Determination of Amines, Amine Metabolites and some Amine Metabolizing Enzymes by High Performance Liquid Chromatography Noirin Nic a’ Bhaird, John M. McCrodden, Anthony M. Wheatley, Michael C. Harrington, James P. Sullivan and Keith F. Tipton* Department of Biochemistry, Trinity College, Dublin 2, Ireland
Some useful high performance liquid chromatographic methods for the determination of amines, amine metabolites and amine metabolizing enzymes are described. These include the separation of tyramine in wines and beers, determination of tryptamine in urine, assay of monoamine oxidase and catechol-0-methyltransferase and analysis of amine-aldehyde condensation products.
Several procedures involving high performance liquid chromatography (HPLC) have been reported for the determination of amines and their metabolites. Rather than attempting to review these in detail, the present account will concentrate on some approaches that we have found to be particularly valuable for our neurochemical and biochemical studies. Determination of tyramine Dietary tyramine is the commonest cause of the acute pressor response that may occur in patients being treated with monoamine oxidase inhibitors as antidepressants (Tipton, 1989). The occurrence of tyramine in a number of foods and drinks has been extensively documented (Marley and Blackwell, 1970; Da Prada et aZ., 1988) and, because of the high tyramine content of some cheeses, this response, which can be fatal, is often known as the ‘cheese reaction’. Although the presence of tyramine in some wines and beers has been described, a report that a patient being treated with a monoamine oxidase inhibitor suffered a ‘cheese reaction’ after consuming non-alcoholic beer (Draper et al., 1984) led us to investigate the tryamine levels in a number of beverages, including alcoholic and non-alcoholic beers (Wheatley and Tipton, 1987; Wheatley et al., 1988). For this purpose we developed a simple HPLC procedure involving electrochemical detection (ECD) for the separation and determination of tyramine in such materials. Full details of the methods have been reported elsewhere (Wheatley and Tipton, 1987). The beverage is deproteinized by centrifugation through an ultrafiltration membrane (Amicon C F 25) and further purified by chromatography on Amberlite CG-50 and eluting with 1 M perchloric acid, after washing the column with water. The recovery of tyramine from these procedures was found to be greater than 97%. Samples from this stage could be stored at -20°C until HPLC analysis. This treatment effectively concentrated the tyramine and removed a number of other components which would otherwise complicate the HPLC elution profile (Wheatley and Tipton, 1987). Author to whom correspondence should be addressed.
Analysis by HPLC involved the use of a Corasil C,, precolumn and a C,, resolving column (both Waters p Bondapak). Isocratic elution was effected by 70 mM NaH,PO,, pH 4.9, containing 1 mM EDTA, 0.75 mM hexane sulfonic acid and 6% methanol, at a flow-rate of 1 mL/min. The eluted amine was determined by ECD with a glassy-carbon electrode (BioAnalytical Systems) at a potential of 0.72-0.85 V. Under these conditions tyramine eluted as a sharp symmetrical peak (retention time: 10.8 min), such that peak height could be used as an satisfactory alternative to area for determining the concentration. Dopamine can be used as an internal standard with a retention time of 7.8min and complete resolution from the tyramine peak. Addition of standard samples of tyramine was routinely used to confirm the identity of the eluted peak and to check the linearity of detector response. Using this procedure the tyramine contents of several beers and wine-coolers were found to vary between 0.44 and 3.21 mg/L. Analysis of the tyramine levels in three different non-alcoholic beers revealed a similar variation (0.58-3.96 mg/L; Wheatley et aZ., 1988). Thus nonalcoholic beers should be subject to the same dietary strictures as their alcoholic counterparts for patients undergoing treatment with monoamine oxidase inhibitory antidepressants. A particularly important result from these studies was that there could be large variations between the tyramine levels in different batches of the same product. Although the tyramine concentrations determined in the same batch of any given beer showed a variation of less than 5%, the values found in three different batches of the same imported beer varied by almost seven-fold (0.191.31 mg/ L). These data suggest that published values of the tyramine contents of beverages should be treated with caution if they were based on the analysis of single samples. Furthermore there is the possibility that a patient may come to believe that it is possible to drink a specific product without adverse reaction, only to suffer a pronounced cheese reaction at some later stage when a sample with a particularly high tyramine content is consumed.
0269-3879/90/0229-0233 $5.00 @ 1990 by John Wiley & Sons, Ltd.
