Alcoholism-Associated Hyperhomocysteinemia and Previous Withdrawal Seizures

BRIEF REPORTS

Alcoholism-Associated Hyperhomocysteinemia and Previous Withdrawal Seizures Kristina Bayerlein, Thomas Hillemacher, Udo Reulbach, Brigitte Mugele, Wolfgang Sperling, Johannes Kornhuber, and Stefan Bleich Background: Higher homocysteine levels were found in actively drinking alcoholics as well as in early abstinent patients. Furthermore, it has been shown that high homocysteine levels predicted first-onset alcohol withdrawal seizures. The aim of the present study was to determine plasma homocysteine levels in actively drinking alcoholics and patients with early abstinence in order to evaluate whether there is an additional association between elevated plasma homocysteine levels and a history of alcohol withdrawal seizures. Methods: Two groups of patients with an established diagnosis of alcohol dependence were studied. Group A comprised 56 consecutively admitted alcoholics who had been abstinent from alcohol between 24 to 72 hours before hospitalization. Group B consisted of 144 consecutively recruited alcoholics who were admitted - acutely intoxicated - for withdrawal treatment. Furthermore, groups were divided into two subgroups: patients with and without a history of alcohol withdrawal seizures. Results: Alcoholics of GROUP B with a history of withdrawal seizures had significantly (p ⬍ .0001) higher homocysteine levels than actively drinking patients without seizures in their history: 42.0 ␮mol/l (SD 26.4) versus 22.5 ␮mol/l (SD 11.4). Using a logistic regression analysis, history withdrawal seizures in Group B but not in Group A patients were best predicted by a high homocysteine level at admission (Wald ␹2 ⫽ 15.5, p ⬍ .0001; odds ratio 1.11, 95% CI 1.05–1.20). Conclusions: Homocysteine levels on admission may be a useful screening method to identify actively drinking patients with a higher risk of alcohol withdrawal seizures. Key Words: Homocysteine, alcoholism, history of withdrawal seizures, NMDA receptors, active drinkers, abstinent drinkers

T

here is increasing evidence that chronic alcoholism, especially in nonabstinent patients, is associated with hyperhomocysteinemia (Bleich et al 2000a; Cravo et al 1996; Hultberg et al 1993). The assumed reason is an impaired metabolism of homocysteine, such as deranged remethylation because of a dysfunction of methionine synthase (MS). This is due to an ethanol-induced vitamin deficiency (folate, vitamine B12) as well as to a direct inhibition of MS caused by acetaldehyde, the product of the oxidative degradation of ethanol (Bleich et al 2000b; Kenyon et al 1998). Homocysteine is an excitatory amino acid that markedly enhances the vulnerability of neuronal cells to excitotoxic and oxidative injury in vitro and in vivo (Kruman et al 2000; Lipton et al 1997). There is evidence obtained from experimental studies that the convulsant action of homocysteine is mediated through its agonism at N-methyl-D-aspartate (NMDA) receptors (Lipton et al 1997). Chronic alcoholism does not only lead to an increase in postsynaptic NMDA receptors, but also to an interference with presynaptic substances, i.e. glutamate. Recently, it has been shown that alcoholism-associated hyperhomocysteinemia is associated with the alcohol-related brain atrophy (Bleich et al 2000c, 2003a, 2003b) and predicts first-onset alcohol withdrawal seizures (Bleich et al 2000d). The aim of the present study was to determine plasma homocysteine levels in actively drinking alco-

From the Department of Psychiatry and Psychotherapy (KB, TH, UR, WS, JK, SB), Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen; Klinikum am Europakanal (BM), Department of Addiction, Erlangen, Germany. Address reprint requests to Dr. K. Bayerlein, M.D., Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany; E-mail: kristina.bayerlein@ psych.imed.uni-erlangen.de. Received June 4, 2004; revised January 25, 2005; accepted January 27, 2005.

0006-3223/05/$30.00 doi:10.1016/j.biopsych.2005.01.046

holics and patients with early abstinence in order to evaluate whether there is an additional association between elevated plasma homocysteine levels and a history of alcohol withdrawal seizures. Taking into account a permanently hyperhomocyteinemia in nonabstinent patients (Bleich et al 2000a) and that previous withdrawal seizures are a risk factor of repeated seizures during withdrawal (Brown et al 1988), the hypothesis was that actively drinking alcoholics with a history of alcohol withdrawal seizures reveal higher homocysteine levels.

