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Cholinesterases in cerebrospinal fluid
Journal of the Neurological Sciences, 1986, 72:121-129 Elsevier
121
JNS 2604
Cholinesterases in Cerebrospinal Fluid Correlations with Clinical Measures in Alzheimer's Disease F. J a c o b H u f f 1'2"3, Jean-Claude Maire 4, John H. G r o w d o n 2"3, Suzanne Corkin 1'2 and Richard J. Wurtman a 1Department of Psychology and 2ClinicalResearch Center, Massachusetts Institute of Technology, Cambridge; 3Department of Neurology, Harvard Medical School at the Massachusetts General Hospital, Boston and 4Laboratory of Neuroendocrine Regulation, Massachusetts Institute of Technology, Cambridge, MA (U.S.A.) (Received 9 April, 1985) (Revised, received 23 August, 1985) (Accepted 26 August, 1985)
SUMMARY
Acetylcholinesterase and butyrylcholinesterase activities in cerebrospinal fluid were measured in 17 patients with Alzheimer's disease and 6 control patients, as potential clinical measures of impaired cholinergic'nenrotransmission in Alzheimer's disease. The activity of butyrylcholinesterase was decreased in patients with Alzheimer's disease compared to that observed in control patients, but there was overlap between values in the 2 groups. Low butyrylcholinesterase activity was correlated with severity of dementia, memory impairment, and language disorder. Acetylcholinesterase activity was significantly correlated with visual contrast sensitivity, but not with dementia severity, and did not differentiate patients with Alzheimer's disease from control cases. These results suggest that cholinesterases in cerebrospinal fluid are related to brain cholinesterases, and indicate that the activities of acetylcholinesterase and butyrylcholinesterase should be distinguished in studies of cerebrospinal fluid.
Key words: Acetylcholinesterase - Alzheimer's disease - Butyrylcholinesterase - Cerebrospinal f l u i d - N e u r o p s y c h o l o g y - Visual contrast sensitivity
This work was supported by NIH grants AG000232, MH32724, MH28783, US Army grant DAAG2983D0045, and by a Swiss Foundation for Fellowship in Medicine and Biology grant to Dr. Maire. Reprint requests to Dr. Huff. Address: 517 Falk Clinic, Departments of Psychiatry and Neurology, Western Psychiatric Institute and Clinic, University of Pittsburgh, 3601 Fifth Avenue, Pittsburgh, PA 15213, U.S.A. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
122 INTRODUCTION Enzymes involved in metabolism of acetylcholine are affected in Alzheimer's disease (AD). Brain levels of choline acetyltransferase and of acetylcholinesterase (ACHE) are decreased and activity of the nonspecific cholinesterase, butyrylcholinesterase (BuChE), is increased (Davies and Maloney 1976; Op den Velde and Stare 1976; Perry et al. 1978; Bowen et al. 1982). These findings have led to studies of AChE and BuChE in cerebrospinai fluid (CSF) as a diagnostic test for AD. Because the direction of alteration of AChE in brain is opposite that of BuChE, it is reasonable to expect that alterations of these enzymes in CSF may differ from one another. Studies in animals suggest a neuronal origin of CSF cholinesterases (ChEs). AChE is secreted into CSF in response to drugs (Bareggi and Giacobini 1978; Greenfield and Shaw 1982), and to electrical stimulation of the brain (Greenfield and Smith 1979), indicating that neuronal activity can result in secretion of ACHE. Studies of molecular forms of ChEs by electrophoresis (Chubb et al. 1976; Scarsella et al. 1979; Greenfield et al. 1983) and of the rate of recovery of ChE activity after irreversible inhibition (Yaksh et al. 1975), suggest that brain tissue and not plasma is the source of CSF ACHE, and probably also CSF BuChE. There is no evidence that ChE activities in blood and CSF are related, and studies of ChE activities in plasma and erythrocytes have produced conflicting results regarding differences between AD and control cases (Chipperfield etal. 1981; Perry etal. 1982; Smith et al. 1982). Several investigators have reported that AChE activity in CSF was lower in AD than in control patients (Appleyard et al. 1983; Tune et al. 1985), while others did not detect a difference (Davies 1979; Deutsch et al. 1983; Lal et al. 1984). Total ChE activity in CSF was observed to be lower in AD than control patients in one study (Soininen et al. 1984). Arendt et al. (1984) reported that the ratio of AChE to BuChE in CSF separated AD and control cases better than either enzyme measurement alone. Dementia severity was reported to correlate weakly with AChE (Tune et al. 1985) and total ChE (Soininen et al. 1984), but other investigators did not find a correlation (Lal et al. 1984). These results are inconclusive, and indicate a need for clarification of methodological issues that may explain discrepancies among different studies. We investigated whether CSF ACHE, BuChE, and the ratio of BuChE to AChE activities can be used to discriminate patients with Alzheimer's disease from nondemented patients with peripheral or central nervous system disorders that are not known to involve altered ChE activities. We also examined correlations between neurochemical measures and clinical neuropsychological measures in the AD patients. It was expected that if differences were observed between AD and control patients in neurochemical measures, then correlations would be found between the neurochemical and neuropsychological abnormalities in AD patients.