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Determination of tryptamine in urine There are two forms of monoamine oxidase (MAO; EC 1.4.3.4)with different substrate specificities and inhibitor sensitivities (Tipton, 1986; Youdim et al., 1988). Inhibitors of the A-form are effective antidepressants whereas those of the B-form are apparently not. Biek et al., (1984) suggested that the determination of urinary tryptamine excretion might provide an index of MAO-A inhibition in uiuo, because that amine was a selective substrate for MAO-A in human kidney. We have found that a similar method to that described above for tyramine may be used to determine the tryptamine concentrations in urine samples. The diagnostic value of such determinations is, however, unclear since our data indicate that tryptamine is a substrate for both forms of M A 0 in human kidney and other tissues (Sullivan et al., 1986). The possibility remains that some type of metabolic compartmentation may result in MAO-A playing the dominant role in tryptamine metabolism in viuo. Studies on the effects of selective M A 0 inhibitors on the urinary excretion of tryptamine in human subjects will be necessary to test this. Determination of monoamine oxidase activity Amongst the many procedures that have been published for the assay of M A 0 (Tipton and Youdim, 1983; Yu, 1986) there are several involving the separation of the reaction products by HPLC. A problem with the use of such assays with crude tissue preparations is that further metabolism of the aldehyde product of the reaction may take place. This can involve oxidation catalysed by aldehyde dehydrogenase (EC 1.2.1.3)in the presence of contaminating NAD', to give the corresponding carboxylic acid, or reduction by NADPH and aldehyde reductase (EC 1.1.1.2), or perhaps by NADH and alcohol dehydrogenase (EC 1.1.1.1),to give the corresponding alcohol (Tipton et al., 1977).This necessitates the separation and quantification of all these possible products if accurate determinations of the true activity of the enzyme are to be obtained. Furthermore the binding of the aldehyde product to crude tissue preparations (Alvisatos and Ungar, 1968) may decrease the yield of products obtained. Because of the relatively high K , values shown by M A 0 towards many of its amine substrates (Youdim et al., 1988) the alternative approach of determining the decrease in amine concentration by HPLC can be insensitive. Green and Haughton (1961)devised a colorimetric assay for M A 0 activity in which the reaction was carried out in the presence of semicarbazide, to trap the aldehyde formed as the corresponding semicarbazone. After stopping the reaction, by the addition of acid, the addition of excess 2,4-dinitrophenylhydrazine resulted in exchange of the aldehyde moiety to form the corresponding hydrazone. This was then extracted and determined from its absorbance at 360 nm. In order to adapt this procedure for HPLC separation we have followed the method of Green and Haughton (1961).The reaction between M A 0 and amine substrates was allowed to proceed, at 37 "C in 0.1 M phosphate buffer (pH 7 . 2 ) , containing 50 mM semicarbazide, for varying times before it was stopped by the addition of 0.5 M acetic acid. The mixture was immediately placed in a boiling waterbath and after 5 min it was centrifuged 230 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990
at 15 000 g for 10 min to remove precipitated protein. The supernatant was mixed with an equal volume of 0.5 mg/mL 2,4-dinitrophenylhydrazineand, after incubation at room temperature for 10 min, applied directly to a C,* (p Bondapak) column which had been equilibrated with water: methanol (30:70,v/v). Elution was carried out under the same conditions with a flow rate of 0.6 mL/min. Under these conditions, which are similar to those used by Fung and Grosjean (1981)for the separation of dinitrophenylhydrazine derivatives, the 2,4-dinitrophenylhydrazonewas well separated from the unreacted hydrazine. Elution could be monitored by the absorbance at 360 nm and used to quantify the M A 0 activity. Although this procedure appears to be robust and free from the potential problems, referred to above, encountered with the direct determination of product formation in the MAO-catalysed reaction, we have yet to investigate its application to the determination of the oxidation products of the catecholamines. Monoamine oxidase is involved in the activation of the neurotoxin l-methyl-.l-pheny1-1,2,3,6-tetrahydropyridine (MPTP), which causes a condition resembling idiopathic Parkinson's disease in humans and some other species. MPTP is oxidized by MAO-B to l-methyl4-phenyl-2,3-dihydropyridine(MPDP), which is then further oxidized, either by M A 0 itself or non-enzymatically, to the 1-methyl-4-phenylpyridinium ion (MPPf), which is the effective neurotoxin (Kinemuchi et al., 1987). Although this process may be followed spectrophotometrically because MPTP, MPDP and MPP+ absorb at different wavelengths (Tipton and Sullivan,
Figure 1. Separation of MPDP and MPP+ by HPLC. Samples (25 p.L, containing 333 p . MPDP ~ and 200 IJ.M MPP+) were applied to a C,, column (0.5x30cm; Waters p. Bondapak) and eluted with v/v) containing acetonitrile: 100 mM potassium phosphate (40:60, 0.1% triethylamine, final pH 7.2. The elution of MPDP (peak 1) was monitored at 340nm (broken line) and that of MPPf (peak 2) at 280 nm (continuous line).
@ 1990 by John Wiley & Sons, Ltd.
HPLC AND AMINES
1990), the results obtained are difficult to evaluate, because MPDP is light-sensitive and rapidly degrades under such conditions (Buckman 8z Eiduson, 1985). As an alternative that avoids this problem, HPLC may be used to separate the products of MPTP oxidation, as shown in Fig. 1, after the enzyme and MPTP have been incubated together in the dark for varying periods of time. The results obtained confirm the time-dependent inhibition of M A 0 by MPTP that had been previously reported (Tipton et al., 1988), indicating that this process was not a photochemical reaction. They are also consistent with the intermediate and final product relationship between MPDP and MPPf which was indicated by the earlier spectrophotometric studies.
Amine-aldehyde condensation products The possibility that amine-aldehyde condensation reactions followed by ring closure to form tetrahydroisoquinoline derivatives may occur in vivo has been widely discussed in connection with the effects of ethanol (Tipton et aL, 1977; Deitrich and Erwin, 1984). Particular attention has been paid to the condensation products involving dopamine. This amine will condense with acetaldehyde to form salsolinol or with 3,4-dihydroxyphenylacetaldehyde (DOPAL), the aldehyde resulting from the MAO-catalysed oxidation of dopamine, to give tetrahydropapaveroline (THP). Since ethanol metabolism forms acetaldehyde and also, directly or indirectly elevates the steady-state concentrations of aldehydes such as DOPAL (Tipton et al., 1977; Weiner, 1980; Collins, 1980), it has been argued that the formation of the isoquinoline derivatives may be important in some aspects of alcoholism (Deitrich and Erwin, 1984). There has, however, been considerable controversy about the endogenous levels of these compounds in the tissues and excreted in the urine, whether they may have a dietary origin or whether the levels are, indeed, affected by ethanol consumption (Dordain et a[., 1984; Weiner, 1980; Collins, 1980). In order to investigate the conditions necessary for the formation of salsolinol and THP in tissue preparations, we have developed HPLC procedures for their separation and quantitation. Incubation of tissue samples was carried out under the conditions previously described for our studies on ethanol metabolism in different intracellular compartments (Harrington et a l , 1988). Following incubation the samples (3 mL) were treated with perchloric acid (0.2 M, final concentration) and centrifuged to remove precipitated protein. Ascorbic acid (100 pL, 5% w/v) and EDTA (100 pL, 0.1% w/v) were added to the supernatants, which were then adjusted to pH 8.0 by the addition of KOH. After centrifugation to remove the precipitated KC104, the supernatants were applied to alumina columns which were then washed successively with water and 0.05 M perchloric acid. 3,4-Dihdroxybenzylamine was taken through the entire procedure as an internal standard. A procedure based on the method of Shier et al. (1983), in which the stepwise gradient of acetonitrile used by those workers was replaced by a non-linear acetonitrile gradient, was found to effect the resolution of dopamine, its metabolites, salsolinol and THP (Fig. 2 ) . However the resolution between dopamine and salsolinol was too small to be adequate for routine work @ 1990 by John Wiley CQ Sons, Ltd.
