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ORIGINAL ARTICLE
Year : 2021  |  Volume : 9  |  Issue : 2  |  Page : 61-67

Brain response to intraperitoneal and oral administration of monosodium glutamate in wistar rats


Department of Biochemistry, Faculty of Basic Medical Sciences, University of Calabar, Calabar P.M.B 1115 Cross River State, Nigeria

Date of Submission17-Sep-2020
Date of Acceptance25-Nov-2020
Date of Web Publication10-Aug-2021

Correspondence Address:
Dr. Uche Stephen Akataobi
Department of Biochemistry, Faculty of Basic Medical Sciences, University of Calabar, P.M.B. 1115, Calabar, Cross River State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_38_20

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  Abstract 


Background: It has been reported that at high concentration monosodium glutamate (MSG) administration induces neurological toxicity caused by increased concentration of glutamate that promotes the production of free radicals and apoptosis. The blood–brain barrier is used by the brain to protect itself against the effect of glutamate and other neurotoxins but its level of protection varies with age. Aim and Objective: In this present study, we examined brain responses to combined intraperitoneal and oral administration of MSG at different doses in Wistar rats. Material and Method: 4 mg/g MSG was administered intraperitoneally to neonates in postnatal days 2, 4, 6, 8, and 10–2 groups and one of the groups was further administered 10 mg/g MSG orally as adult, while the last group received 10 mg/g MSG as adults only for 21 days. At the end of the 21 days, brain tissue was collected and used to determine MSG effect. Results: In the brain tissue, MSG administration caused a significant increase (P < 0.05) in glutamate decarboxylase, glutamate synthase, catalase, and glutathione peroxydase activities in a concentration dependent manner higher in group that received MSG both in neonate and adult. Superoxide dismutase also showed a significant increase (P < 0.05) in the treated groups but higher in group administered as neonates only. Conclusion: The result showed that MSG administration increased the level of neurotransmitters in both neonate and adult groups similarly and in response the brain increase the activity of the respective catabolic enzyme to protect itself against its effect

Keywords: Free radicals, monosodium glutamate, neurotoxicity


How to cite this article:
Akataobi US, Unanaowo BE, Michael OC, Arong WO. Brain response to intraperitoneal and oral administration of monosodium glutamate in wistar rats. Niger J Exp Clin Biosci 2021;9:61-7

How to cite this URL:
Akataobi US, Unanaowo BE, Michael OC, Arong WO. Brain response to intraperitoneal and oral administration of monosodium glutamate in wistar rats. Niger J Exp Clin Biosci [serial online] 2021 [cited 2021 Dec 4];9:61-7. Available from: https://www.njecbonline.org/text.asp?2021/9/2/61/323669




  Introduction Top


One of the discoveries made in the forties by two ophthalmology residents Lucas and Newhouse leads to the understanding of the harmful role of artificial sweeteners like monosodium glutamate (MSG) to the brain, mostly the nerve cells in the inner layer of the retina where it causes severe destruction.[1] Despite the disturbing effects of MSG published in their study, it went unnoticed and the food industries continued the addition of MSG to their products until a decade later when Dr. Olney reported a more shocking discovery which supported the work of Lucas and Newhouse and added that MSG did not only cause severe damage to the neurons in the retina of the eye but further caused destruction in the hypothalamus and other areas of the brain adjacent to the ventricular systems known as the circumventricular organ.[2] The report included that the damage occurs majorly in immature or newborn animals and suggested that this area of the brain was affected severely because it may not have a blood–brain barrier system or the level of development was not strong enough to protect it against toxic substance from the blood such as glutamate obtained from the breakdown of MSG.[2]

