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Abstract

Valproic acid (VPA, 2-propyl pentanoic acid) is a broad-spectrum antiepileptic drug (AED) and is commonly used in the treatment of bipolar disorders and epilepsy. AEDs are known to result in vascular disturbances. Vitamin B6 (Vit B6) is water soluble vitamin essential for normal growth, development, and metabolism. In this study, we aimed to investigate the protective effects of Vit B6 against VPA-induced lens damage in experimental animals. In this study, male 4-month-old, Sprague-Dawley rats were used. The animals were divided into four groups. Group I was intact control animals. Group II rats were administered with Vit B6 (50 mg/kg/day) for 7 days. Group III rats were administered with only VPA (500 mg/kg/day) for 7 days. Group IV was given VPA + Vit B6 (in a same dose and time). Vit B6 was given to rats by gavage and VPA was given by intraperitoneally. On the 8th day of experiment, all of the animals were fasted overnight and then killed under ether anesthesia. Lens tissues were taken from animals, homogenized in 0.9% saline to make up a 10% homogenate. The homogenates was used for glutathione (GSH), lipid peroxidation (LPO), protein levels, and enzyme analysis. In VPA groups, levels of lens GSH and LPO and activities of glutathione-S-transferase, glutathione peroxidase, glutathione reductase, and aldose reductase were increased, while superoxide dismutase activity was decreased. Treatment with Vit B6 reversed these effects. These results demonstrated that administration of Vit B6 is potentially beneficial agent to reduce the lens damage in VPA toxicity, probably by decreasing oxidative stress.

Keywords

Valproic acid vitamin B6 lens oxidative stress antioxidant enzymes

Introduction

Epilepsy is one of the most common neurological disorders, characterized by recurrent seizures, which may increase the content of reactive oxygen species (ROS).¹ There are various antiepileptic drugs (AEDs) that have been used in the treatment of epilepsy. In recent years, alterations of visual function have been extensively studied in epileptic patient treated with different AEDs.²

Valproic acid (VPA, 2-propyl pentanoic acid) is one of these drugs that has been widely used for the treatment of epilepsy and other neuropsychiatric diseases such as bipolar disorders and migraine.^3,4 Long-term treatment with VPA has been known to be associated with polycystic ovary syndrome, hyperammonemic encephalopathy, hyperandrogenism, hyperprolactinemia, hyperinsulinemia, pancreatitis, changes in luteinizing, follicle stimulating, and sexual and thyroid hormones and amenorrhea in epileptic patients.⁵ It was also investigated that VPA caused hepatotoxicity associated with increased ROS formation, which in turn constitutes an important risk factor for tissue damage and organ dysfunction.^6,7 VPA has been demonstrated to cause deficits in color vision after a short treatment period.^8,9

Vitamin B6 (Vit B6) is water soluble and occurs in three closely related compounds with similar physiological actions, pyridoxine, pyridoxal, and pyridoxamine.¹⁰ Vit B6 is essential because of its participation in more than 100 enzymatic reactions, including protein metabolism, conversion of tryptophan to niacin, and neurotransmitter function, among others.¹¹ As a therapeutic agent, Vit B6 and its forms have been described in relation to diabetes,¹² epilepsy,¹³ and cardiovascular disease.¹⁴ The antioxidant and radical scavenging properties of the Vit B6 have long been considered in literature, for example, in reducing oxidative stress markers associated with homocysteinemia¹⁵ or in preventing ROS formation and lipid peroxidation (LPO) in a cellular model.¹¹ AEDs produce a considerable number of metabolic alterations, including changes in serum lipids, hormones, and various vitamin levels.¹⁶ Some studies also suggested that levels of Vit B6 in plasma may be decreased in association with AED treatment.^17,18

No studies of Vit B6 on VPA-induced lens damage have been found in the literature. This is the first study that evaluate the protective effects of Vit B6 against VPA-induced lens damage in experimental animals.

