Department of Psychiatry, School of Medicine and
1 Department of Biochemistry, School of Pharmacy, Hacettepe University, Sihhiye, Ankara 06100, Turkey
Received 11 February 2002; in revised form 19 April 2002; accepted 27 May 2002
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ABSTRACT |
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INTRODUCTION |
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The platelet monoamine oxidase (MAO), an outer mitochondrial membrane enzyme which is responsible for the deamination of biogenic amines, has long been suggested as a possible marker for alcoholism, or for predisposition to it (von Knorring et al., 1985; Sullivan et al., 1990
; Devor et al., 1993
; Hallman et al., 1996
; von Knorring and Oreland, 1996
; Shih et al., 1999
). Of its two isozymes, with different substrate specifities and inhibitor sensitivities, the B form has traditionally been used as a model for brain MAO-B, since platelets share similar biochemical processes with neurons and their MAO-B activity appears to be related to central monoamine turnover (Georgotas et al., 1986
). It is known that the deduced amino acid sequences of MAO-B derived from human platelets and the brain frontal cortex are identical (Chen et al., 1993
).
There have been multiple reports that platelet MAO activity is decreased in alcohol-dependent subjects compared to controls (von Knorring et al., 1985, 1987
; Pandey et al., 1988
; Sullivan et al., 1990
; Hallman et al., 1996
). Lower platelet MAO activity has also been reported in the relatives of alcoholics (Schuckit et al., 1982
). Furthermore, some other studies indicating lower enzyme activity in alcohol-dependent subjects with affected relatives (Major and Murphy, 1978
; Alexopoulos et al., 1983
) than in alcoholics with no positive family history emphasized the notion that MAO might be a marker of genetic susceptibility to alcoholism. When alcoholics are classified by Cloningers model of alcoholism, lower platelet MAO activity is claimed to be a characteristic of the early-onset (type 2) group (von Knorring et al., 1985
; Pandey et al., 1988
; Hallman et al., 1996
). Taken together with the research data suggesting that lower platelet MAO levels are highly associated with impulsivity, sensation seeking and antisocial behaviour (Stalenheim et al., 1997
; Virkkunen and Linnoila, 1997
), this implies that type 2 alcoholics have different neurobiological and personality correlates. However, some other researchers reported that type 1 patients also displayed lower enzyme activity, when compared with healthy control subjects (Sullivan et al., 1990
). In contrast, there are also studies where no such association was observed between platelet MAO enzyme activity and type 1 or type 2 alcoholism (Parsian et al., 1995
; Anthenelli et al., 1998
; Farren et al., 1998
; Whitfield et al., 2000
). Furthermore, some recent reports raised concerns about the determinants of low MAO activity in alcoholics (Helander and Tabakoff, 1997
; Anthenelli et al., 1998
; Whitfield et al., 2000
). This last group of researchers suggested that low MAO activity might have been the result of tobacco use, rather than being associated with alcoholism.
In a parallel research field, an association between platelet MAO activity and outcome scores in some computerized neuropsychological tests, such as reaction time, number of failed inhibitions, and maze checking time, has been demonstrated (af Klinteberg et al., 1990). This finding implies that not only some personality traits, but also some neuropsychological skills, have a relationship with MAO activity. The nature of this relationship is still a fruitful area for further exploration.
The purpose of the present study was to examine a possible differentiated pattern of platelet MAO activity in patients with types 1 and 2 alcoholism classified on the basis of the age of onset of alcoholism. Another aim was to investigate whether platelet MAO activity is related to the psychopathological traits reflected by the Minnesota Multiphasic Personality Inventory-2 (MMPI-2) and the impairment in executive functions.
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SUBJECTS AND METHODS |
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Seventeen type 1 and 16 type 2 male chronic alcoholic patients were recruited for the study. The alcoholic subjects were classified according to the following criteria. The patients whose subjective alcohol problems had started before reaching 21 years of age (20) were categorized as type 2. For inclusion in the type 2 group, the reporting of at least two instances of social complications related to alcoholism before reaching the age of 21 was required (such as job loss, alcohol-related absence from school or work, arrest for intoxicated behaviour, or violence while intoxicated). Those who did not meet these criteria were categorized as type 1 dependent. The time when alcoholic patients satisfied the rule for the social complications of alcoholism was considered the beginning of their alcohol misuse. Patients with major psychiatric, neurological or medical illnesses, or conditions known or suspected to affect platelet MAO activity, and patients who were taking medications known or suspected to affect enzyme activity were not included in the study. In the type 2 group, ages ranged from 27 to 62 years (mean ± SD: 41.6 ± 9.6). In the type 1 group, ages ranged from 34 to 56 years (mean ± SD: 45.1 ± 6.3).
