CSF monoamine metabolites and MRI brain volumes in alcohol dependence

Psychiatry Research: Neuroimaging 122 (2003) 21–35

CSF monoamine metabolites and MRI brain volumes in alcohol dependence Ingrid Agartza,b,*, Susan Shoafa, Robert R. Rawlingsa, Reza Momenana, Daniel W. Hommera a Section on Electrophysiology and Brain Imaging, Laboratory of Clinical Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892-1256, USA b Department of Clinical Neuroscience, Clinical Alcohol and Drug Addiction Research Section and Human Brain Informatics, Karolinska Institute, SE-171 76 Stockholm, Sweden

Received 23 October 2001; received in revised form 8 May 2002; accepted 11 September 2002

Abstract Correlations between cerebrospinal fluid (CSF) concentrations of monoamine metabolites (MAM) and brain structure have been described in schizophrenia, but not in alcoholism. To investigate the relationship between monoaminergic transmission and brain structure in alcoholism, the metabolites of dopamine (homovanillic acid, HVA), norepinephrenine (3-methoxy-4-hydroxyphenylethyleneglycol, MHPG) and serotonin (5-hydroxyindoleacetic acid, 5-HIAA) were measured in lumbar CSF in 54 alcohol-dependent patients and 20 healthy subjects. The volumes of the cerebrum, total grey and white matter, total and ventricular CSF, left and right hippocampus, and corpus callosum area were measured with MRI. MHPG and age were positively correlated in alcoholic women. The MAM concentrations were not significantly correlated with the MRI volumes in the subject categories. There were no differences in MAM across subjects defined by diagnosis and gender, age of onset of alcoholism or comorbidity of psychiatric disorders. Total CSF, cerebrum, and white and grey matter tissue volumes differed between patients and healthy subjects. The greatest difference was the white matter reduction in alcoholic women. In alcoholic women and men, monoaminergic neurotransmission measured by the CSF MAM HVA, MHPG, and 5-HIAA is not significantly correlated with the size of different brain structures. 䊚 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Magnetic resonance imaging; Tissue segmentation; 5-Hydroxyindoleacetic acid; Homovanillic acid; 3-Methoxy-4hydroxyphenylethyleneglycol; Cerebrospinal fluid

*Corresponding author. Department of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, S-171 76 Stolckholm, Sweden. Tel.: q46-8-517-717-58; fax: q46-8-517-717-17. E-mail address: [email protected] (I. Agartz). 0925-4927/03/$ - see front matter 䊚 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 5 - 4 9 2 7 Ž 0 2 . 0 0 0 8 4 - 7

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1. Introduction The concentration of monoamine metabolites (MAM) in the cerebrospinal fluid (CSF) reflects changes in the synthesis and release of the neurotransmitters dopamine, norepinephrine and serotonin in the brain. Deviant concentrations of mainly the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) have been reported in subtypes of alcoholics. Brain imaging and postmortem examinations of alcohol-dependent individuals show structural brain abnormalities. Therefore, it is of interest to explore the relationship between CSF MAM concentrations and brain structure in alcohol dependence. 1.1. CSF MAM in alcoholism Late-onset alcoholism or type 1 is characterized by a late age of onset of heavy drinking, weak heredity and personality traits such as anxiety. Early-onset alcoholism (defined as onset of heavy alcohol consumption before 25 years of age) or type 2 affects 25% of all male alcoholics. Earlyonset alcoholics are frequently characterized by a history of hyperactivity, antisocial personality traits, alcoholic fathers, lack of risk reduction with a functional adoptive family, lower mean platelet monoamine oxidase activity, and a strong urge to consume alcohol in response to an intravenous infusion of the serotonin 2C receptor agonist metachlorophenyl-piperazine (Hommer et al., 1997; George et al., 1997). They have been shown to have a more severe course of alcoholism with a lower mean CSF 5-HIAA concentration, but not homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), or tryptophan, than late-onset alcoholics (Fils-Aime et al., 1996). In the study by Fils-Aime et al., alcoholism in both parents was found to predict particularly low mean CSF 5-HIAA, HVA and tryptophan concentrations. Irwin et al. (1990) found that, regardless of family history, the main factor defining the course and characteristics of an alcoholic seeking treatment was the age of onset. Serotonergic disturbances and loss of control are ˚ common features in suicide attempters (Asberg, ¨ 1997; Traskman-Bendz et al., 1993), violent

