Monoamine Oxidase Assays

Keith F. Tipton1, Gavin Davey1, Martha Motherway1

1 Trinity College, Dublin, null
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 3.6
DOI:  10.1002/0471141755.ph0306s09
Online Posting Date:  May, 2001
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Abstract

Monoamine oxidase catalyzes the oxidative deamination of primary aliphatic and aromatic amines and some secondary and tertiary amines, including the hormone and neurotransmitter amines epinephrine, dopamine, norepinephrine and serotonin. The two isoenzymes, MAO‐A and MAO‐B, differ according to substrate and inhibitor specificities. For example, selective inhibitors of MAO‐A have been shown to be effective antidepressants whereas some MAO‐B inhibitors have been reported to be beneficial in the treatment of Parkinson's and perhaps Alzheimer's diseases. Thus, it is important to have accurate procedures for determining the activities of each MAO isoenzyme in tissues that may contain both. In this unit, several MAO assay procedures are described, along with support protocols for equilibrating ion‐exchange resin, preparing aldehyde dehydrogenase, preparing dose‐response curves for determining the relative proportions of isoenzyme activities, determining concentrations of each isoenzyme, assessing new compounds as putative MAO inhibitors, and determining protein concentration of membrane‐bound proteins.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Radiochemical Determination of MAO Activity by Solvent Extraction
  • Alternate Protocol 1: Radiochemical Determination of MAO Activity Using Epinephrine or Norepinephrine
  • Support Protocol 1: Equilibration of Dowex Ion‐Exchange Resin
  • Basic Protocol 2: Spectrophotometric Determination of Aldehyde Formation
  • Support Protocol 2: Aldehyde Dehydrogenase Preparation
  • Basic Protocol 3: Polarographic Determination of Oxygen Consumption
  • Basic Protocol 4: Fluorometric Determination of Hydrogen Peroxide Formation
  • Basic Protocol 5: Spectrophotometric Determination of the Oxidation of Specific Substrates
  • Support Protocol 3: Dose‐Response Curves with Clorgyline and (–)‐Deprenyl to Assess the Relative Proportions of MAO‐A and MAO‐B Activity
  • Basic Protocol 6: Determination of Absolute MAO Concentrations in Particulate Samples
  • Alternate Protocol 2: Determination of Absolute MAO Concentrations in Soluble Samples
  • Support Protocol 4: Determination of the Concentrations of MAO‐A and ‐B in Preparations Containing Both Isoenzymes
  • Support Protocol 5: Assessing a New Compound as an MAO Inhibitor
  • Support Protocol 6: Determination of Protein Concentration
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Radiochemical Determination of MAO Activity by Solvent Extraction

  Materials
  • 100 mM sodium or potassium phosphate buffer, pH 7.2 ( appendix 2A)
  • MAO preparation (see recipe)
  • Test inhibitors (optional)
  • Substrates (see recipe): e.g.,
  •  Unlabeled benzylamine⋅HCl and [7‐14C]benzylamine⋅HCl,
  •  Unlabeled 5‐hydroxytryptamine (5‐HT) creatinine sulfate and [side chain‐2‐14C]5‐HT creatinine sulfate,
  •  Unlabeled 2‐phenylethylamine⋅HCl (PEA⋅HCl) and [ethyl‐1‐14C]PEA⋅HCl, or
  •  Unlabeled tyramine⋅HCl and [2‐14C]tyramine⋅HCl
  • 2 M citric acid solution
  • Extraction/scintillant mixture (see recipe)
  • 5‐ml plastic or glass scintillation vials

Alternate Protocol 1: Radiochemical Determination of MAO Activity Using Epinephrine or Norepinephrine

  • Substrates (see recipe):
  •  Unlabeled (–)‐norepinephrine (arterinol, noradrenaline) bitartrate (Sigma) and [8‐14C](–)‐norepinephrine bitartrate (Amersham), or
  •  Unlabeled (±)‐epinephrine (adrenaline)⋅HCl (Sigma) and [7‐3H](±)‐epinephrine (Amersham)
  • 100 mM HCl
  • Equilibrated Dowex 50X2‐400 ion‐exchange resin (H form; see protocol 3)
  • 8 mM ascorbic acid, freshly prepared
  • Scintillant: Tritosol solution (for norepinephrine assays; see recipe) or Triton/toluene/PPO solution (for epinephrine assays; see recipe)
  • Assay vials: 10 × 15–cm glass culture tubes or standard 20‐ml plastic scintillation vials

