1. A gluten- & casein-free diet as an intervention for autism spectrum disorder (ASD). Paul Whiteley, Research Fellow Autism Research Unit Department of Pharmacy, Health & Wellbeing Faculty of Applied Sciences, University of Sunderland, UK http://osiris.sunderland.ac.uk/autism

2. Aims of presentation The diet / health relationship. Dietary interventions for ASD. Gluten & casein-free diets. Evidence currently available. ScanBrit trial. Potential mode(s) of action.

3. Caveats Much of the research in this presentation remains under investigation. Based on peer-reviewed research articles (published or in press) – not grey literature. Dietary intervention should be viewed as complimentary to other approaches for ASD. Consultation with your childs physician is advised before embarking on any dietary change.

4. Interventions for ASD Any intervention for ASD needs to bear in mind four very important factors that will affect outcome: (1) ASD (or PDD) is Pervasive. Spread throughout, all encompassing. (2) ASD is Heterogeneous. Not uniform in structure or composition. (3) ASD is often accompanied by co-morbidities. e.g. epilepsy, ADHD, anxiety-related problems (4) People with ASD differentially develop without specific intervention. Age & environment may play a role

5. Diet & health are linked: not just another casual relationship!

6. Diet & health relationship Examples of a relationship between diet & health: Physical health Obesity & malnutrition – significantly increased risk of developing several physical symptoms & health-related diseases (including death). Cognitive-mental health PKU (Phenylketonuria) – a leading causes of learning disability prior to the discovery of Imbecillitas Phenylpyruvica. (Følling,1934) Physical & cognitive-mental health Coeliac (see-lee-ak) disease – reaction to gluten protein causing GI malabsorption, failure to thrive & linked to increased risk of psychiatric co-morbidity (see later). (Aretaeus the Cappadocean, 200AD; Gee, 1887)

7. Schonwald A. (2008) AAP Grand Rounds 19: 17 Diet & ADHD: acknowledgement of a relationship

8. Could diet also affect ASDs?

9. A: Numerous ! Including (individually or combinations of): Gluten / casein-free diets (Cereal and mammalian dairy produce) MSG / aspartame free diets (Flavour enhancer/artificial sweetener) Lutein-free diet (Carotenoid from fruit / vegetables) Feingold diet (Artificial flavourings / colourings) SCD (Complex carbohydrates / starches / processed sugars) Combined with various dietary supplements (ω-3 oils, vitamins). Q: How many types of dietary intervention are being used as an intervention for ASD?

10. A: It depends on who you talk to! * Parents / primary caregivers * Health / education / social care professionals * Researchers * Media * Government (All with varying degrees of persuasiveness!) Q: How much evidence is there to support the use of these dietary interventions in ASD? Autism Research Institute Feb 2008

11. Q: How much scientific evidence is there to support the use of dietary intervention in ASD? A: Some, of varying degrees of objectivity e.g. Ketogenic diet and autism ( fat protein, carb) N=30 children (4-10 years old) 60% of children showed improvement (varying degrees) on CARS. Evangeliou A. et al (2003) Application of a ketogenic diet in children with autistic behaviour: pilot study. Journal of Child Neurology 18: 113-118

12. Q: Which dietary intervention for ASD has been studied the most? A: Gluten and casein-free diets As of 2001: 11 Group studies 2 Surveys 3 Case reports + multiple anecdotal reports Several other studies have been added to the list since. Knivsberg A-M. et al (2001) Reports on Dietary Intervention in Autistic Disorders. Nutritional Neuroscience 4: 25-37

13. Gluten & casein What is gluten ? A mixture of two proteins, gliadin & glutenin that give flour a cohesive, elastic property to turn into dough. Present in: wheat, barley & rye (oats <20% avenin). What is casein ? Primary protein found in mammalian dairy sources. Several variants according to order / species*. Present in: milk, cheese & yogurts. * Kaminski S. et al (2007) Polymorphism of bovine beta-casein and its potential effect on human health. Journal of Applied Genetics 48: 189-198

14. Schizophrenia & psychotic disorders. Improvement in psychiatric symptoms on diet. Dohan FC. et al (1973) Relapsed schizophrenics: earlier discharge from the hospital after cereal-free, milk-free diet. American Journal of Psychiatry 130: 685-688. Vlissides DN. et al (1986). A double-blind gluten-free/gluten-load controlled trial in a secure ward population. British Journal of Psychiatry 148: 447-452 Extrapolation to autism & related ASDs. Several trials suggestive of amelioration of symptoms. History of gf-cf diets in psychiatry

15. Dietary intervention studies Knivsberg A-M. et al (1990) Dietary intervention in autistic syndromes. Brain Dysfunction 3: 315-327 Effect of both a gluten- & casein-free diet (n=15; 6-22 years) Open trial (non-blinded / non-randomised). Improvements reported in several areas: Social interaction Language use / comprehension Sensory / motor abilities [Reduction in peptide-like urinary material] Participants followed-up after 4 years – improvements continued.

