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Signature: Med Sci Monit, 2003; 9(10): CR422-425 PMID: 14523330

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Received: 2002.12.09 Accepted: 2003.06.16 Published: 2003.10.02

Indolyl-3-acryloylglycine (IAG) is a putative diagnostic urinary marker for autism spectrum disorders
Gillian Bull1 cdef, Paul Shattock2 ag, Paul Whiteley2 abdef, Roz Anderson3 ab, Paul W. Groundwater3 ab, John W. Lough3 ab, George Lees3 cdef
1 2

Background:

Material/Methods:

Results:

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R SO O N N A LY L U
Source of support: none.

Authors Contribution: A Study Design B Data Collection C Statistical Analysis D Data Interpretation E Manuscript Preparation F Literature Search G Funds Collection

Sunderland General Hospital, Sunderland, U.K. Autism Research Unit, School of Sciences, University of Sunderland, U.K. 3 Institute of Pharmacy, Chemistry & Biomedical Sciences, University of Sunderland, U.K.

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Summary

Autism is a heterogeneous pervasive developmental disorder with a poorly defined aetiology and pathophysiology. There are indications that the incidence of the disease is rising but still no definitive diagnostic biochemical markers have been isolated. Here we have addressed the hypothesis that urinary levels of trans -indolyl-3-acryloylglycine (IAG) are abnormal in patients diagnosed with autism spectrum disorders (ASD) compared to age-matched controls. Urine samples were collected on an opportunistic basis and analysed for IAG concentration (normalised against creatinine content to account for changes in urinary volume) using reversed phase HPLC with UV detection. Statistical analysis (Mann-Whitney tests) showed highly significant increases (p=0.0002) in the levels of urinary IAG in the ASD group (median 942 V per mmol/L of creatinine [interquartile range 5211729], n=22) compared to asymptomatic controls (331 [163456], n=18). Detailed retrospective analysis showed that gender (boys 625 V per mmol/L of creatinine [2941133], n=29; girls 460 [2821193], n=11: P=0.79) and age (control donor median 10 years [814], n=15; ASD median 9 years [711] n=22: P=0.54) were not significantly correlated with IAG levels in this non-blinded volunteer study. Our results strongly suggest that urinary titres of IAG may constitute an objective diagnostic indicator for ASD. Mechanisms for the involvement of IAG in ASD are discussed together with future strategies to address its specificity.

Autism trans -indolyl-3-acryloylglycine (IAG) diagnosis of autistic spectrum disorders urinary creatinine reversed phase HPLC with UV detection developmental disease markers

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Professor George Lees, PhD MRPharmS, School of Pharmacy, Wharncliffe Str., University of Sunderland, Sunderland SR1 3SD, U.K., email: george.lees@sunderland.ac.uk

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Bull G et al Urinary IAG in Autism

BACKGROUND
Autism, a heterogeneous condition, is categorised as a pervasive developmental disorder usually present within the first three years of infancy. Symptoms include qualitative impairments in language and communication, problems in reciprocal social interaction and the presence of repetitive or stereotyped behaviours [1]. Studies indicating the possibility of a worldwide increase in the numbers of diagnosed cases of autism spectrum disorders [2] (ASD) [35] have further fuelled the need for more research into the causation and proliferation of autism spectrum disorders (ASD). Although much of this increase has been ascribed to improved diagnostic criteria and better awareness of the syndrome, there still remain no reproducible genetic or biological markers, or objective medical tests for the diagnosis of autism.

Samples were taken on an opportunistic basis following parental consent. The University of Sunderland Ethics Committee has ratified protocols for sample collection and analysis. Urinary analysis Urinary analysis was conducted after storage of frozen samples (containing 0.5 g of the preservative thymol) at 20C. Chromatographic equipment consisted of a CR4A Chromatopak integrator, 2 Shimadzu LC10-AD solvent delivery pumps with a Shimadzu SP6-AD UV/VIS detector and a Pye Unicam UV detector. Dyson Instruments Ltd, Houghton-Le-Spring, UK supplied all Shimadzu equipment. The analytical column was a 254.6 mm I.D. Vydac C18 (5 m) supplied by Phenomenex, Macclesfield U.K. Sample injections were made with SGE syringes via a Rheodyne 7125 valve (Anachem, Luton, U.K.). Bond ElutTM SPE cartridges (10 ml, 200 mg C18) were supplied by Phenomenex, Macclesfield, UK. Acetonitrile (HPLC grade) and TFA (trifluoroacetic acid) (all AR grade) were supplied by Sigma, Poole, U.K.

