Double-Blind,
Placebo-Controlled Study of l-Carnosine
Supplementation in Children With Autistic Spectrum Disorders
Michael G.
Chez, MD; Cathleen P. Buchanan, PhD; Mary C. Aimonovitch;
Marina Becker, RN; Karla Schaefer, RN; Carter Black, RPh; Jamie Komen, MA
ABSTRACT
l-Carnosine,
a dipeptide, can enhance frontal lobe function or be neuroprotective.
It can also correlate with g-aminobutyric
acid (GABA)-homocarnosine interaction, with possible anticonvulsive effects.
We investigated 31 children with autistic spectrum disorders in an
8-week, double-blinded study to determine if 800 mg l-carnosine
daily would result in observable changes versus placebo.
Outcome measures were the Childhood Autism Rating Scale, the Gilliam
Autism Rating Scale, the Expressive and Receptive One-Word Picture Vocabulary
tests, and Clinical Global Impressions of Change.
Children on placebo did not show statistically significant changes.
After 8 weeks on l-carnosine,
children showed statistically significant improvements on the Gilliam Autism
Rating Scale (total score and the Behavior, Socialization, and Communication
subscales) and the Receptive One-Word Picture Vocabulary test (all P<.05).
Improved trends were noted on other outcome measures.
Although the mechanism of action of l-carnosine
is not well understood, it may enhance neurologic function, perhaps in the enterorhinal
or temporal cortex. (J Child Neurol 2002;17:833-837).
Autistic
spectrum disorders (autistic disorder, pervasive developmental delay not
otherwise specified, and Asperger’s syndrome) are long-term, developmental
disorders with no known or agreed on nervous system pathology or definitive
genetic site.1,2 Unfortunately,
very few double-blind studies have been performed involving autistic
spectrum-disorders, owing to the different degrees of presentation and the
multiple phenotypes of the disease. The
varying symptoms observed with the autistic spectrum disorder syndrome
indicate more than a single region of injury that results in
an assortment of behaviors.1
With no available cure, treatment is contained to modifying the
course of the disease.
Research in the past several years has shown an association between
sleep electroencephalograms (EEGs) and autism.3-7
One third of autistic children suffer one or more seizures by
adolescence, perhaps linking epileptiform activity with some instances of
autistic spectrum disorders.5
Magnetoencephalographic data have suggested that the percentage may
even be higher; in a recent autistic study, 14% more children were identified
as having epileptiform activity by magnetoencephalography (82%) who were not
identified by concurrent EEG (68%).5
Valproic acid, an anticonvulsant effective in
treating seizure disorders, is hypothesized to increase g-aminobutyric
acid (GABA) concentrations within the central nervous system.8, 9
GABA is the major inhibitory neurotransmitter in the cerebral cortex.10,11
Positive results have been observed in administration of valproic acid
to autistic patients, especially in patients with EEG abnormalities.12-16
Anticonvulsants have also been successful in psychiatric disease use,
particularly bipolar disorder.
Recent work by Petroff and his colleagues at Yale University has
demonstrated that GABA activity can be measured using magnetic resonance
imaging spectroscopy with a higher field strength.17
Although Petroff and colleagues studied cases of generalized myoclonic
epilepsies, his finding is significant because of the correlation established
between levels of GABA and homocarnosine levels.
Even in cases of low GABA function, homocarnosine levels can correlate
with better seizure control. Other
investigators have reported elevated homocarnosine
levels in infants with epilepsy or brain injury.18
Cases of temporal lobe or generalized seizures have also shown seizure
control to correlate with homocarnosine levels.17
Homocarnosine is formed when GABA and
carnosine bind. Carnosine appears
to modulate copper and zinc influx into cells and near GABA receptors, thereby
affecting potential epileptic inhibition.
Further, modulation of zinc and copper can have complementary
antiepileptiform effects in the hippocampus and frontal lobe,19 as
well as being neuroprotective against ischemia.20, 21
Carnosine can offer anticonvulsant protection in vitro and in rats in
prior studies, perhaps by altering homocarnosine levels or by a direct
chelating effect on zinc at GABA receptor sites.22
Because carnosine has been described as accumulating in the
enterorhinal subfrontal cortex, we hypothesize that it might act in a
protective or activating role for the frontal lobe.
