Alcohol and Alcoholism Advance Access originally published online on September 22, 2008
Alcohol and Alcoholism 2008 43(6):653-657; doi:10.1093/alcalc/agn076
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Breath Alcohol Level and Plasma Amino Acids: A Comparison between Older and Younger Chronic Alcohol-Dependent Patients
1 Department of Psychiatry and Psychotherapy, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria
2 1st Faculty of Medicine, Institute of Clinical Biochemistry and Laboratory Diagnostics, Charles University, 128 08 Prague, Czech Republic; and
3 Université Catholique de Louvain, Biologie du Comportement, 1, Croix du Sud, B-1348 Louvain-la-Neuve, Belgium
* Corresponding author: Department of Psychiatry, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. Tel: +43-1-40400-3526; Fax: +43-1-40400-3472; E-mail: Henriette.walter{at}meduniwien.ac.at
Received 15 May 2008; first review notified 30 June 2008; in revised form 11 July 2008; accepted 18 July 2008; advance access publication 22 September 2008
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ABSTRACT |
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Aim: The aim of the present study is to examine the distribution of plasma excitatory and inhibitory amino acids, according to the age and current breath alcohol levels (BrAl±), of alcohol-dependent patients. Participants and Methods: 78 alcohol-dependent patients (mean age = 46.2 ± 11 years, men/women = 54/24) were clinically tested, including the determination of the major excitatory as well as inhibitory amino acids. The independent variables were gender, age and current alcohol consumption measured with the breath alcohol level (BrAl ± status). Results: In comparison to BrAl negatives, BrAl positives had higher plasma levels of glutamic acid (P = 0.01) and proline (P = 0.026), and lower levels of aminobutyric acid (P = 0.002), serine (P = 0.031) and urea (P = 0.01). In the BrAl positives, no age effect was found related to the plasma amino acids. In contrast, the BrAl negatives displayed age-related differences. The older (
50 years) BrAl negative patients had higher plasma levels of cystine, tyrosine, citrulline and urea, and lower histidine levels, compared to the younger group (<50 years). In general, differences in plasma levels of certain amino acids were dependent on gender, BrAl status, age and biochemical markers (GGT, MCV) of alcohol abuse. Conclusions: Abstaining patients (BrAl–/) display age-related differences in AAs’ distribution, while active drinking (BrAl+/) seems to even out those differences, underpinning the hypothesis that drinking mimics changes seen with advanced age. |
Introduction |
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For several years the problem of alcohol dependence in ageing patients has received increased attention. Yet little is known about how amino acids (AAs) might influence neuronal signalling in older alcohol-dependent patients. Being the basis of proteins, AAs are major constituents of life. Protein turnover recycles amino acids and permits adjustments in enzyme and structural proteins, as well as in receptors and modulators. Furthermore the turnover allows for the destruction of damaged proteins (e.g. oxidative damage) and their replacement by functional proteins. With increasing age, however, imperfections of protein synthesis and destruction may occur (Dobson, 2002
). Alterations in neuronal nitric oxide (NO) production may play a role in the origination of diseases, predominantly associated with higher age such as Parkinson's disease, Alzheimer's disease and multiple system atrophy (Kuiper et al., 2000). The biosynthesis of NO depends on the availability of arginine, the substrate for NO synthesis (NOS), and on glutamate, which stimulates NOS via the NMDA receptor. In this process, citrulline is formed.
The major inhibitory amino acids (IAAs) are known to be taurine, glycine, aminobutyric acid and histamine. Taurine protects neurons from excitotoxicity, induced by EAAs (French et al., 1986; Trenkner, 1990). Glycine has NMDA modulating properties and histamine, being derived from histidine, acts via H1 and H2 receptors. These are target receptors for many drugs, such as tricyclic antidepressants or antihistaminic drugs. The imbalance of both, glutamate and aminobutyric acid, are widely discussed as being responsible for at least part of the withdrawal symptoms (Dahchour and De Witte, 2000, 2003a, 2003b; De Witte, 2004). L-glutamate is considered to be an endogenous NMDA agonist, and glycine is considered to be a co-agonist for the NMDA receptor. Only if drinking is ceased, the alcohol-induced NMDA up-regulation (Hoffmann and Tabakoff, 1994; Grant and Lovinger, 1995) and the GABA-A down-regulation (Grant and Lovinger, 1995) will come to an end.
