Department of Psychiatry, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK
Received 24 April 1998; in revised form 9 November 1998; accepted 8 December 1998
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ABSTRACT |
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INTRODUCTION |
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The alcohol-withdrawal syndrome occurs when the blood-alcohol level falls after prolonged alcohol ingestion. It has been suggested that some symptoms may be the result of neurotransmitter overactivity caused by previous receptor up-regulation (Hoffman and Tabakoff, 1996), but many features of this condition remain unanswered. Of interest is what happens to any adaptive membrane rigidification caused by chronic alcohol exposure during the withdrawal period. Relatively few studies have addressed this issue in man and none has compared erythrocyte and platelet membranes. There is some evidence suggesting that the platelet may be a useful model for the neurone, possibly better than the erythrocyte. For example, in Alzheimer's disease, cell membranes in the brain appear to fluidize (Chia et al., 1984
) and this is mirrored in the membrane of the platelet but not the erythrocyte (Hajimohammadreza et al., 1990
).
The aim of the present study is twofold. The first is to see whether membranes of alcohol-dependent patients are more rigid than controls and if so, to monitor any change during subsequent alcohol withdrawal. Erythrocyte and platelet membrane fluidity in alcohol-dependent patients before and after alcohol withdrawal were compared with a social/non- drinking control group.
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MATERIALS AND METHODS |
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Preparation of platelet and erythrocyte membranes
Platelet membranes were prepared by differential centrifugation using the method of Menashi et al. (1981). Whole blood (18 ml) was centrifuged at 200 g for 10 min at room temperature and the platelet-enriched plasma was carefully removed. The remaining pellet was washed three times with acid citrate dextrose (ACD) buffer (36 mM citric acid, 5 mM KC1, 90 mM NaCl, 5 mM glucose, 10 mM EDTA, pH 6.8) and the washings were pooled with the original supernatant. The pooled suspension was centrifuged again at 200 g for 10 min to remove any residual erythrocytes. The platelets were then collected by centrifugation at 2000 g for 20 min and the platelet pellet was washed three times with 10 ml of phosphate buffered saline (PBS; 120 mM NaC1, 15.3 mM Na2HPO4, 1.46 mM KH2PO4, 1.68 mM KCl, pH 6.8). The final pellet was resuspended in 700 µl of PBS and membrane fluidity was determined as soon as possible and usually within 24 h. Erythrocyte ghost membranes were prepared by osmotic lysis essentially using the method of Dodge et al. (1963). Whole blood (9 ml) was centrifuged at 200 g for 10 min and the erythrocyte pellet was washed twice with ice-cold 0.15 M sodium phosphate buffer, pH 7.4. After centrifugation at 2000 g for 10 min, the pellet was resuspended in 4 ml of buffer and cells were lysed by pipetting into 56 ml of ice-cold hypotonic solution (5 mM sodium phosphate buffer pH 7.4). Erythrocyte ghosts were collected by centrifugation at 20 000 g for 1 h at 4°C. Ghosts were washed twice with 5 mM sodium phosphate buffer and twice with PBS. The final pellet was resuspended in 1 ml of PBS and fluorescence polarization was again carried out as soon as possible.
Membrane fluidity determination by fluorescence polarization
Membrane fluidity was measured (in triplicate) by fluorescence polarization using the fluorescent probe 1,6-diphenyl hexatriene. This was made up as a stock solution in tetrahydrofuran and was stored at 20°C until required. Immediately before use, the stock solution was diluted x 1000 with PBS and mixed with membranes giving a final probe concentration of 3 µM. Protein concentration was 60 mg/l. After pre-incubation with the probe for 1 h at 37°C in darkness, measurements were taken with a PerkinElmer LS-3 spectro-photofluorimeter. Samples were excited at 360 nm and emission was measured at 430 nm. Fluorescence anisotropy is a measure of fluidity (high values equating to more rigid membranes) and was calculated as described by Hajimohammadreza et al. (1990).
Statistics
Statistical analysis of the data was carried out using analysis of variance (ANOVA).
