Despite extensive research, the etiology and natural history

Relationship of Dietary Intake to Gastrointestinal Symptoms in Children with Autistic Spectrum Disorders Susan E. Levy, Margaret C. Souders, Richard F. Ittenbach, Ellen Giarelli, Andrew E. Mulberg, and Jennifer A. Pinto-Martin Background: Gastrointestinal (GI) symptoms and abnormalities in stool consistency are frequently reported by parents of children with autism spectrum disorders (ASD). The purpose of this study was to 1) describe dietary intake of a cohort of children with ASD compared with normative data and 2) determine whether GI symptoms and stool consistency are related to dietary intake. Methods: Data from diet diaries of children (3 8 years) with ASD (n 62) were analyzed by a registered pediatric dietician to compare to RDA standards for total calories, protein, carbohydrate, and fat. Dietary intake was correlated with descriptors of stool consistency using cumulative logistic regression methods. Results: Intake of calories, carbohydrates, and fat were in the average range; protein intake was increased (211% of RDA). Reported frequency of GI abnormalities, including abnormal stool consistency (e.g., bulky or loose), was increased (54%). No statistically significant relationships between stool consistency and dietary intake were observed. Conclusions: In this sample, there was a high rate of reported gastrointestinal symptoms, despite lack of medical causes. Intake was adequate for calories and carbohydrates and increased for protein. The children did not exhibit excessive carbohydrate intake. There was no association of nutrient intake to changes in stool consistency. Key Words: Autism, autism spectrum disorders (ASD), gastrointestinal (GI), pervasive developmental disorder not otherwise specified (PDD-NOS), recommended dietary allowance (RDA) Despite extensive research, the etiology and natural history of autism spectrum disorders (ASD) remains poorly understood. It is clear that this collection of disorders, characterized by deficits in communication, social interaction, and behavior, has a large genetic component (Muhle et al 2004; reported in the early 1970s (Goodwin et al 1971). Types of reported GI symptoms include chronic diarrhea, excessive gas, Rapin and Katzman 1998), yet the etiology remains unclear. abdominal discomfort, abdominal distension (Horvath et al Many children with ASD have associated medical disorders or 1999), constipation (Quigley and Hurley 2000), and food intolerance (Wakefield et al 1998). Symptoms may be severe, as in symptoms (Barton and Volkmar 1998; Gillberg and Coleman 1996), including seizures (Tuchman 2000; Tuchman and Rapin constipation, which may lead to megacolon and requiring medical intervention (Afzal et al 2003), or they may involve subclin- 2002), immune system dysregulation (Hornig and Lipkin 2001; Sperner-Unterweger 2005), and gastrointestinal (GI) symptoms ical symptoms such as mild gastroesophageal reflux, which may (D Eufemia et al 1996; Goodwin et al 1971; Horvath et al 1999; only interfere with the volume or variety of oral intake. Roberts et al 2001). Described GI symptoms include diarrhea, Little data are available about the true prevalence of GI gastroesophageal reflux, constipation, abdominal pain, and others. dysfunction in an unselected population of children with ASD. The treatment of children with ASD is complex and timeintensive. Most experts agree that the first line of treatment Several population-based studies report rates of 9% 18% of GI symptoms among children with ASD (Black et al 2002; Fombonne and Chakrabarti 2001; Taylor et al 2002), but these studies should be a comprehensive, intensive program of educational intervention, developmental therapies, and behavioral treatment lacked uniform definitions for GI symptoms (Kuddo and Nelson (Council 2001; Hurth et al 1999; Rogers 1998). Because children with this disorder may show slow progress, many families have pursued alternative medical treatments, either as a substitute to or in conjunction with accepted treatments in an effort to promote recovery from the disorder. These treatments have From the Division of Child Development and Rehabilitation (SEL, MCS), Children s Seashore House of the Children s Hospital of Philadelphia; Biostatistics and Data Management Core (RFI), The Children s Hospital of Philadelphia; University of Pennsylvania School of Nursing (JAP-M); Division of Gastroenterology and Nutrition (AEM), The Children s Hospital of Philadelphia, Philadelphia, Pennsylvania. Address reprint requests to Susan E. Levy, M.D., The Children s Hospital of Philadelphia, 3405 Civic Center Boulevard, Philadelphia, PA 19104; e-mail: levys@email.chop.edu. Received March 17, 