The co-stimulation of NMDA and cholinergic receptors leads to an endogenous release of AAs. They are involved, at least partially, in the tonic and phasic dopamine release, primarily in the ventral tegmental area (VTA) (Karreman et al., 1996). High chronic alcohol intake as well as alcohol withdrawal is related to tonic and phasic dopamine release. AAs such as tyrosine can also enhance the dopamine synthesis in nigrostriatal neurons, if the firing rates of these neurons are accelerated (Melamed et al., 1980).
Chronic alcohol intake, irrespective of age, withdrawal, stress and problems in oxygen saturation, causes an imbalance between excitatory and inhibitory AAs. Yet still little is known about extracellular amino acid levels in relation to gender and age in chronic alcohol-dependent patients (Faingold et al., 1998).
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Objectives |
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Based on the assumption, that alcohol intake, withdrawal and stress cause an imbalance between excitatory and inhibitory AAs (Faingold et al., 1998
), the purpose of the present study was to examine the distribution of a wide spectrum of amino acids in younger and older, BrAl positive and BrAl negative, chronic alcohol-dependent patients. Higher values for EAAs were hypothesized for BrAl positive and older patients, while lower EAA concentrations were hypothesized for sober and younger patients. Furthermore, it can be clinically observed that alcohol-dependent patients appear to have ‘artificially’ aged, and with this study we sought to endorse this subtle observation. |
Subjects and Methods |
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Patients’ description
After clinical ethical approval, 78 alcohol-dependent patients (male/female 54/24 = 69.2%/30.8%), diagnosed according to DSM-IV and ICD-10, who came for medical checkups at our out-patient centre, have consecutively been included into the study. Drinking behaviour was assessed with the time line-follow back method and by measuring breath alcohol (BrAl±) levels, measured in milligram alcohol per litre of breath air (BrAl mg/l). Measurements were taken with the ‘Dräger Alcotest 6510’. Participants were either currently under the influence of alcohol or have been abstinent for a maximum of 2 days. 21 patients (26.9%) were BrAl positive and 57 (73.1%) were BrAl negative (BrAl–/). The BrAl positive (BrAl+/) group comprised 15 (71.4%) male and 6 (28.6%) female patients. The BrAl negative (BrAl–/) group consisted of 39 (68.4%) male and 18 (31.6%) female patients. The mean BrAl value was 1.16 ± 0.79 mg/l. The 15 male patients, who were BrAl positive, had a mean BrAl of 1.07 ± 0.73 mg/l. Higher mean BrAl values were found for the female participants (n = 6; mean BrAl = 1.38 ± 0.96 mg/l).
The mean GGT was 129.04 ± 185.1 (range from 1.0 to 1110.0). BrAl+/ patients had a mean GGT of 237.5 ± 304.9 (range from 23.0 to 1110.0); the BrAl–/ patients had a mean GGT of 88.9 ± 88.1 (range from 11.00 to 449.00) (P = 0.013).
The mean MCV was 92.81 ± (SD 5.878) (range 80.30–111.0). BrAl+/ patients had a mean MCV of 93.56 ± (SD 5.94) (range 81.20–105.00) and the BrAl–/ patients had a mean MCV 92.52 (SD 5.74) (range 80.30–111.00). The groups did not differ significantly.
The mean age was 46.2 ± 11 with a standard deviation of 11 years ranging from 19 to 71 years of age. We defined two age groups (<50 and 50 years of age) in order to be able to give a closer look to the data of an older group of patients. Fifty patients (64.1%) were <50 years of age and 28 (35.9%) were 50 years of age.
The group of <50 years consisted of 35 (44.9%) male and of 15 (19.2%) female patients. The group 50 comprised 19 (24.4%) male and 9 (11.5%) female patients.
Age and BrAl
Twenty-one patients (26.9%) of the whole sample were measured as being BrAl positive. Sixteen of them were in the younger group (<50 years of age) with a mean BrAl of 1.26 ± 0.86 mg/l. Another five BrAl positive (BrAl+/) patients were found in the older group, having a lower mean BrAl value (0.86 ± 0.49 mg/l) than the younger ones (see Table 1).
