Abstract: The aim of the present study was to evaluate the effect of various treatment of glycerol, mannitol, neurotol and neurotol plus on xanthine oxidase, adenylate kinase activities and MDA levels in alcohol induced ischemic rat model. Twenty Wistar rats (weighing 100- 200 g) were divided into four groups, glycerol treated group (20%), mannitol treated group (10%), fixed dose combination of glycerol (10%)+ mannitol (20%) (Neurotol) treated group and neurotol plus treated group (glycerol (10%)+mannitol (10%)). A significant decrease in xanthine oxidase activity, adenylate kinase activity and MDA levels were observed on treatments but decrease was maximum in neurotol plus treated groups suggesting that it has better free radical scavenging activity than glycerol, mannitol and neurotol.
INTRODUCTION
Brain injury is one of the major causes of increased intracranial pressure (ICP), secondary deterioration and death in patients after stroke (Hacke et al., 1996; Muizelaar et al., 1991). Oxidative stress in the brain occurs when the generation of ROS overrides the ability of the endogenous antioxidant system to remove excess ROS subsequently leading to cellular damage. Several cellular features of the brain suggest that it is highly sensitive to oxidative stress. For example, brain is known to possess the highest oxygen metabolic rate of any organ in the body (Maiese, 2002). It consumes approximately 20% of the total amount of oxygen in the body (Shulman et al., 2004). This enhanced metabolic rate leads to an increased probability that excessive levels of ROS will be produced. It has been proposed that toxic metabolites from xanthine oxidase (XO) contribute to the development of injury seen during reperfusion of a variety of ischemic tissues (Patt et al., 1988). Adenylate kinase is also a marker enzyme for brain tissue injury.
Various new therapeutic strategies have been developed to reduce ICP that preserve the integrity of nerve cells and also prevent the accumulation of toxic metabolites. Substances such as glycerol and mannitol have been shown to decrease edema formation in the brain (Kaufmann and Cardoso, 1999; Kofke, 1993; Sheikh et al., 1996). Combination of mannitol and glycerol comprises of two sugars with better osmotic diuretic properties (Freshman et al., 1993; Manno et al., 1999; Takagi et al., 1984). Either glycerol or mannitol can be administered individually, however the addition of glycerol to mannitol avoids rebound edema likely to be observed with the intravenous administration of only mannitol (Berger et al., 1995; Soustiel et al., 2006). This provides a strong rationale for combining glycerol and mannitol in the management of cerebral edema and raised intracranial tension or pressure (Soustiel et al., 2006). This combination strategy is able to enhance the diffusion of water from cerebrospinal fluid back into plasma by elevating the osmolality of the plasma (Yilmaz et al., 2007). It rapidly enters cerebrospinal fluid and brain compartments and favorably affects the recovery from stroke in two ways, one is by redistribution of cerebral blood flow with increase in regional cerebral blood flow and regional cerebral blood volume in ischemic brain and other is by reducing focal cerebral edema (Sacco et al., 2004).
In the present study, we evaluated effect of various treatment of glycerol, mannitol, neurotol and neurotol plus on xanthine oxidase, adenylate kinase activities and MDA levels in alcohol induced ischemic rats.
MATERIALS AND METHODS
Study Conduct
The study was carried out from 10th April 2009 to 25th July 2009 in pre-clinical
unit of Venus Medicine Research Centre, Venus Remedies Ltd. Baddi (India).
Chemicals
All the biochemicals used in the present study were procured from Sigma,
St. Louis, MO, USA. Other chemicals, purchased locally, were of analytical grade.
In vivo Studies
Twenty Wistar rats (weighing 100-200 g) were used in the experiment. The rats
were fed standard pelleted diet and sterile water ad libitum. The test
room was air conditioned with temperature 23±2°C, humidity 65±5%
and with artificial fluorescent light (10 and 14 h of light and dark, respectively).
Experimental rats were intubated intragastrically with single dose of 25% alcohol.
The rats were divided into four groups of six animals each as given below:
| • | Glycerol treated group (20%) |
| • | Mannitol treated group (10%) |
| • | Neurotol treated group (FDC of glycerol (10%)+mannitol (10%)) |
| • | Neutrol plus treated group (FDC of glycerol (10%)+mannitol (20%)) |
The respective drugs were administered intravenously (20 μL g-1 body weight). Blood samples were collected at 30 min and 2 h intervals before and after respective treatment by the retro-orbital plexus into heparinized vials under the light anesthesia.
Plasma Preparation
Citrate containing blood samples were centrifuged at 7600 rpm for 15 min
at 0-4°C and plasma was separated out. It was used for the analysis.