2006; revised June 19, 2006; accepted July 6, 2006. been adopted by parents of children who have widely varying phenotypes without scientific evidence of their appropriateness or efficacy. Because the reported frequency of GI symptoms is increased, treatments targeting the GI tract such as elimination diets (e.g., gluten- or casein-free diets) and use of supplements have been adopted to decrease frequency of symptoms or to regulate gastrointestinal function. Associations between autism and GI symptoms were first 2003). Although Molloy reported a frequency of 24% among children attending a specialized care program (Molloy and Manning-Courtney 2003), others have reported rates of GI symptoms as high as 46% 84% (Goodwin et al 1971; Horvath et al 1999; Wakefield et al 1998). This wide discrepancy of reported rates will need to be resolved by prospective studies of a representative sample of children with ASD and typical control children (Kuddo and Nelson 2003). Despite the lack of specific data about the frequency of GI abnormalities in children with ASD, descriptive data are available about feeding difficulties (including food and textural selectivity, food refusal, oral motor delays, and others) in children with ASD (Ahearn et al 2001; Erickson et al 2005; Field et al 2003; Schreck and Williams 2006; Schreck et al 2004). Feeding difficulties such as selectivity and poor oral intake may be significant, and result in or exacerbate GI symptoms such as constipation. These 0006-3223/07/$32.00 doi:10.1016/j.biopsych.2006.07.013 BIOL PSYCHIATRY 2007;61:492 497 2007 Society of Biological Psychiatry

S.E. Levy et al BIOL PSYCHIATRY 2007;61:492 497 493 3-day (Shearer et al 1982) diet diary. In a group of children n ( 26) referred to a nutrition support service because of feeding dysfunction, 24 of the children (92%) met or exceeded dietary reference values for energy and protein (Bowers 2002). The remaining two had severe food selectivity and did not meet dietary reference values. The serum amino acid patterns of a cohort of children with ASD were examined to compare sufficiency of essential amino acids in children consuming typical and restricted diets (Arnold et al 2003). Of the 36 subjects in this study, 10 consumed a gluten/casein restricted diet and 26 consumed a typical diet. Children consuming typical and restricted diets had frequent essential amino acid deficiencies suggestive of poor protein intake (Arnold et al 2003). The high frequency of reported GI symptoms (Horvath and changes. The exceeding of the absorptive capacity of the small intestine to handle concentrations of osmotically active solutes Perman 2002), concurrent timing of onset of symptoms of ASDwithin the lumen can yield significant increased water content of and GI difficulties (Wakefield et al 1998), and reports of abnormal findings of intestinal endoscopy in a population of childrenstool ph and consistency (Hammer et al 1989; Robayo-Tores et al the stool consistent with diarrhea and concomitant changes in with ASD (Wakefield et al 2002) led to the hypothesis that the GI 2006). pathology may be related to the etiology (White 2003). Similarities have been drawn between the association of celiac disease, a cohort of children with ASD in comparison to normative data The purpose of this study was 1) to describe dietary intake of an immune-mediated enteropathy that results in impaired intestinal permeability, and neurologic or psychiatric disorders are related to dietary intake. We hypothesized that an increased and 2) determine whether GI symptoms and stool consistency (Bushara 2005; Pynnonen et al 2002). The association betweenfrequency of loose stools would be related to abnormal intake of schizophrenia and celiac disease has been examined, and a recent review described an increased risk of developing celiac disease in individuals with schizophrenia (Kalaydjian et al 2006). Multiple clinical trials of treatment with gluten showed little improvement in psychiatric symptoms but suffered from methodologic flaws. Gluten sensitivity has been suggested to contribute to the etiology, but it is not clear whether it is an epiphenomenon (Bushara 2005). A similar connection was postulated with autism that abnormal intestinal permeability in children with ASD results in the absorption of peptides which are the products of incomplete breakdown of proteins such as gluten or casein (D Eufemia et al 1996; Knivsber et al 2001; Levy and reports include data of children referred to feeding programs lymphonodular hyperplasia and microscopically determined enterocolitis of in pediatric patients with autism. Assessment of proin- (Ahearn et al 2001; Field et al 2003) or from a database previous research on feeding problems for