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Carnitine acetyltransferase activity in the human brain and its microvessels is decreased in Alzheimer's disease.

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Carnitine Acetyltransferase
Activity in the Human
Brain and Its Microvessels
Is Decreased in
Alzheimer’s Disease
Rajesh N. Kalaria, PhD, and Sami I. Harik, M D
~~
L-Carnitine and acetyl-L-carnitine facilitate mitochondrial P-oxidation of fatty acids. In the brain, they may
also have a role in acetylcholine synthesis. Carnitine acetyltransferase catalyzes the interchange between L-carnitine and acetyl-L-carnitine.Recently, acetyl-L-carnitine
was reported to have a beneficial effect in patients with
Alzheimer’s disease. We therefore assessed carnitine acetyltransferase activity in selected brain regions and in
isolated cerebral microvessels obtained at autopsy from
patients with Alzheimer’s disease and from age-matched
control subjects. We found a 25 to 40% decrease in carnitine acetyltransferase activity in patients with Alzheimer’s disease, which attained statistical significance in
most brain regions and in cerebral microvessels. These
findings document another neurochemical abnormality
in patients with Alzheimer’s disease and provide a rationale for the use of acetyl-L-carnitine in the treatment of
patients with Alzheimer’s disease.
Kalaria RN,Harik SI. Carnitine acetyltransferase
activity in the human brain and its microvessels is
decreased in Alzheimer’s disease.
Ann Neurol 1992;32:583-586
L-Carnitine plays a vital role in the oxidation of long
chain fatty acids by mitochondria of numerous tissues
111. Reversible acylation of L-carnitine by carnitine
acyltransferase mitochondrial enzymes is important for
the translocation of acyl groups (e.g., acetate), coenzyme A (CoA), and acetyl-CoA across mitochondrial
membranes [l, 2). Chief among carnitine acyltransferase enzymes is carnitine acetyltransferase (CarAT;
acety1CoA:carnitine 0-acetyltransferase, EC 2.3.1.7).
CarAT, carnitine, and acetyl-L-carnitine (ALC) are
widely distributed in mammalian tissues, including the
brain [3-51, where they attain their highest levels in
the hypothalamus 151.
From the Department of Neurology, University Hospitals of Cleveland, and the Departments of Neurology and Neuroscience, Case
Western Reserve University School of Medicine, Cleveland, OH.
Received Mar 10, 1992, and in revised form Apr 13. Accepted for
publication Apr 13, 1992.
Address correspondence to Dr Kalaria, Department of Neurology,
University Hospitals of Cleveland, 2074 Abington Road, Cleveland,
OH 44106.
Because the brain relies almost entirely on glucose
for metabolic fuel, the biological role of carnitine and
ALC in the brain is less clear than in other tissues.
However, carnitine and ALC in the brain are suspected
of facilitating cholinergic neurotransmission either directly or by shuttling acetyl groups from mitochondria
to the cytosol where they can be used for acetylcholine
synthesis 141.
Chronic ALC administration to patients with dementia improved their cognitive functions [b],or was beneficial in preserving cognitive functions and decreasing
the rate of progression of Alzheimer’s disease (AD)
171. The mechanism(s) by which ALC produces its salutary effects remains unknown. One possibility is that
brain CarAT is deficient in patients with AD. In this
study, we assessed CarAT activity in discrete brain regions and in isolated microvessels from the frontal cortex in tissues obtained at autopsy from patients with
AD and from age-matched control subjects.
Methods
Stlbjects
Human brains were sampled at autopsy immediately after
removal. Tissues were obtained from 36 patients with AD
and 24 age-matched control subjects. The age and sex distribution and the postmortem intervals in the two groups are
detailed in Table 1. The diagnosis of A D was clinically suspected and pathologically confirmed. However, precise clinical information concerning the magnitude and duration of
the dementia was not available. Age-matched control subjects were selected based on their age and the lack of clinical
or pathological evidence of brain disease.
