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A routine assay procedure for monoamine oxidase and its application to human blood platelets
357
Clinica Chimica Acta, 70 (1976) 357-369 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CGA 7745
A ROUTINE ASSAY PROCEDURE FOR MONOAMINE APPLICATION TO HUMAN BLOOD PLATELETS
OXIDASE AND 1TS
LOTHAR DEMISCH a, HANS J. BOCHNIK a and NIKOLAUS SEILER b** a Zentrum der Psychiatric im Klinihum der J. W. Goethe Universitiit, Frankfurt/Main and b Max-Planck-Znstitut fiir Hirnforschung, Arbeitsgruppe Neurochemie, Frankfurt/Main (G.F.R.) (Received December 15, 1975)
Summary
In the radiometric assay procedure for monoamine oxidase separation of the reaction products by ion-exchange column chromatography was optimized. A device was constructed that allowed the separation of 48 samples at the same time. This device can be applied for a great variety of analytical methods. With a 0.1 mM substrate concentration in the incubation mixture, reaction products could be determined with a reproducibility of 2-5%, where the standard deviation depends on the substrate. Using /3-phenylethylamine as substrate 20 c(g of platelet protein was sufficient for monoamine oxidase activity determinations. The dependence of human blood platelet monoamine oxidase on age and sex were studied using the method.
Introduction
Monoamine oxidase (amine:oxygen oxidoreductase (deaminating), EC 1.4.3.4) (MAO) has attracted attention for a considerable time now. Although its function as a regulatory enzyme of monoamine metabolism is still far from being clarified, a growing interest has been notable during the last few years. The finding of reduced MAO activities in the blood platelets of schizophrenic patients [1,2] considerably stimulated interest in the field. Since human blood platelets are an accessible source of mitochondrial MAO, clinically relevant problems can be studied by analyzing platelet MAO. Even though platelet MAO does not necessarily reflect mitochondrial MAO of the different organs, a series of problems of high interest, related to psychiatric disorders [ 3-91. migraine [lo] and iron deficiency anemia [ 111 have been raised by studying platelet MAO. * To
whom correspondence should be addressed.
358
Normally a large number of samples have to be analyzed in the experimental study of clinically relevant problems, and even though platelet MAO is considered a uniform enzyme of type B [ 12,131, it is advisible to use several substrates in the assay procednrz. On these premises a routine assay procedure was desirable. Materials and methods Chemicals
Usual laboratory chemicals were of A grade. With the following exceptions, they were purchased from E. Merck, Darmstadt, G.F.R. Tryptamine hydrochloride, serotonin binoxalate, fl-phenylethylamine hydrochloride, Dowex 50 W HCR (50W X 8) (100-200 mesh), and Amberlite CG 50 I (100-200 mesh) were from Serva, Heidelberg, G.F.R. Tranylcypromine (trans-2-amino-l-phenylcyclopropylamine hydrochloride) was from Smith, Kline and French, Philadelphia, Pennsylvania, U.S.A.; Unisolve I liquid scintillation coctail was from Zinsser, Frankfurt, G.F.R., and y-globulin (bovine, ‘fraction II) from Biomol, Ilvesheim, G.F.R. Radiochemicals
l-Amino-2-indoly[2-14C]ethane bisuccinate (tryptamine bisuccinate) (spec. radioactivity 46.5 Ci/mol), l-amino-2-phenyl[l-‘4C]ethane hydrochloride (pphenylethylamine hydrochloride) spec. radioactivity 9.6 Ci/mol), l-amino-2(5hydroxyindolyl) [2-l 4 C] ethane binoxalate (serotonin binoxalate) (spec. radioactivity 27.7 Ci/mol), 1-amino-2-(3,4-dihydroxyphenyl) [l-14C]ethane hydrobromide (dopamine hydrobromide (spec. radioactivity 9.0 Ci/mol) and 5-hydroxyindolyl [ carbonyl 14C] acetic acid (spec. radioactivity 5.2 Ci/mol) were purchased from New England Nuclear Corp., Boston, Mass., U.S.A. [carbonyl’ 4 C] Homovanillic acid (spec. radioactivity 9.0 Ci/mol) and phenyl [ carbonyl14C]acetic acid (spec. radioactivity 9.6 Ci/mol) were obtained by enzymatic oxidative deamination of labelled dopamine and fl-phenylethylamine, respectively. The radioactive amines were incubated (12 h, 38°C) with suspensions of rat liver mitochondria in 66 mM phosphate buffer, pH 7.2. The labelled acids were separated from cationic compounds by ion exchange column chromatography (Dowex 50W HCR, H’ form) using methanol/water (1 : 1, v/v) for elution. Purification was achieved by thin-layer chromatography on silica gel Gr2 s 4 layers of 2 mm thickness. Solvent: n-Butanol/butan-2-one/25% ammonia/water (4 : 3 : 2 : 1, v/v). 3,4,5_Trimethoxy [2,6-3H]phenylacetic acid (spec. radioactivity 51 Ci/mol) was obtained by electrophilic substitution of the non-labelled compound, adopting a method that was published for the labelling of mescaline [ 141. Isolation of blood platelets 5-10 ml samples of venous blood were taken from a total of 37 women
and 24 men of 15-73 years. All subjects were drug free and without signs of mental disorders. The blood samples were immediately mixed with 0.5 ml ACDsolution. Platelets were isolated according to Wyatt et al. [ 151. MAO determinations were carried out not later than 10 days after preparation of the platelets. These were stored at -20°C until use.
359
Isolation of rat liver mitochondria Mitochondria of rat liver were isolated by differential (w/v) homogenates in 0.25 M sucrose by the procedure boom [16].
centrifugation of Schneider
of 1 : 10 and Hoge-
Estimation of monoamine oxidase Incubation mixtures Blood platelets of 5 or 10 ml blood were lysed by freezing and thawing in 0.2 ml of 66 mM Sorensen phosphate buffer, pH 7.2 0.05-ml aliquots of this preparation (corresponding to 0.2-0.4 mg of platelet protein) were mixed with 0.02 ml of substrate solution.‘The stoppered tubes (polypropylene 12 mm X 75 mm) were incubated at 38 i: 0.5”C for 60 min. The samples were shaken at a fixed rate. By addition of 0.2 ml of 0.5 M perchloric acid, the enzymatic reaction was stopped at the end of the incubation period. The samples were stored at 4” C before being analyzed. Routinely blanks were carried through the procedure, which were prepared by addition of 0.2 ml of 0.5 M perchloric acid to the complete incubation mixture immediately after its preparation. Incubation mixtures with mitochondrial suspensions were prepared analogously. Substrate solutions The commercial radioactive amines were used without further purification. [’ 4 C]Tryptamine was mixed with non-labelled tryptamine hydrochloride to yield a specific radioactivity of 7.7 Ci/mol. All other amines were immediately dissolved in 66 mM Sorensen phosphate buffer, pH 7.2, to solutions containing 0.35 pmol/ml. (0.02-ml portions of these solutions in the incubation mixture (total volume 0.07 ml) correspond to an amine concentration of 0.1 mM). The amine solutions were stored in l-ml portions at -20” C. Protein determination Protein determinations of 0.02-ml aliquots of the platelet suspensions were carried out with a modified Lowry method y-globulin as standard.
