393
Clinica
Chimica
0 Elsevier
Acta,
Scientific
62 (1975) 393-400 Publishing Company,
Amsterdam
- Printed
in The Netherlands
CGA 7153
A SENSITIVE NON-ISOTOPIC ASSAY FOR MONOAMINE ACTIVITY IN HUMAN BLOOD PLATELETS
SABURO
TAKAHASHIa
and TADAHIKO
OXIDASE
KARASAWAb
‘Department of Psychiatry and Neurology, Kyoto Prefectural University of Medicine, Kyoto, 602 (Japan) and b Department of Pharmacology, Research Laboratories, Dainippon Pharmaceutical Co., Ltd, Suita, Osaka, 564 (Japan)
(Received
March 11, 1975)
Summary A sensitive and specific procedure for measuring monoamine oxidase (MAO) activity in human platelets is described. Serotonin is used as substrate and formed 5-hydroxyindoleacetic acid (5-HIAA) is separated by a double microcolumn technique on Sephadex G-10 and Amberlite CG-50 and measured fluorimetrically. MAO activities were 5.11 -I- 1.32 (mean k S.D.) nmol/mg protein/hour in male and 7.85 k 1.58 in female healthy adults @ < 0.001). MAO activity measured in 32 depressed patients was in a range of 3.20-10.62 nmol/mg protein/hour, the value not differing from that in the normal subjects. No significant difference was established between three subtypes of depression, in bipolar, unipolar and involutional patients, while significantly higher values were evident in female than in male patients @ < 0.001).
Introduction The widespread therapeutic use of monoamine oxidase inhibitors in psychiatry has required direct and rapid means of measuring the activity of this enzyme in patient materials. Currently radioisotopic techniques using [’ 4 C] tryptamine or [’ 4 C] benzylamine are used for the determination of monoamine oxidase (MAO) activity in human platelets [1,2] . They have prevailed in biological researches, in psychiatry because of their sensitivity and specificity [3-101 and, actually, serial determinations from a psychotic patient require not only analytical methods requiring small blood samples but also concise experimental procedures. However, these methods require expensive radioisotopic materials as substrate and have disadvantages except for limited research purposes. On the other hand, the crucial step in the fluorimetric methods is separation of the deaminated compound from substances which interfere with subsequent fluorimetric analysis.
We believe that it is still of great use to devise a simple assay for the MAO activity in human platelets, as recent pharmacological developments necessitate more biological information for more intelligent treatment of affective disorders. We have developed a simple and specific assay for the MAO activity in human platelets utilizing a purification procedure based on a double microcolumn technique on Sephadex G-10 and Amberlite CG-50 [ 111. 5-Hydroxytryptamine (serotonin) is used as substrate and 5-hydroxyindoleacetic acid (5-HIAA) formed is measured fluorimetrically. Sixty or more assays can be performed within 8 hours. This method is sensitive enough to measure enzyme activity in the platelets from as little as 3-4 ml of blood, an amount obtainable repeatedly from a patient. Experimental Collection
of human platelets
Human platelets were isolated from fresh whole blood collected in disodium ethylenediaminetetraacetate (Na, -EDTA) anticoagulant. Blood. samples were collected by venipuncture into a non-wettable plastic disposable syringe and immediately transferred into a polypropylene centrifuge tube containing a third volume of 0.5% Na, -EDTA solution in saline adjusted to pH 7.4, and inverted gently about ten times. This treatment of blood samples secures the stability of platelets in ice during transportation within 4 hours. Samples were centrifuged at 130 X g for 15 minutes to sediment red cells and leukocytes. The supernatant, platelet rich plasma (PRP), was then recentrifuged at 130 X g for 10 minutes. PRP was transferred to a siliconized glass tube and centrifuged at 1500 X g for 10 minutes. The precipitate was resuspended in 0.1% Na, EDTA in saline, pH 7.4, and recentrifuged at 1500 X g for 5 minutes. Care must be taken in separating platelets from blood to avoid destruction of platelet cells, which causes serious loss of serotonin content as well as MAO activity. Heparin as anticoagulant appeared to be unsatisfactory for protecting the platelet cells during multiple suspensions and centrifugations, whereas Na, -EDTA in the concentration given appears not to affect the MAO activity in the platelets. All operations were carried out at 4°C. Platelets of 0.12-0.15 mg protein were harvested from 1 ml of blood by this collection method. The prepared platelet samples ready for the enzyme assay were stored at -20°C. The enzyme MAO was quite stable for at least 8 weeks, whereas serotonin content in the platelets was liable to decrease under these conditions. Preparation
of columns
Sephadex G-10 (Pharmacia Fine Chemicals, AB, Uppsala, Sweden) was washed and cycled through 0.5 N NaOH, 0.5 N HCl and deionized water and stored as a suspension in water containing 0.01% sodium azide. A suspension corresponding to 1 g dry gel was pipetted into a glass column, 1 cm in diameter and 15 cm in length, which had been plugged at the bottom with a pledget of polypropylene wool. Amberlite ion exchange resin CG-50, type 2 (Rohm and Haas Co., Philadelphia, U.S.A.) was washed with 0.5 N NaOH, 0.5 N HCl, and suspended in
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deionized water. The resin of 130 mg dry weight was packed in a glass column, 0.6 cm in diameter and 15 cm in length, in the same way as described above. The flow rate of solutions through the columns was adjusted to approximately 0.3 ml/min by controlling the tightness of the polypropylene wool plug. Care was taken to prevent an airlock around the plug. The columns were washed with 3-5 volumes deionized water before use [ 111. Enzyme