children with autism flammatory cytokines, interleukin (IL)-6, IL-8, and IL-1beta has (Schreck and Williams 2006), so findings are not yet generaliz-noable to the broader population of children with ASD. developmental disorders and age-matched control subjects. In revealed abnormalities between patients with pervasive Two controlled studies described nutritional intake of children with ASD, reporting adequate (or higher) intake of calories with pervasive developmental disorders than in age-matched general, intestinal levels of IL-6 and IL-8 were lower in patients and nutrients using either a 7-day (Raiten and Massaro 1986) or control subjects. These data fail to support an association between autism and GI inflammation (DeFelice et al 2003). The causality of changes in stool consistency and appearance reported by parents remains unclear and unsubstantiated and potentially related to the intake of carbohydrates or other macronutrients. Disaccharide digestion is normally associated with absorption in the small intestine. When carbohydrates are malabsorbed, the osmotic load produced by the high amount of low molecular weight sugars and partially digested starches in the small intestine can cause abdominal distension, rapid peristalsis, and diarrhea. Sugar intolerance is the presence of abdominal symptoms related to the malabsorption of carbohydrates and subsequent conversion to osmotically active colonic secretory carbohydrates. Methods and Materials This study was part of a larger double-blind, placebo-controlled crossover trial that examined the effect of human synthetic secretin (ChiRhoClin, Silver Spring, Maryland) on behavior in children with ASDs (Levy et al 2003). All parents were informed of the study protocol by the nurse coordinator, and written informed consent was obtained. The Institutional Review Board of The Children s Hospital of Philadelphia approved the study. Hyman 2005; Millward et al 2004; Reichelt et al 1990; Whitely et In the earlier study, children were recruited from a sample of al 1999). These peptide fragments are presumed to act as170 families who volunteered for the secretin trial. Many of the endogenous opioids, crossing the blood brain barrier, resulting families believed that secretin administration would ameliorate in the symptoms or profile of ASD. This opioid theory explains the symptoms of autism and improve GI function. The subjects the potential mechanism of pathophysiology and effect for the (n 62) were randomly assigned to one of two treatment gluten free/casein free (GF/CF) diet (Le Couteur et al 1988; groups, treatment with secretin or placebo. Diagnosis of ASD Panksepp 1979; Reichelt et al 1990; Shattock et al 1990; Shattockwas confirmed by examination of DSM-IV-TR criteria (American and Lowdon 1991; Shattock and Whiteley 2002). Parents andpsychiatric Association 2000) by an experienced clinician, administration clinicians have assumed that increased intestinal permeability of the Autism Diagnostic Interview Revised allows these by-products to move easily across the intestinal basement membrane and exert an effect on the central nervous (ADI-R; Lord et al 1994) to parents or caregivers, and administration of the Childhood Autism Rating Scale (CARS; Schopler et system (Liu et al 2005). Yet the only supporting evidence for al 1988). Group 1 received saline placebo then crossed over at 6 increased intestinal permeability in patients with autism was weeks to receive a single intravenous dose (2 CU/kg to a reported in a small sample of children with autism (D Eufemia et maximum dose of 75 CU) of human synthetic secretin. Group 2 al 1996). Studies of other causes of impaired permeability, suchreceived human synthetic secretin then crossed over and received saline placebo. Before randomization to group member- as ileal lymphoid hyperplasia and an abnormal immune response to GI trace (with abnormal cytokines), have shown inconsistent ship, baseline laboratory data were collected as follows: complete blood count, metabolic panel, celiac panel (IgG, IgA results (Erickson et al 2005). The putative relationship between autism and GI immune anti-gliadin, IgA anti-endomysial antibodies, and serum transglutaminase), and stool analysis (ph measurement, bacterial dysregulation may be related to the macroscopically observed culture,