123 METHODS
Patients The diagnosis of AD was determined clinically in patients with dementia of both presenile and senile onset. Diagnostic criteria (McKhann et al. 1984) included progressive worsening of memory and other cognitive functions and exclusion of other causes of dementia by neurological examination, laboratory studies, CT, and EEG. We excluded patients who were taking psychoactive medication and those with evidence of alcoholism or depression. The AD group comprised 17 patients (7 men and 10 women) with a mean age of 63.8 years (range 52-75). Onset of dementia occurred before age 65 in 12 patients, and at 65 or later in 5. The mean score on the Blessed Dementia Scale (Blessed et al. 1968) was 17.2, with a range of 2-33. Controls comprised 6 patients (3 men and 3 women), 4 undergoing myelography for radicular symptoms with no CSF block on the myelogram, and 2 nondemented patients who had recovered from transient amnesia, one associated with closed head injury. Mean ages of the control (59.0 years, range 44-76) and AD groups did not differ significantly. Control patients were not demented on the basis of neurological examination, but were not given the Blessed Dementia Scale or other neuropsychological tests. Sample collection Patients were recumbent for 12 h before lumbar puncture, which was performed between 9.00 h and 11.00 h. The 3rd and 4th ml of CSF collected were used for assay of ChEs. All specimens were frozen before analysis 1-90 days later. Lumbar puncture and neuropsychological tests were usually performed in the same week, and always within 4 months. Biochemical procedures Activities of AChE and BuChE were determined by a colorimetric method using thiocholine ester substrates (Ellman et al. 1961). Because the activity of BuChE on acetylthiocholine was 25 ~o that of AChE, the BuChE inhibitor ethopropazine was used when assaying ACHE. Cross-reactivity of AChE in the BuChE assay was insubstantial, and no inhibitor was used. Neuropsychological tests Patients in the AD group were given the following 14 neuropsychological tests: The Blessed Dementia Scale (Blessed et al. 1968), a measure of overall severity of dementia; 4 tests of memory ability: immediate memory (Brown 1958), verbal paired-associate learning (Corkin 1982), nonverbal paired-associate learning (Corkin 1982), and verbal recognition memory (Craik and Lockhart 1972); 6 measures of language ability: the Token Test (DeRenzi and Vignolo 1962), the Reporter's Test (DeRenzi and Ferrari 1978), WAIS Vocabularly Test (Wechsler 1955), category fluency test (Huffand Corkin 1984), Boston Naming Test (Kaplan et al. 1978), semantic naming errors (Huff and Corkin 1984);
124 and 3 measures of visuospatial functions: visual form discrimination (Huff and Corkin 1984), matchsticks test (Benson and Barton 1970), and visual contrast sensitivity (Nissen et al. 1985). A score is not available on some of the neuropsychological tests for several of the most impaired patients, who were unable to perform those tests. RESULTS C S F AChE (Table 1) did not differ significantly between AD and control patients (t (21) = 0.86; P = 0.40). BuChE was significantly lower in the A D group (t (21) = 2.33; P < 0.05), as was the ratio of BuChE to AChE activities (t (21) = 2.21; P < 0.05). The range of values in the AD and control groups overlapped for all measures: AChE values for 14 of the 17 AD patients overlapped with values for 5 of the 6 control cases; BuChE values overlapped for 9 A D patients and 4 controls; BuChE/AChE values overlapped for 12 A D cases and 4 controls. In computing correlations involving neuropsychological tests, the signs of scores on tests were set so that a more positive score indicated more impaired performance. The abilities measured by different tests in the battery are not completely independent, and scores on different tests were intercorrelated. The correlations of tests with C S F ChE measures were examined to assess which tests correlated significantly with each biochemical measure. Contrast sensitivity was the only test that was significantly correlated with C S F AChE activity, and stronger correlations were observed at higher spatial frequencies (Table 2). C S F BuChE correlated significantly with severity of dementia as measured by the Blessed Dementia Scale, with 2 memory tests, and 3 language tests. The BuChE/AChE ratio in C S F correlated with dementia severity, 2 memory tests, and 4 language tests. In order to evaluate whether the correlations of memory and language tests with C S F BuChE/AChEwere independent of the correlation of the Blessed Dementia Scale with BuChE/AChE, partial correlations were determined, in which the correlation of Blessed Scale to the BuChE/AChE ratio was eliminated from correlations between TABLE 1 ACETYLCHOLINESTERASE(ACHE)AND BUTYRYLCHOLINESTERASE(BuChE) ACTIVITIES IN CSF FROM CONTROL PATIENTS AND PATIENTS WITH ALZHEIMER'S DISEASE Patient group
AChE (nmol/ml/min)
BuChE (nmol/ml/min)
BuChE AChE
Alzheimer's disease (n = 17)
20.3 _+1.4
7.3 + 0.5*
0.38 +_0.10*
Control (n = 6)
17.1 + 3.1
9.8 _+1.2
0.90 + 1.01
* Significantly different from controls by Student's t-test, P < 0.05.
