About Authors:
Habibur Rahman1, M.C Eswaraiah2
1Department of Pharmacology,
2Dept. of Pharmacognosy,
Anurag Pharmacy College,
Ananthagiri (V), Kodad (M),
Nalgonda (Dt.), A.P-508206, India
ABSTRACT
Brain neurotransmitters like Dopamine, serotonin, Adrenaline, Glutamate and enzymes like Acetyl cholinesterase (AchE), Mono amino-oxidase (MAO-A & MAO-B) levels and antioxidant enzymes like Superoxide dismutase, catalase, peroxidase etc play important roles in different complex neurological disorders like Alzheimer’s disease(AD), Parkinsonism, depression etc. But biochemical estimation of these parameters is very difficult to conduct in simple laboratory. This review is a collection of simple spectroscopic methods to determine the common brain neurotransmitters and antioxidant enzymes.
Reference Id: PHARMATUTOR-ART-1244
Introduction
Neurotransmittersare endogenous chemical that transmit signals from a neuron to a target cell across a synapse. Glutamate is the major excitatory neurotransmitter in the brain and spinal cord and it involved in different disease like anxiety, epilepsy, psychosis, schizophrenia, Alzheimer’s disease and others common neurological disorders [1]. Excessive glutamate release can lead to excitotoxicity causing cell death. GABA is used at the great majority of fast inhibitory synapses in virtually every part of the brain. Many sedative/tranquilizing drugs act by enhancing the effects of GABA [2]. Acetylcholine is distinguished as the transmitter at the neuromuscular junction connecting motor nerves to muscles. Acetylcholine involved in memory, paralysis and others disorders. Dopamine has a number of important functions in the brain. It plays a critical role in the reward system, but dysfunction of the dopamine system is also implicated in Parkinson's disease and schizophrenia.[3] Serotonin is a monoamine neurotransmitter to regulate appetite, sleep, memory and learning, temperature, mood, behavior, muscle contraction, and function of the cardiovascular system and endocrine system. It is speculated to have a role in depression, as some. The brain is uniquely vulnerable to oxidative injury, due to its high metabolic rate and elevated levels of polyunsaturated lipids, the target of lipid per oxidation. Antioxidants appear to prevent oxidative stress in neurons and prevent apoptosis and neurological damage. Antioxidants are also being investigated as possible treatments for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [4, 5].
Estimation of Brain Neurotransmitters
Acetylcholineserase (AChE) enzyme determination
Acetylcholineserase (AChE) enzyme activity was estimated by Elman method [6].
Reagents
1. 0.1M Phosphate buffer
Solution A: 5.22g of K2HPO4 and 4.68g of NaH2PO4 are dissolved in 150 ml of distilled water.
Solution B: 6.2g NaOH is dissolved in 150ml of distilled water.
Solution B is added to solution A to get the desired pH (pH 8.0 or 7.0) and then finally the volume is made up to 300ml with distilled water.
2. DTNB Reagent
39.6 mg of DTNB with 15 mg NaHCO3 is dissolved in 10 ml of 0.1M phosphate buffer (pH 7.0).
3. Acetylthiocholine (ATC)
21.67 mg of acetylthiocholine is dissolved in 1 ml of distilled water.
Procedure
The mice were decapitated; brains are removed quickly and placed in ice-cold saline. Frontal cortex, hippocampus and septum are quickly dissected out on a Petri dish chilled on crushed ice. The tissues are weighed and homogenized in 0.1M Phosphate buffer (pH 8). 0.4ml aliquot of the homogenate is added to a cuvette containing 2.6 ml phosphate buffer (0.1M, pH 8) and 100µl of DTNB. The contents of the cuvette are mixed thoroughly by bubbling air and absorbance is measured at 412 nm in a spectrophotometer. When absorbance reaches a stable value, it is recorded as the basal reading. 20µl of substrate i.e., acetylthiocholine is added and change in absorbance is recorded. Change in the absorbance per minute is thus determined.
|
Reagents |
Sample |
Blank |
|
Phosphate buffer solution |
2.6 ml |
2.7 ml |
|
Supernatant |
0.4 ml |
0.4ml |
|
DTNB |
0.1 ml |
……. |
Calculations
The enzyme activity is calculated using the following formula [7].
