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Inhibitory effect of some monoamine oxidase inhibitors on fluorescence assay for the o-methylated metabolites of the catecholamines
Inhibitory Effect of Some Monoamine Oxidase Inhibitors on Fluorescence Assay for the o-Methylated Metabolites of the Catecholamines
D. G. CALVERLEY,H. R. MCKIM, AND W. G. DEWHURST
INTRODUCTION Determination of brain levels of the o-methylated metabolites of the catecholamines provides important information regarding turnover of the catecholamines. Measurement of these metabolites in brain tissue is particularly of value when animals have been treated with a monoamine oxidase inhibitor (MAOI) that blocks the other major pathway for breakdown of the catecholamines. A number of techniques are available for the separation and quantification of the o-methylated metabolites of the catecholamines in brain tissue. One technique that we have found to be of sufficient sensitivity for analysis of normetanephrine (NM) and 3-methoxytyramine (3-MT) in MAOI-treated rat brain regions is the fluorometric technique of Karasawa et al. (1975). In our work with tranylcypromine (Calverley et al., 1978; McKim et al., 1980; McKim et al., reported elsewhere) we discovered, that when animals were treated with MAOI (20 mg/kg, i.p., 1.5 hours before death), NM and 3-MT could not be reliably measured in the mesencephalon-pons and diencephalon. We therefore tested and report here on possible interference with this assay by a number of MAOls and structurally related compounds, in vitro. METHODS Very briefly, the Karasawa et al. (1975) technique involves running an extract of brain tissue in sequence through aluminum oxide and Amberlite (X-50 columns. The aluminum oxide removes the catecholamines. NM and 3-MT adhere to the Amberlite column and are removed by 0.5 N HCI. A hydroxyindole assay is used with iodine as oxidizing agent and sodium sulfite and EDTA as reducing and stabilizing agents, respectively. Fluorescence is developed under acid conditions. We have found that by using a reaction time of 2 minutes at 100°C for NM and an additional 4 minutes at 100°C for 3-MT, we could shorten the time for production of the measured fluorophore, increase our sensitivity, and not lose any reliability over the original procedure, From the Neurochemical Research Unit, Department of Psychiatry, University of Alberta, Edmonton, Alberta, Canada. Address reprint requests to H. R. McKim, Neurochemical Research Unit, Department of Psychiatry, 1-121 Clinical Sciences Building, University of Alberta, Edmonton, Alberta T6C 2G3, Canada. Received January 8, 1980; revised and accepted September 9, 1980. 179 lournal of Pharmacological D 1981 Elsevier
North
Methods
Holland,
5, 179-181 (1981)
Inc., 52 Vanderbilt
Avenue. New York, NY 10017
0160.540218110179003$02.50
180
D. C. Calverley et al. Drugs tested for an effect on this assay included the MAOls, (t)-tranylcypromine, (+)-pheniprazine, phenelzine, isocarboxazid, nialamide, pargyline, (-)-deprenil, clorgyline, and (t )-a-ethyltryptamine. Related compounds tested included (+ )-amphetamine, (-)-amphetamine, phentermine, P-phenylethylamine, and tryptamine. The effects of different concentrations of these drugs on the reaction that produced the fluorophore from NM and 3-MT were studied. Fluorescence values in fluorescence intensity units (Flus) were obtained for each of the fluorophores produced from 3 x IO-’ M NM and 3-MT. Comparisons were then made to the Flus obtained from an identical reaction in the presence of a series of different concentrations (1O-6 M to 10e4 M) of each of the drugs under study. Dose-response curves were constructed and the concentration of drug that resulted in a 50% inhibition of the production of the fluorophore (IC,,) was determined from the curves. RESULTS AND DISCUSSION All of the MAOls except the structurally related group including pargyline, deprenil, and clorgyline inhibited the formation of the fluorophore. Of the non-MAO1 but related compounds, only tryptamine reduced fluorescence readings. The hydrazines, phenelzine and pheniprazine, and the substituted hydrazines, isocarboxazid and nialamide, all had similar effects on the assay. In the case of all of these drugs the fluorescence values were greater than 90% of control at 2 x lop6 M and decreased to less than 