offenders (Linnoila et al., 1994; Linnoila and Virkkunen, 1997) and alcoholics (Ballenger et al., 1979). Studies of early-onset, violent Finnish alcoholics have shown that impaired impulse control is associated with low mean CSF 5-HIAA (Linnoila et al., 1994; Virkkunen et al., 1996). Among alcoholic violent offenders and fire setters, low 5HIAA and HVA concentrations have been strongly associated with a family history positive for alcoholism (Virkkunen et al., 1996). Recidivist violent offenders and fire setters were predicted by low CSF 5-HIAA and MHPG concentrations. The lower the CSF 5-HIAA and MHPG concentrations, the more likely the offender was to become a recidivist. Limson et al. (1991) found significant negative correlations between aggression measures and CSF 5-HIAA and HVA concentrations but not MPHG or norepinephrine in alcoholic subjects. Depressed alcoholics, compared with neverdepressed alcoholics, have been shown to have a higher daily alcohol intake, more lifetime diagnoses of other anxiety and affective disorders and drug abuse, more suicide attempts, and more reported alcoholism in both parents. They also have significantly lower CSF levels of HVA and gamma-aminobutyric acid (Roy et al., 1991a) but not CSF 5-HIAA. Low mean CSF 5-HIAA concentration was found in depressed alcoholics with alcoholic relatives compared to depressed alcoholics without alcoholic relatives. Among alcoholics, CSF variables may lack seasonal variation (Roy et al., 1991b). Reduced concentrations of arginine–vasopressin and MHPG but not 5-HIAA or HVA or somatostatin in lumbar CSF in amnesic patients with Korsakoff psychosis have been reported (Mair et al., 1986). Altered dopamine function in pathological gamblers reflected in HVA and MHPG (but not 5-HIAA) concentrations has been reported (Bergh et al., 1997) and thought to reflect signs of arousal and changes in reward sensitivity. An important state variable in alcoholism is withdrawal. Elevated CSF 5-HIAA concentrations have been suggested to last a minimum of 2–3 weeks after acute detoxification of alcoholics (Banki, 1981; Borg et al., 1985). Some, but not all, research groups have reported that alcoholics who have been abstinent for 2–4 weeks have

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lower CSF 5-HIAA concentrations. Based on studies on the duration of the biochemical effects of antidepressant withdrawal in humans, it has been commonly judged to be safe to start investigations on neurotransmitter metabolism in alcoholics no sooner than after 21 days of abstinence (Linnoila, 1988).

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et al., 1996, 2001; Agartz et al., 1999). These studies of alcohol-dependent subjects have suggested that alcoholic women have an equal or greater degree of brain damage than alcoholic men despite fewer years of heavy drinking. However, not all studies have found evidence for a gender difference in the vulnerability of the brain to the effects of alcohol (Pfefferbaum et al., 2001).

1.2. Brain structure changes in alcoholism In previous studies our research group and other investigators have demonstrated tissue volume deficits in the brains of chronic alcoholics. Postmortem investigations have shown decreased brain weight (Harper and Blumbergs, 1982), reduced white matter (Badsberg-Jensen and Pakkenberg, 1993; Harper, 1998) and decreased neuronal density of the cortical grey matter with selective neuronal loss in the superior frontal cortex (Kril et al., 1997; Kril and Halliday, 1999; Harper and Kril, 1989, 1991). In a postmortem study of the hippocampus in chronic alcoholics, hippocampal volume reduction was found exclusively in the white matter (Harding et al., 1997). Heavy drinking has been shown to accelerate age-related myelin loss (Wiggins et al., 1988). Brain imaging studies have demonstrated a reduction of cortical grey and white matter volume in alcoholism (Jernigan et al., 1991; Mann et al., 1995; Rosenbloom et al., 1995; Sullivan et al., 1998; Pfefferbaum et al., 2000; Hommer et al., 2001) that becomes further reduced with increasing age (Pfefferbaum et al., 1992). Recovery with abstinence appears greatest in the first weeks of sobriety (Pfefferbaum et al., 1997). Hippocampal volume reductions on MRI have been reported in chronic alcoholics (Sullivan et al., 1995), but the reduction of the hippocampus is proportional to the reduction of the volume of the rest of the brain (Agartz et al., 1999). The reduction of the corpus callosum appears greater in alcohol-dependent women than in alcohol-dependent men (Hommer et al., 1996). Women achieve higher peak blood-alcohol levels than men on the same alcohol dose (Jones and Jones, 1982; Thomasson, 1995). A number of imaging studies have investigated gender-specific vulnerability of the brain to alcohol (Mann et al., 1992; Jacobson, 1986; Kroft et al., 1991; Hommer

1.3. Associations between MAM and brain structure in schizophrenia Among schizophrenic patients, an inverse relationship between ventricular size and CSF MAM has been described by several investigators. Potkin et al. (1983) reported that although no overall difference in CSF 5-HIAA was found between schizophrenic patients and control subjects, schizophrenic patients with enlarged ventricles on computed tomography (CT) had significantly lower 5-HIAA concentrations. Ventricular size correlated ¨ et inversely with 5-HIAA concentrations. Nyback al. (1983) reported no differences in mean CSF levels of MAM between acute schizophrenic patients compared to healthy volunteers, but the patients had significantly wider lateral and third ventricles on CT. The patients, but not the healthy volunteers, demonstrated a significant negative correlation with lateral ventricular size and levels of HVA and 5-HIAA in the CSF. In agreement with this, van Kammen et al. (1983) reported lower CSF levels of HVA and dopamine-b-hydroxylase in schizophrenic patients with evidence of brain atrophy and high ventricle-to-brain ratios compared with patients with normal CT scans. The failure of MHPG levels to correlate with ventricular size was thought to reflect the fact that a large part (25–59%) of CSF MHPG in humans is derived from peripheral norepinephrine metabolism (Kopin et al., 1984). Similarly, Jennings et al. (1984) found that psychotic adolescents had significantly negative correlations between ventricle-brain ratio and CSF HVA and between ventricle-brain ratio and 5-HIAA. They suggested that a relationship between ventricle-brain ratio and MAMs appears to occur in psychoses other than schizophrenia, is present early in the course of the psychotic illness and probably does not represent

