PLATELET MONOAMINE OXIDASE B ACTIVITY AS A STATE MARKER FOR ALCOHOLISM: TREND OVER TIME DURING WITHDRAWAL AND INFLUENCE OF SMOKING AND GENDER

Teresa Coccini1,*, Anna F. Castoldi1, Cristiano Gandini1, Giovanna Randine1, Giovanni Vittadini2, Paola Baiardi3 and Luigi Manzo1,4

1 Toxicology Division,
2 Alcoholism Treatment Division and
3 Biostatistics Unit, IRCCS Salvatore Maugeri Foundation, Institute of Pavia and
4 Department of Internal Medicine and Therapeutics, University of Pavia, Italy

Received 13 November 2001; in revised form 1 March 2002; accepted 20 March 2002


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Aims and methods: The present study evaluated time-related changes in platelet monoamine oxidase B (MAO-B) activity in an Italian cohort of alcohol-dependent subjects (n = 98) during early abstinence, and the effect of potential confounding factors, such as gender and smoking status, on the temporal trend of the enzyme activity. Results: While still under the influence of ethanol (time point T1), the mean value of platelet MAO-B activity in alcoholics was 6.4 ± 3.1 nmol/mg of protein/h. This increased by >40% (to 9.3 ± 4.4 nmol/mg of protein/h) after 8 days of withdrawal (T2), and remained stable thereafter (T3 and T4: 15 and 22 days of abstinence, respectively). In a cohort of 138 healthy subjects, MAO activity levels averaged 9.9 ± 0.9 nmol/mg of protein/h. In the group of alcoholic patients, alcohol intake cessation was confirmed by the progressive decrease of serum % carbohydrate-deficient transferrin (CDT), which was pathologically above the reference limits (6%) at T1 (7.8 ± 3.3%), declined to 6.6 ± 2.1% at T2 and reached physiological values at T3 and T4. In a subgroup of cirrhotic alcoholics, %CDT did not decrease over time, while MAO activity rose after the first week of abstinence, without further change at T3 and T4. During early withdrawal, neither gender nor tobacco smoking affected the temporal pattern of MAO activity. Conclusions: MAO-B can be regarded as a state marker of alcohol consumption. The temporal pattern of platelet MAO-B activity may be used for the diagnostic assessment of alcoholism and early abstinence, regardless of gender and smoking status.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Alcohol consumption has been steadily rising in Western countries, with a parallel increase in the prevalence of alcohol-related diseases. Despite its diffusion, alcohol misuse remains a condition frequently overlooked for several reasons. First, the reliability of personal interviews about alcohol consumption is limited, since alcohol misusers tend to underestimate, or even deny, their drinking. Moreover, the clinical evaluation is rarely useful for the diagnosis, since clinical signs are rather minimal in the early phase of alcohol misuse (Sillanaukee, 1996Go). Furthermore, the commonly used laboratory markers of alcohol misuse, such as serum {gamma}-glutamyltransferase (GGT), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the erythrocyte mean cell volume (MCV), have limited diagnostic accuracy (Cushman et al., 1984Go; Skinner et al., 1984Go; Conigrave et al., 1995Go; Sharpe et al., 1996Go). Although serum carbohydrate-deficient transferrin (CDT) is currently considered a reliable and specific marker for recent excessive alcohol consumption, the ability of this test to reflect cessation of alcohol intake may be reduced in patients affected by certain liver diseases, such as primary biliary cirrhosis, liver cirrhosis and chronic active hepatitis (Stibler and Hultcrantz, 1987Go; Murawaki et al., 1997Go). The intrinsic limitations of these markers have intensified the search for other tests of alcohol misuse, including those reflecting changes in central neurotransmission. One of the tools proposed as a trait marker of alcohol dependence is the enzyme monoamine oxidase type B (MAO-B) in platelets (Farren and Tipton, 1999Go). Monoamine oxidase (MAO; EC 1.4.3.4) is a mitochondrial flavoprotein that catalyses the oxidative deamination of primary amines, including the naturally occurring catecholamines and indolamines, in central presynaptic nerve endings. The enzyme exists in two forms, designated A and B, distinguished by their substrate specificity, inhibitor sensitivity and tissue distribution (Suarez et al., 1995Go). MAO-B is the sole type in human platelets and the primary type in the human brain. The amino acid sequences of MAO-B in both platelets and brain are identical (Chen et al., 1993Go) and the biochemical and pharmacological characteristics of the isoenzyme in the two tissues are also similar (Donnelly and Murphy, 1977Go). For these reasons, platelet MAO has been adopted as a useful surrogate model for the study of aspects of central neuronal function, and it has been extensively investigated in neurological and psychiatric disorders (Wyatt et al., 1973Go; Fowler et al., 1982Go; Whitfield et al., 2000Go). Particular interest in using platelet MAO-B as a peripheral marker of central MAO activity in alcoholism was evoked by the finding of reduced MAO activity in post-mortem brains of alcoholic suicides, relative to non-alcoholic controls (Gottfries et al., 1975Go). Since then, low platelet MAO activity has been widely studied as a trait marker for alcoholism (see Farren and Tipton, 1999Go for review), especially for ‘type 2’, which is associated with early age of onset, a positive family history of alcoholism and personality traits, such as monotony avoidance, impulsiveness, and sensation-seeking behaviour (von Knorring et al., 1985Go; Pandey et al., 1988Go; Sullivan et al., 1990Go; Devor et al., 1993Go). Several investigators, however, failed to find differences between alcoholics and normal controls and/or between the alcoholic subtypes (Tabakoff et al., 1988Go; Anthenelli et al., 1998Go; Farren et al., 1998Go; Farren and Tipton, 1999Go). Indeed, many other variables, some of which are correlated with alcoholism, appear to be associated with platelet MAO activity. For example, one controversial issue relates to the stability of the enzyme activity level during abstinence. State-dependent fluctuations of platelet MAO-B activity during withdrawal have been reported in a few studies performed on a limited number of alcoholics (Takahashi et al., 1976Go; Wiberg, 1979Go; Alexopoulos et al., 1981Go; Major et al., 1981Go; Anthenelli et al., 1995Go; Berggren et al., 2000bGo). Besides alcoholism, several intrinsic and extrinsic factors have been suggested to contribute to differences in platelet MAO-B activities (Anthenelli et al., 1998Go). These include gender and race (Robinson, 1980Go), psychiatric (Fowler et al., 1982Go) and other medical illnesses (Sandler et al., 1981Go), metabolic factors (Sullivan et al., 1980Go), personality traits (von Knorring et al., 1984Go), tobacco smoking (Anthenelli et al., 1995Go, 1998Go; Whitfield et al., 2000Go), medications (Fowler et al., 1982Go) and drug misuse (Faraj et al., 1994Go).