Dysfunction of the frontal lobe is hypothesized to be linked to
expression and behavior; both areas characteristically impaired in autistic
spectrum disorders.23-25
We
therefore designed a study to target some of the purported brain regions of
dysfunction in autistic spectrum disorders via supplementation with a
naturally occurring amino acid that is believed to act on frontal lobe systems
or GABA receptors. We sought to
eliminate the “expectancy effect” of medication augmentation by enrolling
children in study so that both the clinicians and the parents were blind to
group assignment.
METHODS
Children were included for study if they met the following criteria: age
3-12 years and prior diagnoses of autistic spectrum disorder (including either
pervasive developmental disorder or autistic disorder; by the Diagnostic
and Statistical Manual of Mental Disorders –IV-Revised).26
Thirteen subjects had a current abnormal EEG, and 13 were being
maintained on valproic acid. Children
were excluded if they had a family history of seizure disorder, fragile X
syndrome, or other genetic disorder or etiology of their spectrum disorder.
Children were enrolled in the study irrespective of cognitive ability
level. All children were tested
at baseline in the following domains: expressive language (Expressive One-Word
Picture Vocabulary test), receptive language (Receptive One-Word Picture
Vocabulary test), autism severity ratings (Childhood Autism Rating Scale and
Gilliam Autism Rating Scale), and Clinical Global Impressions of Change, which
were completed by the parents. At baseline and 8-week testing, all children
underwent the Childhood Autism Rating Scale, the Gilliam Autism Rating Scale,
the Expressive One-Word Picture Vocabulary test, the Receptive One-Word
Picture Vocabulary test, and the Clinical Global Impression of Change.
Children were tested with their parents in a pediatric neurology clinic
in a room dedicated to assessment. The
parents signed informed written consent (approved by the Lake Forest Hospital
Institutional Review Board) before being randomly assigned to either
active-agent or placebo, and reporting of adverse events was explained per
office emergency policy. Parents,
clinicians, and neurologists were all blinded to placebo versus active
carnosine. At the completion of
the study, the blind code was broken by identifying the patient’s bottle
number with the placebo or carnosine administration.
Because of our prior experience with the
substance in an open label format, we were able to caution regarding the
following potential adverse events: hyperactivity and excitability.
Both placebo and active substances were identical
in powdered appearance, without taste or smell.
All pills were contained by a gelatin capsule; parents were instructed
to mix the powder with either food or drink.
Dosage of carnosine was 400 mg by mouth twice daily.
Every 2 weeks, parents faxed a Clinical Global Impression of Change
regarding their child. The
Clinical Global Impression was a 5-point rating scale covering the following
domains: (1)socialization, interaction with others during play;
(2)spontaneous, expressive language or vocal- izations;
(3)attention span, “focus”/eye contact, alertness level; (4)agitation and
hyperactivity or lethargy, energy level; (5)coordination, body use, gross
motor movements; and (6)anxiety, rigidity, perseveration, adaptation to
change. A score of 0 indicated
“no change,” and a score of 5 indicated a “great improvement”
Parents were not allowed to refer back to faxes from the prior 2-week
period, so that each rating was essentially “blinded” to the week before.
An overall baseline Clinical Global Impression was then compared with
an ending Clinical Global Impression.
Statistical analyses included descriptive statistics and comparisons of
baseline and ending means. Pairwise
t-tests were conducted with
Turkey
’s correction for multiple comparisons.
RESULTS
Thirty-one
children (21 males, 10 females, mean age=7.45; range 3.2-12.5) meeting
inclusion criteria were enrolled in an 8-week, double-blind,
placebo-controlled study. Children
were entered randomly into one of two groups: group 1(n=17) received a
placebo for the first 8 weeks, whereas group 2(n=14) received the
active substance. Children were
assessed at baseline in the domains show in Table 1.
There
were no statistically significant differences in any of the baseline measures
with the exception of the Communication subscale of the Gilliam Autism Rating
Scale. Although the group on the
active substance started out with a higher level on the Communication scale,
there were no statistically significant differences when tested on the
objective language measures, the Receptive One-Word Picture Vocabulary test
and the Expressive One-Word Picture Vocabulary test.
