Impact of lithium alone or in combination with haloperidol on selected oxidative stress parameters in human plasma in vitro

Abstract

Objectives: Lithium may inhibit lipid peroxidation (LP) and protein oxidation, stimulate cell proliferation, increase neurogenesis, and delay cell death. Oxidative stress (OxS) is a state of imbalance between oxidative processes and antioxidant defenses, which may play an important role in the pathophysiology and disease course of bipolar disorder (BD). The aim of this study was to estimate the influence of lithium, administered alone or in combination with haloperidol, on selected OxS parameters in human plasma in vitro.

Methods: The OxS parameters evaluated were thiobarbituric acid reactive substances (TBARS) and total antioxidant capacity (TAC). Plasma samples from healthy volunteers were incubated with drug concentrations used in psychiatry.

Results: Incubation of plasma with lithium or haloperidol alone did not produce statistically significant changes of TBARS levels in comparison with control samples. However, significantly higher TBARS levels were observed in samples incubated with haloperidol plus lithium compared to control, haloperidol, or lithium samples. The TAC value did not differ between samples.

Conclusions: Lithium does not influence OxS parameters in human plasma in vitro during short-term observation when applied at concentrations used in psychiatry. However, lithium increased the TBARS level in the samples when given in combination with haloperidol, which may be one of the mechanisms behind the neurotoxicity associated with combined lithium and haloperidol administration.

Keywords:

• Lithium
• Haloperidol
• Oxidative stress
• Lipid peroxidation
• Toxicity

Introduction

Lithium was introduced to modern psychiatry as long ago as the 1940s. Nowadays, it remains a key element in the treatment of bipolar disorder (BD): a first-line treatment for acute mania and maintenance therapy and a second-line option for acute bipolar depression.¹ The fact that long-term lithium treatment prevents suicide lends it additional advantages over other mood stabilizers.² Previous studies have proposed many sites of action of lithium in the central nervous system (CNS). In the last two decades, a growing body of evidence has shown that lithium has several neuroprotective effects.³

Oxidative stress (OxS) is a state of imbalance between oxidative processes and antioxidant defence.⁴ Increased OxS induces lipid peroxidation (LP) in membranes, proteins, and DNA.⁵ The antioxidant system involves the coordinated effects of antioxidant enzymes, macromolecules and small molecules, with the cumulative effect being referred to as total antioxidant capacity (TAC).⁶ OxS may well play an important role in the pathophysiology, severity of symptoms and disease course of BD.⁷

In the treatment of acute mania, lithium is commonly combined with haloperidol or another first-generation antipsychotic. Importantly, chronic treatment with typical antipsychotics, especially haloperidol, appears to increase the OxS parameters detected in plasma.⁸ However, a systematic review by Lepping et al. reveals no clear conclusion in this regard.⁹ Mood stabilizers, especially lithium, have been demonstrated to influence OxS in cell cultures and animal models but the results differ profoundly.^10–12 Single human studies indicate that lithium possesses antioxidant properties and influences OxS parameters in blood plasma.^13–15 However, these effects demand further investigation, particularly with regard to identifying the specific targets that mediate these unique properties.

The present study investigates the effects of lithium administered alone and in combination with haloperidol on selected OxS parameters in human plasma in vitro. Two different doses of lithium were administered, which resulted in appropriate concentrations within the therapeutic range of the drug as administered to patients: 0.7 and 1.0 mmol/l. The aim of this study was to confirm whether even short-term treatment with lithium can decrease the level of OxS markers in human plasma in vitro, when given either alone or in combination with haloperidol, as a means of preventing the oxidation caused by treatment with haloperidol alone.

Materials and methods

Subjects

Blood samples were taken from 58 healthy subjects ranging in age from 20 to 40 years. The inclusion criteria were a lack of psychiatric or somatic disorders, no history of head injuries, no allergies, no lipid or carbohydrate metabolism disorders, a normal body mass index and abdominal circumference, no pharmacological treatment, no use of any addictive substances or antioxidant supplementation, consumption of a balanced diet, and similar socio-economic conditions to the rest of the group.

All subjects signed an informed consent form for participation in the study, in accordance with the protocol accepted by the Medical University of Lodz Committee for Research on Human Subjects (number RNN/13/08/KE). Mental status was assessed with the M.I.N.I. (Mini International Neuropsychiatric Interview), Polish version 5.0.0. Fasting blood (20 ml) was collected into EDTA tubes between 7:30 and 8:30 a.m. Blood samples were centrifuged twice for 15 min at 4 °C at 1500 g to obtain platelet-poor plasma.

