PLATELET AND ERYTHROCYTE MEMBRANE FLUIDITY CHANGES IN ALCOHOL-DEPENDENT PATIENTS UNDERGOING ACUTE WITHDRAWAL

Paul Thompson*

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


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This study tested the hypothesis that membrane fluidity may alter during the alcohol-withdrawal syndrome. Platelet membranes of alcohol-dependent patients (n = 7) were significantly more rigid than controls (n = 7) at the start of alcohol withdrawal (mean fluorescence anisotropy 203.1 x 10–3 vs 195.5 x 10–3 respectively, P = 0.03), but were significantly more fluid when withdrawal was complete (191.4 x 10–3 vs 199.2 x 10–3, P = 0.03). Consequently platelet membranes of patients adapted to the known acute fluidizing effect of alcohol by becoming more rigid, but underwent a marked fluidization during withdrawal. There were no significant changes in erythrocyte membrane fluidity during withdrawal.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The precise molecular basis of alcohol intoxication, dependency, and withdrawal remains unclear. Evidence is accumulating, however, which suggests that the alcohol molecule can act directly on a range of membrane proteins including {gamma}-aminobutyric acid (GABA) and N-methyl-d-aspartate (NMDA) receptors causing acute changes in activity with secondary adaptive changes (Tsai et al., 1995Go; Nutt, 1996Go). Many studies have also demonstrated an effect of alcohol on the physical properties of biological membranes. Acute exposure causes an almost universal fluidization whatever the membrane type (Harris et al., 1987Go). When laboratory rodents are chronically exposed to alcohol in vivo, synaptic and erythrocyte membranes undergo an adaptive rigidification and there is often an associated resistance to the acute fluidizing effect of alcohol (Taraschi et al., 1986Go; Beaugé et al., 1987Go). Human studies on the chronic effects of alcohol ingestion have focused on the effects on the erythrocyte membrane, with conflicting results. Studies have shown an increase (Hrelia et al., 1986Go), a decrease (Beaugé et al., 1985Go, 1988Go; Stibler et al., 1991Go) and no change (Wood et al., 1987Go; Beaugé et al., 1994Go) on baseline membrane fluidity in alcohol abusers relative to controls. Varying degrees of adaptation have been suggested to account for this discrepancy.

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, 1996Go), 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., 1984Go) and this is mirrored in the membrane of the platelet but not the erythrocyte (Hajimohammadreza et al., 1990Go).

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.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Subjects
Patients were recruited from the Alcohol Treatment Unit, Sunnyside Royal Hospital, Montrose, UK and all satisfied ICD-10 criteria for alcohol-dependence syndrome (World Health Organization, 1992Go). For inclusion into the study, patients had to score at least 30 on the severity of alcohol-dependence questionnaire (maximum score 60) and to have continued drinking until at least the day before admission to the Unit. Patients with more severe dependence were selected in order to maximize the likelihood of having alcohol-induced membrane changes. Age- and sex-matched controls were recruited from medical colleagues. Any person taking psychotropic drugs or any other medication known to interfere with membrane fluidity was excluded. The protocol was approved by the local Ethics Committee and all subjects gave their informed consent. On the day of admission, blood (18 ml) was taken by venepuncture and anti-coagulated with EDTA. Patients were treated with a reducing regime of diazepam titrated according to the severity of the withdrawal symptoms. These were rated with the Clinical Institute withdrawal assessment for alcohol scale (CIWA-Ar) (Sullivan et al., 1989Go). Patients were also commenced on 300 mg of thiamine and two multivitamin tablets/ day. A second blood sample was taken 13 days later after which time withdrawal symptoms had settled and diazepam had been discontinued.

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 Perkin–Elmer 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).


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Table 1Go presents some demographic features of the patient and control groups. Both groups contained six males and one female. Patients had a mean alcohol-dependence rating of 39 (range 30– 48). The mean withdrawal assessment (CIWA-Ar) score during the first 72 h was 21 (range 12–30) falling to 2 (range 0–3) during the final 72 h of the 13-day period. Six out of the seven patients had an abnormal {gamma}-glutamyl transpeptidase (GGT) on admission, with a mean value of 122 U falling to a mean of 88 U 13 days later. The mean amount of diazepam used during withdrawal was 286 mg (range 150–535 mg).