16. Lucarelli S. et al (1995) Food allergy and infantile autism, Panminerva Medica 37: 137-141 Effect of casein-free diet (alone) (n=36; age 8-13 yrs) + control group (n=20) Blind casein-challenge (placebo vs. casein capsules). Significant changes (improvement) after 8 weeks of diet (BSE). (e.g. isolation, verbal communication, cognition) Detrimental changes to scores following dietary challenge. Dietary intervention studies

17. Whiteley P. et al (1999) A gluten free diet as an intervention for autism. Autism 3: 45-65 Effect of a gluten-free diet (alone) (n=22; mean age 4 years). Open trial (non-blinded / non-randomised) + control groups. Significant changes (improvement) after 5 months of diet (BSE): motor (p=0.04) & eating disturbances (p=0.01), attention (p=0.02) Improvement on 3 out of 6 cognitive subtests. Changes to levels of urinary IAcrGly. Dietary intervention studies

18. Behavioural effects [1] From the research trials conducted so far, the behavioural effects of dietary intervention include: Attention & concentration Communication & language Social integration Motor co-ordination Self-injurious behaviours But……

19. Behavioural effects [2] Some indications of significant changes in group results although not universally successful. More detailed analysis of individual results suggested that the younger, more severely affected children were best responders. Parents tended to be more pleased with the results than teachers.

20. Transient negative effects Withdrawal phase is common at the early stages of dietary intervention. Variable & more apparent in younger children. * Anxiety & clinginess * Crying & general whinginess * Staring into space * Dizziness / decrease in movement * Increased frequency of urination / defecation * Flu type symptoms (adults)

21. Breaking the diets Transient but sometimes extreme reactions to the ingestion of even small amounts of gluten / casein. e.g.Increased hyperactivity Spacing out Increased aggression Abatement of reactions following re-adoption of the dietary intervention.

22. Evidence for the existence of somatic features co-morbid to ASD diagnostic features. Preliminary reports of improvement in somatic symptoms following dietary intervention. Whiteley P. et al (1998) Clinical features associated with autism: observations of symptoms outside the diagnostic boundaries of autism spectrum disorders. Autism: 2: 415-422 Whiteley P. (2004) Developmental, behavioural and somatic factors in pervasive developmental disorders: preliminary analysis. Child: Care, Health & Development 30: 5-11 Somatic symptoms in ASD

23. Abnormal bowel habits / conditions One quarter of children with ASD have at least one chronic gastrointestinal (GI) symptom. Molloy CA. et al (2003) Prevalence of chronic gastrointestinal symptoms in children with autism and autistic spectrum disorders. Autism. 7:165-171 Consumption of milk is a strong predictor of constipation. Afzal N. et al (2003) Constipation with acquired megarectum in children with autism. Pediatrics 112: 939-942 Significant in mucosal eosinophil* infiltrate on gluten and casein-free diet. * white blood cell responsible for combating infection & controlling mechanisms involved in allergy Ashwood P. et al (2003) Intestinal lymphocyte populations in children with regressive autism: evidence for extensive mucosal immunopathology. Journal of Clinical Immunology 23: 504-517

24. Nutritional status on diet Not a widely researched area but: No significant differences in energy, protein & nutrient intake whilst on a gluten- & casein-free diet. Cornish E. (2002) Gluten and casein free diets in autism: a study of the effects on food choice and nutrition. Journal of Human Nutrition & Dietetics. 15: 261-269 Stewart PA. et al (2008) Nutritional quality of the gluten-free and casein-free diet. IMFAR poster proceedings Trend towards levels of plasma tryptophan & tyrosine ( tryptophan has been found in non-dietary participants). Arnold GL. et al (2003) Plasma amino acids profiles in children with autism: potential risk of nutritional deficiencies. JADD 39: 449-454

25. Bone thickness & calcium Hediger et al (2008) Reduced bone cortical thickness in boys with autism or ASD. JADD Bones were growing longer but not thicker than normal. Possible explanations: Use of casein-free diet ( calcium & vitamin D intake). Lack of variety in food habits*. GI issues affecting absorption of vitamins / minerals. * Stewart C. Latif A. (2008) Symptomatic nutritional rickets in a teenager with autistic spectrum disorder. Child: Care, Health & Development 34: 276-278 Requires further research on actual bone density (not thickness). Reiterates the need for involvement of dietetic support.