A/min X Vt
Achetylcholinestease activity (M/ml) =…………………………….
ε X b X Vs
Where,
A/min= Change in absorbance per min
ε= 1.361 X104 M-1cm-1
b= pathlenth (1 cm)
Vt= Total volume (3.1 ml)
Vs= sample volume (0.4 ml)
The final reading of enzyme activity is expressed as μ moles/minute/mg tissue.
μ moles /ml sample
μ moles/minute/mg protein = ……………………………
mg protein/ml sample dilution
Estimation of Monoamine Oxidase A and B
The MAO- A and B activity was estimated by the method of Charles and McEwen [8].
Reagents
Sucrose (0.25M)
Tris buffer (0.1 M)
EDTA (0.02M)
Serotonin (4mM)
Sodium phosphate buffer (100 mM, pH 7.4)
Hydrochloric acid (1M)
Butyl acetate/cyclohexane
Preparation of sample
Mouse brain mitochondrial fraction are prepared by cutting the brain sample in to small pieces and rinsed in 0.25M sucrose, 0.1 M tris, 0.02M EDTA (pH 7.41) to remove blood. The pieces were homogenized for 45 sec in a potter-elvehjem homogenizer with 400 ml of the same medium. The homogenate was centrifuged at 800rpm for 10min and the pellets were discarded. The supernatant was then centrifuged at 12,000 rpm for 20 min in the same medium. The precipitate was washed twice more with 100ml of sucrose tris EDTA and resuspended in 50ml of the medium. The protein concentration was adjusted to 1 mg/ml.
Procedure for estimation of MAO- A
250 µl of the homogenate was added to 250 µl of serotonin and 250 µl of buffer. The reaction tube was placed at 37°C for 20 minutes and the reaction was arrested by the addition of 200 µl of 1M HCl. The reaction product was extracted with 5 ml of Butyl acetate. The organic phase was separated and measured at 280 nm using a spectrophotometer. Blank samples were prepared by adding 1M HCl (200 µl) prior to reaction and the reaction was carried out.
The MAO-A is expressed in nmoles/ mg protein.
Procedure for estimation of MAO- B
250 µl of the homogenate was added to 250 µl of serotonin and 250 µl of buffer. The reaction tube was placed at 37°C for 20 minutes and the reaction was arrested by the addition of 200 µl of 1M HCl. The reaction product was extracted with 5 ml of Cyclohexane. The organic phase was separated and measured at 242 nm using a spectrophotometer. Blank samples were prepared by adding 1M HCl (200 µl) prior to reaction and the reaction was carried out.
The MAO-B activity is expressed in nmoles/ mg protein.
Estimation of Dopamine, Adrenaline and Serotonin
Preparation of tissue extracts by method Schlumpf M [9].
Reagents:
1. HCl – Butanol sol. : (0.85 ml of 37% hydrochloric acid in one-litre n-butanol)
2. Heptanes
3. 0.1 M HCl: (0.85 ml conc. HCl upto 100 ml H20)
Procedure:
On the day of experiment mice were sacrificed, whole brain was dissected out and the sub cortical region (including the striatum) was separated. Weight tissue was weight and was homogenized in5ml HCl–butanol for about 1 min. The sample was then centrifuged for 10 min at 2000 rpm. An aliquot supernatant phase (1 ml) was removed and added to centrifuge tube containing 2.5 ml heptane and 0.31ml HCl of 0.1 M. After 10 min of vigorous shaking, the tube was centrifuged under the same conditions as above in order to separate the two phases, and the overlaying organic phase was discarded. The aqueous phase (0.2 ml) was then taken either for 5-HT or NA and DA assay. All steps were carried out at 00C.
(N.B: It taken in between 50-75 mg of tissue for homogenate with 5 ml of HCl-Butanol in correlation of same tissue concentration 1.5-5 mg/0.1 ml of HCl-butanol used in Schlumpf M et al, 1974. This is done to get adequate amount of supernatant liquid for analysis).
Estimation of Noradrenaline and dopamine [10].
Reagents
1. 0.4M HCl: 3.4 ml conc. HCl up to 100 ml H20
2. Sodium acetate buffer (pH 6.9): 2.88 ml of 1M acetic acid (5.7 ml of Glacial acetic acid upto 100 ml with distilled water) +27.33 ml of 0.3M sodium acetate (4.08 g of sodium acetae 100 ml with distilled water) and volume is made up to 100 ml with distilled water). PH is adjusted with sodium hydroxide sol.
