ELEVATED HOMOCYSTEINE LEVELS IN ALCOHOL WITHDRAWAL

S. Bleich*, D. Degner, J. Wiltfang, J. M. Maler, P. Niedmann1, S. Cohrs, A. Mangholz, J. Porzig, R. Sprung2, E. Rüther and J. Kornhuber

Departments of Psychiatry,
1 Clinical Chemistry II and
2 Environmental and Forensic Medicine, Georg-August-University of Göttingen, Germany

Received 23 December 1999; accepted 13 February 2000


    ABSTRACT
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 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Ethanol exerts its behavioural effects largely by interacting with receptors for brain neurotransmitters. However, the molecular mechanisms involving these interactions and the pathogenesis of alcohol-withdrawal symptomatology are still not well understood. Until recently, no data were available about homocysteine (Hcy) levels in acute alcohol intoxication of chronic alcoholics and in patients undergoing withdrawal from alcohol. Hcy, blood-alcohol concentrations, vitamins B6, B12, and folate concentrations were assessed in 29 chronic alcoholics, who underwent withdrawal from alcohol. We observed increased Hcy levels in most patients. Hcy levels steadily decreased during the observation period. We postulate that hyperhomocysteinaemia and excitatory amino acid neurotransmitters, by their agonism at the N-methyl-d-aspartate receptor, may partly mediate alcohol-associated withdrawal symptomatology. The importance of assessing serum Hcy levels in order to detect methylation deficiency in patients with chronic alcoholism and for possible therapeutic strategies is discussed.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Alcohol dependence and alcohol withdrawal are associated with a variety of neuropsychiatric syndromes, including mental symptoms, e.g. depressed mood, sleep disturbance, unstable emotion, feeling of emptiness, anxiety, and physical symptoms such as autonomic dysregulation, loss of libido, sweating, clouding of sensorium, delirium tremens or convulsions. It has been suggested that withdrawal symptomatology could result from increased activity of excitatory mechanisms [e.g. N-methyl-d-aspartate (NMDA)] receptors and from reduced functioning of inhibitory receptors [e.g. {gamma}-amino butyric acid (GABA)A-receptors] (Rommelspacher et al., 1991Go; Spanagel and Kornhuber, 1999Go).

It has been shown that chronic alcoholism is associated with hyperhomocysteinaemia (Hultberg et al., 1993Go; Cravo et al., 1996Go; Halsted et al., 1996Go). However, until recently, no data were available about homocysteine (Hcy) levels in patients undergoing withdrawal from alcohol. Interestingly, excess Hcy can be metabolized to sulphur-containing excitatory amino acid (EAA) neurotransmitters, which may cause seizures and excitotoxic neuronal death (Knöpfel et al., 1987Go; Lipton et al., 1997Go). Hcy and EAA neurotransmitters are endogenous agonists at the NMDA receptors and potentiate excitatory postsynaptic potentials (Knöpfel et al., 1987Go; Lipton et al., 1997Go), and we postulate that excess Hcy and EAA may partly mediate alcohol-associated withdrawal symptomatology. Therefore, the present study was undertaken to determine whether Hcy metabolism is altered in the serum of patients with chronic alcoholism undergoing withdrawal from alcohol.


    METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The study was approved by the Ethics Committee of the Medical Faculty of the Georg-August-University of Göttingen and written, informed consent was obtained from all subjects.

The present open, uncontrolled and un-randomized study included 29 chronic alcoholics (27 men and 2 women) aged 32–66 years (mean: 47 years) [for other details, see Bleich et al. (1999)]. Briefly, all patients were active drinkers and had an established diagnosis of alcohol dependence according to the Diagnostic and Statistical Manual for Mental Disorders (DSM-IV) of the American Psychiatric Association (1994) with a history of alcohol consumption ranging between 6 and 25 years (mean: 14.8 years). Mean daily ethanol intake ranged from 95–380 g (mean: 230 g), this latter quantification was obtained according to Cravo et al. (1996). The patients were all seen as in-patients in our detoxification unit, stopped drinking immediately before admission, and were taking no vitamin supplements or other drugs before being enrolled in the study. In addition, the patients' nutritional assessment according to Baker et al. (1982) revealed no abnormalities (with the exception of four patients with decreased folate levels who were considered malnourished; data not shown). Patients were detoxified in all cases with the same psychotropic medication (clormethiazole). Patients with any commonly known other risk factors for hyperhomocysteinaemia, such as altered nutritional status, medication (i.e. methotrexate), endocrinological conditions, and other diseases (i.e. thromboembolic and cardiovascular diseases) were not included in the study.