and mitochondrial [ 171, using bovine
Separation of the reaction products Anionic and neutral reaction products of the amines were separated by exchange column chromatography. Dowex 50 W HCR (100-200 mesh) and Amberlite CG 50 I (100-200 mesh) were found to be equally suitable. The initial experiments were carried out with small glass columns (5 mm X 60 mm). In a more advanced state of the work, and for routine determinations, a multichannel column chromatographic apparatus was used. Immediately before separation, the acidified samples were mixed with 0.2 ml of water and 0.5 ml of methanol. Separation of the amines from the anionic and neutral components of the reaction mixture was carried out as described below.
360
The multichannel column chromatographic equipment The multichannel column chromatographic equipment consists essentially of three parts: A 48 channel pe~st~tic pump, a block with 48 cylind~c~ holes (the chromatographic columns) and an attachment which tightly connects the column heads with the 48 channels of the pump. The sampling side of the peristaltic pump consists of stainless steel needles with an inner diameter of 0.5 mm. These needles can be lifted, lowered, and inserted in the samples to an adjustable depth. A scheme of the ~ang~ment is given in Fig. 1. This Figure demo~stra~s the column head (H) in working position, when connection of the peristaltic pump and chramatographic column is established by pressing down the column head. The peristaltic pump used in this arrangement has a special drive gear that forces the rollers to rotate against the pumping direction to avoid pulsation of the pumped liquid. The pump also has electronic equipment which allows preselection of flow rate and volume. The arrangement of the column block (B) and the column heads (H) (front view) together with the tray that contains the vials (E) for sample collection is shown in Fig. 2. Tight connection between column heads (H) and colums (C) is achieved by Viton gaskets (V). The column heads are arranged in a device that allows the tigh~ning of all column heads simul~neously using four wing screws (W). The columns are constricted at the lower end, and funnel shaped at the upper end. Loss of adsorbent or ion exchange resin is prevented by polyethylene frits (F).
Fig. 1. Multichannel column chromatographic equipment. Scheme of the arrangement of sample (S), stainless steel needle (N). peristaltic pump (P), column block with drilled column (C). the connecting parts (Teflon tubing (T) and column head (H)) and the vial for eluate collection (E). (V, Viton gasket; PT. silicon rubber puping tube).
361
Fig. 2. Multichannel column chromatographic equipment. Scheme (front view) of the device showing the arrangement of column block (B) and column heads (H). (C. columns; F. polyethylene frit.s: V, Viton gaskets; W. wing screws; E, vials for eluate collection).
For the present purpose columns with 1.2 ml resin volume ware used. However, other column sizes can be used as well. A device consisting of several columns, a peristaltic pump and a sampling unit has been previously suggested for the estimation of triiodothyronine [ 181. Use of the multichannel column chromatographic equipment The columns of a block are filled up to the funnel shaped part with ion exchange resin. Filled blocks can be stored for several days in the refrigerator in a shallow layer of water. The resin-filled block is put in place and the column heads are brought into working position using the wing screws, so that the peristaltic pump is connected to the columns. By pumping about 5 ml of methanol/water (1 : 1, v/v) the columns are equilibrated. Sample preparation is carried out as described above. Normally groups of 12 samples are handled together by placing them in centrifuge tube holders. Up to 48 samples are placed at the sampling side of the peristaltic pump. For chromatography the flow rate is adjusted to 3 ml/h. The entire contents of the sample vessels are pumped through the ion exchange columns. Two 1 ml portions of a methanol/water mixture (1 : 1, v/v) are used to carefully rinse the sample vessels. They are subsequently pumped through the columns, in the same manner as the samples. The column eluates of MAO determinations are immediately used for radioactivity measurements. They are mixed with 15 ml of Unisolve I liquid scintillator for this purpose.
362
By loosening the wing screws, the column heads are lifted. The used column block can be removed and exchanged against another block. After the connection between peristaltic pump and columns has been achieved, the equipment is again ready for chromatography of a further set of 48 samples. Tests with 1 /.Si of 14C-labelled amines per column showed no memory effect in a subsequent separation. Usually at the end of a working day, or if the type of analysis is changed, the tubing of peristaltic pump and column head is rinsed by pumping a solution of a suitable detergent, followed by distilled water. Although it is quite possible to regenerate the ion exchange resin within the columns, practical experience showed that it is less time consuming and preferable to use several colum blocks, and to regenate the whole batch of resin, after removing it from the columns. Both filling columns with ion exchange resin, and emptying used columns are rapid procedures. With the described equipment one can carry out at‘least 3 X 48 MAO assays during a working day. Results Recovery
of acidic reaction products
Recovery of the acidic reaction products of several amines was tested by adding the labelled acids to incubation mixtures that did not contain radioactive amines. The mixtures were usually incubated for 60 min. Then they were separated as described. The water content of the medium that was used for the elution of the acids considerably affected the yields with both Dowex 5OW HCR and Amberlite CG 50 I (both in the H’ form). The different acids differed considerably in their respective recoveries. For instance 60% of Ei-hydroxy-indolylacetic acid, but 83% of 3,4,5-trimethoxyphenylacetic acid were recovered from the same type of column, using water as eluant. Addition of methanol to the eluant increased yields. With methanol/water mixture (1 : 1, v/v) recovery of all acids was about 95%, as is shown for Dowex 50W HCR columns-in Table I. Quite similar results were obtained with Amberlite CG 50 I. Blanks