3. 5M NaOH : 20 g of sodium hydroxide pellets dissolved in distilled water and volume is made up to 100 ml with distilled water)
4. 0.1 M Iodine solution (in Ethanol): 4 g of pot. Iodide +2.6 g of iodine dissolved in ethanol volume is made up to 100 ml)
5. Na2SO3 sol. ((0.5 g Na2SO3 in 2 ml H2O + 18 ml 5 M NaOH)
6. 10M Acetic acid: 57 ml of glacial acetic acid dissolved in distilled water up to 100 ml.
Procedure
To the 0.2 ml of aqueous phase, 0.05 ml 0.4 M HCl and 0.1 ml of EDTA / Sodium acetate buffer (PH6. 9)were added, followed by 0.1 ml iodine solution(0.1 M in ethanol) for oxidation. The reaction was stopped after 2 min by addition of 0.1 ml Na2SO3 solution. 0.1 ml Acetic acid is added after 1.5 min. The solution was then heated to 100°C for 6 min when the sample again reached room temperature, excitation and emission spectra were read from the spectrofluorimeter. The readings were taken at 330-375 nm for dopamine and 395-485 nm for nor-adrenaline.
Estimation of Serotonin
The serotonin content was estimated by the method of Schlumpf[9].
Reagents
1. O-phthaldialdehyde (OPT) reagent: (20 mg in 100 ml conc. HCl)
Procedure
To 0.2 ml aqueous extract 0.25 ml of OPT reagent was added. The fluorophore was developed by heating to 100°C for 10 min. After the samples reached equilibrium with the ambient temperature, readings were taken at 360-470 nm in the spectrofluorimeter. Tissue blanks for Dopamine and nor-adrenaline were prepared by adding the reagents of the oxidation step in reversed order (sodium sulphite before iodine). For serotonin tissue blank, 0.25 ml cont. HCI without OPT was added.Internal Standard: (500 µg/ml each of noradrenaline, dopamine and serotonin are prepared in distilled water: HCl-butanol in 1:2 ratio.
Estimation of Glutamate
The level of Glutamate was estimated by multiple development paper chromatography as described by Raju [11].
Reagents
1.butanol: acetic acid: water: 12: 3: 5
2. Ninhydrin reagent : 0.25%
3. Copper sulphate solution: 0.005%
4. Standard glutamate: 2.942 mg of glutamate in 10 ml distilled water.
Procedure
1.0 ml of the supernatant from brain homogenate was evaporated to dryness at 70°C in an oven and the residue is reconstituted in 100 ml of distilled water. Standard solutions of glutamate and GABA at a concentration of 2mM along with the sample are spotted on Whatman No. 1 chromatography paper using a micropipette. It was placed on a chamber containing butanol: acetic acid: water (12: 3: 5 v/v) as solvent. When the solvent front reached the top of the paper, it was removed and dried. A second run is performed similarly, after which the papers are dried sprayed with ninhydrin reagent and placed in an oven at 100°C for 4 minutes. The portions which carry glutamate corresponding with the standard are cut and eluted with 0.005% CuSo4 in 75% ethanol. Their absorbance is read against blank at 515 nm in spectrophotometer.
Calculation
The levels of glutamate and GABA are calculated by using the following formula;
Unknown OD X Standard in mg (3µg) X 1000
A= ……………………………………………………
Standard OD X Volume spotted (10µl) X W
Where,
A = Aminoacid content in umoles/gram wet weight tissue
1000 = Conversion factor for gram wet weight tissue
W = weight of the tissue in gram
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Estimation of Antioxidant Enzymes
Estimation of Superoxide dismutase (SOD)[12]
Reagents
Carbonate buffer (100mM, pH 10.2)
Epinephrine (3mM)
Procedure
The SOD activity in supernatant was measured by the method of Misra and Fridovich. The supernatant (500 µl) was added to 0.800ml of carbonate buffer (100mM, pH 10.2) and 100 µl of epinephrine (3mM). The change in absorbance of each sample was then recorded at 480 nm in spectrophotometer for 2 min at an interval of 15 sec. Parallel blank and standard were run for determination SOD activity.
