J Neural Transm [GenSect] (1991) 84:103-117

__ Journal o f Neural Transmission 9 Springer-Verlag 1991 Printed in Austria

Neurotransmitter markers in the cerebrospinal fluid of normal subjects Effects of aging and other confounding factors P. Hartikainen 1, H. Soininen 1, K. J. Reinikainen 1, J. Sirvi/~ 2, R. Soikkeli 1, and P. J. R i e k k i n e n 1

1 Department of Neurology, and 2 Neurochemical Research Laboratory, University of Kuopio, Finland Accepted November 23, 1990

Summary. We have investigated neurotransmitter-related markers of the cerebrospinal fluid (CSF) in a carefully screened series of normally aging subjects in standardized conditions in order to find out the influence of age and other confounding factors on CSF measures. The levels of 3-methoxy-4-hydroxyglycol (MHPG) and the activity of acetylcholinesterase (ACHE) also increased with age, while homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5 HIAA) and immunoreactivities of somatostatin (SLI), beta-endorphin (BLI) and adrenocorticotropic hormone (ACTH) were unrelated to age. The gender of subjects had no significant effect on the levels of neurotransmitter markers, while seasonal changes, as well as height and weight of the subjects seemed to cause some variations in the levels of HVA, dopamine-13-hydroxylase (DBH) and ACTH. The study underscores the importance of standardized conditions and matched patient groups in the CSF studies.

Keywords: Normal aging, cerebrospinal fluid, neurotransmitter markers, seasonal variations.

Introduction In normal aging findings of neurotransmitter related measures of the cerebrospinal fluid (CSF) are inconsistent. Acetylcholinesterase (ACHE), a cholinergic marker in the CSF, was reported to increase with age by some authors (Tune et al., 1985; Elble et al., 1987; Atack et al., 1988). The concentration of homovanillic acid (HVA), the main metabolite of dopamine, was found to increase in aged subjects (Gottfries et al., 1971; Bareggi et al., 1982; Palmer et al., 1984; Stahl et al., 1985), but also unaltered levels were reported (Ballenger et al., 1983). No age related changes of the levels of CSF 5-hydroxyindoleacetic acid (5HIAA), a metabolic of serotonin, and of 3-methoxy-4-hydroxyglycol (MHPG), a metabolite of noradrenalin, were shown (Ballenger et al., 1983;

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Palmer et al., 1984; Stahl et al., 1985; Guthrie et al., 1986), although the previous findings referred to increased metabolite concentrations during aging (Bowers et al., 1968; Gottfries et al., 1971). The immunoreactivities of peptides, like somatostatin (SLI), 13-endorphin (BLI) and adrenocorticotropic hormone (ACTH), seemed not to be age-related in normal subjects (Kaiya et al., 1983; Facchinetti et al., 1984; Beal et al., 1986; Sunderland et al., 1987; Atack et al., 1988; Alessio et al., 1988), although the number of studies with a wide range of age is limited. In lumbar CSF investigations, it may also be necessary to pay attention to a number of factors other than age like circadian and seasonal rhythms (Nicoletti et al., 1981; Losonczy et al., 1984), diet, CSF dynamics and gradients, height, weight, medication, physical and psychic state of subjects (Reinikainen and Riekkinen, 1990; Soininen et al., 1981; Post et al., 1973). This study is a part of a project investigating cognitive changes, brain electrical activity and CSF neurotransmitter markers during normal aging, in Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. This paper will report the CSF findings in a series of thoroughly investigated normal subjects under carefully controlled conditions in which the effects of age and other confounding factors on the CSF parameters were examined. In addition, interrelationships between the neurotransmitter systems were studied. Patients and methods Patients