The sensitivity and reproducibility of the method depend on the blank readings. They reflects to a certain degree the purity of the substrates. Three types of blanks were compared: In one group of experiments the enzymatic reaction was prevented by addition of 0.2 ml of 0.5 M perchloric acid to the enzyme preparation, in the second group enzymes were inactivated by heating, and in the third group sample mixtures were prepared from enzyme preparations that were preincubated with a MAO inhibitor (Tranylcypromine). As is shown in Table II, the blank readings were directly proportional to the total amount of the amines in the incubation mixture. Although there were differences in the blank values of the different amines, these differences were The only exception being the [i4C]dopamine substrate. Usually blanks were below 0.4% of total radioactivity. By storage of 0.35 mM amine solutions at -2O”C, blank readings increased by about 20% after six months. The similar results with blanks produced by different inactivation methods
363 TABLE I RECOVERY OF RADIOACTIVE DEAMINATIONS
REACTION PRODUCTS OF MONOAMINE OXJDASE CATALYZED
Incubation, separation on Dowex 50 NCR (100-200 mesh) columns and determination of the radioactive compounds was carried out as described in the methods section. (Mean values ? S.D. of ten determinations). Reaction product
Concn. t&W
~~~~~~l~~~b~~~l-14Cl
7.1
acetic acid 5-Hydroxy-indole[corbonyI14Clacetie acid 3,4-Dihydroxyphcnyltcarbo~y~. ’ 4 Cl acetic acid 3,4,5-Trimethoxu[2,6-3Hlphenylacetic acid
210
Total amount. (nmol)
Recovery (a)
0.5
91.3 i 5
26.7
97.3 +_1
8.7
0.61
95.1 t 2
3.1
0.22
98.4 f 2
allow one to conclude that under the expe~ment~ conditions non-enzymatic deamination does not occur. Since the blanks are of the same order of magnitude as the impurities of the radioactive substrates, it is evident that the separation method used separates completely the cationic substrates from their neutral and acidic reaction products. Purification of the labelled amines by thinlayer chromatography allows one to lower the blanks below 0.1% of total radioactivity. Reproducibility of the blanks was better than &15% under the conditions described in the Methods section. The e~~rna~ic reaction The amount of reaction product formed by platelet MAO of different substrates at identical reaction conditions differed greatly. P-Phenylethylamine was most effectively deaminated, followed by tryptamine. Dopamine and serotonin were unfavourable substrates (Table II). The amount of reaction product was directly proportional to the incubation time, at least up to 90 min. In the range of 0.3-2.2 mg of platelet protein in the reaction mixture, product amounts were, furthermore, linear with the amount of enzyme. O2 availability was not a limiting factor of reaction velocity. Even the increase of the reaction volume from 0.07 ml to 0.65 ml did not influence the linearity of the relation between the amounts of enzyme and reaction product. The sensitivity limit was defined as signal/blank noise = 2. The labelled amines of the present study allowed measurement of the formation of about 10 pmol of reaction product (Table II). This sensitivity was sufficient to measure oxidative deamination by blood platelets of serotonin even at a substrate concentration of 0.06 mM. The standard deviation of the procedure was it2.2% (including sample preparation, incubation, separation and radioactivity measu~ment), as determined from a total of 200 samples using [’ 4C] t~pt~ine and [I4 Cldopamine as substrates, and rat liver mitochondria and bovine blood platelets as enzyme sources.
IX
AND SENSITIVITY OF MONOAMINE OXIDASE DETERMINATIONS
l-Amino-2_(3,4-dihydroxyphenyl)~l-14C1 ethane (Dopaminef
l-Amino-2-phenylll-14C]ethane f@-phenylethylamine)
l-Amino-2-indolyl[2-14Cl~ ethane (Tryptamine) I-Amino-2-f5-hydroxyindolyl)12-14C]etbane (Serotonin)
Substrate Total (dpml
91 f 18 * 297 -i-20 * 305 * 104 * 1693 z+326 * 1749 +_224 ** 2164+ L72*** 400 f. 60 * 1956 .z 433 * 1752 i 66 ** 1530+ 520*** 2805 +_350 *
Total amount (dpm)
44042 188131 244390 1439370 1439370 1439370 86630 545932 545932 545932 144968
Concn. (/.W
39 100 57 100 100 100 57 100 100 100 100
Blank
Substrate
0.21 0.16 0.12 0.12 0.12 0.15 0.46 0.36 0.32 0.28 1.9
+_0.04 t 0.01. * 0.04 ” 0.02 * 0.02 & 0.01 F 0.07 f 0.08 + 0.01 t 0.1 s 0.2
Percent of total radioactivity mg
0.32 ?: 0.02
2 230
4720
t 0.2
7.3
0.70 + 0.02 2.3 i 0.1 0.05 r 0.004
nmolfmg protein/h
47587 i 1400
3500 ?: 98 11313 ?: 660 1197 * 90
dpm/0,33 protein/b
Reaction product
0.011 0.037 0.010 0.055 0.060 0.071 0.037 0.18 0.16 0.14 0.28
Sensitivity nmol/sample
MAO determinations of human blood platelets, using labelled tryptamine (spec. radioactivity 7.7 Cifmol), ~p~enylethy~mine &pee. radioactivity 9.6 Ciimol), serotonln (spec. radioactivity 27.7 Cilmol) and dopamine (spee. radioactivity 9.0 Ci/mol) as substrates. (Mean values of ten determinations i S.D.1. Sensitivity: signal/ blank noise = 2. Three types of blanks were compared: Inactivation of enzyme activity by treatment with 0.5 M percbloric acid (*), preparation of incubation mixtures with boiled enzyme preparation {**) and preincubation of the enzyme preparation with Tranylcypromine (0.1 mM) before addition of substrate (***).
R~FROD~CTIVITY
TABLE
365
Reproducibility was confirmed by numerous determinations of the MAO activity of human platelets. During these experiments it was recognized that MAO activity of platelets that were isolated from blood stored a few hours at room temperature, did not differ from those, isolated at -1-4”C immediately after venipuncture. Storage of the platelets at -20” C, however, caused a gradual loss of MAO activity. After 40 days only 20% of the original activity was measured with /3-phenylethylamine as substrate, and 40% with tryptamine.
MAO determinations with three substrates (P-phenylethylamine, tryptamine and serotonin) were carried out on the platelets from 37 women and 24 men aged 15-73 years. Figs. 3 and 4 shows the results for /3-phenylethylamine, Fig. 5 for tryptamine. Each point represents an individual’s MAO activity. Regression curves of the form
were calculated. The curves of female and male individuals (Fig. 3) shows the same tendency, namely an increase of enzyme activity during the first forty years of age, and then a slight decrease in the enzyme activity, which seems somewhat more pronounced for men. However, the overall change of MAO activity with age is not very significant. The curves of females and males do not differ significantly, even at the 5% level of probability. A remarkable difference of the measurements is the significantly larger scat-
. . . .
