Urinary levels of neopterin and biopterin in Autism

Neuroscience Letters 241 (1998) 17–20

Urinary levels of neopterin and biopterin in autism S. Messahel a , b, A.E. Pheasant a ,*, H. Pall b, J. Ahmed-Choudhury a, R.S. Sungum-Paliwalc, P. Vostanis c a

School of Biochemistry, University of Birmingham, Edgbaston Park Road, Birmingham B15 2TT, UK b Department of Neurology, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK c Department of Child Psychiatry, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

Received 7 August 1997; received in revised form 30 November 1997; accepted 7 December 1997

Abstract The pterins, neopterin and biopterin, occur naturally in body fluids including urine. It is well established that increased neopterin levels are associated with activation of the cellular immune system and that reduced biopterins are essential for neurotransmitter synthesis. It has been suggested that some autistic children may be suffering from an autoimmune disorder. To investigate this further we performed high performance liquid chromatography analyses of urinary pterins in a group of pre-school autistic children, their siblings and age-matched control children. Both urinary neopterin and biopterin were raised in the autistic children compared to controls and the siblings showed intermediate values. This supports the possible involvement of cell-mediated immunity in the aetiology of autism.  1998 Elsevier Science Ireland Ltd.

Keywords: Autism; Neopterin; Biopterin; Autoimmunity

The pterins, neopterin and biopterin, occur normally in body fluids including urine. The biosynthesis of pterins starts from GTP which is converted to 7,8-dihydroneopterin triphosphate by GTP cyclohydrolase. This key intermediate in pteridine biosynthesis is then further metabolised in a series of steps to 5,6,7,8-tetrahydrobiopterin (BH4) [4]. Alternatively 7,8-dihydroneopterin triphosphate can be cleaved by phosphatases to 7,8-dihydroneopterin which leaks from cells, becomes oxidised and gives rise to the neopterin observed in body fluids [6]. It has been known for some time that increased production and release of neopterin and 7,8-dihydroneopterin accompanies immune activation of macrophages both in vitro and in vivo [7]. Interferon gamma (secreted by T-lymphocytes) and tumour necrosis factor alpha are the key cytokines which lead to this immunologically triggered increase in neopterin levels [7] by inducing the activity of GTP cyclohydrolase (the first enzyme of pterin biosynthesis), whilst the remaining enzymes on the pteridine biosynthetic pathway are unaffected. * Corresponding author. Tel.: +44 121 4145409; fax: +44 121 4143982.

The determination of neopterin in body fluids of groups of patients with diverse diseases involving cellular immunity has provided a consistent view that this marker is able to follow the undulating course of disease severity and hence aid in diagnostic and therapeutic decisions [7]. In autoimmune diseases (e.g. rheumatoid arthritis) neopterin levels are raised significantly during exacerbation of the disease and are lower during remission phases thus acting as a marker of disease activity [7]. In tissues other than human macrophages the induction of GTP cyclohydrolase by cytokines leads ultimately to the formation of BH4 which acts as a cofactor for various reactions including NO synthetase and the aromatic amino acid hydroxylases and is therefore required for the biosynthesis of the neurotransmitters dopamine, adrenalin and serotonin [11]. Any disruption of BH4 metabolism can lead to neurological dysfunction. Autism and other pervasive developmental disorders are severe conditions occurring up to 10 times in every 10 000 live births [9]. Despite extensive research into this disease the cause of autism is as yet unknown [3]. Several authors [15,19,21] have suggested that a subgroup of autistic children may be suffering from an autoimmune disorder as