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a dilutional or neuroleptic effect. Elevated CSF alanine in schizophrenic subjects has been found to be highly correlated with ventricular enlargement on CT (Reveley et al., 1987). Weinberger et al. (1988) demonstrated the strong correlation between CSF HVA and 5-HIAA concentrations and relative prefrontal regional cerebral blood flow (rCBF) in chronic schizophrenic subjects primarily under behavioral conditions. Lower concentrations of HVA appeared related to lower prefrontal activity. In this study, cerebral ventricular size was inversely correlated with prefrontal rCBF. Breier et al. (1993) reported the prefrontal volumes of schizophrenic subjects to be significantly smaller than in healthy controls. The schizophrenic patients had significantly greater 2-deoxyglucose (2-DG) induced plasma HVA elevations and showed an inverse relationship between 2-DG induced peak changes in plasma HVA levels and the size of the right and left prefrontal cortex total volumes. 1.4. The present study The aim of the present study was to investigate the association between CSF MAMs and brain structure in alcoholism. The rationale for the investigation was that deviant CSF MAMs, particularly abnormally low levels of 5-HIAA in early-onset alcoholics, have been observed in alcoholism. Brain structure in alcoholics is pathologically changed. Of interest was that associations between deviant CSF MAM levels and brain structure abnormalities have also been found in schizophrenia. We therefore measured 5-HIAA, HVA and MHPG concentrations in lumbar CSF and investigated the relationship with total and regional brain tissue volumes on MRI in chronic alcoholdependent patients and healthy volunteers. To characterize MAM and brain structure in the current subject population, we also investigated potential differences in CSF MAM and MRI brain volume measures between alcoholics and control subjects, men and women. The hypothesis was that alcoholics who had been abstinent for at least 3 weeks would demonstrate mild reductions in 5-HIAA reflecting a decrease in serotonin turnover due to loss of brain tissue. We also hypothesized that in alcoholics,

MAM levels and brain tissue volumes would demonstrate a positive correlation, meaning that alcoholics with smaller relative brain tissue volumes would also have a lower monoamine turnover in the brain. We did not predict this occurrence in the healthy volunteers since we did not hypothesize a general relationship between the size of different brain compartments and CSF MAM concentration. Our alcohol-dependent patients were moderate to severe drinkers. Some were early-onset alcoholics who started to drink heavily before the age of 25 years, and some also had a depressive disorder in addition to alcohol dependence. Since alcoholics with early-onset alcoholism or comorbidity with depression or antisocial personality disorder have been reported to have lower CSF 5-HIAA concentrations, we specifically investigated the importance of comorbidity of psychiatric diagnoses and personality disorders. We also investigated the influence of age, gender, height, weight and measures of alcohol consumption on CSF MAM concentrations and MRI volumes. 2. Methods 2.1. Subject characteristics and demographics As shown in Table 1, 74 subjects (40 alcoholic men, 14 alcoholic women, 17 healthy men and 3 healthy women) participated in the study. The age range was 27–59 years. The patients were selected from recently abstinent alcoholics undergoing research and treatment on the inpatient research ward of the National Institutes of Health Clinical Center in Bethesda, MD, USA, between 1992 and 1997. All subjects were administered the Michigan Alcoholism Screening Test (MAST) (Selzer, 1971). Information on recent and chronic alcohol consumption, as well as alcohol-related behavior, was obtained from structured research questionnaires (Eckardt et al., 1978). Alcohol intake in the past 6 months (recent alcohol intake) was corrected for alcohol distribution volume (total body water, TBW) (Watson et al., 1980). The subjects were interviewed using the Structured Clinical Interview for the Diagnostic and

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Table 1 Demographic and drinking variables of alcoholic men and women and the healthy men and women Variables

Age (yrs) Education (yrs) Height (cm) Weight (kg) BMI (kgym2) TBWa ICV (ml)a Age of onset (yrs) Years of heavy drinking Recent drinking (kg) Recent drinkingyTBW (kgyl)b Lifetime drinking (kg) MAST

Alcoholic men

Alcoholic women

Healthy men

Healthy women

Mean (S.D.)

N

Mean (S.D.)

N

Mean (S.D.)

N

Mean (S.D.)

N

38.2 (7.8) 14.5 (2.4) 174.7 (7.2) 80.0 (11.8) 25.65 (3.06) 44.45 (4.71) 1383.7 (104.7) 26.5 (8.5) 11.7 (6.9) 1.797 (1.524) 0.36 (0.26) 531.5 (499.4) 43.2 (14.3)

40 39 34 34 34 34 40 40 40 40 34 40 40

38.6 (8.9) 14.1 (2.7) 164.4 (5.7) 63.2 (10.3) 23.15 (4.06) 31.07 (2.86) 1202.1 (93.3) 31.2 (10.5) 7.4 (6.2) 2.243 (1.369) 0.73 (0.46) 432.1 (493.1) 38.8 (8.3)

14 14 11 11 11 11 14 14 14 14 11 14 14

39.6 (10.1) 16.6 (2.4) 166.8 (3.2) 81.5 (9.9) 26.1 (3.2) 44.17 (5.24) 1355.0 (74.6) 0 0 0.628 (0.119) 0.21 (0.42) 17.7 (21.2) 0.8 (1.2)

17 16 11 11 17 11 17 12 12 17 11 17 17

37.7 (4.0) 15.0 (1.7) 165.7 (4.0) 70.1 (11.7) 25.43 (3.21) 35.76 (4.59) 1100.7 (101.0) 0 0 0 0 1.5 (2.1) 0

3 3 3 3 3 1 3 2 2 2 2 2 2

TBW: total body water was used to correct for individual differences in alcohol distribution volume (for reference, please see text); age of onset: the current age subtracted from the number of years of heavy drinking; years of heavy drinking: if the number of days drinking in the last month multiplied by the number of drinks in a day in the last 6 months multiplied by type of drink in grams )90, then sum those years; recent drinking: total number of days drinking in the last 6 months multiplied by number of drinks in a day in the last 6 months multiplied by type of drink in grams; lifetime drinking: number of years of drinking multiplied by 12 multiplied by number of days per month multiplied by average number of drinks multiplied by type of drink in grams; MAST: the Michigan Alcohol Screening Test. a Gender effect at P-0.001, ANOVA. b Among alcoholic subjects there was a gender effect at P-0.01, ANOVA.

