Sulphation deficit in “low-functioning” autistic children: a pilot study

Sulphation Deficit in “Low-Functioning” Autistic Children: A Pilot Study Antonino Alberti, Patrizia Pirrone, Maurizio Elia, Rosemary H. Waring, and Corrado Romano Background: Parents of autistic children and autism support groups often report that autistic episodes are exacerbated when the children eat certain foodstuffs such as dairy products, chocolates, wheat, corn sugar, apples, and bananas. The hypothesis that autistic behavior might be related to metabolic dysfunctions has led us to investigate in a group of “low functioning” autistic children and in an age-matched control group each made up of 20 subjects, the sulphation capacity available. Methods: Utilizing the biochemical characteristics of paracetamol we evaluated by high performance liquid chromatography, the urine paracetamol–sulfate/paracetamol-glucuronide (PS/PG) ratio in all subjects following administration of this drug. Results: The PS/PG ratio in the group of autistic subjects gave a significantly lower result than the control group with p ⬍ .00002. Conclusions: The inability to effectively metabolize certain compounds particularly phenolic amines, toxic for the CNS, could exacerbate the wide spectrum of autistic behavior. Biol Psychiatry 1999;46:420 – 424 © 1999 Society of Biological Psychiatry Key Words: Sulphation, autism, HPLC, paracetamol, urine, PS/PG ratio

Introduction

I

t has been hypothesized that autistic children might have metabolic dysfunctions, and that some substances contained in the food they ingest make them behave in an autistic manner. Parents and autism support groups often report that the autistic episodes are exacerbated when the children eat certain foodstuffs such as dairy products, wheat, corn, sugar, apples, bananas, and chocolate. This

From the Departments of Pediatrics (AA, PP, CR) and Neurology (ME), Oasi Institute for Research on Mental Retardation and Brain Aging (IRCCS), Troina, Italy; and the School of Biochemistry (RHW), University of Birmingham, Birmingham, UK. Address reprint requests to Antonino Alberti, MD, Dept. of Pediatrics, Oasi Institute, Via Conte Ruggero, 73, 94018 Troina, Italy. Received August 25, 1997; revised March 2, 1998; revised September 8, 1998; accepted September 28, 1998.

© 1999 Society of Biological Psychiatry

has led to the belief that autism could be allergy induced (Rapp 1978; Rapp 1980; O’Bannion et al 1978; Knivsberg et al 1990). Preliminary studies have demonstrated that these children have a compromised capacity for sulphoconjugation and therefore, are unable to effectively metabolize numerous compounds particularly phenolic amines such as dopamine, tyramine, and serotonin (present in many foodstuffs), which function as neurotransmitters (Ngong 1994). These substances are normally metabolized into nontoxic, water soluble, readily excreted metabolites through a detoxification process of conjugation with sulphate in the gastrointestinal tract and in the liver after absorption, or by monoamine oxidase activity. Other detoxification processes include conjugation with glucuronic acid. Paracetamol (4-acetylaminophenol, acetaminophen) is a well known, widely used analgesic and antipyretic drug. It contains a phenolic grouping that is normally conjugated with glucuronic acid or sulphate before excretion in the urine. The glucuronic acid residue is transferred via UDPGA (uridine diphosphate glucuronic acid) and the microsomal glucuronyl transferase enzymes, while sulphoconjugation (addition of sulphonate, SO32⫺, residue) occurs via PAPS (3⬘-phosphoadenosine-5⬘-phosphosulphate) as an active carrier of sulphate and the cytosolic sulphotransferase enzymes. Both these pathways give metabolites that are more water soluble, more readily excreted, and less toxic than the parent, paracetamol. Because pure reference metabolites are not readily available, paracetamol glucuronide and paracetamol sulphate are measured by following the increase in paracetamol when the conjugates are hydrolysed by ␤-glucuronidase or sulphatase (both hydrolytic enzymes), respectively. In any individual, although absolute amounts of the metabolites may vary over time, the ratio of the paracetamol sulphate excreted to paracetamol glucuronide is constant and the use of the PS/PG ratio enables metabolism to be compared over a population (Bonham Carter et al 1983; Rona et al 1994). Paracetamol has been used as a “probe” drug to estimate the relative contributions of glucuronide and sulphate pathways in a number of clinical dysfunctional states (Bradley et al 1991; Steventon et al 1990; Al0006-3223/99/$20.00 PII S0006-3223(98)00337-0

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Obaidy et al 1996; Dolara et al 1987). The aim of our study was to use the biochemical characteristics of paracetamol to evaluate the sulphation capacity available for the process of detoxification through sulphoconjugation in a significant number of autistic children and in a control group.

