Cerebrospinal fluid kynurenic acid in male and female controls – Correlation with monoamine metabolites and influences of confounding factors

Journal of Psychiatric Research 41 (2007) 144–151

JOURNAL OF PSYCHIATRIC RESEARCH www.elsevier.com/locate/jpsychires

Cerebrospinal fluid kynurenic acid in male and female controls – Correlation with monoamine metabolites and influences of confounding factors Linda K. Nilsson

a,*

, Conny Nordin b, Erik G. Jo¨nsson c, Go¨ran Engberg a, Klas R. Linderholm a, Sophie Erhardt a

a Department of Physiology and Pharmacology, Karolinska Institutet, SE-171 77 Stockholm, Sweden Department of Neuroscience and Locomotion, Psychiatry Section, Linko¨pings Universitet, SE-581 85 Linko¨ping, Sweden Department of Clinical Neuroscience, Psychiatry Section, HUBIN project, Karolinska Institutet and Hospital, R5:00, SE-171 76 Stockholm, Sweden b

c

Received 24 August 2005; received in revised form 21 November 2005; accepted 5 December 2005

Abstract The concentrations of the tryptophan metabolite kynurenic acid (KYNA) and the monoamine metabolites homovanillic acid (HVA), 5-hydroxy-indoleacetic acid (5-HIAA) and 4-hydroxy-3-methoxyphenylglycol (HMPG) were determined in the cerebrospinal fluid (CSF) from 43 healthy volunteers (30 males and 13 females). Healthy female controls displayed higher CSF concentration of KYNA (1.91 nM ± 0.20) compared to healthy male controls (1.06 nM ± 0.07) and lower CSF levels of HMPG (39.2 nM ± 2.0 and 43.4 ± 1.2, respectively). CSF levels of HVA and 5-HIAA did not differ between females (181.3 nM ± 21.9 and 93.7 nM ± 11.4, respectively) and males (138.9 nM ± 12.6 and 74.8 nM ± 5.9, respectively). Positive intercorrelations were found between CSF KYNA, HVA and 5-HIAA while CSF content of HMPG did not correlate with KYNA or the other monoamine metabolites in CSF. A negative correlation was found between back length and CSF concentrations of KYNA, HVA and 5-HIAA and also between CSF KYNA levels and body height. The results of the present study suggest that concentrations of KYNA and the monoamine metabolites in CSF from healthy controls are dependent on gender and back length, which must be taken in consideration when analysing mixed groups of men and women. The higher KYNA concentration found in female controls compared to male might be attributed to a shorter back in women compared to men. Furthermore, these findings suggest that increased KYNA formation is associated with an increased dopamine and serotonin turnover.  2005 Elsevier Ltd. All rights reserved. Keywords: Homovanillic acid; 5-Hydroxy-indoleacetic acid; 4-Hydroxy-3methoxyphenylglycol; Human; Cerebrospinal fluid

1. Introduction During the last decades cerebrospinal fluid (CSF) has been an important resource for assessing neurochemical changes in the brains of living subjects. The total volume of CSF in the cerebrospinal system is estimated to approximately 140 mL and the rate of formation to about 500 mL every 24 h (Geigy Scientific Tables, 1981; *

Corresponding author. Tel.: +46 852 486 706; fax: +46 831 0622. E-mail address: [email protected] (L.K. Nilsson).

0022-3956/$ - see front matter  2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jpsychires.2005.12.001

Kandel et al., 2000), which gives a turnover of 3–4 times a day. CSF is a transparent, colourless liquid consisting of 99% water, however, over 100 compounds have been identified in the remaining 1% (Geigy Scientific Tables, 1981). Several confounding factors may influence the CSF concentration of the measured compound, including age and interindividual factors such as body height, back lengths and body weight (Blennow et al., 1993; Eklundh et al., 1996; Jo¨nsson et al., 1996; Nordin et al., 1987, 1993, 1995; Wode-Helgodt and Sedvall, 1978) as well as methodological factors and external conditions