Xanthine Oxidase Activity
Assay of xanthine oxidase was carried out essentially according to the method
described by Roussos (1967). The assay mixture, in final volume of 3.0 mL, consisted
of 0.30 mL Tris-HCl buffer, 50 mM pH 7.4; 0.30 mL CuSO4, 10 mM; 0.05
mL. Xanthine, 2.58 mM per mL in 0.05 M glycine buffer, pH 7.4; 0.1 mL of diluted
blood and water to make up the volume. Change in absorbance was recorded at
290 nm at 15 sec interval for 1 min. Suitable control was run simultaneously.
One unit of activity has been defined as change in absorbance at 290 nm in 1
min by 1 mL enzyme preparation.
Adenylate Kinase Assay
Adenylate kinase assay was performed by method of Lamprecht and Trautschold
with minor modification (Lampreht, 1963). The reaction mixture 3.0 mL consisted
of ADP 0.30 mL (4.0 mM), 0.55 mL of glucose (10 mM), 0.55 mL of MnCl2 (10
mM) 0.30 mL of NADP (0.2 mM), 0.58 mL of Tris buffer (50 mM, pH 7.4), 10 μg
of hexokinase (10 unit), 10 μg of glucose 6 phosphate dehydrogenase (1
unit) and added water to make up the 3.0 mL. The reaction was started by adding
20 μL of sample. Change in absorbance was recorded at 340 nm at 15 sec
interval for 1 min. Suitable control was run simultaneously. One unit of AK
activity in the forward direction was defined as 1 μmole of ADP removed/min
at 37°C under experimental condition.
Estimation of Malonaldialdehyde Level
Free radical mediated damage was assessed by the measurement of the extent
of lipid peroxidation in the term of malonaldialdehyde (MDA) formed, essentially
according to Ohkawa et al. (1979). It was determined by thio barbituric
acid reaction. The reaction mixture consisted of 100 μL of blood suspension,
0.20 mL of 8.1% Sodium Dodecyl Sulphate (SDS), 1.5 mL of 20% acetic acid, 1.5
mL of 0.8% thio barbituric acid (TBA) and water to make up the volume to 4.0
mL. The tubes were boiled in water bath at 95°C for 1 h, immediately cooled
thereafter under running tap water and 1.0 mL of water and 5.0 mL of mixture
of n-butanol and pyridine (15:1 v/v) was added and vortexed. The tubes were
centrifuged at 3500x g for 30 min. The upper layer was aspirated out and absorbance
measured at 532 nm. The reference used was 1, 1, 3, 3 tetra ethoxy propane.
The study was approved by the institutional animal ethical committee.
RESULTS
Xanthine Oxidase Activity
A significant increase in xanthine oxidase activity was observed 2 h after
the intoxication with alcohol as compared to 30 min after intoxication. Its
activity was significantly reduced on treatment with glycerol, mannitol, neurotol
and neurotol plus as compared to its activity after 30 min of intoxication and
2 h of intoxication, respectively. A significant decrease in xanthine oxidase
activity was observed after 30 min and 2 h of the treatment with the study drugs
as compared to just before the initiation of the treatment i.e., 2 h after the
alcohol intoxication. The percentage decrease in xanthine oxidase activity after
30 min of treatment was 65.8, 61.8, 68.5 and 73.6% in glycerol, mannitol, neurotol
and neurotol plus treated groups respectively as compared to respective control
value before the initiation of the treatment. The percent decrease after 2 h
of the treatment was 74, 70, 73.1 and 85.4% in glycerol, mannitol, neurotol
and neurotol plus treated groups, respectively as compared to respective control
value before the initiation of the treatment (Fig. 1).
Assay of Adenylate Kinase
A significant increase in adenylate kinase activity was observed 2 h after
the intoxication with alcohol as compared to 30 min after intoxication. Its
activity was significantly reduced on treatment with glycerol, mannitol, neurotol
and neurotol plus as compared to its activity after 30 min of intoxication and
2 h of intoxication, respectively. A significant decrease in adenylate kinase
activity was observed after 30 min and 2 h after the treatment with the study
drugs as compared to just before the initiation of the treatment i.e., 2 h after
the alcohol intoxication. The percentage decrease in adenylate kinase activity
after 30 min of the treatments was 12.1, 24.4, 31.6 and 25.6% in glycerol, mannitol,
neurotol and neurotol plus treated groups, respectively as compared to respective
control value before the initiation of the treatment. The % decrease in its
activity after 2 h of the treatments was 21.37, 19.66, 32.5 and 45.2% in glycerol,
mannitol, neurotol and neurotol plus treated groups respectively as compared
to respective control value before the initiation of the treatment. In glycerol
and mannitol treated groups no significant change in adenylate kinase activity
was observed between 30 min after the treatment and 2 h after the treatment,
where as decrease was significant in neurotol and neurotol plus treated groups
(Fig. 2).