T i w e Preparation
Cerebral microvessels were harvested from 50 to 100 grn of
freshly obtained frontal cerebral cortex by bulk isolation E8,
91. Each microvessel preparation was obtained from 1 subject. Microvessel purity was checked by differential interference microscopy and by biochemical assays of their enrichment with the marker enzymes, y-glutamyltranspeptidase
and angiotensin-converting enzyme [8, 91. Samples of the
Table 1. Subjects’ Age, Sex, and Postmortem Interval
n
Age (yr)
Mean SEM
Range
Sex
Postmortem interval
*
0-4
Mean
Range
* SEM
Control Subjects
Patients with A D
24
36
71 5 2
57-83
16 MI8 F
72 1
60-83
15 M/21 F
8.8 rt 1.2
3-20
*
*
7.9 1.0
2-24
Postmortem interval is the time between death and brain tissue acquisition.
AD = Alzheimer’s disease.
Copyright 0 1992 by the American Neurological Association 583
Table 2. CarAT Activity in Brains of Patients with AD and Control Subjects
Region
Control Subjects
n
Patients with AD
Frontal cortex
Temporal cortex
Hippocampus
Cerebellum
21.1 +26.7 +14.1 k
16.3 +-
12
10
13
11
16.1 -C
18.2 2
10.6
10.0
1.4
1.8
1.2
2.1
*
*
1.7
3.1
2.1
2.1
n
P
10
12
11
11
0.04
0.04
0.15
0.04
CarAT activity values, in nanomoles of product formed per milligram of tissue particulate protein per minute, are the mean 2 SEM of the
number of observations (n). Significant differences between the results obtained from patients with AD and age-matched control subjects were
assessed by the Student’s t test, two-tailed.
CarAT = carnitine acetyltransferase; AD = Alzheimer’s disease.
Table 3 . Enzyme Activities in Isolated Microvessels from the Frontal Cerebral Cortex of Patients with AD and Control Subjects
~
Enzyme
Control Subjects
CarAT
(nmoumg of proteidmin)
Choline acetyltransferase
@mol/mg of proteidmin)
y -Glutamyltranspeptidase
(nmoYmg of proteidmin)
Angiotensin-converting enzyme
(nmoYmg of proteidmin)
n
Patients with AD
n
P
16
6.6 t 0.7
15
0.04
0.11
15
1.19 +- 0.16
15
0.2 1
128.3
* 10.0
14
10
0.25
3.32
* 0.36
14
10
0.24
8.8 +: 0.8
0.94
2
113.3 2 8.3
2.71 +: 0.32
Enzyme activity values are the mean 2 SEM of the number of observations (n). Significant differences between the results obtained from patients
with AD and age-matched control subjects were assessed by Student’s t test, two-tailed. Note that the activity of choline acetyltransferases is
expressed as picomoles per milligram of protein per minute, whereas the activity of all the other enzymes is expressed as nanomoles per
milligram of protein per minute.
AD = Alzheimer’s disease; CarAT = carnitine acetyltransferase.
frontal cortex (Brodmann area 9 or lo), temporal cortex
(Brodmann area 2 l), hippocampus, and cerebellar cortex
were also obtained. Cerebral microvessels and brain samples
were stored at - 70°C until assayed.
All procedures were performed at 0 to 4°C unless otherwise stated. Brain samples were homogenized in 10 mM
phosphate buffer, pH 7.0, in a Brinkmann (Westbury, NY)
polytron. The homogenates were centrifuged at 37,000 g for
20 minutes and the pellet resuspended in phosphate buffer
to a concentration of 1 to 3 mg of tissue protein per milliliter.
Aliquots of each sample were taken for Lowry protein assay,
and Triton X-100 was added to the remainder of the homogenates to a final concentration of 0.025% before CarAT
assays were performed.
Isolated cerebral microvessels were homogenized in 0.2 5
M sucrose solution containing 50 mM Tris buffer, pH 7.4,
and 1 mM ethylenediaminetetraacetate (EDTA) in motordriven glass-glass homogenizers. Aliquots from each sample
were taken for assays of y-glutamyltranspeptidaseactivity {9],
angiotensin-converting enzyme activity 191, and choline acetyltransferase activity [lo]. Portions of each microvessel homogenate were then centrifuged at 50,000 g for 20 minutes
and the pellet resuspended in 10 mM phosphate buffer and
prepared for CarAT assay as described above for brain tissue.
