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Microassay for the estimation of monoamine oxidase activity
ANALYTICAL
BIOCHEMISTRY
72,637-642
(1976)
SHORT COMMUNICATIONS Microassay
for the Estimation of Monoamine Oxidase Activity
A simple, sensitive, and specific radiochemical method for analysis of monoamine oxidase activity was developed. Liquid ion exchanger added to the incubation medium removed the remaining labeled substrate. An aliquot of the aqueous phase was placed in a scintillation vial and counted after extraction of the deaminated products into the toluene-based scintillation cocktail. The specificity of the method was indicated by the 90% inhibition of the enzyme activity measured by this method in presence of 5 x 10-5~ pargyline-HCI.
Monoamine oxidase (EC 1.4.3.4.) (MAO) is a central enzyme in the metabolism of monoamine neurotransmitters. Its activity is usually measured by manometric (l-3), fluorimetric (4-7), or spectrophotometric (8) techniques or by methods involving the oxygen electrode (9). These methods are either laborious, insensitive, or both. Recently, Wurtman and Axelrod (10) described a sensitive procedure capable of measuring MAO activity in as little as 5 pg of rat liver. [C14]tryptamine was used as substrate. However, not all the amines give similar results. Some of the labeled substrate amines (p-phenylethylamine and benzylamine) are readily extracted into the organic phase in significant quantities despite acidification prior to the extraction. Many attempts (1 I- 14) have been made to remove the substrate and thus reduce the blank value and improve the sensitivity. One of the criteria for the study of the multiple forms of MAO has been the substrate-specific inhibitors (14-18). We feel that a simple method which is not limited to a few substrates would facilitate research in this area. We have, therefore, developed a simple, sensitive, and specific radiochemical method for MAO based on the removal of labeled substrate by a liquid ion exchanger followed by a direct extraction of deaminated metabolites into a scintillation fluid. Sixty or more samples may be analyzed in a 2 hr period. This method appears to work equally well for benzylamine, P-phenylethylamine, p-tyramine, dopamine, tryptamine, and 5hydroxytryptamine. MATERIALS
AND METHODS
Benzylamine ( [14C]methylene) (4.0 mCi/mmol) ICN Isotope and Nuclear Division, California; 637 CopyrIght 0 1976 by Academx Pres. Inc. All rights of reproduction in any form rewrved.
was purchased from [ l-C14]P-phenylethyl-
638
SHORT
COMMUNICATIONS
amine (7 mCi/mmol) was obtained from New England Nuclear, Massachusetts; [I-C41p-tyramine (44 mCi/m mol), [1-C4]dopamine (56 mCi/ mmol), [3H(G)]tryptamine (535 mCi/mmol), [ I-Cl415hydroxytryptamine (55 mCi/mmol), [l-C14]3-indolyl acetic acid (52 mCi/mmol) and [l-C4]phenylacetic acid (52 mCi/mmol) were purchased from Amersham Searle, Illinois. Diethylhexylphosphoric acid was obtained from Sigma Chemical Co., St. Louis, Missouri. All other chemicals used were analytical grade. Radiochemicals were checked for purity and, if necessary, purified by paper chromatographic procedures. All the substrates were dissolved in 50 mM phosphate buffer at pH 7.2, with the exception of dopamine which was dissolved in dilute acetic acid with 10% ascorbic acid to prevent oxidation. The solutions were kept at -20°C until used. Cold amines were added to the radioactive compounds to a final concentration of approximately 2.2 FM. Freshly excised rat liver was washed in chilled 50 mM phosphate buffer and homogenized in the same