The aims of the present study were to evaluate whether: (1) there were time-related changes in platelet MAO-B activity levels in an Italian cohort of alcohol-dependent subjects during early abstinence; (2) whether potential confounding factors, such as gender, smoking status and liver cirrhosis, influence the temporal pattern of this enzyme activity.


    SUBJECTS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Subjects
The control group consisted of 138 subjects between the ages of 18 and 66 years (mean ± SD: 40 ± 13) enrolled from a blood donor centre of the IRCCS Policlinico S. Matteo Hospital, Pavia, Italy. The mean age ± SD of males (n = 95) was 42 ± 13 years, whereas that of females (n = 43) was 35 ± 12 years. All individuals were ascertained not to be alcohol or drug dependent by direct interview. Laboratory tests were also carried out to rule out liver, renal or metabolic disturbances. The smoking status of the controls was not recorded.

The 98 alcoholic patients were admitted to the Alcoholism Treatment Division of the IRCCS Salvatore Maugeri Foundation Medical Centre, Pavia, Italy for detoxification. The sample (mean age: 46 ± 10 years; range: 26–75) consisted of 69 men (mean age: 46 ± 10 years; range: 27–75) and 29 women (mean age: 45 ± 10 years; range: 26–69) and met the DSM-IV criteria for the diagnosis of alcoholism (American Psychiatric Association, 1994Go). The subjects were not classified as type 1 or type 2 alcoholics according to Cloninger. They had an average length of alcoholism of 16 ± 12 years and all of them reported a daily alcohol consumption level of >80 g of pure ethanol. When admitted for in-patient rehabilitation, they had been abstinent for 0–1 day. All patients were given diazepam in decreasing doses during the first week of hospitalization to prevent withdrawal symptoms, as well as vitamins. Seventy-six out of 98 alcohol-dependent individuals (78%) were current smokers (mean ± SD: 28.5 ± 15 cigarettes/day). Thirty-thee were affected by hepatomegaly, 42 by steatosis, 20 by chronic hepatopathic disease, 22 by chronic persistent hepatitis, five by chronic active hepatitis and six by liver cirrhosis.