Methods and Materials The study was approved by the local Ethics Committee (Institutional Review Board) of the University of ErlangenNuremberg and all patients gave their written informed consent. The present case-control study included 200 chronic alcoholics. All patients had an established diagnosis of alcohol dependence according to the Diagnostic Statistical Manual for Mental Disorders (DSM-IV) with a history of alcohol consumption ranging from 6 to 47 years (mean: 19.2 years). For statistical calculations and risk assessment we divided the patients into two groups taking into account that according to previous studies homocysteine levels were altered in early abstinent alcoholics (Cravo et al 1996) and strongly elevated in actively drinking alcoholics (Bleich et al 2000a; Hultberg et al 1993). Furthermore, groups were divided into two subgroups: patients with (history SZ⫹) and without (history SZ⫺) a history of alcohol withdrawal seizures. Group A (“abstained between 24 to 72 hours”) compromised 56 consecutively admitted alcoholics (46 males, 10 females, aged 23 to 63 years, median 42 years) who had abstained from alcohol between 24 and 72 hours before hospitalization. Group B (“active drinkers”) consisted of 144 consecutively recruited alcoholics (115 males, 29 females aged 22 to 67 years, median 44 years) who were admitted acutely intoxicated for withdrawal treatment with a blood alcohol concentration (BAC) ranging from 22 to 570 mg/dl (median 160 mg/dl). BIOL PSYCHIATRY 2005;57:1590 –1593 © 2005 Society of Biological Psychiatry

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Table 1. Comparison of Variables of Alcoholic Patients With (Group A) and Without Abstinence (Group B) Group A (Abstinent Alcoholics) History SZ⫹ (n ⫽ 19) 34%

History SZ⫺ (n ⫽ 37) 66%

Group B (Active Drinking Alcoholics) t-Test p level

Admission Age HCY BAC %CDT Folate B12 YD LD

46.7 (5.8) 13.8 (6.3) — 6.6 (3.4) 8.3 (3.5) 382 (203) 22.3 (7.9) 2799 (3273)

History SZ⫹ (n ⫽ 36) 25%

df 54 40.2 (9.4) 12.3 (4.9) — 4.3 (2.7) 7.2 (3.9) 379 (345) 16.1 (9.5) 1530 (2610)

.003a .39 — .02a .29 .97 .02a .15

Admission 42.0 (9.1) 42.0 (26.4) 216.6 (123.2) 6.9 (4.6) 6.7 (2.9) 512 (418) 16.7 (9.2) 1423 (825)

After 10 days HCY %CDT Folate B12

10.2 (3.8) 4.3 (1.8) 6.8 (3.5) 345 (134)

History SZ⫺ (n ⫽ 108) 75%

t-Test p level df 142

44.7 (9.0) 22.5 (11.4) 173.6 (92.4) 5.7 (3.4) 7.6 (3.5) 402 (198) 19.1 (11.4) 1634 (1825)

0.13 ⬍.0001a .06 .18 .11 .15 .21 .35

10.0 (3.7) 3.7 (1.7) 7.5 (3.2) 333 (207)

.03a .21 .02a .90

After 10 days 7.9 (2.9) 3.3 (1.6) 6.2 (3.6) 314 (284)

a

.04 .08 .61 .64

11.9 (3.9) 4.3 (2.1) 5.9 (2.9) 328 (176)

Each group is divided into patients with (history SZ⫹) and without a history (history SZ⫺) of alcohol withdrawal seizures. Both Group A (duration of abstinence between 24 and 72 hours) and Group B (active drinkers) were divided into two subgroups: history SZ⫹ and history SZ⫺ (patients with or without a history of alcohol withdrawal seizures). Scores are given as means (SD) of age (in years), blood alcohol concentration (BAC, mg/dl), folate (normal range: 4 –17 ␮g/l), B12 (170 – 850 ng/l), total plasma homocysteine (HCY, 5–15 ␮mol/l) and % Carbohydrate-deficient transferrin (%CDT, reference value ⬍ 2.6%). Lifetime drinking (LD, kg): daily alcohol intake in kg ⫻ 365 ⫻ years of drinking (YD). GROUP B patients had significantly higher plasma homocysteine levels than GROUP A patients, with or without a history of withdrawal seizures (p’s ⬍ .0001). Regarding the patients of Group B, those with a history of seizures had significantly higher HCY levels (t-Test, p ⬍ 0.0001). Statistical details are summarized in the results section. a Statistically significant.