The subjects included in the study were admitted to the Neurological Department of Kuopio University Central Hospital for diagnostic investigations for a 3-7 day period, due to minor neurological symptoms, for example, headache and vertigo. The subjects consented to their participation in the study. The study was approved by the local Ethics Committee. The normal aging groups consited of 34 subjects (age range 20-79 years, 14 males and 20 females). The study battery comprised physical and neurological examination with a measurement of blood pressure, ECG, chest X-ray, EEG, spectral analysis of EEG, somatosensory evoked potentials, visual evoked potentials, brain CT-scan, neuropsychological tests and Hachinski Ischaemic score (Rosen etal., 1980). Laboratory tests consisted of blood cell count, erythrocyte sedimentation rate, blood glucose, serum urea, electrolytes, calcium, phosphate, vitamin B12, erythrocyte folate, liver and thyroid function tests, cholesterol, triglycerides, immunoglobulin (IgG), albumin (alb), cardiolipin, and urine analysis. The routine CSF analysis comprised erythrocytes, leukocytes, total protein, electrophoresis, IgG, alb, IgG/-alb ratio, IgG index = (CSF IgG/alb)/(serum IgG/alb). Metabolic, endocrine and nutritional diseases and central nervous system (CSF) infections and psychiatric illnesses were excluded. Alcohol consumption was reported by the subjects themselves and according to physical status and laboratory tests (mean corpuscular volume of red blood cells and serum alanine amino transferase) any alcohol intake affecting health status was not observed. All subjects had findings within normal limits in the above mentioned investigations. The subjects did not use any psychotropic drugs, and only four subjects received diuretic drugs and two of them beta-blocking medication. The participants were divided into three groups accoring to their age (Table 1). In order to evaluate seasonal variations of the neurochemical measures, we divided the year

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Table 1. Main clinical characteristics of the age groups Age group

I

II

III

Number (M/F) Mean age Age range Height (cm) Weight (kg) SAP (mmHg) DAP (mmHg) fB-glucose

11 (6/5) 29.2 4- 7.0 20-39 172.2 + 10.0 71.7 + 10.8 126.1 4- 8.1 79.0 4- 10.1 4.4 + 0.6

14 (6/8) 52.5 + 4.3 40-59 167.8 + 8.4 # 69.8 + 8.2 134.0 + 12.7 82.3 + 6.8 4.4 + 0.3

9 (2/7) 69.1 + 6.3 60-79 163.1 + 9.7* 70.8 + 8.2 141.1 + 12.8" 83.8 + 10.5 4.5 + 0.5

Values given are the means + SD Significance of difference vs group I: * p < 0.05 Significance of difference vs group I I I : # p < 0.05 SAP systolic arterial pressure; DAP diastolic arterial pressure;fB-glucose fasting blood glucose level

into two p e r i o d s - the light period (spring and summer) and the dark period (autumn and winter), due to the strong seasonal changes in the climate of Scandinavia.

CSF samples Before the lumbar puncture, the subjects were kept on low monoamine diet, an overnight bed rest and they were fasting. The CSF samples were taken betweeen 08.00-10.00 a.m., when the subjects were lying in a lateral decubitus position. The first 4 ml of CSF was collected in 1 ml fractions for the routine analysis and the next 15 ml in 5 ml fractions for other determinations. Specimens were immediately frozen and stored at - 80 ~ until assayed.

Chemical methods The total protein in the CSF was measured by the method of Lowry et al. (1951). The levels of monoamine metabolites were determined by using high performance liquid chromatography (HPLC) with electrochemical detection as described previously (Jolkkonen et al., 1987). The activity of dopamine-[3-hydroxylase (DBH), noradrenaline synthetising enzyme in CSF was determined by a radioenzymatic method as reported by Soininen et al. (1984). The activity of ChE, AChE and butyrylcholinesterase (BChE) in CSF was measured with the use of colorimetric method (Ellman et al., 1961) as described previously by Sirvi6 et al. (1989). The levels of SLI, BLI and A C T H in the CSF were measured by radioimmunological assays (Jolkkonen et al., 1987).

Statistical methods The normal distributions of the analysed variables were tested with Kolmogorov-Smirnov test. Differences between the age groups were evaluated by the analysis of variance (ANOVA/Duncan test) and the effects of other factors to CSF were investigated by the analysis of covariance. Relationships between CSF variables were analysed with the use of Pearson correlation test. The data were presented as the means 4- standard deviation (SD). The result was considered significant at the p < 0.05 level.

