EffEcts of isolatEd vitamin B 6 supplEmEntation on oxidativE strEss and hEart function paramEtErs in ExpErimEntal hypErhomocystEinEmia

Methods: Fifteen male Wistar rats were divided into three groups according to their treatment. Animals received water and food ad libitum and an intragastric probe was used to administer water for 60 days (groups: CB6, HcyT, and HB6). On the 30th day of treatment, two groups were supplemented with VB6 in the drinking water (groups: CB6 and HB6). After 60 days of treatment, homocysteine (Hcy), cysteine, and hydrogen peroxide concentration, nuclear factor (erythroid-derived 2)-like 2 (NRF2) and glutathione S-transferase (GST) immunocontent, and superoxide dismutase (SOD), catalase (CAT), and GST activities were measured.

Homocysteine (Hcy) is recognized as an independent risk factor for atherosclerosis, which is a leading cause of vascular disease worldwide.It may participate in the development of cardiovascular diseases through its effects on smooth muscle and vascular endothelium cells 1 .In addition, ischemic heart disease remains a leading cause of premature adult mortality 2 .
Hcy is a non-protein-forming, sulfur-containing amino acid which is metabolized through two pathways: transsulfuration to cysteine (Cys) and remethylation to Met 3 .Hyperhomocysteinemia (Hhe) can be defined as Hcy plasma concentration > 12 µmol/L, which is considered a risk factor for cardiovascular disease 4,5 .Vitamin B 6 (VB 6 ) participates in the transsulfuration of Hcy as an enzymatic cofactor of CβS (cystationine β synthase) with cysteine as a final product 6 .Mendes et al.Apart of its role in Hcy metabolism, VB 6 consumption decreases the risk for neuropsychiatric disorders, including seizures, migraine, chronic pain, and depression, and cardiovascular diseases, such as atherosclerosis and endothelial cell proliferation 7,8 .
Nutritional factors play a fundamental role on Hcy metabolism.VB 6 is a water-soluble vitamin that can be found in different types of food, including fish, poultry, whole grains, potatoes, vegetables, and nuts.It exists in several forms such as pyridoxal, pyridoxine, pyridoxamine, and its active form, pyridoxal 5'-phosphate (PLP).Low VB 6 concentrations could reflect, thus, an increased consumption of PLP that is associated with an accelerated synthesis of inflammatory cytokines 9 .Additionally, VB 6 appears to control reactive oxygen species (ROS) production similarly to vitamins C and E. It is well established in the literature that increased oxidative stress is associated with VB 6 deficiency [10][11][12] .Moreover, a recent case-control study found that a lower status of B vitamins (B 12 , B 6 , and folate) in the diet and in the serum concentration are involved in the etiology of hyperhomocysteinemia (Hhe), cardiovascular disease, and oxidative stress 13 .
Thus, the aim of this study was to evaluate oxidative stress and heart function parameters after isolated VB 6 supplementation in an experimental Hhe.

Animals and Treatments
The experiment was in accordance with the Guidance for the Description of Animal Research in Scientific Publications from the National Research Council (US) Institute for Laboratory Animal Research 14 .
Fifteen male Wistar rats (30 days old) from the Animal House of the Universidade Federal de São Paulo, maintained in standard conditions (12h/12h light/dark cycle, room temperature at 21ºC, food and water ad libitum), were divided into three groups: 1) CB 6 : rats received water by intragastric probe for 60 days, and from the 30th to the 60th day they also received supplementation with pyridoxine hydrochloride (60 mg/kg) diluted in the water; 2) HcyT: rats received water containing homocysteine thiolactone (L-homocysteine thiolactone hydrochloride, Sigma; 100 mg/kg) by intragastric probe for 60 days; 3) HB 6 : rats received water containing homocysteine thiolactone (100 mg/kg) by intragastric probe for 60 days, and from the 30th to the 60th day they also received supplementation with pyridoxine hydrochloride (60 mg/kg).Animals were weighed at the beginning and at the end of the experiment.After 60 days, animals were euthanized by decapitation, and blood and hearts were collected.
Blood was immediately processed, and hearts were stored at -80ºC for posterior biochemical analysis.

Homocysteine and Cysteine Assay
Blood was centrifuged for 5 minutes at 3,000 g in tubes containing anticoagulant, and plasma was collected.Total homocysteine and cysteine levels were determined by high performance liquid chromatography (HPLC) with fluorescence detection, as performed by Oliveira et al. 15 .