L.K. Nilsson et al. / Journal of Psychiatric Research 41 (2007) 144–151

at time of lumbar puncturing (see Eklundh, 2000). The expression ‘‘back length’’, also known as the neuroaxis distance, is used for the distance measured between the site of puncture and the external occipital protuberance (Eklundh et al., 1996, 2001). The usage of the back length parameter is an attempt to describe a more relevant measure of the length of the spinal compartment than body height, which stem to a great extent from the length of the legs, and less from head and trunk (Wode-Helgodt and Sedvall, 1978). A limitation with studies requiring invasive experiments such as LP is the availability of healthy control subjects and thus, some investigators use patients with neurological and medical complaints as control group. Furthermore, judging from many studies of human CSF it could be questioned to what extent ‘‘healthy volunteers’’ represent a cross-section of the population, since invasive experiments may attract certain personalities (Gustavsson et al., 1997). However, it is difficult to circumvent this problem since all subjects participating for this kind of demanding biological research do so on a voluntary basis. Analyses of CSF concentrations of the neurotransmitter metabolites are commonly used to study various neuropsychiatric disorders in humans, since the actual neurotransmitters are present in low concentrations in the CSF and are therefore difficult to measure (see Scheinin, 1985). The most frequently studied metabolites in the CSF are homovanillic acid (HVA), the main metabolite of dopamine (DA); 5-hydroxy-indoleacetic acid (5HIAA), the main metabolite of 5-hydroxytryptamine (5HT; serotonin); and 4-hydroxy-3-methoxyphenylglycol (HMPG), the main metabolite of noradrenaline (NA). Another compound of interest in neuropsychiatry is the tryptophan metabolite kynurenic acid (KYNA). The presence of KYNA in human brain was first discovered in the late 1980s (Moroni et al., 1988a; Turski et al., 1988) and in recent years our knowledge about its physiological significance has become clearer. KYNA, synthesized in and released from brain astrocytes, is an endogenously occurring antagonist of glutamate, preferentially N-methyl-D-aspartate (NMDA) receptors, and nicotinic receptors (Birch et al., 1988; Ganong and Cotman, 1986; Hilmas et al., 2001; Kessler et al., 1989; Parsons et al., 1997). Growing evidence, including results from electrophysiological (Erhardt et al., 2001a; Erhardt and Engberg, 2002; see Erhardt et al., 2003; Nilsson et al., 2005a; Schwieler et al., accepted for publication) and behavioural (Erhardt et al., 2004) experiments in rats, suggest that endogenous KYNA may participate in the pathophysiology of schizophrenia. In consonance, increased CSF (Erhardt et al., 2001b; Nilsson et al., 2005b) and post-mortem prefrontal cortex (Schwarcz et al., 2001) levels of KYNA has been found to be elevated in patients with schizophrenia. Furthermore, previous studies regarding CSF have revealed that KYNA concentration is higher in patients with amyotrophic lat-

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eral sclerosis compared to controls (Ilzecka et al., 2003), and decreased in patients with relapsing-onset multiple sclerosis (Rejdak et al., 2002) as well as eating-disordered patients (Demitrack et al., 1995). Since analysis of KYNA in the CSF is a rather new invention, our knowledge concerning confounding factors is restricted. Hence, studies regarding relationships between CSF concentration of KYNA and CSF concentrations of monoamine metabolites as well as confounding factors, including gender, in healthy controls should be an important contribution to the field of neuropsychiatric diseases. Thus, in the present study we examine possible relationships between CSF concentration of different compounds such as KYNA, HVA, 5-HIAA and HMPG in male and female healthy controls. Furthermore, all compounds were tested for correlation with age, back-lengths and body height.

2. Materials and methods 2.1. Subjects KYNA concentration was measured in CSF samples from 56 healthy volunteers (43 males and 13 females), mean age 26.8 years ±0.73; range 19–44. In 43 of the healthy volunteers (30 males and 13 females) CSF content of the three monoamine metabolites, HVA, 5HIAA and HMPG was analysed. The volunteers were mainly recruited from students and hospital staff members. Informed consent was obtained from all subjects after written and verbal information about the procedure and the purpose of the study. All volunteers were in good physical health and prior to the study the subjects were interviewed, physically examined and blood and urine tested based on the following exclusion criteria: having either neurological, cardiovascular, hepatic, renal, hematopoetic, gastrointestinal, metabolic or psychiatric dysfunction, alternatively receiving medication on a regular basis. Accordingly, all subjects were found to be free from current signs of psychiatric morbidity or difficulties in social adjustment at time of sampling and had no family history of major psychosis or suicide in first or second-degree relatives. KYNA data from the male controls and monoamine metabolite data from both male and female controls have previously been included in a larger sample of subjects in which the mean CSF concentrations of KYNA (Nilsson et al., 2005b) and HVA, 5-HIAA and HMPG (Ha¨rnryd et al., 1984; Oxenstierna et al., 1984, 1996; Jo¨nsson, 1997) were evaluated. 2.2. Cerebrospinal fluid sampling CSF was obtained by LP (L4-L5) and performed between 8 and 9 am. To reduce the influence of physical