I
4 0
L
1
15
0
30
Time (minl
Figure 2. Separation of dopamine, its oxidation products, salsolinol and THP by HPLC. Samples were applied to a C,, column ( 0 . 5 ~ 30cm. Waters Bondapak) and eluted with a 1-15% (v/v) gradient of acetonitrile in 0.1 N triethylammonium phosphate, pH 2.25. Elution was monitored by the absorbance at 254nm. Details of sample preparation are given in the text. DA: dopamine; DHPET: 3.4-dihydroxyphenylethanol; DOPAC: 3.4-dihydrozyphenylacetic acid; SALS: salsolinol; THP: tetrahydropapaveroline.
with tissue samples. As an alternative, separate systems were used for the resolution of salsolinol and THP. A decreasing gradient of pH was used for the former compound, as shown in Fig. 3, whereas the system shown
0.06-
T
I
I
,
I -
I 0
I
I
10
Time
20 ( min
1
Figure 3. Resolution of salsolinol by HPLC. Details are as given in Fig. 2, except that elution was effected in 0.1 N triethylammonium phosphate with a linear gradient from pH 9.0-2.25.
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990 231
N. NIC. a' BHAIRD ET AL.
in Fig. 2 was used for THP. These systems have been used to show that salsolinol is formed when a cytosolic fraction from rat or human liver is incubated with dopamine (1 mM) and ethanol (30 mM), . In experiments in which I4C-labelled ethanol and 3H-labelled dopamine were used, the appearance of both labels in the separated product confirmed it to be salsolinol. The formation of THP was not detected when cytosolic fractions from rat liver were used. However the admixture of the mitochondrial fraction, where the M A 0 activity is localized (Tipton, 1986) resulted in the formation of this compound. These results show that salsolinol and THP can be formed in such tissue preparations in vitro, and further work will be directed towards defining the conditions necessary for the formation of these compounds more precisely and relating them to those likely to occur in vivo.
Assay of catechol-0-methyltransferase (COMT) activity A number of radiochemical assays, based on the incorporation of the labelled methyl group from S-adenosylmethionine (AdoMet) into a catechol substrate, have been developed for the assay of COMT (EC2.1.1.6) (Borchardt, 1981; Gulliver and Tipton, 1978; Zurcher and Da Prada, 1982). However, the necessity of ensuring adequate separation of labelled substrate and product may restrict the use of such procedures with some substrates. A spectrophotometric assay, based on the change in absorbance when S-adenosylhomocysteine (AdoHcy) is converted to S-inosylhomocysteine in the presence of adenosine deaminase (Coward and Wu, 1973), is applicable to a wide range of catechol substrates, but its low sensitivity makes it unsuitable for determining the activity of the enzyme in crude tissue preparations. As an alternative we have investigated the use of HPLC separation of the methylated product from the parent catechols as a convenient and generally applicable assay procedure. The activity towards 3,4-dihydroxyphenylacetic acid (DOPAC) was assayed in 100 mM potassium phosphate buffer and at 37 "C by the method of Gulliver and Tipton (1978). Parallel assays under the same conditions were terminated after fixed times by the addition of 1 M perchloric acid and the mixtures were centrifuged to remove precipitated protein before the supernatants were analysed by HPLC (Murai et al., 1988). Samples of 20-50 p.L were applied to a CIScolumn (Waters p Bondapak) and eluted with 20 mM sodium acetate, 12.5 mM citric acid, pH 3.92, containing 0.033% (w/v) heptanesulfonic acid and 0.1 mM EDTA. The eluted products were detected electrochemically at an applied voltage of 0.83 V. This procedure gave good resolution of DOPAC from its methylated product and comparison of kinetic data obtained by this method and the radiochemical procedure indicated close agreement between the two. Good agreement was also obtained between the results of this HPLC procedure and a method based on the separation of the AdoHcy formed in the reaction from AdoMet (Molloy e l al., 1990). Alternative elution conditions would, however, be required for adrenaline and noradrenaline as substrates, since these were found not to be adequately resolved from their 0-methylated 232 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990
derivatives by the present system. Furthermore it is possible that para-methylation might occur, in addition to meta-methylation, under some conditions (Creveling et aL, 1972) and that would necessitate resolution of both products. In a study of the application of the HPLC procedure to monitor the methylation of catechol the product, guaiacol, was found to be well separated from catechol by the procedure described above. Comparison of the performance of this assay with the results of a radiochemical assay (Zurcher and Da Prada, 1982) or the HPLC assay based on AdoHcy formation revealed it to give significantly lower (about 30%) rates than the other two procedures. The reasons for this discrepancy remain obscure, since the HPLC method was calibrated with standard product solutions and the K , value for catechol given by this procedure was in close agreement with that obtained when the radiochemical method was used. Since the HPLC and radiochemical results correlated well when DOPAC was used as substrate, it is unlikely that the discrepancy observed with catechol could be due to different enantiomeric compositions of the radioactive and unlabelled AdoMet samples. Until the causes of this discrepancy are resolved caution would be necessary in using the HPLC procedure to give absolute rate measurements.
