from www.AutismResearchInstitute.com
Fall DAN!TM 2003 Conference
*** Portland, Oregon *** October 3-5, 2003
Why Use the Gluten-Free and Casein-Free Diet in
Autism and What the Results have Shown so Far
Peptides and Autism
Karl Reichelt, MD, PhD1 and A. M. Knivsberg PhD2
1
2
Institute of Pediatric Research, Univ. of Oslo, Rikshospitalet ,N-0027 Oslo, Norway
Centre for Reading Research, N-4068 Stavanger , Norway
Key words: Autism, Exorphins, Peptide, Serotonin uptake, Peptidase
Abstract:
Objective: The reasons for using diet in autism should also reasonably explain the
disorders patho-physiology. Changes ought to be found in rating scales, peptide levels
and learning ability after intervention.
Method: Peptides have been separated by HPLC, co-chromatography, immune assay
and mass spectrometry and IgA antibodies to food proteins measured. Because a
double blind was impossible to carry through (people know what they eat), an open
study over 4 years and a single blind controlled pair wise matched intervention with
Gluten free/Casein free diet have been used.
Conclusion: An increase in urine peptides and specific peptide like compounds can be
demonstrated. Furthermore IgA antibody increases relative to controls point to certain
food proteins as being important. Because of the indicated limitations p values lower
than 0.01 two tailed were generally chosen as limit in the open dietary experiments.
Even so clear-cut behavioral changes can be measured after one and four years.
Abbreviations: 5HT: serotonin. CSF: cerebrospinal fluid. Cm: Casomorphin
Introduction
Several laboratories have found increase in urinary peptides (Fig 1 - 3) in autism
(table 1) and that some of these are probably exorphins (1-5). The definite structure of
these has been obtained by mass spectrometry and fragmentation mass spectrometry
(5, 6). Furthermore co-chromatography and immune assay for peptides have been
performed. The presence of rare d-amino acid containing exorphins has also been
confirmed (5). Furthermore increase of opioids have been found in serum and CSF (7,
8), some of which are bovine casomorphins (1, 4, 5). IgA antibody increase against
gliadin, gluten and casein have been found (4, 9-11). Therefore diet free of these
proteins have been used with behavioural testing before and after intervention.
Methods:
Diagnosis: based on ICD 10 or DSM IV (almost the same).
Urine: 10 ml of the first morning urine has been the basis of analysis .It was frozen
sent in Styrofoam boxes to our laboratory. The urine had creatinine measured and 600
µ l urine was filtered by centrifugation at 3000 x g for 20 min. using Spin-X Costar
centrifuge Tube filters (0.22µlm cellulose acetate) in 2 ml polypropylene tubes.
(Corning Inc, Corning, NY 14831, USA). Filtered urine equivalent to 250 nanomoles
of creatinine was routinely used for analysis.
Urine analysis: With an automatic sample applicator and thermostatic control set at
30o C. and HPLC machine from Hewlet Packard (Agilent series 1100) was used and
Vydac C-.18 reversed phase column (0.4x 20 cm). Peptide bonds were measured at
215 nano-meters wave length and aromatic side groups at 280 nano-meters. The latter
is important because many drugs contain aromatic groups and can thus be found.
Flow rate was 1 ml per minute and gradients of trfluoroacetic acid (10mM) against
acetonitril was run (for details see 12)
Antibody analysis: was analysed in serum taken from venous blood sample in the
cubital vein. IgA and IgG antibodies were measured against gliadin and gluten and
IgA against lact-albumin, lacto-globulin, casein and ov-albumin were measured using
Elisa as described (13, 14)
Dietary intervention: Because we have not been able to run dietary intervention
double blind (people must know what they eat) two strategies for gluten free/casein
free diet were used.
Group A: It is exceedingly difficult to have a control group on diet and given gluten
and casein blind. The reason for this is that the effort involved excludes willingness to
continue if no improvement is registered.
We therefore ran one experiment on 15 participants for 4 years to rule out possible
placebo in an open study.
Group B: In another experiment run for 1 year matched pairs were randomly assigned
to diet or control group and studied single blind. All other treatments were unchanged.
p values lower than 0.01 increases the probability of a likely effect (13 ).
