The safe threshold for gluten contamination in gluten-free products. Can trace amounts be accepted in the treatment of coeliac disease?

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1 Aliment Pharmacol Ther 2004; 19: doi: /j x The safe threshold for gluten contamination in gluten-free products. Can trace amounts be accepted in the treatment of coeliac disease? P. COLLIN*, L. THORELL, K. KAUKINEN* & M. MÄKIà *Department of Medicine, Tampere University Hospital, and Medical School, University of Tampere, Tampere, Finland; Arla Foods, Innovation Centre, Stockholm, Sweden; àdepartment of Paediatrics, Tampere University Hospital, and Medical School, University of Tampere, Tampere, Finland Accepted for publication 19 March 2004 SUMMARY Background: Gluten contamination in gluten-free products cannot totally be avoided. The safe threshold for gluten remains obscure. Aim: The purpose was to estimate a reasonable limit for residual gluten, based on current literature and measurement of gluten in gluten-free products on the market. Methods: The gluten content of 59 naturally gluten-free and 24 wheat starch-based gluten-free products were analysed by enzyme-linked immunosorbent assay. The daily intake of flours was calculated in 76 adults on gluten-free diet, and the intake compared with mucosal histology. Results: A number of both naturally gluten-free (13 of 59) and wheat starch-based gluten-free (11 of 24) products contained gluten from 20 to 200 ppm (¼mg/ kg). The median daily flour consumption was 80 g (range: ). Within these limits, the long-term mucosal recovery was good. Conclusions: The threshold for gluten-contamination can safely be set at 100 ppm. Provided that the daily flour intake is even 300 g, a level of 100 ppm results in 30 mg of gluten intake. This has been shown to be safe, when correlated to histology, in clinical and challenge studies. The level can be achieved by the industry, and does not make the diet too cumbersome. INTRODUCTION Coeliac disease is characterized by small intestinal inflammation, villous atrophy and crypt hyperplasia after ingestion of gluten in genetically susceptible patients. The mucosal lesion recovers when the gluten-containing cereals wheat, rye and barley are withdrawn. A lifelong gluten-free diet is the only effective treatment of the disorder. An unanimous view is that gluten-free dieting should be as strict as possible. However, a diet completely devoid of gluten would be difficult if not impossible to Correspondence to: Dr P. Collin, Medical School, FIN University of Tampere, Tampere, Finland. pekka.collin@uta.fi maintain. The products on the market may contain trace amounts contaminating gluten. The current standards of the Codex Alimentary Commission for a gluten-free diet go back to As at that time no sensitive and specific methods for gluten determination were available, a threshold value of 0.05 g nitrogen per 100 g dry matter was set for wheat starch. This limit is valid for wheat starch only, as many other gluten-free products contain endogenous proteins, which invalidate the nitrogen measurement. In the Codex Alimentarius Commission, there is an ongoing discussion regarding the safe threshold for residual gluten in gluten-free products, an issue which evidently needs to be considered. 2, 3 One approach to the problem has been to carry out a gluten challenge and demonstrate how much gluten has to be taken before intestinal mucosal Ó 2004 Blackwell Publishing Ltd 1277