125 TABLE 2 CORRELATIONS BETWEEN NEUROPSYCHOLOGICAL MEASURES AND CSF CHOLINESTERASES (ChEs) IN PATIENTS WITH ALZHEIMER'S DISEASE Neuropsychological measure
Correlation (r) with CSF ChE AChE
BuChE
BuChE AChE
Severity of dementia; Blessed Dementia Scale (n = 17) Memory: Immediate memory (n = 15) Verbal paired associates (n = 16) Nonverbal paired associates (n = 16) Verbal recognition memory (n = 12) Language: Token test (n = 11) Reporter's test (n = 11) WAIS vocabulary test (n = 12) Category fluency (n = 16) Boston naming test (n = 17) Semantic naming errors (n = 16) Visuospatial functions: Form discrimination (n = 15) Matchsticks test (n = 13) Contrast sensitivity (n = 12) 0.5 cpd 1.0 cpd 2.0 cpd 4.0 cpd 8.0 cpd
0.01 0.14 0.08 - 0.25 0.28 0.21 0.09 0.38 0.07 0.09 0.19
-0.54*
-0.52*
-0.49* -0.26 - 0.64** - 0.36
-0.65** -0.34 - 0.33 - 0.71"
-
-
0.32 0.62* 0.48 0.56* 0.54* 0.48
0.03 - 0.20
- 0.13 - 0.51
-
-
0.27 0.46 0.54 0.62* 0.67*
0.23 0.01 0.06 0.13 0.43
0.44 0.56 0.82** 0.66** 0.64** 0.72**
- 0.21 - 0.39 0.10 0.53 0.65* 0.52 0.27
* P < 0.05; ** P < 0.01. cpd = cycles per degree of visual angle. B u C h E / A C h E a n d o t h e r t e s t s ( M c N e m a r 1962). F i v e o f t h e m e m o r y a n d l a n g u a g e t e s t s r e m a i n e d s i g n i f i c a n t l y c o r r e l a t e d w i t h B u C h E / A C h E in t h i s a n a l y s i s ; t h e c o r r e l a t i o n f o r category fluency was nonsignificant. DISCUSSION W e o b s e r v e d n o d i f f e r e n c e in C S F
AChE
activity b e t w e e n A D a n d c o n t r o l
p a t i e n t s . D a v i e s (1979), D e u t s c h et al. (1983), a n d L a l et al. (1984) a l s o r e p o r t e d n o d i f f e r e n c e in C S F A C h E b e t w e e n A D a n d c o n t r o l c a s e s , b u t A p p l e y a r d et al. (1983) a n d T u n e et al. (1985) r e p o r t e d l o w e r C S F A C h E in A D r e l a t i v e t o c o n t r o l c a s e s . A p o s s i b l e e x p l a n a t i o n f o r t h e a b s e n c e o f a d i f f e r e n c e b e t w e e n A D a n d c o n t r o l g r o u p s in t h e p r e s e n t s t u d y is t h e h e t e r o g e n e i t y o f t h e c o n t r o l g r o u p , w h i c h i n c l u d e d p a t i e n t s w i t h transient a m n e s i a and others with s y m p t o m s o f r a d i c u l o p a t h y . However, values for A D c a s e s o v e r l a p p e d t h e r a n g e o f v a l u e s f o r b o t h t y p e s o f c o n t r o l p a t i e n t s . A l t h o u g h all
126 control cases studied by Tune et al. (1985) had myelograms for low back pain, half had CSF AChE values that overlapped the range for AD patients in their study. Thus, AChE values did not discriminate AD and control cases, despite the difference in mean values for the two groups. Appleyard et al. (1983) observed differences in AChE between AD and control cases in ventricular CSF obtained at postmortem. A difference in CSF AChE may be more difficult to detect in lumbar than ventricular CSF because AChE from spinal cord, or other extraneous factors, may affect lumbar CSF measurements. Another explanation for the discrepancies among studies may be differences in techniques for measuring ChE activities. In investigations using the colorimetric method (Ellman et al. 1961), specific inhibitors are necessary in order to differentiate AChE and BuChE activities. Soininen et al. (1984) measured total CSF ChE activity colorimetrically, and observed lower activity in AD than in control cases. Some investigators (Lal et al. 1984; Tune et al. 1985) report measuring AChE colorimetrically without using inhibitors. A contribution of BuChE in their AChE measurements is likely, and what is reported as "ACHE" would be more appropriately decribed as "total ChE", as was done by Soininen et al. BuChE may account for 10-15 ~o oftotai ChE measured in this way. We observed decreased CSF BuChE in AD patients, and the difference in CSF ChE between AD and control cases reported by Tune et al. and Soininen et al. probably reflect abnormalities of CSF BuChE as well as ACHE. Formation of the BuChE/AChE ratio eliminates possible variance in AChE and BuChE levels due to CSF volume dilution associated with different degrees of cerebral atrophy in different patients. This reduction in variance may account for the strong correlations between the BuChE/AChE ratio and clinical measures. Another reason for examining the CSF BuChE/AChE ratio is that brain activities of BuChE and AChE in AD deviate differently from normal values: BuChE activity is high, whereas AChE activity is low (Perry et al. 1978). If abnormalities in CSF BuChE and AChE activities in AD also differ in direction relative to normal, computing the BuChE/AChE ratio may enhance discrimination between AD and control cases. We observed that the ratio of BuChE to AChE in CSF was lower in AD than in control cases, whereas Arendt et al. (1984) reported a difference in the opposite direction. They observed decreased AChE and a tendency toward increased BuChE in AD cases, but we observed decreased BuChE and a tendency toward increased AChE in AD cases. These discrepancies are difficult to explain, although differences in patient populations may account for them. Arendt et al. did not provide clinical information about their patients, such as severity of dementia and use of psychoactive medications that may have influenced CSF ChE activities (Bareggi and Giacobini 1978; Greenfield and Shaw 1982). Our observation that greater severity of dementia correlated with lower CSF BuChE supports our observation of lower BuChE in AD than control cases. Because severity of dementia has been observed to correlate with increased BuChE in brain in AD (Perry et al. 1978), our observation of a correlation of clinical severity with decreased BuChE in CSF is surprising. It is possible that a shift in the relative proportions of soluble and insoluble forms ofBuChE (Massoulie and Bon 1982) in AD results in accumulation of insoluble BuChE in the brain, while decreased soluble BuChE is reflected in CSF.