Testing before and after:
The following tests were used because they have been standardized against
Norwegian/Danish children and were therefore chosen.
A: The group followed for four years, and tested after 1 and 4 years on diet.
1: DIPAB (14) testing especially for social isolation and bizarre behaviour. This was
compared with Tafjord test (15), Ravens Progressive Matrices (16) and to compensate
for natural maturation: Illinois test of psycholinguistic ability (17) and the peptide
levels.
After four years The Illinois test of Psycholinguistic ability and Ravens progressive
matrices were checked (18).
B: In the single blind 1-year intervention with a control group on randomly assigned
paired children we used DIPAB, as above. Information on autistic traits, cognitive
level, language , and motor skills were also obtained using the tests described ( 13).
Thus linguistic ability (ITPA) standardized for Norwegian children between 4 and 10
years old (17) or Reynells language test between 1.5 and six years of age (19), non
verbal cognitive level (20), and Motor problem registration(21) were used. Profiles of
each child were obtained before and after and the children were pair wise matched on
severity of autistic symptoms and age (13) and randomly assigned to diet or control
group. All other interventions were unchanged.
Results:
In Fig 1 the normal urine pattern and level is shown running urine equivalent to 250
nano-moles of creatinine on HPLC. Fig 2 shows a late onset regressive autistic
pattern, while fig 3 shows an infantile autistic .Indolyl acryloyl glycine (IAG) found
by the Sunderland group
(22) seems to be the best marker for infantile autism .
In table the level of peptides eluting after hippuric acid in autistic syndromes
measured as area under the UV 215nm curve is presented and controls likewise. The
difference is statistically highly significant. That the peaks are peptide like has been
extensively documented (23) and is based on release of amino acids on hydrolysis,
peptidase treatment and isolation of peptides after purification ( 24 ) .
Some of the peptide peaks show opioid activity (1,13), cochromatography on HPLC
after purification and on spiking with standards, react with specific antibodies, and by
mass spectrometry indicates that some of these are exorphins . (Table 2) (1,10,13),
which agrees with data from other groups (4, 5).
Furthermore the peptide levels decrease on diet (4,18), as expected if they had a
dietary origin.
Immune assay: As can be seen in table 3 the IgA antibodies are significantly different
from controls for gliadin, gluten and casein. This indicates increased permeability for
these proteins without celiac disease (negative endomyzium or transglutaminase.
Some specificity is present however, because IgA antibodies against lact-albumin,
lacto-globulin and ovalbumin ere not statistically different in autistic syndromes and
controls.
Results of diet:
Group A followed for 1 and 4 years to compensate for the placebo effect even if no
change was induced in training /schooling .
The details can be seen in table 4. There are very statistically significant changes and
Haracopos scheme and Tafjord scheme. By matching each other closely this increases
the probability that an effect is seen. The improvement in language in the Tafjord test
is also reflected in ITPA.
Three children quit the diet for various reasons such as break up of the family. After
four years these three show not significant changes. The difference for the three that
broke the diet to those that stay on diet has a Mann Whitney U statistic =0.0 and U’=
36 and p= 0.004; or a very significant difference. As can be seen in the original
publication those that quit diet do in fact show clear cut regression.(18). This is thus
an unplanned control of the changes.
Group B:
In the controlled single blind study the changes measured can be seen in table 5. For
the measures taken the diet group improved with decrease in autistic behaviour ,
increase in non-verbal cognitive level and decrease in motor problems. There was in
this 1-year intervention no difference in linguistic age (13) and this may reflect that
the children chosen were linguistically relatively high functioning to start with.
The changes noticed are similar to those published ( 4,8,9,25,26) and as outlined (24)
the found peptides can indeed explain the disorder. Furthermore the measurable
regression in those that quit diet as well as the lack of changes in the control group in
the single blind series, clearly indicates that diet is effective. A recent Internet study
also points to this conclusion.(27 )
Because neuroactive peptides may show long term effects after early exposure ( 28,29
) and several of the opioids have trophic effects ,( 30,31) and are epileptogenic ( 32 )
,we think it is important to get started as early as possible with diet . Given
supplements of vitamins , enough other proteins and cod liver oil it is furthermore not
hurtful and after many years clearly quite safe. This is not the case for most
medications.
