Difference in monoamine oxidase activity measured by either liquid ion exchange or ion exchange resin chromatography in rat and cat brain

ANALYTICAL BIOCHEMISTRY 149, 183-190 (1985)

Difference in Monoamine Oxidase Activity Measured by Either Liquid Ion Exchange or Ion Exchange Resin Chromatography in Rat and Cat Brain

M. JOHNSON AND A. G. ROBERGE

Departement de Biochimie (Medecine) and Departement de Nutrition Humaine (F.S.A.A.), Universith Laval, Quebec GlK 7P4. Canada

Received October 12, 1984

Cat and rat brain monoamine oxidase (MAO) activity was measured with a radioisotopic procedure and two extraction methods. Results indicated an underestimation of MAO activity when liquid ion exchange chromatography (LIEC) was used instead of an ion exchange chromatographic method (IEC) to separate the different products of the deaminated tyramine, phenylethylamine, or serotonin. MAO produced aldehydic products which may be found in the incubation medium and may be extracted with the substrate in the chloroform phase by the LIEC method. In cat brain, the resulting underestimation of the MAO activity was prevented by the addition of nicotinamide adenine dinucleotide ( 10e3 M) in the incubation medium or by allowing a 2-h period between the end of incubation and the LIEC extraction procedure. In the rat brain, the same result was obtained by the addition of an equimolar mixture of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate in reduced form (NAD-NADPH, IO-’ M). Using the IEC method, the NAD decreased only the deamination of tyramine and serotonin in rat brain. This study suggests that the use of an IEC method to evaluate MAO activity is more accurate for the estimation of the enzymatic activity. 0 1985 Academic Press. Inc.

KEY WORDS: brain MAO; S-hydroxytryptamine; phenyiethylamine; tyramine; cat; rat.

Manometric (I), spectrophotometric (2,3), fluorometric (4), and radiochemical tech- niques (5) have been successively proposed to measure monoamine oxidase activity (monoamine; O2 oxidoreductase, EC 1.4.3.4; MAO).’ Radiochemical methods have been found to be simple, sensitive and more specific, and are now commonly used (6,7). In those methods, the deaminated products are usually separated from their respective substrates by either an organic extraction procedure (5,6,8), an ion exchange resin chromatography [IEC (9)], or a liquid ion exchange chromatography [LIEC (7)].

In the rat brain, MAO catalyzes the deamination of monoamines to aldehyde products (10) which are further metabolized by alde-

’ Abbreviations used: MAO, monoamine oxidase activity; IEC, ion exchange resin chromatography; LIEC, liquid ion exchange chromatography.

hyde dehydrogenase (EC 1.2.1.3) (11) or aldehyde reductase (EC 1.1.1.2) ( 12) to acidic and alcoholic metabolites, respectively. Ac- cording to Breese et al. (13) and Tabakoff (14), those intermediate enzymes require nicotinamide derivatives as cofactors.

Wurtman and Axelrod (5) reported a low activity of aldehyde dehydrogenase in the human buccal mucosa which caused the accumulation of aldehyde products when MAO activity was measured “in vitro.” Thus, several products must be separated from the substrate. Preliminary results showed a difference in MAO activity when an ion exchange resin chromatography (9) and a liquid ion exchange chromatography (7) were used. The purposes of the present paper are (a) to compare the measurement of MAO activity in cat and rat brain using two chromatographic methods when the metabolism of aldehydes is stimulated by the presence of

183 0003-269?/85 $3.00 Copyright 0 1985 by Academic PRSS. Inc. All rights of reproduction in any form reserved.

184 JOHNSON AND ROBERGE

NAD and NADPH cofactors in the incubation medium, and (b) to define the accuracy of the two methods for different substrates and animal species.