Statistical Manual of Mental Disorders (SCID for DSM-III-R) (Spitzer et al., 1986) patient edition (SCID-P with psychotic screen) for axis I (clinical syndromes). The Structured Clinical Interview for DSM-III-R Personality Disorders was used to assess axis-II disorders. The results are presented in Table 2. The alcoholic patients met the DSM-III-R criteria for alcohol dependence (American Psychiatric Association, 1987). Patients who met the criteria for alcohol abuse, who suffered from a somatic disease (also as a complication of alcoholism), who had a previous history of delirium tremens or psychotic disorders, who had results on neuropsychological testing demonstrating an intelligence quotient of less than 80, or who had signs of early onset dementia or Korsakoff disease were excluded from the study. No subjects were thiamine deficient. Subjects with a history of intravenous drug use at any time during their life or any substance abuse disorder, other than alcohol or tobacco abuse or dependence, in the 6 months preceding admission were excluded. The healthy

non-alcoholic subjects had no psychiatric disorder meeting DSM-III-R criteria. On the basis of the subject’s history, physical examination, blood chemistry, and a negative urinary drug screen, all subjects were judged to be medically healthy. Weights were collected within 1–3 days from the MRI examination. The intracranial volume (ICV) was obtained as a volumetric measure calculated from MR images. Nutritional status was assessed by measuring the total protein, albumin, transferrin, and mean corpuscular volume in serum at the time of admission and MRI. The values were all within the reference range. None of the subjects had a history of head injury requiring hospitalization. The majority of the patients were actively drinking up to their hospitalization and were detoxified at the NIH Clinical Center. Patients who had been directly transferred from another hospital where they had been detoxified were scanned at least 3 weeks after their last alcohol use. Both MRI scans and CSF samples were obtained after at least 3 weeks supervised abstinence. The subjects that participated in the

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Table 2 Psychiatric comorbidity in 54 alcoholic patients defined by DSM-III-R and DSM-IV All patients

Alcoholic men

Alcoholic women

Axis-I clusters Mood disorders Substance dependenceyabuse PTSD Anxiety disorders Other axis-I diagnoses Sum

24 (1); 14 (2); 1 (3) 12 (1); 8 (2); 5 (3); 1 (4) 11 10 (1); 2 (2); 3 (3) 6 97

19 (1); 8 (2); 1 (3) 9 (1); 8 (2); 3 (3); 1 (4) 6 7 (1); 2 (3) 5 69

5 (1); 6 (2) 3 (1); 2 (3) 5 3 (1); 2 (2); 1 (3) 1 28

Personality disorders (axis-II) NOS No axis-II diagnosis (V71.09) Narcissistic Avoidant Obsessive–compulsive Borderline Dependent Passive–aggressive Antisocial Schizoid andyor schizotypal

20 15 14 13 11 9 8 5 5 5

12 7 11 6 5 4 4 2 5 1

8 8 3 7 6 5 4 3 0 4

Sum

105

57

48

The table presents in order of frequency the number of alcoholic subjects who received one or several diagnoses with the axis-1 clusters and the axis-2 disorders. The number within parentheses signifies the number of diagnoses within the axis-1 cluster that a certain number of subjects received.

current study have in part participated in previous MRI studies. They were selected for the current study because they had CSF MAM collected at the same time as an MRI scan. However, there was no a priori selection as to which subject would have both lumbar puncture and MRI and which subject would not. The protocol was approved by the IRB at the NIH. The study was conducted in accordance with the principles of the declaration of Helsinki (1964). All subjects provided written informed consent before the start of the study. 2.2. Magnetic resonance scan acquisition and analysis The subjects were examined in a 1.5 T MRI scanner (GE Medical Systems, Milwaukee, WI) using a fast spoiled-GRASS sequence. The brain was scanned in a gap-less series of high-contrast 2-mm-thick T1-weighted coronal images (pulse repetition time: 25 ms; pulse echo time: 5 ms). The images were acquired using a 256=256

matrix with a 240=240 mm2 field of view. Each volumetric brain originally consisted of 124 coronal slices. The voxel size was 0.9375 = 0.9375 = 2.0 mm3. A hand-driven cursor was used to de-skull the intracranial tissue on coronal sections. The ICV included the cerebrum and CSF spaces but excluded the cerebellum. The de-skulled volume was automatically segmented into CSF, brain grey matter and white matter. The algorithm for the segmentation of intracranial tissues utilizes information from the histogram of pixel intensities of the intracranial image (Momenan et al., 1997). From the segmented volume, the volumes of total brain, grey and white matter, and total and ventricular CSF were obtained. With the current MR image contrast resolution the hippocampus is practically isointense with some of the surrounding tissues and cannot be reliably segmented. Therefore, it was manually outlined. The coronal slices were resampled (Unser et al., 1991) to 1-mm-thick sagittal slices. The sagittal projection allowed the visualization of the