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Assessment of amino acids
In the present study we measured the following amino acids: aspartic acid, glutamic acid, glutamine, glycine, alanine, citrulline, aminobutyric acid, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, lysine, histidine, L3-methyhistidine, proline, serine, threonine, phosphoethanolamine, taurine, phosphoserine and urea.
Blood specimens were taken in the morning after overnight fasting from the antecubital vein into lithium heparine tubes, centrifuged immediately at 3000 rpm for 15 min. The plasma obtained was stored at –20°C. All biochemical tests were performed using plasma stored at –20°C. 250 µl of plasma was mixed with 250 µl seraprep; after 15 min incubation the 500 µl was centrifuged at 13,000 rpm; the supernatant was then filtered using a 0.22 µm filter. Plasma AAs were analysed using an automated HPLC technique (Pharmacia Biotech Biochrome 20 system) with UV detection after reaction with the ninhydrin reagent. The concentrations of aspartic acid, glutamic acid, glutamine, glycine, alanine, citrulline, aminobutyric acid, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, ornithine, lysine, histidine, methylhistidine, proline, serine, threonine, phosphoethanolamine, taurine, phosphoserine and urea were measured.
Statistical assessment
Data are expressed as mean ± SD. For statistical assessment we used the Mann–Whitney U-test for not normally distributed data. To measure correlations, we used the two-sided Pearson correlation. All statistical tests were two sided, and a P-value of <0.05 was considered to be significant.
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Results |
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AAs and age
Both age groups showed significant differences in cystine and tyrosine plasma levels being distinctly higher in the older group (P = 0.009 and P = 0.020, respectively) (Table 2).
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AAs and BrAl±
In comparison to BrAl–/, the BrAl+/ patients had higher plasma levels of glutamic acid (P = 0.001) and proline (P = 0.026), and lower levels of aminobutyric acid (P = 0.002), serine (P = 0.031) and urea (P = 0.0010) (Table 3).
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AAs, BrAl and age
No significant AA differences between the age groups could be found for the BrAl+/ patients. In contrast, the BrAl–/ patients displayed significant age-related differences. Patients belonging to the BrAl–/ older group (males and females) had significantly higher levels of cystine (P = 0.006), tyrosine (P = 0.007), citrulline (P = 0.017) and urea (P = 0.037). Only histidine was significantly lower in the older BrAl–/group, compared to the younger BrAl–/ group (P = 0.037) (Table 4).
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AAs, sex and BrAl
Histidine differs significantly (P = 0.015) between male and female participants. Male patients had higher levels in both the BrAl+/ as well as the BrAl–/ group. In the BrAl+/ group only glutamine differed significantly (P = 0.044) between male and female participants, with female participants having higher levels.
In the BrAl–/ group (n = 57) cystine (P = 0.018) and histidine differed significantly (P = 0.024) between men and women (Table 5).
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Sex comparison within the age groups
Sex comparisons in the older group of
50 years of age did not show significant differences. In the younger group, men and women differed significantly in valine (P = 0.038) and urea levels (P = 0.014), both being higher for male patients (Table 6).
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AAs, sex, age and BrAl
For BrAl+/ men and women, no significant difference between the age groups, regarding all AAs, could be found.
In the BrAl–/group, however, men <50 years had significantly lower citrulline levels (P = 0.043) and lower cystine levels (P = 0.016), compared to men 50 years. The same could be found for tyrosine, where males <50 showed a significantly lower average level than male participants 50 years (P = 0.001). For histidine only, the <50 male group had higher mean values (P = 0.045).
For BrAl–/female patients only urea differed significantly between the age groups, with higher urea for the older group (<50 years: urea/mean 1743 ± 491; 50 years: urea/mean 2609 ± 375; P = 0.007) (Table 7).
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Age and GGT
In the younger age group, low GGT correlated with high glutamine (r = –0.561), isoleucine (r = –0.340) and lysine (r = –0.372) levels. A positive correlation between GGT and glutamic acid (r = 0.549) was observed.
In the older group a positive correlation between GGT and aspartic acid (r = 0.508), glutamic acid (r = 0.670), valine (r = 0.522), methionine (r = 0.537), isoleucine (r = 0.469), leucine(r = 0.570), tyrosine(r = 0.540), phenylalanine (r = 0.569), ornithine (r = 0.513) and lysine (r = 0.471) was found.