One unit of SOD is defined as the amount of enzyme required to produce 50% inhibition of epinephrine auto oxidation.
|
Reagents |
Uninhibited(Standard) |
Inhibited(Sample) |
Blank |
|
Carbonate buffer |
0.900 ml |
0.800 ml |
1.0 ml |
|
Supernatant |
…… |
0.1 ml |
…… |
|
Epineprine |
0.1 ml |
0.1 ml |
--------- |
Calculation
?A480nm/min Uninhibited - ?A480nm /min inhibited
% Inhibition=……………………………………………………..X 100
?A480nm /min Uninhibited - ?A480nm /min Blank
% Inhibition X Vt
Units/ml enzyme = …………………..
(50%) X Vs
Units/ml enzyme
Units/mg protein = ……………………………
mg protein/ml enzyme
Estimation of Catalase (CAT)[13]
Reagents
1. Phosphate buffer solution (50 mM)
A) Dissolving 6.81 gm of KH2PO4 in 1000 ml distilled water.
B) Dissolving 6.9 gm of Na2HPO4 in 1000ml distilled water.
390 ml from solution (A) are mixed with 610 ml from solution (B), the pH is adjusted to 7.
2. Hydrogen peroxide (H2O2) 30 mM
0.34 ml of 30% H2O2 is diluted with phosphate buffer to 100 ml.
Procedure
Catalase activity was measured by the method of Aebi. 0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate buffer (pH 7.0). Reaction was started by the addition of 1.0ml of freshly prepared 30 mM H2O2. The rate of decomposition of H2O2 was measured spectrophotometrically from changes in absorbance at 240 nm. Activity of catalase was expressed as units/mg protein. A unit is defined as the velocity constant per second.
|
Reagents |
Sample |
Blank |
|
Phosphate buffer solution |
1.9 ml |
2..9 ml |
|
Supernatant |
0.1 ml |
0.1 ml |
|
H2O2 |
1 ml |
--------- |
The reaction occurs immediately after the addition of H2O2.
Solutions are mixed well and the first absorbance (A1) is read after 15 seconds (t1) and the second absorbance (A2) after 30 seconds (t2).The absorbance is read at wave length 240 nm.
Calculation
Vt 2.3 A1
K = -------- x -------- x Log -------- x 60
Vs Δt A2
Where,
K= Rate constant of the reaction.
Δ t= (t2 – t1) =15 seconds.
A1= absorbance after 15 seconds.
A2= absorbance after 30 seconds.
Vt = total volume (3 ml).
Vs = volume of the sample (0.1ml).
Estimation of Lipid peroxidase (LPO)
The level of Lipid peroxides was estimated by Thio barbituric acid reaction method described by Ohkawa et al.[14].
Reagents
1. Sodium dodecyl sulphate (SDS) (8.1 %)
2. Acetic acid (20%; pH 3.5)
3. Thiobarbituric acid (TBA) (0.8%)
4. n-butanol/pyridine mixture (15:1, v/v)
Procedure
To 0.2 ml of test sample, 0.2 ml of SDS, 1.5 ml of acetic acid and 1.5 ml of TBA were added. The mixture was made up to 4 ml with water and then heated in a water bath at 95°C for 60 minutes. After cooling, 1 ml of water and 5 ml of n-butanol/pyridine mixture were added and shaken vigourously. After centrifugation at 4000 rpm for 10 minutes, the organic layer was taken and its absorbance was read at 532 nm. The level of lipid peroxides was expressed as nmoles of MDA released/ g wet tissue
|
Reagents |
Sample |
Blank |
|
SDS |
0.2 ml |
0.2 ml |
|
Supernatant |
0.2 ml |
……… |
|
DDW |
1.6ml |
1.8 ml |
|
Acetic acid |
1.5 ml |
1.5ml |
|
TBA |
1.5 ml |
1.5ml |
|
n-butanol/pyridine mix |
5ml |
5ml |
Calculation
Absorbance at 532 nm
The concentration of MDA = ---------------------------------- x D
L x ?
Where,
L: light path (1 cm).
? : extinction coefficient 1.56 x 105 M-1.Cm-1.