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activity and stress, the subjects had at least 8 h of supervised bedrest in the hospital abstaining from food and smoking. Samples of approximately 12–18 mL CSF were drawn according to a standardised sampling proce˚ sberg, 1984). dure (Sedvall et al., 1980; Bertilsson and A All samples were immediately frozen, coded and sent blindly to Karolinska Institutet. 2.3. Policy and ethics The work described in the present study was carried out in accordance with ‘‘The code of ethics of the world medical association (declaration of Helsinki) for experiments including humans: http://www.wma.net/e/policy/ b3.htm’’. The study was approved by the Ethics Committees of the University Hospital in Linko¨ping and Karolinska Institutet. 2.4. Analysis of kynurenic acid Kynurenic acid is a stable compound and is not degraded even by repeated thawing (Heyes and Quearry, 1990). It was analysed with an isocratic reversedphase high-performance liquid chromatography (HPLC) system, including a dual piston, high liquid delivery pump (Bischoff, Leonberg, Germany), a ReproSil-Pur C18 column (4 · 150 mm, Dr. Maisch GmbH, Ammerbuch, Germany) and a fluorescence detector (Jasco Ltd., Hachioji City, Japan) with an excitation wavelength of 344 nm and an emission wavelength of 398 nm (18 nm bandwidth). A mobile phase of 50 mM sodium acetate pH 6.20 (adjusted with acetic acid) and 7.0% acetonitrile was pumped through the reversed-phase column at a flow rate of 0.5 mL/min. Samples of 25 lL were manually injected (ECOM, Prague, Czech Republic). Zinc acetate (0.5 M, not pH adjusted) was delivered postcolumn by a peristaltic pump (P-500, Pharmacia, Uppsala, Sweden) at a flow rate of 0.10 mL/min. The signals from the fluorescence detector were transferred to a computer for analysis with Datalys Azur (Grenoble, France). The retention time of kynurenic acid was approximately 13 min, and the detection limit of the method was approximately 0.125 pmol (signal: noise ratio 5:1). Initially the sensitivity of the fluorescence method was evaluated by injection of a standard mixture of kynurenic acid, with concentrations from 1.25 to 60 nM. This resulted in a standard plot, which was used to relate the heights of the peaks in the chromatogram to the correct concentration of kynurenic acid in the samples. 2.5. Analysis of monoamine metabolites 5-HIAA, HVA and HMPG concentrations were measured by mass fragmentography with deuterium

labelled internal standards (Swahn et al., 1976). For determination of CSF concentrations of 5-HIAA, HVA and HMPG 2 mL of CSF was used. Before any further processing the following amounts of standards were added: 2800 pmol of HVA-d2, 2300 pmol of 5-HIAA-d2, and 950 pmol of HMPG-d2. Standard solutions were prepared containing the mentioned amounts of standards and a known amount of the authentic substances that ranged between 0 and 1600 pmol for HVA, 0–800 pmol for 5-HIAA, and 0– 500 pmol for HMPG. The pH of the samples was adjusted to about 2 with 4 M formic acid. NaCl was added to saturate the solutions before extraction with 3 portions of diethyl ether (4 mL). The diethyl ether was removed and evaporated under a stream of nitrogen. The residue was transferred to a small conical test tube with 2 portions of methanol (0.3 mL), which was subsequently removed with nitrogen. When the solvent was completely removed, derivatives were prepared by the addition of a mixture of purified pentafluoropropionic anhydride (PFPA) and 2,2,3,3,3-pentafluoropropan-1-ol (PFPOH; 4:1; 50 lL). The tubes were sealed with a ground glass stopper and allowed to react for 15 min at 75 C. The reagent was again evaporated and the residue was dissolved in ethyl acetate containing 1% PFPA (30 lL). About 2 lL of the final solution was used for analysis. The analysis was performed on a Finnigan 3200 GC– MS system. The instrumental conditions were as follows. The gas chromatograph was equipped with an OV-17 column (1.5 m · 2 mm ID), operated at about 150 C. The injector temperature was 180 C. The flow of helium was about 30 mL/min. From the mass spectra of the derivatives, the following fragment pairs were selected to be monitored: 458; 461; (HMPG), 460; 462 (HVA), and 438; 440 (5-HIAA). The separator temperature was 275 C and the electron energy 50 eV. For the simultaneous recording of the metabolites the programmable multiple ion monitor unit was run in the current amplifier mode, with a sample time of 100 ms, filters on 0.05 Hz, and the preamplifier sensitivity at 10 8. The other settings were adjusted for optimal resolution and sensitivity. 2.6. Statistical analysis All values are given as means ± SEM. Differences regarding CSF levels of the compounds between males and females were established using Kruskal–Wallis analysis of variance followed by Mann–Whitney U-test. Linear regression analysis was performed to study interrelationships between CSF concentration of KYNA, HVA, 5-HIAA and HMPG and also to study correlations of the monoamine metabolites and KYNA with age, body height and back length. Significance was assumed for all values where p < 0.05.

