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Cerebrospinal Fluid Choline Levels Are Decreased in Parkmson’s Disease

in untreated Parkinson’s disease patients and in those who were treated with carbidopa-levodopa and carbidopa-levodopa plus amantadine, which is known to have anticholinergic activity.

Bala V. Manyam, MD,+ Ezio Giacobini, MD, PhDJ and Jerry A. Colliver, PhD$

We examined acetylcholinesterase (AChE) activity and choline levels in cerebrospinal fluid (CSF) in 16 patients with idiopathic Parkinson’s disease and 9 control subjects of corresponding age: 8 were untreated Parkinson’s patients; 4 were treated with carbidopa-levodopa (1001 1,000 mg/day) for 20 3 months; and 4 were treated with carbidopa-levodopa (110/1,100 mg/day) for 28 & 18 months plus amantadine (200 mg/day) for 16 8 months. CSF choline levels (nmoUml) were 2.97 & 0.79 (control subjects); 1.31 0.29 (untreated patients); 1.00 0.29 (carbidopa-levodopa treated); and 1.26 0.19 (carbidopa-levodopdamantadinetreated). Choline levels were significantly lower in untreated and treated patients compared to control subjects (p = 0.0001).AChE activity did not differ in Parkinson’s disease patients as compared to control subjects. The reduced level of choline in CSF may reflect a deficit in choline transport into the brain or a decrease of choline-phospholipid output from the brain.

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Manyam BV, Giacobini E, Colliver JA. Cerebrospinal fluid choline levels are decreased in Parkinson’s disease. Ann Neurol 1990;27:683-685

Patients and Methods Sixteen men with idiopathic Parkinson’s disease were included in the study after informed consent was obtained. The clinical profile of patients is presented in Table 1. Objective recording of mental status was not undertaken, hence, cognitive state was not compared. The control group consisted of 9 men with a mean ( * SD) age of 64 & 9 years who had no known neurological or mental disease.

Lumbar Puncture CSF was collected via lumbar puncture in all patients following overnight bed rest and fasting for 12 hours. In patients who were treated for Parkinson’s disease, the interval between the last dose of medication and CSF collection was at least 12 hours. The details of CSF collection, preparation, and storage conditions are described elsewhere fbf. CSF used for measurement of choline and AChE activity from both control subjects and the experimental group was taken from the first 10 ml of CSF drawn. For study of gradient effect on AChE activity and choline, CSF was collected in 4-ml sequential aliquots in 6 patients with Parkinson’s disease. Choline levels and AChE activity were measured in the first, third, and fifth 4-ml samples.

Assay of Acetylcholinesterase Activity The radiometric method of Johnson and Russell 171was used to determine AChE activity with 3H-acetylcholineiodide as a substrate (90 mCdmmo1, sp. act.). The details are described elsewhere f4}.

Choline Assay There is both biochemical and pharmacological evidence for the role of cholinergic function in Parkinson’s disease [l]. Choline is a precursor in the synthesis of acetylcholine (ACh) and neuronal membrane phospholipids. Acetylcholinesterase (AChE), the enzyme that inactivates ACh, is neuronal in origin and is secreted into cerebrospinal fluid (CSF) following electrical stimulation of the brain 121 or treatment with drugs 131. CSF choline levels and AChE activity may be important biochemical markers of cholinergic dysfunction and neuronal damage in Alzheimer’s disease 14, 51. We measured CSF choline and AChE activity

From the *Division of Neurology, Department of Medicine; tDepartment of Pharmacology; and the $Division of Statistics and Research Consulting, Southern Illinois University School of Medicine, Springfield, IL. Received Jun 23, 1989, and in revised form Oct 5 and Nov 27. Accepted for publication Nov 27, 1989. Address correspondence to Dr Manyam, Division of Neurology, D411, SIU School of Medicine, PO Box 19230, Springfield, IL 62794-9230.

For the assay of CSF choline, 1 ml of CSF was mixed with 10 ml 1 N formic acid-acetone (15535) and centrifuged at 3,OOOg for 20 minutes to remove protein. The levels of choline were measured in 15-(*.1aliquots of CSF by the radiometric enzymatic assay [ S ] .