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We have refined methods that have revealed the presence of trans-indolyl-3-acryloylglycine (IAG) in the urine of patients with autism spectrum disorders [6]. Although IAG is unequivocally present in the urine of asymptomatic subjects [7], it is elevated in some disease states such as Hartnup disease [8] and light sensitive dermatitis [9,10]. Others have reported marked fluctuations in urinary titres with age: Marklova et al. [7] observed that maximum urinary IAG excretion was found in subjects aged between 2-7 years. Here we address the important hypothesis that the levels of IAG in the urine of children with autism spectrum disorders are abnormal. As surrogate measures we examine whether gender, or subtle variations in age, are important determinants of urinary titres of this substance.

MATERIAL AND METHODS

Subjects

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Samples were collected unblinded from two groups: a) Autism Spectrum Disorder (ASD) diagnosed using DSM-IV/ICD-10 criteria (n=22); b) Control (no diagnosis) (n=18). Subjects were recruited as follows: Patients were required to have received a formal diagnosis of ASD made by a qualified clinician using standardised DSM-IV or ICD-10 diagnostic criteria. Subjects (median age 9 years, range 622) were not on any medication or special dietary intervention at the time of urine testing. Samples were collected from patients (before noon, between July 1998 September 1998). Patients reports did not indicate any connection between symptom onset and the administration of the combined Measles-Mumps-Rubella (MMR) vaccination. Control (median donor age 10 years, range 518) samples taken from asymptomatic guests at a birthday parties (early afternoon between August 1998 October 1998) not diagnosed with any Pervasive Developmental Disorder or involved on any specific dietary/drug intervention.

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The research was carried out in accordance with the Declaration of Helsinki and with the authorisation of the University of Sunderland Ethics Committee.

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R SO O N N A LY L U
Statistical analysis

The samples were subjected to a preliminary clean-up process using a 10 ml Bond ElutTM SPE cartridge. Final urine collection was in 1 ml of 40% acetonitrile in 0.1% TFA; 10 l of the sample was injected into the system. Initial mobile phase conditions were: acetonitrile 0.1% v/v aqueous TFA (5:95 v/v) for 8 min, followed by a gradient of 550% v/v acetonitrile in 0.1% v/v aqueous TFA over 840 min, flow rate: 2.0 mlmin1. UV detection was at 215 nm and 326 nm. Area under the IAG peak was analysed against levels of urinary creatinine to correct for fluctuation in urine volume. Creatinine concentration was determined by a trained blinded technician at the Chelsea & Westminster Hospital, London, UK.

Data are reported as median (interquartile range or IQR) unless otherwise stated. All statistical analyses and tests were conducted using Prism 3.0 software (GraphPad California, USA). The alpha value for statistical significance in (two-tailed) hypothesis or correlation testing was P<0.05.

RESULTS
IAG titres, normalised for creatinine content (to eliminate fluctuations in urine volume) were clearly not normally distributed (Figure 1A). Control median levels of IAG were 331.3 (IQR 163456) V per mmol/L compared to 941.9 (5211729) V per mmol/L. A Mann Whitney 2-tailed ranks test showed that there was a highly significant difference in quantitative levels of urinary IAG between ASD and control groups (P=0.0002, see Figure 1A). Due to the skewing of sex ratios in autism (predominantly boys 4:1) it was important to establish if there was any connection between gender and levels of IAG excreted. To test the hypothesis that males excrete significantly different amounts of IAG to females, subjects

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Clinical Research
A. 5000 4000 3000 2000 1000 0 Control Subjects Autistic Subjects

Med Sci Monit, 2003; 9(10): CR422-425

Area under urinary curve (V/mmol/L creatinine)

Area under urinary curv (V/mmol/L creatinine)

5000

n=22 P=0.0002 4517

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2500

n=18 1101

(942)

Control Subjects Autistic Subjects

B. 30 30

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Age (Years)
P=0.54 n=15 20 18 10 (10) 5 0

Age (Years)

20

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n=22 22 6 P=0.79 Females

(331)138

10

(9)

Control Subjects Autistic Subjects

Control Subjects Autistic Subjects

Area under urinary curve (V/mmol/L creatinine)

C. 5000 4000 3000 2000 1000 0

Area under urinary curve (V/mmol/L creatinine)

D.