buffer containing 0.2% Triton X-100 (J. T. Baker Chem. Co.). In a typical assay, 50 ~1 of the liver homogenate was mixed with 140 ~1 50 mM phosphate buffer at pH 7.2 and 10 ~1 of substrate (2.4 FM p-tyramine, 2.5 ,UM /3-phenylethylamine and benzylamine, 2.2 PM dopamine or 2.2 FM 5-hydroxytryptamine and tryptamine) in a 15 ml centrifuge tube and incubated at 37°C for 30 or 60 min with shaking. For the incubation with a smaller quantity of tissue, 0.22 PM of p-tyramine and 0.22 PM tryptamine were used. The reaction was terminated by immersing the tubes in an ice bath followed by the addition of 500 ~10.1 M diethylhexylphosphoric acid liquid ion exchanger dissolved in chloroform to remove the unreacted substrate. This mixture was shaken for 1 min in a Vortex mixer followed by centrifugation at 3000g for 2 min. An aliquot of 100 ~1 of the aqueous phase was then removed and placed in a counting vial. To this was added 500 ~1 of 0.8 N perchloric acid. After shaking (10 set in a Vortex mixer) with 10 ml of liquid scintillation fluid (toluene containing 5 g/liter solimix ICN, California), this was then counted in a liquid scintillation spectrophotometer. Counting efficiencies were greater than 90% for carbon-14 and 43% for tritium. Boiled tissue processed in a similar manner served as a blank. Recovery of phenylacetic acid and indolyl acetic acid was determined by adding known amounts of these isotopes to tissue blanks. Pargyline HCl was added to the incubation medium containing 200 pg of rat liver homogenate to final concentrations of 5 ‘X 10W4,5 x 10p5, 5 x lO+j, and 5 x lop7 M. RESULTS
AND DISCUSSION
The reaction was linear over a period of 60 min (see Fig. 1) when 400 pg of rat liver homogenate was incubated with approximately 2.2 pM 5-hydroxytryptamine and dopamine; p-phenylethylamine, tryptamine, however, withp-tyramine and benzylamine, it was linear for only 30 min. It
SHORT
639
COMMUNICATIONS
L 0
LO
20 TIME
60
(minut*)
FIG. I. Rat liver homogenate 400 pg was incubated with approximately 2.2 pM of [‘“Clp-tyramine (0). [YIP-phenylethylamine (0). [3H(G)]tryptamine (A), [laC]benzylamine (XL [W]S-hydroxytryptamine (0). and [Wldopamine (0) at 37°C over a period of 0 to 60 min. The results are the average of two experiments.
is also clear from Fig. 1, that p-tyramine, phenylethylamine, and tryptamine are deaminated with a faster rate than the other three amines. Similar findings have been previously reported (19-21). The reaction was linear over the range O-400 pug rat liver for all the amines tested during the course of incubation. The MAO activity in 100 pg rat liver homogenate is approximately seven times that of the tissue blanks when p-tyramine, p-phenylethylamine, and tryptamine were used as substrate (see Fig. 2). When rat liver homogenate was incubated with 0.22 PM tryptamine or p-tyramine for 60 min, the reaction was linear from 0 to 100 pg rat liver. As little as 10 ,ug rat liver tissue is sufficient to give a significant determination. This method is simplified by using the scintillation fluid as the extraction solvent and performing the extraction directly in the scintillation
640
SHORT
COMMUNICATIONS
RAT
LIVER
,pgj
FIG. 2. Approximately 2.2 PM of [V]p-tyramine (0). [14C]/3-phenylethylamine (O), [3H(G)]tryptamine (A),[Y?]S-hydroxytryptamine (x). [‘“C]benzylamine (0, and [“Cldopamine (0) were incubated with O-400 pg of rat liver homogenate at 37°C for a period of 60 min. With the exception of [14Clp-tyramine and [‘%]benzylamine which were allowed to proceed for only 30 min. The results are the average of two experiments.