Study design, laboratory methods and nutritional assessments were performed as previously described (Bleich et al 2000d). All patients had been informed and had given their written consent. Characteristics of the two study groups such as age and lifetime drinking (LD, daily alcohol intake in kg ⫻ 365 ⫻ years of drinking) are listed in the results section (Table 1). Sociodemographic data such as period of alcohol consumption, last alcohol intake, daily intake of alcohol and withdrawal seizures in medical history were taken using a modified semi-structured interview according to Wetterling et al (1999) All patients were seen as inpatients in a closed detoxification unit and were not taking any vitamin supplements (verified by a semi-structured interview following an exclusion of 5 patients) or other drugs (including withdrawal medication) before being enrolled in the study. In total, 205 patients were screened. The above mentioned 5 patients with a regular intake of vitamins such as folate and vitamin B12 in their history were excluded. Folate supplementation interferes with homocysteine metabolism which might bias the study. However, since a folate administration by physicans was excluded and since folate is not fortificated in most European countries including Germany, the probability of an additional folate supplementation in the included patients seems unlikely, especially a self-administration in patients with alcohol dependence. Abstinence during withdrawal was monitored by alcohol and drug screening. With the exception of 9 patients with decreased folate but not different homocysteine levels who were considered as suffering from malnutrition (data not shown), the patients’ nutritional assessment using a general clinical assessment including a physical examination (according to Baker et al 1982) revealed no abnormalities. In all cases, patients were detoxified after admission with the same psychotropic medication (clomethiazole and carbamazepine in daily mg doses up to 10 days after admission). Patients with other known risk factors

for hyperhomocysteinemia such as nutritional status, medication (i.e. methotrexate), endocrinological conditions and other diseases (i.e. thromboembolic and cardiovascular diseases) as well as patients with any other psychiatric comorbidity including other substance abuse were not included in the study. For all analyses, blood was drawn (within one hour) as soon as patients were admitted to the hospital for treatment for alcohol withdrawal. Furthermore, fasting blood was drawn after 1, 2 (data not shown) and 10 days of withdrawal treatment to demonstrate the known decrease of initially elevated homocysteine levels during withdrawal (Bleich et al 2000a). Fasting total homocysteine in plasma (reference value ⬍ 15 ␮mol/L) was measured by an enzyme-linked immunosorbent assay (Axis® Homocysteine EIA, Germany/Norway, IBLNo.: AX 513 01) according to Frantzen et al (1998). Plasma homocysteine was taken at admission and after 10 days. Blood samples of vitamins (folate, vitamin B12) and blood alcohol concentrations (BAC) were also taken at admission. All fasting blood samples were centrifuged immediately after collection. Carbohydrate-deficient transferrin (%CDT) at admission and after 10 days was assessed in order to identify chronically heavy alcohol consumption and monitoring abstinence. 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. Statistical Analysis Comparisons were made using the Student’s t-test (twotailed). Results are presented in the form of mean values (SD). Logistic regression analysis was performed using a logistic procedure with SCORE and STEPWISE options. A p-value of less www.sobp.org/journal

1592 BIOL PSYCHIATRY 2005;57:1590 –1593 than .05 was considered significant. The data were analysed using SPSSTM for Windows 11.5 (SPSS, Inc., Chicago, IL).