Support Protocol 1: Equilibration of Dowex Ion‐Exchange Resin

  Materials
  • Dowex 50X2‐400 (H‐form) ion‐exchange resin (Sigma)
  • 10 M NaOH
  • 6 M HCl

Basic Protocol 2: Spectrophotometric Determination of Aldehyde Formation

  Materials
  • 100 mM sodium or potassium phosphate buffer, pH 7.2 ( appendix 2A)
  • 10 mg/ml β‐nicotinamide‐adenine dinucleotide (NAD+), prepared fresh
  • MAO preparation (see recipe)
  • 80 mM pyrazole
  • 100 µM rotenone in methanol
  • Aldehyde dehydrogenase (see protocol 5)
  • Substrate stock solutions (see recipe): e.g.,
  •  4 mM Tyramine⋅HCl
  •  13.32 mM Benzylamine⋅HCl
  •  800 µM 2‐Phenylethylamine⋅HCl
  •  4 mM 5‐Hydroxytryptamine creatinine sulfate
  •  2.22 mM Dopamine⋅HCl
  • Test inhibitors (optional)
  • UV/visible spectrophotometer with chart recorder or computer output and cuvette temperature regulation
  • 1‐cm standard quartz spectrophotometer cuvettes or disposable plastic cuvettes with satisfactory transmission characteristics at 340 nm

Support Protocol 2: Aldehyde Dehydrogenase Preparation

  • Acetone
  • Ox liver, fresh and kept on ice
  • 3.6 mM Na 2EDTA buffer, adjust pH to 7.0 with 50 mM HCl
  • 96% (v/v) ethanol, ice cold
  • 0.1 M acetic acid
  • 50 mM sodium pyrophosphate buffer, pH 8.8
  • 3.0 mM acetaldehyde
  • −20°C freezer with spark‐proof electrical system and thermostat
  • Blender (e.g., Waring) with acetone‐resistant vessel and sealed motor to avoid danger of spark ignition
  • Large Büchner funnel and flask
  • Whatman no.1 filter paper
  • Vacuum pump (water)
  • Vacuum dessiccator with silica gel (oven‐dried, if necessary)
  • Polypropylene centrifuge bottles
  • Cold bath, −10°C (commercial cold bath or propanol/dry ice bath)
  • Dialysis tubing (Visking tubing, MWCO 12,400, boiled in 5 to 10 mM EDTA and thoroughly rinsed with water before use)
NOTE: Enzyme solutions should be made fresh, kept on ice, and used on the day of preparation.

Basic Protocol 3: Polarographic Determination of Oxygen Consumption

  Materials
  • 100 mM sodium or potassium phosphate buffer, pH 7.2 ( appendix 2A)
  • Saturated sodium dithionite solution, fresh
  • 500 mM semicarbazide
  • 200 mM KCN
  • 500 U/ml catalase (EC 1.11.1.6; Sigma)
  • MAO preparation (see recipe)
  • Substrates (see recipe): e.g.,
  •  2.5 mM Tyramine⋅HCl
  •  8.325 mM Benzylamine⋅HCl
  •  500 µM 2‐Phenylethylamine⋅HCl
  •  2.5 mM 5‐Hydroxytryptamine creatinine sulfate
  •  1.375 mM Dopamine⋅HCl
  • Oxygen electrode assembly (e.g., Rank Brothers): Rank‐ or Clark‐style with a water‐jacketed reaction vessel stirred from below using a magnetic stirring bar
  • Chart recorder set to full‐scale input recommended by manufacturers of the electrode assembly (1 mV to >1 V, depending on amplification circuitry used)
  • 100‐µl Hamilton syringe