CarAT Assay
CarAT activity was assayed by a modification of the method
of Sterri and Fonnum [I I]. The assay incubation volume of
20 p1 had 10 mM sodium phosphate buffer, p H 7.0, 150
mM NaC1,0.5 mM EDTA, 1 mM eserine salicylate, 0.4 mM
584 Annals of Neurology
Vol 32 No 4 October 1992
C’*C]acetyl-CoA, 2 mM L-carnitine, and 10 to 30 pg of tissue
protein. The reaction took place for 5 minutes at 37°C and
was stopped by the addition of 5 ml of ice-cold 10 mM
sodium phosphate buffer, pH 4.0, containing excess unlabeled ALC (50 mg/L). This was followed by 2 ml of acetonitrile containing 1.5% tetraphenylboron. ALC was extracted
into the organic phase and the radioactivity counted at an
efficiency of approximately 90%. Assays were performed in
triplicate and the blanks were tissues that were heated in
boiling water for 10 minutes. CarAT activity was expressed
as nanomoles of ALC produced per milligram of tissue protein per minute. In preliminary experiments, we showed that
the amount of tissue protein and the incubation time that we
used were within the linear range.
Analysis of Data
Differences between the results obtained from patients with
AD and control subjects were assessed by the Student’s t
test, two-tailed. Significance was considered at p < 0.05.
Results
There were no differences in CarAT activity between
sexes within each group of subjects. There were only
modest regional variations in CarAT activity in the human brain; activity in the frontal and temporal cortex
was higher than in the hippocampus and cerebellum
(Table 2). Also, CarAT activity was two- to threefold
higher in the cerebral cortex than in cerebral microvessels (Tables 2, 3). CarAT activity values that we report
in human cerebral microvessels were similar to those
of rat and pig cerebral microvessels (results not shown).
Despite the lack of differences in age, postmortem
interval, or duration of storage of frozen tissues between patients with AD and control subjects (see Table
l), there was consistently less CarAT activity in tissues
from patients with AD. The differences were significant in the frontal and temporal cerebral cortex and
cerebellum, but not in the hippocampus (see Table
2). There was also significantly less CarAT activity in
cerebral microvessels from patients with AD than in
control subjects, although choline acetyltransferase activity and the activities of the two marker enzymes
of brain microvessels, y-glutamyltranspeptidaseand angiotensin-converting enzyme, were similar in both
groups (see Table 3).
Discussion
Our results confirm the presence of CarAT activity in
the human brain at autopsy in the same range reported
previously 112, 131. CarAT activity is apparently stable
in postmortem brain and can withstand stormy agonal
states 1131. We found severalfold higher CarAT activity in the human cerebral cortex obtained at autopsy
many hours after death than in the rat cerebral cortex
that was processed immediately after decapitation. We
also report, for the first time, the presence of CarAT
activity in cerebral microvessels, but at a lower level
than in the cerebral cortex from which the microvessels
were isolated (see Tables 2, 3). CarAT may have an
important role in regulating the oxidative metabolism
of brain microvessels, which, unlike the rest of the
brain, oxidize fatty acids in preference to glucose [14}.
In AD, CarAT activity is significantly reduced in
several brain regions and in isolated cerebral microvessels. It is unlikely that this is secondary to a lower
density of brain mitochondria in patients with AD because other mitochondrial enzymes, such as monom i n e oxidase, are increased (rather than decreased)
in patients with AD 1153. Perry and colleagues [12)
reported lack of a significant decrease in CarAT activity
in brain samples from patients with AD, but they measured CarAT activity in the amygdaloid and caudate
nuclei only.
The lower CarAT activity in cerebral microvessels
from patients with AD than in control microvessels
cannot be attributed to impurities because contamination of isolated brain microvessels with other brain elements would increase, rather than decrease, CarAT
activity. Also, activities of the enzyme markers of
brain microvessels (e.g., y-glutamyltranspeptidase and
angiotensin-converting enzyme) were not significantly
altered in microvessels from patients with AD (see Table 3). Thus, the lower CarAT activity should be added
to the growing list of abnormalities in cerebral microvessels isolated from patients with AD 19, 161.
There are chemical similarities between carnitine
and choline. Also, the assays for both CarAT and choline acetyltransferase use acetyl-CoA as acetyl donor
{lo, 113. Although CarAT is primarily a mitochondrial
enzyme whereas choline acetyltransferase is a cytosolic enzyme, care must be used in enzyme assays of
total tissue homogenates to differentiate the activities
of the two enzymes. Such interference may explain the
unusually h g h choline acetyltransferase activities that
were reported in nonneural tissues such as sperm and
muscle [17}. We suspect that similar interference may
underlie the widely differing reports on choline acetyltransferase activity in isolated cerebral microvessels
[18-231. We have used choline acetyltransferase activity as an indicator of contamination of isolated brain
microvessel preparations by brain parenchyma because
we reported a 30-to-1 ratio in human brain homogenates to isolated microvessels (see Table 1 of IS]).