127 In the present study, impairments on 6 tests of language and memory correlated significantly with low BuChE/AChE ratio in CSF. Correlations of 5 of these tests with BuChE/AChE were significant with Blessed Dementia Scale partialled out. It is possible either that these tests measure severity of dementia more reliably than the Blessed Scale, or that cognitive abilities measured by these tests are more strongly related to CSF and brain ChE activity than is the Blessed Scale. These correlations do not establish a role of BuChE in cognition and behavior: cognitive deficits and altered BuChE activity may be correlated only because of their shared association with the pathological process of AD. The function of BuChE in the brain is not clear. BuChE is present in glial cells and neurons (Graybiel and Ragsdale 1982) and is associated with senile plaques (Friede 1965; Struble et al. 1984). BuChE hydrolyzes substance P (Lockridge 1982) as well as acetylcholine, and may therefore be involved in catabolism of multiple neurotransmitters. There is no direct evidence that BuChE activity is involved in the neurochemical processes underlying cognition. Visual contrast sensitivity, particularly to high spatial frequencies, correlated with CSF ACHE. AChE is present in neurons of primate primary visual cortex (Graybiel and Ragsdale 1982), and abnormalities of contrast sensitivity to high spatial frequencies have been observed in patients with lesions of the parietal and occipital lobes (BodisWoUner and Diamond 1976). The anticholinesterase drug physostigmine differentially affects response to low and high spatial frequencies in the cat (Harding et al. 1983). Contrast sensitivity deficits occur in AD (Nissen et al. 1985), and it is possible that contrast sensitivity is affected early in the course of AD, but does not change with progression to more severe stages of dementia. However, the lack of correlation with dementia severity suggests that the relation of contrast sensitivity function to CSF AChE may not be exclusive to dementia or AD. Although the present study indicates that AD and control cases cannot be reliably distinguished by measurement of either CSF AChE or BuChE activities, such differentiation may be possible by concurrent measurement of ChEs and other neurochemical markers, such as somatostatin-like immunoreactivity (Soininen et al. 1984). Because activities of AChE and BuChE in CSF appear to be affected differently in AD, separate determination of the 2 enzymes may improve discriminative precision in studies of CSF neurochemical measures. ACKNOWLEDGEMENTS The authors thank T. John Rosen, and Chris Collins, for statistical analyses; Allison Feeley and Marguerite Randolph for testing subjects; and Darlene Bumford for manuscript preparation. REFERENCES Appleyard,M. E., A.D. Smith, G. K. Wilcockand M.M. Esiri (1983)DecreasedCSF acetylcholinesterase activity in Alzheimer's disease, Lancet, ii: 452.