Temperature effects Ionization processes and hydrophobic interactions are temperature dependent. Indeed such effects have frequently been applied to improve chromatographic separations. Figure 4 shows the effects of temperature on the retention time of dopamine under the HPLC conditions described above. Clearly such variations could have profound effects on peak identification and resolution. It is therefore unfortunate that many HPLC systems are supplied without temperature control as a standard facility.
6.5
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Column temperature ( C) Figure 4. The effects of temperature on the elution of dopamine. The column (&, Waters p Eondapak) was equilibrated to the indicated temperature before sample application. Other details are given in the text.
@ 1990 by John Wiley & Sons, Ltd.
HPLC AND AMINES
REFERENCES Alivisatos, S. G. A. and Ungar. F. (1968). Biochemistry 2, 285. Biek, P. R., Nilsson, E., Schick, C.. Waldmeier, P. C. and Lauber, J. (1984). In Neurobiology of the Trace Amines, (ed. by Boulton, A. A., Baker, G. B., Dewhurst, W. G. and Sandler, M., pp. 525-533. Humana Press, New York. Borchardt, R. T. (1981). Meth. Enrymol 77, 267. Buckman, J. D. and Eiduson, S. (1985). J. Biol. Chern. 260, 11899. Collins, M. A. (1980). In BiologicalEffects ofAlcoholed. by Begleiter, H., pp. 87-102. Plenum Press, New York. Coward, J. K. and Wu, F. Y.-H. (1973). Anal Biochem. 55, 406. Creveling. C. R., Morris, N., Shimizu. H., Ong, H. H. and Daly, J. (1 972). Mol. Pharmacol. 8, 398. Da Prada, M., Zurcher, G.. Wuthrich, I. and Haefely, W. E. (1988). J. Neural. Transm. Suppl. 26, 31. Deitrich, R. A. and Erwin, V. G. (1984). In Monoamine Oxidase and Disease, ed. by Tipton, K. F., Dostert, P. and Strolin Benedetti, M.. pp. 275-289. Academic Press, London. Dordain, G., Dostert, P., Strolin Benedetti, M. and Rovei, V. (1984). In Monoamine Oxidase and Disease, ed. by Tipton, K. F., Dostert, P. and Strolin Benedetti, M., pp. 417426. Academic Press, London. Draper, R. J., Sandler, M. and Walker, P. L. (1984). Brit. Med. J. 289, 308. Fung, K. and Grosjean, D. (1971). Anal. Chem. 53, 168. Green, A. L. and Haughton, P. M. (1961). Biochem. J.78. 172. Gulliver, P. A. and Tipton, K. F. (1978). Biochem. Pharmacol. 27, 773. Harrington, M. C., Henehan, G. T. M. and Tipton, K. F. (1988). Biochem. SOC.Trans. 16, 239. Kinemuchi, H., Fowler, C. J. and Tipton. K. F. (1987). Neurochem. lnt. 11, 359. Marley, E. and Blackwell, B. (1970). Adv. Pharmacol. Chemother. 8, 185. Mollay, A. M., Weir, D. G., Kennedy, G., Kennedy, S. and Scott, J. M. (1990). Biomed. Chrornatogr. 4, 257-260.
@ 1990 by John Wiley & Sons, Ltd.
Murai, S., Saitoh. H., Matsuda, Y. and Itoh, T. (1988). J. Neurochem. 50,473. Shier, W. T., Kock, Y. and Bloom, F. E. (1983). Neuropharmacol. 22, 278. Sullivan, J. P., McDonnell, L., Hardiman, 0. L., Farrell. M. A.. Phillips, J. P. and Tipton, K. F. (1986). Biochem. Pharmacol. 35,3255. Tipton, K. F. (1986). Cell Biochem. Funct. 4, 79. Tipton K. F. (1989). In Biochemical and Pharmacological Aspects of Depression, ed. by Tipton, K. F. and Youdim, M. B. H., pp. 1-24. Taylor and Francis, London. Tipton, K. F. and Sullivan, J. P. (1990). Proc. Roy. lrish Acad., in press. Tipton, K. F. and Youdim, M. B. H. (1983). In Methods in Biogenic Amine Research, ed. by Parvez, S., Nagatsu, T. and Parvez, H., pp. 441465. Elsevier, Amsterdam. Tipton, K. F., Houslay, M. D. and Turner, A. J (1977). Essays Neurochem. Neuropharmacol. 1, 103. Tipton, K. F., McCrodden, J. M. and Youdim, M. B. H. (1988). Biochem. J. 240, 379. Wiener, H. (1980). In Enzymatic Basis of Detoxication. ed. by Jakoby, W., pp. 261-280. Academic Press, New York. Wheatley. A. M. and Tipton, K. F. (1987). J. food Biochem. 11, 133. Wheatley, A. M., McCrodden, J. and Tipton, K. F. (1988). In Frontiers of Flavor, ed. by Charalambous, G., pp. 743-751. Elsevier, Amsterdam. Youdim, M. B. H., Finberg. J. P. M. and Tipton, K. F. (1988). In Handbook o f Experimental Pharmacology, ed. by Trendelenburg, H. U. and Weiner. N,, Vol. 90, pp. 119-192. SpringerVerlag. Berlin. Yu, P. H. (1986). In Neuromethods, Vol. 5, ed. by Boulton, A. A,, Baker, G. B. and Yu, P. H., Vol. 5, pp. 235-272. Humana Press, New Jersey. Zurcher, G. and Da Prada, M. (1982). J. Neurochem. 38, 191. Received 9 April, 1990; accepted 4 May, 1990
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990 233
Some useful high performance liquid chromatographic methods for the determination of amines, amine metabolites and amine metabolizing enzymes are described. These include the separation of tyramine in wines and beers, determination of tryptamine in urine, assay of monoamine oxidase and catechol-0-methyltransferase and analysis of amine-aldehyde condensation products.