Incubation with drugs

All the reagents and chemicals used in these experiments were of the highest analytical grade of purity. Lithium carbonicum and haloperidol (4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butan-1-one) were obtained from Sigma–Aldrich in the form of active substances. Lithium salt was dissolved in distilled water and haloperidol in dimethylsulfoxide (DMSO) to form stock solutions (100 mmol/l for lithium carbonicum and 1 mg/ml for haloperidol). Drug solutions were added to samples containing 0.5 ml of plasma to obtain final concentrations corresponding with those used in psychiatry: 0.67 and 1.00 mmol/l for lithium (prophylactic and therapeutic concentrations used to manage BD) and 10 ng/ml for haloperidol.¹⁶ For each experiment, a control sample without the drug was prepared with plasma and DMSO. The final concentration of DMSO in all samples was 0.01%. The plasma samples from each of the volunteers were labeled as follows: control, lithium prophylactic (Li0.67), lithium therapeutic (Li1.00), haloperidol (hpl), lithium prophylactic + haloperidol (Li0.67 + hpl), and lithium therapeutic + haloperidol (Li1.00 + hpl). The plasma samples were incubated for 24 hours at 37°C. The OxS parameters evaluated were LP and TAC content.

Selected oxidative stress parameters

Thiobarbituric acid reactive substances (TBARS) level is a direct index of cell LP. The concentration of TBARS was measured according to the Rice-Evans method.⁴ To assess TAC, an improved ABTS radical cation decolorization assay was used.⁵ All the experiments were performed in triplicate.

Statistical analysis

All calculations were performed using Statistica v.10.0 software (Statsoft, Inc.). As the normal values of TBARS and TAC seen in humans vary considerably, all measurements were converted to percentages of controls. Mean values, standard deviation (SD) and standard error of the mean were calculated. The data was analyzed with the one-way analysis of variance. Statistical significance was set at 5% (alpha = 0.05). Post hoc comparisons were carried out with the Tukey test. The results are shown in bar graphs.

Results

The levels of TBARS in plasma incubated with prophylactic and therapeutic concentrations of lithium were very similar to those observed in controls. Similarly, no differences were observed between the samples from subjects administered 0.67 and 1.00 mmol/l lithium (Fig. 1). Nonetheless, the post hoc analyses showed that the TBARS levels in the Li0.67 + hpl samples were significantly higher than those seen in controls (P < 0.05). TBARS levels in the Li0.67 + hpl sample were also significantly higher than in the Li1.00 (P < 0.01) or hpl (P < 0.05) samples, and significantly higher in the Li1.00 + hpl samples than the hpl samples (P < 0.01) (Fig. 1).

Figure 1 The comparison of influence of lithium (prophylactic and therapeutic concentration), haloperidol and their combination on lipid peroxidation, expressed as TBARs levels, in human plasma (in vitro). The results are expressed in percentages of control and shown as mean ± SD.

The level of TAC did not differ between samples.

Discussion

The present findings showed that lithium did not affect the level of LP markers (TBARS) or TAC in human plasma in vitro compared with controls and haloperidol samples.

Similarly, Abdalla et al. did not report any influence of short-term (7 days) or long-term (1 month) treatment with lithium on the concentration of LP or antioxidative defense parameters in the erythrocytes and brain tissue of examined healthy rats.¹¹ Similarly, chronic therapeutic concentrations of lithium have also not been shown to have any impact on TBARS levels in the liver and kidneys of mice.¹⁷ The fact that the present study and the two discussed above all use tissue from healthy subjects may account for the similar results; lithium was also shown not to prevent OxS in ALS (amyotrophic lateral sclerosis) mice, despite blocking apoptosis.¹⁸

Contrary to the results of the first part of our study, many previous studies have already demonstrated that lithium treatment may attenuate OxS. Chronic treatment with therapeutic doses of lithium significantly inhibited the increase of peroxidation markers induced by glutamate, H₂O₂ and high lithium concentrations, and were associated with reduced cytotoxicity in primary cultured neuronal cells.^12,19,20 The main difference between the cited studies and the present one seems to be the pretreatment OxS level. Similarly, lithium was seen to exert a protective influence against increased oxidative status in the brain tissue of the tested animals, but not the blood, liver, or kidneys.^21–23 It may be assumed that the protective effects of lithium influence neuronal cells rather than other tissues. Interestingly, chronic lithium treatment reversed some of the OxS parameter levels to normal values in animal models of depression,²⁴ mania,^25,26 or sepsis²⁷ which again, might indicate that lithium acts under pathological rather than neutral conditions. On the other hand, Vasconcellos et al. note that lithium treatment in rats induced an antioxidant effect in the hippocampus, but it was not able to prevent a stress-induced increase in damage to macromolecules such as LP.²⁸

The lack of agreement between the results of the first part of our study and those of studies demonstrating the antioxidative properties of lithium may be explained by short time of incubation, low level of primary OxS (using blood only from healthy volunteers and not bipolar patients), and type of peripheral tissue examined (blood). Moreover, some of the peripheral antioxidant effects may be secondary to CNS effects and thus might be seen only in in vivo experiments.

