Gender-Related Differences in the Relationship between Homovanillic Acid in the Cerebrospinal Fluid and Clinical Symptoms in Suicide Attempters

Introduction: Decreased dopaminergic activity – as reflected by lower levels of the major metabolite homovanillic acid (HVA) in cerebrospinal fluid (CSF) – may be involved in the pathophysiology of attempted suicide. An inverse association has also been found between dopaminergic activity and clinical symptoms of depression and anxiety in non-suicidal individuals. The aim of this study was to assess the relationship between CSF-HVA and clinical symptoms associated with an increased risk of suicide in individuals who attempted suicide. Methods: Ninety-five people (52 women; 43 men) who had recently attempted suicide received lumbar punctures to analyse levels of HVA in the CSF. They were also evaluated with the Comprehensive Psychopathological Rating Scale, from which scores on the Montgomery-Åsberg Depression Rating Scale (MADRS), the Brief Scale of Anxiety (BSA), and an item on suicidal thoughts were analysed. Results: Among female participants, CSF-HVA was significantly and negatively correlated with BSA total scores, after adjusting for covariates (beta = −0.442, p = 0.002), but not with scores on the MADRS or suicidal thought item. No significant correlations were observed between CSF-HVA and symptoms among male participants. Conclusion: Our findings suggest that lower dopaminergic activity may be associated with clinical symptoms of anxiety among women who have recently attempted suicide.


Introduction
Suicide is a major public health issue worldwide and one of the leading causes of death among young people [1]. Every year, 700,000 persons die as a result of suicide, and there are many more people who attempt suicide. However, based on results of previous studies, above 2% of the traffic accidents are actually suicidal behaviours, indicating that this is underreported [2]. Suicide is a complex, multicausal phenomenon, involving several psychiatric, psychosocial, demographic, genetic, and cultural components. Among psychiatric factors, major depressive disorder (MDD) or more severe depressive symptoms have been repeatedly linked to suicide [3]. There is also some evidence that pharmacological treatments like antidepressant and physical exercise known to improve depressive symptoms linked may decrease the risk of suicide in psychiatric patients or in the general population [3].
karger@karger.com www.karger.com/nps A suicide risk factor found being independent of psychiatric diagnosis is the history of attempted suicide, making individuals who have attempted suicide an important group for suicide research [4][5][6]. To develop new treatments and methods of identifying persons with a high risk of completed suicide, researchers have begun exploring the neurobiological mechanisms underlying attempted suicide. Despite extensive searching, a reliable biomarker has not been found; one reason may be that the mechanisms involve various biological and psychological systems, including the dopaminergic system.
Dopamine is a neurotransmitter that plays a key role in the modulation of human emotions, including reward motivation and the ability to experience pleasure [7]. Multiple lines of evidence indicate that dopaminergic system dysfunction is involved in the pathophysiology of attempted suicide. Specifically, lower levels of dopamine and its main metabolite homovanillic acid (HVA) in urine or cerebrospinal fluid (CSF) have repeatedly been found in individuals with a recent suicide attempt, as compared to controls [8,9]. For instance, a meta-analysis of 22 studies showed that individuals with attempted suicide experience lower dopaminergic activity, as indicated by levels of CSF-HVA [8]. To better understand the role of the dopaminergic system in attempted suicide, several authors have examined the relationship between CSF-HVA and known risk factors for attempted suicide like use of a violent method; history of attempted suicide; clinical diagnoses of MDD, dysthymic disorder, or adjustment disorder; or the severity of depressive symptoms [10][11][12][13][14][15]. However, none of these studies found a link between CSF-HVA and these risk factors [10][11][12][13][14][15].
Promising evidence has been found in non-suicidal populations, where lower CSF-HVA may be correlated with more severe psychiatric symptoms. A negative correlation was observed between lower CSF-HVA and the severity of depressive symptoms among depressed individuals and in individuals with HIV [16][17][18][19]. Low levels of CSF-HVA have also been linked to more severe cognitive symptoms in people with schizophrenia, as well as to more severe anxiety in people with post-traumatic stress disorder [20,21]. These findings are interesting because more severe clinical symptoms are linked with an increased risk of completed suicide among individuals who have attempted suicide [22].
The aim of this study was to assess the relationship between CSF-HVA and clinical symptoms of depression, anxiety, and suicidal ideation, all of which are known risk factors of attempted suicide. As both CSF-HVA changes in attempted suicide and suicidal behaviour appear to be independent of the psychiatric diagnosis [8], we chose to investigate our hypotheses regardless of clinical diagnosis. The specific aims of the study were as follows: 1. To investigate the possible association between CSF-HVA and clinical symptoms in individuals who attempted suicide 2. To investigate the possible association between CSF-HVA and specific clinical symptoms such as anxiety and depressive symptoms in individuals who attempted suicide 3. To investigate whether there is a relationship between CSF-HVA and suicidal thoughts in individuals who attempted suicide