Total volume (10 ml)
D: dilution factor =---------------------------------
Vol of the sample (0.2ml)
Estimation of Glutathione peroxidase[15]
Reagents
1. Phosphate buffer, pH 7.0 (75 mM)
2. Glutathione reductase (60 mM)
3. Sodium azide (0.12 M)
4. Di-sod. EDTA (0.15 mM)
5. NADPH(3 mM)
6. H202 (7.5 mM)
Procedure
3-ml cuvette containing 2.0 ml of phosphate buffer(75 mmol/L, PH 7.0) , 50µl of (60mmol/L) glutathione reductase solution, 50µL of (0.12 mol/L) NaN3, 0.1 ml of (0.15mmol/L) Na2 EDTA ,100µL of (3.0 mmol/L) NADPH, and 100µL of tissue supernatant was added. Water was added to make a total volume of 2.9 ml. The reaction was started by the addition of 100µL of (7.5 mmol/L) H202, and the conversion of NADPH to NADP was monitored by a continuous recording of the change of absorbance at 340 nm at 1-min interval for 5 min. Enzyme activity of GSHPx was expressed in terms of mg of proteins.
|
Reagents |
Sample |
Blank |
|
Phosphate buffer solution |
2.0 ml |
2..9 ml |
|
Glutathione reductase |
0.05 ml |
0.05 ml |
|
Sodium azide |
0.05 ml |
0.05 ml |
|
Di-sodium EDTA |
0.1ml |
0.1 ml |
|
NADPH |
0.1 ml |
0.1 ml |
|
Tissue supernatant |
0.1 ml |
0.1 ml |
|
H2O2 |
0.1 ml |
--------- |
|
DDW |
0.5 ml |
0.6 ml |
Calculations
A340/min X Vt
Enzyme activity (M/min/ml) = …………………….
ε X d X Vs
Where, ε=6.22 X 10 6 M-1cm-1
d=1cm
Vt= Total volume (3.0)
Vs= Sample volume (0.1 ml)
Estimation of Glutathione reductase (GRD)
Glutathione reductase was assayed by the method of Stahl et al.)[16]
Reagents
1. Phosphate buffer (0.3 M; pH 6.5)
2. EDTA( 0.25M)
3. Glutathione oxidized, GSSG (0.012 M)
4. NADPH (0.003 M); Nicotinamide Adenine Dinucleotide Phosphate Reduced Tera Sod. Salt, NADPHNa4 (Mw. 833.35).
Procedure
The reaction mixture containing 1 ml phosphate buffer, 0.5 ml EDTA, 0.5 ml GSSG and 0.2 ml of NADPH was made up to 3 ml with distilled water. After the addition of 0.1 ml of tissue homogenate, the change in optical density at 340 nm was monitored for 2 minutes at 30 seconds interval.
One unit of the enzyme activity was expressed as nmoles of NADPH oxidized / min/ mg protein.
|
Reagents |
Sample |
Blank |
|
Phosphate buffer solution |
1.0 ml |
1.5 ml |
|
EDTA |
0.5 ml |
0.5ml |
|
GSSG |
0.5 ml |
……. |
|
NADPH |
0.2ml |
0.2ml |
|
Supernatant |
0.1 ml |
0.1 ml |
|
DDW |
0.8 ml |
0.8 ml |
Calculation
A340/min X Vt
Enzyme activity (M/min/ml) =…………………….
ε X d X Vs
Where,
ε=6.22 X 10 6 M-1cm-1
d=1cm
Vt= Total volume (3.1)
Vs= Sample volume (0.1 ml).
Estimation of ascorbic acid (vitamin c)
The level of vitamin C was determined by the method of Omaye et al[17].
Reagents
1. Tri-chloro acetic acid (TCA): 5%
2. Sulphuric acid: 65%
3. DTC reagent : 3 gm of DNPH, 0.4 gm of thiourea and0.05 gm of copper sulphate were dissolved in 100 ml of 9N sulphuric acid.
4. Standard Ascorbic acid
Procedure
To 0.5 ml of homogenate, 0.5 ml of water and 1.0 ml of TCA were added, mixed thoroughly and centrifuged. To 1.0 ml of the supernatant, 0.2 ml of DTC reagent was added and incubated at 37°C for 3 hours. Then 1.5 ml of sulphuric acid was added, mixed well and the solutions were allowed to stand at room temperature for another 30 minutes. The colour developed was read at 520 nm in a UV spectrophotometer.