Several procedures involving high performance liquid chromatography (HPLC) have been reported for the determination of amines and their metabolites. Rather than attempting to review these in detail, the present account will concentrate on some approaches that we have found to be particularly valuable for our neurochemical and biochemical studies. Determination of tyramine Dietary tyramine is the commonest cause of the acute pressor response that may occur in patients being treated with monoamine oxidase inhibitors as antidepressants (Tipton, 1989). The occurrence of tyramine in a number of foods and drinks has been extensively documented (Marley and Blackwell, 1970; Da Prada et aZ., 1988) and, because of the high tyramine content of some cheeses, this response, which can be fatal, is often known as the ‘cheese reaction’. Although the presence of tyramine in some wines and beers has been described, a report that a patient being treated with a monoamine oxidase inhibitor suffered a ‘cheese reaction’ after consuming non-alcoholic beer (Draper et al., 1984) led us to investigate the tryamine levels in a number of beverages, including alcoholic and non-alcoholic beers (Wheatley and Tipton, 1987; Wheatley et al., 1988). For this purpose we developed a simple HPLC procedure involving electrochemical detection (ECD) for the separation and determination of tyramine in such materials. Full details of the methods have been reported elsewhere (Wheatley and Tipton, 1987). The beverage is deproteinized by centrifugation through an ultrafiltration membrane (Amicon C F 25) and further purified by chromatography on Amberlite CG-50 and eluting with 1 M perchloric acid, after washing the column with water. The recovery of tyramine from these procedures was found to be greater than 97%. Samples from this stage could be stored at -20°C until HPLC analysis. This treatment effectively concentrated the tyramine and removed a number of other components which would otherwise complicate the HPLC elution profile (Wheatley and Tipton, 1987). Author to whom correspondence should be addressed.
Analysis by HPLC involved the use of a Corasil C,, precolumn and a C,, resolving column (both Waters p Bondapak). Isocratic elution was effected by 70 mM NaH,PO,, pH 4.9, containing 1 mM EDTA, 0.75 mM hexane sulfonic acid and 6% methanol, at a flow-rate of 1 mL/min. The eluted amine was determined by ECD with a glassy-carbon electrode (BioAnalytical Systems) at a potential of 0.72-0.85 V. Under these conditions tyramine eluted as a sharp symmetrical peak (retention time: 10.8 min), such that peak height could be used as an satisfactory alternative to area for determining the concentration. Dopamine can be used as an internal standard with a retention time of 7.8min and complete resolution from the tyramine peak. Addition of standard samples of tyramine was routinely used to confirm the identity of the eluted peak and to check the linearity of detector response. Using this procedure the tyramine contents of several beers and wine-coolers were found to vary between 0.44 and 3.21 mg/L. Analysis of the tyramine levels in three different non-alcoholic beers revealed a similar variation (0.58-3.96 mg/L; Wheatley et aZ., 1988). Thus nonalcoholic beers should be subject to the same dietary strictures as their alcoholic counterparts for patients undergoing treatment with monoamine oxidase inhibitory antidepressants. A particularly important result from these studies was that there could be large variations between the tyramine levels in different batches of the same product. Although the tyramine concentrations determined in the same batch of any given beer showed a variation of less than 5%, the values found in three different batches of the same imported beer varied by almost seven-fold (0.191.31 mg/ L). These data suggest that published values of the tyramine contents of beverages should be treated with caution if they were based on the analysis of single samples. Furthermore there is the possibility that a patient may come to believe that it is possible to drink a specific product without adverse reaction, only to suffer a pronounced cheese reaction at some later stage when a sample with a particularly high tyramine content is consumed.
0269-3879/90/0229-0233 $5.00 @ 1990 by John Wiley & Sons, Ltd.