. . *
f*
Fig.3. Sex and age dependence of monoamine oxidase activity of human blood platelets. Substrate: pof phenol 114Cl-ethylamine. Dots, females; triangles. males. - - - - - -, regression CUN~ (Y = a + blx + b2x2) females:. . . . . ., regression curve of m&es.
366
OJ15. Fig.
4.
Age
Fig. mine.
5.
an
1
,
15
20
Age
of
1
,
, 30
curve
I
10
monoamine curve
individual’s
dependence
Regression
an individual’s
1
Regression
represents
oJ
r
30
dependence
tl‘%lethylamine. Point
r
I
20
I
oxidase
,
,
,
LO
oxidase
,
,
activity.
, 60
oxidase f 62x*)
1
activity shaded
human
of
alI
The
,
f
,
blood
platelets.
measurements
activity.
shaded
Substrate:
irregardless area
@henyl
of
corresponds
sex. with
Each fl.
70 years of human
oi 311 measurements The
n
b
70 years Age of
+ b2x2)
oxidase
50
of monoamine
I
60
+3ivity
(Y = a + blx
monoamine
(Y = a + blx
monoamine
I
50
blood
PlateMa.
irregardless
area corresponds
with
Substrate:
of sex. @.
Each
[‘*Cl point
trypta-
represents
361
tering of the values for females (variance of the computed curve s$ = 6.2) than for males (si,, = 3.9). The curve computed from all estimations, irregardless of sex (Fig. 4) had a variance of sz,, = 5.5. The shaded areas in Figs. 4 and 5 correspond to @, i.e. approximately to standard deviation. The age dependent increase of platelet MAO activity using tryptamine as substrate was similar to that using P-phenylethylamine. However, the decrease of enzyme activity with age (Fig. 3) was not observed with tryptamine. Because of the scattering values obtained using serotonin as substrate no clear relationship could be found to age and sex. Discussion Quite a number of methods have been described for the estimation of MAO [ 19-211, some were specially adapted for platelet MAO [ 22-241. The advantage of radiometric methods was first recognized by Wurtman and Axelrod [ 251. Since no special structural features are prerequisites, except for a label of a suitable isotope, radiometric methods allow the use of any type of substrates, including physiological substrates. They are usually highly sensitive. Aldehyde dehydrogenase is not always present in excessive amounts in MAO preparations. The reaction may therefore not proceed quantitatively to the acid that corresponds to the amine substrate. Aldehyde and its derivatives may be produced in significant amounts. In procedures applying solvent extraction as a separation method [ 25-271, non-anionic reaction products might not be separated from the substrate [ 281. Procedures using cation exchange resins for separation meet the requirement that anionic and neutral reaction products are eluted from these resins, whereas cations are retained. Our assay procedure is essentially a combination of advantageous details of known analytical and technical means, in that essential parts [29,30] have not been applied previously to MAO determination. The procedure allows one to lower blank readings and to increase recovery of reaction products, thus incresing the sensitivity and reproducibility of MAO determinations. The efficiency of the method is demonstrated by the large number of samples that can be analyzed per day. The low blank readings allow the measurement of the reaction products of MAO of only 20 pg of platelet protein, if 50 PM solutions of /3phenylethylamine (specific radioactivity 9.6 Ci/mol) are used as substrate. About 5000 dpm are still measured under this condition after 60 min of incubation. With this substrate, the platelets of 10 ml of human blood are sufficient for about 50 MAO estimations, including one protein assay. But even with serotonin as substrate, 3-4 MAO estimations can be carried out under the mentioned conditions with a reproducibility of S.D. = *7.5X It is well known that MAO activity of platelets is not sufficiently characterized if only one substrate is used [ 5,12,13,22,31], though platelet MAO seems to be less complex than tissue MAO. On the basis of the inhibitor spectrum and the preference for certain substrates it is believed that platelet MAO isoftheBtype[13]. The knowledge of age and sex dependencies of enzymes is crucial in clinically oriented problems. Genetic [6,7,13,32], and hormonal [33-351 influences on human platelet MAO activity have been demonstrated. Age dependence of
368
platelet MAO has also been claimed to exist [6,36]. Even if a statistical difference would be demonstrable on the basis of a larger number of estimations, clinically relevant sex-dependent differences of human platelet MAO activity do not exist (Fig. 3), in agreement with some published data [ 5,7]. The estrogen state of the females was not taken into consideration in our study. The broader scattering of MAO activity of platelets of women could reflect hormonal influences. On the other hand, decline of platelet MAO activity above the age of 50 was less pronounced in females than in males. According to Figs. 3-5, changes of platelet MAO activity were small during aging, both in females and males. The curves in these figures differ greatly from those published previously 16,361. With fl-phenylethalamine as substrate MAO activity decreased, after a slight initial increase, at ages above 50, although this decrease was only small. In the former work the steepest increase of the curves was observed (with benzylamine as substrate) at the period, when decreased MAO activity was noted in this work. Besides differences in the substrate there was a difference in the mode of evaluation of the experimental results, which may also add up to the observed differences of age dependence of platelet MAO: Mean values and standard deviations of age groups were calculated and plotted in a diagram in the previous papers [6,36 1. In contrast, the curves in Figs. 3-5 were obtained by calculation of non-linear regression curves, using a Hewlett Packard 9821 A calculator. Acknowledgement We would like to thank Mr. U. Demisch, Max-Planck-Institut Frankfurt, for computer calculations.