Material and methods

Animals and experimental design

The experiments were reviewed and approved by the Animal Care and Use Institute’s Committee of Istanbul University, Istanbul, Turkey. Male Sprague-Dawley rats weighing 90–130 g were used. The animals were 4 months old and clinically healthy. The rats were housed in metal cages at room temperature and fed with balanced diet. The animals were randomly divided into the following four groups: group I: intact control animals (n = 5); group II: animals administered with Vit B6 (n = 5; 50 mg/kg/day); group III: animals administered with only VPA (500 mg/kg/day; n = 5); group IV: animals administered with VPA + Vit B6 (n = 5; in same dose and time). Vit B6 was given to rats by gavage and VPA was given intraperitoneally. On the 8th day of experiment, all the animals were fasted overnight and then killed under ether anesthesia.

Biochemical assays

Lens tissue samples were taken and frozen until needed for study. The tissues were homogenized in cold saline using a glass homogenizer to make up a 10% (w/v) homogenate. The homogenates were centrifuged at 10,000g for 20 min and the clear supernatants were used for GSH, LPO levels, tissue enzyme activities, and protein analysis.

The GSH of lens tissue was determined by the method of Beutler,¹⁹ and the results were expressed as nanomoles of GSH per milligram of protein. The malondialdehyde (MDA) levels were measured by the method of Ledwozyw et al.²⁰ for products of LPO and results were expressed as nanomoles of MDA per milligram of protein.

Superoxide dismutase (SOD) was determined according to the Mylorie et al,²¹ glutathione-S-transferase (GST) according to the Habig and Jacoby,²² glutathione peroxidase (GPx) according to the method of Paglia and Valentine²³ and modified by Wendel,²⁴ glutathione reductase (GR) activity was determined according to the Beutler,²⁵ aldose reductase (AR) according to the Hayman and Kinoshita.²⁶ Protein level of lens tissue was measured by the method of Lowry using bovine serum albumin as standard.²⁷

Statistical analysis

Biochemical results were evaluated using an unpaired t test and analysis of variance using the NCSS statistical computer package. The values were expressed as mean ± SD. p < 0.05 was considered as significant.

Results

GSH and LPO levels of lens tissue were shown in Table 1. There was a significant increased in GSH levels of VPA groups compared with control rats (p < 0.0001). Administration of Vit B6 significantly decreased GSH levels in VPA group (p < 0.0001). LPO levels of lens tissue were significantly increased in VPA group compared to control group (p < 0.05). Vit B6 significantly decreased LPO level in VPA group (p < 0.05; Table 1).

Table 1.

Effect of Vit B6 on GSH and LPO levels of the lens tissue of all the groups.^a

Groups	GSH (nmol /mg protein)	LPO (nmol/mg protein)
Control	4.62 ± 1.03	0.05 ± 0.01
Vit B6	3.28 ± 0.63^d	0.07 ± 0.01
VPA	15.43 ± 2.40^b	0.19 ± 0.08^d
VPA + Vit B6	3.24 ± 2.54^c	0.09 ± 0.03^e
p _ANOVA	0.0001	0.004

Vit B6: vitamin B6; GSH: glutathione; LPO: lipid peroxidation; VPA: valproic acid; ANOVA: analysis of variance.

^aValues are given as mean ± SD.

^b p < 0.0001 compared with the control group.

^c p < 0.0001 compared with the VPA group.

^d p < 0.05 compared with the control group.

^e p < 0.05 compared with the VPA group.

Activities of SOD, GST, GPx, GR, and AR of lens tissue were shown in Table 2. SOD activity of lens tissue was significantly decreased in VPA group compared with the control group (p < 0.05), where SOD activity of lens tissue insignificantly increased in the Vit B6-treated VPA group. GST, GPx, and GR activities were significantly increased in VPA group as compared to the control group (p < 0.05), whereas these enzyme activities in lens tissue decreased in the Vit B6-treated VPA group. AR activity was insignificantly increased in VPA group. However, AR activity was insignificantly decreased by Vit B6 administration (Table 2).

