.

Study of immune cell responses in the reaction of blast transformation of lymphocytes

The reaction of blast transformation of lymphocytes (RBTL) method allows assessing the proliferative response of the lymphocytes. Here, we evaluated spontaneous and phytohemagglutinin (PHA) induced transformation levels under the influence of tested substances. Lymphocytes were obtained from a healthy donor blood and suspended with RPMI 1640 medium (with 20% fetal bovine serum and antibiotic) at a concentration of approximately 2 × 10⁶ cells/mL. Aliquots of 0.1 mL of cell suspension were dispatched in cell culture plates. All lithium salts dissolved in RPMI medium were added to cell suspension in varying concentrations (0.6–1.2 µmol/mL of Li⁺) with or without PHA. The same amount of RPMI 1640 medium without lithium salts was added to the control group with and without PHA. All samples were sealed and incubated for 72 h at 37°C. Lymphocyte blast transformation was detected by standard RBTL method.[13]

Statistical analysis

The differences between mean values were assessed by the Student’s t-test. Differences were considered as significant at P value less than 0.05. Statistical analysis was performed with Statistica software (StatSoft), version 12. Experiments were repeated three times. Results were presented as mean ± standard deviation.

RESULTS

Antioxidant activity of lithium salts of Krebs cycle substrate is provided in Table 1. This parameter reflects the ROS scavenging by antioxidants. Lithium salts of succinate, fumarate, and pyruvate express comparatively higher level of antioxidant activity.

Table 1.

Coefficients of antioxidant activity of Krebs cycle substrate lithium salts

Antioxidant activity of equal weight mixes of tested lithium salts is provided in Table 2. This parameter shows the interaction of salts in water solution and the possible synergetic or antagonistic effects. Combination of malate/fumarate and malate/pyruvate provides more expressed antiradical action against ROS.

Table 2.

Coefficients of antioxidant activity of lithium salt mixes (equal mass ratio, 1:1; concentration, 0.1%)

Results of RBTL under action of lithium compounds are given in Table 3. Relatively high level of cell proliferation was detected under the influence of fumarate, but all tested salts showed close parameters. Lithium salts displayed no toxic influence on spontaneous and stimulated lymphocyte proliferation in therapeutic dosage (calculated for lithiumions).

Table 3.

Influence of Krebs cycle substrate's lithium salts on immune blood cells in reaction of blast transformation of lymphocytes

