Autism/PDD
See Events and Conferences planned by the Great Plains
Laboratory, Inc.
1996 Autism Society National
Conference Proceedings
What follows are
proceedings from the Autism Society's national conference held July 10-13,
1996 in Milwaukee, Wisconsin. The material included here addresses some
of the scientific and technical issues relevant in organic acid testing
for children with autism.
Organic
Acid Testing to Evaluate Abnormal
Microbial Metabolies in Urine of Children with Autism
Experience with Organic
Acid Testing to Evaluate Abnormal Microbial
Metabolites in the Urine of Children With Autism
For the past two years, we
have evaluated by gas-chromatography mass-spectrometry biochemical abnormalities
that appear to be of microbial origin in urine samples of children with
autism and other developmental disorders. Our interest in this phenomenon
began when we found that certain putative microbial metabolites appeared
in higher than normal values in urine samples of two brothers with autism
(1).
These findings were of especial
interest to us because of a report that autistic children have a greater
incidence of ear infections than age-matched peers; that lower functioning
autistic children had an earlier onset of ear infections than their higher
functioning autistic peers; and that the ears of children with autism
were anatomically positioned differently than those of normal children,
perhaps leading to greater ear infection susceptibility (2). Intestinal
overgrowth of yeast and anaerobic bacteria are well documented sequelae
of the common oral antibiotics used to treat ear infections (3-10).
Therefore
we considered the possibility that the biochemical products of abnormal
microorganisms may play a role in the etiology of autism just as abnormal
elevations of phenylalanine and its metabolites cause PKU. Certain
of the metabolites that had been previously identified in urine samples
include tartaric and citramalic acids. Other compounds that we identified
for the first time in urine samples included arabinose(a carbohydrate),
3-methylmalic acid, 3-oxoglutaric acid, phenylcarboxylic acid, and carboxycitric
acid (1).
Since that time we have identified
several additional compounds as commonly increased in the urine samples
of children with autism including dihydroxyphenylpropionic acid, furandicarboxylic,
hydroxymethylfuroic, and furancarbonylglycine. We suspected that most
of the above compounds were of microbial origin based on reports that
demonstrated the presence of these compounds or closely related biochemicals
in the culture media of yeast,fungi ,or bacteria (1, 11-13).
During this period of time,
we have gathered information about these compounds and their possible
role in autism:
By conducting a formal
uncontrolled clinical trial of antifungal drug therapy which involved
23 children with autism who were seen as outpatients at the hospital;
By offering reference laboratory
testing to physicians who used this testing to evaluate possible
standard inborn errors of metabolism as well as putative abnormal microbial
metabolites;
By testing culture media
of different microorganisms to determine which compounds were produced
by different species.
if the
urinary excretion of abnormal Krebs cycle metabolites and/or abnormal
carbohydrates are biochemical characteristics in autistic children
and
if antifungal
treatment results in decrease or elimination of abnormal Krebs cycle
metabolites, metabolites such as dihydroxyphenylpropionic acid,phenylcarboxylic
acid, and/or carbohydrates in autistic children and/r improvement
in autistic symptoms.
The study was approved
by the Investigational Review Board Of Children’s Mercy Hospital.
Funding for the studies was provided by grants from the Katherine
B. Richardson Foundation , from Pfizer Pharmaceutical Corporation,
and the parents of an autistic child.
Twenty-three autistic
children were enrolled for the study. Each child was classified
as autistic according to the latest criteria proposed by the American
Psychiatric Association DSM-IV (1994). After informed consent, a
random urine sample was collected without special preparation for
organic acid analysis by gas chromatography/mass spectrometry.
If the urine showed the
presence of abnormal Krebs cycle metabolites and/or elevated abnormal
carbohydrates , the child was offered treatment of suspected yeast
infection with Mycostatin (Nystatin) 100,000 units q.i.d. orally
for 10 days and another random urine sample obtained and analyzed
for organic acids. If the second urine sample still showed the presence
of abnormal metabolites, the child was offered a second course of
treatment with Mycostatin for 2 months and another urine sample
tested. If this still showed abnormal organic acids, the child was
offered to enter treatment with fluconazole (Diflucan) 2 mg/kg/day
as a single dose daily for 2 weeks. A second two-week course of
treatment was offered if abnormal metabolites were still present
in the urine and liver function was satisfactory based on serum
transaminase activities before and after fluconazole therapy. A
random urine sample was obtained two weeks later and analyzed for
organic acids. An additional urine sample was collected four weeks
after the end of therapy to determine the duration of any biochemical
abnormality associated with drug therapy.
Each of the patient's
baseline urine values served as controls. Urine from 20 normal children
of laboratory employees served as additional normal controls. An
additional 50 normal controls have been collected and data will
be available for the meeting. An assessment of the severity of autistic
behaviors was done by both a staff psychologist working with a parent
and by a teacher (if child was in school or preschool) using the
CARS scale (Childhood Autism Rating Scale). This assessment was
done twice: prior to starting therapy and at the end of therapy.
The use of two evaluators was employed to increase the reliability
of this test. Furthermore, parents and teachers are the individuals
who have usually observed the child longer than any other people.
As indicated in Table
1, the mean values of all of the above compounds with the exception
of the phenylcarboxylic acid compound decreased following 10 days of Nystatin
therapy. The mean value of the phenylcarboxylic compound actually increased
by 89.3% following Nystatin therapy (p=0.05 by paired t-test).