MATERIAL AND METHODS

Five male and female mongrel cats weighing 2.7 & 0.9 kg were kept together for 2 weeks in a room containing perches. They were fed Purina Cat Chow and received water ad libitum. Five Sprague-Dawley female rats, weighing 250 f 10 g, were kept in individual cages, fed Purina Rat Chow, and had water ad libitum. Rooms were main- tained at a constant temperature (2 1 “C) and humidity (55%) with background music. The photoperiod was fixed between 0600 and 1800 h. Cats and rats were decapitated and their brains were rapidly dissected. Cat brain cortex and whole rat brain were stored at -80°C until biochemical assays were performed.

Monoamine oxidase assay. Brain MAO activity was measured according to the conditions described by Wu and Dyck (7) with slight modifications. The incubation medium contained 200 ~1 of 5 mM phosphate buffer (pH 7.5), the enzyme preparation, and one of the following: 1.5 mM [14C]tyramine (0.18 mCi/mmol), 0.5 mM [‘4C]SerOtOniII (0.5 IIICi/

mmol), and either 0.02 and 0.2 mM [P-

14C]phenylethylamine (10.3 or 1 .O mCi/ mmol) for cat and rat brain. Radiochemicals were purified by thin-layer chromatography on silica gel using a mixture of 1-butanol, acetic acid, and water (25:4:10, v/v) as sol- vent. Tissues were homogenized in a 5 mM phosphate buffer (pH 7.5) containing 0.5% Cutscum to give a 10% (w/v) solution, and the homogenate was shaken for 30 min at 4°C. The incubation medium contained 3 10 and 240 pg of total protein for cat and rat brain, respectively, as enzymatic source. When specified, the incubation medium contained lop3 M of either P-nicotinamide adenine dinucleotide, reduced P-nicotinamide

adenine dinucleotide phosphate, or a mixture of these two cofactors.

The incubation medium was preincubated 10 min and the reaction initiated by addition of the substrate. Incubation was carried out at 37°C for 30 min, except for the deamination of serotonin in cat brain which was done at 20°C and the reaction was ended at 4°C in an ice bath.

Blanks were set up by the addition of 80 ~1 of 0.4 N HC104 or 20 ~1 of 0.3 N NaOH to the incubation medium before the addition of the substrate and kept at 4°C in an ice bath. p-Hydroxyphenylacetic acid, [ “C]phenylacetic acid (0.50 mCi/mmol), and [5-‘4C]hydroxy-3-indoleacetic acid (0.36 mCi/ mmol) were used as standards. p-Hydroxy- phenylacetic acid concentrations were determined by an Aminco-Bowman spectropho- tofluorometer, American Instrument Com- pany, at 280 nm excitation and 310 nm emission.

Liquid ion exchange procedure. The separation of amines from their respective deaminated products as described by Wu and Dyck (7) was slightly modified. At the end of the incubation, 20 ~1 of 0.3 N NaOH was added and samples were kept at 4°C in an ice bath for 2 h, unless specified; then 500 ~1 of 0.1 M di-(2-ethylhexyl)phosphoric acid solution in chloroform was added to the incubation medium and the mixture was shaken immediately for 1 min. The test tubes were centrifuged for 30 min at 2500g at 4°C in order to break the emulsion. An aliquot of 110 ~1 of the aqueous phase was added to a scintillation mixture (2,5-diphenyloxazole, 1,4-bis( 5-phenyloxazol-2-yl)benzene, Triton X-100, and toluene). Recoveries were 79, 89, and 85% for p-hydroxyphenylacetic acid, 5- hydroxyindoleacetic acid, and phenylacetic acid, respectively. Counting efficiency for i4C was 84%.

Zon exchange resin chromatography. Ion exchange chromatography was performed with a Bio-Rad Econo-Column measuring 0.7 X 10 cm or 0.7 X 4 cm and containing

CHROMATOGRAPHY OF BRAIN MONOAMINE OXIDASE ACTIVITY 185

either a 5.5- or 4-cm length of Amberlite CG-50 resin when @-phenylethylamine or tyramine and serotonin, respectively, were used as substrates. Amberlite resin was pre- pared as described by Pisano (15).

