Neuroscience Letters, 141 (1992) 9-12
9
© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 08715
Concentrations of serotonin and its related substances in the cerebrospinal fluid in patients with Alzheimer type dementia Hideo Tohgi, Takashi Abe, Satoshi Takahashi, M u n e t a k a Kimura, J u n k o Takahashi and Takahiko Kikuchi Department of Neurology, lwate Medical University, Morioka (Japan) (Received 28 February 1992; Accepted 27 March 1992)
Key words: Alzheimer type dementia; Serotonin; 3-Hydroxykynurenine; 5-Hydroxyindoleacetic acid; Cerebrospinal fluid We studied concentrations of free and total serotonin (5-hydroxytryptamine, 5-HT) and its related substances in the cerebrospinal fluid from patients with Alzheimer type dementia (ATD) compared with controls. In ATD patients, concentrations of total 5-HT, tryptophan, 5-hydroxytryptophan (5-HTP), melatonin, kynurenine, and 3-hydroxykynurenine decreased significantly. The rate of concentration of tryptophan metabolites to that of tryptophan was significantly reduced for total 5-HT and 3-hydroxykynurenine only. The 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was significantly larger in ATD patients than in controls. The greater reduction in the 3-hydroxykynurenine concentration (81% vs. controls) than in the total 5-HT concentration (51% vs. controls) suggests that the metabolism of tryptophan to 5-HT and 3-hydroxykynurenine is in favor of 5-HT.
Previous studies have demonstrated that the serotonergic system, originating primarily from brainstem midline raphe nuclei, is affected in Alzheimer type dementia (ATD) [Alzheimer's disease (AD) and senile dementia of the Alzheimer type (SDAT)]; brain serotonin (5-hydroxytryptamine; 5-HT) content [2, 6, 19] and serotonergic cell density in the raphe nuclei [14, 25] are reduced. How the serotonergic system changes in ATD is particularly of clinical interest, because evidence has been accumulated that it plays an important role in memory, learning, and sleep regulation which are impaired in ATD patients. However, previous results concerning the concentrations of 5-hydroxyindole-3-acetic acid (5HIAA), the major metabolite of 5-HT, in the cerebrospinal fluid (CSF) in ATD have been controversial. The 5-HIAA concentration has been described to be lower in some reports in the CSF from ATD patients [3, 11, 23], and correlated with severity of dementia [3], while unaltered in others [4, 22]. There have been only a limited number of reports on 5-HT concentrations in the CSF which may better reflect brain serotonergic function than 5-HIAA [5]. This has been mainly because of technical difficulties derived from the low concentration of free 5-HT in the CSF. Previous studies using the amperometric and coulometric systems have yielded different free
Correspondence: H. Tohgi, Department of Neurology, Iwate Medical University, 19-1 Uchimaru, Morioka, 020 Japan.
5-HT concentrations [22, 23], but reported reduced free 5-HT concentrations in ATD using the coulometric system [23]. As far as we know, there has been no report concerning the total (conjugated and free) 5-HT concentrations in the CSF. We therefore studied concentrations of free as well as total 5-HT, because the concentration of total 5-HT in the CSF was reported to be about 8 times higher than that of free 5-HT [21]. We also studied the concentrations of free tryptophan (the precursor of 5-HT), 5-hydroxytryptophan (5-HTP; the precursors of 5-HT), 5HIAA and melatonin (metabolites of 5-HT), kynurenine and 3-hydroxykynurenine (3-OH kynurenine) (metabolites of tryptophan) in the CSF of ATD patients, and their possible correlations with the severity of dementia. We studied 14 untreated patients (68.4 + 10.1 years) with ATD and 10 controls (68.5 + 6.1 years). In patients with ATD, the onset of the disease was before 65 years in 6 