Methods and Materials We examined a group of 60 patients (49M, 11F) aged 2.7 to 15.0 years (mean age 10.3, SD 3.61) affected by autistic disorder. The other types of pervasive developmental disorders were excluded from the study. Diagnosis was based on DSM-IV criteria; interview with the families; direct observation of the patients during the various moments of the day; and application of CARS scale. Griffith developmental scales, Brunet-Lezine, Vineland, and PEP-R tests. We also examined 20 volunteers aged 3.2 to 14.2 years (14M, 6F; mean age 8.1, SD 3.39) as control group, and then, in order to make a correct statistical comparison (i.e., two-sided t test for independent samples), we selected 20 age-matched subjects (15M, 5F; mean age 8.0, SD 3.38) from our sample of 60 autistic patients. All subjects enrolled were free of gastrointestinal diseases and inborn errors of metabolism. One hour before starting the test, each patient interrupted all medication intake. After a light breakfast, we asked them to empty the bladder and a 500 mg tablet of paracetamol was given to those aged 12 to 15 years, and 250 mg to the younger patients. All urine was collected for the next 8 hours. The total volume was measured and the amount noted. An aliquot of 20 mL was retained and used for the paracetamol analysis. The first procedure consisted in evaluating free urine paracetamol, as measured by high performance liquid chromatography (HPLC), in absence of hydrolysis. Therefore, 1.2 mL of .05 molar acetate buffer solution was added to 1 mL of urine. In the second procedure, to the same quantity of urine and buffer solution, we added the enzyme beta-glucuronidase (Sigma: beta-glucuronidase from bovine liver G-0251 624000 U/gr., solid dessicate) for the detection of paracetamol freed from glucuronide-paracetamol conjugates. No phosphate incubation buffer was employed as the sulphatase activity in the betaglucuronidase is usually less than .5 percent. In the third procedure, to the same quantity of urine and buffer solution, we added the enzyme sulphatase (Sigma:sulphatase powder S-9626 100000 units) and D-saccharic acid 1,4 lactone (Sigma D-saccharic acid 1,4 lactone n° S-0375), the first for the detection of paracetamol freed from sulphatase-paracetamol conjugates, the latter to inhibit beta-glucuronidase activity. The urine samples, prepared in duplicate as described, were then incubated at 37°C for 18 hours in a water bath with constant agitation and temperature. Subsequently, each sample was filtered with a .45 ␮m diameter HA type Millipore filter, and a 10 ␮L volume was injected directly onto the HPLC column equipped with a 10 ␮L Rheodyne loop following Howie’s method (Howie et al 1997); (Gilson pump 305 and 307, detector UV/VIS, Techsphere C18 column with internal diameter 250 ⫻ 4.6). A 50 ␮g/mL paracetamol solution was used as standard (Sigma: 4-acetamidophenol).

Figure 1. Mean and SD of the four groups. Column A: autistic patients (n ⫽ 60); Column B: autistic patients (n ⫽ 40); Column C: autistic patients (n ⫽ 20); and Column D: control group (n ⫽ 20).

Elutions were carried out at room temperature using a mobile phase of 1% (vol/vol) acetic acid/methanol/ethyl acetate (90/15/ .1% vol/vol), at a flow rate of 1.2 mL/min, and detection at a wavelength of 250 nm. Integration was performed by a Hewlett Packard (HP3395) integrator. All data obtained for each subject for the three different biochemical determinations, expressed in milligram equivalent of paracetamol, are reported in terms of paracetamol-sulfate/paracetamol-glucuronide (PS/PG) ratios, with a normal range established at 1.5 or greater (Ngong 1994).