DISCUSSION

Despite decades of application, lithium is considered as a unique element, the medical potential of which is not fully realized. Here, we proposed Krebs cycle substrates as the anion component of salt, which increase antioxidant properties, stimulate cell metabolism, and eventually decrease the undesirable side effects of the treatment. Substrates of Krebs cycle possess antioxidant activity and take part in energy metabolism of the cell, during which ROS is physiologically generated. The voltammetry results of antioxidant activity confirmed high antioxidant properties of all tested substances [Table 1]. The synergetic antioxidant action could also contribute to the potential benefits of prospect lithium salts. The synergetic effects of antioxidant in mixes were shown previously.[14] Krebs cycle metabolites in the form of lithium salts provide radical-scavenging action. Moreover, mitochondrial alteration of Krebs cycle under ischemic condition leads to preferential succinate oxidation and plays a crucial role in oxidative brain injury. Substrates of TCA cycle possess significant cytoprotective action, linked to their metabolic effects. Pyruvate and other mitochondrial substrates reduced cell death of astrocyte and neuron in cultures from approximately 70% to near basal levels after DNA alkylating agent treatment, meanwhile excess of glucose had negligible effects.[1] In turn, lithium was established as a neuroprotective agent, possessing indirect antioxidant properties.[15] At the same time, the concentrations of lithium ions remain within the recommended clinically effective doses. Our results show very close parameters of antioxidant action for all tested lithium salts at maximal therapeutic concentration of 1.2 µmol/mL of Li⁺. Relatively higher level of antioxidant action was observed for lithium succinate and lithium fumarate. Differences in the results were more significant at minimal concentration [Table 1]. Values of antioxidant activity of lithium salts could be comparable with the standard antioxidant ascorbic acid at low concentration range. It should be noted that lithium ascorbate also possesses expressed antioxidant action compared to other tested substances. Lithium citrate showed relatively low activity, but it is much higher than that of lithium carbonate (widely used lithium drug).[12] Combination of lithium and antioxidants enhance protective effects of resulting normothymic molecules. Investigation of different mixes of lithium salts revealed theoretically expected level of antioxidant activity except the combination lithium malate/fumarate. This combination showed slight synergetic action [Table 2]. In addition, lithium can prevent DNA damage, free radical formation, and lipid peroxidation, and reduce negative consequences of oxidative stress.[15,16] It provides a solid base for in vivo experiment as normothymic antioxidant. Different oxidative mechanisms may unify common pathogenic pathways in psychiatric disorders and should be considered as targets for novel therapies.[9] Comparison of activity of Krebs cycle salts in different concentrations showed moderate increase in antioxidant action from 0.6 mmol/L to 1.2 mmol/L. Lithium in this dosage could stimulate immunity and probably have specific antiviral properties against herpes virus, as patients taking prophylactic doses of lithium to avoid affective disorder recurrences had labial herpes rarely.[17] The mechanisms of this action are still not clear enough. One of the ways of such action is indirect influence through immunomodulating effects. Lithium enhances interleukin-2 production at concentrations greater than 1 mM and increases IgG and IgM production.[18] There is a proposition that the antiviral and immunomodulatory actions of lithium in vivo may regard to the mood-stabilizing effects of lithium. Other words lithium salt could enhance antiviral immunity and indirectly affect on mood status due to possible role of viruses and immune dysfunction in pathways of affective illness.[17] Obtained results showed less influence of lithium salts on human lymphocytes [Table 3]. Lithium fumarate revealed slight stimulation effects; contrary to this, citrate caused decrease in spontaneous transformation of lymphocytes in both concentrations. In PHA-stimulated blast transformation of lymphocytes, all salts showed insignificant stimulation compared to the control group. Obtained results in RBTL reflect low toxicity and sensibilizing properties of tested lithium antioxidants. On the contrary, some studies indicated that lithium enhanced the proliferation of immune cells, including lymphocytes in response to mitogens and phagocytosis by macrophages.[18] The same effect is assumed for lithium salts of the Krebs cycle substrates in case of phagocytic activity. Reportedly, lithium treatment stimulates the proliferation of rat neural stem cells and the generation of dopaminergic neurons,[19] but the influence of anionic component in case of lithium salts application was not considered. Some studies showed reduced rates of cancer among patients having bipolar affective disorder.[20,21] This effect could be interpreted as nondirected lithium effects on cell proliferation. It is supported by the fact that lymphoma and metastatic cancer are less likely to occur in persons with bipolar disorder.[22] It is also well consistent with the antioxidant effects of lithium salts. However, here, we established no significant suppressive effects on the stimulated lymphocytes of healthy donors in vitro. Lithium likely has modulatory effects in patients because of its effect on immune cells.[23] The influence on immune cells is linked not only with lithium ions but also with anionic component of the tested substance. Lithium antioxidants could protect lipids and proteins of blood against oxidative damage.[24] Components of Krebs cycle act here as direct antioxidant. However, the main advantage of lithium salts (normothymic agents) is cytoprotection, especially neuroprotection. It also includes lithium intracellular effects, as antiapoptotic agent through the inhibition of GSK-3β and phosphoinositide 3-kinase. Despite numerous controversial data, the internal ways of lithium effect relates to the changing activity of kinases, both inhibitory and stimulatory in some cases.[25,26] Recent data explained most lithium effects by the accumulation of beta-catenin and beta-catenin-dependent gene transcriptional events. Lithium is also effective as a preventive medication and in the therapy of not only manic but also depressive episodes.[27] This properties are also associated with changing activity of GSK-3β. Cell response changes in oxidative stress conditions. Peroxidation of biomolecules, DNA damage, and neuronal dysfunction are some of the fundamental consequences of oxidative stress. It plays an important role in the pathogenesis of mental disorder, which provides the perspective of developing novel therapeutic approaches.[28] Lithium antioxidants affect several pathways simultaneously, including initial oxidation stages, radical scavenging, and intracellular signaling pathway. Substrates of Krebs cycle have multiple influences on cell metabolism, including roles of messenger or signaling agent that activate cells.[29] It makes a solid base for Krebs cycle lithium salts as antioxidant, immunoprotective, and mood-stabilizing agents.

CONCLUSION

Lithium salts of Krebs cycle substrates could be considered as prospective psychotropic antioxidants for further studies. All tested lithium salts of Krebs cycle substrates showed antioxidant properties in therapeutic dose range. We revealed no suppressive influence on the proliferation of human blood lymphocytes. The high antioxidant and cytoprotective properties of these lithium compounds have been found to justify their use as neuroprotectors and hemoprotectors. The effect on the key kinases of cellular metabolism confirms a wide range of biological activities of these compounds. Lithium antioxidants have an etiotropic effect on oxidative stress. This expands the range of their application in pathologies associated with oxidative stress. Thus, antioxidants correct oxidative stress, but the inclusion of lithium ion into their structure provides neuroprotective and normothymic effects, which is important in the complex therapy of mental pathologies.

Financial support and sponsorship

This study was supported by the Russian Science Foundation (project no.: 17-75-20045).

Conflicts of interest

There are no conflicts of interest.