Life Sciences, Vol. 29, pp. 2511-2518 Pergamon Press Printed in the U.S.A. LOW PLATELET MONOAMINE OXIDASE ACTIVITY IN CIGARETTE SMOKERS Lars Oreland 1, Christopher J. Fowler 2 and Daisy Schalling 3. Department of Pharmacology, University of Ume~, S-901 87 Ume~, Sweden 1. Department of Biochemistry, Trinity College, Dublin 2, Ireland 2. Department of Psychiatry, Karolinska Hospital, S-104 01 Stockholm, Sweden 3. (Received in final form October 19, 1981) SUMMARY The activity of platelet monoamine oxidase was found to be significantly lower in normal female subjects who smoked at least 5 cigarettes per day than in non-smokers. The platelet MAO activity of individuals who had given up smok- ing was not significantly different from the activity for non-smokers. The difference in activities between smokers and non-smokers, due entirely to a Vma x rather than a K m change, was not due to a direct effect of nicotine upon the platelet MAO. In addition, platelet-poor plasma from smok- ers activated platelet MAO in an identical manner to that from non-smokers. The significance of these results are discussed in terms of personality characteristics such as impulsivity and sensation seeking, that may be related to both smoking and to low MAO activity. The activity of human platelet monoamine oxidase (MAO, monoamine 02: oxidoreductase, EC 1.4.3.4) has been suggested to be a marker for vulnerability to psychiatric disease (i). Low platelet MAO activities have been found in patients suffering from alcoholism (2-5), cycloid psychosis (6), schizophrenia with hallucinations (but not in schizophrenia without hallucinations) (7,8) and in patients with suicidal tendencies (9,10). However, in most of these studies, the patients were under long-term treatment with agents that may affect the rates of synthesis and degradation (and hence observed activities) of MAO. In normal healthy individuals, the range of platelet MAO activit- ies found are similar to those found in the various psychiatric groups. Since psychological disturbances have been reported in the families of subjects with low MAO activities (see e.g. I,ii), it has been suggested that low platelet MAO activity is not relat- ed to the psychiatric disorder per se, but rather to a constitut- ional vulnerability that may predispose individuals to undergo an acute breakdown in the presence of other factors, such as stress or somatic disease (1). Such a hypothesis is of considerable clinical importance, in view of the observation that platelet MAO activity is under genetic control (12,13), and has a high test- 0024-3205/81/242511-08502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
Transcript
Life Sciences, Vol. 29, pp. 2511-2518 Pergamon Press Printed in the U.S.A.
LOW PLATELET MONOAMINE OXIDASE ACTIVITY IN CIGARETTE SMOKERS
Lars O r e l a n d 1, C h r i s t o p h e r J . Fowle r 2 and Daisy S c h a l l i n g 3.
Department of Pharmacology, University of Ume~, S-901 87 Ume~, Sweden 1.
Department of Biochemistry, Trinity College, Dublin 2, Ireland 2.
Department of Psychiatry, Karolinska Hospital, S-104 01 Stockholm, Sweden 3.
(Received in final form October 19, 1981)
SUMMARY
The activity of platelet monoamine oxidase was found to be significantly lower in normal female subjects who smoked at least 5 cigarettes per day than in non-smokers. The platelet MAO activity of individuals who had given up smok- ing was not significantly different from the activity for non-smokers. The difference in activities between smokers and non-smokers, due entirely to a Vma x rather than a K m change, was not due to a direct effect of nicotine upon the platelet MAO. In addition, platelet-poor plasma from smok- ers activated platelet MAO in an identical manner to that from non-smokers. The significance of these results are discussed in terms of personality characteristics such as impulsivity and sensation seeking, that may be related to both smoking and to low MAO activity.
The activity of human platelet monoamine oxidase (MAO, monoamine 02: oxidoreductase, EC 1.4.3.4) has been suggested to be a marker for vulnerability to psychiatric disease (i). Low platelet MAO activities have been found in patients suffering from alcoholism (2-5), cycloid psychosis (6), schizophrenia with hallucinations (but not in schizophrenia without hallucinations) (7,8) and in patients with suicidal tendencies (9,10). However, in most of these studies, the patients were under long-term treatment with agents that may affect the rates of synthesis and degradation (and hence observed activities) of MAO.