vial. The labeled deaminated metabolites can subsequently be determined by liquid scintillation counting at high efficiency in a biphasic aqueous: toluene scintillation mixture similar to that described by Fonnum (22) for determination of choline acetyltransferase. The ion exchange process, however, does not completely remove all the substrate; therefore to prevent the solvent (toluene) extraction of this small amount of amine into the scintillation cocktail and also to increase the efficiency of the extraction of acidic metabolites, perchloric acid is added to the aqueous phase. Extraction of incompletely oxidized metabolites should be similar and, if not, then one can count the aqueous phase without toluene extraction in a scintillation cocktail compatible with that volume of water. Such a procedure has been shown, however, to increase the blank value. Recoveries of labeled phenylacetic acid and 3-indolyl acetic acid added to tissue blank were 88 _+ 1% (n = 4) and 82 + 12% (n = 6) respectively. Pargyline hydrochloride (5 x 10e5~) added to the incubation medium inhibited over 90% of the observed MAO activity (see Fig. 3). The presence of the drug does not appear to interfere with the ion exchange or
SHORT
NONE
COMMUNICATIONS
5x10
641
L
PARGYLINE-HCL
( M 1
FIG. 3. Rat liver homogenate (200 pg) was incubated in the medium containing 2.2 FM [‘*C]~-phenyl~thylamine and 5 x lo-‘M, 5 x ~O+M, 5 x 10-5~, and 5 x ~O+M of pargyline-HCI. The results are expressed as mean +: SE of five experiments in percentage of remaining monoamine oxidase activity.
solvent extraction. This also indicated the method is very specific for measuring MAO activity. The simplicity and sensitivity of the method permitted the assessment of MAO activity in the nervous tissue of snail and insects (in preparation). ACKNOWLEDGMENTS We thank the Department of Health, Province of Saskatchewan for continuing financial support and Drs. A. A. Boulton and H. A. Robertson for helpful discussion. Pargyhne hydrochloride was donated by Abbott Laboratories, Chicago.
REFERENCES 1. 2. 3. 4. 5. 6.
Blaschko, H., Richter, D., and Schlossmann, H. (1937) S&hem. J. 31, 2187. Quastel, J. J-J., and Wheatley, A. H. M., (1933) Biochem. J. 27, 1699. Creasy, N. H. (1956) BiocAem. J. 64, 178. Karjl, M. (1965) Biochem. Pharmac. 14, 1684. Century, B., and Ripp, K. L. (1968) Biochem. Pharmac. 17, 2012. Dvomik, D., Kraml, M., Dubuc, J., Tom, H., and Zsoter, T., (1963) B&hem. Pharmac.
7. Lovenberg,
12, 229.
W.. Levine,
R. J. and Sjoerdsma,
A., (1962) J. Pharmac.
Ehp. Ther.
135, 7.
8. 9. 10. 1 I.
Tabor. C. W,, Tabor, H., and Rosenthal, S. M. (1954)~. B&l. C&m. 208, 645. Williams, C. H. (1974) Biochem. Pharmac. 23, 615. Wurtman, R. J., and Axelrod, J., (1963) Biochem. Pharmac. 12, 1439. Robinson, D. S., Lovenberg, W., Keiser, H., and Sjoerdsma, A., (1968) Biochem. Pharmac.
17, 109.
642
SHORT
COMMUNICATIONS
12. Jain, J., Sands, F., and Von Koti, R. W. (1973) Anal. Biochem. 52, 542. 13. Izumi, F., Oka, M., Yashida, H., and lmiazumi, R., (1969) Biochem. Pharmac. 18, 1739. 14. Goridis, C. and Neff, N. H. (1971) Neuropharmacology 10, 557. 15. Johnston, J. P., (1968) Biochem. Pharmac. 17, 1285. 16. Squires, R. F., (1968) B~ochem. Phurmuc. 17, 1401. 17. Goridis, C. and Neff, N. H., (1971) Brft. J. Pharmacol. 43, 814. 18. Fuller, R. W., (1972)Adv. Biochem. Psychopharmac. 5, 5, 339. 19. Weissbach, H., Smith, T. E., Daly, J. W., Witkop, B., and Udenfriend, S. (196O)J. Biol. Chem. 235, 1160. 20. Hope, D. B. and Smith, A. D. (1960) Biochem. J. 74, 101. 21. McCaman, R. E., McCaman, M. W., Hunt, J. M., and Smith, M. S. (l%S) J. ~eurochem. 12, 15. 22. Fonnum, F. (1975) J. Neurochem. 24,407.