L.K. Nilsson et al. / Journal of Psychiatric Research 41 (2007) 144–151

3. Results The mean concentrations (±SEM) of KYNA and the monoamine metabolites in CSF from the healthy volunteers are shown in Table 1. Females were found to have significantly higher concentration of KYNA in the CSF than males and lower CSF concentration of HMPG (see Table 1). A positive correlation was found between CSF KYNA concentration and CSF HVA concentration (r = 0.69, p < 0.0001; see Fig. 1A). This correlation remained when analysing males and females separately (r = 0.68, p < 0.0001 and r = 0.68, p = 0.01, respectively). CSF KYNA was also positively correlated to CSF concentration of 5-HIAA (r = 0.72, p < 0.0001, see Fig. 1B). Also this correlation remained when analyzing males and females separately (r = 0.70, p < 0.0001 and r = 0.74, p = 0.004, respectively). A positive correlation was observed between CSF HVA and 5HIAA (r = 0.82, p < 0.0001) in all healthy controls (see Fig. 1C), as well as in both male and female separately (r = 0.78, p < 0.0001 and r = 0.85, p = 0.0003, respectively). No correlations were found between HMPG content in the CSF and the concentration of KYNA or the other monoamine metabolites in CSF (data not shown). A negative correlation was found between KYNA CSF concentration and back length (r = 0.54, p < 0.0001; see Fig. 2A), however, when analysing males and females separately the correlation was only significant in female controls. A negative correlation was also seen between CSF KYNA and body height in all the healthy controls (r = 0.41, p = 0.0017, see Fig. 2B) but not in males and females when analysed separately. No correlation was found between age and CSF KYNA concentration in either groups (data not shown). Also CSF levels of HVA and 5-HIAA were negatively correlated with back length in healthy controls (r = 0.37, p = 0.017 vs. r = 0.41, p = 0.007, respectively, data not shown), when divided into male and female the correlation was only significant in females (r = 0.64, p = 0.025 vs. r = 0.75, p = 0.005, respectively). CSF concentration

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of HMPG did not correlate with back length (data not shown). No correlations were found between CSF HVA, 5-HIAA or HMPG levels and body height or age (data not shown).

4. Discussion CSF KYNA concentration in healthy human controls was positively correlated to concentrations of 5HIAA and HVA. Furthermore, we confirm a robust positive correlation between 5-HIAA and HVA concentrations, one of the most consistent findings in studies ˚ gren using human CSF data (Jibson et al., 1990; A et al., 1986). The NA metabolite HMPG did not correlate with KYNA, HVA or 5-HIAA in the CSF, which might be related to a contribution of HMPG from blood/plasma (Kopin et al., 1983, 1984). Thus, the neutrally charged HMPG can diffuse more freely over the blood brain barrier (BBB; Wolfson and Escriva, 1976) as compared to KYNA, 5-HIAA and HVA. The CSF level of HMPG is therefore probably more sensitive to fluctuations of the compound in the plasma. Since KYNA, 5-HIAA and HVA are acids, access from the plasma to the brain over the BBB is rather restricted (Fukui et al., 1991; see Scheinin, 1985), suggesting that significant proportions of these compounds in CSF derive from the brain. Indeed, studies of post-mortem humans have demonstrated that CSF HVA and 5HIAA reflect brain HVA and 5-HIAA concentrations (Knott et al., 1989; Stanley et al., 1985; Wester et al., 1990). However, CSF concentration of KYNA in healthy human (ranging between 0.97 and 3.60 nM; Demitrack et al., 1995; Erhardt et al., 2001b; Heyes and Quearry, 1990; Heyes et al., 1992, 1994; Nilsson et al., 2005b; Rejdak et al., 2002) is considerable lower than human frontal cortex concentration (approximately 150–290 nM; Moroni et al., 1988a, Turski et al., 1988). The large gradient between tissue and CSF levels might be explained by the fact that astrocytes (in which the synthesis and release of KYNA occurs) encapsulate terminal boutons and their adjacent postsynaptic complex in such an intimate manner (Harris