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Table 2. Levels of protein, monoaminergic and cholinergic markers and neuropeptides of the CSF in normal aging

Age group Age range N

I 20-39 years 11

II 40-59 years 12-14

III 60-79 years 9

Protein (mg/1) MHPG (nmol/1) DBH (pmol/ml/min) 5HIAA (nmol/1) HVA (nmol/1) ChE (nmol/ml/min) AChE (nmol/ml/min) BChE (nmol/ml/min) SLI (pmol/1) ACTH (pmol/1) BLI (pmol/1)

400.5 -4- 59.5 31.0 + 3.4 13.8 4- 9.9 120.0 4- 18.6 179.1 + 46.9 26.4 4- 3.7 21.8 + 3.3 10.2 + 1.5 44.0 + 9.2 20.5 • 4.0 17.0 + 1.7

449.7 • 109.8 34.9 + 5.4* 13.4 + 7.8 146.3 4- 56.9 225.8 4- 92.4 32.3 + 10.0 27.8 4- 6.6 10.6 4- 2.8 52.5 4- 15.1 20.0 4- 3.9 17.8 + 2.5

482.2 4- 100.7 36.0 -4- 3.7* 16.3 4- 8.0 136.0 4- 42.0 223.6 -4- 67.2 32.4 -4- 8.9 26.9 4- 7.4 10.6 + 3.2 48.5 + 13.5 19.8 4- 2.6 18.8 + 2.1

Values given are the means + SD Significance of difference vs group I: * p < 0.05 (Oneway analysis of variance/Duncan test) Abbreviations: M H P G 3-methoxy-4-hydroxyphenylglycol;D B H dopamine-13-hydroxylase; 5 H I A A 5-hydroxyindoleacetic acid; H V A homovanillic acid; ChE cholinesterase; A C h E acetylcholinesterase; BChE butyrylcholinesterase; S L I somatostatin-like immunoreactivity; A C T H adrenocorticotropic hormone; B L I [3-endorphin-like immunoreactivity

Results

The m a i n clinical characteristics o f the subjects are shown in Table 1. F o r age related c o m p a r i s o n s the subjects were divided into three groups according to their age (Table 1). W o m e n were significantly smaller t h a n m e n (p < 0.001) a n d the w o m e n in the group III t e n d e d to be smaller t h a n the w o m e n in the two other groups. M e n were equally tall in all age groups. The weight o f m e n was significantly higher t h a n the weight o f w o m e n in all age groups (p -- 0.03). A n age-related increase o f systolic blood pressure values was observed (r -- 0.53, p = 0.001), and systolic blood pressure differed significantly between the oldest and youngest group, but the significantly different systolic blood pressure values o f the oldest age group did n o t influence any o f the C S F variables measured. The CSF levels o f proteins, M H P G , D B H , 5 H I A A , H V A , ChE, ACHE, BChE, SLI, A C T H , BLI are s h o w n in Table 2. The CSF p a r a m e t e r s did not differ significantly between gender groups. The levels o f M H P G and A C h E showed significant differences between the age groups (Table 2). F u r t h e r m o r e , a correlation analysis o f all age groups together showed a significant positive correlation between age and the levels o f M H P G (r = 0.44, p = 0.01) (Fig. 1) a n d A C h E (r -- 0.34, p -- 0.05) (Fig. 2) in the CSF, w h e n the activity o f A C h E was presented per v o l u m e unit. W h e n the activity o f A C h E was adjusted for protein c o n t e n t o f CSF, the levels o f A C h E did n o t correlate with age. The

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Table 3. Seasonal variation in CSF protein, monoaminergic and cholinergic markers and neuropeptides during normal aging

Protein (mg/1) MHPG (nmol/1) DBH (pmol/ml/min) 5HIAA (nmol/1) HVA (nmol/1) ChE (nmol/ml/min) AChE (nmol/ml/min) BChE (nmol/ml/min) SLI (pmol/1) ACTH (pmol/1) BLI (pmol/1)

Light season (April-September)

Dark season (October--March)

Group effecta

SIG

414.5 4- 91.7 (15) 33.2 4- 4.8 (15) 9.2 4- 5.0 (13) 137.5 4- 47.7 (14) 188.8 4- 60.3 (13) 31.4 + 9.6 (15) 25.1 4- 6.9 (14) 10.1 4- 3.0 (14) 51.0 4- 17.0 (15) 22.8 4- 2.9 (15) 18.6 4- 2.0 (14)

464.4 4- 97.0 (19) 34.3 + 4.7 (19) 19.3 + 9.8 (19) 132.7 4- 41.3 (19) 223.0 4- 79.7 (19) 29.7 4- 7.5 (19) 25.7 4- 6.2 (19) 10.8 4- 2.0 (18) 46.8 4- 9.2 (19) 18.0 4- 2.3 (19) 17.2 4- 2.2 (19)