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Table 1. Demographic features of the patient and control groups
 
Platelet membranes of patients were significantly more rigid than controls at the start of withdrawal [mean fluorescence anisotropy of patients and controls was 203.1 x 10–3 and 195.5 x 10–3 respect-ively, ANOVA F(1, 23) = 5.18; P = 0.03], but were significantly more fluid than controls 13 days later [mean fluorescence anisotropy of patients and controls was 191.4 x 10–3 and 199.2 x 10–3 respectively, ANOVA F(1, 23) = 5.37; P = 0.03] (Fig. 1aGo). The increase in membrane fluidity in patients during the 13-day period was highly significant [ANOVA F(1, 12) = 14.35; P = 0.003]. In contrast, platelet membranes of control subjects showed no significant change in fluidity over this period [ANOVA F(1, 12) = 1.44; P = 0.25]. There was no correlation between the severity of withdrawal symptoms and the size of the fluidity change seen in platelet membranes of patients (data not shown). Erythrocyte membrane fluidity showed no statistically significant interactions (with a probability of 0.05) between patients, controls or time [mean fluorescence anisotropy at t = 0 days for patients and controls was 210.4 x 10–3 and 211.3 x 10–3 respectively and at t =13 days 209.8 x 10–3, and 211.7 x 10–3 respectively, ANOVA F(1, 12) = 0.14; P = 0.71] (Fig. 1bGo).



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Fig. 1. Membrane fluidity changes during alcohol withdrawal.

The mean fluorescence anisotropy for patient (n = 7) and control (n = 7) groups before and after alcohol withdrawal is given. Error bars represent SEM. A lower fluorescence anisotropy is equivalent to a more fluid membrane.

 
No ethanol or diazepam (or metabolites) were detectable in representative samples of erythrocyte and platelet membranes. This confirmed the proposal that either all traces had been eliminated from the patient prior to venepuncture or that they were washed away during membrane preparation.


    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animal experiments on the chronic effects of alcohol on membranes have identified two phenomena, namely a baseline rigidification and a resistance to the acute fluidizing effect of alcohol. It is of interest to know over what time period such changes develop and how quickly they normalize with abstinence, if at all, as this may help in understanding the biochemical basis of dependency and withdrawal. Beaugé and colleagues have reported that rigidification of rat synaptic membranes persists beyond 5 days after stopping alcohol (Beaugé et al., 1987Go; Beaugé, 1991Go), whereas Samynathan et al. (1995) showed that it disappeared after 16 h. Resistance to the acute fluidizing effect of alcohol has been shown to be lost after 5 days (Beaugé et al., 1987Go; Beaugé, 1991Go), 4 days (Nie et al., 1989Go), and 1 to 2 days (Taraschi et al., 1986Go).

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., 1989Go). In agreement with others (Gilsanz et al., 1992Go; Beaugé et al., 1994Go), 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., 1992Go).

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., 1993Go) and indeed in the past this mechanism has been hypothesized to account for both general anaesthesia (Janoff et al., 1981Go) and alcohol-withdrawal seizures (Tan and Weaver, 1997Go). 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, 1996Go). 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.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
I thank F. Haut, K. Peden, P. M. Rice (Tayside Alcohol Problem Service, Sunnyside Royal Hospital, Montrose), C. Stewart, I. C. Reid (Department of Psychiatry, University of Dundee, Dundee) for advice and support, and F. M. Corrigan (Argyll & Bute Hospital, Lochgilphead) for critically reading the manuscript. Mr G. Lauder (Department of Biochemical Medicine, University of Dundee, Dundee, UK) kindly performed the alcohol and diazepam assays.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Address for correspondence: Carnegie Clinic, Sunnyside Royal Hospital, Montrose, Angus DD10 9JP, UK. Back


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 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
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