MSG effect has also been reported to cause brain damage in offspring of exposed pregnant rhesus monkeys,[7],[2] making it a serious danger to pregnant women who can endanger their offspring when glutamate from MSG from the mother's blood enter blood of the unborn baby according to Dr. Olney.[2] Glutamate a neurotransmitter and the major component of MSG is found normally in the brain and spinal cord as a common transmitter chemical may become a deadly toxins to the neurons with glutamate receptors as well as the nerve cells connected to these neurons when their concentration increases to a critical level.[4] This thus means that glutamate may destroy not only the neurons having glutamate receptors but can kill all neurons connected to it that uses other receptors[3] by causing neurons to be extremely excited and in large dose may cause the cells to degenerate and die.[5] As a result, the nervous system places a mechanism that carefully controls the amount of glutamate present in the fluid around it known as extracellular space using different methods, one of which include a special pumping system that removes and return glutamate to circulation from the brain,[2] this pumping system is reported to be very effective a reason why it takes large doses of MSG to actually damage the neurons of the experimental animal according to the report of Dr. Olney.[2],[9] In recent studies it has been noted that many cells mostly neurons contain channels involved in regulating calcium entry which play important role in normal functioning of the neurons including their activation and ability to transmit impulses,[6] binding of a neurotransmitter to the receptor on the neuron fiber membrane causes the calcium channel to open allowing an inflow of calcium which triggers the neurons to be activated and fire in a carefully regulated manner.[7] It has been suggested that glutamate works by opening the calcium channel via certain receptors[8] and when neurotransmitters come in contact with receptor in too high concentration or for a long period causes the calcium channels to take a permanent open position which allows calcium to enter in large amount in the cell and triggering a protective mechanism,[2] the inflow of calcium pump uses ATP which is in continues supply and calcium channel appears to play vital role in certain clinical conditions such as brain injuries.[9] Entry of calcium into the cell activates a messenger known as protein kinase C a runner which in turn causes calcium to be released from endoplasmic reticulum and more calcium entry into the cell.[10] According to Russell[2] protein kinase C alters membrane calcium channel structure causing it to be stuck in the open position allowing a continuous pouring of calcium into the cell which activates an enzyme called phospholipase C that breaks down fatty acid composition of the cell membrane and released of arachidonic acid. This released fatty acid is attacked by two different enzymes lipoxygenase and cylooxygenase resulting in series of destructive reactions and production of chemical products that causes cell death.[7]

It has been proven that, the continuous entry of calcium and the reactions that follow lead to the production of free radicals (known for containing an atom molecule or group of atoms with an unpaired electron in it outer orbit). The free radicals can be viewed as a red hot particle with the ability to damage anything it comes in contact with,[11] once produced the free radicals take a position inside the call and react with other chemicals present in the cell which results in the destruction of the cell.[12] The bodies way of protecting itself against the activities of these free radicals involves the use of compounds that act as scavengers of these particles called antioxidants.[4] These antioxidants are known for the ability to absolve these harmful chemicals (particles) and have an advantage that allows them to be able to pass the blood–brain barrier into the brain cells where they are really needed most.[2] Studies have reported that the brain uses a system to guard itself against free radicals that involve three enzymes “superoxide dismutase, catalase and glutathione peroxidase[13] also it has been recorded that the brain contains little quantity of these enzymes, which makes it vulnerable the free radical attacks as a result of increased level of toxins (like glutamate from MSG), injuries and diseases.[3] This study is aimed at evaluating the brain response to a combined intra-peritoneal and oral administration of MSG at different concentrations to provide an understanding on the level and role of glutamate and antioxidants in the brain of exposed Wistar rats. This study also considered the age of exposure to MSG with regards to the level of development of the blood–brain barrier and its protective role against toxic substances, MSG in this case to determine if a difference exists between the effects of MSG on Wistar rats either as a neonate or as adult.