patients, and after 65 years in 8 patients. However, we combined these patient groups, because there were no significant differences in their results. The average duration of the disease in ATD patients was 3.7 + 3.5 years. The diagnosis of ATD was made based upon the DSMIII-R [1], Hachinski's ischemic score [12], the criteria of the NINCDS-ADRDA Work Group [16], and CT and MRI findings. The severity of dementia was assessed using the Mini-Mental State (MMS) test [8]. The average of MMS scores was 12.9 + 8.0 in ATD patients. Control CSF samples were obtained from patients who under-
10
went lumbar puncture for spinal anaesthesia. Informed consent was obtained from all patients. Lumbar CSF was obtained with the patients in the lateral decubitus position between 09.00 and 10.00 h after overnight bed-rest and fasting. An initial 3 ml of the CSF was used for routine examination. The additional 2 ml of the CSF was placed on ice immediately after lumbar puncture and stored at -80°C. We did not deproteinize the CSF before storage. Concentrations of free tryptophan, 5-HTP, 5-HIAA, melatonin, kynurenine, 3-OH kynurenine, and 5-HT were determined by injection of 80/.tl volumes of the CSF into a reverse phase C~8 column (Neuro Column, Niko Bioscience, Tokyo). Sample analysis was performed using high-performance liquid chromatography (HPLC) with a 16-sensor Neurochemical analyser (ESA Inc., Bedford, MA) [15] as described previously [20]. The mobile phase A consisted of 0.1 M sodium phosphate and 10 mg/l sodium dodecyl sulfate at pH 3.35; the mobile phase B consisted of 50% methanol/0.1 M sodium phosphate and 50 mg/1 sodium dodecyl sulfate at pH 3.45. Both were obtained directly from ESA as final reagents. The 16 serial electrodes were set in an incremental 60 mV array from 0 to 900 mV. The column and electrodes were maintained at 37°C. All standards were obtained from
the Sigma Chemical Company. The detection limit was 20 pg/ml and the recovery rate was nearly 100%. For the determination of the concentration of total (conjugated and free) 5-HT [21], 500/.d of sample was mixed with 50 Ill of 3 M perchloric acid, 5/.d of 12.5 mM sodium thiosulfate, and N-methylserotonin as an internal standard (2 ng/ml). The aliquot was boiled for 30 min to hydrolyze the conjugated amines, and centrifuged. 100 ¢tl of the supernatants was applied to HPLC (NBS C~8 reversed phase column, 150 mm x 4.6 mm) with an electrochemical detector (Model 5200 A, Niko Bioscience). The electrode potentials were maintained at 0.15 V for the guard cell, 0 V for detector I and 0.15 V for detector II. The elution buffer containing 0.1 M sodium citrate, 6.6 mM sodium octanesulfonate, and 2 mM disodium EDTA was adjusted to pH 5.0. Recovery of authentic standard of 5-HT was 85-90%. The detection limit was 10 pg/ml. Our control values were similar to the reported values of Volicer et al. [23] for 5-HTP and 5-HIAA concentrations, but were about four times lower than their values for free 5-HT (Table I). The free 5-HT concentration was detectable in 9 out of 10 controls (P=0.011 in a binomial test), and in 12 out of 14 patients with ATD (P--0.006 in a binomial test). Within the range of such low concentrations (only 4 times the detection limit on average), we did
TABLE I C O N C E N T R A T I O N S ( M E A N _+ S.D.) O F S E R O T O N I N ( F R E E A N D TOTAL) A N D ITS R E L A T E D SUBSTANCES (FREE) IN T H E CSF (ng/ml) IN PATIENTS W I T H A L Z H E I M E R TYPE D E M E N T I A (ATD) C O M P A R E D W I T H C O N T R O L S ( U P P E R C O L U M N ) , A N D T H E RATE O F T R Y P T O P H A N M E T A B O L I T E S TO T R Y P T O P H A N C O N C E N T R A T I O N S (× l0 3) ( L O W E R C O L U M N ) . Correlation coefficients between each parameter and Mini-Mental State Test (MMST) scores in ATD patients and their significance are also shown.