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990 229
N. NIC. a' BHAIRD ET AL.
Determination of tryptamine in urine There are two forms of monoamine oxidase (MAO; EC 1.4.3.4)with different substrate specificities and inhibitor sensitivities (Tipton, 1986; Youdim et al., 1988). Inhibitors of the A-form are effective antidepressants whereas those of the B-form are apparently not. Biek et al., (1984) suggested that the determination of urinary tryptamine excretion might provide an index of MAO-A inhibition in uiuo, because that amine was a selective substrate for MAO-A in human kidney. We have found that a similar method to that described above for tyramine may be used to determine the tryptamine concentrations in urine samples. The diagnostic value of such determinations is, however, unclear since our data indicate that tryptamine is a substrate for both forms of M A 0 in human kidney and other tissues (Sullivan et al., 1986). The possibility remains that some type of metabolic compartmentation may result in MAO-A playing the dominant role in tryptamine metabolism in viuo. Studies on the effects of selective M A 0 inhibitors on the urinary excretion of tryptamine in human subjects will be necessary to test this. Determination of monoamine oxidase activity Amongst the many procedures that have been published for the assay of M A 0 (Tipton and Youdim, 1983; Yu, 1986) there are several involving the separation of the reaction products by HPLC. A problem with the use of such assays with crude tissue preparations is that further metabolism of the aldehyde product of the reaction may take place. This can involve oxidation catalysed by aldehyde dehydrogenase (EC 1.2.1.3)in the presence of contaminating NAD', to give the corresponding carboxylic acid, or reduction by NADPH and aldehyde reductase (EC 1.1.1.2), or perhaps by NADH and alcohol dehydrogenase (EC 1.1.1.1),to give the corresponding alcohol (Tipton et al., 1977).This necessitates the separation and quantification of all these possible products if accurate determinations of the true activity of the enzyme are to be obtained. Furthermore the binding of the aldehyde product to crude tissue preparations (Alvisatos and Ungar, 1968) may decrease the yield of products obtained. Because of the relatively high K , values shown by M A 0 towards many of its amine substrates (Youdim et al., 1988) the alternative approach of determining the decrease in amine concentration by HPLC can be insensitive. Green and Haughton (1961)devised a colorimetric assay for M A 0 activity in which the reaction was carried out in the presence of semicarbazide, to trap the aldehyde formed as the corresponding semicarbazone. After stopping the reaction, by the addition of acid, the addition of excess 2,4-dinitrophenylhydrazine resulted in exchange of the aldehyde moiety to form the corresponding hydrazone. This was then extracted and determined from its absorbance at 360 nm. In order to adapt this procedure for HPLC separation we have followed the method of Green and Haughton (1961).The reaction between M A 0 and amine substrates was allowed to proceed, at 37 "C in 0.1 M phosphate buffer (pH 7 . 2 ) , containing 50 mM semicarbazide, for varying times before it was stopped by the addition of 0.5 M acetic acid. The mixture was immediately placed in a boiling waterbath and after 5 min it was centrifuged 230 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990
at 15 000 g for 10 min to remove precipitated protein. The supernatant was mixed with an equal volume of 0.5 mg/mL 2,4-dinitrophenylhydrazineand, after incubation at room temperature for 10 min, applied directly to a C,* (p Bondapak) column which had been equilibrated with water: methanol (30:70,v/v). Elution was carried out under the same conditions with a flow rate of 0.6 mL/min. Under these conditions, which are similar to those used by Fung and Grosjean (1981)for the separation of dinitrophenylhydrazine derivatives, the 2,4-dinitrophenylhydrazonewas well separated from the unreacted hydrazine. Elution could be monitored by the absorbance at 360 nm and used to quantify the M A 0 activity. Although this procedure appears to be robust and free from the potential problems, referred to above, encountered with the direct determination of product formation in the MAO-catalysed reaction, we have yet to investigate its application to the determination of the oxidation products of the catecholamines. Monoamine oxidase is involved in the activation of the neurotoxin l-methyl-.l-pheny1-1,2,3,6-tetrahydropyridine (MPTP), which causes a condition resembling idiopathic Parkinson's disease in humans and some other species. MPTP is oxidized by MAO-B to l-methyl4-phenyl-2,3-dihydropyridine(MPDP), which is then further oxidized, either by M A 0 itself or non-enzymatically, to the 1-methyl-4-phenylpyridinium ion (MPPf), which is the effective neurotoxin (Kinemuchi et al., 1987). Although this process may be followed spectrophotometrically because MPTP, MPDP and MPP+ absorb at different wavelengths (Tipton and Sullivan,
Figure 1. Separation of MPDP and MPP+ by HPLC. Samples (25 p.L, containing 333 p . MPDP ~ and 200 IJ.M MPP+) were applied to a C,, column (0.5x30cm; Waters p. Bondapak) and eluted with v/v) containing acetonitrile: 100 mM potassium phosphate (40:60, 0.1% triethylamine, final pH 7.2. The elution of MPDP (peak 1) was monitored at 340nm (broken line) and that of MPPf (peak 2) at 280 nm (continuous line).
@ 1990 by John Wiley & Sons, Ltd.
HPLC AND AMINES
1990), the results obtained are difficult to evaluate, because MPDP is light-sensitive and rapidly degrades under such conditions (Buckman 8z Eiduson, 1985). As an alternative that avoids this problem, HPLC may be used to separate the products of MPTP oxidation, as shown in Fig. 1, after the enzyme and MPTP have been incubated together in the dark for varying periods of time. The results obtained confirm the time-dependent inhibition of M A 0 by MPTP that had been previously reported (Tipton et al., 1988), indicating that this process was not a photochemical reaction. They are also consistent with the intermediate and final product relationship between MPDP and MPPf which was indicated by the earlier spectrophotometric studies.
Amine-aldehyde condensation products The possibility that amine-aldehyde condensation reactions followed by ring closure to form tetrahydroisoquinoline derivatives may occur in vivo has been widely discussed in connection with the effects of ethanol (Tipton et aL, 1977; Deitrich and Erwin, 1984). Particular attention has been paid to the condensation products involving dopamine. This amine will condense with acetaldehyde to form salsolinol or with 3,4-dihydroxyphenylacetaldehyde (DOPAL), the aldehyde resulting from the MAO-catalysed oxidation of dopamine, to give tetrahydropapaveroline (THP). Since ethanol metabolism forms acetaldehyde and also, directly or indirectly elevates the steady-state concentrations of aldehydes such as DOPAL (Tipton et al., 1977; Weiner, 1980; Collins, 1980), it has been argued that the formation of the isoquinoline derivatives may be important in some aspects of alcoholism (Deitrich and Erwin, 1984). There has, however, been considerable controversy about the endogenous levels of these compounds in the tissues and excreted in the urine, whether they may have a dietary origin or whether the levels are, indeed, affected by ethanol consumption (Dordain et a[., 1984; Weiner, 1980; Collins, 1980). In order to investigate the conditions necessary for the formation of salsolinol and THP in tissue preparations, we have developed HPLC procedures for their separation and quantitation. Incubation of tissue samples was carried out under the conditions previously described for our studies on ethanol metabolism in different intracellular compartments (Harrington et a l , 1988). Following incubation the samples (3 mL) were treated with perchloric acid (0.2 M, final concentration) and centrifuged to remove precipitated protein. Ascorbic acid (100 pL, 5% w/v) and EDTA (100 pL, 0.1% w/v) were added to the supernatants, which were then adjusted to pH 8.0 by the addition of KOH. After centrifugation to remove the precipitated KC104, the supernatants were applied to alumina columns which were then washed successively with water and 0.05 M perchloric acid. 3,4-Dihdroxybenzylamine was taken through the entire procedure as an internal standard. A procedure based on the method of Shier et al. (1983), in which the stepwise gradient of acetonitrile used by those workers was replaced by a non-linear acetonitrile gradient, was found to effect the resolution of dopamine, its metabolites, salsolinol and THP (Fig. 2 ) . However the resolution between dopamine and salsolinol was too small to be adequate for routine work @ 1990 by John Wiley CQ Sons, Ltd.