In normal healthy individuals, the range of platelet MAO activit- ies found are similar to those found in the various psychiatric groups. Since psychological disturbances have been reported in the families of subjects with low MAO activities (see e.g. I,ii), it has been suggested that low platelet MAO activity is not relat- ed to the psychiatric disorder per se, but rather to a constitut- ional vulnerability that may predispose individuals to undergo an acute breakdown in the presence of other factors, such as stress or somatic disease (1). Such a hypothesis is of considerable clinical importance, in view of the observation that platelet MAO activity is under genetic control (12,13), and has a high test-
2512 Low Platelet MAO Activity in Smokers Vol. 29, No. 24, 1981
retest reliability (5,14).
A number of studies have demonstrated a correlation between platelet MAO activities and certain stable personality character- istics. A relationship has been found between low platelet MAO activity and high scores on the Zuckerman Sensation Seeking Scale (15-17). Correlations have also been found between low platelet MAO activity and the scales of Impulsivity and Monotony Avoidance (18,19). Platelet MAO activity of monkeys is also associated with a number of behavioural parameters (20).
One group of individuals who are often included in "healthy control" groups are those individuals who smoke, since this form of abuse is considered to be socially acceptable. However, many studies have shown that male heavy smokers tend to be highly impulsive and show sensation seeking behaviour, as judged from personality scales (21-23). It might therefore be expected that smokers have low platelet MAO activity. In the present study, platelet MAO activity has been measured in a group of female cigarette smokers and compared with the activities of non-smokers and of women who have given up smoking. In addition, experiments have been carried out to determine whether or not smoking has a direct effect on platelet MAO activity.
Methods.
Study 1
Samples of blood (5 ml), drawn into Vacutainer R tubes containing sodium citrate, were taken from 72 women who were all attending a nurses training course. They were asked about smoking habits by questionnaire. 'Smokers' were defined as those people who smoked at least 5 cigarettes per day. The red blood cells were allowed to self-sediment and the platelet concentration in the platelet-rich plasma counted by Coulter Counter. The plasma was then frozen. Before use, samples were sonicated to render them more homogeneous. Such a procedure has been found to disrupt virtually all the platelets (24), and, in the rat liver, is without effect upon the substrate-specificity or kinetic propert- ies of MAO (25). Samples were assayed for MAO activity in random order and single blind. The sample size was sufficient to study the platelet MAO activities in the 'smokers', 'non-smokers' and 'ex-smokers' separately, but it was not sufficiently large to determine whether or not there was any relationship between the platelet MAO activity and the number of cigarettes smoked per day.
Study 2
Samples of blood (450 ml) were also taken from three individuals, all of them non-smokers, and centrifuged at 200 g for 10 min to remove the red cells. The cells were washed twice with 0.15 M NaCI and the combined supernatants further centrifuged at 3,000 g for 15 min to produce platelet preparations, which were washed (by resuspension followed by recentrifugation) twice before being re- suspended in 0.25 M sucrose, 10 mM potassium phosphate, pH 7.8, at a protein concentration of 1.5 mg.ml -I. The platelets were frozen. Before each use, the platelet preparations were sonicated. Protein concentrations were determined by the method of Markwell et al. (26).
Vol. 29, No. 24, 1981 Low Platelet MAO Activity in Smokers 2513
Study 3
Samples of blood (5 ml) were taken from 6 non-smokers (4 female, 2 male) and 9 smokers (7 female, 2 male) and the red cells allowed to self-sediment. The platelet-rich plasma was then centrifuged at 3,000 g for 15 min and the supernatant ('platelet-poor plasma'), which had no more than 1.5% of the platelet concentration of the platelet-rich plasma, and no measurable MAO activity, was stored frozen. Before use, the platelet-poor plasma was sonicated.
Monoamine oxidase activity was assayed as described elsewher@4(27 ) l with 14C-tyramine, 14C-B-phenethylamine, 14C-tryptamine and C- dopamine (all obtained from New England Nuclear, Boston, MA, USA) as substrates. Unless otherwise specified, assays were carried out under an atmosphere of air with a substrate concentration of 50 ~M.