Basic Protocol 4: Fluorometric Determination of Hydrogen Peroxide Formation

  Materials
  • 100 mM sodium or potassium phosphate buffer, pH 7.2 ( appendix 2A)
  • 4 mg/ml 4‐hydroxy‐3‐methoxybenzoic acid (homovanillic acid; Sigma), prepare fresh and keep at 0° to 4°C, protected from light
  • 1 U/ml horseradish peroxidase (e.g., Sigma Type II) in 100 mM phosphate buffer
  • MAO preparation (see recipe)
  • Substrates (see recipe): e.g.,
  •  2.5 mM Tyramine⋅HCl
  •  8.325 mM Benzylamine⋅HCl
  •  500 µM 2‐Phenylethylamine⋅HCl
  • H 2O 2
  • Spectrophotofluorometer (e.g., Perkin‐Elmer) with temperature‐controlled cuvette compartment, attached to a strip‐chart recorder or interfaced to a computer
  • Fluorometer cuvette

Basic Protocol 5: Spectrophotometric Determination of the Oxidation of Specific Substrates

  Materials
  • 100 mM sodium or potassium phosphate buffer, pH 7.2 ( appendix 2A)
  • MAO preparation (see recipe)
  • Substrates (see recipe): e.g., 10 mM benzylamine⋅HCl or 1 mM kynuramine⋅2HBr
  • UV/visible spectrophotometer with chart recorder or computer output and cuvette temperature regulation
  • 1‐cm standard quartz spectrophotometer cuvettes or disposable plastic cuvettes with satisfactory transmission characteristics at 250 nm (for benzylamine) or at 314 or 360 nm (for kynuramine)

Support Protocol 3: Dose‐Response Curves with Clorgyline and (–)‐Deprenyl to Assess the Relative Proportions of MAO‐A and MAO‐B Activity

  Materials
  • 10 mM clorgyline⋅HCl (e.g., Sigma or Research Biochemicals)
  • 10 mM (–)‐deprenyl⋅HCl (e.g., Sigma or Research Biochemicals)
  • Substrates (see recipe): e.g.,
  •  Tyramine⋅HCl or 5‐hydroxytryptamine creatinine sulfate (for MAO‐A)
  •  Benzylamine⋅HCl or 2‐phenylethylamine⋅HCl (for MAO‐B)
  • Additional reagents and equipment for MAO activity assays (see protocol 1Basic Protocols 1 to protocol 85 or see protocol 2)

Basic Protocol 6: Determination of Absolute MAO Concentrations in Particulate Samples

  Materials
  • MAO preparation (see recipe): crude homogenate or mitochondrial preparation
  • 100 mM sodium or potassium phosphate buffer, pH 7.2 ( appendix 2A)
  • 18 mM unlabeled pargyline⋅HCl (Sigma)
  • [3H]Pargyline: [phenyl‐3, benzyl‐3H]pargyline‐HCl (NEN Life Sciences) in water with unlabeled pargyline⋅HCl to yield 50 Ci/mol at 4 µM
  • Triton/toluene/PPO solution (see recipe)
  • 20‐ml glass or plastic scintillation vials
  • Additional reagents and equipment for determining protein concentration (see protocol 14)

Alternate Protocol 2: Determination of Absolute MAO Concentrations in Soluble Samples

  • MAO preparation (see recipe): solubilized
  • 2% (w/v) activated charcoal (acid washed; Sigma) in 100 mM phosphate buffer, pH 7.2 ( appendix 2A)
  • 4% (w/v) bovine serum albumin (BSA) in 100 mM phosphate buffer, pH 7.2 ( appendix 2A)

Support Protocol 4: Determination of the Concentrations of MAO‐A and ‐B in Preparations Containing Both Isoenzymes

  Materials
  • 11 µM Clorgyline⋅HCl (e.g., Sigma or Research Biochemicals)
  • 5.5 µM (–)‐Deprenyl⋅HCl (e.g., Sigma or Research Biochemicals)
  • Additional reagents and equipment for MAO activity assays (see protocol 1Basic Protocols 1 to protocol 85 or see protocol 2)

Support Protocol 5: Assessing a New Compound as an MAO Inhibitor

  • Test compound
  • Dialysis tubing (MWCO 12,400)
  • Additional reagents and equipment for inhibition analysis (see protocol 9)