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boundary between the hippocampus and the amygdala, thus ensuring that the entire hippocampal volume could be measured (Agartz et al., 1999). Each contour in a slice was calculated as a volume, and all the volumes were added to include the total volume of the hippocampus on the right and left side. Since the lateral extension of the corpus callosum is difficult to determine on MRI, this structure was measured from the mid-sagittal slice only. The contour of the corpus callosum in the mid-sagittal slice was semi-automatically detected to separate it from the rest of the white matter. The corpus callosum area in this slice was then automatically calculated. 2.3. CSF MAM analysis: procedures The standard procedure for obtaining CSF samples consisted of at least 3 weeks of supervised abstinence from alcohol on a locked research ward and a minimum of 3 days on a low-monoamine diet. The low-monoamine diet excluded certain beverages (e.g. chocolate drinks), fruits (e.g. bananas), nuts (e.g. walnuts), vegetables (e.g. tomatoes), meats (e.g. chicken livers), diary products (e.g. aged cheese), fish (e.g. smoked fish), and soft drinks sweetened with aspartame. Lumbar puncture was performed between 9.00 and 10.00 h, following overnight bed rest and fast. Approximately 32 ml of CSF was collected while the patient was in the left lateral decubitus position. The first 12 ml of CSF was collected as a single aliquot into a tube on wet ice, mixed thoroughly, aliquoted to 1-ml test tubes, and stored immediately at y70 8C. None of the samples was ever thawed before analysis. Concentrations of the major CSF MAM of norepinephrine (MHPG), dopamine (HVA), and serotonin (5-HIAA) were quantified with highperformance liquid chromatography (HPLC) by means of electrochemical detection (ECD) (Scheinin et al., 1983). Samples were thawed, 200-ml aliquots and 20 ml of the internal standard, F-HVA (650 pmol), were placed in 10 000 mW cut-off filters (Amicon, Beverly, MA), which were pre-rinsed with water to remove the glycerol, and centrifuged at 10 600=g for 20 min at 4 8C.

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CSF was analyzed using HPLC with ECD similar to that of Scheinin et al. (1983). The column used was a Microsorb Short-One, C-18 (Rainin, Woburn, MA) with a Guard-Pak precolumn containing Nova-Pak, C-18 (Waters, Milford, MA). The buffer was 0.05-M sodium acetate, 0.021-M citrate and 0.25-mM EDTA, pH 4.4. The organic was 5.5% (vyv) acetonitrile and 2% (vyv) methanol. The flow rate was 0.6 mlymin. Two liters of mobile phase was recirculated. Column temperature was 30 8C. The interday variation for each compound, MHPG, HVA and 5-HIAA, was less than 5%. The intraday variation was less than 3%. Standards of MHPG-hemipiperazinium salt, HVA and 5-HIAA were purchased from Sigma (St. Louis, MO). F-HVA was a gift from Kenneth Kirk (NIDDK, Bethesda, MA). 2.4. Statistical analysis A two-way analysis of variance (ANOVA) with gender and diagnosis as factors was performed on the demographic and descriptive measures. A oneway ANOVA with a gender factor was performed for alcoholics only on the drinking measures. Chisquared tests were performed on psychiatric diagnoses for alcoholics with gender and diagnosis as factors. Two-way ANOVAs were performed on the relative MRI volume measures (ratio of absolute MRI volume to ICV) to adjust for head size differences. Correlations between body mass index (BMI) and absolute MRI volume measures as well as between BMI and CSF MAM were performed. Since BMI was not correlated with CSF MAM, we did not use any correction factor for CSF MAM. One-way ANOVAs, with gender as a factor, were performed for alcoholics only for the MAM and relative MRI volume measures. Two-way ANOVAs with gender and posttraumatic stress disorder (PTSD) as factors were used to examine the effect of PTSD on CSF MAM, absolute and relative MRI volumes. Many of the above analyses were repeated with covariate adjustments.

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Table 3 Number of subjects (N) and mean, (standard deviation, S.D.) of absolute brain volumes in milliliters of alcoholic and healthy men and women Volumes

Alcoholic men

Total CSF Ventricles Total brain (cerebrum) Grey matter White matter Right hippocampus Left hippocampus Corpus callosuma a

Alcoholic women

Healthy men

Healthy women

Mean (S.D.)

N

Mean (S.D.)

N

Mean (S.D.)

N

Mean (S.D.)

N

278.88 (43.57) 20.82 (9.94) 1104.79 (93.97) 581.34 (49.74) 523.54 (46.68) 3.665 (0.500) 3.555 (0.605) 594.55 (85.06)

40 40 40 40 40 40 40 40

291.92 (48.30) 18.97 (8.15) 910.22 (71.92) 483.18 (39.31) 427.03 (34.840) 3.057 (0.327) 3.271 (0.425) 495.43 (83.66)

14 14 14 14 14 14 14 14

262.98 (28.20) 19.79 (6.45) 1090.04 (74.85) 573.77 (41.07) 518.27 (35.20) 3.873 (0.360) 3.702 (0.482) 612.417 (99.56)

17 17 17 17 17 17 17 17

211.46 (73.32) 9.88 (3.71) 889.28 (35.91) 478.74 (11.44) 410.55 (31.27) 2.966 (0.204) 2.952 (0.365) 584.33 (53.98)

3 3 3 3 3 3 3 3

Corpus callosum is an area measured in mm2.