For the control group, 17 healthy males were recruited through local advertisements. They were selected on the basis of their similarity to the patients in terms of age, years of education and smoking status. Exclusion criteria included neurological, psychiatric and major medical illnesses, a history of alcohol/drug dependence and concurrent use of medications known or suspected to affect enzyme activity. Their ages ranged from 29 to 56 years (mean ± SD: 40.3 ± 8.4).
Personality traits were investigated by the restandardized version of the MMPI-2 (Butcher et al., 1989; Sava
ir and Çulha, 1996
). In addition to its clinical scales, MMPI-2 includes 15 content scales which enable clinicians to assess personality traits and major problem areas more comprehensively than its original version (Butcher and Williams, 1992
). Executive skills were investigated by the Wisconsin Card Sorting Test (WCST) (Grant and Berg, 1948
). Both WCST and MMPI-2 were administered to each patient 1521 days after their entry into the detoxification programme, when the withdrawal syndrome had ceased and all the psychotropic medications had been stopped for
1 week. Platelet samples were obtained on the fifteenth abstinent day of the alcoholic patients.
Platelet MAO measurement
Venous blood samples (10 ml) were collected in stoppered laboratory vacutainer tubes containing sodium citrate as anticoagulant. Platelet-rich plasma (PRP) was prepared by sequential centrifugation: blood was centrifuged at 200 g for 15 min at room temperature. The supernatant was aspirated and recentrifuged at 1200 g for 10 min. The supernatant was kept and the pellet was resuspended in 0.5 ml of Tris buffer (15 mM TrisHCl, 140 mM NaCl, 10 mM EDTA), pH 7.4, and centrifuged at 10 000 g for 10 min. The supernatants were pooled. Platelet counts were determined on aliquots of pooled PRP diluted in Isoton II and counted twice on a Coulter Thrombocounter. All of the PRP samples contained 1.5 x 108 to 3 x 108 platelets/ml.
Platelet MAO-B activity was determined according to the method of Holt et al. (1997) by using benzylamine as substrate. In short, the assay mixture contained a chromogenic solution which consisted of 1 mM vanillic acid, 500 mM 4-aminoantipyrine, 4 U/l peroxidase in 0.2 M potassium phosphate buffer, pH 7.6 and 450 mM benzylamine. The mixture was preincubated at 37°C for 15 min. A reaction was initiated by the addition of PRP samples and the increase in absorbance was monitored at 498 nm, at 37°C for 1 h. The molar absorption coefficient was 4654 M1 cm1. Results were expressed as nmol/108 platelets/h.
All subjects provided written informed consent for participation in the study, which was approved by the local ethics committee (99-7).
Data analysis
Platelet MAO levels, MMPI-2 and WCST scores of the study groups were compared by KruskalWallis analysis of variance. Significant differences were followed up with two-way comparisons using the MannWhitney U-test. For dichotomous variables, the differences between the groups were analysed by the 2-analysis. Possible relationships between platelet MAO levels, MMPI-2 subscale scores, neuropsychological measures and some clinical variables, such as the lifetime severity of alcohol consumption, were assessed by the Pearson product-moment correlation analysis. P > 0.05 was considered non-significant.
MMPI-2 scores were represented as standardized T scores. Their statistical analysis included only those subjects for whom valid responses (L <65, F <90 and K <65) were available.
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RESULTS |
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Correlation between platelet MAO activity, MMPI-2 scores, and neuropsychological measures
Of the clinical variables, the duration of misuse/dependence and the lifetime severity of alcohol dependency scores did not correlate with MAO enzyme activity. Additional analysis between enzyme activity, neurocognitive measures and MMPI-2 scores revealed no significant relationships.