Support Protocol 6: Determination of Protein Concentration

  Materials
  • 10 mg/ml bovine serum albumin (BSA) stock solution or an alternative protein calibration standard ( appendix 3A)
  • Buffer used to prepare the enzyme sample
  • Enzyme sample (MAO preparation, see recipe) containing 50 to 250 µg protein
  • Markwell‐Lowry reagents (see recipe)
  • Spectrophotometer and 1‐cm cuvettes (disposable plastic)
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Figures

  •   FigureFigure 3.6.1 The aldehyde dehydrogenase–coupled assay of monoamine oxidase. The aldehyde product of amine oxidative deamination is further oxidized by aldehyde dehydrogenase with the concomitant reduction of NAD+ to NADH, which can be followed by the increase in absorbance at 340 nm.
  •   FigureFigure 3.6.2 Inhibition of rat liver MAO by clorgyline (A) and human brain MAO by (–) ‐ deprenyl (B). Tissue homogenates were incubated for 30 min at 37°C before activity was determined using a radiochemical assay () towards 10 µM 2‐phenylethylamine (squares), 100 µM tyramine (closed circles), or 100 µM 5‐HT (open triangles). Each point is the mean value ± SEM of triplicate determinations. Representative data from authors' unpublished work.
  •   FigureFigure 3.6.3 Determination of monoamine oxidase concentration by the binding of [3H]pargyline. (A) Binding to a rat liver mitochondrial preparation according to . (B) Binding to an ox liver preparation that had been rendered soluble and partially purified according to . Total binding (circles), specific binding (triangles), nonspecific binding (squares). Representative data from authors' own unpublished work.
  •   FigureFigure 3.6.4 Substrate preferences of MAO‐A and MAO‐B. Note: MPTP and its substituted 2′ derivatives are highly toxic compounds and should be handled with extreme caution.
  •   FigureFigure 3.6.5 Some irreversible inhibitors of monoamine oxidase.
  •   FigureFigure 3.6.6 Some reversible inhibitors of monoamine oxidase.
  •   FigureFigure 3.6.7 Some assay procedures for the monoamine oxidases. The bold numbers refer to the specific assay protocols described in the text. Other procedures are described in Tipton and Youdim ().
  •   FigureFigure 3.6.8 The oxidation of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) by monoamine oxidase. Note: MPTP is a highly toxic compound and should be handled with extreme caution.
  •   FigureFigure 3.6.9 Suicide substrates for MAO. These compounds act as substrates and inhibitors of MAO. All reactions also yield hydrogen peroxide. Note: MPTP is highly toxic compound and should be handled with extreme caution. The product of MPTP oxidation can be further oxidized to the pyridinium ion (see Fig. ).
  •   FigureFigure 3.6.10 Reaction scheme for a substrate that also acts as a time‐dependent irreversible inhibitor. The partition ratio, which is defined as moles product formed per mole enzyme irreversibly inhibited, is given by the ratio k+3/ k+4.