Table 2.

Effect of Vit B6 on SOD, GST, GPx, GR, and AR activities of the lens tissue of all the groups.^a

Groups	SOD (U/mg protein)	GST (U/g protein)	GPx (U/g protein)	GR (U/g protein)	AR (U/g protein)
Control	1.31 ± 0.37	16.44 ± 5.50	6.84 ± 2.61	0.64 ± 0.17	0.98 ± 0.23
Vit B6	1.04 ± 0.23	15.11 ± 2.07	10.46 ± 1.50	0.77 ± 0.34	0.48 ± 0.19
VPA	0.57 ± 0.16^b	24.01 ± 4.24^b	13.16 ± 1.65^b	2.23 ± 0.64^b	1.20 ± 0.02
VPA + Vit B6	0.72 ± 0.32	17.84 ± 5.29	11.89 ± 3.33	1.18 ± 0.44^c	0.80 ± 0.18
p _ANOVA	0.020	0.028	0.017	0.001	0.007

Vit B6: vitamin B6; SOD: superoxide dismutase; GST: glutathione-S-transferase; GPx: glutathione peroxidase; GR: glutathione reductase; AR: aldose reductase; VPA: valproic acid; ANOVA: analysis of variance.

^aValues are given as mean ± SD

^b p < 0.05 compared with the control group.

^c p < 0.05 compared with the VPA group.

Discussion

Epilepsy and seizure disorders affect 50 million people around the world and contribute to morbidity and mortality.²⁸ AEDs are commonly used in the treatment of epilepsy, psychiatric diseases, and pain disorders. Epilepsy is a neurologic disorder that may be associated with visual alterations. Several drugs used for neurological disorders have been associated with ocular complications. The eye has a rich blood supply and relatively small total mass, making it highly susceptible to drugs that penetrate the central nervous system. Drugs entering the eye can be deposited at various sites. Prolonged treatment with some of antiepileptic agents can result in irreversible vision loss. Sodium valproate causes ocular congenital malformation.²⁹ In epileptic patients visual disturbances may be caused by either the epilepsy per se or by the anticonvulsant therapy used to control the seizures.² Valproate increases oxygen-connected tissue damage with some mechanisms.^30,31

Free radical load of the body is increased when valproate is metabolized and, at the same time, if damage occurs in the antioxidant system. Drug toxicity was also reported to increase due to changes in antioxidant enzyme activities. VPA toxicity is associated with increased ROS formation, which in turn constitutes an important risk factor for tissue damage and organ dysfunction.³²

GSH is an antioxidant that is present in high amounts in cells and an important mediator that protects cells against various xenobiotics.³³ GSH redox cycle has a very important role among enzymatic or nonenzymatic cellular antioxidant mechanisms for protection against oxidative damage. GSH, which is highly concentrated in the lens and helps to reduce proteins,³⁴ contains a side chain of sulfhydryl –SH residue that enables it to protect cells against oxidants. In our study, lens GSH levels were increased in VPA group. Vit B6 decreased GSH levels in the lens. The present investigation found that Vit B6 significantly restored the levels of GSH in VPA-induced rats. The increase in cellular GSH levels may be as a result of fast GSH synthesis and this occurred as a defense mechanism against toxic stimulation. Several studies reported that VPA can increase the level of GSH in brain.^35,36

LPO is plausibly the most extensively investigated process induced by free radical and hence regarded as excellent index of oxidative stress. Free radical generation or oxidative stress develops where there is an imbalance between pro-oxidants and antioxidants ratio, leading to the generation of ROS.³⁷ LPO has been hypothesized to be a major mechanism of cell damage by free radicals. In the present study, we have observed that malondialdehyde (MDA) formation, the index of LPO, was increased in VPA group. The obtained data revealed that the significant increase in the level of LPO may be due to its poor antioxidant defence or the inactivation of antioxidant enzymes due to oxidative stress. However, administration of Vit B6 prevented these changes. This may be attributed to its free radical scavenging properties³⁸ and antioxidative potential.³⁹