Because of the number of tests performed, an alpha level of 0.01 was used in all of the analyses. Model adequacy was examined. Appropriate data transformations were performed to assure model adequacy. 3. Results 3.1. Demographic and descriptive data and drinking measures Of the demographic and descriptive variables (Table 1), TBW was lower in women (Fs78.40, d.f.s1, 55, P-0.001), by ANOVA, and the ICV was larger in men (Fs40.35, d.f.s1, 70, P0.001). There were no diagnosis effects for TBW or ICV. Other demographic measures did not differ significantly between alcoholic and healthy subjects, men and women. With respect to drinking measures among alcoholics, recent drinking corrected for TBW was

higher in women (P-0.01, Table 1). There were no significant gender differences for alcoholics for age of onset, recent drinking, years of heavy drinking, lifetime drinking or MAST. Recent drinking corrected for TBW by age regression in women alcoholics was not significant. Of the psychiatric diagnoses, PTSD only was significantly associated with gender in the alcoholic subjects (x2s7.0, d.f.s1, P-0.01), i.e. 15% (6y40) of the alcoholic men and 36% (5y14) of the alcoholic women had PTSD. Psychiatric comorbidity in the alcoholic subjects is presented in Table 2. 3.2. Correlations between CSF MAM and absolute MRI volumes and age No significant correlations between any of the absolute or relative MRI volumes (Table 3) and CSF MAM concentrations (Table 4) were found (Table 5). The subjects (ns74) were analyzed on a group (group categories: diagnosis and gender)

Table 4 Number of subjects, mean (standard deviation, S.D.), and range of CSF MAM (pmolyml) in alcoholic and healthy men and women Metabolites

N

Mean (S.D.)

Range

N

Mean (S.D.)

Range

MHPG HVA 5-HIAA

39 40 40

Alcoholic men 33.08 (8.00) 192.80 (89.84) 94.48 (33.82)

19.00–51.00 67.00–390.00 44.00–164.00

12 14 14

Alcoholic women 33.42 (10.00) 167.00 (77.02) 95.57 (45.07)

20.00–53.00 75.00–303.00 46.00–193.00

MHPG HVA 5-HIAA

17 17 17

Healthy men 38.71 (9.56) 207.29 (73.10) 86.18 (28.13)

20.00–59.00 100.00–405.00 47.00–137.00

2 3 3

Healthy women 37.50 (10.61) 256.67 (162.29) 111.67 (48.52)

30.00–45.00 140.00–442.00 64.00–161.00

Subject group

Alcoholic women Absolute MRI volumes

Relative MRI volumes

Alcoholic men Absolute MRI volumes

Relative MRI volumes

Healthy men Absolute MRI volumes

Relative MRI volumes

*

MAM

MRI volumes (r)

Area (r)

CSF

Ventricles

Brain

Grey matter

White matter

Right hippocampus

Left hippocampus

Corpus callosum

MHPG HVA 5-HIAA MHPG HVA 5-HIAA

y0.36 y0.48 y0.39 y0.13 y0.40 y0.31

y0.08 y0.33 y0.29 0.05 y0.29 y0.23

y0.39 y0.16 y0.14 0.07 0.35 0.30

y0.24 y0.06 y0.03 0.36 0.41 0.38

y0.54 y0.26 y0.26 y0.51 0.25 0.11

y0.59* y0.32 y0.24 y0.45 y0.11 y0.07

y0.03 y0.01 0.01 0.34 0.27 0.23

y0.57 y0.14 y0.15 y0.40 0.00 y0.01

MHPG HVA 5-HIAA MHPG HVA 5-HIAA

y0.11 y0.07 0.05 y0.11 y0.16 y0.13

0.00 y0.26 y0.11 0.01 y0.29 y0.17

y0.01 0.17 0.13 0.12 0.20 0.13

y0.01 0.16 0.29 0.09 0.13 0.07

y0.01 0.17 0.32* 0.13 0.22 0.20

y0.06 0.11 0.13 y0.02 0.03 y0.05

y0.14 0.14 0.15 y0.11 0.07 y0.02

0.02 0.09 0.07 0.07 0.05 y0.04

MHPG HVA 5-HIAA MHPG HVA 5-HIAA

y0.21 0.04 y0.22 y0.10 0.05 y0.04

y0.13 y0.21 y0.25 y0.13 y0.20 y0.20

y0.11 y0.03 y0.27 0.11 y0.06 0.02

y0.15 0.03 y0.21 y0.02 0.08 0.14

y0.06 y0.10 y0.35 0.29 y0.26 y0.16

y0.36 0.05 0.00 y0.21 0.05 0.19

y0.15 y0.16 y0.37 y0.06 y0.16 y0.22

y0.46 y0.33 y0.34 y0.41 y0.22 y0.15

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Table 5 Correlations (r) (Pearson) between CSF MAM and absolute and relative CSF, ventricular, brain, grey matter, white matter, and right and left hippocampus MRI volumes and the corpus callosum area in each of the three subject groups

Significant at P-0.05.