TABLE I
Platelet Monoamine Oxidase Activities in Smokers, Non-Smokers and Ex, Smokers.
Values are given as means + SD. Platelet MAO activities and plasma platelet concentrations were determined in duplicate for each sample.
i. Concentrations given as x 109 platelets.l -I
2. Activities given as nmol (of substrate metabolised). (109 platelets)-l.min -I.
**, p < 0.05; *, p < 0.06; otherwise not significantly different from values for non-smokers (two-tailed t-test).
Results and Discussion
Study 1
The activity of human platelet MAO was significantly lower in smokers than in non-smokers with all three substrates tested (Table I).
The results shown above have been confirmed in a separate study, with a different MAO assay method used (D. Schalling, unpublished
2514 Low Platelet MAO Activity in Smokers Vol. 29, No. 24, 1981
results). Coursey et al. (28) have also found that cigarette consumption was heavier among students with low MAO activity than among those with high MAO activity. Individuals who had given up smoking, however, had MAO activities close to the non-smokers (Table I). These differences in activity appeared to be due entirely to differences in the Vma x values of the MAO towards the substrate tested, with no difference in the K m values (Table II).
TABLE II
Kinetic Parameters of Platelet MAO Activities Towards Tryptamine in Non-Smokers, Smokers and Ex-Smokers.
K (~M) V (non-smoker mean m max
set to 100%)
Non-Smokers (n = 32) 2.1 + 1.0 i00 + 32
Smokers (n = 20) 2.2 + 1.1 77 + 17"
Ex-Smokers (n = ii) 2.0 + 1.2 91 + 30
K and V values were calculated from single determinations of .ma
M~O actlvl~y at 4 - 6 substrate concentrations (range 2.5 - 50 ~M~ The activities were plotted as S/v against S, and the K m and Vma x values calculated by linear regression analysis. In all cases, the correlation coefficients of the regression lines were higher than r = 0.91. Values in the table are given as means + SD.
*, p < 0.005, otherwise not significantly different from the non- smoker values (two-tailed t-test).
The low platelet MAO activity in the group of cigarette smokers could be explained in three ways:-
i. One of the components released into the blood when a cigarette is smoked acts directly upon platelet MAO, producing inhibition.
2. Chronic cigarette smoking results in an inhibition of synthesis of MAO, or else an increased breakdown of the enzyme, resulting in an apparent decreased enzyme activity.
3. Individuals with low platelet MAO activity are more prone to cigarette smoking.
The normal MAO values found in the group of ex-smokers, in con- trast to the low values found for the smokers (Table I), could be interpreted to mean that cigarette smoking produces a reversible inhibition of MAO. However, another possible explanation is that those smokers who were able to stop smoking were more casual in their habit, and do not belong to the impulsive sensation- seeking personality type, prone to low MAO. Recent experiments have suggested (D. Schalling, unpublished results) that ex-smokers tend to have higher scores in a neuroticism personality test than smokers.
Vol. 29, No. 24, 1981 Low Platelet MAO Activity in Smokers 2515
Study 2
In order to explore the first hypothesis suggested above, a study was performed to determine whether or not one of the constituents of cigarettes released into the blood might have an effect upon the platelet MAO. The effect of nicotine sulphate (obtained from Sigma Chemical Company, Saint Louis, Mo., USA), upon platelet MAO was determined. After 30 min of incubation between platelets and nicotine sulphate solutions at 37 ° , inhibition of MAO activity was observed (Fig. i). However, the concentration of nicotine required for 20% inhibition of platelet MAO activity (i.e. the concentration that would give the decrease in platelet MAO activity observed in Tables I and II), was about 5 x 10 -4 M, a concentration about 2,000 times higher than that found in the blood of heavy smokers. Thus, the low activity of MAO in platelets of smokers is not due to a direct inhibitory effect of nicotine upon this enzyme.