Conclusion:
Peptides are excreted in increased amounts in autism and some of these are opioids.
The excreted peptides may explain the social isolation as expected form opioids (33).
Also trophic changes may be due to opioid peptides, as such compounds to have
effects on brain maturation (30,31 ). Furthermore Fos antigen induction is found in
key nuclei such as N Accumbens , and the superior and inferior colliculus after IV
casomorphin 1-7(bovine) (34). Because immunocompetent cells have receptors for
opioids ( 35 ) immunological changes are to be expected as found. Although not
double blind the result of our dietary intervention strongly indicates that gluten free,
gliadin free and casein free diet is an effective treatment. This is furthermore
supported by the effect of oral peptidase treatment to increase protein and peptide
break down ( 36).
The anatomical changes found in autistic children in brain (37,38) lends some
urgency to the treatment as early as possible , the more so because of their
behavioural effects(39).
References:
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forlag a/s,Herning ,Denmark.
15. Tafjord M. Observasjon av forutsetninger for lek og aktivitet;
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.Universitetsforlaget, Oslo , 1975
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explained by the nature and the discovered urine peptides? Nutr. Neuroscience
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25. Whiteley P, Rodgers J, Savery D, Shattock P. A gluten-free diet as an
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26. Kniker WT, Andrews A, Hundley A, Garver C. The Possible role of
Intolerance to Milk/dairy and wheat/gluten foods in older children and adults
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autism Res Unit, Sunder land Univ.: edit) 2002; pp77-84
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Basel.1990; 8:pp205-214
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proliferation in the developing rat brain. Brain Res 1987; 412: 68-72
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32. Siggins GR, Henriksen SJ, Chavkin C and Gruol D . Opioid peptides and
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reduce separation distress in chickens. Peptides 1978; 5: 829-31
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immunoreactivity in discrete brain regions relevant to schizophrenia and
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Walckman F, W, Walckman M, Pangborn J, Buchholz I.Enzyme-based
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Text to Figures
Fig 1: Shows the HPLC separation of peptides from a normal boy, based on the
principles published ( ). C –18 reverse phase column is used and absorption read at
215 and 280 nm giving us a purity index. That most of the material after hippuric acid
is peptidic has been discussed extensively ( ), and is based on amino acid release by
hydrolysis and also peptidase treatment followed by amino acid analysis and mow
also Mass spectrometry.
Fig 2: The urinary profile from a 6-year-old boy with autism. Comparing this with fig
1 the difference is striking. Both runs are based on urine volumes equivalent to 250
nanomoles creatinin.
Fig 3: Another boy with the same diagnosis and CARS (Childhood autism rating
scale) score as in fig 2. Notice the different pattern in spite of having the same
diagnosis, sex and age. This indicates that different enzyme defects are probably
present in different families, but with overlaps.
Table 1: The quantitative aspects of autism in prepubertal children found from 1995
to 1998 from 8 different countries in our lab.
Diagnosis
Age range
N
Peptide level (mean)
SD
95% CI
Lower value
Higher
autism normal
2- 14
3-14
315
143
720
346
471
108
560
773
329
365
The units are Abs units in µm2 under the UV 215 nm trace of the peaks eluting after
hippuric acid, and based on urine = 250 nanomoles creatinin. Only patients that have
been diagnosed by certified psychiatrists are included. Ratio of UV 215/ UV 280 was
used to check of puritiy of individual peaks. Many drugs have high UV 280 nm peaks
due to aromatic ring structures. The autistic children are different from controls with a
p= 0.001. Samples from 8 different countries and MD’s did not differ statistically
indicating that diagnosis has become quite standardized through the use of DSM III to
IV.
Table 2: Different compounds found in autism and the frequency of appearance.
Cochromat.