However, other investigators [18, 21, 231 reported
much higher choline acetyltransferase activity in isolated brain microvessels, which sometimes exceeded
that of the cerebral cortex. The meager activity of choline acetyltransferase that we now report in isolated
cerebral microvessels from control subjects and patients with AD is consistent with our previous report
181 and is more than 5,000-fold lower than CarAT
activity (see Table 3).
There are no data on the concentrations of carnitine
and ALC, or on CarAT activity, in the human brain in
senescence. In senescent rates, there was no change in
brain CarAT activity [24}, although decreased levels of
carnitine and acylcarnitines were reported [2 51. Furthermore, the level of brain carnitines in senescent rats
was increased by the ALC administration {25f. Because CarAT is the enzyme that catalyzes the interconversion of carnitine and ALC, our finding of decreased
CarAT activity in the brains of patients with AD offers
an explanation for the reported salutary effect of ALC
administration to patients with AD [b, 71.
This research was supported by Public Health Service Grants AG08012 and HL-35617, by a grant from Sigma Tau Pharmaceutical
Company, and by the David S. Ingalls Sr Fund.
Presented at the 1992 Annual Meeting of the American Academy
of Neurology in San Diego, CA.
We thank Susan M. Sromek for technical assistance and Jeanette
Barnhart for manuscript preparation.
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Brief Communication: Kalaria and Harik: Decreased Brain CarAT in A D
585
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Extraocular Muscles are
Spared in Advanced
Duchenne Dystrophy
Henry J. Kaminski, MD,”S Mazen Al-Hakim, MD,t
R. John Leigh, MD,*S M. Bashar Katirji, MD,IS
and Robert L. Ruff, MD, PhD’S
Fast-twitch extremity muscle fibers are preferentially affected in Duchenne’s and Becker’s muscular dystrophy.
Since saccades are thought to be mediated by fast-twitch
fibers, saccadic velocities would be expected to be decreased in these patients. Using infrared oculography,
we found that the peak velocities of saccades in 3 patients with advanced Duchenne’s or Becker’s muscular
dystrophy were normal. Clinical findings in 7 other patients with these forms of dystrophy were normal. This
investigation is the first study of ocular motility in Duchenne’s and Becker’s muscular dystrophy. It demonstrates that extraocular muscle function is preserved and
suggests that fast-twitch fibers in extraocular muscles
possess properties that protect against degeneration.
Kaminski HJ, Al-Hakim M, Leigh RJ, Katirji MB,
Ruff RL..Extraocular muscles are spared in
advanced Duchenne dystrophy.
Ann Neurol 1992;32:5 86- 5 88
Duchenne’s (DMD) and Becker’s muscular dystrophy
(BMD) are caused by an abnormality of dystrophin
f 11. Using monoclonal antibodies to differentiate muscle fiber types, Webster and colleagues {21 and Minetti
and associates 133 found that type IIb fibers are the
first to degenerate during the course of D M D and are
not detectable in patients older than 5 years. Type IIb
fibers are faster contracting and subjected to higher
neuronal firing frequencies than are type I fibers, placing greater mechanical stress on these fibers. The investigators 12, 31 hypothesized that dystrophin-deficient
fibers may be particularly susceptible to mechanical
stress.
Extraocular muscle (EOM) is composed of a heterogeneous mixture of muscle fiber types that differ anatomically and physiologically from their extremity muscle counterparts. The most rapid eye movements,
From the “Department of Neurology, Department of Veterans Affairs Medical Center, tUniversity Hospitals of Cleveland, and $Case
Western Reserve University School of Medicine, Cleveland, OH.
Received Mar 3, 1992, and in revised form Apr 16. Accepted for
publication Apr 18, 1992.
Address correspondence to Dr Kaminski, Department of Neurology, Department of Veterans Affairs Medical Center, 10701 East
Boulevard, Cleveland, OH 44106.
586 Copyright 0 1992 by the American Neurological Association
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