26. The British Dietetic Association Effective Practice Bulletin Issue 37 November 2003

27. Methodological issues Issues yet to be addressed: Open-trials not controlled trials. Confirmation of diagnosis (ADI-R, ADOS-G). Clarity on why the diet/s are working. Recommendations of the Cochrane Review report (2004) & MRC report on autism research (2001). Millward C. et al (2004; 2008) Gluten and casein-free diets for autistic spectrum disorder. CD003498

28. Elder et al (2006) The gluten-free, casein-free diet In autism: results of a preliminary double blind clinical trial. JADD Effect of a gluten-free /casein-free diet (n=15; age 2-6 yrs). Double-blind cross-over trial. 12 week study (6 weeks on diet / 6 weeks off). No significant changes to scores or peptide levels but some reports of improvements on diet (real / placebo?). Author criticism: Low study power / not long enough on diet? Double-blind study

29. ScanBrit dietary study Effect of a gluten-free & casein-free diet (n=72; age 4-11 yrs). (ClinicalTrials.gov ID: NCT00614198) Single-blind, randomised-controlled, matched-pair trial. Comprehensive assessments (SCQ, ADOS, VABS, GARS, ADHD-IV) Additional measures (dietary habits, somatic symptoms, urine profiles, anthropometric data, quality of life). Adaptive design with interim analysis based on pre-defined thresholds of positive improvement (A: 24 months, B: 12 months).

30. Why might these diets work for people with ASD?

31. The role of classical allergy? Classical allergy response by the immune system. Production of antibodies called Immunoglobulins (Ig-) to an allergen (inflammatory response). Various isotypes / classes of Ig- (IgA, IgE, IgG, IgM). IgE (Type-1 hypersensitivity) = classical allergy. Some indication of possible allergy to milk in ASD. (Lucarelli et al, 1996) Contradictory evidence from skin prick tests. (Bakkaloglu et al, 2008) Male D. et al (1996) Immunology (7 th Edition). Mosby. (Prof. J. Brostoff as co-author) Lucarelli S. et al (1995) Food allergy and infantile autism, Panminerva Medica 37: 137-141 Bakkaloglu B. et al (2008) Atopic features in early childhood autism. European Journal of Paediatric Neurology

32. Autoimmune-type GI disease » genes & environment. Most prevalent in the West of Ireland (potato famine ca.1845). Familial risk factor (approx. 4.5% prevalence amongst 1° relatives) Can be clinically silent (no serious overt symptoms). Various somatic symptoms (present or not present) including: Functional bowel habit problems Fatigue / muscle wasting Dyspepsia (pain / discomfort in upper GI tract) Malabsorption Anaemia ( Fe or folate)* Walker-Smith J. & Murch S. (1999) Diseases of the small intestine in childhood (4 th edition). Isis Medical Media. Lessons from coeliac disease?

33. Interesting parallels in somatic symptoms but: Co-morbidity of CD & ASD = rare?? (no routine screening!). People with CD do not necessarily present with autism. Untreated CD is however associated with the appearance of various behavioural / psychological symptoms including: Depression (Ciacci et al, 1998) Disruptive behaviours (Pynnönen et al, 2004) Anxiety (Addolorato et al, 2001) Learning disorders (Zelnik et al, 2004) Schizophrenia (De Santis et al, 1997) A peripheral association between CD & ASD cannot be ruled out.

34. A role for opiates? Opioids – class of natural & synthetic compounds that bind to opioid receptors found in the CNS and gastrointestinal tract (agonists). Morphine is an exogenously derived opiate. Endorphins & enkephalins are endogenous opiates. Opioid peptides (chains of amino-acids) formed as a consequence of the digestion of proteins. e.g. gluten (cereal produce) – gluten exorphins Zioudrou C. et al (1979) Opioid peptides derived from food proteins: the exorphins. Journal of Biological Chemistry 254: 2446-49 Terenius L. et al (1986) Opioid peptides in the cerebrospinal fluid of psychiatric patients. Progress in Brain Research 65: 207-219 Teschemacher H, Koch G. (1991) Opioids in the milk. Endocrine Regulations 25: 147-150 Fukudome S. et al (1997) Release of opioid peptides, gluten exorphins by the action of pancreatic elastase. FEBS 412: 475-479