Participants
Participants were recruited between 1988 and 2001 at the University Hospital in Lund, shortly after a suicide attempt. The criteria for inclusion were as follows: • Admitted to the Medical Emergency and Intensive Care Unit (MEIU) after an attempted suicide. Suicide attempts were considered as "situations in which a person has performed an actually or seemingly life-threatening behaviour with the intent of jeopardizing his/her life, or to give the appearance of such an intent but which has not resulted in death" [23] • Referred to the psychiatric research ward from the MEIU and admitted for at least 1 week The criteria for exclusion were as follows: • Discharged before being given information and opportunity to consent to the study • Exhibited an urgent need for psychiatric treatment (i.e., washout was not possible): examples for conditions with urgent need are psychotic symptoms with immediate need for antipsychotic medication or severe withdrawal symptoms with substance abuse disorders like delirium tremens • Did not receive successful lumbar puncture • Had missing information on smoking status, the Comprehensive Psychopathological Rating Scale (CPRS) or washout period Ninety-five participants fulfilled these criteria. All participants gave their informed consent to participate in this study. This study was approved by the Lund University Medical Ethics Committee (LU14-1988).

Procedures
Psychiatric diagnoses were set by consensus by 2 trained psychiatrists after they independently diagnosed the participants according to the Diagnostic and Statistical Manual of Mental Disorders, Third Edition-Revised (DSM-III-R) [24]. All participants were given a general physical examination, including measurement of height and weight to calculate body mass index (BMI). A complete medical history was taken, including questions about current diseases and smoking status. No participant had a severe medical illness (e.g., autoimmune diseases or cancer). Participants did not receive any antidepressant, antipsychotic, attention-deficit/hyperactivity disorder, or mood stabilizing medication during a washout period of 15 ± 8 days (mean ± standard deviation [SD]) after the suicide attempt. Occasional doses of benzodiazepines were permitted; however, no participant received a benzodiazepine for at least 9 h before the lumbar punctures. At the end of the washout period, CSF samples were collected in a standardized manner. On the same day, participants were evaluated with the CPRS [25]. The clinical and demographic characteristics of the sample are presented in Table 1.

Comprehensive Psychopathological Rating Scale
On the day of the lumbar punctures, participants' psychiatric symptoms were evaluated using the CPRS [25]. CPRS is developed to assess the current psychiatric state and rating the severity of psychiatric symptoms and observed behaviour. The CPRS consists of 65 items, of which 40 are self-reported and 25 are clinicianreported (observed) [25]. The items are rated on a scale of 0-3 (with half-point intervals) and assessing symptoms such as psychotic symptoms, depressive and anxiety symptoms [25]. Rating is performed through a flexible interview where the psychiatrist asks open and general questions, and the participant is encouraged to describe his/her condition in his/her own words. The rating takes about 15-20 min. From the CPRS, more specific measures of psychopathology can be derived. The Montgomery-Åsberg Depression Scale (MADRS) can be derived to assess the severity of depressive symptoms [26]. The scale consists of 10 items presented in Table 2. To assess anxiety symptoms, the Brief Scale of Anxiety (BSA) can be derived [27] which also consists of 10 items. The items are presented in Table 2. For the MADRS and BSA, the 3point CPRS scales are converted to 6-point scales (thus, every item is rated from 0 to 6). To assess suicidal ideation (Table 2), a single item from the CPRS (suicidal thoughts) was used; participants were grouped according to whether they had 0 points (no suicidal thoughts) or >0 points (had suicidal thoughts) on this item.
Lumbar Puncture and HVA Analysis Lumbar punctures were performed after a mean washout period of 15 ± 8 days (mean ± SD). A 12 ± 6 mL sample of CSF was collected in a standardized manner. Spinal taps were performed in a sitting position in the morning between 8:00 and 9:00 a.m. after one night of fasting and bed rest. The CSF was immediately centrifuged and stored in 2 mL portions at −80°C until assayed for HVA. The same portion was used for analyses. HVA was analysed in CSF according to the method described in an earlier study [12].