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129 McKhann, G., D. Drachman, M. Folstein, R. Katzman, D. Price and E. M. Stadlan (1984) Clinical diagnosis of Alzheimer's disease - - Report of the NINCDS-ADRDA work group under the auspices of Health and Human Services Task Force on Alzheimer's Disease, Neurology (Cleveland), 34: 939-944. McNemar, Q. (1962) Psychological Stat~_tics, 3rd edition, Wiley, New York, pp. 164-168. Massoulie, J. and S. Bon (1982) The molecular forms of cholinesterase and acetylcholinesterase in vertebrates, Ann. Rev. Neurosci., 5: 57-106. Nissen, M.J., S. Corkin, F. S. Buonanno, J.H. Growdon, S.H. Wray and J. Bauer (1985) Spatial vision in Alzheimer's disease - - General findings and a case report, Arch. Neurol., 42: 667-674. Op den Velde, W. and F.C. Stam (1976) Some cerebral proteins and enzyme systems in Alzheimer's presenile and senile dementia, J. Amer. Geriat. Soc., 1: 12-16. Perry, E.K., B.E. Tomlinson, G. Blessed, K. Bergrnann, P.H. Gibson and R.H. Perry (1978) Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia, Brit. Med. J., II: 1457-1459. Perry, R. H., I. D. Wilson, M.J. Bober, J. Atack, G. Blessed, B. E. Tomlinson and E. K. Perry (1982) Plasma and erythroeyte acetylcholinesterase in senile dementia of Alzheimer type, Lancet, i: 174-175. Scarsella, G., G. Toschi, S.R. Bareggi and E. Giacobini (1979) Molecular forms of cholinesterases in cerebrospinal fluid, blood plasma, and brain tissue of the beagle dog, J. Neurosci. Res., 4: 19-24. Smith, R.C., B.T. Ho, L. Hsu, G. Vroulis, J. Claghorn and J. Schoolar (1982) Cholinesterase enzymes in the blood of patients with Alzheimer's disease, Life Sci., 30: 543-546. Soininen, H.S., J.T. Jolkkonen, K.J. Reinikainen, T.O. Halonen and P.J. Riekkinen (1984) Reduced cholinesterase activity and somatostatin-like immunoreactivity in the cerebrospinal fluid of patients with dementia of Alzheimer type, J. Neurol. Sci., 63: 167-172. Struble, R. G., J. C. Hedreen, L. C. Cork and D. L. Price (1984) Acetylcholinesterase activity in senile plaques of aged macaques, Neurobiol. Aging, 5: 191-198. Tune, L., S. Gucker, M. Folstein, L. Oshida and J.T. Coyle (1985) Cerebrospinal fluid acetylcholinesterase in dementia of the Alzheimer type, Ann. Neurol., 17: 46-48. Wechsler, D. (1955) Manual for the WechslerAdult Intelligence Scale, Psychological Corporation, New York. Yaksh, T. L., M. G. Filbert, L. W. Harris and H. I. Yamamura (1975) Acetylcholinesterase turnover in brain, cerebrospinal fluid and plasma, J. Neurochem., 25: 853-860.
121
JNS 2604
Cholinesterases in Cerebrospinal Fluid Correlations with Clinical Measures in Alzheimer's Disease F. J a c o b H u f f 1'2"3, Jean-Claude Maire 4, John H. G r o w d o n 2"3, Suzanne Corkin 1'2 and Richard J. Wurtman a 1Department of Psychology and 2ClinicalResearch Center, Massachusetts Institute of Technology, Cambridge; 3Department of Neurology, Harvard Medical School at the Massachusetts General Hospital, Boston and 4Laboratory of Neuroendocrine Regulation, Massachusetts Institute of Technology, Cambridge, MA (U.S.A.) (Received 9 April, 1985) (Revised, received 23 August, 1985) (Accepted 26 August, 1985)
SUMMARY
Acetylcholinesterase and butyrylcholinesterase activities in cerebrospinal fluid were measured in 17 patients with Alzheimer's disease and 6 control patients, as potential clinical measures of impaired cholinergic'nenrotransmission in Alzheimer's disease. The activity of butyrylcholinesterase was decreased in patients with Alzheimer's disease compared to that observed in control patients, but there was overlap between values in the 2 groups. Low butyrylcholinesterase activity was correlated with severity of dementia, memory impairment, and language disorder. Acetylcholinesterase activity was significantly correlated with visual contrast sensitivity, but not with dementia severity, and did not differentiate patients with Alzheimer's disease from control cases. These results suggest that cholinesterases in cerebrospinal fluid are related to brain cholinesterases, and indicate that the activities of acetylcholinesterase and butyrylcholinesterase should be distinguished in studies of cerebrospinal fluid.
Key words: Acetylcholinesterase - Alzheimer's disease - Butyrylcholinesterase - Cerebrospinal f l u i d - N e u r o p s y c h o l o g y - Visual contrast sensitivity
This work was supported by NIH grants AG000232, MH32724, MH28783, US Army grant DAAG2983D0045, and by a Swiss Foundation for Fellowship in Medicine and Biology grant to Dr. Maire. Reprint requests to Dr. Huff. Address: 517 Falk Clinic, Departments of Psychiatry and Neurology, Western Psychiatric Institute and Clinic, University of Pittsburgh, 3601 Fifth Avenue, Pittsburgh, PA 15213, U.S.A. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
122 INTRODUCTION Enzymes involved in metabolism of acetylcholine are affected in Alzheimer's disease (AD). Brain levels of choline acetyltransferase and of acetylcholinesterase (ACHE) are decreased and activity of the nonspecific cholinesterase, butyrylcholinesterase (BuChE), is increased (Davies and Maloney 1976; Op den Velde and Stare 1976; Perry et al. 1978; Bowen et al. 1982). These findings have led to studies of AChE and BuChE in cerebrospinai fluid (CSF) as a diagnostic test for AD. Because the direction of alteration of AChE in brain is opposite that of BuChE, it is reasonable to expect that alterations of these enzymes in CSF may differ from one another. Studies in animals suggest a neuronal origin of CSF cholinesterases (ChEs). AChE is secreted into CSF in response to drugs (Bareggi and Giacobini 1978; Greenfield and Shaw 1982), and to electrical stimulation of the brain (Greenfield and Smith 1979), indicating that neuronal activity can result in secretion of ACHE. Studies of molecular forms of ChEs by electrophoresis (Chubb et al. 1976; Scarsella et al. 1979; Greenfield et al. 1983) and of the rate of recovery of ChE activity after irreversible inhibition (Yaksh et al. 1975), suggest that brain tissue and not plasma is the source of CSF ACHE, and probably also CSF BuChE. There is no evidence that ChE activities in blood and CSF are related, and studies of ChE activities in plasma and erythrocytes have produced conflicting results regarding differences between AD and control cases (Chipperfield etal. 1981; Perry etal. 1982; Smith et al. 1982). Several investigators have reported that AChE activity in CSF was lower in AD than in control patients (Appleyard et al. 1983; Tune et al. 1985), while others did not detect a difference (Davies 1979; Deutsch et al. 1983; Lal et al. 1984). Total ChE activity in CSF was observed to be lower in AD than control patients in one study (Soininen et al. 1984). Arendt et al. (1984) reported that the ratio of AChE to BuChE in CSF separated AD and control cases better than either enzyme measurement alone. Dementia severity was reported to correlate weakly with AChE (Tune et al. 1985) and total ChE (Soininen et al. 1984), but other investigators did not find a correlation (Lal et al. 1984). These results are inconclusive, and indicate a need for clarification of methodological issues that may explain discrepancies among different studies. We investigated whether CSF ACHE, BuChE, and the ratio of BuChE to AChE activities can be used to discriminate patients with Alzheimer's disease from nondemented patients with peripheral or central nervous system disorders that are not known to involve altered ChE activities. We also examined correlations between neurochemical measures and clinical neuropsychological measures in the AD patients. It was expected that if differences were observed between AD and control patients in neurochemical measures, then correlations would be found between the neurochemical and neuropsychological abnormalities in AD patients.