assay procedures
MAO activity in platelets containing as little as 0.1 mg protein is, in practice, measurable and, in a typical assay, a platelet pellet obtained from 8 ml of blood is used for duplicate determinations. A thawed platelet pellet was agitated in 1.2 ml of physiological saline using a Pasteur pipette and made into a suspension which contained approximately 1 mg platelet protein. Duplicate samples of 0.6 ml were placed in polycarbonate centrifuge tubes with 0.2 ml of 0.3 M Na, HP04 /KHZ PO4 buffer, pH 7.7, and, after preincubation at 37°C for 5 minutes in the shaking water bath, the reaction was initiated by the addition of 0.2 ml of serotonin creatinine sulphate solution, 17 pmol/ml, adjusted to pH 7.7 with NaOH, as substrate. The incubation was carried out at 37°C for 30 minutes. The reaction was terminated by the addition of 2 ml of 0.3 N perchloric acid solution and placed in ice for 10 minutes in order to precipitate the protein. They were centrifuged at 12 000 X g at 0°C for 15 minutes. Supernatant was passed through the column of Sephadex G-10, followed by 27 ml of 0.02 N acetic acid solution for wash. After draining, each of the Sephadex G-10 columns was placed above a column of Amberlite CG-50, which was previously washed with 3 ml of 0.02 N NH4 OH solution containing 0.01% cysteine, so that the effluent from the former flowed into the latter. 5 ml of 0.02 N NH4 OH solution containing 0.01% cysteine was passed through the pairs of columns, so that adsorbed 5-HIAA tias eluted from the upper columns and through the lower ones while excessive serotonin was completely trapped on the Amberlite resin. 3 ml of eluate was then mixed with 1 ml of cone HCl containing 1% cysteine and determined spectrofluorometrically according to the method of Udenfriend et al. [12]. Fluorescence intensity was read at 540 nm while exciting at 295 nm on a Hitachi MPF-2A fluorescence spectrophotometer. A corection was made for the column blank value by performing the entire procedure without addition of incubated assay mixtures. Protein concentrations were measured by the method of Lowry et al. [ 131 using bovine serum albumin as standard. Enzyme activity has been expressed in nmoles of substrate per mg platelet protein per hour at 37°C and a pH value of 7.7. Subjects
The subjects were 32 depressed patients and 50 normal control subjects. The normal control subjects included staff physicians (N = 16), nurses (N = lo), nursing students (N = 8), attendants (N = 8) and healthy volunteers (N = 8) ranging in age from 20 to 66. The control values reported represented the mean of samplings on two different days at 2-3 week interval. The depressed patients were studied, since they may exhibit an abnormality of MAO activity in their platelets [3,10]. They were newly admitted
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patients at Kyoto Prefectural University Hospital, on a psychiatric ward. They consisted of 15 men and 17 women between the ages of 25 and 68 years. There were six patients with bipolar depression, 18 with unipolar depression and eight with involutional depression. The clinical characteristics of these patient groups have been described elsewhere [ 141. The diagnosis was established on the basis of interviews with the patient and his family and reports from previous physicians, as well as our own medical records. The bipolar patients were identified by their history of one or more typical manic episodes of sufficient severity to require hospitalization. Only samples from drug-free patients were included in this study, since the prominent effects of psychoactive drugs on monoamine metabolism in platelets may possibly induce some effects on MAO activity. Fasting blood samples for the platelet studies were obtained at 8:00 a.m. during the pretreatment period. No patients received drugs other than occasional sleep inducers, nitrazepam 5-10 mg, use of which was allowed in patients who complained of sleep disturbances. When the psychoactive drugs had already been given before admission, patients underwent a wash-out period of at least seven days before blood samples were drawn for determination of MAO activity. Two depressed patients were studied on several separate days before and during the administration of the MAO inhibitor nialamide, 100 mg daily. Results As shown in Fig. 1, MAO activity in human platelets, measured using serotonin as substrate, is most active at pH 7.7. The MAO activity measured at pH 7.7 is approximately 25% higher than at pH 7.4, the value selected in methods utilizing tryptamine or benzylamine as substrate previously described [1,2,15]. There may be substrate preference for the optimum pH, and when tryptamine is used as substrate V, ax is described to be 10% higher than the value with serotonin at pH 7.4 [2]. Nevertheless, we have obtained results of higher MAO activity by this procedure with serotonin (Table I) than those reported with radioisotopic tryptamine as substrate [ 3,5,9,16] . Care was taken
Fig. used
1. pH-activity for
the
final
curve volume
of monoamine of
9
8
7
6
1.0
ml
of
oxidase the
ph
in human
incubation
platelets.
mixture.
0.2
ml
of
0.3
M phosphate
buffer
was
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TABLE
I
MONOAMINE
Group
OXIDASE
No.
of
subjects
ACTIVITY
Age
IN HUMAN
PLATELETS
FROM
MAO
in years
NORMAL
activity*
___ Range
Mean
k S.D.
Range
Male
26
26-66
40.7
f 10.8
2.89-
Female
24
20-58
32.1
i
5.49-10.81
* nmoles/mg **
p