Statistical Analysis of Results Computations were done on an IBM 4341 computer. The Statistical Analysis System (SAS) procedure was used to estimate means, standard deviations, and significance. The mean choline levels and AChE activity for the four groups (normal control subjects, untreated, carbidopallevodopatreated, and carbidopallevodopaplus amantadine treated) were compared with analysis of covariance, using age as the covariate to adjust for difference among the four groups due to age. Follow-up tests were used for pairwise comparison of the four groups. All group means were reported as the mean 2 1 standard deviation and significance expressed as a p value. Results

The mean ( * SD) CSF choline levels adjusted for age were significantly lower (p = 0.0001) in patients with untreated Parkinson’s disease than in control subjects

Table 1. Clinical Summary of Patients with Parkinson's Disease" ~~~

~~

Treatment None No. of patients Age (yr) Duration of illness (mo) Carbidopa-levodopa (mg/day) Duration of treatment (mo) Amantadine (mglday) Duration of treatment (mo) "All values are expressed as mean

Carbidopa-Levodopa

65 t 7 123 2 57 110/1, 100 28 t 18 200 19

*

... ... ...

... ...

...

+ Amantadine

4

4 53 ? 12 53 36 100/1,000 20 t 3

8 57 ? 15 22 c 16

?

Carbidopa-Levodopa

SD.

Table 2. Parkinson's Disease: CSF AChE Activity and Choline Levels" Category

No.

Age (yr)

Choline (nmoyml)

AChE (dmVhr)

Normal controls Parkinson's disease N o treatment Carbidopa-Ievodopa treated Carbidopa-levodopa and amantadine treated

9

64 c 9

2.97

0.81

8

57

1.31 ? 0.2gb

0.83 t 0.19'

4

53 t 12

1.00 ? 0.29b

0.90 t 0.43'

1.26

1.19

~~~~

~~

4 ~

65 ~~

+-

?

~~~

5

7

0.79

?

0.19b

?

* 0.28

?

0.20'

~~

"All values are expressed as mean f SD. bSignificant compared to controls (p = 0.0001) by analysis of covariance with age as covariate. 'Not significant when compared to controls.

CSF = cerebrospinal fluid; AChE = acetylcholinesterase.

Table 3. Efect of Gradient on CSF Choline Leveh and AChE Activity (n

=

6)"

Order of CSF Aliquots (4 ml) Withdrawn

3rd

1st

Choline (nmoVml) AchE (pmoVmVhr) Protein (mg/dl) Albumin (mg/dl)

* 0.03 * 0.06 6 *3

1.23 1.09 52 ? 26

1.07 +. 1.15 t 47 +23 -t

5th

0.04b O.OSb 6' 3'

1.29 ? O.OSb 1.11 f O.Obb

44 c

5d

22 c 3d

"All values are expressed as mean 2 SEM. bNot significant. 'Significant at p < 0.01 by paired Student's t test. dSignificant at p < 0.005 by paired Student's t test.

CSF

=

cerebrospinal ffuid; AChE = acetylcholinesterase.

(Table 2). No difference in the mean CSF AChE activity adjusted for age was seen between control subjects and untreated Parlunson's disease patients. No significant difference was seen between untreated Parkinson's disease patients, carbidopa-levodopa treated patients, and carbidopa-levodopa plus amantadine treated patients for mean choline levels and CSF AChE activity adjusted for age, showing that the treatment did not have any effect. The correlation coefficient between CSF AChE activity and choline levels and duration of illness, sever684 Annals of Neurology Vol 27 N o 6 June 1990

ity of the disease, and effect of treatment did not show any significant relationships. CSF choline levels and AChE activity in sequential aliquots of CSF are shown in Table 3. Whereas CSF protein and albumin levels decreased significantly in serial aliquots, choline levels and AChE activity showed no significant change.

Discussion The relationship between age and CSF choline has previously been investigated; two studies have found no variation of CSF choline levels with advancing age { S ,