123 METHODS
Patients The diagnosis of AD was determined clinically in patients with dementia of both presenile and senile onset. Diagnostic criteria (McKhann et al. 1984) included progressive worsening of memory and other cognitive functions and exclusion of other causes of dementia by neurological examination, laboratory studies, CT, and EEG. We excluded patients who were taking psychoactive medication and those with evidence of alcoholism or depression. The AD group comprised 17 patients (7 men and 10 women) with a mean age of 63.8 years (range 52-75). Onset of dementia occurred before age 65 in 12 patients, and at 65 or later in 5. The mean score on the Blessed Dementia Scale (Blessed et al. 1968) was 17.2, with a range of 2-33. Controls comprised 6 patients (3 men and 3 women), 4 undergoing myelography for radicular symptoms with no CSF block on the myelogram, and 2 nondemented patients who had recovered from transient amnesia, one associated with closed head injury. Mean ages of the control (59.0 years, range 44-76) and AD groups did not differ significantly. Control patients were not demented on the basis of neurological examination, but were not given the Blessed Dementia Scale or other neuropsychological tests. Sample collection Patients were recumbent for 12 h before lumbar puncture, which was performed between 9.00 h and 11.00 h. The 3rd and 4th ml of CSF collected were used for assay of ChEs. All specimens were frozen before analysis 1-90 days later. Lumbar puncture and neuropsychological tests were usually performed in the same week, and always within 4 months. Biochemical procedures Activities of AChE and BuChE were determined by a colorimetric method using thiocholine ester substrates (Ellman et al. 1961). Because the activity of BuChE on acetylthiocholine was 25 ~o that of AChE, the BuChE inhibitor ethopropazine was used when assaying ACHE. Cross-reactivity of AChE in the BuChE assay was insubstantial, and no inhibitor was used. Neuropsychological tests Patients in the AD group were given the following 14 neuropsychological tests: The Blessed Dementia Scale (Blessed et al. 1968), a measure of overall severity of dementia; 4 tests of memory ability: immediate memory (Brown 1958), verbal paired-associate learning (Corkin 1982), nonverbal paired-associate learning (Corkin 1982), and verbal recognition memory (Craik and Lockhart 1972); 6 measures of language ability: the Token Test (DeRenzi and Vignolo 1962), the Reporter's Test (DeRenzi and Ferrari 1978), WAIS Vocabularly Test (Wechsler 1955), category fluency test (Huffand Corkin 1984), Boston Naming Test (Kaplan et al. 1978), semantic naming errors (Huff and Corkin 1984);
124 and 3 measures of visuospatial functions: visual form discrimination (Huff and Corkin 1984), matchsticks test (Benson and Barton 1970), and visual contrast sensitivity (Nissen et al. 1985). A score is not available on some of the neuropsychological tests for several of the most impaired patients, who were unable to perform those tests. RESULTS C S F AChE (Table 1) did not differ significantly between AD and control patients (t (21) = 0.86; P = 0.40). BuChE was significantly lower in the A D group (t (21) = 2.33; P < 0.05), as was the ratio of BuChE to AChE activities (t (21) = 2.21; P < 0.05). The range of values in the AD and control groups overlapped for all measures: AChE values for 14 of the 17 AD patients overlapped with values for 5 of the 6 control cases; BuChE values overlapped for 9 A D patients and 4 controls; BuChE/AChE values overlapped for 12 A D cases and 4 controls. In computing correlations involving neuropsychological tests, the signs of scores on tests were set so that a more positive score indicated more impaired performance. The abilities measured by different tests in the battery are not completely independent, and scores on different tests were intercorrelated. The correlations of tests with C S F ChE measures were examined to assess which tests correlated significantly with each biochemical measure. Contrast sensitivity was the only test that was significantly correlated with C S F AChE activity, and stronger correlations were observed at higher spatial frequencies (Table 2). C S F BuChE correlated significantly with severity of dementia as measured by the Blessed Dementia Scale, with 2 memory tests, and 3 language tests. The BuChE/AChE ratio in C S F correlated with dementia severity, 2 memory tests, and 4 language tests. In order to evaluate whether the correlations of memory and language tests with C S F BuChE/AChEwere independent of the correlation of the Blessed Dementia Scale with BuChE/AChE, partial correlations were determined, in which the correlation of Blessed Scale to the BuChE/AChE ratio was eliminated from correlations between TABLE 1 ACETYLCHOLINESTERASE(ACHE)AND BUTYRYLCHOLINESTERASE(BuChE) ACTIVITIES IN CSF FROM CONTROL PATIENTS AND PATIENTS WITH ALZHEIMER'S DISEASE Patient group
AChE (nmol/ml/min)
BuChE (nmol/ml/min)
BuChE AChE
Alzheimer's disease (n = 17)
20.3 _+1.4
7.3 + 0.5*
0.38 +_0.10*
Control (n = 6)
17.1 + 3.1
9.8 _+1.2
0.90 + 1.01
* Significantly different from controls by Student's t-test, P < 0.05.