Separation of the amines from their respective deaminated products, described by Robinson et al. (9), was slightly modified. To the incubation medium, 80 ~1 of 0.4 N

HC104 was added and the whole mixture was centrifuged for 10 min at 2500g at 4°C and brought to pH 6.5 with 80 ~1 of 1 M

sodium acetate buffer. The supernatant was eluted through Amberlite CG-50 with 7 ml of water. The effluent containing the deaminated products was recovered in 10 ml of scintillation mixture. Counting efficiency was 75%. Recoveries were 95, 89, and 99% for p- hydroxyphenylacetic acid, phenylacetic acid, and 5-hydroxy-3-indoleacetic acid, respectively.

Protein determination. Protein concentration was determinated by Bradford’s method ( 16) using bovine serum albumin as standard.

Statistical analysis. Mean, standard devia- tion, standard error of the mean, and Stu- dent’s t test were calculated according to Lison (17). Variance analysis, Duncan multiple range test, and Bartlett test were performed according to Kramer (18), Duncan (19) and Snedecor and Cochran (20), respectively.

Chemicals. Tyramine-HCl, P-phenylethylamine-HCl, p-hydroxyphenylacetic acid 5- hydroxytryptamine creatinine sulfate salt, 5-hydroxyindoleacetic acid dicyclohexylam- monium salt, di-(2-ethylhexyl)phosphoric acid, and P-nicotinamide adenine dinucleotide were purchased from Sigma Chemical Com- pany (St. Louis, MO.). /3-Nicotinamide adenine dinucleotide phosphate in reduced form was purchased from Boehringer-Mannheim (Dorval, Canada). Cutscum was purchased from Fisher Scientific Company (Fair Lawn, N. J.), Bio-Rad Econo-Columns (i.d. 7 X 40 mm and 7 X 100 mm) were from Bio- Rad Laboratories (Richmond, Va.), and Am-

berlite (CG-SO(H), 200 mesh) was from BDH Chemicals Ltd, (Poole, England). [ 14C]- Tyramine [p- hydroxyphenyl[2 - 14C]ethyla- mine HCl (50 mCi/mmol)], [5-14C]hydroxytryptamine [5-hydroxy-3-indolyl[ l- “C]ethyl-2-amine creatinine sulfate mono- hydrate (58 mCi/mmol)], and phenyl[l- i4C]acetic acid (50 mCi/mmol) were purchased from Amersham Corporation (Oak- ville, Canada). [5-Carboxyl-‘4C]hydroxy-3- indoleacetic acid (15.2 mCi/mmol) and ,& [ethyl-l-‘4C]phenylethylamine (50 mCi/ mmol) were purchased from New England Nuclear (Boston, Mass.).

RESULTS

Cat Brain MAO Activity

The accumulation of deaminated products (expressed in dpm X lo3 versus time plot) obtained with liquid ion exchange chromatography (LIEC) and with ion exchange resin chromatography (IEC) is shown in Fig. 1. A pool of three cat brain cortices was used to deaminate either tyramine, P-phenylethylamine, or serotonin. The extraction was performed immediately after the incubation, At zero time, mean blank values expressed in disintegrations per minute were, respectively, 594 t- 9, 267 f 33, and 528 + 39 with tyramine, P-phenylethylamine, and serotonin as substrates when the extraction is performed with IEC, whereas these values were 5739 + 291, 697 + 98, and 5027 + 55, respectively, with the LIEC extraction. In both methods, a linear accumulation of deaminated products was observed for at least 45 min when tyramine, &phenylethylamine, and serotonin were used as substrates. With the IEC method, the correlation coefficients of linear regression evaluated for tyramine, P-phenylethylamine, and serotonin were 0.99, 0.99, and 0.99, respectively. However, with the LIEC method, these values were lower, being 0.85, 0.96, and 0.9 1 for tyramine, @phenylethylamine, and serotonin, respectively.