Being medicated with valproic acid or having a current abnormal EEG did
not make a difference in terms of group differences.
Table
1. Baseline
Measures for Children in Blinded Carnosine Study
Baseline
8 Weeks
Measure
Mean (SD)
Mean (SD)
Significance
| Age
(mo) |
92.47
(28.95) |
85.69
(24.57) |
.NS |
| Clinical
Global Impression |
12.94
(4.18) |
14.50
(3.65) |
NS |
| Expressive
One-Word Picture Vocabulary test (raw) |
30.65
(26.28) |
35.36
(20.87) |
NS |
| Expressive
One-Word Picture Vocabulary test (age adjusted) |
35.41
(29.94) |
40.71
(23.44) |
NS |
| Receptive
One-Word Picture Vocabulary test (raw) |
34.29
(28.56) |
38.00
(23.67) |
NS |
| Receptive
One-Word Picture Vocabulary (age adjusted) |
29.65
(27.91) |
40.57
(24.55) |
NS |
| Childhood
Autism Rating Scale |
34.85
(6.69) |
31.71
(6.55) |
NS |
| Gilliam
Autism Rating Scale |
50.88
(16.96) |
55.50
(16.35) |
NS |
|
Behavior
Scale |
17.17
(8.71) |
15.71
(6.65) |
NS |
|
Socialization
Scale |
18.47
(6.40) |
18.14
(6.30) |
NS |
|
Communication
Scale |
15.23
(6.68) |
21.64
(7.99) |
NS |
NS = not
significant
After 8
weeks, children returned for repeat testing.
The scores for the follow-up test appear in Table 2.
As may be seen form Table 2, any changes that occurred within the
placebo group did not result in any statistically significant changes after 8
weeks.
Table
2.
Changes in Children after 8 Weeks on Placebo
Baseline
8 Weeks
Measure
Mean (SD)
Mean (SD)
Significance
| Clinical
Global Impression (baseline vs. 8 wk) |
12.94
(4.18) |
14.25
(4.51) |
.NS |
| Clinical
Global Impression (2 wk vs 6 wk |
4.82
(5.30) |
4.71
(5.02) |
NS |
| Expressive
One-Word Picture Vocabulary test (raw) |
30.65
(26.28) |
31.65
(29.19) |
NS |
| Expressive
One-Word Picture Vocabulary test (age adjusted) |
35.41
(29.94) |
37.12
(33.38) |
NS |
| Receptive
One-Word Picture Vocabulary test (raw) |
34.29
(28.56) |
37.11
(30.89 |
NS |
| Receptive
One-Word Picture Vocabulary (age adjusted) |
39.65
(27.91) |
41.65
(30.46) |
NS |
| Childhood
Autism Rating Scale |
34.85
(6.69) |
33.76
(6.54) |
NS |
| Gilliam
Autism Rating Scale |
50.88
(16.96) |
49.88
(16.80) |
NS |
|
Behavior Scale |
17.17
(8.71) |
15.82
(7.74) |
NS |
|
Socialization
Scale |
18.47 (6.40) |
17.18
(7.76) |
NS |
|
Communication
Scale |
15.23
(6.68) |
16.88
(6.48) |
NS |
It
is important from examination of Table 3 that there were significant changes
across several measures in multiple domains for those children who were given
the carnosine for 8 weeks.
Significant improvements with carnosine
compared with the placebo were seen in the 2-week versus 6-week faxed Clinical
Global Impression ratings (P =.04), Receptive One-Word Picture
Vocabulary test scores (P =.01), and Gilliam Autism Rating Scale scores
(P =.01), including the Behavior (P =.04), Socialization (P
=.01). and Communication (P =.03) subscales.
The baseline to 8-week Clinical Global Impression and Childhood Autism
Rating scale testing measures also showed improving trends, although not
statistically significant (P =.06, P =.07, respectively).
Pairwise t-tests performed on the placebo
group showed that there were no significant changes on any of the measures
after 8 weeks on the inert pills with a confidence level of P<.05.
Table
3.