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Fig. 1. MHPG by age in alcoholic women. The positive correlation with age was significant (rs0.74, ns12, Ps0.006).

basis. The non-alcoholic female group (ns3) was disregarded because of the small sample size. With the alpha level of 0.01, there was a significant positive correlation between MHPG, and age in the alcoholic women (rs0.74, ns12, Ps0.006) (Fig. 1). In the alcoholic women, white matter was negatively correlated with MHPG, but the correlation was not significant. 3.3. Correlations between body measures and CSF MAM and absolute MRI volumes There were no significant correlations between BMI and CSF MAM or between BMI and absolute MRI values. 3.4. Intercorrelations between CSF MAM HVA and 5-HIAA were highly correlated in all subjects (rs0.82, ns74, P-0.001). For each of the subject categories except for the healthy women (too small sample size to be analyzed), the correlations were similar (alcoholic men: rs0.85, ns39, P-0.001; alcoholic women: rs0.86, ns

12, P-0.001; healthy men: rs0.85, ns17, P0.001). 3.5. Correlations between body measures and CSF MAM and absolute MRI volumes There were no significant correlations between BMI and CSF MAM or between BMI and absolute MRI values. Height and CSF MAM were not significantly correlated. 3.6. Diagnosis and gender differences in CSF MAM There were no significant effects (main effects or interactions) when CSF MAM were analyzed by two-way ANOVA with gender and diagnosis as factors. 3.7. Diagnosis and gender differences in relative MRI volumes Only the relative MRI volumes (absolute brain volume adjusted for ICV) were compared. Relative

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CSF volumes were significantly greater for the alcoholics (Fs9.47, d.f.s1, 70, Ps0.003). Relative brain (cerebrum), grey matter and white matter volumes were significantly smaller in the alcoholics (Fs9.47, d.f.s1, 70, Ps0.003; Fs 7.70, d.f.s1, 70, Ps0.007; and Fs9.00, d.f.s1, 70, Ps0.004, respectively). The gender effect for relative white matter volume was highly significant (Fs18.65, d.f.s1, 70, P-0.001) with smaller white matter volumes in women. Relative corpus callosum area was smaller in the alcoholics (Fs 9.31, d.f.s1, 70, Ps0.003). No significant effects were found for relative ventricular volume or for right and left hippocampus volumes. The same results were obtained if the absolute MRI volumes were analyzed with ICV as a covariate. 3.8. CSF MAM and relative MRI volumes in alcohol-dependent patients Among alcoholics, age at MRI, age of onset of heavy drinking and the drinking measures were analyzed as covariates. The only covariates that were significantly correlated with the dependent variables (MAM and relative MRI volumes) were the number of years of heavy drinking and age of onset. We then performed a one-way ANOVA with gender as a factor and covaried for the number of years of heavy drinking and age of onset. There were no significant effects for MHPG, HVA and 5-HIAA. The CSF volume ratio was greater in alcoholic women than in alcoholic men (Fs26.11, d.f.s1, 50, P-0.001). Relative brain, grey matter and white matter volumes were smaller in alcoholic women (Fs26.11, d.f.s1, 50, P-0.001; Fs11.09, d.f.s1, 50, Ps0.002; and Fs51.04, d.f.s1, 50, P-0.001, respectively). 3.9. Effect of PTSD on CSF MAM and MRI volumes in alcohol-dependent patients Two-way ANOVAs for alcoholic subjects only showed no statistically significant effects of PTSD on CSF MAM, or on absolute or relative MRI volume measures.

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4. Discussion In the present study, we did not find any significant relationship between MRI volumes (absolute and relative) and concentrations of the CSF MAM 5-HIAA, HVA and MHPG. The positive correlation between MHPG and age in the alcoholic women indicates higher noradrenergic activity at higher ages. We did not find the same relationship in the alcoholic or non-alcoholic men. During alcohol withdrawal, there is noradrenergic overactivity (Linnoila, 1988; Borg et al., 1980, 1986; Hawley et al., 1981, 1985) while low adrenergic activity has been reported in long-term abstinent alcoholics (Borg et al., 1983). Both central and peripheral noradrenergic overactivity is associated with symptoms of alcohol withdrawal (Borg et al., 1983). Elevated levels of MHPG decline from week 1 to week 3 of alcohol abstinence (Borg et al., 1981). One possible explanation for the increased noradrenergic response may be that women at higher ages (here in the range of 36–51 years) had been drinking greater amounts before detoxification, which might have led to more severe withdrawal from alcohol. However, the older women did not have a significantly higher recent alcohol consumption nor did they have a history of more withdrawal seizures than the younger women. We found no effects on 5HIAA, although 5-HIAA has been reported to change during withdrawal (Borg et al., 1985). The results suggest that at higher ages, the combination of reduced brain plasticity and the accumulated toxic effects of alcohol adversely affect the brain in such a way that it takes longer for MHPG to return to baseline values with abstinence. To confirm the observation in alcoholic women, the sample should be enlarged and the age span increased in the upper range (above 51 years, which was the age of our oldest female subject). To our knowledge, no previous study has addressed the recovery with abstinence from alcohol in alcoholic women, although there are studies on brain recovery in men (Pfefferbaum et al., 1995; Agartz et al., in press 2002). A second possible explanation is that women at higher ages have higher baseline MPGH levels than younger women have. Due to the small sample size of healthy women in