Table 1 shows the values of MAO activity


t TYRAMlNE t PHENYLETHYLAMINE

t

OOW

TINE (mln)

m- 2 ; 50- / 2 A

D z z - / iec E

:: t z

-1 - r=o .99

% A

x

TlME(min1

0 20 40 - TIME (min)

FIG. 1. Accumulation of the deaminated products produced by cat brain MAO for tyramine, & phenylethylamine, and serotonin using liquid ion exchange chromatography and ion exchange resin chromatography. A pool of cat brains was used as enzymic source and triplicates (x f SE) were done. Correlation coefficients (r) are expressed for each time plot.

expressed in pmol * mg protein-’ * min-’ ob- methods when tyramine was used as substrate served in Fig. 1 with both extraction methods. (Table 2, control column). A significant dif- Whatever the substrate used, a lower and ference was observed when P-phenylethylam- significant MAO activity (P < 0.001) was ine (P < 0.001) and serotonin (P < 0.01) were measured with the LIEC method as compared used as substrates. The significant difference to the IEC method, if the extraction was between the LIEC and IEC methods in the performed immediately after the incubation. evaluation of MAO activity disappeared when However, when the LIEC extraction proce- the incubation medium contained NAD or dure was performed 2 h after the end of the a NAD-NADPH mixture at a concentration incubation, no significant difference in the of 10m3 M. A lower concentration of nicotin- MAO activity was observed between the two amide cofactors did not correct the discrep-

TABLE 1

CAT BRAIN MAO ACTIVITY ESTIMATED WITH A LIQUID ION EXCHANGE AND AN ION EXCHANGE RESIN CHROMAT~GRAPHIC PROCEDURE

Extraction procedure

IEC (9) LIEC (9)

Tyramine

445 f 16 211 * 19***

Monoamine oxidase activity

@-Phenylethylamine

161 k2.6 28 f 1.9***

Serotonin

129 k3.1 61 f 3.0***

Note. The number in parentheses represents the number of determination done on a pool of three cat brain cortices. Extractions were performed immediately after the incubation. Results are expressed in pmolamg protein-’ * min- ’ (,%? -t SE). The student t test was performed to compare MAO activity evaluated by LIEC and IEC chromatographic methods.

*** P < 0.001.


TABLE 2

E~CTS OF LIQUID ION EXCHANGE EXTRACTION AND ION EXCHANGE RESIN CHROMATOGRAPHY ON MAO MEASUREMENT IN CAT BRAIN 2 h AVER INCUBATION

Monoamine oxidase activity (pmol - mg protein-‘. min-’ f SE)

Extraction NAD NADPH NAD-NADPH procedure Control (lo-’ M) (lo-’ M) ( 1o-3 M)

Tyramine IEC”,’ (5) 553 +_40 553 Ik 54 565 +41 549 +_43 LIEC (5) 410 k63 605 + 53t 475 *44 618 241

Phenylethylamine IEC (5) 180 f 18 219 +20 181 + 17 223 f21 LIEC (5) 45 + 4*** 185 rf: 16ttt 75 f 9*** 184 f 17***

Serotonin IEC (5) 131 + 9 1284 4 125 f 6 133 t 7 LIEC (5) 64 + 19” 132 +-24t 91 f 8** 123 k14t

‘Statistical analysis between LIEC and IEC determinations was done using Student’s t test. The number of animals used appears in parentheses. *P < 0.05; **P < 0.01; ***P < 0.001.

b Statistical analysis between control medium and those containing NAD, NADPH, or a mixture of both was done using an ANOVA followed by a Duncan’s test. tP < 0.05; ttP < 0.01; tttp < 0.001.

ancy between the two methods when /3- phenylethylamine was used as substrate (unpublished results). A multiple range test did not reveal any significant difference between the control and nicotinamide incubation media, whatever the substrate used, when the IEC method was used to determine the MAO activity. However, with the LIEC method, the incubation media containing NAD or NAD-NADPH showed a significantly higher MAO activity (P < 0.05; P < 0.001) when the values were compared to their corre- sponding control incubation medium, whatever the substrate used. A similar MAO activity was measured with the LIEC method in the presence or absence of NADPH into the incubation medium. However, the results obtained by this method with NADPH were lower than the results of the IEC method when ,&phenylethylamine and serotonin were used as substrates (NADPH column, Ta- ble 2).