125 TABLE 2 CORRELATIONS BETWEEN NEUROPSYCHOLOGICAL MEASURES AND CSF CHOLINESTERASES (ChEs) IN PATIENTS WITH ALZHEIMER'S DISEASE Neuropsychological measure
Correlation (r) with CSF ChE AChE
BuChE
BuChE AChE
Severity of dementia; Blessed Dementia Scale (n = 17) Memory: Immediate memory (n = 15) Verbal paired associates (n = 16) Nonverbal paired associates (n = 16) Verbal recognition memory (n = 12) Language: Token test (n = 11) Reporter's test (n = 11) WAIS vocabulary test (n = 12) Category fluency (n = 16) Boston naming test (n = 17) Semantic naming errors (n = 16) Visuospatial functions: Form discrimination (n = 15) Matchsticks test (n = 13) Contrast sensitivity (n = 12) 0.5 cpd 1.0 cpd 2.0 cpd 4.0 cpd 8.0 cpd
0.01 0.14 0.08 - 0.25 0.28 0.21 0.09 0.38 0.07 0.09 0.19
-0.54*
-0.52*
-0.49* -0.26 - 0.64** - 0.36
-0.65** -0.34 - 0.33 - 0.71"
-
-
0.32 0.62* 0.48 0.56* 0.54* 0.48
0.03 - 0.20
- 0.13 - 0.51
-
-
0.27 0.46 0.54 0.62* 0.67*
0.23 0.01 0.06 0.13 0.43
0.44 0.56 0.82** 0.66** 0.64** 0.72**
- 0.21 - 0.39 0.10 0.53 0.65* 0.52 0.27
* P < 0.05; ** P < 0.01. cpd = cycles per degree of visual angle. B u C h E / A C h E a n d o t h e r t e s t s ( M c N e m a r 1962). F i v e o f t h e m e m o r y a n d l a n g u a g e t e s t s r e m a i n e d s i g n i f i c a n t l y c o r r e l a t e d w i t h B u C h E / A C h E in t h i s a n a l y s i s ; t h e c o r r e l a t i o n f o r category fluency was nonsignificant. DISCUSSION W e o b s e r v e d n o d i f f e r e n c e in C S F
AChE
activity b e t w e e n A D a n d c o n t r o l
p a t i e n t s . D a v i e s (1979), D e u t s c h et al. (1983), a n d L a l et al. (1984) a l s o r e p o r t e d n o d i f f e r e n c e in C S F A C h E b e t w e e n A D a n d c o n t r o l c a s e s , b u t A p p l e y a r d et al. (1983) a n d T u n e et al. (1985) r e p o r t e d l o w e r C S F A C h E in A D r e l a t i v e t o c o n t r o l c a s e s . A p o s s i b l e e x p l a n a t i o n f o r t h e a b s e n c e o f a d i f f e r e n c e b e t w e e n A D a n d c o n t r o l g r o u p s in t h e p r e s e n t s t u d y is t h e h e t e r o g e n e i t y o f t h e c o n t r o l g r o u p , w h i c h i n c l u d e d p a t i e n t s w i t h transient a m n e s i a and others with s y m p t o m s o f r a d i c u l o p a t h y . However, values for A D c a s e s o v e r l a p p e d t h e r a n g e o f v a l u e s f o r b o t h t y p e s o f c o n t r o l p a t i e n t s . A l t h o u g h all
126 control cases studied by Tune et al. (1985) had myelograms for low back pain, half had CSF AChE values that overlapped the range for AD patients in their study. Thus, AChE values did not discriminate AD and control cases, despite the difference in mean values for the two groups. Appleyard et al. (1983) observed differences in AChE between AD and control cases in ventricular CSF obtained at postmortem. A difference in CSF AChE may be more difficult to detect in lumbar than ventricular CSF because AChE from spinal cord, or other extraneous factors, may affect lumbar CSF measurements. Another explanation for the discrepancies among studies may be differences in techniques for measuring ChE activities. In investigations using the colorimetric method (Ellman et al. 1961), specific inhibitors are necessary in order to differentiate AChE and BuChE activities. Soininen et al. (1984) measured total CSF ChE activity colorimetrically, and observed lower activity in AD than in control cases. Some investigators (Lal et al. 1984; Tune et al. 1985) report measuring AChE colorimetrically without using inhibitors. A contribution of BuChE in their AChE measurements is likely, and what is reported as "ACHE" would be more appropriately decribed as "total ChE", as was done by Soininen et al. BuChE may account for 10-15 ~o oftotai ChE measured in this way. We observed decreased CSF BuChE in AD patients, and the difference in CSF ChE between AD and control cases reported by Tune et al. and Soininen et al. probably reflect abnormalities of CSF BuChE as well as ACHE. Formation of the BuChE/AChE ratio eliminates possible variance in AChE and BuChE levels due to CSF volume dilution associated with different degrees of cerebral atrophy in different patients. This reduction in variance may account for the strong correlations between the BuChE/AChE ratio and clinical measures. Another reason for examining the CSF BuChE/AChE ratio is that brain activities of BuChE and AChE in AD deviate differently from normal values: BuChE activity is