  Methods Top


Distribution and exposure to monosodium glutamate

Neonates of co-habited Wistar rats were used as experimental animals in the study according to the method described by Rotimi et al.,[14] following the day of delivery, pups were divided into four groups of seven animals each and administered a single dose of 4 mg/g of MSG (reconstituted in normal saline) intraperitoneally on postnatal day 2, 4, 6, 8 and 10 to two groups (group 2 and 3). The animals were grown for 32 weeks, groups 1 were given normal saline throughout the study period, at the end of 32 weeks, group 3 and 4 were given 10 mg/g MSG orally as adult using an oral gavage for 28 days [Table 1]. All the animals had free access to feed (rat chow) and water throughout the study.
Table 1: Experimental design

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Termination of the experiment and collection of samples

At the end of the study, all groups were fasted overnight and sacrificed under ketamine and the animals were decapitated and the skull was opened and the whole brain collected and suspended in an ice-cold phosphate buffer saline of pH 7.4 in preparation for biochemical assays.

Preparation of brain homogenate

The whole brain was thoroughly homogenized in phosphate buffer of pH 7.4, using a laboratory mortar and pestle, and centrifuged at 2000 rpm for 30 min. After centrifugation, the supernatant was withdrawn into a plain tube and used for biochemical analysis.

Glutamate decarboxylase activity assay

Glutamate decarboxylase catalyzed the conversion of glutamate to γ-amino butyrate (GABA). The enzyme activity was measured using the method described by Miester et al.[15] 20 μl of the supernatant was measured into a test tube followed by 100 μl reagent of 0.01M imidazole-HCl, 25 mM β-mercaptoethanol, 50 mM-sodium L-glutamate, 10 mM Na-ATP, 125 mM hydroxylamine, There after the mixture was shaken thoroughly and the pH adjusted to 6.8 using NaOH, and incubated for 15 min at 37°C and allowed to cool. Then 0.75 ml of cold solution mixture of 0.37M FeCl2, 0.67MHCl and 0.20M TCA was added. The reaction mixture was then centrifuged for 10 min at 3000 rpm and absorbance read at 535 nm.

Glutamate synthetase activity assay

Glutamate synthetase catalyzes the conversion of glutamate to glutamine in the glutamate–glutamine cycle. The activity of this enzyme was analyzed using the method described by Dao et al.,[16] 50 μl supernatant was measured into a test tube 100 μL of 50 mM imidazole-HCl (pH 6.8), l00 μL of 0.5 mM EDTA, 50 μl ml 1 mM dithiothreitol were added and supplemented with protein inhibitor and protein concentration of the supernatant was adjusted to 1 mg/ml. An aliquot of 150 ml was used, in a solution containing 1 ml 50 mM imidazole-HCl (pH 6.8), 10 ml 50 mM Gin, 2 ml 25 mM-Hydroxylamine, 2 ml 25 mM sodium arsenate, 2 ml 2 mM MnCl2, and 0.16 ml ADP and incubated for 30 min at 37°C. The reaction mixture was then stopped by adding 2 ml solution of 2.42% ferric chloride (FeCl3) and 1.45% TCA in 1.88% HCl. To remove the precipitate the tube was centrifuged for 5 min at 2000 rpm and supernatant read at 540 nm.

Superoxide dismutase activity assay

This was done according to the method described be Hacioglu, Ayse, and Imran,[17] involving the use of xanthine and xanthine oxidase to generate superoxide radicals which reacted with 2-4– idophenyl– 3-4 nitrophenol-5– phenylterazolium chloride (INT) to form red formazan dye, and the degree of inhibition of this reaction was used to measure enzyme activity where a unit of enzyme activity is that which caused 50% inhibition of the rate of INT reduction under the condition of the assay. Reagents used include R1a (mixed substrates), R1b (buffer), R2 (Xanthine oxidase), and Standard, exactly 15 μl of homogenate, standard and distilled water was added into 3 test tubes labeled test, standard and blank and 500 μl of R1 was added into each of the test tubes shaken to properly mixed, 75 μl R2 was then added into the tubes and mixed and first absorbance taken at 505 nm. After absorbance was taken every 30 s for 3 min and units Superoxide dismutase activity (SOD) per gram protein were determined from a standard curve thus;