When the extraction procedures were performed immediately after the incubation of

tyramine with three different cat brain cortices, a significant difference (P < 0.01) in the MAO activity was observed between the two methods (IEC: 580 f 50, pmol . mg protein-’ . min-’ vs LIEC: 3 17 + 25). The presence of NADPH in the incubation medium did not affect these results. However, addition of NAD or the NAD-NADPH mixture raised the MAO ‘activity measured by the LIEC method to the level of the IEC determination (IEC: 580 + 50 pmol . mg of protein-’ * min-’ vs LIEC plus NAD: 466 _+ 61, LIEC plus NAD-NADPH: 594 f 25). These last two determinations were significantly higher (P < 0.05 and P < 0.01, respectively) than that of the LIEC control.

Rat Brain MAO Activity

Using tyramine and P-phenylethylamine as substrates (Table 3, control column), a significantly lower activity by the LIEC method was observed when the results obtained by the two methods are compared.


TABLE 3

EFFECTS OF LIQUID ION EXCHANGE EXTRACTION AND ION EXCHANGE RESIN CHROMATOGRAPHY ON MAO MEASUREMENT IN BAT BRAIN 2 h AFTER INCUBATION

Monoamine oxidase activity (pmol . mg protein-’ - min-’ + SE)

Extraction procedure Control

NAD NADPH (lo-- M) (lo-’ M)

NAD-NADPH (lo-’ M)

Tyramine IEC”* (5) LIEC (5)

Phenylethylamine IEC (5) LIEC (5)

Serotonin IEC (5) LIEC (5)

5540 2314 4536 k292 5476 +353 5236 k245 3860 &229*" 3058 + 166*+-t 3988 &154** 4642 k259tt

860 f 54 874 f 52 885 + 29 889 k 55 289 f 29*** 389 + 25***st 356 f ll*** 768 f 3377-f

3566 +165 2640 + 1Oltt 3466 +172 2996 +I211 3284 + 64 2248 zk 5O***ttt 3338 &I26 3128 f 82

‘Statistical analysis between LIEC and IEC determinations was done using Student’s f test. The number of animals used appears in parentheses. *P -c 0.05; **P < 0.01; ***P < 0.001.

*Statistical analysis between control medium and those containing NAD, NADPH, or a mixture of both was done using an ANOVA followed by a Duncan’s test. tP < 0.05; ttP < 0.01; tttP i 0.001.

There was no significant difference in the MAO activity measured with the IEC and the LIEC methods when serotonin was used, even if the extraction was performed immediately after the incubation (unpublished results). The significant difference observed between MAO activities, evaluated with the LIEC and IEC methods when tyramine and /3-phenylethylamine were used, disappeared with the addition of a NAD-NADPH mixture in the incubation medium. Addition of a NAD-NADPH mixture decreased the MAO activity (P < 0.05) evaluated with the IEC method using serotonin as substrate.

The addition of NADPH did not change the results obtained in the control conditions with either method. However, the presence of NAD decreased the MAO activity measured with the LIEC method when the results were compared with those of the IEC method, or with the control value of the LIEC method, when tyramine and serotonin were used as substrates. NAD decreased the MAO activity measured with the IEC method for tyramine (P < 0.05 with a Student’s t test) and serotonin (P < 0.01).

DISCUSSION

The present findings demonstrate that (a) the ion exchange resin chromatography (IEC) is a more specific and accurate method to estimate cat or rat brain MAO activity than liquid ion exchange chromatography (LIEC); (b) the LIEC underestimates the MAO activity by the extraction of the intermediary deaminated products, such as aldehydes, in the chloroform phase; (c) the presence of NAD or a mixture of NAD-NADPH pre- vents the underestimation of cat and rat brain MAO activity, respectively, and, in rat brain, type A MAO may be inhibited by NAD. Moreover, using tyramine as substrate, the time at which the LIEC extraction is performed affects the evaluation of cat brain MAO activity.