P. H. WV L. E. DYCK Psychiatric Research Unit University Hospital Saskatoon, Saskatchewun Canada S7N 0 W8 Received June 23, 1975; accepted December 18, 1975
BIOCHEMISTRY
72,637-642
(1976)
SHORT COMMUNICATIONS Microassay
for the Estimation of Monoamine Oxidase Activity
A simple, sensitive, and specific radiochemical method for analysis of monoamine oxidase activity was developed. Liquid ion exchanger added to the incubation medium removed the remaining labeled substrate. An aliquot of the aqueous phase was placed in a scintillation vial and counted after extraction of the deaminated products into the toluene-based scintillation cocktail. The specificity of the method was indicated by the 90% inhibition of the enzyme activity measured by this method in presence of 5 x 10-5~ pargyline-HCI.
Monoamine oxidase (EC 1.4.3.4.) (MAO) is a central enzyme in the metabolism of monoamine neurotransmitters. Its activity is usually measured by manometric (l-3), fluorimetric (4-7), or spectrophotometric (8) techniques or by methods involving the oxygen electrode (9). These methods are either laborious, insensitive, or both. Recently, Wurtman and Axelrod (10) described a sensitive procedure capable of measuring MAO activity in as little as 5 pg of rat liver. [C14]tryptamine was used as substrate. However, not all the amines give similar results. Some of the labeled substrate amines (p-phenylethylamine and benzylamine) are readily extracted into the organic phase in significant quantities despite acidification prior to the extraction. Many attempts (1 I- 14) have been made to remove the substrate and thus reduce the blank value and improve the sensitivity. One of the criteria for the study of the multiple forms of MAO has been the substrate-specific inhibitors (14-18). We feel that a simple method which is not limited to a few substrates would facilitate research in this area. We have, therefore, developed a simple, sensitive, and specific radiochemical method for MAO based on the removal of labeled substrate by a liquid ion exchanger followed by a direct extraction of deaminated metabolites into a scintillation fluid. Sixty or more samples may be analyzed in a 2 hr period. This method appears to work equally well for benzylamine, P-phenylethylamine, p-tyramine, dopamine, tryptamine, and 5hydroxytryptamine. MATERIALS
AND METHODS
Benzylamine ( [14C]methylene) (4.0 mCi/mmol) ICN Isotope and Nuclear Division, California; 637 CopyrIght 0 1976 by Academx Pres. Inc. All rights of reproduction in any form rewrved.
was purchased from [ l-C14]P-phenylethyl-
638
SHORT
COMMUNICATIONS
amine (7 mCi/mmol) was obtained from New England Nuclear, Massachusetts; [I-C41p-tyramine (44 mCi/m mol), [1-C4]dopamine (56 mCi/ mmol), [3H(G)]tryptamine (535 mCi/mmol), [ I-Cl415hydroxytryptamine (55 mCi/mmol), [l-C14]3-indolyl acetic acid (52 mCi/mmol) and [l-C4]phenylacetic acid (52 mCi/mmol) were purchased from Amersham Searle, Illinois. Diethylhexylphosphoric acid was obtained from Sigma Chemical Co., St. Louis, Missouri. All other chemicals used were analytical grade. Radiochemicals were checked for purity and, if necessary, purified by paper chromatographic procedures. All the substrates were dissolved in 50 mM phosphate buffer at pH 7.2, with the exception of dopamine which was dissolved in dilute acetic acid with 10% ascorbic acid to prevent oxidation. The solutions were kept at -20°C until used. Cold amines were added to the radioactive compounds to a final concentration of approximately 2.2 FM. Freshly excised rat liver was washed in chilled 50 mM phosphate buffer and homogenized