fur Biophysik,
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119 Palm, D. and Magnus, U (1968) Klin. Wochenschr. 46. 720-728 Zeller. E.A., Harinath Babu, B., Cavanaugh, M.J. and Strauch, G.J. (1969) Pharmacol. Res. Commun. 1, 20-24 Wurtman, R.J. and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439-1440 Otsuka, S. and Kobayashi. Y. (1964) Biochem. Pharmacol. 13.995-1006 McCaman, R.E., McCamen, M.W., Hunt, J.M. and Smith, M.S. (1965) J. Neurochem. 12, 15-23 Southgate, J. and Collins, C.G.S. (1969) Biochem. Pharmacol. 18, 2285-2287 Seiler, N. and Demisch, L. (1971) Biochem. Pharmacol. 20, 2485-2493 Jandem, P. and Churacek, J. (1973) J. Chromatogr. 86. 351421 Youdim, M.B.H., Holzbauer, M. and Woods, H.F. (1974) in Psychopharmacology of Monoamies and Their Regulatory Enzymes (Usdin, E. ed.) pp. 11-28, Raven Press, New York Nies. A., Robinson, D.S., Lambom, K.R. and Lampert, R.P. (1973) Arch. Gen. Psychiatr. 28, 834838 Sandier, M. and Youdim. M.B.H. (1972) Pharmacol. Rev. 24. 331-348 Belmaker, R.H.. Murphy, D.L., Wyatt, R.J. and Loriaux, D.L. (1974) Arch. Gen. Psychiatr. 31, 553556 Klaiber, E.L.. Broverman, D.M., Vogel. W., Kobayashi, Y. and Moriatry, D. (1972) Am. J. Psychiatr. 128.1492-1498 Robinson. D.S., Davis, J.M., Nies, A., Ravaris, C.L. and Sylvester, D. (1971) Arch. Gen. Psychiatr. 24, 536-539
Clinica Chimica Acta, 70 (1976) 357-369 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CGA 7745
A ROUTINE ASSAY PROCEDURE FOR MONOAMINE APPLICATION TO HUMAN BLOOD PLATELETS
OXIDASE AND 1TS
LOTHAR DEMISCH a, HANS J. BOCHNIK a and NIKOLAUS SEILER b** a Zentrum der Psychiatric im Klinihum der J. W. Goethe Universitiit, Frankfurt/Main and b Max-Planck-Znstitut fiir Hirnforschung, Arbeitsgruppe Neurochemie, Frankfurt/Main (G.F.R.) (Received December 15, 1975)
Summary
In the radiometric assay procedure for monoamine oxidase separation of the reaction products by ion-exchange column chromatography was optimized. A device was constructed that allowed the separation of 48 samples at the same time. This device can be applied for a great variety of analytical methods. With a 0.1 mM substrate concentration in the incubation mixture, reaction products could be determined with a reproducibility of 2-5%, where the standard deviation depends on the substrate. Using /3-phenylethylamine as substrate 20 c(g of platelet protein was sufficient for monoamine oxidase activity determinations. The dependence of human blood platelet monoamine oxidase on age and sex were studied using the method.
Introduction
Monoamine oxidase (amine:oxygen oxidoreductase (deaminating), EC 1.4.3.4) (MAO) has attracted attention for a considerable time now. Although its function as a regulatory enzyme of monoamine metabolism is still far from being clarified, a growing interest has been notable during the last few years. The finding of reduced MAO activities in the blood platelets of schizophrenic patients [1,2] considerably stimulated interest in the field. Since human blood platelets are an accessible source of mitochondrial MAO, clinically relevant problems can be studied by analyzing platelet MAO. Even though platelet MAO does not necessarily reflect mitochondrial MAO of the different organs, a series of problems of high interest, related to psychiatric disorders [ 3-91. migraine [lo] and iron deficiency anemia [ 111 have been raised by studying platelet MAO. * To
whom correspondence should be addressed.
358
Normally a large number of samples have to be analyzed in the experimental study of clinically relevant problems, and even though platelet MAO is considered a uniform enzyme of type B [ 12,131, it is advisible to use several substrates in the assay procednrz. On these premises a routine assay procedure was desirable. Materials and methods Chemicals
Usual laboratory chemicals were of A grade. With the following exceptions, they were purchased from E. Merck, Darmstadt, G.F.R. Tryptamine hydrochloride, serotonin binoxalate, fl-phenylethylamine hydrochloride, Dowex 50 W HCR (50W X 8) (100-200 mesh), and Amberlite CG 50 I (100-200 mesh) were from Serva, Heidelberg, G.F.R. Tranylcypromine (trans-2-amino-l-phenylcyclopropylamine hydrochloride) was from Smith, Kline and French, Philadelphia, Pennsylvania, U.S.A.; Unisolve I liquid scintillation coctail was from Zinsser, Frankfurt, G.F.R., and y-globulin (bovine, ‘fraction II) from Biomol, Ilvesheim, G.F.R. Radiochemicals
l-Amino-2-indoly[2-14C]ethane bisuccinate (tryptamine bisuccinate) (spec. radioactivity 46.5 Ci/mol), l-amino-2-phenyl[l-‘4C]ethane hydrochloride (pphenylethylamine hydrochloride) spec. radioactivity 9.6 Ci/mol), l-amino-2(5hydroxyindolyl) [2-l 4 C] ethane binoxalate (serotonin binoxalate) (spec. radioactivity 27.7 Ci/mol), 1-amino-2-(3,4-dihydroxyphenyl) [l-14C]ethane hydrobromide (dopamine hydrobromide (spec. radioactivity 9.0 Ci/mol) and 5-hydroxyindolyl [ carbonyl 14C] acetic acid (spec. radioactivity 5.2 Ci/mol) were purchased from New England Nuclear Corp., Boston, Mass., U.S.A. [carbonyl’ 4 C] Homovanillic acid (spec. radioactivity 9.0 Ci/mol) and phenyl [ carbonyl14C]acetic acid (spec. radioactivity 9.6 Ci/mol) were obtained by enzymatic oxidative deamination of labelled dopamine and fl-phenylethylamine, respectively. The radioactive amines were incubated (12 h, 38°C) with suspensions of rat liver mitochondria in 66 mM phosphate buffer, pH 7.2. The labelled acids were separated from cationic compounds by ion exchange column chromatography (Dowex 50W HCR, H’ form) using methanol/water (1 : 1, v/v) for elution. Purification was achieved by thin-layer chromatography on silica gel Gr2 s 4 layers of 2 mm thickness. Solvent: n-Butanol/butan-2-one/25% ammonia/water (4 : 3 : 2 : 1, v/v). 3,4,5_Trimethoxy [2,6-3H]phenylacetic acid (spec. radioactivity 51 Ci/mol) was obtained by electrophilic substitution of the non-labelled compound, adopting a method that was published for the labelling of mescaline [ 141. Isolation of blood platelets 5-10 ml samples of venous blood were taken from a total of 37 women
and 24 men of 15-73 years. All subjects were drug free and without signs of mental disorders. The blood samples were immediately mixed with 0.5 ml ACDsolution. Platelets were isolated according to Wyatt et al. [ 151. MAO determinations were carried out not later than 10 days after preparation of the platelets. These were stored at -20°C until use.