Catalase activity assay

The enzyme activity assay was done according to the method described Okwudiri et al.[18] involving reduction of dichromate in acetic acid to chromic acetate when heated in the presence of hydrogen peroxide with the formation of per chromic acid as an unstable intermediate. Spectrophotometric determination of the chromic acid formed was done at 570 nm and the intermediate was allowed to split and H2O2 measured. Reagents used include phosphate buffer 0.01M (pH 7), hydrogen peroxide 0.2M, potassium dichromate 5%, dichromate acetic reagent; potassium dichromate and glacial acetic acid was properly mixed in the ratio of 1:3. Exactly 0.9 ml distilled water and 0.1 ml homogenate was measured into a test tube, mixed with 2 ml H2O2 and 2 ml phosphate buffer, and activated the reaction by mixing 2 ml of dichromate acetic acid reagent with 1 ml of the prepared mixture. Absorbance was taken in 30 s interval for 2 min and enzyme activity expressed as U/ml of plasma (U–micromoles of H2O2 utilized/s). Abs1 = absorbance at t = 0 s and Ab2 = absorbance at t = 30 s.



Glutathione peroxidase activity assay

This was done as described by King and Wootton,[19] exactly 0.1 ml of homogenate was mixed with 0.9 ml of distilled water. 0.02 ml of sodium sulfate was then added and the mixture allowed to stand for 2 min, and 0.02 ml (20%) lithium sulfate, 20% 0.2 ml of NaCO3, and 0.2 ml phosphor-18-tungstic acid was added to the mixture. The solution was made to stand for 4 min as the color develop after which 2.5 ml 20% sodium sulfate was also added and absorbance measured within 10 min and 0.1 ml of H2O in place of homogenate was set up and glutathione concentration was calculated from a standard curve.




  Results Top


Effect of monosodium glutamate treatment on glutamate decarboxylase activity

[Figure 1] shows MSG effect on glutamate decarboxylase activity. From the result, MSG administration caused a significant (P < 0.05) increase in glutamate decarboxylase activity in all the administered groups significantly in the group administered both as neonate and adult. This is followed by the group-administered as neonate only while the adult-only group showed the lowest glutamate decarboxylase activity.
Figure 1: Effect of monosodium glutamate administration on glutamate decarboxylase activity

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Values were expressed as mean ± standard error of mean (SEM), n = 7, 4 mg/g neonate plus 10 mg/g adult significantly higher (P < 0.05) versus normal control.

Effect of monosodium glutamate treatment on glutamate synthase activity

[Figure 2] shows the effect of MSG on glutamate synthase activity. The result indicated an increase in enzyme activity following MSG administration significant (P < 0.05) in the adult-only administered group versus normal control group while neonate-only and neonate plus adult administered groups did not indicate any significant difference in glutamate synthase activity between them.
Figure 2: Effect of monosodium glutamate administration on glutamate synthase activity

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Values were expressed as mean ± SEM, n = 7, 4 mg/g Neonate significantly (P < 0.05) different versus 10 mg/g adult; 10 mg/g adult significantly different (P < 0.05) higher versus normal control.

Effect of monosodium glutamate treatment on catalase activity

[Figure 3] shows MSG effect on catalase activity. The result indicated that MSG administration caused a significant (P < 0.05) increase in catalase activity in the administered groups versus normal control group. Neonate plus adult administered group showed a higher increase in enzyme activity followed by neonate only while the adult only group showed the least activity not significant (P > 0.05) versus normal control.
Figure 3: Effect of monosodium glutamate administration on catalase activity

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Values were expressed as mean ± SEM, n = 7, 4 mg/g neonate plus 10 mg/g adult significantly (P < 0.05) higher versus normal control.

Effect of monosodium glutamate treatment on glutathione peroxidase activity

[Figure 4] shows the effect of MSG on glutathione peroxidase activity. The result indicated that MSG administration caused a significant (P < 0.05) increase in glutathione peroxidase activity in the test groups versus normal control. The neonate plus an adult administered group showed a higher increase in enzyme activity while the adult and neonate only groups did not indicate any significant difference in the enzyme activity between them.
Figure 4: Effect of monosodium glutamate administration on glutathione peroxidase activity

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Values were expressed as mean ± SEM, n = 7, 4 mg/g Neonate plus 10 mg/g adult significantly (P < 0.05) higher versus normal control.