Table 1 CSF concentrations of kynurenic acid, homovanillic acid, 5-hydroxy-indoleacetic acid and 4-hydroxy-3-methoxyphenylglycol in male and female healthy controls Data on control subjects Subject

KYNA (nmol/L)

HVA (nmol/L)

5-HIAA (nmol/L)

HMPG (nmol/L)

All controls Male controls Female controls

1.26 ± 0.09 (n = 56) 1.06 ± 0.07 (n = 43)a 1.91 ± 0.20*** (n = 13)

151.7 ± 11.2 (n = 43)b 138.9 ± 12.6 (n = 30)b 181.3 ± 21.9 (n = 13)

80.5 ± 5.5 (n = 43)b 74.8 ± 5.9 (n = 30)b 93.7 ± 11.4 (n = 13)

42.1 ± 1.0(n = 43)b 43.4 ± 1.2(n = 30)b 39.2 ± 2.0* (n = 13)

a b * ***

Samples from Nilsson et al. (2005b). Samples from Ha¨rnryd et al. (1984), Jo¨nsson (1997), Oxenstierna et al. (1984, 1996). p < 0.05 compared to concentration in males. p < 0.001 compared to concentration in males.

KYNA (nM)

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KYNA (nM)

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A

KYNA (nM)

KYNA (nM)

A

B

5-HIAA (nM)

B

C Fig. 1. Linear regression analysis between CSF concentration (nM) of: (A) kynurenic acid vs. homovanillic acid (r = 0.69; p < 0.0001, y = 0.006x + 0.41), (B) kynurenic acid vs. 5-hydroxy-indoleacetic acid (r = 0.72; p < 0.0001; y = 0.13x + 0.51), and (C) 5-hydroxy-indoleacetic acid vs. homovanillic acid (r = 0.82; p < 0.0001; y = 0.396x + 20.49) in healthy male and female controls.

and Rosenberg, 1993, see Coyle and Schwarcz, 2000) that diffusion of KYNA from the synaptic cleft to the CSF only occur to a minor extent.

Fig. 2. Linear regression analysis between CSF concentration of kynurenic acid (nM) and (A) back length (cm; r = 0.54; p < 0.0001, y = 0.053x + 4.42), (B) body height (cm; r = 0.41; p = 0.0017; y = 0.029x + 6.38).

The origin and relevance of the commonly found correlation between HVA and 5-HIAA in human CSF remains a subject of ongoing discussion (Hsiao et al., 1987, 1993a,b; see Hsiao et al., 1993a,b; Jibson et al., ˚ gren et al., 1986). The ques1990; Risby et al., 1987; A tion has been raised whether the correlation reflects a real important interrelationship between DA and serotonin in the central nervous system or if it arises due to a common active transport mechanism along the spinal cord, which creates a CSF correlation that would not be indicative of brain turnover of the transmitter. However, the predominant theory is that the correlation between 5-HIAA and HVA in the CSF might at least in part reflect functional interactions among the monoamine system in the brain, since a widespread serotonergic influence on DA neuronal activity has been demonstrated in several brain regions (Dray et al., 1976; Fuenmayor and Bermudez, 1985; Jibson et al., ˚ gren et al., 1986). This is the 1990; Park et al., 1982; A first study investigating correlations between concentrations of endogenous KYNA and monoamine metabo-