359
Isolation of rat liver mitochondria Mitochondria of rat liver were isolated by differential (w/v) homogenates in 0.25 M sucrose by the procedure boom [16].
centrifugation of Schneider
of 1 : 10 and Hoge-
Estimation of monoamine oxidase Incubation mixtures Blood platelets of 5 or 10 ml blood were lysed by freezing and thawing in 0.2 ml of 66 mM Sorensen phosphate buffer, pH 7.2 0.05-ml aliquots of this preparation (corresponding to 0.2-0.4 mg of platelet protein) were mixed with 0.02 ml of substrate solution.‘The stoppered tubes (polypropylene 12 mm X 75 mm) were incubated at 38 i: 0.5”C for 60 min. The samples were shaken at a fixed rate. By addition of 0.2 ml of 0.5 M perchloric acid, the enzymatic reaction was stopped at the end of the incubation period. The samples were stored at 4” C before being analyzed. Routinely blanks were carried through the procedure, which were prepared by addition of 0.2 ml of 0.5 M perchloric acid to the complete incubation mixture immediately after its preparation. Incubation mixtures with mitochondrial suspensions were prepared analogously. Substrate solutions The commercial radioactive amines were used without further purification. [’ 4 C]Tryptamine was mixed with non-labelled tryptamine hydrochloride to yield a specific radioactivity of 7.7 Ci/mol. All other amines were immediately dissolved in 66 mM Sorensen phosphate buffer, pH 7.2, to solutions containing 0.35 pmol/ml. (0.02-ml portions of these solutions in the incubation mixture (total volume 0.07 ml) correspond to an amine concentration of 0.1 mM). The amine solutions were stored in l-ml portions at -20” C. Protein determination Protein determinations of 0.02-ml aliquots of the platelet suspensions were carried out with a modified Lowry method y-globulin as standard.
and mitochondrial [ 171, using bovine
Separation of the reaction products Anionic and neutral reaction products of the amines were separated by exchange column chromatography. Dowex 50 W HCR (100-200 mesh) and Amberlite CG 50 I (100-200 mesh) were found to be equally suitable. The initial experiments were carried out with small glass columns (5 mm X 60 mm). In a more advanced state of the work, and for routine determinations, a multichannel column chromatographic apparatus was used. Immediately before separation, the acidified samples were mixed with 0.2 ml of water and 0.5 ml of methanol. Separation of the amines from the anionic and neutral components of the reaction mixture was carried out as described below.
360
The multichannel column chromatographic equipment The multichannel column chromatographic equipment consists essentially of three parts: A 48 channel pe~st~tic pump, a block with 48 cylind~c~ holes (the chromatographic columns) and an attachment which tightly connects the column heads with the 48 channels of the pump. The sampling side of the peristaltic pump consists of stainless steel needles with an inner diameter of 0.5 mm. These needles can be lifted, lowered, and inserted in the samples to an adjustable depth. A scheme of the ~ang~ment is given in Fig. 1. This Figure demo~stra~s the column head (H) in working position, when connection of the peristaltic pump and chramatographic column is established by pressing down the column head. The peristaltic pump used in this arrangement has a special drive gear that forces the rollers to rotate against the pumping direction to avoid pulsation of the pumped liquid. The pump also has electronic equipment which allows preselection of flow rate and volume. The arrangement of the column block (B) and the column heads (H) (front view) together with the tray that contains the vials (E) for sample collection is shown in Fig. 2. Tight connection between column heads (H) and colums (C) is achieved by Viton gaskets (V). The column heads are arranged in a device that allows the tigh~ning of all column heads simul~neously using four wing screws (W). The columns are constricted at the lower end, and funnel shaped at the upper end. Loss of adsorbent or ion exchange resin is prevented by polyethylene frits (F).
Fig. 1. Multichannel column chromatographic equipment. Scheme of the arrangement of sample (S), stainless steel needle (N). peristaltic pump (P), column block with drilled column (C). the connecting parts (Teflon tubing (T) and column head (H)) and the vial for eluate collection (E). (V, Viton gasket; PT. silicon rubber puping tube).
361
Fig. 2. Multichannel column chromatographic equipment. Scheme (front view) of the device showing the arrangement of column block (B) and column heads (H). (C. columns; F. polyethylene frit.s: V, Viton gaskets; W. wing screws; E, vials for eluate collection).
For the present purpose columns with 1.2 ml resin volume ware used. However, other column sizes can be used as well. A device consisting of several columns, a peristaltic pump and a sampling unit has been previously suggested for the estimation of triiodothyronine [ 181. Use of the multichannel column chromatographic equipment The columns of a block are filled up to the funnel shaped part with ion exchange resin. Filled blocks can be stored for several days in the refrigerator in a shallow layer of water. The resin-filled block is put in place and the column heads are brought into working position using the wing screws, so that the peristaltic pump is connected to the columns. By pumping about 5 ml of methanol/water (1 : 1, v/v) the columns are equilibrated. Sample preparation is carried out as described above. Normally groups of 12 samples are handled together by placing them in centrifuge tube holders. Up to 48 samples are placed at the sampling side of the peristaltic pump. For chromatography the flow rate is adjusted to 3 ml/h. The entire contents of the sample vessels are pumped through the ion exchange columns. Two 1 ml portions of a methanol/water mixture (1 : 1, v/v) are used to carefully rinse the sample vessels. They are subsequently pumped through the columns, in the same manner as the samples. The column eluates of MAO determinations are immediately used for radioactivity measurements. They are mixed with 15 ml of Unisolve I liquid scintillator for this purpose.
