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Urinary
profiling of people with autism
Autism
as a Metabolic Disorder.
The research undertaken at the Autism Research Unit subscribes to the
theory that autism is a
consequence of a metabolic disorder, whereby certain biologically
active “peptides” and other related compounds (derived mainly but
not exclusively from dietary gluten and/or casein) are not metabolised
correctly. These compounds, together with the additional problem of
abnormal permeability of the gastrointestinal membrane, are present in
much greater quantities than would normally be expected and are
subsequently able to exert an effect that may interfere with normal
neural processes. The presence of these compounds in the blood would
mean that they would tend to be collected in the kidneys and then
excreted in urine. Hence,
the content of urine would be to some extent, be reflective of the
content of the blood.
Various
analytical methods for detecting, and to some extent determining, these
compounds in the urine are available today.
One of the most popular methods is HPLC (High Performance Liquid
Chromatography). HPLC uses a liquid mobile phase to separate the
compounds of a mixture. Mixtures (samples) are first dissolved in a
solvent, and then forced to flow through a chromatographic column under
high pressure. Inside the column, the mixture is separated into its
components, as it passes [elutes] down the column. Positioned
immediately after the column is a detector, which emits a response as a
function of the components of the sample passing through it. There are
several types of detector used for this purpose; the one currently used
in this analysis is a ultra-violet (UV) detector. A UV detector measures
the ability of a sample to absorb UV light. The majority of organic
compounds can be analysed using this kind of detector. Depending on time
taken for the compound to elute through the column, and the ability of a
compound to absorb UV radiation, we are provided with data about the
type of compound being detected and a general idea of how much of the
compound is present (see published
references for HPLC protocols).
More
information about HPLC can be at the following websites:
University
of Kentucky | University
of Athens (includes graphics)
The
qualitative results and interpretations of example urine profiles can be
viewed in the following articles: Autism
as a Metabolic Disorder and Urinary
Profiles from people with Autism (apologies for the poor quality of
the results shown). The
University
of
Sunderland
Ethics Committee
has approved all research relating to the biochemical study of autism
undertaken by the Autism Research Unit.
A
control urine sample spiked with casomorphin peptides (215nm).
Protocol
for urinary profiling
The analysis undertaken at the Unit represents the culmination of over
10 years of work identifying and examining the role of these urinary
compounds. At present we have a 2-stage system for qualitative urinary
analysis. Firstly,
the collection of background information about participants, using a
specially designed questionnaire (ARU Autism Information Checklist).
Secondly the collection of a urine
sample for biochemical analysis. The questionnaire is only available
from the Autism Research Unit (it is not currently available on
this website).
(a)
Background
information
Continuing studies show that the pattern of urinary profiles can differ
from individual to individual, and group to group. As we have previously
described in the article Urinary
Profiles from people with Autism, there are very characteristic
differences in the profiles obtained from people with for example
differing diagnoses (e.g. classical autism, Asperger syndrome, etc) or
differing severity of problems. There are also early indications that
other factors may also influence the results (e.g. problems at birth,
history of viral infections such as meningitis / encephalitis). To take
these factors into account, we rely on the background information given
in the questionnaire, which in the majority of cases enables us to
interpret any obscure results. Details sought from the questionnaire
include: official diagnosis and the availability of any conformational
material (if any), pre-, peri-, &
post-natal conditions, current medical condition (including any
accompanying medical conditions), feeding habits, and incidence of other
adverse events. In addition, details of the participant's physician
(General Practitioner) are requested because copies of urinary results
are also sent to them as a matter of routine.
The
normal timescale of events means that participants’ (or participant
carers) complete and return the questionnaire back to us at the Unit
before any sample is submitted. Please note all details taken from the
questionnaire are subject to the strictest confidence, and are entered
on to our research database (access to the database is by authorised
personnel only). This has been ratified by the
University
of
Sunderland
Ethics Committee
.
(b)
Urinary
test kit
Normally, upon receipt of the completed questionnaire back to the Unit,
a urinary test kit is issued containing: (i)
a 30 ml plastic tube in protective container (with preservative), and
(ii) instructions for the collection of the urine sample (shown below).
Instructions
for the collection of a urine sample for HPLC analysis (v3.0)
Autism
Research Unit (ARU), School of Health, Natural & Social Sciences,
University of Sunderland,
Sunderland
SR1 3SD Tel: 0191 510 8922 Fax: 0191 567 0420 email:
autism.unit@sunderland.ac.uk
IMPORTANT:
The transport of organic / biochemical products by the postal service
demands that strict health and safety procedures are followed regarding
sample packaging and clear labelling of goods carried. It is your
responsibility to ensure that the sample is packaged correctly and
returned to the ARU in a non-hazardous condition. Please read
through the following instructions for safe management and transport of
the sample.
1.