ROS Evaluation
Hydrogen peroxide was measured through the horseradish peroxidase (HRPO)-mediated oxidation of phenol red.Slices of left ventricular (LV) tissue were incubated for 30 minutes at 37°C in 10 mmol/L phosphate buffer (140 mmol/L NaCl and 5 mmol/L dextrose).Supernatants were transferred to tubes with 0.28 mmol/L phenol red and 8.5 U/mL HRPO.After 25 minutes of incubation, 1 mol/L NaOH was added and the absorbance of the solution was measured at 610 nm.Results were expressed in nmoles H 2 O 2 per g of tissue 16 .

Western Blot Analysis
Thirty micrograms of protein were submitted to a 12% one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 17 .The separated proteins were transferred to nitrocellulose membranes using a buffer containing 20 mmol/L Tris, 150 mmol/L glycine, 20% (v/v) methanol, 0.1% (w/v) SDS, pH 8.2, in a cooled Bio-Rad TransBlot unit.Non-specific protein-binding sites were blocked with non-fat milk in Tris-buffer for 1 hour 18 .The membranes were processed for immunodetection using rabbit anti-NFR2 (52kDa) and mouse anti-GST (26kDa) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) incubated at 4°C.Bound primary antibodies were detected using rabbit anti-goat and rabbit anti-mouse, and membranes were developed using chemiluminescence.Autoradiographic images were scanned and analyzed using an image software (Image master VDS CI, Amersham Biosciences Europe, IT).Results of each membrane were normalized through Ponceau S staining 19 .

Determination of Protein Concentration
Protein was measured by the method of Lowry et al. (1951) using a standard solution of bovine serum albumin 20 .

Preparation of Erythrocytes
Heparinized venous blood samples were washed in a solution of 9 g/L sodium chloride and centrifuged three times at 3000 g for 10 minutes at Heart function, oxidative stress and vitamin B 6 room temperature.White cells were discarded by aspiration.Erythrocytes were diluted 1/10 in 1 mM acetic acid and 4 mM magnesium sulfate, placed on an ice bath for 10 minutes, and centrifuged at 4200 g for 20 minutes at 0ºC.Supernatant was used for enzyme assays 21 .

Determination of Antioxidant Enzyme Activities in Erythrocytes
The quantification of superoxide dismutase (SOD) activity, expressed as units per milligram of protein, was based on the inhibition of superoxide radical reaction with pyrogallol 22 .Catalase (CAT) activity was determined by following a decrease in hydrogen peroxide (H 2 O 2 ) absorbance at 240 nm.It was expressed as nanomol of H 2 O 2 reduced per minute per milligram of protein 23 .Glutathione-S-transferase (GST) activity, expressed as nanomol per milligram of protein, was measured by the rate of formation of dinitrophenyl-S-glutathione at 340 nm 24 .

Echocardiographic Evaluation
A transthoracic echocardiography was performed in all experimental animals at the end of treatment using a SEQUOIA 512 equipment (ACUSON, Mountain View, CA, USA), with a 13-MHz linear transducer.Rats were anesthetized with a combination of ketamine (80 mg/kg, ip) and xylazine (10 mg/kg, ip).Images were obtained with the transducer placed on the animal's shaved chest in the supine position and were optimized using a transmission gel.All measurements were based on an average of three consecutive cardiac cycles.Echocardiographic indices were obtained according to the recommendations of the American Society of Echocardiography.Ejection fraction (EF) was calculated from M-mode recordings to assess the following parameters: (1) morphometric: LV mass, LV diastolic diameter corrected by weight (LVDD/kg), and relative wall thickness (RWT); (2) systolic function: EF; (3) diastolic function: LV isovolumic relaxation time (IVRT) and ratio of peak velocity of early (E) and late (A) diastolic filling (E/A ratio); and (4) overall function: myocardial performance index (MPI), as previously described 25 .

Statistical Analysis
Data are expressed as mean ± standard deviation (SD).Data were tested for normal distribution using the Shapiro-Wilk test, and the one-way analysis of variance followed by the Tukey test was used to compare groups.Pearson's correlation was used to study the association between heart function parameters and CAT (pmol/mg protein) and GST (nmol/mg protein).Significance level was established at P ≤ 0.05.Data were analyzed using SPSS, version 19.0.