494 BIOL PSYCHIATRY 2007;61:492 497 S.E. Levy et al culture for ova and parasites, and measurement of reducing substances). These laboratory studies were completed to determine whether alternative medical conditions (such as inflammation or infection) existed to explain findings of abnormal GI permeability or malabsorption. Data Collection Data were collected using parent-completed reports of symptoms, biochemical analyses, and physical measurements. Each child s parents or caregivers completed a baseline 3-day diet record and provided stool samples for reducing substances, bacterial culture, ova and parasites, and ph measurement. A physical examination with measurement of growth parameters was completed at each visit. Height and weight were plotted on National Center for Health Statistics (in collaboration with the National Center for Chronic Disease Prevention and Health Promotion, www.cdc.gov/growthcharts, 2000) standard graphs providing a percentile for each measurement according to age and sex. Parents were asked to rate their child in the areas of GI symptoms, including consistency and volume of stools, presence of abdominal pain, emesis, and food selectivity. Parents completed a descriptive daily log of GI symptoms of abdominal pain or emesis and stool output for the 12-week duration of the secretin study (Levy et al 2003). The nurse research coordinator instructed parents how to complete the daily GI log and stool output records. Parents were given a choice of three descriptions of stool consistency to describe their child s stool output: loose, bulky, or solid. The nurse coordinator provided definitions of stool consistency to provide a reliable way for parents to describe their child s stool output. A solid stool was defined as a stool that was formed in a log and if cut with a knife would retain its shape. A bulky stool was defined as soft, squishy and could have lumps. A loose stool was thin, spreadable and could be slimy but did not have visible water loss. Parents were also instructed that the definition of diarrhea was a watery stool. Families recorded all food and fluid intake for 3 days. Three-day diet diaries have been shown to have reasonable accuracy (Buzzard 1998) to allow us to examine the relationship between dietary intake and GI symptoms, because it was possible that administration of secretin would alter GI symptoms. Data Analysis Descriptive statistics were computed for all variables in the data set as well as for three groups of children classified by their stool consistency as loose, bulky. or solid on the basis of the research team s evaluation of specimens. Indices of nutritional status for all children in our sample were computed for percent calories, percent carbohydrates, percent fat, and percent protein and then compared with population norms using a series of four one-sample t tests, one for each type of nutrient. In addition, nonparametric measures of association were computed between stool consistency and each of the four aforementioned nutritional status variables using the Spearman s rho rank-order coefficient. Because of the exploratory nature of this study, no attempts were made to adjust alpha beyond the traditional.05 level. All data were analyzed using SAS 9.0 (SAS Institute, Cary, North Carolina). With the assistance of a computerized program, a registered dietician analyzed the 3-day diet diaries completed by parents, converting the results to a percentage of the RDA (Recommended Dietary Allowance; National Academy of Sciences 1989) intake for calories, protein, carbohydrate and fat. The weekly stool diary was analyzed for frequency of abnormal stool consistency, grouping into three broad categories of stool consistency: bulky (excessively large), loose, or solid. A research assistant coded each stool diary for consistency. The types of daily stool consistency were recorded and categorized into one of the descriptive categories described. If there was variability of the stool consistency type, the category representing 75% of the time of data collection was selected. Results Records of 62 subjects who participated in the secretin trial (Levy et al 2003) were analyzed. All subjects had growth parameters, 52 subjects had complete diet histories, and 59 subjects had complete stool diaries. Subjects ranged from 44 to 104 months old, with a mean age of 73 months. The sample included 50 boys (89.6%) and was 90% Caucasian, 5% African American, and 5% Latino. No subjects had weight measurements below the 5th percentile. Six subjects (9.7%) had weight measurements over the 95th percentile. Laboratory markers for celiac disease (gliadin IgA, gliadin IgG, tissue transglutaminase, and endomyseal antibodies) were within normal range for all subjects. There were no positive bacterial stool cultures or samples for ova and parasites. Stool ph was 5.5 or higher for all but two samples. Two patients had ph measurements of 4.5 and 5.0, and both had normal stool consistency. No stool samples had positive reducing substances. Thus, other potential medical causes of GI symptoms were ruled out. Dietary Intake Patterns The investigators analyzed