Blood samples for vitamins (folate, B12, and B6), blood-alcohol concentrations (BACs) and Hcy were taken at admission (day 0), followed by Hcy blood analyses on the first (day 1) and third (day 3) days of treatment. Fasting blood samples were collected in ethylenediaminetetraacetic acid-containing tubes and were promptly centrifuged following collection. Plasma was stored at –80°C. Vitamin B12 and serum folate concentrations were measured by chemiluminescence using Chiron kits (Chiron Diagnostics Corporation/Fernwald, Germany) on a Chiron ACS: 180 automated analyser. Vitamin B6 was determined by high-pressure liquid chromatography (HPLC) (Ubbink et al., 1985Go), using an Immunodiagnostik kit (Immunodiagnostik/Bensheim, Germany). Hcy was determined by HPLC (Ling et al., 1991Go) using a Bio-Rad kit (Bio-Rad/Munich, Germany). BAC was determined by head-space gas chromatography (Bleich et al., 1998Go). Genotyping for the thermolabile methylenetetrahydrofolate reductase variant was done by the polymerase chain reaction (PCR) and consecutive restriction enzyme digestion according to von Ahsen et al. (1999). Genomic DNA for use in the PCR was isolated with a QIAamp Blood kit (Quiagen/Hilden, Germany). Association between variables was assessed by regression analysis (linear multiple regression analysis) and the results are presented as means ± SEM. A P-value of less than 0.05 was considered significant.


    RESULTS
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 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
As shown in Fig. 1Go, plasma samples from patients with alcohol dependence were found at admission (day 0) to have increased endogenous Hcy levels (normal range: 5–15 µmol/l). Thus, the mean value (±SEM) at day 0 was 33.6 ± 25.2 (median: 26.4; range: 9.7–119.0 µmol/l). BAC at admission (day 0) is also shown in Fig. 1Go, the mean ± SEM of which was 177 ± 68 mg/dl (median: 176; range: 50–340 mg/dl). As can be seen in Fig. 1Go, both BAC and Hcy levels at admission could be correlated positively, also as summarized in Table 1Go.



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Fig. 1. Concentration of total plasma homocysteine (Hcy) and blood-alcohol concentration (BAC) in alcoholic subjects at admission (day 0).

Significant correlation between Hcy and BAC (P < 0.001); r2 = 0.7662 through origin.

 

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Table 1. Statistical results of the stepwise forward multiple regression analysis
 
Table 1Go also shows a significant negative correlation between plasma Hcy and folate levels at admission. The latter were on average 9.51 ± 4.77 µg/1 (mean ± SEM) (median: 9.2; range: 2.5–19.3 µg/l) against a normal range of 4–17 µg/l.

Plasma Hcy concentration fell steadily during the observation period (Fig. 2Go). Thus, the mean value (±SEM) of 33.6 ± 25.2 µmol/l observed at day 0 was decreased to 20.6 ± 12.8 on day 1 (median: 16.2; range: 6.6–56.1 µmol/l) and to 13.9 ± 8.8 on day 3 (median: 11.0; range: 6.3–44.0 µmol/l).



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Fig. 2. Plasma homocysteine (Hcy) levels (over a period of 3 days) during alcohol withdrawal.

Hcy levels steadily decreased during the observation period and were in normal range (5–15 µmol/l) after day 3 of withdrawal treatment (statistical details are summarized in the Results section). Values are means ± SEM (bars) and are also given as median and range. {circ}, Outliers; *, extremes.