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lites in CSF. The present finding, that KYNA in CSF was positively correlated with 5-HIAA and HVA, suggest that increased KYNA formation is associated with an increased DA and serotonin turnover. In support of this, recent preclinical studies demonstrate that KYNA acts as a physiologically significant modulator that tonically controls glutamatergic neurotransmission and thereby interacts with the dopaminergic system. Thus, decreased endogenous levels of KYNA in the rat brain are associated with reduced activity of midbrain DA neurons (Schwieler et al., accepted for publication). In consonance, acutely as well as chronically elevated levels of endogenous rat brain KYNA produce an increased activity of DAergic neurons (Erhardt and Engberg, 2002; Erhardt et al., 2001a; Nilsson et al., 2005a), hereby linking increased CSF (Erhardt et al., 2001b; Nilsson et al., 2005b) and post-mortem prefrontal cortex (Schwarcz et al., 2001) levels of KYNA in patients with schizophrenia with a hyperdopaminergic activity. Today it is generally accepted that the symptoms of schizophrenia are related to DAergic systems in the brain; nevertheless, the primary cause of the disease remains to be revealed. The positive correlation between KYNA and HVA in CSF from healthy controls supports the notion that hyperdopaminergic activity in schizophrenia is caused by elevated levels of endogenous kynurenic acid (Erhardt et al., 2001b; Nilsson et al., 2005b). A direct physiological interaction between KYNA and the serotonergic system has not yet been investigated, however, the correlation between CSF KYNA and 5-HIAA might be related to tryptophan, which is the common precursor for both serotonin and KYNA. We found no correlations between CSF KYNA or monoamine metabolite concentrations and age. Previous studies have shown an age-related increase of endogenous KYNA in rat brain (Gramsbergen et al., 1992; Moroni et al., 1988b) and in CSF from male patients with schizophrenia (Erhardt et al., 2001b; Nilsson et al., 2005b), as well as in patients with acute headache (Kepplinger et al., 2005). No such correlation was found in CSF from healthy controls (Erhardt et al., 2001b; Nilsson et al., 2005b). Studies investigating the influence of age on monoamine metabolite concentration in CSF have revealed conflicting results and some investigators have found correlations with age and others have not (e.g., Blennow et al., 1993; see Eklundh, 2000). The discrepancy in this regard might be related to the fact that some investigators study patients with various diseases, while others use healthy subjects. Also differences in age range occurred between studies, which may contribute to the heterogenous picture regarding age and various compounds in the CSF. An absence of correlation between age and the CSF compounds in the healthy controls in the present study might be related to the relatively young age of the controls and the narrow age range (19–44 years).

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A negative correlation between monoamine metabolite concentrations and body height is a common finding in CSF studies, however, not always consistent between studies and for all metabolites (Blennow et al., 1993; Jo¨nsson et al., 1996; Koslow et al., 1983; Nordin et al., 1996; Stanley et al., 1985; Wode-Helgodt and Sedvall, 1978). It has been assumed that a taller person exhibits a larger surface for resorption from the CSF, due to a longer spinal compartment, which results in lower concentrations of monoamine metabolites (Blen˚ gren et al., 1986). However, since now et al., 1993; A the body height is mostly related to the length of the legs the parameter back length (or neuroaxis distance) has been introduced as a more applicable measure for the length of the spinal compartment (Jo¨nsson et al., 1996; Nordin et al., 1993; Wode-Helgodt and Sedvall, 1978). In support of a relatively great impact of back length on the CSF composition we found that KYNA, HVA and 5-HIAA concentrations were negatively correlated to back length, while only KYNA showed a negative correlation to body height. With regard to this, the higher concentration of KYNA found in females compared to males might be related to the fact that women in general have shorter back (and body height) compared to men, which could tentatively result in a smaller dilution gradient. However, the correlation between KYNA and back length (and body height) was only evident in women when males and females were analysed separately. Thus, we cannot exclude a real gender difference with regard to CSF KYNA concentration. Previous studies of CSF from healthy controls have revealed that women have higher concentrations of HVA and/or 5-HIAA than men (Blennow et al., 1993; ˚ gren et al., 1986). In the present Nordin et al., 1996; A study, the gender differences in HVA and 5-HIAA concentrations tended to reach statistical significance. In summary, our results show that CSF concentration of KYNA in healthy controls is dependent on gender and back length, which must be taken in consideration when analysing CSF from mixed groups of men and women. The higher KYNA concentration found in female controls compared to male might be attributed to a shorter back in women compared to men. Furthermore, these findings suggest that increased KYNA formation is associated with an increased DA and serotonin turnover.

Acknowledgements This study was supported by Ha˚llstens Forskningsstiftelse, Swedish Brain Foundation, Swedish Research Council (No. 529-2004-6488 (S.E.), No. K2003-04X07484-18A (G.E.) and No. K-2004-21X-15078-01A ˚ hle´nsstiftelsen, (E.J.)), Svenska Schizofrenisa¨llskapet, A Fredrik och Ingrid Thurings stiftelse, Svenska

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La¨karesa¨llskapet, Stiftelsen Apotekare Hedbergs fond fo¨r Medicinsk forskning, Svenska Lundbecksstiftelsen, the Karolinska Institutet, So¨derstro¨m-Ko¨nig Foundation, Research Foundation of the University Hospital in Linko¨ping and FoU and the HUBIN project. We thank Mrs Alexandra Tylec, Department of Clinical Neuroscience for technical assistance.

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