Effect of monosodium glutamate treatment on superoxide dismutase activity

[Figure 5] shows MSG effect on SOD. The result shows that MSG administration caused a significant (P < 0.05) increase in SOD in the administered groups versus control group. The highest enzyme activity was recorded in the group administered as neonate only followed by neonate plus adult group while the adult-only group indicated a lowest enzyme activity.
Figure 5: Effect of monosodium glutamate administration on superoxide dismutase activity

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Values were expressed as mean ± SEM, n = 7, 4 mg/g neonate higher significantly (P < 0.05) versus Normal control; 4 mg/g neonate plus 10 mg/g adult significantly (P < 0.05) lower versus 4 mg/g neonate and higher versus 10 mg/g adult.


  Discussion Top


Brian protection against toxins from blood is an active mechanism which prevent certain toxic substances entry into the brain carried out by the blood–brain barrier in a well-regulated manner,[2] it has also been shown that there exist areas of the brain with little or less protection of blood–brain barrier permitting access to the toxins or during brain development, studies have shown that the blood–brain barrier offers little protection to the brain.[10]

In this, the brain is seen to react differently to toxic against from the blood such as glutamate from MSG which in normal condition act as an important neurotransmitter but conditions were the brain barrier is ineffective glutamate and other toxins concentrations are not properly regulated and their accumulation occurs,[9] evidenced in this current study which showed elevation in glutamate decarboxylase [Figure 1] and glutamate synthase [Figure 2] activities which suggest accumulation of glutamate following MSG exposure and increased activity levels of its metabolic enzymes as a method of response by the brain to protect itself, this result may also be an indication of glutamate being utilized.[11] Furthermore, the result indicated that the elevation occurred similarly in both neonate and adult exposed groups but were higher in the group exposed both as neonate and adult, thus suggest the free entry of the toxic substance during brain development with little blood–brain barrier protection in neonate and through unprotected regions of the brain in adult according to the report of.[17]

As a result of this series of reactions involving increased level of glutamate, calcium channels are place in an open fixed position which allows more calcium to enter the brain cells and production of free radicals[2] that leads to the destruction of the cells known as apostosis.[14] In response to this, the brain cells use defense mechanisms involving antioxidants, which play the role of converting the generated free radicals to form that are less harmful to it thus prevent apoptosis or cell destruction,[12] these antioxidants are reported to be in less quantity in the brain[3] but can easily cross the brain barrier into the brain to make up for it concentration and perform their defensive role.[4]

In this current study, result obtained indicated that MSG administration caused a significant increase in concentrations of Catalase activity [Figure 3] in both neonatal and adult administered groups when compared to the control, this thus suggests that despite the effective role of the brain barrier glutamate entry into the brain cells triggered the production of hydrogen peroxide (hyroxy radical) and activation of the enzyme activity to reduce it to water and prevent respiratory burst in the cell that can lead to cell death.[12] This increase in enzyme activity occurred dose-dependently but significantly higher in the group exposed both as neonate and as adult while no difference between its activity in neonate and adult only exposed groups indicated that ability of glutamate to enter that brain cells either during and after the development of blood–brain barrier.[2]

Glutathione peroxidase [Figure 3] activity recorded in this current study indicated that following MSG exposure there was increased production of reactive species in the cells in both age of exposure similarly though the result clearly showed that the increase was more in the group exposed in both neonate and adult when compared to the groups exposed either as neonate or as adult respectively glutathione peroxidase are involve in the reaction that reduces hydrogen peroxide using sulfhydryl group to water.[7] Glutathione (γ-glutamylcysteinylglycine) is one of the body's principal means of protecting itself against oxidative damage,[14] this current study suggests that in response to increased level of free radical production in brain cells, elevation of the enzyme activity were used as a means of converting and reducing the level of radicals to a level that are not harmful to the cells.[2],[6],[14]