 
No significant correlation was observed between Hcy levels and age, gender or vitamins B12 and B6 (P = 0.25–0.98). Levels of these two vitamins (means ± SEM) were 461 ± 157 ng/1 (normal range: 170–850 ng/1) and 18.6 ± 14.2 µg/l (normal range: 4–20 µg/l) respectively. Overall, we did not observe decreased vitamin levels in these patients undergoing withdrawal from alcohol, except for four malnourished subjects with subnormal folate levels.

The patients received chlormethiazole in daily mg doses as follows: day 0: 72.8 ± 148.8 (median: 0.00; range: 0–384); day 1: 2562 ± 953 (median: 2304; range: 960–4992); day 3: 907 ± 580 (median: 768; range: 0–2304). Chlormethiazole treatment therefore did not correlate with plasma Hcy levels. Additionally, in all patients, the genotyping for the thermolabile methylenetetrahydrofolate reductase variant revealed no abnormalities (data not shown).


    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Moderate hyperhomocysteinaemia was observed in chronic alcoholics who underwent withdrawal from alcohol. In addition, there was a strong correlation between the Hcy and BAC at admission, while Hcy levels steadily decreased during the observation period. All patients suffering from alcohol-withdrawal symptoms (i.e. sweating, tachycardia, tremor, depressive mood, anxiety, and sleep disturbances) completely recovered after 6 days. Four patients suffered from a withdrawal seizure. What are the reasons for the observed hyperhomocysteinaemia?

First, Hcy is a thiol-containing amino acid derived from methionine, an essential amino acid. Hcy is produced entirely from the methylation cycle, as it is totally absent from any dietary source (Finkelstein, 1990Go). Hcy is metabolized by remethylation to methionine; this conversion is catalysed by methionine synthase (MS) or by transsulphuration to cysteine. The latter reaction is catalysed by cystathionine- ß-synthase (CBS). Vitamins B12 and B6 are essential co-factors and folic acid (folate) is an essential co-substrate for these processes (Kang et al., 1987Go). Because folate is required for the remethylation of Hcy, it is plausible that tissue folate concentration is closely associated with the regulation of Hcy metabolism and Hcy concentrations in plasma are inversely correlated with the above-mentioned vitamins (Kang et al., 1987Go; Curtis et al., 1994Go). Various studies have emphasized that serum or plasma vitamin B12 and folate levels are not an entirely reliable guide for tissue B12 and folate deficiency and it has been shown that total Hcy concentrations may be a sensitive functional indicator of intracellular folate and B12 deficiency (Curtis et al., 1994Go). Therefore, the results of our study showing increased levels of Hcy and normal vitamin B6, B12, and folate levels (only 4 patients had decreased serum folate levels) are not inconsistent with this notion. For example, 19% of the subjects with subnormal serum folate had more than a three-fold increase of Hcy (Kang et al., 1987Go). We postulate that the observed hyperhomocysteinaemia in these patients is partly due to their decreased intracellular folate levels. In addition, it has been known for many years that ethanol has an effect on folate metabolism, which cannot be explained by an alcohol-induced low intake of folate (Sullivan and Herbert, 1964Go). The aetiology of folate deficiency in alcoholism can be ascribed to several causes, such as low dietary intake, poor absorption, decreased hepatic uptake and retention, and increased urinary excretion of folate (Halsted et al., 1971Go; Halsted and Keen, 1990Go).