362
By loosening the wing screws, the column heads are lifted. The used column block can be removed and exchanged against another block. After the connection between peristaltic pump and columns has been achieved, the equipment is again ready for chromatography of a further set of 48 samples. Tests with 1 /.Si of 14C-labelled amines per column showed no memory effect in a subsequent separation. Usually at the end of a working day, or if the type of analysis is changed, the tubing of peristaltic pump and column head is rinsed by pumping a solution of a suitable detergent, followed by distilled water. Although it is quite possible to regenerate the ion exchange resin within the columns, practical experience showed that it is less time consuming and preferable to use several colum blocks, and to regenate the whole batch of resin, after removing it from the columns. Both filling columns with ion exchange resin, and emptying used columns are rapid procedures. With the described equipment one can carry out at‘least 3 X 48 MAO assays during a working day. Results Recovery
of acidic reaction products
Recovery of the acidic reaction products of several amines was tested by adding the labelled acids to incubation mixtures that did not contain radioactive amines. The mixtures were usually incubated for 60 min. Then they were separated as described. The water content of the medium that was used for the elution of the acids considerably affected the yields with both Dowex 5OW HCR and Amberlite CG 50 I (both in the H’ form). The different acids differed considerably in their respective recoveries. For instance 60% of Ei-hydroxy-indolylacetic acid, but 83% of 3,4,5-trimethoxyphenylacetic acid were recovered from the same type of column, using water as eluant. Addition of methanol to the eluant increased yields. With methanol/water mixture (1 : 1, v/v) recovery of all acids was about 95%, as is shown for Dowex 50W HCR columns-in Table I. Quite similar results were obtained with Amberlite CG 50 I. Blanks
The sensitivity and reproducibility of the method depend on the blank readings. They reflects to a certain degree the purity of the substrates. Three types of blanks were compared: In one group of experiments the enzymatic reaction was prevented by addition of 0.2 ml of 0.5 M perchloric acid to the enzyme preparation, in the second group enzymes were inactivated by heating, and in the third group sample mixtures were prepared from enzyme preparations that were preincubated with a MAO inhibitor (Tranylcypromine). As is shown in Table II, the blank readings were directly proportional to the total amount of the amines in the incubation mixture. Although there were differences in the blank values of the different amines, these differences were The only exception being the [i4C]dopamine substrate. Usually blanks were below 0.4% of total radioactivity. By storage of 0.35 mM amine solutions at -2O”C, blank readings increased by about 20% after six months. The similar results with blanks produced by different inactivation methods
363 TABLE I RECOVERY OF RADIOACTIVE DEAMINATIONS
REACTION PRODUCTS OF MONOAMINE OXJDASE CATALYZED
Incubation, separation on Dowex 50 NCR (100-200 mesh) columns and determination of the radioactive compounds was carried out as described in the methods section. (Mean values ? S.D. of ten determinations). Reaction product
Concn. t&W
~~~~~~l~~~b~~~l-14Cl
7.1
acetic acid 5-Hydroxy-indole[corbonyI14Clacetie acid 3,4-Dihydroxyphcnyltcarbo~y~. ’ 4 Cl acetic acid 3,4,5-Trimethoxu[2,6-3Hlphenylacetic acid
210
Total amount. (nmol)
Recovery (a)
0.5
91.3 i 5
26.7
97.3 +_1
8.7
0.61
95.1 t 2
3.1
0.22
98.4 f 2
allow one to conclude that under the expe~ment~ conditions non-enzymatic deamination does not occur. Since the blanks are of the same order of magnitude as the impurities of the radioactive substrates, it is evident that the separation method used separates completely the cationic substrates from their neutral and acidic reaction products. Purification of the labelled amines by thinlayer chromatography allows one to lower the blanks below 0.1% of total radioactivity. Reproducibility of the blanks was better than &15% under the conditions described in the Methods section. The e~~rna~ic reaction The amount of reaction product formed by platelet MAO of different substrates at identical reaction conditions differed greatly. P-Phenylethylamine was most effectively deaminated, followed by tryptamine. Dopamine and serotonin were unfavourable substrates (Table II). The amount of reaction product was directly proportional to the incubation time, at least up to 90 min. In the range of 0.3-2.2 mg of platelet protein in the reaction mixture, product amounts were, furthermore, linear with the amount of enzyme. O2 availability was not a limiting factor of reaction velocity. Even the increase of the reaction volume from 0.07 ml to 0.65 ml did not influence the linearity of the relation between the amounts of enzyme and reaction product. The sensitivity limit was defined as signal/blank noise = 2. The labelled amines of the present study allowed measurement of the formation of about 10 pmol of reaction product (Table II). This sensitivity was sufficient to measure oxidative deamination by blood platelets of serotonin even at a substrate concentration of 0.06 mM. The standard deviation of the procedure was it2.2% (including sample preparation, incubation, separation and radioactivity measu~ment), as determined from a total of 200 samples using [’ 4C] t~pt~ine and [I4 Cldopamine as substrates, and rat liver mitochondria and bovine blood platelets as enzyme sources.
IX
AND SENSITIVITY OF MONOAMINE OXIDASE DETERMINATIONS
l-Amino-2_(3,4-dihydroxyphenyl)~l-14C1 ethane (Dopaminef
l-Amino-2-phenylll-14C]ethane f@-phenylethylamine)
l-Amino-2-indolyl[2-14Cl~ ethane (Tryptamine) I-Amino-2-f5-hydroxyindolyl)12-14C]etbane (Serotonin)
Substrate Total (dpml
91 f 18 * 297 -i-20 * 305 * 104 * 1693 z+326 * 1749 +_224 ** 2164+ L72*** 400 f. 60 * 1956 .z 433 * 1752 i 66 ** 1530+ 520*** 2805 +_350 *
Total amount (dpm)
44042 188131 244390 1439370 1439370 1439370 86630 545932 545932 545932 144968
Concn. (/.W
39 100 57 100 100 100 57 100 100 100 100
Blank
Substrate
0.21 0.16 0.12 0.12 0.12 0.15 0.46 0.36 0.32 0.28 1.9
+_0.04 t 0.01. * 0.04 ” 0.02 * 0.02 & 0.01 F 0.07 f 0.08 + 0.01 t 0.1 s 0.2
Percent of total radioactivity mg
0.32 ?: 0.02
2 230
4720
t 0.2
7.3
0.70 + 0.02 2.3 i 0.1 0.05 r 0.004
nmolfmg protein/h
47587 i 1400
3500 ?: 98 11313 ?: 660 1197 * 90
dpm/0,33 protein/b
Reaction product
0.011 0.037 0.010 0.055 0.060 0.071 0.037 0.18 0.16 0.14 0.28
Sensitivity nmol/sample
MAO determinations of human blood platelets, using labelled tryptamine (spec. radioactivity 7.7 Cifmol), ~p~enylethy~mine &pee. radioactivity 9.6 Ciimol), serotonln (spec. radioactivity 27.7 Cilmol) and dopamine (spee. radioactivity 9.0 Ci/mol) as substrates. (Mean values of ten determinations i S.D.1. Sensitivity: signal/ blank noise = 2. Three types of blanks were compared: Inactivation of enzyme activity by treatment with 0.5 M percbloric acid (*), preparation of incubation mixtures with boiled enzyme preparation {**) and preincubation of the enzyme preparation with Tranylcypromine (0.1 mM) before addition of substrate (***).