in the same buffer containing 0.2% Triton X-100 (J. T. Baker Chem. Co.). In a typical assay, 50 ~1 of the liver homogenate was mixed with 140 ~1 50 mM phosphate buffer at pH 7.2 and 10 ~1 of substrate (2.4 FM p-tyramine, 2.5 ,UM /3-phenylethylamine and benzylamine, 2.2 PM dopamine or 2.2 FM 5-hydroxytryptamine and tryptamine) in a 15 ml centrifuge tube and incubated at 37°C for 30 or 60 min with shaking. For the incubation with a smaller quantity of tissue, 0.22 PM of p-tyramine and 0.22 PM tryptamine were used. The reaction was terminated by immersing the tubes in an ice bath followed by the addition of 500 ~10.1 M diethylhexylphosphoric acid liquid ion exchanger dissolved in chloroform to remove the unreacted substrate. This mixture was shaken for 1 min in a Vortex mixer followed by centrifugation at 3000g for 2 min. An aliquot of 100 ~1 of the aqueous phase was then removed and placed in a counting vial. To this was added 500 ~1 of 0.8 N perchloric acid. After shaking (10 set in a Vortex mixer) with 10 ml of liquid scintillation fluid (toluene containing 5 g/liter solimix ICN, California), this was then counted in a liquid scintillation spectrophotometer. Counting efficiencies were greater than 90% for carbon-14 and 43% for tritium. Boiled tissue processed in a similar manner served as a blank. Recovery of phenylacetic acid and indolyl acetic acid was determined by adding known amounts of these isotopes to tissue blanks. Pargyline HCl was added to the incubation medium containing 200 pg of rat liver homogenate to final concentrations of 5 ‘X 10W4,5 x 10p5, 5 x lO+j, and 5 x lop7 M. RESULTS
AND DISCUSSION
The reaction was linear over a period of 60 min (see Fig. 1) when 400 pg of rat liver homogenate was incubated with approximately 2.2 pM 5-hydroxytryptamine and dopamine; p-phenylethylamine, tryptamine, however, withp-tyramine and benzylamine, it was linear for only 30 min. It
SHORT
639
COMMUNICATIONS
L 0
LO
20 TIME
60
(minut*)
FIG. I. Rat liver homogenate 400 pg was incubated with approximately 2.2 pM of [‘“Clp-tyramine (0). [YIP-phenylethylamine (0). [3H(G)]tryptamine (A), [laC]benzylamine (XL [W]S-hydroxytryptamine (0). and [Wldopamine (0) at 37°C over a period of 0 to 60 min. The results are the average of two experiments.
is also clear from Fig. 1, that p-tyramine, phenylethylamine, and tryptamine are deaminated with a faster rate than the other three amines. Similar findings have been previously reported (19-21). The reaction was linear over the range O-400 pug rat liver for all the amines tested during the course of incubation. The MAO activity in 100 pg rat liver homogenate is approximately seven times that of the tissue blanks when p-tyramine, p-phenylethylamine, and tryptamine were used as substrate (see Fig. 2). When rat liver homogenate was incubated with 0.22 PM tryptamine or p-tyramine for 60 min, the reaction was linear from 0 to 100 pg rat liver. As little as 10 ,ug rat liver tissue is sufficient to give a significant determination. This method is simplified by using the scintillation fluid as the extraction solvent and performing the extraction directly in the scintillation
640
SHORT
COMMUNICATIONS
RAT
LIVER
,pgj
FIG. 2. Approximately 2.2 PM of [V]p-tyramine (0). [14C]/3-phenylethylamine (O), [3H(G)]tryptamine (A),[Y?]S-hydroxytryptamine (x). [‘“C]benzylamine (0, and [“Cldopamine (0) were incubated with O-400 pg of rat liver homogenate at 37°C for a period of 60 min. With the exception of [14Clp-tyramine and [‘%]benzylamine which were allowed to proceed for only 30 min. The results are the average of two experiments.