10% of control by 5 x 1O-6 M. I&values were approximately 4 x lO-‘j M. Tranylcypromine, on the other hand, affected the reaction differently in that fluorescence values remained greater than 90% of control to 5 x 1O-6 M, then declined to less than 10% of control by 1.2 x lop5 M. The I&, value of tranylcypromine (7.5 x 10e6 M) was much higher than the IC50 for the other MAOls. Results with a-ethyltryptamine and tryptamine were quite erratic with I& values ranging from 3 x 1O-6 M to 1.3 x 1O-5 M for a-ethyltryptamine and from 2 to 5 x lop6 M for tryptamine. Concentrations of these compounds appear, at first glance, high in relation to the concentration of the amines, but we have reported brain levels of tranylcypromine of 12.1 to 15.3 kg/g in brain regions 1.5 hours after a single 20 mg/kg i.p. injection (Calverley et al., 1979; reported elsewhere). With brain weights approximately as follows: hippocampus 0.1 g, corpus striatum, 0.1 g, mesencephalon-pons, 0.5 g, and diencephalon 0.3 g; 100% extraction of the tranylcypromine with the NM and 3-MT wou,ld result in concentrations of the drug eluates from the Amberlite columns of 4 x 10S6 M, 3.3 x 10e6 M, 1.5 x 10e5 M, and 1.0 x 10e5 M respectively for the four brain regions. In our original work (Calverley et al., 1978; McKim et al., 1980; McKim et al., reported elsewhere) on the effects of tranylcypromine on the regional concentrations of brain amines, we found no decrease in expected fluorescence values from authentic NM and 3-MT added as internal standards to hippocampus and corpus striatum extracts. However, in extracts obtained from the mesencephalon-pons and diencephalon of these same animals drastic reductions in fluorescence values were noted. These differences would be predicted from the
Inhibitory Effect on Fluorescence Assay
results reported here showing the inhibitory effects this drug has on the fluorophore reaction. Tests were carried out to determine at what point in the assay these compounds exert their inhibitory effect. All of the effective drugs were found to inhibit the oxidation step of the reaction. Since the oxidation step is dependent on the concentration of iodine in the reaction mixture, we found that we could overcome the inhibition of the fluorophore reaction by increasing the iodine concentration used in the reaction. We tested this finding in vivo and were successful in preventing the inhibitory effect of tranylcypromine in mesencephalon-pons and diencephalon from rats treated with this drug at 20 mg/kg 1.5 hours before death. The effects of tranylcypromine, phenelzine, pheniprazine, isocarboxazid, and nialamide were tested in vitro on the noradrenaline and dopamine fluorometric hydroxyindole assays as described by Karasawa et al. (1975). At 6 x lop6 M none of the drugs had any effect on either assay. It is probable that the much higher concentrations of oxidizing agents in these two assays are sufficient to overcome any inhibitory effect of these compounds. It is evident from our findings that erroneous results may arise in studies of the effects of MAOls on NM and 3-MT concentrations in brain tissue if the Karasawa et al. (1975) technique is used to measure these amines. This problem can be rectified if the iodine concentration used in the procedure is increased or if one uses a smaller amount of tissue in the extraction. The authors wish to thank Dr. C.B. Baker who suggested and made available to us a number of the drug tests. Financial support was provided by an Alberta Mental Health Research Grant.
REFERENCES Calverley DG, McKim HR, Baker GB, Dewhurst WC (1978) Studies on the interaction of the monoamine oxidase inhibitor tranylcypromine (Parnate) with dopamine, noradrenaline and 5-hydroxytryptamine in rat brain regions. Can 1 fharm Sci 13:99. Calverley DC, Baker GB, Martin I, Dewhurst WC, McKim HR (1979) A gas chromatographic technique for measurement of tranylcypromine in rat brain. Proc Can Fed Biol Sot 22:lOO.
McKim HR, Baker GB, Calverley DG, Dewhurst WG (1980) Effects of tranylcypromine and phenelzine on dopamine in corpus striatum. Proc Can Fed Biol Sot 23 :284. Karasawa T, Furukawa K, Yoshida K, Shimizu M (1975) A double column procedure for the simultaneous estimation of norepinephrine, normetanephrine, dopamine, 3-methoxytyramine and 5-hydroxytryptamine in brain tissue. lap / Pharmacol27:727.