The values were expressed as µg/ g wet tissue.
|
Reagents |
Standard |
Sample |
Blank |
|
Supernatant |
1.0 ml |
….. |
……. |
|
Standard Ascorbic acid |
…… |
1.0 ml |
…… |
|
DTC reagent |
0.2 ml |
0.2 ml |
1.0 ml |
|
TCA |
------ |
------ |
0.1 ml |
|
H2SO4 |
1.5 ml |
1.5 ml |
1.5 ml |
|
DDW |
0.7 ml |
0.7 ml |
0.7 ml |
Calculation
Abs Tesr X Std. dilution
Amount of ascorbic acid (µg/mg of tissue) =…………………………… X1000
Abs Std X test dilution
Where,
Abs Tesr= Test sample absorbance at 520 nm
Abs Std = Standard sample absorbance at 520 nm
Conclusion:
Estimation of brain neurotransmitter and antioxidants from laboratory animals like mice are some difficult task. Although biochemical, sophisticated methods can give more accurate and more precise results, but it is not easy to use in common laboratory due to high cost and lack of chemicals. This review is the ultimate collection of simple spectroscopic methods to estimate the brain neurotransmitters and antioxidant enzymes. This will be helpful for academic research for estimation of neurotransmitters and antioxidant enzymes.
References:
1. McEntee W. & Crook T. Glutamate: its role in learning, memory, and the aging brain. Psychopharmacology, 1993,111 (4): 391–401.
2. Li K, Xu E. The role and the mechanism of gamma-aminobutyric acid during central nervous system development, Neurosci Bull, 2008, 24 (3): 195–200.
3. Merims D, Giladi N, Dopamine dysregulation syndrome, addiction and behavioral changes in Parkinson's disease. Parkinsonism Relat. Disord. 2008, 14 (4): 273–80.
4. Di Matteo V, Esposito E. Biochemical and therapeutic effects of antioxidants in the treatment of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, Curr Drug Targets CNS Neurol Disord , 2003, 2 (2): 95–107.
5. Rao A, Balachandran B. Role of oxidative stress and antioxidants in neurodegenerative diseases, Nutr Neurosci, 2002, 5 (5): 291–309.
6. Ellman GL, Courtney KD, Anders U, Feather stone RM, A new and rapid colorimetric determination of acetyl cholinesterase activity, Biochem Pharmacol, 1961; 7: 88-95.
7. Srikumar BN, Ramkumar K, Raju TR and Shankaranarayana Rao BS, Assay of acetylcholinesterase activity in the brain, National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:142-144.
8. Charles M, McEwen J, Tabor H et al (eds). Methods of Enzymologists. 1977. Academic press. New York.
9. Margret Schlumfjf, Walter Lichtensteiger, Heinrich Langemann, Peter G. Waser And Franz Hefti, A Fluorometric Micromethod For The Simultaneous Determination of Serotonin, Noradrenaline and Dopamine in Milligram Amounts of Brain Tissue, Pergamon Press, Printed M Great Britain. Biochemical Pharmacology. 1974, 23: 2337-2446.
10. Dilip Kumar Pal, Determination of Brain Biogenic Amines in Cynodon Dactylon Pers. and Cyperus Rotundus L. Treated Mice, International Journal Of Pharmacy And Pharmaceutical Sciences, 2009, 1(1):190-197.
11. Raju TR, Kutty BM, Sathyaprabha TN, Shankarnarayana Rao BS. Brain and behavior. National Institute of Mental Health and neurosciences, Bangalore. 2004. Pp. 134-138.
12. Misra, H.P, Fridovich I. Biochemistry, 1967, 15: 681.
13. Aebi H. Methods of enzymatic analysis, ed., New York, Academic Press, 1974, 2: 674.
14. Ohkawa H, Ohisi N and Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem, 1979; 95: 351-358.
15. Wood, J.L.: In: Metabolic Conjugation and Metabolic Hydrolysis (Fishman W. H., Ed), Academic Press, New York. 1970, Vol. II: 261-299
16. Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med, 1963; 61:882-8.
17. Oayama H, Measurement of antioxidants inhuman blood plasma, Methods Enzymol, 1994; 234: 269-279.
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