vial. The labeled deaminated metabolites can subsequently be determined by liquid scintillation counting at high efficiency in a biphasic aqueous: toluene scintillation mixture similar to that described by Fonnum (22) for determination of choline acetyltransferase. The ion exchange process, however, does not completely remove all the substrate; therefore to prevent the solvent (toluene) extraction of this small amount of amine into the scintillation cocktail and also to increase the efficiency of the extraction of acidic metabolites, perchloric acid is added to the aqueous phase. Extraction of incompletely oxidized metabolites should be similar and, if not, then one can count the aqueous phase without toluene extraction in a scintillation cocktail compatible with that volume of water. Such a procedure has been shown, however, to increase the blank value. Recoveries of labeled phenylacetic acid and 3-indolyl acetic acid added to tissue blank were 88 _+ 1% (n = 4) and 82 + 12% (n = 6) respectively. Pargyline hydrochloride (5 x 10e5~) added to the incubation medium inhibited over 90% of the observed MAO activity (see Fig. 3). The presence of the drug does not appear to interfere with the ion exchange or
SHORT
NONE
COMMUNICATIONS
5x10
641
L
PARGYLINE-HCL
( M 1
FIG. 3. Rat liver homogenate (200 pg) was incubated in the medium containing 2.2 FM [‘*C]~-phenyl~thylamine and 5 x lo-‘M, 5 x ~O+M, 5 x 10-5~, and 5 x ~O+M of pargyline-HCI. The results are expressed as mean +: SE of five experiments in percentage of remaining monoamine oxidase activity.
solvent extraction. This also indicated the method is very specific for measuring MAO activity. The simplicity and sensitivity of the method permitted the assessment of MAO activity in the nervous tissue of snail and insects (in preparation). ACKNOWLEDGMENTS We thank the Department of Health, Province of Saskatchewan for continuing financial support and Drs. A. A. Boulton and H. A. Robertson for helpful discussion. Pargyhne hydrochloride was donated by Abbott Laboratories, Chicago.
REFERENCES 1. 2. 3. 4. 5. 6.
Blaschko, H., Richter, D., and Schlossmann, H. (1937) S&hem. J. 31, 2187. Quastel, J. J-J., and Wheatley, A. H. M., (1933) Biochem. J. 27, 1699. Creasy, N. H. (1956) BiocAem. J. 64, 178. Karjl, M. (1965) Biochem. Pharmac. 14, 1684. Century, B., and Ripp, K. L. (1968) Biochem. Pharmac. 17, 2012. Dvomik, D., Kraml, M., Dubuc, J., Tom, H., and Zsoter, T., (1963) B&hem. Pharmac.
7. Lovenberg,
12, 229.
W.. Levine,
R. J. and Sjoerdsma,
A., (1962) J. Pharmac.
Ehp. Ther.
135, 7.
8. 9. 10. 1 I.
Tabor. C. W,, Tabor, H., and Rosenthal, S. M. (1954)~. B&l. C&m. 208, 645. Williams, C. H. (1974) Biochem. Pharmac. 23, 615. Wurtman, R. J., and Axelrod, J., (1963) Biochem. Pharmac. 12, 1439. Robinson, D. S., Lovenberg, W., Keiser, H., and Sjoerdsma, A., (1968) Biochem. Pharmac.
17, 109.
642
SHORT
COMMUNICATIONS
12. Jain, J., Sands, F., and Von Koti, R. W. (1973) Anal. Biochem. 52, 542. 13. Izumi, F., Oka, M., Yashida, H., and lmiazumi, R., (1969) Biochem. Pharmac. 18, 1739. 14. Goridis, C. and Neff, N. H. (1971) Neuropharmacology 10, 557. 15. Johnston, J. P., (1968) Biochem. Pharmac. 17, 1285. 16. Squires, R. F., (1968) B~ochem. Phurmuc. 17, 1401. 17. Goridis, C. and Neff, N. H., (1971) Brft. J. Pharmacol. 43, 814. 18. Fuller, R. W., (1972)Adv. Biochem. Psychopharmac. 5, 5, 339. 19. Weissbach, H., Smith, T. E., Daly, J. W., Witkop, B., and Udenfriend, S. (196O)J. Biol. Chem. 235, 1160. 20. Hope, D. B. and Smith, A. D. (1960) Biochem. J. 74, 101. 21. McCaman, R. E., McCaman, M. W., Hunt, J. M., and Smith, M. S. (l%S) J. ~eurochem. 12, 15. 22. Fonnum, F. (1975) J. Neurochem. 24,407.
P. H. WV L. E. DYCK Psychiatric Research Unit University Hospital Saskatoon, Saskatchewun Canada S7N 0 W8 Received June 23, 1975; accepted December 18, 1975