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00976- 2

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various cell-mediated immunological abnormalities have been found. These include cell-mediated immune responses to myelin basic protein [21] and autoantibodies against the serotonin 5HT1A receptor [15,19,24]. Indirect evidence of immune activation has been reported by Singh et al. [16] who found increased concentrations of interleukin-2 and soluble T8 antigen indicating that the activation of a subpopulation of T-cells occurs in some children with autism. If an autoimmune disorder is a causal factor in autism neopterin levels would be affected. A preliminary study in these laboratories found raised levels of both neopterin and biopterin in the urine of a sample of children with a broad range of autistic-like disorders [10] with the most marked differences being apparent in the younger children. The study described here targeted children of pre-school age and was designed to confirm the previous observations on a clinically homogenous sample of autistic children selected according to established diagnostic criteria, using a standardised procedure for sample collection. Urine samples from children with autism (aged 3–5 years, 12 male, two female) were obtained through the Language Unit of Parkview Clinic in Birmingham (regional child and adolescent psychiatry service for the West Midlands). All children were diagnosed as autistic using ICD-10 criteria [20]. Only typical autism (code F84.0) was included. Other types (e.g. atypical autism and Asperger’s syndrome) were not included in this sample to preserve its homogeneity. Diagnostic criteria were: (1) the presence of abnormal and/or impaired development that is manifest before the age of 3 years and the characteristic type of abnormal functioning in (2) social interaction, (3) communication and (4) restricted, repetitive behaviour. Control urine samples were provided by well-functioning children at a local preschool nursery (aged 3–5 years, eight male, eight female). Urine samples were also collected from the unaffected siblings of the autistic children (age range 1–19 years, 10 males, 11 females). Urine samples obtained were early morning specimens where possible and were protected from light, frozen within 3 h of collection and stored at −20°C until analysis. Urinary pterins were analysed by high performance liquid Table 1 Urinary neopterin and biopterin levels in autistic children, their siblings and control children

Autistic children (n = 14) Siblings (n = 21) Control children (n = 16)

Neopterin (mmol/ mol creatinine)

Biopterin (mmol/ mol creatinine)

3116 ± 686*

3691 ± 882**

1490 ± 346 908 ± 201

2923 ± 626** 359 ± 80

Data are the mean ± SEM. Significantly different from controls: *P , 0.01; **P , 0.001.

Fig. 1. Urinary neopterin levels in autistic children, their siblings and control children. The mean value for each group is designated by a line.

chromatography using fluorescence detection essentially by the method of Fukushima and Nixon [8], the mobile phase used being 1% methanol, 1% acetonitrile and 0.01% acetic acid. Analyses of ‘native’, fully oxidised neopterin and biopterin present in the urine were performed without pretreatment of samples. Neopterin and biopterin were identified by retention times (and co-chromatography in some cases) and quantitated using peak areas by comparison to calibration curves prepared using standard pterins (Dr. B. Schircks, Wetzikan, Zurich). Urinary creatinine was determined using a Sigma Diagnostics Creatinine kit and the results were expressed as mmol pterin/mol creatinine. Table 1 and Figs. 1 and 2 show the urinary levels of neopterin and biopterin respectively for control children, autistic children and their siblings. Autistic children had significantly higher urinary neopterin (P = 0.01) and biopterin (P , 0.001) compared to control children. Moreover the levels of both pterins were raised in the urine of the sibling group compared to the controls (neopterin P = 0.16: biopterin P = 0.001). The neopterin level for the siblings lay between the control value and that of the autistic group whereas there was no difference in biopterin excretion by the autistic children and their siblings. (The same conclusions were reached when the data were analysed by Student’s t-test and the Mann–Whitney test.) The observed increase in urinary native neopterin in autism agrees with our previous observations and indicates activation of cellular immunity in these children thus supporting the possible involvement of autoimmunity in the pathogenesis of autism. The apparent discrepancy between these results and those of Eto et al. [5] who found no change in biopterin excretion and an apparent fall in neopterin excretion, may be explained by differences in methodology. Eto et al. [5] measured total pterins following oxidation of the reduced forms present in urine. When this method was employed Harrison and Pheasant [10] also found no differences in total urinary pterins although ‘native’ urinary pterins were increased in autistic children. There is also a difference in the age of the children studied. Eto et al. [5] used autistic children from 6–18 years old whereas we concentrated on younger subjects. We have previously observed much greater changes in children less than 6