On the day of hospitalization, all subjects underwent a thorough physical examination and underwent clinical laboratory tests, including liver-function tests, haematological indices and routine urine examinations. At the same time point (T1: under the influence of alcohol), as well as once weekly during the 3-week hospitalization (T2, T3 and T4: 8, 15 and 22 days of withdrawal, respectively), blood was drawn to determine serum %CDT and platelet MAO-B activity. The routine blood tests were repeated before hospital discharge. To ensure the patients’ sobriety, the alcoholics were carefully supervised by members of staff. Informed consent was obtained from all subjects before enrolment in the study.

Determination of platelet MAO-B activity
Blood (5 ml) was collected in EDTA tubes and centrifuged (100 g for 10 min) to obtain platelet-rich plasma (PRP). After the addition of 10% dimethylsulphoxide (v/v), PRP was frozen at –80°C for 24 h and thereafter stored in liquid nitrogen. On the day of the analysis, PRP was thawed and the platelets were isolated by centrifugation at 500 g for 10 min, resuspended in Na+/K+ phosphate buffer (5 mM), pH 7.4, and counted by Coulter Counter (Instrumentations Laboratory). Following centrifugation at 16 000 g for 10 min, the pellet was resuspended in the same Na+/K+ phosphate buffer supplemented with 5% bovine serum albumin, 10 mM dithiothreitol and 2.5 mM EDTA, and was homogenized for 20 s, and further diluted with buffer to a concentration of 20 x 106 platelets/0.4 ml.

The activity of MAO-B was determined radiochemically as described by Young et al.(1986)Go using 10 µM [14C]PEA (ethyl-1-14C-phenylethylamine hydrochloride, 41.8 mCi/mmol; Du Pont de Nemours, Florence, Italy) as the substrate. Each assay was run in duplicate. Specific MAO-B activity was determined in the presence of 100 µM pargyline hydrochloride (Sigma–Aldrich, Milan, Italy). The reaction mixture contained 0.4 ml of tissue homogenate in a final assay volume of 1 ml. The reaction was started by addition of the labelled substrate and stopped by adding 0.25 ml of 4 M HCl after a 10-min incubation at 37°C. Deaminated reaction products were toluene-extracted and the radioactivity contained in a 1-ml aliquot of the organic phase was counted in a liquid scintillator counter (Packard 1900 CA). Protein content was determined by the method of Lowry et al.(1951)Go and the activity of the enzyme was expressed as nmol/mg of protein/h. The mean platelet protein concentrations in platelet homogenate were essentially the same for the controls (0.187 ± 0.029 mg/ml) and the alcoholics (0.186 ± 0.023 mg/ml).

Internal quality control of MAO-B activity
As a measure of quality assurance, MAO-B activity was determined in a platelet preparation obtained from pooled human PRP (stored at –80°C). To build a reference chart, the pooled sample was analysed in 20 different analytical series, and the average values and limits (3 SD) were reported on a plot. This standard platelet MAO preparation was assayed with each group of samples and the reference results were recorded on the chart and then appraised as to their position in comparison with the chosen limits defined by ± SD.

CDT assay
Serum CDT determinations were carried out in duplicates by the commercially available test kit %CDT turbidimetric immunoassay (Biorad Laboratories S.r.l., Milan, Italy). This method is based on the separation of transferrin isoforms on an ion-exchange chromatography column followed by the reaction with the anti-transferrin antibody and turbidimetric measurement. The cut-off limit was that recommended by the manufacturer (6%).

Conventional markers of alcohol misuse
Liver function parameters (serum AST, ALT, GGT) and MCV were determined by routine clinical chemistry methods according to the recommendations of the European Committee for Clinical Laboratory Standards. Cut-off levels were: AST >38 (males) and 32 (females) U/l; ALT >41 (males) and 31 (females) U/l; GGT >50 (males) and 32 (females) U/l; MCV >91 fl.