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Literature Cited

Literature Cited
   Anderson, M.C., Hasan, F., McCrodden, J.M., and Tipton, K.F. 1993. Monoamine oxidase inhibitors and the cheese effect. Neurochem. Res. 18(11):1145‐1149.
  Bensadoun, A. and Weinstein, D. 1976. Assay of proteins in the presence of interfering materials. Analyt. Biochem. 70:241‐250.
   Cesura, A.M. and Pletcher, A. 1992. The new generation of monoamine oxidase inhibitors. Prog. Drug. Res. 38:171‐297.
   Dawson, R.M.C., Elliott, D.C., Elliott, W.H., and Jones, K.M. 1986. Data for Biochemical Research, 3rd edition., pp. 417‐443. Oxford Science Publications, Oxford.
   Delumeau, J.C., Bentue‐Ferrer, D., Gandon, J.M., Amrein, R., Belliard, S., and Allain, H. 1994. Monoamine oxidase inhibitors, cognitive functions and neurodegenerative diseases. J. Neural Trans. Suppl. 41:259‐266.
   Dostert, P.H., Strolin Benedetti, M., and Tipton, K.F. 1989. Interactions of monoamine oxidase with substrates and inhibitors. Med. Res. Rev. 9:45‐89.
   Fowler, C.J., Tipton, K.F., McKay, A.V.P., and Youdim, M.B.H. 1982. Human platelet monoamine oxidase—A useful enzyme in the study of psychiatric disorders. Neuroscience 7:1577‐1594.
   Houslay, M.D. and Tipton, K.F. 1974. A kinetic evaluation of monoamine oxidase activity in rat liver mitochondrial outer membranes. Biochem. J. 139:645‐652.
   Krueger, M.J., Mazouz, F., Ramsay, R.R., Milcent, R., and Singer, T.P. 1995. Dramatic species differences in the susceptibility of monoamine oxidase B to a group of powerful inhibitors. Biochem. Biophys. Res. Commun. 206:556‐562.
  Lyles, G.A. 1996. Mammalian plasma and tissue‐bound semicarbazide sensitive amine oxidases: Biochemical, pharmacological and toxicological aspects. Int. J. Biochem. Cell. Biol. 28:259‐274.
   Markwell, M.A., Haas, S.M., Bieber, L.L., and Tolbert, N.E. 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87:206‐210.
   Merck Index. Any recent edition.
   O'Brien, E.M., Dostert, P., and Tipton, K.F. 1995. Species differences in the interactions of the anticonvulsant milacemide and some analogues with monoamine oxidase‐B Biochem. Pharmacol. 50:317‐324.
   O'Carroll, A.M., Bardsley, M.E., and Tipton, K.F. 1986. The oxidation of adrenaline and noradrenaline by the two forms of monoamine oxidase from human and rat brain. Neurochem. Int. 8:493‐500.
   O'Carroll, A.‐M., Anderson, M.C., Tobbia, I., Phillips, J.P., and Tipton, K.F. 1989. Determination of the absolute concentrations of monoamine oxidase A and B in human tissues. Biochem. Pharmacol. 38:901‐905.
   Salach, J.I. and Weyler, W. 1987. Preparation of the flavin‐containing aromatic amine oxidases of human placenta and beef liver. Methods Enzymol. 142:627‐37.
  Tipton, K.F. 1985. Enzyme assay and kinetic studies. In Techniques in the Life Sciences. B1/II (Suppl.), (K.F. Tipton, ed.) B5113, pp. 1‐61. Elsevier Science Publishing, Shannon (New York).
   Tipton, K.F. 1996. Patterns of enzyme inhibition. In Enzymology LabFax (P.C. Engel, ed.) pp. 115‐174. BIOS Scientific Publishers, Oxford, and Academic Press, San Diego.
   Tipton, K.F., McCrodden, J.M., and Youdim, M.B. 1986. Oxidation and enzyme‐activated irreversible inhibition of rat liver monoamine oxidase‐B by MPTP. Biochem J. 240:379‐383.
   Tipton, K.F. and Singer, T.P. 1993. The radiochemical assay for monoamine oxidase activity: Problems and pitfalls. Biochem. Pharmacol. 46:1311‐1316.
   Tipton, K.F. and Youdim, M.B.H. 1993. Determination of the monoamine oxidases. In Methods in Neurotransmitter and Neuropeptide Research, part 2 (S.H. Parvez, M. Naoi, T. Nagatsu, and S. Parvez, eds.) pp. 61‐91. Elsevier/North‐Holland, Amsterdam.
   Urban, P., Andersen, J.K., Hsu, H.‐P.P., and Pompon, D. 1991. Comparative membrane locations and properties of active monoamine oxidases expressed in yeast. FEBS Lett. 286:142‐146.
   Youdim, M.B.H., Finberg, J.P.M., and Tipton, K.F. 1988. Monoamine oxidase. In Handbook Experimental Pharmacology (U. Trendelenburg and N. Weiner, eds.) 90:119‐192. Springer‐Verlag, Berlin.
   Yu, P.H. 1986. Monoamine oxidase. Neuromethods 5:235‐272.
Key Reference
   Lieberman, A., Olanow, C.W., Youdim, M.B.H., and Tipton, K.F. 1994. Monoamine Oxidase Inhibitors in Neurological Diseases, Marcel Dekker, New York.
  Contains authoritative articles on the properties and pharmacology of MAO.
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