Second, elevated Hcy levels may occur as a result of inherited disorders and/or non-genetic factors that alter enzyme activity in the transsulphuration and remethylation pathways. Moderate hyperhomocysteinaemia has been related to genetic or non-genetic risk factors. The prevalence of moderate Hcy in the population has been estimated to be 5–7% (Kang et al., 1987Go; Kang, 1995Go). If a genetic defect is essential in causing a moderate elevation of plasma Hcy, homozygous thermolabile methylenetetrahydrofolate reductase (MTHFR) and heterozygous CBS and MTHFR deficiency are the most probable aetiological factors (Kang, 1995Go; Kang and Wong, 1996Go). However, we could not observe such genetic defects. In addition, it has been shown that long-term ingestion of large quantities of ethanol causes inhibition of liver MS activity due to its breakdown product acetaldehyde (Barak et al., 1991Go, 1996Go; Kenyon et al., 1998Go) and it has been proposed that elevated blood levels of ethanol, rather than nutritional factors, are responsible for changes in methionine metabolism (Barak et al., 1991Go). This conversion of Hcy to methionine is a crucial reaction from the standpoint of conserving methionine and detoxifying Hcy, which is possibly insufficient in patients suffering from alcohol dependence (active drinkers).

It has been suggested that withdrawal symptomatology could result from increased activity of excitatory mechanisms (e.g. NMDA receptors) and from reduced functioning of inhibitory receptors (e.g. GABAA receptors). Following chronic ethanol ingestion, the number of NMDA receptor–ion channel complexes in certain brain areas (e.g. the hippocampal) is increased (Hoffman et al., 1990Go) and it has been suggested that this increase may contribute to the generation of ethanol withdrawal seizures (Bleich and Degner, 2000Go; Bleich et al., 2000Go). In addition to the excitatory role played by the amino acid transmitters glutamate and aspartate in the central nervous system, their sulphur-containing analogues homocysteic acid (HCA) and cysteine sulphinic acid may also play a similar role. These latter compounds are oxidation products of Hcy and cysteine, are putative neurotransmitters and are endogenous agonists at the NMDA receptor (Cuenod et al., 1990Go). In addition, it has been shown that Hcy itself acts as an agonist at the glutamate binding site of the NMDA receptor (Lipton et al., 1997Go) and leads to an imbalance in excitatory: inhibitory neurotransmission in the hippocampus caused by alterations in extracellular levels of neuroexcitatory (e.g. aspartate) and neuroinhibitory (e.g. GABA) transmitters, which could be an explanation of the epileptic effect of Hcy (Bleich et al., 2000Go). Hence, it has been shown that due to its highly excitatory action HCA can induce seizures (Mares et al., 1997Go; Parsons et al., 1998Go). It has been found that NMDA receptor antagonists (i.e. flupiritine) can counteract the influence of NMDA and HCA (Osborne et al., 1994Go). Thus, the clinical use of flupiritine in patients undergoing withdrawal from alcohol might be beneficial.

In summary, there is growing evidence that chronic alcoholism is associated with a derangement in this sulphur amino acid metabolism. Ethanol-induced hyperhomocysteinaemia with subsequent accumulation of excitatory neurotransmitters may partly mediate the variety of symptoms which are seen in alcohol withdrawal. Excess Hcy can be metabolized to sulphur-containing EAA neurotransmitters, which may cause seizures and excitotoxic neuronal death. Hyperhomocysteinaemia is a treatable condition taking into account that folate therapy will reliably reduce plasma Hcy levels. Furthermore, the administration of taurine or taurine-like agents acting as inhibitory neurotransmitters and/or NMDA receptor antagonists might be beneficial in patients undergoing alcohol withdrawal (Li et al., 1994Go; Kornhuber et al., 1999Go; Bleich and Degner, 2000Go). This study reports the importance of assessing serum Hcy levels in order to detect methylation deficiency in patients with chronic alcoholism. Nevertheless, further investigations and controlled studies are needed to clarify the role of Hcy in patients with alcoholism.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
We wish to thank Dr N. von Ahsen (Georg-August-University of Göttingen, Clinical Chemistry) and Dr Y. Barak (Abarbanel Mental Health, Psychogeriatric Department, Tel-Aviv University, Israel) for genetic analysis and helpful discussions.


    FOOTNOTES
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* Author to whom correspondence should be addressed at: Georg-August-University, Department of Psychiatry, Von-Siebold-Str. 5, D-37075 Göttingen, Germany. Back


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