The percent decrease for dihydroxyphenylpropionic
acid and furancarbonylglycine is relatively small and is or marginal statistical
significance. The percent decrease for the additional compounds is larger
(36.3-69.8%) with greater degrees of statistical significance by the paired
t-test. Please note that four of the patient results are not included
since these results were late arriving but will be included in the final
analysis.
Following 70
days of Nystatin therapy, mean values for ten of the thirteen compounds
decreased. The mean values for 3-oxoglutaric, VMA analog, and phenylcarboxylic
acid compound increased following 70 days of Nystatin therapy.
The percent
decrease for dihydroxyphenylpropionic was slight (11.8%) and was of
marginal statistical significance. The percent change of mean values
compared to baseline ranged from 39.9-87.9 for the remaining compounds
with p values of the paired t-test ranging from 0.02-0.13. (The p value
is the probability that the decreased values are due to chance.)
The marginal
decrease in dihydroxyphenylpropionic acid led to the suspicion that
this compound was not of fungal origin. Testing was performed on several
patients at the attending physician’s request who had suspected or confirmed
Clostridia infections and were treated with metronidazole (Flagyl).
We tested several of these patients before and after metronidazole therapy
and found a dramatic decrease in the concentration of this compound
from baseline in these patients after drug therapy.
As shown in
Table 3, there is a dramatic decrease in the urinary concentration of
dihydroxyphenylpropionic acid following the administration of standard
doses of the antibiotic Flagyl. In all four patients, the concentrations
of dihydroxyphenypropionic acid decreased 99% or more after two to three
weeks on this drug. In the first patient in the above series, dihydroxyphenylpropionic
acid rapidly increased following the cessation of Flagyl treatment.
Table
3 -Effect of Flagyl Therapy on Urinary
Excretion of Dihydroxyphenylprpionic Acid
Diagnosis
& Sex
Age
Length
of time (Days) from start of Flagyl Therapy
Urinary dihydroxyphenylpropionic acid*
Autism,
male
4
0
435
6
184
16
1
21
(stopped Flagyl)
5
24
2
43
236
93
274
Previous
C. difficile infection and uncontrolled diarrhea, female
Thirteen of
the parents and/or teachers of six of the autistic children completed
CARS evaluations both before and after therapy with Nystatin.
The mean CARS
score prior to therapy was 37.3 (sd= 4.2 ) which is a rating of "severe
autism" while the mean CARS score after therapy was 32.6 (sd=5.1),
a rating of "mild to moderate autism."
This difference
was rated as extremely significant by the paired t-test (p< .001).
CARS
Score
Diagnosis
15
- 30.0
Non-autistic
30.1
- 37.0
Mildly
to moderately autistic
37.1
- 60
Severely
autistic
Improvements cited by parents and teachers include decreased hyperactivity,
more eye contact, increased vocalization , better sleep patterns, better
concentration, increased imaginative play, reduced stereotypical behaviors
(such as spinning objects), and better academic performance.
We were unaware
of a possible role of metabolites of anaerobic bacteria in autism until
our formal research study was complete. The overgrowth of anaerobic
bacteria may be a complicating factor in the use of antifungal therapy
for treatment of autism.
In several cases
, concentrations of dihydroxyphenylpropionic acid increased following
antifungal therapy. It is possible that anaerobic bacteria may have
proliferated when yeasts and/or fungi were reduced in the microbial
ecosystem.
If both yeast/fungal
products and products of anaerobic bacteria are involved in the mechanism
of autism, more complex antimicrobial therapies may be necessary to
restore a balanced microbial ecology to the gastrointestinal tract,
perhaps by "reseeding" the gastrointestinal tract with beneficial bacteria
like Lactobacillus acidophilus. Such therapy has proved effective in
the treatment of individuals with recurrent Clostridia difficile infections
(14).
The marked decrease
in dihydroxyphenylpropionic acid following treatment with Flagyl, an
antibacterial agent with specificity toward anaerobic bacteria and no
antifungal properties (15,16) is consistent with the production of this
compound by one or more species of anaerobic bacteria.
Phenylpropionic
acid and monohydroxyphenylpropionic acid which are very closely related
biochemically to this compound are produced by several species of Clostridia
(17 ). However we failed to identify this compound in multiple culture
media samples in which multiple species of Clostridia were cultured.
Our failure
to isolate dihydroxyphenylpropionic acid* from pure cultures of Clostridia
could be due to multiple reasons:
A precursor
of the compound such as monohydroxyphenylpropionic acid may be produced
by the anaerobic bacteria and then be converted to dihydroxyphenylpropionic
acid by another microorganism and/or by human metabolism.
The anaerobic
bacteria producing this compound may be difficult to grow in vitro.
The culture
media for this organism may not provide the nutrients needed for the
biosynthesis of this compound by this organism. We were very interested
in a possible role in the mechanism for autism for this compound because
it is related biochemically to the neurotransmitters dopamine and
norepinephrine and because it is an inhibitor of dopamine decarboxylase,
the enzyme responsible for the conversion of DOPA to dopamine (18).
* This compound has recently
been definitiely identified as 3-(3-hydroxyphenyl)-3-hydroxy propionic
acid.
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