181
D. G. CALVERLEY,H. R. MCKIM, AND W. G. DEWHURST
INTRODUCTION Determination of brain levels of the o-methylated metabolites of the catecholamines provides important information regarding turnover of the catecholamines. Measurement of these metabolites in brain tissue is particularly of value when animals have been treated with a monoamine oxidase inhibitor (MAOI) that blocks the other major pathway for breakdown of the catecholamines. A number of techniques are available for the separation and quantification of the o-methylated metabolites of the catecholamines in brain tissue. One technique that we have found to be of sufficient sensitivity for analysis of normetanephrine (NM) and 3-methoxytyramine (3-MT) in MAOI-treated rat brain regions is the fluorometric technique of Karasawa et al. (1975). In our work with tranylcypromine (Calverley et al., 1978; McKim et al., 1980; McKim et al., reported elsewhere) we discovered, that when animals were treated with MAOI (20 mg/kg, i.p., 1.5 hours before death), NM and 3-MT could not be reliably measured in the mesencephalon-pons and diencephalon. We therefore tested and report here on possible interference with this assay by a number of MAOls and structurally related compounds, in vitro. METHODS Very briefly, the Karasawa et al. (1975) technique involves running an extract of brain tissue in sequence through aluminum oxide and Amberlite (X-50 columns. The aluminum oxide removes the catecholamines. NM and 3-MT adhere to the Amberlite column and are removed by 0.5 N HCI. A hydroxyindole assay is used with iodine as oxidizing agent and sodium sulfite and EDTA as reducing and stabilizing agents, respectively. Fluorescence is developed under acid conditions. We have found that by using a reaction time of 2 minutes at 100°C for NM and an additional 4 minutes at 100°C for 3-MT, we could shorten the time for production of the measured fluorophore, increase our sensitivity, and not lose any reliability over the original procedure, From the Neurochemical Research Unit, Department of Psychiatry, University of Alberta, Edmonton, Alberta, Canada. Address reprint requests to H. R. McKim, Neurochemical Research Unit, Department of Psychiatry, 1-121 Clinical Sciences Building, University of Alberta, Edmonton, Alberta T6C 2G3, Canada. Received January 8, 1980; revised and accepted September 9, 1980. 179 lournal of Pharmacological D 1981 Elsevier
North
Methods
Holland,
5, 179-181 (1981)
Inc., 52 Vanderbilt
Avenue. New York, NY 10017
0160.540218110179003$02.50
180
D. C. Calverley et al. Drugs tested for an effect on this assay included the MAOls, (t)-tranylcypromine, (+)-pheniprazine, phenelzine, isocarboxazid, nialamide, pargyline, (-)-deprenil, clorgyline, and (t )-a-ethyltryptamine. Related compounds tested included (+ )-amphetamine, (-)-amphetamine, phentermine, P-phenylethylamine, and tryptamine. The effects of different concentrations of these drugs on the reaction that produced the fluorophore from NM and 3-MT were studied. Fluorescence values in fluorescence intensity units (Flus) were obtained for each of the fluorophores produced from 3 x IO-’ M NM and 3-MT. Comparisons were then made to the Flus obtained from an identical reaction in the presence of a series of different concentrations (1O-6 M to 10e4 M) of each of the drugs under study. Dose-response curves were constructed and the concentration of drug that resulted in a 50% inhibition of the production of the fluorophore (IC,,) was determined from the curves. RESULTS AND DISCUSSION All of the MAOls except the structurally related group including pargyline, deprenil, and clorgyline inhibited the formation of the fluorophore. Of the non-MAO1 but related compounds, only tryptamine reduced fluorescence readings. The hydrazines, phenelzine and pheniprazine, and the substituted hydrazines, isocarboxazid and nialamide, all had similar effects on the assay. In the case of all of these drugs the fluorescence values were greater than 90% of control at 2 x lop6 M and decreased to less than 10% of control by 5 x 1O-6 M. I&values were approximately 4 x lO-‘j M. Tranylcypromine, on the other hand, affected the reaction differently in that fluorescence values remained greater