1.16 0.01 10.24 0.13 4.29 0.90 0.008 0.57 1.30 26.60 5.07

0.29 0.91 0.004 0.72 0.05 0.35 0.93 0.46 0.26 0.000 0.03

Values given are the means + SD. Number of subjects is in parenthesis a ANOVA all variables adjusted for age, gender, season and height Abbrevations as in Table 2 effect o f blood c o n t a m i n a t i o n on A C h E levels in C S F was insignificant (r -- 0.027, p = 0.884). Height did not affect other C S F p a r a m e t e r s t h a n H V A ; the negative correlation with height was observed even after the effects o f sex, season and age on the H V A levels were t a k e n into a c c o u n t (F = 4.1, p = 0.05). Similarly, the correlation between C h E a n d height (r -- - 0.37, p = 0.04) was s h o w n to be insignificant, w h e n the d a t a was adjusted for age, season and sex (F = 2.4, p = 0.13). The season h a d a significant influence on the C S F levels o f H V A , D B H a n d A C T H (Table 3). Weight was significantly related only to SLI (r = 0.42, p = 0.03) (Table 5). The correlation between A C T H and weight was d e m o n s t r a t e d insignificant, w h e n A C T H was corrected for seasonal variability. The storage time o f the specimens did n o t influence on the C S F measures. Since height a n d weight are intercorrelated, we t o o k into a c c o u n t a n o t h e r measure, n a m e l y b o d y mass index (BMI), which did n o t correlate significantly with the CSF measures (Table 5). The b l o o d - b r a i n barrier (BBB) function was evaluated by a correlation with age and C S F / s e r u m a l b u m i n ratio (r = - 0.20, p = 0.41). A n u m b e r o f significant intercorrelations (15 f r o m 55) were f o u n d between C S F variables particularly between H V A a n d 5 H I A A (r = 0.66, p = 0.000), SLI and cholinesterases A C h E (r = 0.37, p = 0.04) and B C h E (r = 0.39, p = 0.03), a n d BLI and A C T H (r = 0.49, p = 0.004) (Table 4).

Discussion The m a i n aim o f the present study was to find the possible effects o f aging a n d other c o n f o u n d i n g factors on the c o m m o n l y used n e u r o t r a n s m i t t e r m a r k e r s in

0.67 p = 0.000 (32)

0.18 p = 0.31 (32)

0.64 p = 0.000 (32)

0.05 p = 0.79 (32)

0.05 p = 0.78 (33)

- 0.04 p = 0.80 (34)

0.32 p = 0.07 (34)

- 0.06 p = 0.73 (33)

BChE

MHPG

DBH

HVA

5HIAA

ACTH

SLI

BLI

0.25 p = 0.17 (33)

0.57 p = 0.000 (34)

0.02 p = 0.92 (34)

- 0.12 p = 0.52 (33)

0.16 p = 0.38 (32)

0.30 p = 0.10 (32)

0.21 p = 0.25 (32)

0.80 p = 0.000 (32)

0.86 p = 0.000 (33)

ChE

0.31 p = 0.09 (32)

0.37 p = 0.04 (33)

0.10 p = 0.58 (33)

0.08 p = 0.65 (32)

0.19 p = 0.31 (31)

0.34 p = 0.06 (32)

0.40 p = 0.02 (32)

0.75 p = 0.000 (32)

AChE

0.06 p = 0.73 (31)

0.39 p = 0.03 (32)

0.02 p = 0.92 (32)

- 0.19 p = 0.30 (31)

0.06 p = 0.74 (30)

0.45 p = 0.02 (31)

0.07 p = 0.72 (31)

BChE

0.35 p = 0.05 (32)

0.19 p = 0.30 (32)

0.000 p = 0.99 (32)

0.33 p = 0.06 (32)

0.28 p = 0.13 (31)

0,10 p = 0.59 (32)

MHPG

- 0.31 p = 0.09 (31)

0.16 p = 0.38 (32)

- 0.45 p = 0.009 (32)

- 0.05 p = 0.77 (32)

0.14 p = 0.45 (31)

DBH

0.09 p = 0.61 (32)

- 0.009 p = 0.96 (32)

- 0.25 p = 0.17 (32)

0.66 p = 0.000 (32)