Statistical Analyses
All statistical analyses were conducted using IBM SPSS Statistics 21.0. Because there is evidence of gender differences in suicide risk factors and dopaminergic activity, we tested our hypothesis in subgroups according to gender [28][29][30]. Data regarding age, washout period, and BMI were not normally distributed even after log-transformation in both female and male participants. To be able to use these variables in covariance analyses, we chose to dichotomize them, using the median as the cut-off (age: 39.0 years; washout period: 14.0 days; BMI: 23.5 kg/m 2 ). To evaluate NOS, not otherwise specified; SD, standard deviation; MDD, major depressive disorder; BMI, body mass index; CPRS, Comprehensive Psychopathological Rating Scale; MADRS, Montgomery-Åsberg Depression Rating Scale; BSA, Brief Scale of Anxiety; CSF, cerebral spinal fluid; HVA, homovanillic acid. a Cyclothymia (n = 1), obsessive compulsive disorder (n = 1). b Data on axis II disorders in participants are missing. c Trombyl (n = 1), insulin (n = 1), atenolol (n = 1), furantadine (n = 1), iron tablet (n = 2). d Terbutaline (n = 1), omeprazole (n = 1), metoprolol (n = 1).

CSF-HVA and Clinical Symptoms in Attempted Suicide
Neuropsychobiology 2023;82:179-185 DOI: 10.1159/000529803 differences between male and female participants in clinical symptoms, CSF-HVA, age, BMI, washout period, and smoking status, we used Student's t test (linear and dichotomous data) and the χ 2 test (dichotomous data) that were performed.
To examine the correlation between CSF-HVA and clinical symptoms, we used linear regression analyses with CSF-HVA as the dependent variable and clinical symptoms as the independent variable. Age, BMI, washout period, smoking status, and MDD were entered into the regression analyses as independent variables when assessing the relationship between CSF-HVA and the total scores of the CPRS, MADRS, and suicidal thoughts. To evaluate the relationship between CSF-HVA and BSA scores, the presence of anxiety disorders was also entered into the regression analyses as an independent variable. Two-tailed significance levels were used, and the alpha level of significance was set at p < 0.05. Because we performed multiple statistical analyses with the same variables, we used Bonferroni corrections in evaluating the relationship between CSF-HVA and clinical symptoms to reduce the risk of falsepositive results; thus, p < 0.006 was considered significant.

Comparison of CSF-HVA, Confounders, and Clinical Symptoms between Male and Female Participants
No significant differences were found between female and male participants with respect to CSF-HVA (p = 0.103), BMI (p = 0.490), age (p = 0.435), washout period, smoking status (p = 0.665), clinical diagnosis (p = 0.501), or clinical symptoms as measured by the total scores of the Anger, hostility, and aggressive feelings regardless of whether they are acted on or not Worrying over trifles Apprehension and undue concern over trifles, which is difficult to stop and out of proportion to the circumstances Aches and pains Representing reports of bodily discomfort, aches, and pains

CSF-HVA and Clinical Symptoms
Among female participants, CSF-HVA level was significantly correlated with the total BSA scores (p = 0.002), after adjustment for covariates (Table 3). No significant correlations were found between CSF-HVA level, suicidal thoughts, total scores on the MADRS (p > 0.006), or CPRS among women (Table 3). No significant correlations between CSF-HVA level and any of the clinical symptoms were observed among male participants (p > 0.006) ( Table 3).