Statistical analysis
All analyses were carried out by means of the SAS/STAT Guide for Personal Computers (SAS Institute, 1989Go). Descriptive statistics (mean ± SD) were performed on the whole sample and according to subgroups identified by gender and smoking status. A repeated-measures analysis of variance (ANOVA) was carried out to determine differences over time for all the recorded variables. For MAO evaluation, an ANOVA with one or two factors (gender, smoking status or both) was performed to investigate different trends over time between males and females, smokers and non-smokers. Differences in MAO activity between male and female controls were assessed using an unpaired t-test.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The mean platelet MAO-B activity among controls (n = 138) was 9.9 ± 0.9 nmol/mg of protein/h (range 1.8–33.3; coefficient of variation: 9.15%). Within the control group, females (n = 43) displayed a statistically significantly higher enzyme activity than males (n = 95), as the mean value of platelet MAO activity was 12.4 ± 0.9 in the former, and 8.8 ± 0.9 nmol/mg of protein/h in the latter, gender subgroup (P < 0.001).

Figure 1Go shows the temporal pattern of platelet MAO-B activity in the whole sample of alcoholic subjects during early withdrawal. Upon hospital admission (T1), the mean value of platelet MAO-B activity in these patients was 6.4 ± 3.1 nmol/mg of protein/h (Fig. 1Go). Values were >40% higher (9.3 ± 4.4 nmol/mg of protein/h) after 1 week of withdrawal (T2), and remained stable after 15 (T3) and 22 days (T4) of abstinence (9.9 ± 4.8 and 9.7 ± 4.6 nmol/mg of protein/h respectively). In parallel, serum %CDT mean levels decreased over time, confirming ethanol withdrawal. %CDT, which was pathologically above the cut-off limit at T1 (Table 1Go), decreased to 6.6 ± 2.1 and 5.9 ± 1.7%, respectively, 8 and 15 days after, and was in the physiological range at T4 (Table 1Go).



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Fig. 1. Temporal pattern of platelet monoamine oxidase B (MAO-B) activity in 98 chronic alcoholic patients undergoing in-patient detoxification for alcoholism. Serial determinations of platelet MAO-B activity were performed on blood samples drawn on the day of hospital admission (T1), as well as after 8 (T2), 15 (T3) and 22 days (T4) of hospitalization. Enzyme activity is expressed as nmol of product/mg of protein/h and the data represent the mean ± SD (bars). The hatched line represents the mean value of platelet MAO-B activity in healthy male and female controls (n = 138). *P < 0.005, as compared to T1. Compared to the control value, platelet MAO-B activity was significantly lower (P < 0.001) at T1.

 

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Table 1. Values of laboratory markers of alcohol misuse in 98 alcohol-dependent patients undergoing detoxification at the beginning and end of the treatment
 
The mean values of conventional laboratory markers of alcohol misuse at patients’ admission and discharge are reported in Table 1Go. AST, ALT and GGT were elevated at T1, significantly decreased during hospitalization, but still remained above the reference values at the patients’ discharge. MCV values, which exceeded the normal limits at T1, did not significantly change at T4 (Table 1Go).

One of the primary objectives of this study was to examine the influence of gender (Fig. 2aGo) and tobacco smoking (Fig. 2bGo) on the temporal pattern of MAO-B activity in early abstinent alcoholics. Among alcoholics, female subjects (n = 29) displayed higher platelet MAO activity than their male counterparts (n = 69) at all time points, but statistically significant differences were detected only at T4 (11.9 ± 4.7 vs 9.0 ± 4.4 nmol/mg of protein/h, respectively). The temporal pattern of platelet MAO-B activity did not differ between males and females, as evaluated by repeated-measures ANOVA with one factor (gender) (Fig. 2aGo). When the subjects overall were divided according to smoking status, there were no differences between smokers and non-smokers at the single time points as well as in the temporal pattern. Irrespective of the smoking status, the platelet enzyme activity significantly rose after the first week of alcohol intake cessation and remained stable thereafter (Fig. 2bGo). In view of the disproportionate representation of males and females among the smoker (79% males and 21% females) and non-smoker (41% males and 59% females) alcoholic cohorts, the temporal trend was analysed by ANOVA with two factors (gender and smoking). Once again, no different trends were detected between male smokers and non-smokers and between female smokers and non-smokers.