Estimation of Malonaldialdehyde Level in Blood
A significant increase in MDA levels were observed 2 h after the intoxication
with alcohol as compared to 30 min after intoxication. A significant decrease
in MDA levels were observed after 30 min and 2 h after the treatment with the
study drugs as compared to just before the initiation of the treatment i.e.,
2 h after the alcohol intoxication.
| Fig. 1: | Effect of various treatments on xanthine oxidase activity |
| Fig. 2: | Effect of various treatments on adenylate kinase activity |
| Fig. 3: | Effect of various treatments on MDA levels |
The percentage decrease in MDA levels observed were 7.6, 2.2, 20.0 and 48.4% in glycerol, mannitol, neurotol and neurotol plus treated groups, respectively as compared to respective control value before the initiation of the treatment. The percentage decrease after 2 h of the treatment was 42.2, 24.4, 52.6 and 70.6% in glycerol, mannitol, neurotol and neurotol plus treated groups, respectively as compared to respective control value before the initiation of the treatment. In glycerol and mannitol treated groups no significant change in a MDA levels was observed between 30 min after the treatment and 2 h after the treatment, where as decrease was significant in neurotol and neurotol plus treated groups (Fig. 3).
DISCUSSION
Acute ischemic stroke is a major cause of mortality (Ikeda and Long, 1990). Stroke generally refers to a local interruption of blood flow to the brain and is the leading cause of long-term disability, third leading cause of death (Young et al., 2007). Approximately 12% of strokes are hemorrhagic (rupture of a cerebral blood vessel), whereas the remaining 88% are ischemic and result from occlusion of a cerebral artery (either thrombolic or embolic). Blockage of a cerebral artery results in interruption of the blood flow and supply of nutrients, glucose and oxygen to the brain. Collins et al. (1998) has been established alcohol intoxication to cause ionic imbalances leading to increase in ICP and brain edema, which is further known to be associated with oxidative stress, lipid peroxidation and neurodegenration.
Oxidative stress is the one of the cause of neuronal injury during brain ischemia/reperfusion (Ozkul et al., 2007; Reynolds et al., 2007; Taylor and Crack, 2004). It was accompanied by increase in free radicals, including superoxide anion (O2●B), hydroxyl radical (●OH) and hydrogen peroxide (H2O2), along with simultaneous decrease in endogenous antioxidant system, including antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase, or antioxidants, glutathione (GSH), Vitamin (Vit) C and Vit E (α-tocopherol). The highly reactive ●OH is formed from H2O2 in the presence of divalent metal ions, especially Fe2+ and Cu2+ via the Fenton reaction. In addition, during ischemia, xanthine dehydrogenase undergoes irreversible proteolytic conversion to XO, producing O2●B and H2O2 in the presence of oxygen (Ozkul et al., 2007). O2●B does not directly induce LPO but can react with ●NO to form cytotoxic peroxynitrite (ONOO¯) (Bromont et al., 1989; Collins et al., 1998).
In the present study, a significant decrease in xanthine oxidase activity was observed on treatments but decrease was maximum in neurotol plus treated groups suggesting that it has better free radical scavenging activity than glycerol, mannitol and neurotol. Steinberg et al. (1996) reported that adenylate kinase is a marker enzyme for ischemic brain injury and its activity has been reported to increase on injury. Similarly, a significant decrease in adenylate kinase activity was observed in all treated groups but maximum decrease was found that in neurotol plus treated group as compared to all other treated groups.
Tien et al. (1981) reported that mannitol produced more than 95% inhibition of lipid peroxidation initiated by Fenton reaction generated hydroxyl radical. Glycerol has also been reported to reduce lipid peroxidation induced by lipid polysaccharides. There other reports suggesting glycerol as oxygen radical scavenger. In the present study, a significant decrease in MDA levels were observed in all the treated groups as compared to respective controls and decrease in MDA levels were much more in neurotol plus treated group as compared to other treated groups clearly indicating that neurotol plus has been most effective in treatment of alcohol induced ischemic brain injury.
In conclusion, these results establish that neurotol plus was more effective and fast free radical scavenger than glycerol, mannitol and neurotol in the treatment of ischemic brain injury.
ACKNOWLEDGMENTS
Authors are thankful to Mr. Parveen Kumar (Technical Assistant) to support in experiment handling as well as financial department of Venus Medicine Research Centre for financial support.