Endogenous antioxidant mechanisms exist to protect against the oxidative injury associated with normal metabolism. In the literature, there are numerous studies concerning the effect of AEDs on oxidant and antioxidant enzymes.⁴⁰Antioxidant enzymes such as SOD, CAT, and GSH-Px are present in all parts of the lens.⁴¹

SOD is a major defense system against toxic effects of oxygen radicals in aerobic cells. The enzyme rapidly removes superoxide anions and prevents cells from its direct toxic effect. In our study, SOD activity is lowered by administration of VPA but Vit B6 treatment increased SOD activity. Yüksel et al. reported that SOD activity was increased in epileptic children on in VPA therapy compared with the control group.⁴² Sołowiej and Sobaniec observed that SOD activities were decreased in epileptic children receiving VPA in comparison to relevant levels in nonepileptic patients.⁴³

GST is a metabolizing enzyme that plays a prominent role in the detoxification of oxidized metabolites and may serve as antioxidant.³⁷ In the present study, GST enzyme activity increased in the lenses of VPA-treated rats group compared with control group. Administration of Vit B6 restored GST activity to normal level. A decrease in lens GST activity indicates improvement in lens injury.

GPx plays an important role in the detoxification of peroxides. This may be critical in protecting cells against oxidants. The effects of VPA on antioxidant enzymes are also conflicting. Sobaniec et al. and Yüksel et al. found lower levels in patients receiving VPA.^44,45 On the other hand, Kürekçi et al.⁴⁶ and Cengiz et al.⁴⁷ reported higher levels of GPx in patients treated with VPA. In our study, GPx activity was higher in VPA group compared with control group in lens tissue. Administration of Vit B6 decreased lens GPx activity in rats treated with VPA.

GR, one of the enzymes of the GSH redox cycle, has a critical role in maintaining a high cellular reduced GSH to oxidized GSH ratio. In our study, GR activity was higher in VPA group compared with control group in lens tissue. Administration of Vit B6 decreased lens GR activity in rats treated with VPA.

Similar to our study, it was revealed that AEDs may lead to the development of oxidative stress.⁴⁸ In our study, the results indicate that VPA induced lens damage is associated with increased oxidative stress.

AR is a monomeric-reduced, NADPH-dependent enzyme and a member of aldo–keto reductase superfamily.⁴⁹ This enzyme is key enzyme in the polyol pathway. The activation of this pathway could lead to the depletion of NADPH, thereby perturbing the cellular redox poise, leading to the formation of oxidative stress. In this study, VPA-treated rats showed increase in the activities of AR compared to control rats. However, administration of Vit B6 decreased lens AR activity in rats treated with VPA. The decrease in those increasing AR activity shows that Vit B6 prevented damage in the lens tissue.

In conclusion, the results indicate that VPA induced lens damage is associated with increased oxidative stress. Elevated and decreased lens enzyme activity might be considered as VPA toxicity. The results of the present study show that Vit B6 effectively ameliorated VPA-induced oxidative stress. The protective effect of Vit B6 may be due to its antioxidant property. Vit B6 possesses therapeutic value in preventing complications associated with VPA-induced oxidative stress and may be used clinically VPA-induced lens damage during epilepsy disease.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

This work was supported by Scientific Research Projects Coordination Unit of Istanbul University, Istanbul, Turkey (project number: YADOP-29289).

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The effects of vitamin B6 on lens antioxidant system in valproic acid-administered rats

Abstract

Keywords

Introduction

Material and methods

Animals and experimental design

Biochemical assays

Statistical analysis

Results

Discussion

Footnotes

Conflict of interest

Funding

References