Discussion
We investigated the relationship between the dopamine metabolite HVA in CSF and clinical symptoms associated with an increased risk of suicide in individuals who had recently attempted suicide. We found that lower CSF-HVA levels were significantly correlated with higher scores on the BSA after adjustment for BMI, age, washout period, smoking status, MDD, and anxiety disorders among female participants, but not among male participants.
As far as we know, there is only one previous work to compare our results with. In that study, in line with our results, Jokinen and colleagues did not observe any significant correlation between CSF-HVA and the total scores of the MADRS among 15 men with a recent suicide attempt [13]. Interestingly, among non-suicidal individuals, Saloner et al. [18] did not find a significant association between the total scores of depressive symptoms assessed by the Beck Depression Inventory-II (BDI-II) and CSF-HVA in cohorts with male dominance (90%) of HIV-seropositive and HIVseronegative individuals. However, in contrary to these results, Faustman et al. [16] involving 21 male patients with MDD observed a significant negative correlation between CSF-HVA and the depression subscale of the Minnesota Multiphasic Personality Inventory (MMPI). A possible explanation for this discrepancy may be due to the difference in the methodology of the measurement of depressive symptoms. Specifically, in studies that did not observe significant correlation between depressive symptoms and CSF-HVA, depressive symptoms were assessed by psychometric instruments developed to specifically measure the symptom severity of depression (MADRS and BDI-II) and the total scores of these scales were used. Faustman et al. used the depression subscale of the MMPI, which was developed to assess personality traits [16]. Taken together, we think that it is possible to conclude that CSF-HVA may not be involved in the pathophysiology of depressive symptoms in attempted suicide among men. Neither did we find an association between depressive symptoms and CSF-HVA in women with attempted suicide. As far as we know, this is the first study to investigate this association among women, and therefore, there is no previous study to compare our result with. Before concluding the absence of the association between CSF-HVA in women who attempted suicide, further studies are needed.
To the best of our knowledge, this is the first study to investigate the correlation between CSF-HVA and anxiety symptoms among individuals with attempted suicide. Our results indicate that lower CSF-HVA may be associated with more severe anxiety independently of covariates among women who attempted suicide but not among men. The gender difference in the association between CSF-HVA and anxiety symptoms may be not surprising, given that psychiatric disorders are known to show significant gender differences in susceptibility, prevalence, and presentation [31]. Anxiety-related disorders are known to be more common among women than among men [31]. On the other hand, multiple factors explain the gender-related differences in suicidal behaviour, including psychosocial life stressors, sexual abuse, and the duration of the suicidal process [30]. In addition, pre-clinical and clinical studies indicate that the structure and functioning of the dopaminergic systems are intrinsically different between men and women [28]. Stress-related alterations in the dopaminergic system also appear to differ according to gender [28]. Taken together, the gender difference in the relationship between lower CSF-HVA (reflecting lower dopaminergic activity) and anxiety among women with attempted suicide is plausible. Summarizing our results, CSF-HVA may be correlated to the severity of anxiety symptoms but not to other clinical symptoms in attempted suicide. As well, the correlation between CSF-HVA and anxiety symptoms may be limited to female attempted suicide. Our study may have some implications for researchers. Firstly, our findings raise the question whether CSF-HVA could be used as a biological marker of the severity of anxiety symptoms among women who attempted suicide. Another research question may be whether pharmacological treatments with the effect to increase dopaminergic activity, like bupropion, could decrease anxiety among women with attempted suicide. Clinical studies to test this hypothesis are warranted.

Limitations and Strengths
There are several limitations in this study. The main limitations are the small sample size and the absence of healthy controls. However, the stress experienced by individuals with a recent suicide attempt is of a magnitude that makes them unsuited for comparison with healthy individuals for the purposes of detecting within-group differences. There is also a substantial risk of other confounding effects that we did not take into account, such as physical activity, which has been suggested to influence dopaminergic function [30]. On the other hand, an important strength of this study is the fact that participants were not undergoing psychopharmacological treatment. This is important because several psychiatric medications can influence CSF-HVA levels [32,33]. Furthermore, generalizability of the results to a wider population of individuals who attempted suicide is limited as this sample consists only of individuals who were admitted to a MEIU as a consequence of attempted suicide. As patients who exhibited an urgent need for psychiatric treatment were excluded, our findings cannot be applied to this group, which is a further limitation of our study.

Conclusions
Our findings suggest that lower dopaminergic activity may be involved in the pathophysiology of anxiety in female individuals who have attempted suicide. Future studies can further examine and clarify the relationship between dopamine and anxiety symptoms among female suicidal individuals.