HPLC
Aminoacid analysis
after hydrolysis
IAG*:
+
+
CM (b)
1-8*
+
+
CM 1-3
+
+
CM (b)
1-4*
+
+
CM 1-4
NH2*
+
+
CM 1-7*
+
+
Glu M
A4*
+
+
Gli M*
+
+
Receptor
binding
Antibody Freq.
binding
%
92
+
+
85.7
27.4
+
41.7
28.5
+
+
23.7
66.6
+
30.9
The preliminary opioid receptor assay was carried out by Prof L Terenius, Stockholm
and the immune-assays by professor Dr H Teschemacher from Giessen, who kindly
provided us with antibodies for later work. Only peaks that are statistically larger than
in controls are included. CM = Casomorphin , GluM : Glutemorphin. Gli M:
Gliadinomorphin. The mass spectra (MS) and the fragmentation (MS/MS) spectra
have been identified for urinary components and published (5). We identified these by
purification as described (1,9), amino acid analysis after hydrolysis and sequencing
(9). Antibody binding to antibodies against casomorphine 1-8 was used (1) as
guideline for 1-8 and 1-7. Finally cochromatography with synthetic samples in at least
two different HPLC systems was routinely used. Compounds run on MS are marked
*.
Table 3. Immune data in autistic syndromes.
GliadinIgA
Mean
GlutenIgA CaseinIgA
Aut.
Cont Aut. Cont Aut.
Cont
0.29
0.05 0.29 0.05 .47
0.10
Median 0.085 0.05 0.09 0.04 0,145 0.075
N
60
34
60
34
62
34
Min
0.00
0.00 0.00 0.00 0.00
0.00
Max
2.57
0.14 2.10 0.14 2.40
0.44
95%CI
P
0.048
0.009
0.01
CI = confidence interval – Antibodies measured by ELISA in serum as described.
Table 4: 1 and 4 year intervention (18)
TEST
INITIAL
SCORE
6.8±2.8
25.7±5.5
1 YEAR
¶-score
+8.6±2.8
+2.7±2.5
4 YEARS
¶ -score
+8.6± 3.2
+6.1±2.8
N P
C-RAVEN:
12 0.005
ITPA:
10 0.005
TAFJORD SHEME:
1.Social interaction
53.7±15.2
+12.1±5.9
14 0.005
2: Language
71.0± 15.2
+8.7±6.5
14 0.005
3: Structure ability
56.7±17.1
+9,1±5.4
14 0.001
4:Sensory / Motor
72.9±12.3
+ 7.2± 4.5
14 0-001
DIPAB:
A: Social Isolation
8.5±3.3
-6.1±2.7
14 0.005
B: Bizarre traits
5.3±2.2
-5.3±1.22
14 0.005
PEPTIDE LEVELS as µmoles /24h diuresis and hydrolysis released amino acids.
26.89±12
-10.7±13
14 0.016
Notice that Raven C reaches a maximum level after 1 year while Illinois test of
psycholinguistc ability (ITPA) improves even more after 4 years, probably reflecting
the more complex nature of advanced learning (language). DIPAB = Haracopos
scheme: ” Diagnosis of Psychotic Behaviour in Children”). Peptide levels decreased
and followed the decrease in symptoms. Tafjord is a Norwegian scheme for
registering play, interaction, structural ability and sensory motor behaviour during
play. For details see (18). Mann Whitney U test used throughout.
Table 5. Single blind paired and controlled dietary intervention. (13)
Autistic traits
Diet
group
Cont.
group
Before
After
Diet
Difference
/control
Signific.
Signific.
12.5±2.2
5,6±2,4
0.005
11.5±3.9
11.2±5
0.798
0.001
N
10
10
Non-verbal cognitive
level
Diet
group
Cont
81.0±35.9
86.7±38,5
0.03
84.6±36.6
74.3±31.4
0.5
9
0.004
9
group
Linguistic age
Diet
group
Cont
group
26.3±11,5
29.3±13.1
0.161
24.7±14.6
27.8±12.2
0.123
10
0.04
Non parametric MannWhitney U test after 1 year, two tailed.
Corresponding author:
KL Reichelt, MD, PhD
Inst. of Pediatric Res. Univ of Oslo
Rikshospitalet, N-0027 Oslo, Norway
Phone:+ 47 23 07 29 85
Fax:+ 47 23 07 27 80
Email: K.L.Reichelt@klinmed.uio.no
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