I00
~ 6 0
2 0
0
W l
5 X 10 -7 5 X 10 -5 5 X 10 -3
[Nicotine Sulphate] M
FIG. 1
The effect of nicotine sulphate upon the in vitro activity of human platelet MAO towards 50 pM tryptamine (~ and 50 ~M B- phenethylamine (O). Platelet preparations from non-smokers (pre- incubation protein concentration 0.75 mg.ml -I) were preincubated for 30 min at 37°C with the appropriate concentration of nicotine sulphate, and then incubated with substrate in the presence of the same concentration of nicotine sulphate (to ensure that both pre- incubation and incubation concentrations of nicotine were the same~ Abscissa: concentration of nicotine sulphate in M; ordinate, % activity of MAO remaining with respect to preparations preincubated for 30 min at 37°C with distilled water. All points represent means + S.D. of duplicate determinations in three platelet preparations.
Stud~ 3 An additional experiment was performed on the effects of smoking
2516 Low Platelet MAO Activity in Smokers Vol. 29, No. 24, 1981
on MAO activity. Platelet poor plasma from non-smokers and smokers was added to a preparation of platelets from a non-smoker. The addition of platelet-poor plasma produced, in each case, an activation of the MAO activity towards tyramine and dopamine, as reported previously (29). The activation, although dependent upon the substrate used to assay for activity, was identical for men and women (30), and.for smokers and non-smokers (Table III). These data are consistent with the notion that the plasma from smokers behaves towards MAO in a way identical to the plasma from non-smokers, and thus the low platelet MAO activity found in smokers is not due to a direct effect of the plasma upon the MAO activity.
Platelets are heterogeneous in nature, and different platelet populations with different specific MAO activities can be separ- ated by density gradient centrifugation (31). Thus, an effect of nicotine on platelet formation might give rise to the low platelet MAO found in this study. Although this seems unlikely, since the platelet counts of the smokers and non-smokers were the same (Table I), a more detailed study is necessary before such an explanation can be discarded.
TABLE III
The effect of the Addition of Platelet-Poor Plasma from Non-Smokers and Smokers upon the Activity of Human Platelet Monoamine Oxidase.
Non-Smokers Smokers
Number of samples 6 9
Age (years) 34 + 14 29 + 12
% Activation of p!atelet MAO
Tyramine 203 + 13 203 + 14
~-Phenethylamine 85 + 4 85 + 3
Dopamine 353 + 49 350 + 27
Tryptamine iii + 6 114 + i0
25 ~i platelet-poor plasma from non-smokers or smokers was added to 25 ~i platelet preparation (in 0.25 M sucrose, i0 mM potassium phosphate, pH 7.8, at a protein concentration of 1.5 mg.ml -I) and 50 ~i substrate (dissolved in 100 mM potassium phosphate, pH 7.8) and assayed for activity under an atmosphere of oxygen. For control samples, 25 ~i of 0.9% NaCI was added in place of the platelet-poor plasma, and the activation calculated as activity in presence of platelet-poor plasma/activity in absence of the platelet-poor plasma, and expressed as a percentage. None of the platelet-poor plasma samples had any detectable MAO activity. Values in the table are given as means + SD for duplicate deter- minations.
The data presented in this paper would suggest that the low MAO activity found in smokers, which has also been reported for
Vol. 29, No. 24, 1981 Low Platelet MAO Activity in Smokers 2517
chronic marijuana smokers (32), is due either to a long-term effect of one or more of the constituents of cigarettes upon the synthesis or breakdown of platelet MAO, or alternatively that individuals with low platelet MAO activity are more prone to smoking. The latter hypothesis is consistent with the higher impulsivity and sensation seeking scores often obtained in groups of smokers (21-23). The reverse is also true: in a recent study (33) there were significantly more smokers in a group of volunt- eers with high impulsivity scores (62%) than in a low impulsivity group (17%). However, it should be stressed that most earlier studies have been made with male subjects, whereas the present subjects were female.
Acknowledgements
The authors would like to thank Elizabeth Olofsson and Ingrid Persson for their expert technical assistance. This study was supported by grants from the Swedish Medical Research Council (04X-4145 and 04X-5733), and by grants from Frederick & Ingrid Thurings Stiftelse, and Magnus Bergvalls Stiftelse.
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