Results Among the 20 age-matched “low-functioning” autistic subjects, 18 out of 20 had a PS/PG ratio ⱕ to 1.5, with a mean of 1.1109 and SD 0.7449 (Figure 1, column C); whereas in the control group, 19 out of 20 had a PS/PG ratio ⱖ to 1.5 (Table 1), with a mean of 3.1515 and SD 1.674 (Figure 1, column D). The two groups, each made up of 20 subjects, were statistically compared. After a preliminary evaluation of the distribution of normal values, utilizing Kolgomorov-Smirnov, Lilliefors, and Shapiro-Wilk tests, we analyzed the differences of the mean between the two groups utilizing the 2-sided t test for independent samples. The PS/PG ratio in the group of autistic subjects gave a significantly lower result than the control group with p ⬍ .00002, t value ⫺4.9783, and degrees of freedom 38. Among the 40 autistic subjects examined, but not compared, 37 out of 40 had a PS/PG ratio ⱕ1.5 (Table 2), with a mean of 0.905 and SD ⫽ 0.5616 (Figure 1, column A); therefore in the total group of 60 autistic patients, 55 out of 60 had a PS/PG ratio ⱕ1.5 with a mean of 0.9504 and SD 0.5571 (Figure 1, column B).

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Table 1. Age (yrs), Gender, PS/PG Ratios, Comorbid Conditions, and Pharmacologic Therapy in 20 Autistic Children and in AgeMatched Control Subjects Autistic subjects

Control subjects

Subject number

Age

Gender

PS/PG

CC

PT

Age

Gender

PS/PG

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

2.7 3.0 4.0 5.0 5.0 6.0 6.0 6.5 6.7 7.3 8.4 8.5 9.3 9.6 10.2 10.5 11.2 12.6 13.8 14.1

m m m f f m m m f m m m m m m f m m m m

1.152 .943 .879 1.033 3.055 1.121 3.132 1.053 1.018 1.318 .567 .308 .312 1.011 .994 .755 .879 .322 1.428 .938

None None None Epilepsy None None None None None None None Epilepsy None None None CP None None None None

No No No CBZ No No No No No No No VPA, LMT, FNT No No HLP, NTZ Imipramine HCl No No No BPD, CTP, CPM

3.2 3.4 3.7 4.1 5.4 6.1 6.2 6.7 6.8 7.0 8.1 8.6 9.6 9.8 10.6 10.7 11.3 13.2 13.2 14.2

m m m m f f m f m f m m m m f m m m m m

1.430 2.090 2.720 1.890 3.100 7.250 2.690 3.160 5.600 2.300 1.900 2.190 6.100 5.370 2.280 4.600 1.800 2.000 2.100 2.460

CC, comorbid conditions; PT, pharmacological therapy; CP, cerebral palsy; CBZ, carbamazepine; VPA, valproic acid; LMT, lamotrigine; FNT, flunitrazepam; HLP, haloperidol; NTZ, nitrazepam; BPD, biperiden HCl; CTP, clotiapine; and CPM, chlorpromazine HCl.

The unpaired t tests between the group of autistic patients (n ⫽ 60) versus the group of control (n ⫽ 20) gave a t value of ⫺8.89673 and p ⬍ .000013.

Discussion Phenolic amines are normally conjugated through a process of sulphation in the CNS. In humans, this pathway appears to be more significant than glucuronidation (Tyce et al 1986). Sulphotransferase enzymes catalyse sulphation of phenols (P-form) and amines (M-form), though both variants will sulphate the alternative substrate at higher concentration (Coughtrie 1996). There are no gender effects; there is a small age effect (controlled for in the present study) where sulphation is slightly greater in children than in adults. Paracetamol is a good substrate for both forms. Tables 1 and 2 include the comorbid conditions and the pharmacologic treatment of our subjects. Neuroleptics, antidepressants, and anticonvulsants do not play any inhibiting role on both P- and M- forms of sulphotransferase. High doses of aspirin and other NSAIDS are known to inhibit the P-form, although this effect lasts only 1 hour in vivo. Some vegetables of the Brassica family, not present in the diet of our patients, can also inhibit this form (Harris et al 1996). Decreases in formation of paracetamol sulphate therefore reflect reduced activity of phenolsulphotransferase or reduced