S. Messahel et al. / Neuroscience Letters 241 (1998) 17–20

years old compared to those greater than 6 years old (Fig. 3). This suggests that any inappropriate cell-mediated immune activity that may be occurring takes place early in life leaving damaged areas in the brain which give rise to functional abnormalities. A similar pattern of neopterin excretion is observed in children with autoimmune diabetes where increased neopterin levels are seen during the acute phase of autoimmune destruction but diabetic children in the later course of the disease return to normal neopterin production [13]. The results obtained with biopterin species paralleled those for neopterin. It has been found previously that periods of immune activation lead to not only an increase in neopterin in plasma and urine but also of biopterin due to an increase in BH4 biosynthesis [1]. Many types of cell will produce BH4 on stimulation with interferon gamma [22] and could thus be the source of the biopterin. The observation of raised fully oxidised biopterin in urine, particularly in the absence of an increase in total biopterins, implies increased loss of biopterins. This is consistent with lower activity of dihydropteridine reductase (the enzyme that ‘salvages’ quinonoid BH2 during metabolism) reported in young (,12 years) autistic children [12] and suggests that the levels of reduced biopterins, including BH4, may be low in autistic children. This may contribute to the neurological deficits observed and offer an explanation for the beneficial effects of administering BH4 in some cases [18]. The observation that the siblings of autistic children also have raised levels of urinary neopterin and biopterin is interesting and may indicate a common underlying pathway within the families. This could be due to either a genetic or environmental factor. There is considerable evidence of a strong genetic component in the aetiology of autism from twin studies [2] and epidemiology [17]. Autism now appears to be among the most heritable psychiatric disorders with a calculated heritabilty of 91–93% [2,14]. Moreover higher rates of cognitive and social interaction disorders have been reported in the siblings of children with autism [3]. The results reported here demonstrate a measurable biochemical change which could be related to this genetic link. Further investigations of differences in immunological function could shed light on which gene(s) are involved. Siblings

Fig. 2. Urinary biopterin levels in autistic children, their siblings and control children. The mean value for each group is designated by a line.

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Fig. 3. Urinary neopterin levels and their variance with age in autistic and control children.

are also exposed to the same environmental factors including diet and infectious agents and it may be that one of these is also a contributory factor to the autism and triggers the changes in pterin metabolism observed. Further investigations using control groups which account for learning disability and behavioural disturbance are necessary to determine how wide spread the changes observed are in childhood developmental disorders or if some facets are unique to autism. Similarly we also need to study these parameters within the autistic spectrum of disorders to look for any variation [23]. [1] Andondonskaja-Renz, B. and Zeitler, H.J., Biochemical aspects of pteridines during interaction between activated T-lymphocytes and monocytes/macrophages in patients. In B.A. Cooper and V.M. Whitehead (Eds.), Chemistry and Biology of Pteridines, Walter de Gruyter, Berlin, 1986, pp. 431–441. [2] Bailey, A., Le Couteur, A., Gottesman, I., Bolton, P., Simonoff, E., Yuzda, E. and Rutter, M., Autism as a strongly genetic disorder: evidence from a British twin study, Psychol. Med., 25 (1995) 63–78. [3] Bailey, A., Phillips, W. and Rutter, M., Autism: towards an integration of clinical, genetic, neuropsychological, and neurobiological perspectives, J. Child Psychol. Psychiatr., 37 (1996) 89– 126. [4] Curtius, H.Ch., Takikawa, S., Niederwieser, A. and Ghisla, S., Tetrahydrobiopterin synthesis in man. In B.A. Cooper and V.M. Whitehead (Eds), Chemistry and Biology of Pteridines, Walter de Gruyter, Berlin, 1986, pp. 141–149. [5] Eto, I., Bandy, M.D. and Butterworth, C.E. Jr., Plasma and urinary levels of biopterin, neopterin and related pterins and plasma folate in infantile autism, J. Autism Dev. Disord., 22 (1992) 295–308. [6] Fuchs, D., Milstien, S., Kramer, A., Reibnegger, G., Werner, E.R., Goedert, J.J., Kaufman, S. and Wachter, H., Urinary neopterin concentrations vs total neopterins for clinical utility, Clin. Chem., 35 (1989) 2305–2307. [7] Fuchs, D., Weiss, G. and Wachter, H., Neopterin, biochemistry and clinical use as a marker for cellular immune reactions, Int. Arch. Allergy Immunol., 101 (1993) 1–6. [8] Fukushima, T. and Nixon, J.C., Analysis of reduced forms of biopterin in biological tissue and fluids, Anal. Biochem., 102 (1980) 176–188. [9] Gillberg, C., Autism and pervasive developmental disorders, J. Child Psychol. Psychiat., 31 (1990) 99–119. [10] Harrison, K.L. and Pheasant, A.E., Analysis of urinary pterins in autism, Biochem. Soc. Trans., 23 (1995) 603S.

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