Changes in Children after 8 Weeks on Active Carnosine
Baseline
8 Weeks
Measure
Mean (SD)
Mean (SD)
Significance
| Clinical
Global Impression (baseline vs. 8 wk) |
14.50
(3.65) |
16.39
(4.36) |
.06 |
| Clinical
Global Impression (2 wk vs 6 wk |
2.03
(2.24) |
4.92
(5.69) |
.04 |
| Expressive
One-Word Picture Vocabulary test (raw) |
35.36
(20.87) |
37.28
(25.66) |
NS |
| Expressive
One-Word Picture Vocabulary test (age adjusted) |
40.71
(23.44) |
43.78
(28.22) |
NS |
| Receptive
One-Word Picture Vocabulary test (raw) |
38.00
(23.68) |
44.64 (26.56) |
.01 |
| Receptive
One-Word Picture Vocabulary (age adjusted) |
40.57 (24.55) |
47.86 (28.37) |
.01 |
| Childhood
Autism Rating Scale |
31.71
(6.55) |
29.75
(7.53) |
.07 |
| Gilliam
Autism Rating Scale |
55.50
(16.35) |
44.35
(14.93) |
.01 |
|
Behavior Scale |
15.71
(6.65) |
12.86 (5.95) |
.04 |
|
Socialization Scale |
21.64
(7.99) |
18.14
(6.27) |
.03 |
|
Communication Scale |
18.14
(6.30) |
13.36
(6.58) |
01 |
NS = not
significant.
DISCUSSION
The results of this study suggest that supplementation with carnosine
can significantly improve receptive speech, socialization, and behavior with
autistic spectrum disorders. These
gains are observable both by parents and clinicians blinded to study group, as
evidenced by the scores on the Clinical Global Impression.
Although not statistically significant, improved trends in extreme
speech, baseline versus 8-week Clinical Global Impression ratings, and the
Childhood Autism Rating Scale scores were noted, correlating with subjective
reports of improvement by parents to the neurologists.
The extra data points of the Clinical Global Impression between weeks 2
and 6 do reach significance, further supporting overt clinical observation.
Moreover, improvements on objective standardized rating scores such as
the Receptive One-Word Picture Vocabulary test, tests for which practice
effects are not of concern to reliability, also
validate these observations. Several
patients had individually noticeable expressive speech improvement on the
Expressive One-Word Picture Vocabulary test, but, as a group, only a trend was
obtained. This was because of
some individuals who, owing to the heterogeneous
nature of autistic spectrum disorders, had very low scores at
baseline.
Not a single child had to discontinue the
study because of side effects. Parents
reported only sporadic hyperactivity, which was alleviated by decreasing the
dose. Previous studies involving carnosine
have indicated a very low toxicity level; among 691 cases of adult clinical
trials, no symptomatic side effects were reported.27
The
mechanism of action of carnosine is not well understood.
Carnosine is recognized for its antioxidant20, 28-30 and
proton buffer20, 28, 31 properties, protection against oxidative
stress, and resistance toward senescence.30, 32-35
More significantly, it will complex with endogenous transitional metals
of biological significance.19, 27, 36-38
Zinc and copper have been found in pools in brain regions (hippocampus,
locus ceruleus, hypothalamus, olfactory bulb, and cortex) at concentrations
that allow them to exert neuromodulator effects.19
Although these transition metals are required for normal functioning in
the nervous system, they have also been found to be neurotoxic.19, 37
Shifts in zinc and copper may be involved in the neuropathology
of Alzheimer’s disease, ischemic stroke, and seizures.37
Further, zinc and copper have been shown to suppress GABA-mediated
inhibitory synaptic transmission. Petroff
and colleagues’ work has shown a critical relationship between homocarnosine
and GABA acid levels in epilepsy control.17
Carnosine can also prevent the influx of copper and zinc and enhance
GABA function.37
A majority of our subset of children with
autistic spectrum disorders were those who had a positive history of abnormal
EEG or partial response to valproic acid therapy.
If, indeed, carnosine acts to affect GABA bioavailability, it may
likely alter the subclinical seizure threshold or GABA function.
Carnosine is the first dietary supplement that may alter the neuronal
function in children with autistic-spectrum disorders that can be attributed
in a double-blind controlled fashion. Further
study is needed to investigate the mechanism of carnosine supplementation on
brain biochemistry. Our
observations regarding the autistic spectrum population suggest improved
receptive speech and improved social attention, less apraxia, and overall
global improvements.
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