levels of inorganic sulphate; the clinical interest is that neurotransmitter amines are metabolized by the same pathway, which is essential to eliminate excess free catecholamines from the brain (Meek et al 1972). Defects in paracetamol sulphation therefore imply aberrant inactivation of neurotransmitters. The inability to carry out this biochemical process could cause a cerebral increase of catecholamines, which could lead, after being further metabolized, to the formation of neurotoxic substances with psychedelic effects (Ngong 1994). The apparent incapacity for carrying out the process of sulphoconjugation could be due to a deficit of the enzyme phenosulphotransferase, or to a conformationally related reduced activity, or even to a block in the genetic code. Recently, low sulphotransferase levels have been reported in a subset of autistic children (Waring et al 1997). A lack of sulphate anions could also be the limiting factor in the synthesis of sulphate conjugates. Reduced amounts of inorganic sulphate and an abnormal increase of sulphite, thiosulphate, and taurine have also been reported in the urine of these patients (Klovrza et al 1995). Furthermore increased levels of urine peptides, which could have toxic effects on the CNS, have also been found (Reichelt et al 1986). It is not certain where the peptides come from, however it has been hypothesized that it could be due to an increased absorption from the gut following disruption of the intestinal vascular and

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Table 2. Age (years), Gender, PS/PG Ratio, Comorbid Conditions, and Pharmacologic Therapy in 40 Autistic Patients Screened but Not Compared with the Control Group Subject number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Age (years)

Gender

PS/PG ratio

CC

PT

5.0 5.0 6.4 6.4 7.4 7.5 7.8 7.8 8.4 9.0 9.0 9.1 9.2 10.0 10.0 10.1 11.8 12.0 12.0 12.1 12.2 12.4 12.5 12.5 12.5 14.1 14.3 14.4 14.5 14.6 14.6 14.7 14.8 14.8 14.8 14.9 15.0 15.0 15.0 15.0

m m m m m m m m m m m m m f m m m m f m m m m m m m m m f m m f m m m m m m m m

1.024 1.112 .515 .419 .279 2.511 .209 .963 .394 .341 .718 .296 1.576 1.652 1.237 .725 1.023 .730 .878 .232 .882 1.332 .513 1.312 .762 .660 2.898 .955 .823 .288 .691 .474 1.195 .726 .669 1.238 1.390 .939 .969 .652

None None None None None None 18q-syndrome None None CP None None None None None None None None None None None None None TS, Epilepsy None None None None None None None None None None Epilepsy None None None None Epilepsy

No No No No No No TRD No No No No No No No No No No No No No No No No VPA, CNZ HLP No HLP, FXT No No HLP, FXT No No No No ETX, PCZ, LMT No LSP HLP No CBZ

CC, comorbid conditions; PT, pharmacological therapy; CP, cerebral palsy; TS, tuberous sclerosis; TRD, thioridazine HCl; VPA, valproic acid; CNZ, clonazepam; HLP, haloperidol; FXT, fluoxetine HCl; ETX, ethosuximide; PCZ, periciazine; LMT, lamotrigine; LSR, levosulpiride; and CBZ, carbamazepine.

connective tissue (Murch et al 1993). This has been correlated with reduced sulphation of the mucous proteins which regulate gastrointestinal tract function. Low plasma sulphate could indirectly be responsible for the protein leakage as it is known that sulphation of substrates is regulated by anion supply (Vantrappen et al 1993). Our data show that autistic children have a PS/PG ratio below the age-matched control group, which proves that they have a decreased ability to form sulphated metabolites. In fact, a statistically significant difference exists

between group A (autistic), and group B (normal): p ⬍ .00002. There may be a subset of autistic children with normal sulphation values (children 5 and 7, table 1; children 6 and 27, table 2), but a different cause may be involved here. As both autism and mental retardation are heterogeneous conditions, there may be some overlap within the groups. There seems no obvious reason why mental retardation per se should affect sulphation capacity, although in individual cases, low sulphation could be an additive factor in dysfunction. Low sulphation capacity, therefore, appears to be

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strongly associated with autism; however, a cause-effect relationship is not necessarily involved, but probably the consumption of certain foods containing high quantities of phenolic amines may highlight the metabolic dysfunction, exacerbating the autistic behavior.