RESULTS
Total Hcy plasma concentration (μmol/L) was higher (98%) in the HcyT group after treatment compared to the CB6 group, demonstrating the efficiency of our experimental protocol.Cysteine concentration and body weight were not affected by the treatment with HcyT (table 1).
Regarding antioxidant enzyme activities, there was a decrease in SOD activity (U/mg protein) and GST activity (nmol/mg protein) in erythrocytes of the HB 6 group compared to the CB 6 group.Conversely, the HB 6 group showed an increase in GST activity compared to the HcyT group.Furthermore, CAT activity (pmol/mg protein) was lower in the HcyT group compared to the CB 6 group (table 2).
There was an increase (62%) in H 2 O 2 concentration in the HcyT group compared to the CB 6 group.Mendes et al.
However, H 2 O 2 concentration in the HB6 group was similar to that in the CB 6 group (figure 1).
The HcyT group showed a significant decrease (46%) in NRF2 immunocontent in relation to the CB 6 group (figure 2).Furthermore, the HB 6 group showed an increase in GST immunocontent (51%) compared to the HcyT group (figure 3).
In relation to heart function, the HB 6 group showed increased LVDD/Kg in relation to the CB 6 and HcyT groups.Moreover, RWT is decreased in HB 6 group in relation to the HcyT group.Considering LV systolic function variables, EF was significantly decreased (9.5%) in the HB 6 group compared to the CB 6 group, as well as in the HcyT group compared to the CB 6 (20%) and HB 6 (10%) groups.Global LV function assessment with MPI was increased in the HcyT group in relation to the CB 6 (29.5%) and HB 6 (30%) groups.E/A ratio was enhanced in the HcyT group compared to the CB 6 group.Conversely, the HB6 group showed decreased IVRT in relation to the CB 6 and HcyT groups (table 3).
Correlations were found between increased CAT activity and increased MPI (r = 0.71; P = 0.006), as well as in increased E/A ratio (r = 0.6; P = 0.01).
Systolic cardiac function, measured by echocardiogram and represented by EF, was correlated with increased concentration of GST activity (r = 0.62; P = 0.02).