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RESULTS |
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DISCUSSION |
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Less work has been done on the time-course of alcohol-induced membrane changes in man. The present study has shown that platelet membranes of patients were more rigid than controls at the start of withdrawal, indicating adaptation to the chronic presence of alcohol. After patients had experienced the alcohol-withdrawal syndrome, membranes fluidized not just to control values, but beyond, to end up significantly more fluid than controls. This finding is in contrast to a previous study in man, which showed that rigidification persisted for several months after abstinence, although erythrocytes were used and not platelets (Beaugé et al., 1989). In agreement with others (Gilsanz et al., 1992
; Beaugé et al., 1994
), this study failed to detect any significant change in erythrocyte baseline membrane fluidity in alcohol-dependent patients, nor were there any fluidity changes during withdrawal. Although Wood et al. (1987), in an electron-spin resonance study, also found no significant change in baseline erythrocyte membrane fluidity, they did report a resistance to the acute fluidizing effect of alcohol, which persisted for 5 weeks after withdrawal. The lack of an association between the two membrane phenomena is interesting and suggests that rigidification is not responsible for resistance.
Some caution is required in extrapolating fluidity data from membrane preparations to the in vivo situation. Membrane properties, including fluidity, may change during cell preparation and this is especially true when preparing ghosts from erythrocytes, as cells are lysed and this may disrupt membrane-cytosolic links. Indeed differences in ghost preparation techniques may be partly responsible for the reported variation in ghost baseline membrane fluidity in chronic drinkers. This may be a further reason why the platelet is a better cell to study as the preparation procedure is less disruptive.
Although dependence on alcohol and its sudden removal is the most likely reason for the fluidity changes in platelet membranes, alternative explanations need to be considered. Patients, but not controls, were treated with a reducing course of diazepam. Little is known about membrane adaptation to drugs so it is uncertain whether the course of diazepam was sufficiently long to influence the data. Also patients, but not controls, were treated with multivitamins and thiamine so these could conceivably have altered platelet fluidity. An effect of predominantly water-soluble vitamins on membrane fluidity would be surprising and indeed two control subjects were given a course of these vitamins with no change in platelet or erythrocyte fluidity (data not shown). An effect of liver dysfunction on membrane fluidity needs to be considered, especially as six out of the seven patients had an elevated GGT, which fell once drinking stopped. Gilsanz et al. (1992) have shown that, although liver cirrhosis increases erythrocyte membrane fluidity, an elevated GGT in the absence of cirrhosis has no effect on fluidity. No patients in this study had liver cirrhosis. Finally changes in platelet membrane fluidity in the absence of any changes in erythrocyte fluidity rather argue against effects caused by improved nutrition, a course of diazepam, vitamin therapy or liver dysfunction.
It is accepted that the number of patients in this study was small. Patients were recruited over an 8-month period and only seven out of approximately 120 admissions satisfied the entry criteria. Psychotropic medication, especially major tranquillizers, antidepressants, and benzodiazepines was the most common reason for exclusion. All three classes of drugs have been shown to affect human erythrocyte membrane fluidity in vitro (Lejoyeux et al., 1992).
Membrane fluidity changes associated with alcohol use have been described for a number of years without a clear idea of the significance at the molecular level. It is known, however, that acute changes in membrane fluidity can perturb receptor and ion-channel functioning (Collins et al., 1993) and indeed in the past this mechanism has been hypothesized to account for both general anaesthesia (Janoff et al., 1981
) and alcohol-withdrawal seizures (Tan and Weaver, 1997
). As fluctuating levels of consciousness occur in acute organic reactions, including severe alcohol-withdrawal states (delirium tremens), it is possible that fluidity changes may be contributory. Yoshida et al. (1995) described hepatocyte and renal cell membrane fluidity changes in septicaemic rats, but the key question is whether similar changes occur in the brain. The lack of correlation between the severity of withdrawal and the size of the change in platelet fluidity in this study may possibly be due to the small sample size. Certainly it would be interesting to see whether larger changes in platelet fluidity occur during more severe withdrawal states.
It is interesting to note that alcohol can profoundly affect platelet function albeit in a complex way. Acute alcohol ingestion inhibits platelet aggregation in response to challenges by agonists, such as thrombin and collagen, whereas when chronic drinkers stop alcohol there is a rebound increase in aggregation to such agonists (Renaud and Ruf, 1996). At present, however, it is premature to attribute any pathophysiological significance to the changes in platelet membrane fluidity seen here and it is even more speculative to extrapolate changes seen in peripheral cells to the brain.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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