Results Group A Alcoholics who had abstained from alcohol between 24 and 72 hours (“early abstinence”) had plasma homocysteine levels at admission within the normal range (5–15 ␮mol/l). There was neither a significant difference [mean (SD)] in history SZ⫹ patients [13.8 ␮mol/l (6.3)] nor history SZ⫺ [12.3 ␮mol/l (4.9)] (df 54, t ⫽ ⫺.87, p ⫽ .39). Significant differences between the two subgroups were found for age (t ⫽ ⫺3.2, p ⫽ .003), % CDT (t ⫽ ⫺2.5, p ⫽ .02) and years of drinking (t ⫽ ⫺2.6, p ⫽ .02), Table 1. After 10 days of withdrawal treatment plasma homocysteine levels significantly decreased in both subgroups (paired t-test, p‘s ⬍ .01). Patients with history SZ⫹ had a slower decrease and therefore significantly higher plasma homocysteine levels [10.2 (3.8) vs. 7.9 (2.9), p ⫽ .037, Table 1]. However, none of the history SZ⫹ patients in this group suffered from an active seizure. Group B Actively drinking alcoholics in Group B with a history of alcohol withdrawal seizures (history SZ⫹) had significantly higher plasma homocysteine levels at admission than actively drinking history SZ⫺ patients (df ⫽ 142, t ⫽ ⫺4.3, p ⬍ .0001) 42.0 ␮mol/l (26.4) vs. 22.5 ␮mol/l (11.4). Furthermore, BAC concentrations did not differ between the two latter subgroups [(df ⫽ 142, t ⫽ ⫺1.9, p ⫽ .061) 216.6 mg/dl (123.2) vs. 173.6 mg/dl (92.4)]. After 10 days of withdrawal treatment, plasma homocysteine levels significantly decreased in both subgroups (paired t-test, p‘s ⬍ .001) whereas history SZ⫹ patients had significantly higher plasma homocysteine levels [11.9 (3.9) vs. 10.0 (3.7), p ⫽ .03, Table 1]. No further significant difference was observed between the two subgroups.

K. Bayerlein et al Group A versus Group B At admission, total plasma homocysteine levels in alcoholics of Group A were found to be significantly (t-test, df ⫽ 198, t ⫽ ⫺8.6 to ⫺5.3, p‘s ⬍ .0001) lower in comparison to both subgroups of Group B, i.e. patients with or without a history of seizures [13.8 ␮mol/l (6.3) vs. 42.0 ␮mol/l (26.4); 12.3␮mol/l (4.9) vs. 22.5 ␮mol/l (11.4)]. Figure 1 illustrates these findings of significantly higher plasma homocysteine in Group B compared to Group A patients with or without a history of withdrawal seizures. After 10 days of withdrawal, plasma homocysteine levels steadily decreased in both groups (see above and Table 1). However, in Group B patients the decrease of homocysteine levels was significant for both subgroups (paired t-test, p ⬍ .0001). There were a significantly slower decrease and higher homocysteine levels in actively drinking history SZ⫹ patients [(t ⫽ ⫺2.2, p ⫽ .037) 11.9␮mol/l (3.9) vs. 10.2␮mol/l (3.8)]. No significant differences in relation to years of drinking (YD), lifetime drinking (LD) or vitamin levels (folate, B12) were observed between the two groups (t ⫽ ⫺1.5–2.4, p ⫽ .09 –.83). Furthermore, there were no significant differences in the variables listed above when comparing these two groups after a withdrawal period of 10 days. Logistic Regression Analysis As an adjustment for possible confounding variables influencing the dependent variable “history SZ⫹” we performed a logistic regression analysis for both Groups. Therefore, all variables in Table 1 were entered into a logistic regression model. Using the SCORE and STEPWISE procedures, the model that suited best was tested. In Group B no variable such as age, sex, BAC, vitamins, LD, YD with the exception of homocysteine levels and %CDT met the .05 significance level for entry into the model. Thus, in this model, the criterion, history SZ⫹’ was best predicted by a high homocysteine level at admission (Wald ␹2 ⫽ 15.5, p ⬍ .0001; odds ratio ⫽ 1.11, 95% CI 1.05–1.20) and less pronounced by %CDT (Wald ␹2 ⫽ 3.9, p ⫽ .046; odds ratio ⫽ 1.15, 95% CI 1.00 –1.31). In patients with an early abstinence (Group A) the criterion “history SZ⫹” was best predicted by %CDT values (Wald ␹2 ⫽ 5.5, p ⫽ .018; odds ratio ⫽ 1.33, 95% CI 1.05–1.69). No further significant results were observed.