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Fig. 2. Influence of gender (a) and cigarette smoking (b) on the temporal pattern of platelet monoamine oxidase B (MAO-B) activity in chronic alcoholic patients undergoing in-patient detoxification for alcoholism. Serial determinations of platelet MAO-B activity were performed on blood samples drawn on the day of hospital admission (T1), as well as after 8 (T2), 15 (T3) and 22 days (T4) of hospitalization. Enzyme activity is expressed as nmol of product/mg of protein/h and the data represent the means ± SD (bars). In (a), data from 69 ({blacksquare}) males and 29 females ({blacklozenge}) are presented. The two horizontal lines represent the reference values of platelet MAO-B activity in healthy males (solid line; n = 95) and females (dashed line; n = 43), respectively. In (b), results from 76 smokers (•) and 22 non-smokers ({blacksquare}) are shown. The horizontal line represents the mean reference value of platelet MAO-B activity in healthy controls (n = 138). Compared to the control value, platelet MAO-B activity was significantly lower (P < 0.001) at T1.

 
The results of serial measurements of %CDT and platelet MAO-B activity in a subgroup of six alcohol-dependent subjects affected by liver cirrhosis are reported in Table 2Go. Despite alcohol abstinence, %CDT values were always above the 6% cut-off level, while MAO activity was significantly increased at T2 as compared with T1, and did not further change at T3 and T4 (Table 2Go).


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Table 2. Values of serum % carbohydrate-deficient transferrin (CDT) and platelet monoamine oxidase B (MAO-B) activity in six alcoholic patients affected by hepatic cirrhosis at different time points during alcohol withdrawal
 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The present study shows that platelet MAO-B activity underlies state-dependent changes in recently abstinent alcohol-dependent subjects and that potential confounding factors such as gender, smoking status and liver cirrhosis do not influence this temporal trend. On acute alcohol withdrawal, namely at time point T1, the alcoholic patients displayed a significantly lower platelet MAO-B activity than after 8 days of abstinence (T2). The enzyme activity did not further change at least up to the day of hospital discharge (3 weeks of abstinence). Alcohol abstinence was indeed confirmed in this cohort of alcoholic patients by the progressive decrease in serum %CDT (with the exception of those affected by liver cirrhosis), measured at the same time points as MAO-B, as well as by the reduced levels of the other laboratory markers of alcohol misuse (AST, ALT and GGT) at T4 as compared to T1.

To date, temporal fluctuations in platelet MAO-B activity have been previously reported in alcohol-dependent subjects during the first weeks of alcohol withdrawal. Wiberg (1979)Go and Major et al.(1981)Go showed that platelet MAO activity significantly increased during the first few days of alcohol withdrawal, with a gradual return to the initial lower levels associated with abusive drinking over the next 3–4 weeks. Takahashi et al.(1976)Go reported that, 4 weeks after withdrawal of alcohol, the reduced MAO activity in the alcoholic population was restored to normal levels. Rommelspacher et al.(1994)Go found that, on day 1 of abstinence, alcoholics had a lower enzyme activity than sober controls, while 7 days, 3 and 6 months later, MAO activity did not differ from that of non-alcoholics. More recently, Berggren et al. (2000b)Go reported a transient increase in the platelet MAO-B activity of 13 male alcoholics from 2 to 6 weeks after the end of alcohol intake, while the enzyme activity was significantly lower in the alcohol-dependent subjects as compared to controls at 1 week and 2 months after the end of alcohol intake.

Our data agree with those of Wiberg (1979)Go, Major et al.(1981)Go, Rommelspacher et al.(1994)Go and Berggren et al. (2000b)Go with regards to the early increases in platelet MAO activity during withdrawal, namely peaks are detected as early as after 1–2 weeks of alcohol cessation. Moreover, our results extend those of the other studies, in that: (1) the present investigation was performed on a larger group of alcoholic patients; (2) the same temporal pattern was observed in males and females, in smokers and non-smokers, as well as in cirrhotic and non-cirrhotic patients.