Tryptophan 5-HTP Free 5-HT Total5-HT 5-HIAA Melatonin Kynurenine 3-OH-kynurenine 5-HTP/tryptophan Free 5-HT/tryptophan Total 5-HT/tryptophan 5-HIAA/tryptophan Melatonin/tryptophan Kynurenine/tryptophan 3-OH-kynurenine/tryptophan
Controls (n= 10)
ATD (n= 14)
Correlation coefficients with MMST
430 _+115 1.45 +_ 0.63 0.090_+ 0.044 (n=9) 0.35 + 0.03 10.0 _+ 4.2 1.67 + 0.50 10.3 _+ 3.3 1.42 _+ 0.79
311 _+ 85 ~ 0.89 _+ 0.4ff ' 0.086-+ 0.023 01= 12) 0.17 ,+ 0.05 ~ 9.7 ,+ 4.2 1.12 ,+ 0.51 ' 5.4 + 1.9' 0.27 + 0.08 ~
0.08 (P=0.78) 0.55 (P
9
© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 08715
Concentrations of serotonin and its related substances in the cerebrospinal fluid in patients with Alzheimer type dementia Hideo Tohgi, Takashi Abe, Satoshi Takahashi, M u n e t a k a Kimura, J u n k o Takahashi and Takahiko Kikuchi Department of Neurology, lwate Medical University, Morioka (Japan) (Received 28 February 1992; Accepted 27 March 1992)
Key words: Alzheimer type dementia; Serotonin; 3-Hydroxykynurenine; 5-Hydroxyindoleacetic acid; Cerebrospinal fluid We studied concentrations of free and total serotonin (5-hydroxytryptamine, 5-HT) and its related substances in the cerebrospinal fluid from patients with Alzheimer type dementia (ATD) compared with controls. In ATD patients, concentrations of total 5-HT, tryptophan, 5-hydroxytryptophan (5-HTP), melatonin, kynurenine, and 3-hydroxykynurenine decreased significantly. The rate of concentration of tryptophan metabolites to that of tryptophan was significantly reduced for total 5-HT and 3-hydroxykynurenine only. The 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was significantly larger in ATD patients than in controls. The greater reduction in the 3-hydroxykynurenine concentration (81% vs. controls) than in the total 5-HT concentration (51% vs. controls) suggests that the metabolism of tryptophan to 5-HT and 3-hydroxykynurenine is in favor of 5-HT.
Previous studies have demonstrated that the serotonergic system, originating primarily from brainstem midline raphe nuclei, is affected in Alzheimer type dementia (ATD) [Alzheimer's disease (AD) and senile dementia of the Alzheimer type (SDAT)]; brain serotonin (5-hydroxytryptamine; 5-HT) content [2, 6, 19] and serotonergic cell density in the raphe nuclei [14, 25] are reduced. How the serotonergic system changes in ATD is particularly of clinical interest, because evidence has been accumulated that it plays an important role in memory, learning, and sleep regulation which are impaired in ATD patients. However, previous results concerning the concentrations of 5-hydroxyindole-3-acetic acid (5HIAA), the major metabolite of 5-HT, in the cerebrospinal fluid (CSF) in ATD have been controversial. The 5-HIAA concentration has been described to be lower in some reports in the CSF from ATD patients [3, 11, 23], and correlated with severity of dementia [3], while unaltered in others [4, 22]. There have been only a limited number of reports on 5-HT concentrations in the CSF which may better reflect brain serotonergic function than 5-HIAA [5]. This has been mainly because of technical difficulties derived from the low concentration of free 5-HT in the CSF. Previous studies using the amperometric and coulometric systems have yielded different free
Correspondence: H. Tohgi, Department of Neurology, Iwate Medical University, 19-1 Uchimaru, Morioka, 020 Japan.
5-HT concentrations [22, 23], but reported reduced free 5-HT concentrations in ATD using the coulometric system [23]. As far as we know, there has been no report concerning the total (conjugated and free) 5-HT concentrations in the CSF. We therefore studied concentrations of free as well as total 5-HT, because the concentration of total 5-HT in the CSF was reported to be about 8 times higher than that of free 5-HT [21]. We also studied the concentrations of free tryptophan (the precursor of 5-HT), 5-hydroxytryptophan (5-HTP; the precursors of 5-HT), 5HIAA and melatonin (metabolites of 5-HT), kynurenine and 3-hydroxykynurenine (3-OH kynurenine) (metabolites of tryptophan) in the CSF of ATD patients, and their possible correlations with the severity of dementia. We studied 14 untreated patients (68.4 + 10.1 years) with ATD and 10 controls (68.5 + 6.1 years). In patients with ATD, the onset of the disease was before 65 years in 6 patients, and after 65 years in 8 patients. However, we combined these patient groups, because there were no significant differences in their results. The average duration of the disease in ATD patients was 3.7 + 3.5 years. The diagnosis of ATD was made based upon the DSMIII-R [1], Hachinski's ischemic score [12], the criteria of the NINCDS-ADRDA Work Group [16], and CT and MRI findings. The severity of dementia was assessed using the Mini-Mental State (MMS) test [8]. The average of MMS scores was 12.9 + 8.0 in ATD patients. Control CSF samples were obtained from patients who under-