The
30ml tube (enclosed in the protective plastic postal container) you have
been provided with will contain a small quantity of a preservative* (thymol).
*The
preservative may not be visible with the naked eye, although does have a
characteristic smell.
2.
Collect
approximately 20ml of urine from the first production urine sample in
the morning, mid-stream (if possible**). Do not exceed the 20ml
mark on the tube or the sample tube may burst whilst frozen. Make
sure the cap on the tube is firmly closed, and the name and date of
birth of the person from whom the sample was taken is written on the
tube.
**
If there are serious problems in getting a sample at this time, samples
taken at other times may be of some analytical use.
If problems persist, contact your GP / Health Visitor who may be able to
provide special urine collection nappies or bags.
3.
As
soon as the sample is taken, place the tube in a freezer for at least 24
hours.
4.
Place
the frozen sample tube in the protective plastic container provided.
At this point we also recommend that the protective container be
securely fastened with an elastic band*** to prevent opening during
transit. Fill out the form shown at the bottom of this sheet.
*** Please do not use cellotape or packaging
tape to seal the protective container.
5.
Place
the protective container in a padded envelope (a jiffy bag is ideal) and
mail first-class post to us at the address given above, clearly marking
the package as containing a MEDICAL SAMPLE FOR ANALYSIS. Mailing
is best done at the beginning of the week to allow for receipt of the
delivery. Please note the ARU is not open to receive samples at
weekends or bank holidays.
Results
are performed on a batch system with a current waiting time of 1 week
(subject to alteration). A
copy of the results will be sent to both parents
\ carers and the patient’s GP.
Please
do not send a sample unless you have completed and returned the
questionnaire.
Any
sample received without the proper authorisation will not be processed.
Results
of urinary analysis
Completed results are returned to both participants/carers and
physicians. Results come in graphical form with each peak corresponding
to a particular compound detected in the urine (see Figure 1). Results
are accompanied by a general interpretative sheet (shown below) and
written reports specific for individual results.
Update
October 2003: Due
to an upgrade of technical equipment, the ARU urine profiling service is
now able to offer more confirmatory power regarding the analysis of
urine samples. We are now using a HP1100
series system with UV-DAD. New protocols for presentation of results
have been implemented. Further
examples of the type of results obtained from our method are shown here
(please note this is a Microsoft Word document v2000).
Typical profile for a person with Asperger syndrome
Interpretation
of Urinary Profiles V.3 (Oct 2003)
This
information sheet is designed to give a general overview of the urinary
results obtained using HPLC analysis (High Performance Liquid
Chromatography) as part of the research carried out at the Autism
Research Unit at the
University
of
Sunderland
.
This sheet is to be used in conjunction with the graphical set of
results produced for each patient and the sheet providing specific
details of the results for that person. Automated analysis of SPE sample
fragments is conducted on a HP1100 series HPLC with UV-DAD.
We must stress several important points from the outset:
1. Our methodology is still experimental.
2. Any decision to act upon these findings must reside
entirely with the parents and the person with autism.
3. This test does not constitute a diagnosis or
confirmation of a diagnosis of autism or associated spectrum disorder.
4. We suggest the agreement of the physician involved
with each individual case be obtained before commencement of dietary
intervention.
5. The involvement of a registered dietitian /
nutritionist is recommended in the production and implementation of any
dietary
interventions.
The profiles have been prepared in accordance with our published
protocols.
The x-axis (along the base) represents time (in minutes). The
y-axis (vertical) represents the amount of absorbance (mAU,
absorbance units), giving an indication of the percentage amount of each
compound present as a function of the whole sample.
The current HPLC method used is not quantitative at this time (i.e. does
not give a precise result as to the amount of each compound present in
the urine). It provides an overview of the types of compounds present in
the sample.
We supply 2 versions of the results, labelled as graph (a) and (b).
Graph (a) and (b) represent analysis of the sample at two different
wavelengths. Graph (a) represents the profile obtained following our
original published methods (215nm). Graph (b) represents detection
carried out at a different wavelength (326nm). The reason for this is to
confirm the presence of our key marker compound in the sample.
The significant area is, we believe, between 15-30 minutes. Previous
studies have indicated that the main biologically active peptides with
opioid effects will appear in this region. The peak considered to be of
particular relevance typically appears between 18-20 minutes on both
graphs. The structure has been identified as being trans–indolyl-3-acryloylglycine
(IAG). IAG is not thought to be an opioid peptide directly derived from
dietary sources. The exact aetiology and role of IAG is the subject of
continuing research although several preliminary observations have been
made:
a. IAG is present in significant quantities in
approximately 75-85% of people with autism spectrum disorders.
b. IAG is present in significant quantities in
approximately 50% of first-degree relatives, fathers, mothers, brothers
and sisters
c. IAG is thought to be an abnormal metabolite of the
amino-acid tryptophan.
d. IAG (or the precursor compound of IAG) has been
linked to abnormal permeability of various membranes throughout the body
including the
intestinal wall.
e. The use of a gluten-free diet may reduce the levels
of IAG present in urine. This is a preliminary finding that is
under investigation.