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the present study, we do not know if healthy women at higher ages show the same baseline levels as younger women or not. To our knowledge, there is no evidence for gender-dependent age correlations for MHPG in the scientific literature. Gender differences in MHPG have been investigated and not found (Thogi et al., 1993; Hartikainen et al., 1991). The reports on MHPG and age dependence are divergent. Thogi et al. (1993) reported that although total dopamine and norepinephrine among other monoamines and amino acid transmitters in the CSF increased significantly with age, concentrations of other monoamine precursors and metabolites (including MHPG) were unchanged. Hartikainen et al. (1991) found a positive correlation between MHPG and age, but they commented on the fact that a large portion of MHPG may be peripherally derived and thus not reflective of CSF conditions. In a review article, Davis (1989) cited nine studies of which four report an MHPG increase with increasing age, one reports a decrease, and the rest find no correlation. However, from studying 18 references on HVA and age, Davis concluded that there appears to be a tendency toward increasing concentration of HVA with increasing age. A third possible explanation is that a variability that we have not accounted for in the alcoholic population may be responsible for the positive correlation between MHPG and age in the alcoholic women. Significant differences in MAM concentrations between the subject groups were not found. Since there was no correlation between BMI and CSF MAM, we did not find it meaningful to correct CSF MAM for BMI. We did not find significant relationships between MAM and demographic variables (except between MHPG and age in alcoholic women) or with drinking measures. We also did not find significant associations between CSF MAM and the personality disorders. For instance, five alcoholic men had both antisocial personality disorder and an age of onset before 25 years of age but had CSF MAM concentrations within the normal range. PTSD was not related to CSF MAM values or MRI volume measures. A high positive correlation between HVA and 5-HIAA values is

˚ normally found (Agren et al., 1986) and was confirmed in the present study. The size of the ICV did not significantly differ between alcoholic subjects and healthy control subjects but was smaller in women than men. Therefore, in the comparisons of MRI volumes between the different subject groups, we examined the relative MRI volumes only. We found significant diagnosis effects for relative CSF, total brain, grey and white matter volumes with smaller brain tissue volumes and larger CSF volumes in the alcoholic subjects. This is in accordance with the results of previous investigators (Hommer et al., 2001). We found a diagnosis effect for the relative area of the corpus callosum (previously shown by Hommer et al., 1996) but not for relative ventricular and relative hippocampal volumes, although hippocampal volume reductions have been previously reported (Sullivan et al., 1995; Agartz et al., 1999). Ventricular volume is not markedly increased in alcoholism. Among alcoholics, relative brain, grey and white matter volumes were smaller in the alcoholic women and relative CSF volumes larger. The pronounced gender effect for the white matter volumes, with smaller volumes in alcoholic women, is interesting. It is known that the white matter is adversely affected by alcohol toxicity (Harper, 1998). In the present study, we carefully controlled for alcohol distribution volume. With this correction our alcoholic women were found to have significantly higher recent alcohol consumption than the alcoholic men. The white matter volume reduction among the alcoholic women may be explained either by the higher recent alcohol consumption or the fact that white matter in women is more vulnerable to the toxic effects of alcohol or a combination of both. This issue needs to be specifically addressed in further studies. The strength of this study is that it reports for the first time on the lack of relationship between the size of different brain structures and MAM concentrations in the CSF. MRI and MAM data were collected at the same point in time, which is advantageous considering the state-dependent characteristics of MAM concentrations in the CSF (Eklundh, 2000). To be able to maintain alcoholic patients in a strictly supervised abstinence from

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alcohol over at least a 3-week period is an advantage. A limitation is the small sample size (ns3) of healthy women. This makes within-gender comparisons impossible. In conclusion, there were no significant correlations between the studied MRI volumes and CSF MAM among middle-aged alcoholic women and men with moderate to severe alcoholism. Differences due to gender and diagnosis were found with respect to the MRI volumes, but contrary to several other investigators, we found no intergroup differences in MAM. Although MHPG did not differ between the groups, there was an increase in MHPG with increasing age among alcoholic women, which was not found in alcoholic men or healthy men. This finding suggests that alcoholic women at higher ages may be more vulnerable to changes in neurotransmission associated with withdrawal from alcohol than younger women but needs to be further investigated in a larger number of subjects. Acknowledgments We thank Erica Kaiser for performing the corpus callosum measures and the deshelling part of the segmentation procedure. The Fredrik and Ingrid Thurings Foundation and the Swedish MRC are acknowledged for generous financial contributions. References Agartz, I., Momenan, R., Rawlings, R., Kerich, M.J., Hommer, D.W., 1999. Hippocampal volume in patients with alcohol dependence. Archives of General Psychiatry 56, 356–363. Agartz, I., Hammarberg, A., Svinhufvud, K., Franck, J., Okugawa, G., Bergman, H. MR volumetry during acute alcohol withdrawal and abstinence: a descriptive study. Alcohol and Alcoholism, in press, 2002. ˚ Agren, H., Mefford, I.N., Rudorfer, M.V., Linnoila, M., Potter, W.Z., 1986. Interacting neurotransmitter systems. A nonexperimental approach to the 5-HIAA-HVA correlation in human CSF. Journal of Psychiatric Research 20, 175–193. American Psychiatric Association, 1987. Diagnostic and Statistical Manual of Mental Disorders, 3rd ed. rev. American Psychiatric Press, Washington, DC. ˚ Asberg, M., 1997. Neurotransmittors and suicidal behavior. The evidence from cerebrospinal fluid studies. Annals of the New York Academy of Sciences 836, 158–181. Badsberg-Jensen, G., Pakkenberg, B., 1993. Do alcoholics drink their neurons away? Lancet 342, 1201–1204.

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