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DISCUSSION |
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From the personality standpoint, type 2 alcoholism has been linked to high scores on sensation seeking (von Knorring et al., 1985, 1987
; Schalling et al., 1987
). Moreover, the correlation between platelet MAO and extroversion- and impulsiveness-related personality traits has been confirmed in numerous studies using a variety of personality inventories (von Knorring et al., 1987
; Hallman et al., 1996
). In addition to these findings, our results showed that type 2 alcoholics tend to have higher scores on the various scales of MMPI-2, which signifies a more severe psychopathology than that of type 1 individuals. Higher scores on F, Psychopathic Deviancy, Schizophrenia and Psychasthenia are among the well-verified MMPI characteristics of alcoholic populations (Graham and Strenger, 1988
). Additionally, our results showed that type 2 individuals have higher scores on the Bizarre Mentation and Anger content scales. The Bizarre Mentation scale was developed to assess psychotic-like thought processes, while Anger is a measure of loss of control in anger expression (Butcher et al., 1990
). Considering the fact that, in the MMPI-2 scales, higher scores indicate a more severe pathology, these findings provide evidence that type 2 alcoholics are more inclined to have thought disturbance such as paranoid ideation and problems in anger expression. Furthermore, our results also suggest that platelet MAO activity is not correlated with any personality trait as assessed by the MMPI-2.
From the neuropsychological standpoint, our results showed that, when compared with healthy subjects, both alcoholic groups had more perseverative responses and errors. Furthermore, unlike type 1 patients, type 2 alcoholics completed a lower number of categories and made more attempts to complete the first category. Collectively, these findings indicate that there is a trend towards a more severe impairment in the type 2 group in frontal lobe skills, such as concept formation or planning. However, in contrast to the findings of a previous study (af Klinteberg et al., 1990), our results suggest that platelet MAO activity is not related to performance scores in executive skills.
As mentioned above, a general view of platelet MAO studies of alcoholic patients in the literature showed some inconsistencies between findings. A number of confounding factors might be responsible for the discrepancies in such findings. A major factor would be the diversity in the definitions of alcoholic subtypes (Lamparski et al., 1991; Anthenelli et al., 1994
). Alternatively, the relative percentage of patients who have antisocial personality disorder in the alcoholic subgroups might partially be responsible for the discrepancies in findings between studies; however, Anthenelli et al. (1995)
failed to demonstrate any difference in MAO activity between the primary antisocial personality disorder with secondary alcoholism and primary alcoholism subgroups.
Another confounding variable that might jeopardize the interpretation of MAO activity in alcoholics is the smoking status. The inhibiting effect of smoking on MAO activity has been confirmed in studies both on males and females (Norman et al., 1987; Yu and Boulton, 1987
; Fowler et al., 1996
). Furthermore, the findings of some recent reports strongly indicate that decreased platelet MAO activity is not a trait marker of alcoholism, rather it is a state marker of cigarette smoking (Anthenelli et al., 1998
; Whitfield et al., 2000
). From this perspective, literature data suggesting low platelet MAO enzyme activity in alcoholics appeared to be an artefact of cigarette smoking. Thus, any attempt aimed at evaluating platelet MAO activity in alcohol dependence should take into consideration the smoking variable. Smoking status, defined as regular smoking or not smoking, was one of the variables considered in our study. However, we did not collect information on the quantity, frequency and duration of smoking for each individual. Such an attempt would give us more accurate data.
A major limitation of this study is the relatively small number of subjects in each group, which may reduce the validity of the findings. This is especially true when interpreting the MMPI-2 and WCST data. Another methodological limitation of our study is related to the timing of the blood samples taken from alcoholic patients. A number of studies in the literature consistently reported that platelet MAO-B activity tends to increase transiently during the early weeks of abstinence (Alexopoulos et al., 1981; Major et al., 1981
; Berggren et al., 2000
). According to these studies, the preferential time of taking blood samples should be somewhere around 2 months after cessation of alcohol consumption. Due to the fact that a considerable percentage of alcoholic patients cannot maintain sobriety for 2 months, Berggren et al. (2000)
suggested that 1 week after the end of alcohol intake might be used instead, as they did not observe any significant difference between week 1 and 8 values. In the present study, the blood samples were taken on the fifteenth day of abstinence on average. This might have concealed differences regarding enzyme activity between the study groups. Nevertheless, our findings still demonstrated significant differences in MAO enzyme activity between the groups.
In conclusion, our results confirm previous evidence that platelet MAO activity is a useful measure for the subtyping of alcoholics. They also support the notion that the age at onset of alcoholism symptoms may delineate subtypes of alcoholics with significant neurobiological differences.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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