Typically, each patient result will have an indication as to whether IAG
is present or not based on analysis of the sample results by UV-DAD
(Ultra Violet Diode Array Detection). This will normally comprise
of a separate sheet (labelled c) showing two graphical plots overlaid on
top of each other and a percentage correlation co-efficient value based
on the similarities of the plots.
The peaks that we believe to be due to the presence of dietary peptides
occur predominantly after (to the right hand side of) the IAG peak.
The majority of these peaks have not yet been identified.
We have, using the current analytical system, managed to identify
several potential peptide fragments. Most of these are thought to
be derived from casein (the protein from milk). The most common of these
peptides is beta-casomorphin 1-7 (βC1-7). This is an opioid
peptide, which results from the incomplete breakdown of the milk
protein. Synthetic standards of BC1-7 typically appear in the
region of 24-25 minutes on graph (a) only. Its occurrence seems to be
less frequent than that of IAG. Suspicious peaks indicating the possible
presence of BC1-7 will be indicated and a further sheet (labelled d)
will present any results of analysis using UV-DAD.
Other
groups
Although our research is geared predominantly towards biochemical
analysis of urine samples from people with autistic spectrum disorders,
we have been conducting research on several other conditions that appear
to show very similar biochemical anomalies to the autism group. These
groups include: veterans of the 1990 Persian war presenting with
"Gulf War Syndrome"; Chronic Fatigue Syndrome (CFS / ME); Organo-Phosphate
poisoning. We would advise anyone suffering from any of these conditions
to contact us should they wish to find out more about the work.
Cost
of testing
The research carried out at the Autism Research Unit does not receive
any direct funding from any external funding body source. The
University
of
Sunderland
kindly provide us with amenities for the day-to-day running of the
Unit's office and facilities for our biochemical investigations.
Calculations suggest that each sample analysis performed costs
approximately £60 sterling (including labour, materials and VAT), and between
1991-2000, no charge had been attached to any analysis we
conduct. This has however had to change (as of Jan 2001) due to
increasing financial difficulties in the previous years. Further
information is available upon contacting the Unit.
Comment
(
8
March 2005
)
An
article has recently appeared in the scientific literature regarding the
relevance of IAG to autism spectrum disorder (Wright et
al, 2005*). The article
reports that quantitative levels of urinary IAG, although elevated in
autism compared to controls, were not significantly different from urine
samples taken from control (non-autistic) participants.
These findings are in contrast to those we have previously
reported in the scientific literature regarding quantitative levels of
IAG (Bull et al, 2002) [see
a list of publications by
the Autism Research Unit].
Whilst
these recent findings are important, they do not impact significantly on
the method of analysis used to produce the current results.
This is based on a qualitative method (not specifically designed
to measure the amount of IAG present in the urine sample) to ascertain
the appearance of IAG relative to other compounds in the important
biological area (18-30 minutes on graph a).
This method showed significant differences in the presence of IAG
in autism samples compared to non-autism and learning disability
controls (Alcorn et al,
2004).
On
the issue of a possible relationship between urinary IAG and the use of
dietary intervention, this recent paper does not provide any further
details in addition to what we have previously published in the
scientific literature (Whiteley et
al, 1999).
As
a precautionary measure, we feel we must reiterate the following points:
(a)
Our methodology is still experimental.
(b)
Any decision to act upon these findings must reside entirely with
the parents and the person with autism.
(c)
This test does not constitute a diagnosis or confirmation of a
diagnosis of autism or associated spectrum disorder.
(d)
We suggest the agreement of the physician involved with each
individual case be obtained before commencement of dietary intervention.
(e)
The involvement of a registered dietitian / nutritionist is
recommended in the production and implementation of any dietary
interventions.
*
Wright B. et al. (2005) Is the presence of urinary
indolyl-3-acryloylglycine associated with autism spectrum disorder? Dev
Med Child Neurol. 47 (3): 190-192
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Address:
Autism Research Unit
School
of
Health
,
Natural & Social Sciences
University
of
Sunderland
Sunderland
SR1
3SD
,
UK
|
Telephone:
0191
510 8922
Fax:
0191
567 0420
Email:
autism.unit@sunderland.ac.uk
|
Our
telephone lines are regularly very busy.
Anyone wishing to contact us is advised to
contact us by either email or post. The web-pages
included on this site remain the exclusive property of
the Autism Research Unit and the
University
of
Sunderland
.
They are freely available to view or print out
for personal use only.
This
page
http://osiris.sunderland.ac.uk/autism/hplc.htm
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