A significant positive correlation between GGT and MCV (r = 0.422) was found for the older group of patients only (Table 8).
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Age and MCV
In the younger age group no significant correlation between MCV and AAs was found. In the older group positive correlations between MCV and phosphoserine (r = 0.543) and citrulline (r = 0.626) were found. Conversely negative correlations of MCV with phosphoethanolamine (r = –0.596) and lysine (r = –0.528) were found.
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Discussion |
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The high levels of glutamic acid and proline in BrAl+/ patients can be attributed to drinking. In the glutathione cycle, proline and glutamate are related insofar as 5-oxoproline is converted to glutamate by an ATP-dependent reaction. High citrulline levels in older male BrAl–/ patients might be a sign for a more active NO biosynthesis in older alcohol-dependent patients. Citrulline is formed during the biosynthesis of NO, depending on the availability of glutamate, which stimulates the NOS via the NMDA receptor. In the present study, plasma citrulline levels further correlated positively with high MCV values, suggesting that high MCV might be partially due to impairment of NO metabolism. Oxidative damage to cells has been postulated to be a major contributor to the ageing of diverse organisms. Removal of damaged proteins declines with increasing age, thus contributing to the accumulation of damaged proteins and amino acids in the plasma. Furthermore an intense urea cycle produces many AAs as metabolites, like citrulline, ornithine and aspartic acid. Based on our results, it can be hypothesized that alcohol might act as a substance, which artificially accelerates the ageing process.
Glutamic acid is involved in a variety of metabolic processes. Its neurotoxic effects seem to be due to overstimulation of the ion channel receptors, leading to excessive intracellular levels of Ca2+ as well as to mitochondrial damage (Sriram et al., 1998). These glutamate-induced neurotoxic effects occur during alcohol withdrawal and are held responsible for some typical withdrawal symptoms, like anxiety, seizures, hyperexcitability and neuronal death (Dahchour and De Witte, 2003b). Furthermore, it has been reported that homocysteine induces neuronal cell death by stimulating N-methyl-D-aspartate receptors as well as by producing free radicals. Elevated plasma homocysteine levels have been shown to be related to cognitive deficits in patients during early alcohol withdrawal (Wilhelm et al., 2006). Low levels of aminobutyric acid were found in all BrAl+/ patients, but only female BrAl+/ patients had higher glutamine levels, indicating a sex difference. This finding may hint to one of the factors that contribute towards the increased damage alcohol causes in women compared to men. In the <50 years group, male participants had higher levels of valine than female participants. This may be due to the presence of high glutamic acid levels, whereby glutamate is the donor of an amino group to valeric acid, thus forming valine. Histidine was lower in all females and in the older BrAl–/ patients. This might represent a sex and an age effect, suggesting a smaller buffering capacity.
Older male BrAl–/ patients showed not only higher citrulline levels but also higher cystine and tyrosine. Cystine is related to methionine (in the older group positively related to high GGT) and serine (higher in all BrAl+/). The cysteine homologue homocysteine enhances the risk of withdrawal seizures (Bleich et al., 2000a, 2000b; Bayerlein et al., 2005). Homocysteine was further found to enhance the vulnerability of neuronal cells to excitotoxic and oxidative injury in vitro and in vivo and has already been described to be elevated in actively drinking alcohol-dependent patients and in social drinkers (Bleich et al., 2001, 2003). Raised plasma levels of homocysteine are associated with hippocampal atrophy in alcoholism (Bleich et al., 2003). AAs such as tyrosine can enhance the dopamine synthesis in nigrostriatal neurons, if the firing rates of these neurons are accelerated (Melamed et al., 1980). This finding might be related to the short abstinence of maximum 2 days, though the active drinking group did not show this difference, neither in men nor in women. The finding is robust, especially given that BrAl+/ and BrAl–/ participants combined show higher cystine and tyrosine in the 50 group.
Correlations of GGT with glutamic acid, glutamine and aspartic acid are all indicators of alcohol-induced liver damage, ammonia formation and urea cycle, forming other AAs such as e.g. citrulline, ornithine and isoleucine, which were all found to be high in the older patients group.
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References |
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