Unlike the response in catalase and glutathione peroxidase, SOD in this study occurred more in the groups exposed at neonatal stage in response to MSG administration. Superoxide dismutase act as a primary defense against oxidative stress been a strong initiator of series of reactions that convert superoxide anions to hydrogen peroxide and O2 in a reaction method known as dismutation.[7] The result obtained suggests the role of blood–brain barrier in preventing the formation of superoxide anion more in the brain of adult exposed rats than in the neonate, thus a reduction in the enzyme activity.[14] The developing brain is mostly vulnerable to toxins,[11] because the blood brain barrier at this point is still developing thus permeable to toxins than the matured brain,[2] in agreement with the result recorded in superoxide dismutase which indicated more enzyme activity in groups exposed as neonate than in adult.


  Conclusion Top


From the result of this study, it may be concluded that age of exposure does not determine how the brain responds to the effect of MSG, which have been considered in studies in which administration was done at the neonatal stage to create a model in adult.[14] Furthermore, the route of exposure according to the result of this study does not necessarily account for the bioavailability of MSG on the brain of exposed Wistar rats, which indicated an elevation in the enzyme activities in both intraperitoneal and oral administered.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Akataobi US. Key enzymes of glutamate metabolism in the brain of neonatal and adult rats exposed to monosodium glutamate. Asia Pac J Clin Trials Nev Syst Dis 2020; 5 (4):51-57.  Back to cited text no. 2
    
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Pavlovic V, Pavlovic D, Kocic G, Sokolovic D, Jevtovic-Stoimenov T, Cekic S, et al. Effect of monosodium glutamate on oxidative stress and apoptosis in rat thymus. Mol Cell Biochem 2007;303:161-6.  Back to cited text no. 4
    
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Singh A, Kukreti R, Saso L, Kukreti S. Oxidative stress: A key modulator in neurodegenerative diseases. Molecules 2019;24:1583.  Back to cited text no. 5
    
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Igwebuike UM, Ochiogu IS, Ihedinihu BC, Idika II. The effects of oral administration of monosodium glutamate (MSG) on the testicular morphology and caudaepididymal sperm reserves of young and adult male rats. Vet Arch 2011;81:525-34.  Back to cited text no. 10
    
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Akataobi US. Effect of monosodium glutamate (MSG) on behavior, body and brain weight of exposed rats. Environ Dis 2020; 5: 3-8.  Back to cited text no. 11
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Collison KS, Makhoul NJ, Zaidi MZ, Al-Rabiah R, Inglis A, Andres BL, et al. Interactive effects of neonatal exposure to monosodium glutamate and aspartame on glucose homeostasis. Nutr Metab (Lond) 2012;9:58-79.  Back to cited text no. 12
    
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Dawn BM, Allan DM, Colleen MS. Basic Medical Biochemistry. A Clinical Approach. 2nd edition; 2017. p. 190-210.  Back to cited text no. 13
    
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Rotimi OA, Olayiwola IO, Ademuyiwa O, Balagun EA. Effects of fiber-enriched diet on tissue lipid profile of MSG obese rats. J Food Chem Toxicol 2012;50:4062-7.  Back to cited text no. 14
    
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Hacioglu G, Senturk A, Ince I, Alver A. Assessment of oxidative stress parameters of brain-derived neurotrophic factor heterozygous mice in acute stress model. Iran J Basic Med Sci 2016;19:388-93.  Back to cited text no. 15
    
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Okwudiri OO, Sylvanus AC, Ihtuge AP. Monosodium glutamate induces oxidative stress and effect of glucose metabolism in kidney of rats. Int J Biochem Res Rev 2012;2:1-11.  Back to cited text no. 16
    
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King EJ, Wootton ID. Advance in clinical chemistry. Acta Physiol Sci 1959;47:115-23.  Back to cited text no. 17
    
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Bruce PL. The impart of toxin on the developing brain. Ann Rev Public Health 2015;36:211-30.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
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