the available dietary histories (Table 1). Average recorded daily fluid intake was 978 ml (range 398 1908 ml). The dietary analysis reflected average percentage of RDA for calories, protein, carbohydrate, and fat intake consumed over a 3-day period. As noted, mean values for calories, carbohydrates, and fats were in the average range (95% 101%). Protein intake was high with mean RDA of 211% (range 67% 436%). The source of calories for nutritional intake averaged 11% protein, 59% carbohydrate, and 30% fat for subjects as a group. Two subjects with high carbohydrate intake contributing to calories ( 80%) had lower caloric intake and a history of food selectivity. Subjects were compared with normative RDA values for percent intake of calories, carbohydrates, fat, and protein. In each case, children with ASD consumed a significantly higher percentage of nutrients than RDA values (p.001) when using 77% of the RDA intake as the normative standard. The RDA is set at an intake level that exceeds the requirement of 97% 98% of individuals. Using 77% of the RDA as a cutoff value provides a conservative estimate of nutrient inadequacy based on a coefficient of variation for the nutrient of 15% about the Estimated Average Requirement (EAR; Gibson 2005). Table 1. Three-Day Diet Analysis Mean % RDA (SD) No. Below 80% RDA No. Above 120% RDA Calories 95 (25) 16 (31%) 9 (17%) Protein 211 (80) 2 (4%) 40 a (77%) Carbohydrates 100 (33) 12 (24%) 23 (43%) Fats 97 (32) 16 (31%) 7 (13%) n 52 (10 missing diet diaries). RDA, Recommended Dietary Allowance. a Twenty-seven subjects had RDA for protein 200%.

S.E. Levy et al BIOL PSYCHIATRY 2007;61:492 497 495 Table 2. Stool Consistency Categories Categories n % Bulky Stools 9 15 Loose or Mushy 17 29 Solid 33 56 N 59, three missing stool diaries. Frequency of Gastrointestinal Symptoms and Relationship to Dietary Intake We tracked frequency of GI symptoms in the subjects as part of the clinical trial (Levy et al 2003). Average daily stool frequency was 1 stool/day (range 0 4). The overall frequency of GI complaints (including abdominal pain, constipation, food selectivity, and stool abnormalities) was high with 32 of 59 subjects (54%) reporting symptoms. One child experienced abdominal pain within 24 hours of administration of secretin infusion but had no subsequent complaint of abdominal pain. No other parents reported symptoms of abdominal pain or emesis during the treatment trial. Other past histories included two patients with constipation (grouped in the solid stool category) and two subjects who had a history of severe food selectivity or refusal. The two subjects with food selectivity had low caloric intake reported (52% and 62% of RDA) but both sustained growth in the normal range. The stool consistency of children in our sample was grouped into three categories: bulky (large and soft stools), loose, and solid stools (Table 2). Comparisons were made between the subjects in the three groups of stool consistency and their nutritional intake of calories and nutrients of interest (including carbohydrates, fats, and proteins). Forty-four percent (n 26) of subjects with complete stool diaries (n 59) experienced abnormal stool patterns. The relationship between nutritional intake and stool consistency was positive but low for three of the measures: r.27 (% calories), r.26 (% carbohydrates), and r.28 (% protein). In the case of % fat, the relationship was very low (r.12). Even in the case of the % protein, for which the relationship was strongest, r 2 was only 8%, suggesting little shared variability between the two measures. Conversely, the coefficient of determination for % fat was r 2.01, suggesting that only 1% of the variability in stool consistency is accounted for by % fat. Discussion There is anecdotal clinical evidence, reports from families, and some support in the literature of increased GI symptoms in particular, loose stools among children with ASD. From discussion with clinical colleagues with expertise in pediatric gastroenterology, the working theory was that children with ASD often had selective diets, with a predominance of carbohydrates (hence the term great white diet ) and that this resulted in diarrhea because of the increased osmotic load (Hammer et al 1989). Whether the changes in stool appearance frequently reported by parents of children with ASD were related to aberrations of dietary intake (i.e., specific macronutrient intake) was the focus of this study. The cohort of children for this study consumed average amounts of calories, fats, and carbohydrates, with high protein compared with the expected RDA. Their carbohydrate intake was in the upper range but was generally within normal limits (only 4 of 52 subjects