The reason why the alcoholics display a lower enzyme activity when still under the influence of alcohol (T1) is at present unclear. Ethanol itself has been found to inhibit platelet MAO activity in vitro and even more markedly in alcoholics (Tabakoff et al., 1988Go). Rommelspacher et al.(1994)Go hypothesized that the low platelet MAO activity does not result from the actual acute ingestion of alcohol, but rather from chronic changes, as supported by the finding that platelet MAO-B activity was unchanged in a group of controls following an ethanol load (Rommelspacher et al., 1994Go). Since in our study mean platelet protein concentration was found to be essentially the same for the control and alcoholic groups, this other possible explanation for the initially low levels can be excluded.

Whether low peripheral MAO-B activity mirrors an impairment of central brain MAO-B activity is a matter of debate. Brain and platelet MAO-B activity have been found to be highly correlated by means of positron emission tomography (Bench et al., 1991Go), whereas no correlation was found in another study (Young et al., 1986Go). Platelet MAO-B activity has been proposed as a marker of dopamine function in alcoholics: recently, alcohol-dependent patients with decreased dopamine D2 receptor function (assessed by the growth hormone response to apomorphine) were shown to have significantly lower platelet MAO-B activity in comparison to those patients with normal D2 receptor function (Berggren et al., 2000aGo). According to these latter authors, the reduced MAO activity in the presynaptic neuron would result in an increased availability of the neurotransmitter at the synapse and would therefore represent a compensatory mechanism to overcome the down-regulated postsynaptic D2 receptor function (Berggren et al., 2000aGo). Although this hypothesis is intriguing, another study failed to find evidence for altered MAO-B activity in alcoholics with reduced dopamine D2 receptor function (Farren and Dinan, 1996Go).

The early increase in platelet MAO-B activity during abstinence has been proposed to stem from increased adrenergic activity during acute alcohol withdrawal or to thrombocytosis which often occurs on cessation of drinking to rectify thrombocytopenia, a common consequence of alcoholism: the newly formed platelets are larger and thus contain more MAO-B activity when expressed per platelet (Major et al., 1981Go; Ferguson and Goldberg, 1997Go). The latter hypothesis could also apply to our study, since most of our patients showed a significant increase in the number of platelets at T4, as compared to T1 (data not shown).

Irrespective of the mechanism(s) involved in the changes in platelet MAO-B activity during withdrawal, our results demonstrate that this proposed trait marker also underlies state-dependent changes in alcoholic patients. It is noteworthy that platelet MAO-B activity levels did not significantly differ between controls and abstinent alcoholics at time points T2, T3 and T4. Therefore, the time point at which a blood sample is drawn for the determination of MAO-B activity is critical for the appropriate diagnosis of alcoholism. This is further supported by the disagreement of literature data in terms of stability of the enzyme activity during prolonged periods of abstinence (Takahashi et al., 1976Go; Brown, 1977Go; Major and Murphy, 1978Go; Sullivan et al., 1978Go; Wiberg, 1979Go; Tabakoff et al., 1988Go; Rommelspacher et al., 1994Go; Suarez et al., 1995Go). For example, some investigators reported that the early increase in MAO activity detected during the first days of alcohol withdrawal is then followed by a decrease after periods of abstinence longer than 4 (Wiberg, 1979Go; Major et al., 1981Go; Anthenelli et al., 1995Go), 6 (Alexopoulos et al., 1981Go) and 8 weeks (Berggren et al., 2000bGo), respectively. Other researchers, however, did not detect significant differences in MAO activity between controls and alcoholics, when the latter had abstained from alcohol for more than 3 weeks (mean: 16.7 weeks; Farren et al., 1998Go), 3 months (Rommelspacher et al., 1994Go), 6 months (Brown, 1977Go; Rommelspacher et al., 1994Go) or 12 months (Tabakoff et al., 1988Go). Altogether, these results indicate that the length of the abstinence period can markedly influence the results. Nevertheless, our finding indicating that platelet MAO-B in alcoholics increased within 1 week of alcohol withdrawal points to its potential value as an early clinical marker of alcohol cessation. Compared to MAO-B, serum CDT levels normalized more slowly during abstinence, accordingly with its biological half-life of 2 weeks: at T2 %CDT levels were still in the pathological range. Although evaluations of the other laboratory markers of alcohol misuse were performed only at T1 and T4, it is known that GGT, AST and ALT have a biological half-life of 2–3 weeks, whereas MCV of ~3 months (Sillanaukee, 1996Go). On the basis of our data, serial determinations of MAO activity may be successfully applied to monitor the early phases of alcohol detoxification.