5000 4000 3000 2000 1000 0

r=0.069 p=0.68

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10

20

30

Males

Age (years)

Figure 1. A. IAG titres. The scatter plot (left) shows that data are clearly non-parametric. The box and whisker plot (right) shows the median values (in brackets) and range for each dataset. Details of the interquartile range (width of box) are given in text. Note the highly significant increase in IAG levels in the patient group. B. Age matching. Again the data were skewed (left) but there were no significant differences when descriptive statistics were compared (right). C. Lack of Correlation with age within the study. Spearman summary statistics are boxed. D. Gender. Autism is more prevalent in males but Mann-Whitney tests revealed no significant difference between the median levels of IAG in boys and girls across the study (irrespective of symptomology).

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were divided into subgroups containing males and females irrespective of disease state and the data analysed using a Mann Whitney test. The results (Figure 1D) showed that there was no significant difference between gender and IAG levels irrespective of disease state: male median 626 (2941133), n=29 compared to female median 460 (2821193), n=11: P=0.79. There were no significant differences in the age of the donors in each group: control median 10 (interquartile

range 814) versus ASD 9 years (711); P=0.54, Figure 1B. Spearmans correlation statistic (two-tailed test) was used to test for any relationship between small age differences at time of sampling and urinary IAG. Results (regression coefficient=0.069; P=0.68; Figure 1C) showed that at the 95% significance level, no relationship was found between age and IAG titres for all the donors (across patient and control groups) prepared to reveal their age.

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Med Sci Monit, 2003; 9(10): CR422-425

Bull G et al Urinary IAG in Autism

Despite the small sample sizes, it seems that neither age nor gender appears to contribute to the overall observed differences between ASD and control groups.

DISCUSSION
Our own unpublished preliminary observations have indicated that urinary IAG levels were elevated in subjects diagnosed with ASD compared to control groups. This quantitative study has shown highly significant differences in the levels of urinary IAG as a function of creatinine content between these groups. Urine samples are relatively easy to obtain in paediatric illness and hence represent an ideal diagnostic test. Despite this, the sampling regime used here contained minor flaws. Control urine samples were collected later in the day than those donated by patients. The use of creatinine to normalise for urine concentration helps to overcome this and it has been shown that dietary factors are relatively unimportant in dictating IAG levels in small human cohorts [10]. IAG levels have been reported to fluctuate slightly in a seasonal manner [11] but our samples were collected within a closely overlapping time period (none of the samples were taken within the winter period when IAG concentrations were elevated). The magnitude of the shifts here are much greater than those which might be attributable to dietary, metabolic or sampling vagaries in earlier studies. However, the use of urine has intrinsic flaws as an indicator of pathophysiological markers. We intend to seek evidence that the titres of IAG are abnormal in the systemic circulation too. The availability of patient serum for studies (the autism genetic resource exchange, founded by Cure Autism Now will release genetic information and serum samples from affected family groups) of this nature will allow us to measure titres of both IAG and its non-conjugated precursor indole-3-acrylic acid in the systemic circulation. In order to confirm the relevance of IAG as a possible diagnostic marker for autism spectrum disorders, we intend to conduct double blind trials using much larger patient cohorts. Further research is needed as to the exact source and role of IAG in autism and the relationship, if any, between IAG and the severity of symptoms observed in the autistic syndrome.