R~FROD~CTIVITY
TABLE
365
Reproducibility was confirmed by numerous determinations of the MAO activity of human platelets. During these experiments it was recognized that MAO activity of platelets that were isolated from blood stored a few hours at room temperature, did not differ from those, isolated at -1-4”C immediately after venipuncture. Storage of the platelets at -20” C, however, caused a gradual loss of MAO activity. After 40 days only 20% of the original activity was measured with /3-phenylethylamine as substrate, and 40% with tryptamine.
MAO determinations with three substrates (P-phenylethylamine, tryptamine and serotonin) were carried out on the platelets from 37 women and 24 men aged 15-73 years. Figs. 3 and 4 shows the results for /3-phenylethylamine, Fig. 5 for tryptamine. Each point represents an individual’s MAO activity. Regression curves of the form
were calculated. The curves of female and male individuals (Fig. 3) shows the same tendency, namely an increase of enzyme activity during the first forty years of age, and then a slight decrease in the enzyme activity, which seems somewhat more pronounced for men. However, the overall change of MAO activity with age is not very significant. The curves of females and males do not differ significantly, even at the 5% level of probability. A remarkable difference of the measurements is the significantly larger scat-
. . . .
. . *
f*
Fig.3. Sex and age dependence of monoamine oxidase activity of human blood platelets. Substrate: pof phenol 114Cl-ethylamine. Dots, females; triangles. males. - - - - - -, regression CUN~ (Y = a + blx + b2x2) females:. . . . . ., regression curve of m&es.
366
OJ15. Fig.
4.
Age
Fig. mine.
5.
an
1
,
15
20
Age
of
1
,
, 30
curve
I
10
monoamine curve
individual’s
dependence
Regression
an individual’s
1
Regression
represents
oJ
r
30
dependence
tl‘%lethylamine. Point
r
I
20
I
oxidase
,
,
,
LO
oxidase
,
,
activity.
, 60
oxidase f 62x*)
1
activity shaded
human
of
alI
The
,
f
,
blood
platelets.
measurements
activity.
shaded
Substrate:
irregardless area
@henyl
of
corresponds
sex. with
Each fl.
70 years of human
oi 311 measurements The
n
b
70 years Age of
+ b2x2)
oxidase
50
of monoamine
I
60
+3ivity
(Y = a + blx
monoamine
(Y = a + blx
monoamine
I
50
blood
PlateMa.
irregardless
area corresponds
with
Substrate:
of sex. @.
Each
[‘*Cl point
trypta-
represents
361
tering of the values for females (variance of the computed curve s$ = 6.2) than for males (si,, = 3.9). The curve computed from all estimations, irregardless of sex (Fig. 4) had a variance of sz,, = 5.5. The shaded areas in Figs. 4 and 5 correspond to @, i.e. approximately to standard deviation. The age dependent increase of platelet MAO activity using tryptamine as substrate was similar to that using P-phenylethylamine. However, the decrease of enzyme activity with age (Fig. 3) was not observed with tryptamine. Because of the scattering values obtained using serotonin as substrate no clear relationship could be found to age and sex. Discussion Quite a number of methods have been described for the estimation of MAO [ 19-211, some were specially adapted for platelet MAO [ 22-241. The advantage of radiometric methods was first recognized by Wurtman and Axelrod [ 251. Since no special structural features are prerequisites, except for a label of a suitable isotope, radiometric methods allow the use of any type of substrates, including physiological substrates. They are usually highly sensitive. Aldehyde dehydrogenase is not always present in excessive amounts in MAO preparations. The reaction may therefore not proceed quantitatively to the acid that corresponds to the amine substrate. Aldehyde and its derivatives may be produced in significant amounts. In procedures applying solvent extraction as a separation method [ 25-271, non-anionic reaction products might not be separated from the substrate [ 281. Procedures using cation exchange resins for separation meet the requirement that anionic and neutral reaction products are eluted from these resins, whereas cations are retained. Our assay procedure is essentially a combination of advantageous details of known analytical and technical means, in that essential parts [29,30] have not been applied previously to MAO determination. The procedure allows one to lower blank readings and to increase recovery of reaction products, thus incresing the sensitivity and reproducibility of MAO determinations. The efficiency of the method is demonstrated by the large number of samples that can be analyzed per day. The low blank readings allow the measurement of the reaction products of MAO of only 20 pg of platelet protein, if 50 PM solutions of /3phenylethylamine (specific radioactivity 9.6 Ci/mol) are used as substrate. About 5000 dpm are still measured under this condition after 60 min of incubation. With this substrate, the platelets of 10 ml of human blood are sufficient for about 50 MAO estimations, including one protein assay. But even with serotonin as substrate, 3-4 MAO estimations can be carried out under the mentioned conditions with a reproducibility of S.D. = *7.5X It is well known that MAO activity of platelets is not sufficiently characterized if only one substrate is used [ 5,12,13,22,31], though platelet MAO seems to be less complex than tissue MAO. On the basis of the inhibitor spectrum and the preference for certain substrates it is believed that platelet MAO isoftheBtype[13]. The knowledge of age and sex dependencies of enzymes is crucial in clinically oriented problems. Genetic [6,7,13,32], and hormonal [33-351 influences on human platelet MAO activity have been demonstrated. Age dependence of
368
platelet MAO has also been claimed to exist [6,36]. Even if a statistical difference would be demonstrable on the basis of a larger number of estimations, clinically relevant sex-dependent differences of human platelet MAO activity do not exist (Fig. 3), in agreement with some published data [ 5,7]. The estrogen state of the females was not taken into consideration in our study. The broader scattering of MAO activity of platelets of women could reflect hormonal influences. On the other hand, decline of platelet MAO activity above the age of 50 was less pronounced in females than in males. According to Figs. 3-5, changes of platelet MAO activity were small during aging, both in females and males. The curves in these figures differ greatly from those published previously 16,361. With fl-phenylethalamine as substrate MAO activity decreased, after a slight initial increase, at ages above 50, although this decrease was only small. In the former work the steepest increase of the curves was observed (with benzylamine as substrate) at the period, when decreased MAO activity was noted in this work. Besides differences in the substrate there was a difference in the mode of evaluation of the experimental results, which may also add up to the observed differences of age dependence of platelet MAO: Mean values and standard deviations of age groups were calculated and plotted in a diagram in the previous papers [6,36 1. In contrast, the curves in Figs. 3-5 were obtained by calculation of non-linear regression curves, using a Hewlett Packard 9821 A calculator. Acknowledgement We would like to thank Mr. U. Demisch, Max-Planck-Institut Frankfurt, for computer calculations.
fur Biophysik,
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