10
went lumbar puncture for spinal anaesthesia. Informed consent was obtained from all patients. Lumbar CSF was obtained with the patients in the lateral decubitus position between 09.00 and 10.00 h after overnight bed-rest and fasting. An initial 3 ml of the CSF was used for routine examination. The additional 2 ml of the CSF was placed on ice immediately after lumbar puncture and stored at -80°C. We did not deproteinize the CSF before storage. Concentrations of free tryptophan, 5-HTP, 5-HIAA, melatonin, kynurenine, 3-OH kynurenine, and 5-HT were determined by injection of 80/.tl volumes of the CSF into a reverse phase C~8 column (Neuro Column, Niko Bioscience, Tokyo). Sample analysis was performed using high-performance liquid chromatography (HPLC) with a 16-sensor Neurochemical analyser (ESA Inc., Bedford, MA) [15] as described previously [20]. The mobile phase A consisted of 0.1 M sodium phosphate and 10 mg/l sodium dodecyl sulfate at pH 3.35; the mobile phase B consisted of 50% methanol/0.1 M sodium phosphate and 50 mg/1 sodium dodecyl sulfate at pH 3.45. Both were obtained directly from ESA as final reagents. The 16 serial electrodes were set in an incremental 60 mV array from 0 to 900 mV. The column and electrodes were maintained at 37°C. All standards were obtained from
the Sigma Chemical Company. The detection limit was 20 pg/ml and the recovery rate was nearly 100%. For the determination of the concentration of total (conjugated and free) 5-HT [21], 500/.d of sample was mixed with 50 Ill of 3 M perchloric acid, 5/.d of 12.5 mM sodium thiosulfate, and N-methylserotonin as an internal standard (2 ng/ml). The aliquot was boiled for 30 min to hydrolyze the conjugated amines, and centrifuged. 100 ¢tl of the supernatants was applied to HPLC (NBS C~8 reversed phase column, 150 mm x 4.6 mm) with an electrochemical detector (Model 5200 A, Niko Bioscience). The electrode potentials were maintained at 0.15 V for the guard cell, 0 V for detector I and 0.15 V for detector II. The elution buffer containing 0.1 M sodium citrate, 6.6 mM sodium octanesulfonate, and 2 mM disodium EDTA was adjusted to pH 5.0. Recovery of authentic standard of 5-HT was 85-90%. The detection limit was 10 pg/ml. Our control values were similar to the reported values of Volicer et al. [23] for 5-HTP and 5-HIAA concentrations, but were about four times lower than their values for free 5-HT (Table I). The free 5-HT concentration was detectable in 9 out of 10 controls (P=0.011 in a binomial test), and in 12 out of 14 patients with ATD (P--0.006 in a binomial test). Within the range of such low concentrations (only 4 times the detection limit on average), we did
TABLE I C O N C E N T R A T I O N S ( M E A N _+ S.D.) O F S E R O T O N I N ( F R E E A N D TOTAL) A N D ITS R E L A T E D SUBSTANCES (FREE) IN T H E CSF (ng/ml) IN PATIENTS W I T H A L Z H E I M E R TYPE D E M E N T I A (ATD) C O M P A R E D W I T H C O N T R O L S ( U P P E R C O L U M N ) , A N D T H E RATE O F T R Y P T O P H A N M E T A B O L I T E S TO T R Y P T O P H A N C O N C E N T R A T I O N S (× l0 3) ( L O W E R C O L U M N ) . Correlation coefficients between each parameter and Mini-Mental State Test (MMST) scores in ATD patients and their significance are also shown.
Tryptophan 5-HTP Free 5-HT Total5-HT 5-HIAA Melatonin Kynurenine 3-OH-kynurenine 5-HTP/tryptophan Free 5-HT/tryptophan Total 5-HT/tryptophan 5-HIAA/tryptophan Melatonin/tryptophan Kynurenine/tryptophan 3-OH-kynurenine/tryptophan
Controls (n= 10)
ATD (n= 14)
Correlation coefficients with MMST
430 _+115 1.45 +_ 0.63 0.090_+ 0.044 (n=9) 0.35 + 0.03 10.0 _+ 4.2 1.67 + 0.50 10.3 _+ 3.3 1.42 _+ 0.79
311 _+ 85 ~ 0.89 _+ 0.4ff ' 0.086-+ 0.023 01= 12) 0.17 ,+ 0.05 ~ 9.7 ,+ 4.2 1.12 ,+ 0.51 ' 5.4 + 1.9' 0.27 + 0.08 ~
0.08 (P=0.78) 0.55 (P