DISCUSSION
The main findings of the present study were that isolated VB 6 supplementation reduced plasma concentration of Hcy, restored antioxidant enzyme activities in erythrocytes, reduced H 2 O 2 concentration,   We observed that Hcy plasma concentration decreased significantly after the CB 6 and HB 6 groups received isolated VB 6 supplementation for 30 days compared to the HcyT group.Previous studies have demonstrated that VB 6 deficiency induced a significant Hhe.We also observed that folate supplementation improves Hhe induced by VB 6 deficiency 26 .Conversely, epidemiological studies have demonstrated that VB 6 has an important role in the prevention of atherosclerosis regardless of the reduction of plasma homocysteine concentration, but the exact mechanism responsible for this remains unclear [27][28][29] .One plausible Heart function, oxidative stress and vitamin B 6 mechanism is the endogenous production of H2S by the transsulfuration pathway that mediates cysteine production from catabolism of Hcy.H2S is a gaseous signaling molecule that modulates physiologic actions, such as relaxation of smooth muscle, and attenuates myocardial ischemia-reperfusion injury by protecting mitochondrial function 30 .These findings, together with ours, suggest that VB 6 is important for cerebrovascular protection and that the mechanism involved in this process could be related to the transsulfuration pathway.
Furthermore, VB 6 supplementation did not change the body weight of the animals.This result demonstrates that VB 6 supplementation did not influence the metabolic control of animals (data not shown).
The groups that received VB 6 supplementation (CB 6 and HB 6 ) showed a significant reduction in H 2 O 2 , a signaling molecule, indicating that VB 6 might act as a ROS quencher.A recent study reported that a VB 6 deficient diet seemed to mediate the oxidative stress in connection with the redistribution of glutathione from liver to plasma, but did not further affect glutathione-related enzyme activities in mice with homocysteine-induced oxidative stress 31 .Another previous study with Japanese women demonstrated that VB 6 plays a role against oxidative DNA damage, but not folate and homocysteine 32 .One of the possible reasons for our results is the ability of VB 6 to inhibit xanthine oxidase activity, which is an enzyme responsible for the formation of uric acid and hydrogen peroxide 33 .
The detoxification of xenobiotics seems to be related with the GST enzyme and is regulated by NRF2.In the present study, GST immunocontent increased significantly in the groups that received VB 6 supplementation, and the HB 6 group showed the highest level of GST immunocontent.One plausible explanation for this result is that VB 6 supplementation preserved GST immunocontent.Conversely, the depletion of GST occurs during Hhe due to the role of this enzyme in cysteine metabolism.
In the present study, the HB6 group showed an increase in NRF2 immunocontent compared to the HcyT group.The method used for this evaluation is suitable for the cytoplasmic portion; however, it is known that when activated NRF2 is in the nuclear portion.It is a possible explanation for an increased NRF2 immunocontent in the cytoplasmic portion.Other study using hepatoma cell line demonstrated that Hcy activates NRF2 transcription (from the cytoplasmic portion to the nuclear portion) inducing antioxidant-related genes 34 .
Regarding antioxidant enzyme activities in erythrocytes, we observed that SOD and GST activities decreased in the HcyT group compared to the CB 6 group.We also found that CAT activity increased in the HcyT group when compared to the CB 6 and HB 6 groups.Additionally, GST activity increased in the HB 6 group compared to the HcyT group.The relevance of measuring blood oxidative stress parameters by standardized methods may be useful to define the role of oxidative stress in different diseases and also for clinical diagnosis 21,35 .
The exact mechanism by which VB 6 acts as an antioxidant is not clear yet.One possible direct mechanism might be that VB 6 has both hydroxyl and amine groups as substitutes on a pyridine ring, which may react with peroxy radicals and thereby scavenge radicals and lipid peroxides.Moreover, an indirect mechanism by which VB 6 compounds play the role of antioxidant may be through serving as a coenzyme in the glutathione enzyme system 36 .
In the present study, LV mass and LVDD/kg was increased in the HB 6 group when compared to the other groups.A previous study with chronic uremic patients demonstrated a direct correlation between total plasma homocysteine level and LV mass index 37 .One possible reason for this result is the duration of isolated VB 6, which was not long enough to protect cardiac morphological against the Hhe effect.Conversely, concerning systolic function, EF was decreased in the HB 6 group compared to the CB 6 group.However, the HB 6 group showed a significant improvement in relation to the HcyT group.A previous study investigated the effect of micronutrient supplementation (calcium, magnesium, zinc, copper, selenium, vitamin A, thiamine, folate, vitamin B 12 , C, E, D, and coenzyme Q10, riboflavin, VB 6 ) for 9 months on elderly patients with chronic cardiac failure and observed a significant improvement in EF 38 .This result, in agreement with ours, reinforces the hypothesis of association between redox balance and cardiac function.
In relation to LV diastolic function, IVRT was reduced in the HB 6 group in relation to the CB6 and HcyT groups.This finding might be suggestive of diastolic dysfunction, probably by the HcyT effect of increased endothelial-myocyte uncoupling, resulting in hypertension 39 .E/A ratio was increased in the Hcy group compared to the CB 6 group.In agreement with our findings, other study investigated the effects of plasma Hcy on LV diastolic function of Chinese patients with essential hypertension and found a lower E/A ratio and significant correlations between LV diastolic function indices and Hcy levels 40 .Our results suggest that the period used for VB 6 supplementation was not enough to protect diastolic function from Hhe.
LV global function by pulsed-wave MPI was increased in the HcyT group in relation to the CB 6 group.
However, the HB 6 group showed a decrease in MPI, as expected.MPI has prognostic values in different diseases, including chronic obstructive pulmonary disease and pulmonary artery hypertension 41,42 .We found positive correlations between increased CAT activity and increased MPI, which could be explained by the high production of H 2 O 2 in the body that will need to be converted by CAT.H 2 O 2 toxicity will probably act negatively on heart function.Considering the increase in E/A ratio, we suggest that exists an association between increased H 2 O 2 production, which is converted by CAT, and the myocardial stiffness caused by increased E/A ratio.EF was also correlated with an increase in GST activity, indicating a protective role of this enzyme in systolic heart function.This correlation reinforces the association of oxidative and heart function parameters in this experimental model.
Collectively, these findings demonstrated that isolated supplementation of VB 6 seems to modulate antioxidant enzyme activities and some heart function parameters.

Figure 2 :
Figure 2: Nrf2 immunocontent in cardiac tissue: Protein expression of Nrf2 was measured by western blot technique in cardiac tissue of CB6 (n=5), HT (n=5) and HTB6 (n=5) animals.Image represents a blot containing Nrf2 immunocontent of three animals per group.Data are represented by mean ± SD of mega pixels of each experimental.† p<0.05 vs. CB6 and ‡ p<0.05 vs. HT (p<0.05).

Figure 3 :
Figure 3: Glutathione S-Transferase immunocontent in cardiac tissue: Protein expression of GST was measured by western blot technique in cardiac tissue of CB6 (n=5), HT (n=5) and HTB6 (n=5) animals.Image represents a blot containing GST immunocontent of three animals per group.Data are represented by mean ± SD of mega pixels of each experimental.† p<0.05 vs. CB6 and ‡ p<0.05 vs. HT (p<0.05).

Table 1 :
Metabolic evaluations in studied groups.

Table 2 :
Antioxidant enzyme activities in erythrocytes.

Table 3 :
Parameters on echocardiography of experimental groups.