35. Opiate effects overlap with ASD Psychological desire for social contact & diminished clinging behaviour. [opioids inhibit the release of oxytocin – the social hormone] stereotypic behaviours (effects of apomorphine). Impaired developmental, behavioural & organisational abilities. Somatic Altered EEG patterns. tolerance to pain (analgesia). Functional bowel habit problems (e.g. constipation). Physiological / psychological effects following withdrawal. Urca G. et al (1977) Morphine and enkephalin: analgesic and epileptic properties. Science 4298: 83-86 Kalat JW. (1978) Speculations on similarities between autism and opiate addiction. JACS 8: 477-479 Panksepp, J. (1979) A neurochemical theory of autism. Trends in Neurosciences 2: 174-177 Mihatsch WA. et al (2005) Hydrolysis of casein accelerates gastrointestinal transit via reduction of opioid receptor agonists released from casein in rats. Biology of the Neonate 87: 160-163 Martindale: The complete drug reference (2007) Pharmaceutical Press

36. levels of endorphin fragments in CSF samples. Gillberg C. et al (1985) Endorphin activity in childhood psychosis. Spinal fluid levels in 24 cases. Archives of General Psychiatry 42: 780-783 in plasma samples and family members - broader phenotype? Leboyer M. et al (1999) Whole blood serotonin and plasma beta-endorphin in autistic probands and their first-degree relatives. Biological Psychiatry 45: 158-163 in blood of neonates who went on to develop ASD*. * Still under investigation following replication of these findings (Nelson et al, 2006) Nelson K. et al (2001) Neuropeptides and neurotrophins in neonatal blood of children with autism or mental retardation. Annals of Neurology 49: 597-606 Clinical use of opioid antagonists (e.g. Naloxone / Naltrexone)**. ** Low dose naltrexone (LDN) also potentially effective for the treatment of inflammatory bowel disease (Crohns disease) Elchaar GM. et al (2006) Efficacy and safety of naltrexone use in pediatric patients with autistic disorder. Annals of Pharmacotherapy 40: 1086-1095 Opiate-related findings in ASD

37. Autism as a metabolic disorder? Body – Brain – Mind – Behaviour GI tract & brain derived from the same embryonic tissue. Initial indications of GI permeability (40%). Walker-Smith J, Andrews J. (1972) Alpha-1-antitrypsin, autism & coeliac disease. Lancet 7782: 883-884 DEufemia P. et al (1995) Abnormal intestinal permeability in children with autism. Acta Paediatrica 85:1076-1079 transport of peptides (& other material) across to the CNS. Gardner ML. (1988) Gastrointestinal absorption of intact proteins. Annual Review of Nutrition 8: 329-350 Indications of abnormal GI conditions (bacteria, enzymes, etc). Finegold SM. et al (2002) Gastrointestinal microflora studies in late-onset autism. Clinical Infectious Diseases 35: S6-S16 Parracho H. et al (2005) Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children. Journal of Medical Microbiology 54: 987-991 Amelioration of some symptoms by physical therapy. Shattock P, Whiteley P. (2002) Biochemical aspects in autism spectrum disorders: updating the opioid-excess theory and presenting new opportunities for biomedical intervention. Expert Opinion on Therapeutic Targets 6:175-183

38. Conclusions Exclusion diets may be helpful in ameliorating some of the core and/or secondary symptoms of PDD for some people. Balancing a constraining intervention with quality of life. Further data/research required on long-term safety. Theoretical basis to the diets is still under investigation although may include a GI element. Requirement for clinical support when using diets. Evidence for the use of diet as good as most other interventions (specialised education/behavioural plans).

39. Acknowledgments: ScanBrit partners: Dr. Demetrious Haracopos 1 Prof. Ann-Mari Knivsberg 2 Dr. Kalle (Tiny) Reichelt 3 Dr. Judith Jacobsen 4 Dr. Anders Seim Dr. Lennart Pedersen 1 Paul Shattock 5 Sarah Parlar-Lorentzen 1 Maja Schondel 1 Maureen Pilvang 1 Jonna Deibjerg 1 Charlotte Mathiesen 1 Prof. Stefan Samuelsson 6 Partner affiliations: 1 The Center for Autisme, Herlev, Denmark 2 National Centre for Reading Education & Research, University of Stavanger 3 Faculty of Medicine, University of Oslo 4 Statcon ApS 5 Faculty of Applied Sciences, University of Sunderland 6 Department of Behavioural Sciences, Linköping University Thanks also to our study funders: The Center for Autisme The Nils O. Seim Family Fund for Medical Research The Eric Birger Christensen Fond The Norwegian Protein Intolerance Association The Robert Luff Foundation A huge thank you to a very important group: The families and children who participated in the study.

40. As we know, there are known knowns. There are things we know we know. We also know there are known unknowns. That is to say we know there are some things we do not know. But there are also unknown unknowns, the ones we don't know we don't know" Donald Rumsfeld, February 12 th 2002 http://osiris.sunderland.ac.uk/autism The final word on not knowing: Paul Whiteley, Research Fellow Autism Research Unit Department of Pharmacy, Health & Wellbeing Faculty of Applied Sciences, University of Sunderland, UK