Moreover, in contrast to the present results, some studies associate lithium with pro-oxidative activity. Long-term treatment with therapeutic levels of lithium caused a significant increase in the levels of LP and an imbalance of SOD, reduced glutathione, catalese (CAT) and glutathione-S-transferase in rats. Changes in OxS parameters correlated with functional damage, and were seen in the liver, kidneys, lung tissue, and erythrocytes.^11,29–31

In addition, individual in vivo studies also contradict the present findings with regard to the influence of lithium on OxS parameters in humans. It was demonstrated that acute treatment with lithium significantly reduced the increased levels of TBARS seen in unmedicated manic patients, normalized the antioxidant enzyme activity ratio (SOD/CAT) and increased TAC levels.^13,14,32 Lithium was also shown to have antioxidative activity in non-pathological conditions (in the plasma of healthy volunteers).¹⁵

To summarize, a number of different factors may explain the heterogeneity of the results observed in the literature regarding the influence of lithium on OxS parameters. The key factors may include lithium concentration, duration of treatment, tissue type (CNS vs. peripheral tissue) and the primary state of the oxidative balance. In addition, single antioxidant enzyme measurements and their ratios may seem to be more relevant than TAC level in assessing the prevention of OxS by neuropsychiatric medications. However, the present study had been designed before this knowledge emerged.

Our hypothesis that lithium carries antioxidant properties demands supplementary information. It may be concluded that chronic treatment with therapeutically effective lithium concentrations can decrease the vulnerability of neuronal cells to induced OxS.

The results of the second part of the study show that OxS parameters in the lithium + haloperidol samples were significantly higher than those in other samples. Despite the fact that both medications were used in therapeutic concentrations and none of them alone caused increased LP, the combination should be used with particular caution. These findings suggest that, contrary to our primary hypothesis, lithium may not prevent but rather potentiate haloperidol-induced oxidation.

Lithium has been administered concomitantly with neuroleptic drugs, particularly haloperidol, for the treatment of some mental disorders for hundreds of years. Despite the very high therapeutic potential of this combination, especially in manic episodes, physicians and patients have noted its severe adverse effects. Neurotoxicity associated with the combination of lithium and high-potency neuroleptics was first reported by Cohen and Cohen in 1974.³³ Subsequently, multiple authors have tried to clarify the incidence, risk factors and symptomatology of this adverse effect. The resultant neurotoxicity is characterized by variable combinations of delirium, extrapyramidal symptoms, and cerebellar symptoms.³⁴ In 1996, a review of spectrum cases of lithium + neuroleptic neurotoxicity recorded in the Spontaneous Reporting System Database of the U.S. FDA, together with other extant literature, indicted that the plasma level of lithium and combination therapy with haloperidol were significant factors in the development of sequelae.³⁵

The precise mechanism by which the lithium + haloperidol combination induces toxicity is, however, poorly understood, and various mechanisms have been proposed to account for this. Nemes et al. report that patients treated with a lithium + haloperidol combination had significantly higher brain and plasma levels of the neuroleptic than a group treated with haloperidol alone.³⁶ Another study showed that the haloperidol + lithium combination was associated with significantly more frequent photic and other EEG changes than the combination of lithium with other neuroleptics.³⁷ As long ago as the 1970s and 1980s, it was proposed that OxS was responsible for combined haloperidol + lithium toxicity. In 1979, Guynn and Faillace studied the brain metabolism of rats treated with lithium, haloperidol or the combination of both medications. The only significant differences found concerned brain redox parameters: a reduction in both the cytoplasmic free [NAD+]/[NADH][H+] and [NADP+]/[NADPH] ratio in the lithium + haloperidol group compared with the control group.³⁸ A few years later, Sawas and Gilbert demonstrated that the neurotoxic and nephrotoxic effects of lithium carbonate and haloperidol they observed in rats might be a consequence of increased LP in brain and kidney tissues.³⁹ This observation is in accordance with the results of our study.

A review of extant literature indicates that this is the first study since 1985, and only the third one to examine possible mechanism of lithium + haloperidol toxicity in terms of OxS. Consequently, the increased LP level in lithium + haloperidol plasma samples seems to be the most interesting and valuable result of our study, and may suggest that the extracellular matrix plays an independent role in mediating neuronal toxicity of the medications.

Limitations

The results of the present study have some limitations. A significant weakness of our experimental design is the difficulty in extrapolation of these findings to in vivo conditions. An important limitation of the study is using only blood from healthy volunteers and not from patients with BP (depressive, manic, or euthymic states). Another apparent limitation of the present study is the short incubation time with the active substances and the low level of primary OxS. In spite of this, the results may be important for future in vivo studies.

Conclusions

In conclusion, the primary finding of our study was the increased LP level in human samples treated with a lithium + haloperidol combination, likely to be one of the causes of the toxicity of the drug combination. Lithium was not found to have any influence on OxS parameters in human plasma in vitro when administered alone.

Disclaimer statements

Contributors None.

Funding Supported by grant 502-11-756 from the Medical University of Lodz, Poland, and by grant N402 456138 from the Polish Ministry of Science and Higher Education.

Conflict of interest There is no conflict of interest.

Ethics approval All subjects examined in the study signed an informed consent form for participation in the study in accordance with the protocol accepted by the Committee for Research on Human Subjects of the Medical University of Lodz (number RNN/13/08/KE).

Acknowledgments

The authors are thankful to the Head of Department of Pharmaceutical Biochemistry, Medical University of Lodz, Prof. Marek Mirowski for providing the facilities required to carry out the research work.