I
4 0
L
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Time (minl
Figure 2. Separation of dopamine, its oxidation products, salsolinol and THP by HPLC. Samples were applied to a C,, column ( 0 . 5 ~ 30cm. Waters Bondapak) and eluted with a 1-15% (v/v) gradient of acetonitrile in 0.1 N triethylammonium phosphate, pH 2.25. Elution was monitored by the absorbance at 254nm. Details of sample preparation are given in the text. DA: dopamine; DHPET: 3.4-dihydroxyphenylethanol; DOPAC: 3.4-dihydrozyphenylacetic acid; SALS: salsolinol; THP: tetrahydropapaveroline.
with tissue samples. As an alternative, separate systems were used for the resolution of salsolinol and THP. A decreasing gradient of pH was used for the former compound, as shown in Fig. 3, whereas the system shown
0.06-
T
I
I
,
I -
I 0
I
I
10
Time
20 ( min
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Figure 3. Resolution of salsolinol by HPLC. Details are as given in Fig. 2, except that elution was effected in 0.1 N triethylammonium phosphate with a linear gradient from pH 9.0-2.25.
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990 231
N. NIC. a' BHAIRD ET AL.
in Fig. 2 was used for THP. These systems have been used to show that salsolinol is formed when a cytosolic fraction from rat or human liver is incubated with dopamine (1 mM) and ethanol (30 mM), . In experiments in which I4C-labelled ethanol and 3H-labelled dopamine were used, the appearance of both labels in the separated product confirmed it to be salsolinol. The formation of THP was not detected when cytosolic fractions from rat liver were used. However the admixture of the mitochondrial fraction, where the M A 0 activity is localized (Tipton, 1986) resulted in the formation of this compound. These results show that salsolinol and THP can be formed in such tissue preparations in vitro, and further work will be directed towards defining the conditions necessary for the formation of these compounds more precisely and relating them to those likely to occur in vivo.
Assay of catechol-0-methyltransferase (COMT) activity A number of radiochemical assays, based on the incorporation of the labelled methyl group from S-adenosylmethionine (AdoMet) into a catechol substrate, have been developed for the assay of COMT (EC2.1.1.6) (Borchardt, 1981; Gulliver and Tipton, 1978; Zurcher and Da Prada, 1982). However, the necessity of ensuring adequate separation of labelled substrate and product may restrict the use of such procedures with some substrates. A spectrophotometric assay, based on the change in absorbance when S-adenosylhomocysteine (AdoHcy) is converted to S-inosylhomocysteine in the presence of adenosine deaminase (Coward and Wu, 1973), is applicable to a wide range of catechol substrates, but its low sensitivity makes it unsuitable for determining the activity of the enzyme in crude tissue preparations. As an alternative we have investigated the use of HPLC separation of the methylated product from the parent catechols as a convenient and generally applicable assay procedure. The activity towards 3,4-dihydroxyphenylacetic acid (DOPAC) was assayed in 100 mM potassium phosphate buffer and at 37 "C by the method of Gulliver and Tipton (1978). Parallel assays under the same conditions were terminated after fixed times by the addition of 1 M perchloric acid and the mixtures were centrifuged to remove precipitated protein before the supernatants were analysed by HPLC (Murai et al., 1988). Samples of 20-50 p.L were applied to a CIScolumn (Waters p Bondapak) and eluted with 20 mM sodium acetate, 12.5 mM citric acid, pH 3.92, containing 0.033% (w/v) heptanesulfonic acid and 0.1 mM EDTA. The eluted products were detected electrochemically at an applied voltage of 0.83 V. This procedure gave good resolution of DOPAC from its methylated product and comparison of kinetic data obtained by this method and the radiochemical procedure indicated close agreement between the two. Good agreement was also obtained between the results of this HPLC procedure and a method based on the separation of the AdoHcy formed in the reaction from AdoMet (Molloy e l al., 1990). Alternative elution conditions would, however, be required for adrenaline and noradrenaline as substrates, since these were found not to be adequately resolved from their 0-methylated 232 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 6, 1990
derivatives by the present system. Furthermore it is possible that para-methylation might occur, in addition to meta-methylation, under some conditions (Creveling et aL, 1972) and that would necessitate resolution of both products. In a study of the application of the HPLC procedure to monitor the methylation of catechol the product, guaiacol, was found to be well separated from catechol by the procedure described above. Comparison of the performance of this assay with the results of a radiochemical assay (Zurcher and Da Prada, 1982) or the HPLC assay based on AdoHcy formation revealed it to give significantly lower (about 30%) rates than the other two procedures. The reasons for this discrepancy remain obscure, since the HPLC method was calibrated with standard product solutions and the K , value for catechol given by this procedure was in close agreement with that obtained when the radiochemical method was used. Since the HPLC and radiochemical results correlated well when DOPAC was used as substrate, it is unlikely that the discrepancy observed with catechol could be due to different enantiomeric compositions of the radioactive and unlabelled AdoMet samples. Until the causes of this discrepancy are resolved caution would be necessary in using the HPLC procedure to give absolute rate measurements.