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CONCLUSIONS

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The aetiology of ASD remains an enigma. In the absence of marked morphological defects in post-mortem brain bank tissues from autistic patients, signalling defects in neurotransmitter or neuromodulatory pathways are now attracting considerable attention. For example, GABA levels are elevated in ASD serum [12]; several opiate-like peptides (besides IAG) are elevated in ASD urine [e.g. 13] and serotonergic pathways have been implicated too [14]. Serotonin uptake inhibitors (SSRIs) are used by paediatricians and clinical psychologists to treat the symptoms of ASD although more rigorous/large controlled clinical trials are needed [reviewed in 15]. The exact route of formation for IAG is, as yet, unclear but it may be linked to the conversion of tryptophan to 5-hydroxy tryptamine (5HTP) [16]. If IAG is to be used as a diagnostic factor for autism it is crucial that future studies address the specificity of the molecule for this disease. Linkage to Hartnups disease

R SO O N N A LY L U
Acknowledgements

Here we report a sensitive bioassay method applicable to readily accessible urine samples that can detect (highly significant) abnormally high levels of IAG in ASD. Further studies are urgently required to assess the specificity & relevance of this substance as a diagnostic marker and whether it is a determinant of disease severity in autism.

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Thanks go to Carol Vinter at the University of Sunderland for her expert technical assistance. Thanks to the Autism Research Trust for financial support (G.L.)

REFERENCES:

1. American Psychiatric Association: Diagnostic criteria from DSM-IV. American Psychiatric Association. Washington, 1994 2. Wing L: Autism Spectrum Disorders. BMJ, 312: 327-328 3. Chakrabarti S, Fombonne E: Pervasive developmental disorders in preschool children. JAMA, 2001; 285: 3093-3099 4. Magnusson P, Saemundsen E: Prevalence of autism in Iceland. J Autism and Develop Dis, 2001; 31: 153-163 5. Fombonne E: The epidemiology of autism: a review. Psychological Medicine, 1999; 29: 769-786 6. Anderson RJ, Bendall DJ, Garnett I et al: Identification of Indolyl3-acryloylglycine (IAG) in the urine of people with autism. J Pharm and Pharmacol, 2002; 54: 295-298 7. Marklova E, Hais IM: Age variations of indolyl-3-acryloylglycine excretion in healthy children. Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove. 1978; 21: 521-5 8. Halvorsen K, Halvorsen S: Hartnup Disease. Pediatrics, 1963: 29 9. Verhagen A, Burbach J: Indolyl-3-acryloyl-glycine and the Light Band in Chronic Polymorphous Light Dermatosis. Dermatologica, 1966; 133: 349-365

10. Szeinberg A, Bar-Or R, Pollak S et al: Observations on urinary excretion of indolylacryloyl glycine. Clin Chim Acta, 1965; 11: 506-511 11. Marklova E, Malina L, Hais M: Urinary excretion of indolyl-3-acryloylglycine in some skin affections. Clin Chem Acta, 1975; 64: 273-280 12. Dhossche et al: Elevated plasma GABA levels in autistic youngsters; stimulus for a GABA hypothesis of autism. Med Sci Monitor, 2002; 8(8): PR1-6 13. Reichelt KL, Hole K, Hamberger A et al: Biologically Active Peptide Containing Fractions in Schizophrenia and Childhood Autism. Adv Bio Psychopharm, 1981; 28: 627-643 14. Pedersen OS, Liu Y, Reichelt KL: Serotonin uptake stimulating peptide found in plasma of normal individuals and in some autistic urines. J Peptide Res, 1999; 53: 641-646 15. Posey DJ, McDougle CJ: Pharmacotherapeutic management of autism. Exp Opin in Pharmacother, 2001; 2(4): 587-600 16. Anderson RJ, Carr K, Cairns D et al: Putting Tryptophan in the Spotlight. J Child Neurol, 2002; 17(suppl.): 35-38

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and polymorphous light dermatosis has been established [8,9] (although these themes have not been revisited for some years) but no systematic studies have been conducted on other paediatric diseases of the CNS. In this respect, we have heard anecdotal reports (based on very small patient cohorts or individuals submitting samples prior to definitive diagnosis) that urinary IAG is elevated in Attention-Deficit Hyperactivity Disorder (ADHD) and in dyslexia. We plan to garner some hard statistics on these issues but we will need support from both paediatricians and parents to make this possible

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