97, whereas others [lo, 111 reported a modest agedependent increase in CSF choline levels. In our study, the mean age of control subjects and that of Parkinson’s disease patients treated with carbidopdlevodopa plus amantadine was identical, whereas the untreated group and the patients treated with carbidopdlevodopa had a slightly lower mean age. The values in CSF choline levels adjusted for age showed a significant difference between the control subjects and the Parkinson’s disease patients. Aquilonius and colleagues [9], Welch and colleagues 1127, and Flentge and colleagues [lo} measured choline levels in CSF of Parkinson’s disease patients using radioenzymatic or gas chromatographidmass spectrometric methods. These authors did not find significant changes in CSF choline levels. The significantly lower mean age of the control subjects used in those three studies could explain the difference with our results. We found no evidence of caudocranial gradient for CSF choline levels or for AChE activity, although CSF protein and albumin values measured from the same aliquots fell significantly (see Table 3). Welch and associates 1127, taking an “early” and a “late” aliquot of CSF when a pneumonoencephalogram or myelogram was done, reported a ventricular-lumbar gradient for CSF choline, with ventricular fluid levels being higher than lumbar CSF levels. A similar finding was also reported by Bowers C131, but our data (see Table 3 ) with sequential CSF collection during lumbar puncture, with measurement of CSF protein and albumin as controls, failed to show a gradient for choline, Dietary intake is not a factor to be considered, as none of our patients were on any dietary restriction. Although we did not measure plasma choline levels, we performed lumbar puncture at the same time of the day (between 8:OO AM and 9:OO AM) in both the control group and the experimental group. We postulate that low plasma choline levels do not explain the low CSF choline levels in Parkinson’s disease patients reported here. We propose that a decline in CSF choline may be due to a defect in choline transport into the brain or a decrease of choline-phospholipid output from the brain. The origin of CSF choline is twofold; plasma choline and choline from brain extracellular fluid. Thus, a reduced level of CSF choline may be due primarily to a lower uptake into the brain or secondarily to a lower release from the membrane cholinephospholipid pool. Alternatively, increased active transport of choline out of CSF into the blood could explain the decreased levels of this substance in CSF. Our finding of lower lumbar CSF choline in Parkinson’s disease is of interest in view of the reported loss of cholinergic neurons in the basal forebrain and deficiencies in cortical cholinergic enzymes seen in these patients 114, 151. However, the contribution of altered ACh metabolism to choline changes in CSF

may be small. Further investigation into the specificity and mechanism of our finding may provide insight into choline metabolism and transport in Parkinson’s disease.

This work was supported in part by National Institute of Aging grant AG05416. We wish to thank D. Linville, MS, and E. Williams, BS, for their technical assistance.

References 1. Lloyd KG. Neurotransmitter interactions related to central dopamine neurons. In: Youdim MBH, Lovenberg W, Sharman DF, et al, eds. Essay in neurochemistry and neuropharmacology, vol 3. New York John Wiley, 1978:129-207 2. Desarno P, Giacobini E. Release of acetykholinesterase from the caudate nucleus in the rat. J Neurosci Res 1987;18:578590 3. Bareggi SR, Giacobini E. Acetylcholinesterase activity in ventricular and cisternal CSF of dogs: effect of chlorpromazine. J Neurosci Res 1978;3:335-339 4. Elble R, Giacobini E, Scarsella GF. Cholinesterases in cerebrospinal fluid: a longitudinal study in Alzheimer disease. Arch Neurol 1987;44:403-407 5. Elble RJ, Giacobini E, Higgins C. Choline levels are increased in cerebrospinal fluid of Alzheimer patients. Neurobiol Aging 1989;10:45-50 6. Manyam NVB, Hare TA, Katz L. Effect of isoniazid on cerebrospinal fluid and plasma GABA levels in Huntington’s disease. Life Sci 1980;26:1303-1308 7. Johnson CD, Russell RL. A rapid, simple radiometric assay for cholinesterase. Analyt Biochem 1975;64:229-238 8. McCaman RE, Stetzler J. Radiochemical assay for acetylcholine: modification for sub-picomoles measurements. J Neurochem 1977;28:669-671 9. Aquilonius SM, Nystrom B, Schuberth J, Sundwall A. Cerebrospinal fluid choline in extrapyramidal disorders. J Neurol Neurosurg Psychiatry 1972;35:720-725 10. Flentge F, van der Meulen WMH, Lakke JPWF, Teelken AW. CSF choline levels in groups of patients with cranial trauma or extrapyramidal disorders. J Neurol Neurosurg Psychiatry 1984; 471207-209 11. Glen AI, Yates CM, SimpsonJ, et al. Choline uptake in patients with Alzheimer pre-senile dementia Psycho1 Med 1981;ll: 469-476 12. Welch MJ, Markham CH, Jenden DJ. Acetylcholine and choline in cerebrospinal fluid of patients with Parkinson’s disease and Huntington’s chorea. J Neurol Neurosurg Psychiatry 1976;39:367-374 13. Bowers MB. Choline in cerebrospinal fluid. Life Sci 1967;6: 1927-1933 14. Nakano I, Hirano A. Pakinson’s disease: neuron loss in the nucleus basalis without concomitant Alzheimer’s disease. Neurology 1984;15:4 15-418 15. Whitehouse PJ, Hedreen JC, White CL, Price DL. Basal forebrain neurons in the dementia of Parkinson’s disease. Ann Neur01 1983;13:243-248

Brief Communication: Manyam et al: CSF Choline Levels in PD 685

Cerebrospinal fluid choline levels are decreased in Parkinson's disease.

We examined acetylcholinsterase (AChE) activity and choline levels in cerebrospinal fluid (CSF) in 16 patients with idiopathic Parkinson's disease and...
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