had levels over 150% of the RDA). This contradicts anecdotal reports of high carbohydrate consumption among children with ASD and supports other reports of adequate nutritional intake. Our data did not support the presumption that increased frequency of loose stools is related to a diet high in carbohydrates. In fact, when considering the single measures of association, our cohort showed a trend of high carbohydrate intake associated with firmer stools, not loose (p.06). A limitation of this study was the use of a convenience sample. In addition, dietary data collection was based on parental report for 3 days. This may have not been a sufficiently long period to collect data that accurately reflect the association between diet and symptomatology. For school-age children, the diet history was completed by the child s school teachers for nutritional intake during the period. Items were often recorded without the exact serving size of protein for example, one hamburger or one grilled cheese sandwich. This may overestimate protein content of lunchtime meals. Concurrent measurement of serum amino acids, to examine essential amino acids in particular, would have allowed for further exploration of the adequacy of protein intake. Not all parents were compliant with completion of the diet diaries (n 52 supplied diaries) or stool diaries (n 59 supplied diaries), which decreased the size of the sample for analysis (Tables 1 and 2). Although the reduced sample size does increase the potential for a Type II error, this study is a secondary analysis of data drawn from a randomized, double-blinded, crossover clinical trial on the use of secretin that was sufficiently powered for its primary endpoint. The secondary analysis of data presented here is primarily a descriptive study of the relationship between dietary intake and GI symptoms. Yet even for the one set of inferential tests conducted here (four one-sample t tests), the current sample size of n 52 is sufficiently large enough to detect a.5 difference between groups under the current alpha, well below the differences observed here. In this particular sample, GI symptoms were common (54%). Subjects were recruited from the Delaware Valley area and invited to participate in a randomized crossover trial investigating the efficacy of synthetic human secretin. In 1999, many parents were under the impression that children with ASD and GI problems might benefit from secretin administration, and this might have influenced families participation in the study. As a result, the study sample might include an overrepresentation of children with GI abnormalities. Conclusions This study describes increased frequency of GI symptoms in a well-characterized cohort of children diagnosed with ASD. Results suggest that GI symptoms are not significantly related to abnormal patterns of dietary intake of macronutrients specifically, carbohydrates, proteins, or fats. The GI symptom of abnormal stool pattern such as loose stools or bulky volume is unlikely due to other factors that include an intrinsic, as yet uncharacterized GI disease specific to autism spectrum disorder, because none of the children in our study had positive laboratory evidence for celiac disease, infectious gastroenteritis, or symptoms of malabsorption. The cohort of children had adequate caloric intake, did not demonstrate excessive carbohydrate intake, and had high protein content in their diet. We suggest that further study is needed to describe the relationship between nutritional intake and GI symptoms, determine nutritional risk factors for children with ASD and selective diets, and determine the etiology of GI dysfunction in children with ASD. This study was carried out with support from the Maternal and Child Health Bureau (Grant No. 2T73 MC 00035 09), the

496 BIOL PSYCHIATRY 2007;61:492 497 S.E. Levy et al General Clinical Research Center (GCRC) of the Children s Hospital of Philadelphia (NIH Grant No. RR00240), Mental Retardation and Development Disabilities Research Center (MRDDRC; NIH Grant Number 3P30 HD26979-04S2), and Centers for Disease Control Grant No. 541247. The authors thank the GCRC nursing staff, Denise DePaul, RN, and Ellyn Rebecca, RN; GCRC medical assistant, Elizabeth Sanchez; and Amy Schwartz for assistance with data entry. The authors thank the ChiRhoClin Corporation for donating the human synthetic secretin used for this study. Most important, we thank the families who allowed their children to participate in this clinical investigation. Afzal N, Murch S, Thirrupathy K, Berger L, Fagbemi A, Heuschkel R (2003): Constipation with acquired megarectum in children with autism. Pediatrics 112:939 942. 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