Several authors have shown that the deaminated product of MAO is an aldehyde (lo,2 1,22) which is transformed into an acidic metabolite by aldehyde dehydrogenase (1 l), or into alcohol by aldehyde reductase (12,23). In some organic extraction procedure (5) or IEC method (24) the intermediary metabo-


lites might be taken up with the end products, thus favoring a true estimation of MAO activity. However, the use of a LIEC method underestimates the MAO activity when the results are compared to IEC evaluations. The presence of the aldehyde products in the incubation medium, and their extraction in the chloroform phase, may be responsible for this underestimation. The addition of NAD (cofactor of aldehyde dehydrogenase), and NADPH (cofactor of alcohol reductase) (14) to the incubation medium potentiates the transformation of the aldehydic products into acids or alcohols. In cat brain cortex (Table 2), the addition of NAD corrected the discrepancy between the two methods. A low concentration of the aldehyde reductase in brain tissue or a lack of cofactor used in the incubation medium could explain the difference between MAO activity evaluated by the two methods in the presence of NADPH.

In rat brain, the addition of both NAD and NADPH in the incubation medium corrected the discrepancy between the two methods of evaluation of MAO activity. However, those effects are not additive since the individual addition of NAD or NADPH did not increase MAO activity. These results underline the importance of having NAD and NADPH as cofactors in a certain pro- portion in the incubation medium to elimi- nate aldehyde products. The transformation of one cofactor into the other which would increase its concentration and stimulate the transformation of the aldehyde could be an- other explanation. Using serotonin as substrate, results in Table 3 suggest that 5- hydroxy-3-indoleacetaldehyde was metabolized rapidly to an acid metabolite under the control conditions as no difference of MAO activity was noted for the two methods. MAO activity, determined by IEC, was not affected by the addition of NADPH. How- ever, in the presence of NAD, a low activity was measured in rat brain with tyramine and serotonin as substrates. The results remain unexplained. As the deamination of /3-phenylethylamine was not affected, only type A

MAO is suspected to be inhibited. Johnston (25) has shown the existence of two types of MAO activity. It appears that type A MAO preferentially deaminates serotonin whereas /3-phenylethylamine is deaminated by type B (8). Tyramine, on the other hand, is deaminated by both forms.

Aldehyde produced from deamination is generally presumed to be immediately transformed into its acid or alcohol metabolites. In vivo, this is made possible by the generation of NAD for aldehyde dehydrogenase or NADPH for aldehyde reductase. In vitro, Wurtman and Axelrod (5) demonstrated the presence of an aldehyde product in the incubation medium after deamination of [‘4C]tryptamine by MAO from human buccal mucosa and eliminated this product by the addition of aldehyde dehydrogenase and its cofactor. They concluded that the presence of the aldehyde product is not necessarily caused by a lack of its enzyme in the incubation medium. The results of our experiments tend to demonstrate that presence of an aldehyde product is not necessarily caused by a lack of an aldehyde dehydrogenase or reductase, but rather by the lack of their cofactors.

The results of the experiments described herein allow us to conclude that the difference between both methods may be eliminated by the addition of a NAD-NADPH mixture to the incubation medium and that the IEC method yields lower blank values for the three substrates than does the LIEC method. It would appear, then, that the IEC procedure is more suitable for evaluating the true MAO activity in both rat and cat brain, without the presence of NAD-NADPH in the incubation medium, since more than 95% of aldehydic and acidic products are recovered in the eluate (26).

ACKNOWLEDGMENTS

The authors express their gratitude to Dr. John A. Zee for his helpful discussion and revision of manuscript, to Huguette Duquet for her expert secretarial support, and to Nicole Seoane for revising the style of the final


manuscript. This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (Grant A-7946) and by CRESAQ.

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Difference in monoamine oxidase activity measured by either liquid ion exchange or ion exchange resin chromatography in rat and cat brain