Temperature effects Ionization processes and hydrophobic interactions are temperature dependent. Indeed such effects have frequently been applied to improve chromatographic separations. Figure 4 shows the effects of temperature on the retention time of dopamine under the HPLC conditions described above. Clearly such variations could have profound effects on peak identification and resolution. It is therefore unfortunate that many HPLC systems are supplied without temperature control as a standard facility.
6.5
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Column temperature ( C) Figure 4. The effects of temperature on the elution of dopamine. The column (&, Waters p Eondapak) was equilibrated to the indicated temperature before sample application. Other details are given in the text.
@ 1990 by John Wiley & Sons, Ltd.
HPLC AND AMINES
REFERENCES Alivisatos, S. G. A. and Ungar. F. (1968). Biochemistry 2, 285. Biek, P. R., Nilsson, E., Schick, C.. Waldmeier, P. C. and Lauber, J. (1984). In Neurobiology of the Trace Amines, (ed. by Boulton, A. A., Baker, G. B., Dewhurst, W. G. and Sandler, M., pp. 525-533. Humana Press, New York. Borchardt, R. T. (1981). Meth. Enrymol 77, 267. Buckman, J. D. and Eiduson, S. (1985). J. Biol. Chern. 260, 11899. Collins, M. A. (1980). In BiologicalEffects ofAlcoholed. by Begleiter, H., pp. 87-102. Plenum Press, New York. Coward, J. K. and Wu, F. Y.-H. (1973). Anal Biochem. 55, 406. Creveling. C. R., Morris, N., Shimizu. H., Ong, H. H. and Daly, J. (1 972). Mol. Pharmacol. 8, 398. Da Prada, M., Zurcher, G.. Wuthrich, I. and Haefely, W. E. (1988). J. Neural. Transm. Suppl. 26, 31. Deitrich, R. A. and Erwin, V. G. (1984). In Monoamine Oxidase and Disease, ed. by Tipton, K. F., Dostert, P. and Strolin Benedetti, M.. pp. 275-289. Academic Press, London. Dordain, G., Dostert, P., Strolin Benedetti, M. and Rovei, V. (1984). In Monoamine Oxidase and Disease, ed. by Tipton, K. F., Dostert, P. and Strolin Benedetti, M., pp. 417426. Academic Press, London. Draper, R. J., Sandler, M. and Walker, P. L. (1984). Brit. Med. J. 289, 308. Fung, K. and Grosjean, D. (1971). Anal. Chem. 53, 168. Green, A. L. and Haughton, P. M. (1961). Biochem. J.78. 172. Gulliver, P. A. and Tipton, K. F. (1978). Biochem. Pharmacol. 27, 773. Harrington, M. C., Henehan, G. T. M. and Tipton, K. F. (1988). Biochem. SOC.Trans. 16, 239. Kinemuchi, H., Fowler, C. J. and Tipton. K. F. (1987). Neurochem. lnt. 11, 359. Marley, E. and Blackwell, B. (1970). Adv. Pharmacol. Chemother. 8, 185. Mollay, A. M., Weir, D. G., Kennedy, G., Kennedy, S. and Scott, J. M. (1990). Biomed. Chrornatogr. 4, 257-260.
@ 1990 by John Wiley & Sons, Ltd.
Murai, S., Saitoh. H., Matsuda, Y. and Itoh, T. (1988). J. Neurochem. 50,473. Shier, W. T., Kock, Y. and Bloom, F. E. (1983). Neuropharmacol. 22, 278. Sullivan, J. P., McDonnell, L., Hardiman, 0. L., Farrell. M. A.. Phillips, J. P. and Tipton, K. F. (1986). Biochem. Pharmacol. 35,3255. Tipton, K. F. (1986). Cell Biochem. Funct. 4, 79. Tipton K. F. (1989). In Biochemical and Pharmacological Aspects of Depression, ed. by Tipton, K. F. and Youdim, M. B. H., pp. 1-24. Taylor and Francis, London. Tipton, K. F. and Sullivan, J. P. (1990). Proc. Roy. lrish Acad., in press. Tipton, K. F. and Youdim, M. B. H. (1983). In Methods in Biogenic Amine Research, ed. by Parvez, S., Nagatsu, T. and Parvez, H., pp. 441465. Elsevier, Amsterdam. Tipton, K. F., Houslay, M. D. and Turner, A. J (1977). Essays Neurochem. Neuropharmacol. 1, 103. Tipton, K. F., McCrodden, J. M. and Youdim, M. B. H. (1988). Biochem. J. 240, 379. Wiener, H. (1980). In Enzymatic Basis of Detoxication. ed. by Jakoby, W., pp. 261-280. Academic Press, New York. Wheatley. A. M. and Tipton, K. F. (1987). J. food Biochem. 11, 133. Wheatley, A. M., McCrodden, J. and Tipton, K. F. (1988). In Frontiers of Flavor, ed. by Charalambous, G., pp. 743-751. Elsevier, Amsterdam. Youdim, M. B. H., Finberg. J. P. M. and Tipton, K. F. (1988). In Handbook o f Experimental Pharmacology, ed. by Trendelenburg, H. U. and Weiner. N,, Vol. 90, pp. 119-192. SpringerVerlag. Berlin. Yu, P. H. (1986). In Neuromethods, Vol. 5, ed. by Boulton, A. A,, Baker, G. B. and Yu, P. H., Vol. 5, pp. 235-272. Humana Press, New Jersey. Zurcher, G. and Da Prada, M. (1982). J. Neurochem. 38, 191. Received 9 April, 1990; accepted 4 May, 1990
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