than 90% of control to 5 x 1O-6 M, then declined to less than 10% of control by 1.2 x lop5 M. The I&, value of tranylcypromine (7.5 x 10e6 M) was much higher than the IC50 for the other MAOls. Results with a-ethyltryptamine and tryptamine were quite erratic with I& values ranging from 3 x 1O-6 M to 1.3 x 1O-5 M for a-ethyltryptamine and from 2 to 5 x lop6 M for tryptamine. Concentrations of these compounds appear, at first glance, high in relation to the concentration of the amines, but we have reported brain levels of tranylcypromine of 12.1 to 15.3 kg/g in brain regions 1.5 hours after a single 20 mg/kg i.p. injection (Calverley et al., 1979; reported elsewhere). With brain weights approximately as follows: hippocampus 0.1 g, corpus striatum, 0.1 g, mesencephalon-pons, 0.5 g, and diencephalon 0.3 g; 100% extraction of the tranylcypromine with the NM and 3-MT wou,ld result in concentrations of the drug eluates from the Amberlite columns of 4 x 10S6 M, 3.3 x 10e6 M, 1.5 x 10e5 M, and 1.0 x 10e5 M respectively for the four brain regions. In our original work (Calverley et al., 1978; McKim et al., 1980; McKim et al., reported elsewhere) on the effects of tranylcypromine on the regional concentrations of brain amines, we found no decrease in expected fluorescence values from authentic NM and 3-MT added as internal standards to hippocampus and corpus striatum extracts. However, in extracts obtained from the mesencephalon-pons and diencephalon of these same animals drastic reductions in fluorescence values were noted. These differences would be predicted from the
Inhibitory Effect on Fluorescence Assay
results reported here showing the inhibitory effects this drug has on the fluorophore reaction. Tests were carried out to determine at what point in the assay these compounds exert their inhibitory effect. All of the effective drugs were found to inhibit the oxidation step of the reaction. Since the oxidation step is dependent on the concentration of iodine in the reaction mixture, we found that we could overcome the inhibition of the fluorophore reaction by increasing the iodine concentration used in the reaction. We tested this finding in vivo and were successful in preventing the inhibitory effect of tranylcypromine in mesencephalon-pons and diencephalon from rats treated with this drug at 20 mg/kg 1.5 hours before death. The effects of tranylcypromine, phenelzine, pheniprazine, isocarboxazid, and nialamide were tested in vitro on the noradrenaline and dopamine fluorometric hydroxyindole assays as described by Karasawa et al. (1975). At 6 x lop6 M none of the drugs had any effect on either assay. It is probable that the much higher concentrations of oxidizing agents in these two assays are sufficient to overcome any inhibitory effect of these compounds. It is evident from our findings that erroneous results may arise in studies of the effects of MAOls on NM and 3-MT concentrations in brain tissue if the Karasawa et al. (1975) technique is used to measure these amines. This problem can be rectified if the iodine concentration used in the procedure is increased or if one uses a smaller amount of tissue in the extraction. The authors wish to thank Dr. C.B. Baker who suggested and made available to us a number of the drug tests. Financial support was provided by an Alberta Mental Health Research Grant.
REFERENCES Calverley DG, McKim HR, Baker GB, Dewhurst WC (1978) Studies on the interaction of the monoamine oxidase inhibitor tranylcypromine (Parnate) with dopamine, noradrenaline and 5-hydroxytryptamine in rat brain regions. Can 1 fharm Sci 13:99. Calverley DC, Baker GB, Martin I, Dewhurst WC, McKim HR (1979) A gas chromatographic technique for measurement of tranylcypromine in rat brain. Proc Can Fed Biol Sot 22:lOO.
McKim HR, Baker GB, Calverley DG, Dewhurst WG (1980) Effects of tranylcypromine and phenelzine on dopamine in corpus striatum. Proc Can Fed Biol Sot 23 :284. Karasawa T, Furukawa K, Yoshida K, Shimizu M (1975) A double column procedure for the simultaneous estimation of norepinephrine, normetanephrine, dopamine, 3-methoxytyramine and 5-hydroxytryptamine in brain tissue. lap / Pharmacol27:727.
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