high, whereas AChE activity is low (Perry et al. 1978). If abnormalities in CSF BuChE and AChE activities in AD also differ in direction relative to normal, computing the BuChE/AChE ratio may enhance discrimination between AD and control cases. We observed that the ratio of BuChE to AChE in CSF was lower in AD than in control cases, whereas Arendt et al. (1984) reported a difference in the opposite direction. They observed decreased AChE and a tendency toward increased BuChE in AD cases, but we observed decreased BuChE and a tendency toward increased AChE in AD cases. These discrepancies are difficult to explain, although differences in patient populations may account for them. Arendt et al. did not provide clinical information about their patients, such as severity of dementia and use of psychoactive medications that may have influenced CSF ChE activities (Bareggi and Giacobini 1978; Greenfield and Shaw 1982). Our observation that greater severity of dementia correlated with lower CSF BuChE supports our observation of lower BuChE in AD than control cases. Because severity of dementia has been observed to correlate with increased BuChE in brain in AD (Perry et al. 1978), our observation of a correlation of clinical severity with decreased BuChE in CSF is surprising. It is possible that a shift in the relative proportions of soluble and insoluble forms ofBuChE (Massoulie and Bon 1982) in AD results in accumulation of insoluble BuChE in the brain, while decreased soluble BuChE is reflected in CSF.
127 In the present study, impairments on 6 tests of language and memory correlated significantly with low BuChE/AChE ratio in CSF. Correlations of 5 of these tests with BuChE/AChE were significant with Blessed Dementia Scale partialled out. It is possible either that these tests measure severity of dementia more reliably than the Blessed Scale, or that cognitive abilities measured by these tests are more strongly related to CSF and brain ChE activity than is the Blessed Scale. These correlations do not establish a role of BuChE in cognition and behavior: cognitive deficits and altered BuChE activity may be correlated only because of their shared association with the pathological process of AD. The function of BuChE in the brain is not clear. BuChE is present in glial cells and neurons (Graybiel and Ragsdale 1982) and is associated with senile plaques (Friede 1965; Struble et al. 1984). BuChE hydrolyzes substance P (Lockridge 1982) as well as acetylcholine, and may therefore be involved in catabolism of multiple neurotransmitters. There is no direct evidence that BuChE activity is involved in the neurochemical processes underlying cognition. Visual contrast sensitivity, particularly to high spatial frequencies, correlated with CSF ACHE. AChE is present in neurons of primate primary visual cortex (Graybiel and Ragsdale 1982), and abnormalities of contrast sensitivity to high spatial frequencies have been observed in patients with lesions of the parietal and occipital lobes (BodisWoUner and Diamond 1976). The anticholinesterase drug physostigmine differentially affects response to low and high spatial frequencies in the cat (Harding et al. 1983). Contrast sensitivity deficits occur in AD (Nissen et al. 1985), and it is possible that contrast sensitivity is affected early in the course of AD, but does not change with progression to more severe stages of dementia. However, the lack of correlation with dementia severity suggests that the relation of contrast sensitivity function to CSF AChE may not be exclusive to dementia or AD. Although the present study indicates that AD and control cases cannot be reliably distinguished by measurement of either CSF AChE or BuChE activities, such differentiation may be possible by concurrent measurement of ChEs and other neurochemical markers, such as somatostatin-like immunoreactivity (Soininen et al. 1984). Because activities of AChE and BuChE in CSF appear to be affected differently in AD, separate determination of the 2 enzymes may improve discriminative precision in studies of CSF neurochemical measures. ACKNOWLEDGEMENTS The authors thank T. John Rosen, and Chris Collins, for statistical analyses; Allison Feeley and Marguerite Randolph for testing subjects; and Darlene Bumford for manuscript preparation. REFERENCES Appleyard,M. E., A.D. Smith, G. K. Wilcockand M.M. Esiri (1983)DecreasedCSF acetylcholinesterase activity in Alzheimer's disease, Lancet, ii: 452.
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