HVA

N u m b e r s i n d i c a t e P e a r s o n ' s c o r r e l a t i o n coefficients a n d c o r r e s p o n d i n g p - v a l u e s ( t w o - t a i l p r o b . ) N u m b e r o f p a t i e n t s is i n p a r e n t h e s i s A b b r e v i a t i o n s as i n T a b l e 2

-

0.56 p = 0.001 (33)

AChE

-

0.28 p = 0.10 (34)

ChE

Proteins

0.03 p = 0.85 (33)

- 0.05 p = 0.78 (33)

- 0.02 p = 0.91 (33)

5 HIAA

0.49 p = 0.004 (33)

0.06 p = 0.75 (34)

ACTH

0.02 p = 0.92 (33)

SLI

T a b l e 4. C o r r e l a t i o n s b e t w e e n C S F p r o t e i n , m o n o a m i n e m e t a b o l i t e , c h o l i n e s t e r a s e s a n d n e u r o p e p t i d e v a l u e s i n n o r m a l a g i n g s u b j e c t s

t~

~Z

0.07 p = 0.66 (33)

- 0.07 p = 0.69 (34)

0.01 p = 0.95 (27)

- 0.12 p = 0.50 (34)

Height

Weight

BMI

S-Time

- 0.13 p = 0.48 (33)

0.26 p = 0.20 (27)

- 0.08 p = 0.69 (28)

- 0.37 p = 0.04 (33)

0.30 p = 0.09 (34)

ChE

- 0.05 p = 0.77 (32)

- 0.05 p = 0.83 (26)

- 0.09 p = 0.65 (27)

- 0.15 p = 0.39 (32)

0.34 p = 0.05 (33)

AChE

- 0.03 p = 0.88 (31)

0.09 p = 0.68 (25)

0.05 p = 0.80 (26)

- 0.0003 p = 0.99 (31)

0.07 p = 0.35 (32)

BChE

- 0.14 p = 0.45 (32)

0.04 p = 0.85 (25)

0.04 p = 0.81 (26)

0.01 p = 0.93 (31)

0.44 p = 0.01 (32)

MHPG

- 0.06 p = 0.73 (32)

- 0.09 p = 0.67 (25)

0.33 p = 0.10 (25)

0.24 p = 0.20 (30)

0.18 p = 0.34 (31)

DBH

- 0.20 p = 0.27 (32)

0.20 p = 0.34 (25)

- 0.15 p = 0.45 (26)

- 0.41 p = 0.02 (31)

0.28 p = 0.12 (32)

HVA

- 0.03 p = 0.88 (33)

- 0.04 p = 0.83 (26)

- 0.17 p = 0.39 (27)

- 0.16 p = 0.38 (32)

0.09 p = 0.59 (33)

5 HIAA

- 0.29 p = 0.09 (34)

- 0.05 p = 0.82 (27)

- 0.40 p = 0.03 (28)

- 0.30 p = 0.03 (33)

- 0.14 p = 0.43 (34)

ACTH

- 0.11 p = 0.52 (34)

- 0.34 p = 0.08 (27)

0.42 p = 0.03 (28)

0.07 p = 0.66 (33)

0.18 p = 0.31 (34)

SLI

Numbers indicate Pearson's correlation coefficients and corresponding p-values (two-tail prob.) N u m b e r o f p a t i e n t s is i n p a r e n t h e s i s . B M I b o d y m a s s i n d e x ; S-Time s t o r a g e t i m e . O t h e r a b b r e v i a t i o n s a s i n T a b l e 2

-

0.38 p = 0.03 (34)

Age

Proteins

BLI

- 0.24 p = 0.17 (33)

- 0.11 p = 0.58 (26)

- 0.34 p = 0.07 (27)

- 0.27 p = 0.13 (32)

0.29 p = 0.09 (33)

T a b l e 5. C o r r e l a t i o n s b e t w e e n C S F v a r i a b l e s a n d age, h e i g h t , w e i g h t , B M I a n d S C F s t o r a g e t i m e i n t h e w h o l e s t u d y g r o u p

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Neurotransmitter markers in the cerebrospinal fluid of normal subjects. Effects of aging and other confounding factors.

We have investigated neurotransmitter-related markers of the cerebrospinal fluid (CSF) in a carefully screened series of normally aging subjects in st...
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