You are hereSimple spectroscopic Methods for estimating Brain Neurotransmitters, Antioxidant Enzymes of Laboratory animals like Mice: A review

Simple spectroscopic Methods for estimating Brain Neurotransmitters, Antioxidant Enzymes of Laboratory animals like Mice: A review


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

---------

The reaction mixtures are diluted 1/10 just before taking the readings in spectrophotometer.

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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dpsingh's picture
dpsingh
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Joined: 2014-01-18
MAO A and B Calculation

Please send the calculation of MAO-A and MAO-B activity in nmol/mg protein.
I have absorbance for the same.
 
Thanks.

Nadir (not verified)
Thank You !

Calculations are the biggest headache for rookie researcher. Your instructions are easy to comprehend. Thanks a lot...

Anonymous (not verified)
To know the calculation formula for the MAO-A & B

I am a student of M.Sc. Semester IV and i am doing my dissertation work on activity of neurotransmitters on chicken brain intoxicated by fugal food. in that my one protocol is study of MAO-A and B , i have fulfill my this protocol by the method of Charles, M. and J. McEwen, 1977. Mao Activity in Rabbit Serum. In: Methods in Enzymology, XVIIB, Tabor, H. and C.W. Tabor (Eds.). Academic Press, New York and London, pp: 692-698. in this method i have got my readings but i do not know the formula to convert it into nmoles/mg protein. so, if u will email me formula for that and related articals it will help me in my dissertation work & i will be thank full to you for that.

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