Alcoholism is a very complex syndrome, which cannot be characterized by just using a single clinical or biochemical diagnostic tool. Indeed, the integrated and continuous use of several markers of diagnosis is needed for an appropriate evaluation. Moreover, there is a lack of an objective gold standard by which the pathology would be defined and alcoholics are commonly diagnosed using the DSM-IV criteria. Thus, valuable state markers have the potential to assist physicians with an appropriate diagnosis of current alcohol use and to help monitor abstinence in response to treatment. Differently from the conventional biochemical tests of alcohol misuse, platelet MAO-B activity is not a marker of hepatic damage. Accordingly, the temporal trend of this enzyme activity during withdrawal in cirrhotic patients did not differ from that of abstinent alcoholics. On the other hand, despite alcohol abstinence, serum CDT did not decrease over time in the few patients affected by alcoholic cirrhosis.

Another important finding of the present study is that neither gender nor tobacco smoking affected the pattern of platelet MAO-B activity over time during early withdrawal. This is of particular interest, in that it extends the applicability of this test as a marker of recent alcohol consumption in the clinical setting. With regards to gender, men had lower platelet MAO activity than women within both the control and alcoholic groups. Gender-related differences in MAO activity are in agreement with previous reports (Takahashi et al., 1976Go; Alexopoulos et al., 1981Go; Meltzer and Zureick, 1987Go; Faraj et al., 1994Go; Anthenelli et al., 1998Go).

In our alcoholic population, no significant differences were detected at single time points as well as in the temporal trend of MAO activity between smokers and non-smokers. To rule out that the ‘gender effect’ could mask differences between the smoking and the non-smoking alcoholics in terms of enzyme activity over time, due to the unequal distribution of males and females in the two subgroups, we simultaneously assessed the effect of gender and smoking habit on MAO-B temporal trend by two-factor analysis of variance. The latter analysis confirmed the absence of significant differences in the MAO activity trend over time between male smokers and non-smokers, and between female smokers and non-smokers. Indeed, a limitation of the present study was the unavailability of data concerning the smoking status of the control population. Some literature data on healthy subjects indicate that smokers have lower MAO activity than non-smokers (Anthenelli et al., 1998Go; Whitfield et al., 2000Go). However, other reports have not confirmed such differences in sober individuals (Ward et al., 1987Go; Farren et al., 1998Go) as well as in alcoholics (Farren et al., 1998Go).

In summary, the present study supports the notion that platelet MAO-B activity can be regarded as a state marker of alcohol consumption. The temporal pattern of platelet MAO-B activity may be used for the diagnostic assessment of alcoholism and early abstinence regardless of gender, smoking habit and liver cirrhosis. Repeat measures of platelet MAO-B may be helpful, in combination with other biochemical markers, in identifying subjects who can be classified as alcoholics and in monitoring alcohol withdrawal.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Mr Davide Acerbi is kindly acknowledged for his excellent technical assistance. This work was supported in part by grants QLK4-CT-1999-01356 and QLK4-CT-2001-00186 from the European Commission.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed at: Toxicology Division, IRCCS Salvatore Maugeri Foundation, Via Ferrata 8, 27100 Pavia, Italy. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Alexopoulos, G. S., Lieberman, K. W., Frances, R. and Stokes, P. E. (1981) Platelet MAO during the alcohol withdrawal syndrome. American Journal of Psychiatry 138, 1254–1255.[Abstract]

American Psychiatric Association (1994) Diagnostic and Statistical Manual of Mental Disorders, 4th edn. American Psychiatric Association, Washington, DC.

Anthenelli, R. M., Smith, T. L., Craig, C. E., Tabakoff, B. and Schuckit, M. A. (1995) Platelet monoamine oxidase activity levels in subgroups of alcoholics: diagnostic, temporal and clinical correlates. Biological Psychiatry 38, 361–368.[ISI][Medline]

Anthenelli, R. M., Tipp, J., Li, T.-K., Magnes, L., Schuckit, M. A., Rice, J., Daw, W. and Nurnberger, J. I., Jr (1998) Platelet monoamine oxidase activity in subgroups of alcoholics and controls: results from the collaborative study on the genetics of alcoholism. Alcoholism: Clinical and Experimental Research 22, 598–604.[ISI][Medline]

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