Discussion

Figure 1. Plasma homocysteine levels in Group A (abstained between 24 and 72 hours) and Group B (active drinkers) alcoholics (mean ⫾ SEM ⫾ SD). Group A and Group B are divided into those with (history SZ⫹) and without (history SZ⫺) a history of withdrawal seizures (SZ). (#) Significantly higher homocysteine levels in actively drinking history SZ⫹ patients when compared to both, actively drinking history SZ- alcoholics and to patients within Group A (t-Test, p ⬍ .0001). Statistical details are summarized in the results section.

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The present study provides further evidence that alcohol consumption in actively drinking alcoholics is associated with elevated homocysteine levels. This was, however, not the case in patients with early abstinence as observed in recent studies (Cravo et al 1996). Patients with an active drinking pattern and with a history of alcohol withdrawal seizures were found to have significantly higher raised homocysteine concentrations than 1) actively drinking alcoholics without a history of seizures and 2) patients with early abstinence who showed slightly increased or normal homocysteine values. Our results are therefore in line with a recent observation which comes to the conclusion that elevated homocysteine levels predict first-onset alcohol withdrawal seizures in patients with alcoholism (Bleich et al 2000d; Kurth et al 2001). In the present study, none of the patients suffered from an alcohol withdrawal seizure during hospitalization because all patients were prophylactically treated with antiseizure medication (clomethiazole and carbamazepine). In patients with early abstinence the %CDT value was associated with the occurrence of history seizures. Since homocysteine levels are positively correlated to the blood alcohol concentration (Bleich et al 2000a), this marker of early abstinence

K. Bayerlein et al might reflect the sustained effects of ethanol related disturbances in homocysteine metabolism: an alcohol-induced impairment of remethylation of homocysteine by a direct inhibition of methionine synthase by acetaldehyde (Kenyon et al 1998). The exact mechanisms of hyperhomocysteinemia causing seizures in patients in the withdrawal process are still unknown. Our hypothesis is that hyperhomocysteinemia caused by chronic alcohol consumption is followed by an up-regulation of the NMDA receptor system. Withdrawal of alcohol may lead to an overstimulation of NMDA receptors by excitatory amino acids such as glutamate and homocysteine. The simultaneous removal of the inhibitory effect of ethanol may intensify this effect. The result is an overstimulation of the excitatory glutamatergic system causing withdrawal symptoms, i.e. seizures (Bleich et al 2000b; Bleich et al 2004). Course measurements during an alcohol withdrawal treatment showed a continuous reduction in plasma homocysteine levels, which return more or less to within the normal range after around 1–3 days (Hultberg et al 1993; Bleich et al 2000a). The present results showing a decrease and normal homocysteine levels after 10 days of alcohol withdrawal are in line with these recent findings. As shown in previous studies systemic infusion of homocysteine generated tonic-clonic seizures in rats during ontogenesis (Kubová et al 1995). The underlying mechanism might be the agonistic effect of homocysteine and its catabolic decay products (i.e. homocysteic acid) at the NMDA receptors (Lipton et al 1997). Experimental studies with NMDA and nonNMDA antagonists that showed a protective effect against homocysteine-induced seizures support this hypothesis (Folbergrová et al 1997; Folbergrová et al 2000). Homocysteine plays a role in a shared biochemical cascade. This involves overstimulation of NMDA receptors, oxidative stress, activation of caspases, DNA damage and mitochondrial dysfunction. These mechanisms are believed to be crucial in the pathogenesis of excitotoxicity leading to neuronal cell loss respectively seizures (Lipton et al 1997; Bleich et al 2004; Maler et al 2003). Taking into account the relatively high potential of homocysteine in inducing seizures we would like to recommend the possible usefulness of NMDA receptor antagonists as an additional approach in alcohol withdrawal treatment. Furthermore, hyperhomocysteinemia is a condition that can be treated taking into account that folate therapy will reliably reduce plasma homocysteine levels. Thus, the administration of B vitamins such as folate might as well be useful in patients undergoing alcohol withdrawal. This is why homocysteine levels on admission may be a useful screening method to identify patients exposed to the risk of withdrawal seizures. These patients could benefit from an anticonvulsant treatment at an early stage and from special monitoring. Therefore patients who do not belong to this high risk group avoid the potentially side effects of antiepileptic medication. We gratefully acknowledge grant support (to SB) from Axis Shield, Norway (CT-H2003). None of us have a financial or personal conflict of interest. The sponsor had no role in study design, data collection, data analysis, data interpretation, or writing of the report.

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