Knivsberg AM, Wiig K, Lind G, Nodland M, Rechelt KL (1990): Dietary intervention in autistic syndromes. Brain Dysfunct 3:315–327.

References

Murch SH, MacDonald TT, Walker-Smith FA, Levin M, Lionetti P, Klein NJ (1993): Disruption of sulphated glycosaminoglycans in intestinal inflammation. Lancet 341:711–731.

Al-Obaidy SS, Mc Kievnan PJ, Li Wan Po A, Glasgow JF, Collins PS (1996): Metabolism of paracetamol in children with chronic liver disease. Eur J Clin Pharmacol 50(1–2): 69 –76. Bonham Carter SM, Rein G, Glover V, Sandler M, Caldwell J (1983): Human platelet phenolsulphotransferase M and P: Substrate specificities and correlation with in vivo sulphoconjugation of paracetamol and salicylamide. Br J Clin Pharmacol 15(3):323–330. Bradley H, Waring RH, Emery P, Arthur V (1991): Metabolism of low-dose paracetamol in patients with rheumatoid arthritis. Xenobiotica 21(5):689 – 693. Coughtrie MWH (1996): Sulphation catalysed by human cytosolic sulphotranferases-chemical defence or molecular terrorism. Hum Exp Toxicol 15:547–555. Dolara P, Lodovici M, Salvadori M, Zaccara G, Muscas GC (1987): Urinary 6-␤-OH-cortisol and paracetamol metabolites as a probe for assessing oxidation and conjugation of chemicals in humans. Pharmacol Res Comm 19(4):261–273. Harris RM, Waring RH (1996): Dietary modulation of human platelet phenol sulphotransferase activity. Xenobiotica 12: 1241–1247. Howie D, Adriaenssens PI, Prescott LF (1977): Paracetamol metabolism following overdosage: Application of high performance liquid chromatography. J Pharm Pharmacol 29: 235–237. Klovrza L, Waring RH, Williams AC (1995): Sulphur-containing amine levels in neurological dysfunction. Toxicol Lett 78(1): 47.

Meek JL, Neff NH (1972): Acid and neutral metabolites of norepinephrine: Their metabolism and transport from brain. J Pharmacol Exp Ther 181(3):457– 462.

O’Banion D, Armstrong B, Cummings RA, Strange J (1978): Disruptive behaviour: A dietary approach. J Aut Child Schizophr 8:325–333. Ngong JM (1994): Drug Metabolism in Clinical Dysfunction. PhD thesis University of Birmingham. Rapp DJ (1980): Allergies and the Hyperactive Child. New York: Simon & Schuster. Rapp DJ (1978): Does diet affect hyperactivity. J Learn Disabil 11:56 – 62. Reichelt KL, Saelid G, Lindback T, Boler JB (1986): Childhood autism: A complex disorder. Biol Psychiatry 21:1279 –1290. Rona K, Ary K, Szuts L, Kovacs, Gachalyi B (1994): Polymorphic paracetamol conjugation: Phenotyping in a Hungarian population. Acta Medica Hungarica 50(1–2):65–74. Steventon GB, Heafield MTE, Waring RH, Williams AC, Sturman S, Green M (1990): Metabolism of low dose paracetamol in patients with chronic neurological disease. Xenobiotica 20(1):117–122. Tyce GM, Messick JM, Yaksh TL, Byyer DE, Danielson DR, Rorie DK (1986): Amine sulphate formation in the central nervous system. Fed Proc 45(8):2247–2253. Vantrappen G, Geboes K (1993): Glycosaminoglycans and the gut. Lancet 341:730 –731. Waring RH, Ngong JM, Klovrza L, Green S, Sharp H (1997): Biochemical parameters in autistic children. Dev Brain Dysfunct 10:40 – 43.