2 1278 P. COLLIN et al. inflammation and villous shortening ensue. However, such mainly short-term studies have yielded somewhat divergent results, 4 7 and they have not been randomized. A second concern is that the histological responses to gluten withdrawal or challenge are extremely variable in patients with coeliac disease. Small intestinal mucosal recovery on gluten-free diet may take from a couple of months to many years. 8, 9 Similarly, the intestinal mucosal deterioration after gluten challenge occurs in some patients within a few weeks, whereas in some more than 10 years normal diet is required for the mucosal relapse. 10 Further, it is possible that the minor inflammatory response after inadvertent gluten ingestion such as inevitably occurs daily in most coeliac disease patients is not harmful at all. The question of tolerable amounts of gluten in coeliac disease can also be settled in the context of everyday clinical practice. It is logical to assume that such gluten contamination, which has taken place for many years along with successful treatment of coeliac disease, would be safe enough. In other words, provided that we can show products containing a given trace amount of gluten contamination to be safe in long-term use, we can allow such contamination. Wheat starch-derived gluten-free products may, after purification, still contain trace amounts of gluten. They have been allowed for decades for coeliac disease patients in Finland, Sweden and the United Kingdom, and complete intestinal mucosal recovery has been evident both in children and adults. 9, 11 Dietary compliance has also been good 9 and the rates of coeliac complications such as malignant development are low in patients adhering to such a diet. 12 The first randomized study comparing wheat starch-based gluten-free to naturally gluten-free products showed that the morphological and clinical responses were equally good by both treatments. 13 Recent research in the matter of determining trace amounts of gluten have focused on development of immunological methods with high sensitivity and specificity. Until recently, there has been no commercially available enzyme-linked immunosorbent assay (ELISA) for gluten determination which meets all the requirements for a suitable method. 14 However, a promising new ELISA has been developed 15 and made commercially available. The monoclonal antibody used recognizes the pentapeptide QQPFP, and homologous sequences that occur repetitively in the prolamins from wheat, rye and barley. 15 Furthermore, this system has been tested in a collaborative trial, 16 and is proposed by the Working Group on Prolamin Analysis and Toxicity for further evaluation by the Codex Committee on Methods of Analysis and Sampling. 2 In connection with the clinical data available, there is now evidence to provide recommendations as to the safe threshold for residual gluten in gluten-free products. PATIENTS AND METHODS Food samples The gluten content was determined in the most widely used naturally gluten-free and wheat starch-based gluten-free flours and baked products. Twenty-seven naturally gluten-free and nine wheat starch-based flours available on the market were collected in the year 2000, and again 32 (13 baked) and 15 (seven) products in Sandwich enzyme-linked immunosorbent assay Gluten content was determined with a recently developed ELISA, 15 available as a kit (RIDASCREEN Gliadin, Art. No. R7001) produced by R-Biopharm AG, Darmstadt, Germany. The monoclonal secalin antibody R5 used in the test detects gliadin fractions of wheat and corresponding prolamins from rye and barley. Prolamins from oats, maize and rice are not detected. Furthermore, the antibody has no crossreactivity with glutenin. Heat-processed samples were extracted with a cocktail solution (provided with the kit) by the procedure described in the leaflet. All other samples were extracted with 60% ethanol solution. The detection limit used in this study was 10 ppm gluten; the interassay coefficient of variation was 9.8%. This coefficient of variation is close to what has been published by others (8.7%). 15 A content of 10 ppm gluten corresponds to 10 mg gluten/1 kg product and roughly to 5 mg gliadin/1 kg. The daily use of gluten-free flours and the effect of the diet Apart from knowledge of the trace amounts of gluten in gluten-free products, the amount of these products consumed daily must be taken into account. Daily use was therefore estimated from 4-day food records in 76 consecutive adults and 16 children with coeliac disease

3 GLUTEN IN GLUTEN- FREE PRODUCTS 1279 adhering to a strict gluten-free diet for 1 10 years (median 2 years); 28 adults were taking naturally gluten-free, 48 adults and all children wheat starchbased gluten-free products; the diets did not include oats. The daily intake of flour was compared with the small intestinal mucosal histology and antiendomysial antibodies. Mucosal morphology was evaluated blindly from well-orientated small intestinal biopsy samples by measuring the villous height/crypt depth ratio (Vh/CrD). The density of intraepithelial CD3+ lymphocytes was determined as cells per millimetre epithelium, as previously described. 9 Serum immunoglobulin A (IgA) class endomysial antibodies were determined by an indirect immunofluorescence method using human umbilical cord as antigen; 17 a dilution of 1: 5 was considered positive. Statistical analysis The correlation between the daily intake of flours and the small intestinal histology was estimated by twotailed Pearson correlation test. Ethics The study protocol was accepted by the Ethical Committee of Tampere University Hospital. RESULTS Residual gluten in gluten-free products Trace amounts of gluten were found in both naturally gluten-free and wheat starch-derived flours, 42 of 59 and two of 24 were free of gluten contamination (<10 ppm), respectively. Five naturally gluten-free and two wheat starch-based products contained more than 100 ppm gluten; none exceeded 200 ppm (Figure 1). The daily use of flours and clinical outcome Figure 1. Gluten contamination in naturally gluten-free and wheat starch-based gluten-free products measured by enzymelinked immunosorbent assay (ELISA). 15 Gluten-free flours are indicated by closed and gluten-free baked products by open squares. The median daily use of flours was 80 g (range: ) in adults. The correlation between the use of flours and intestinal mucosal histology in adults is depicted in Figure 2. No significant correlation was seen. One patient adhering to a naturally gluten-free and one to a wheat starch-based gluten-free diet proved positive for endomysial antibodies after 1 year on a gluten-free diet, the titres being 1:5 and 1:50, respectively. In 16 children, the mean daily use of flours was 60 g (range: ); they all had normal small bowel morphology at follow-up. DISCUSSION Evidently a gluten-free diet completely devoid of gluten is unrealistic. Coeliac disease patients are exposed to products containing trace amounts of gluten, even when the products are sold as naturally gluten-free (Figure 1). In order to estimate the safe and rational threshold for daily gluten intake, the amount of residual gluten in gluten-free products and the total intake of these products must be considered. Provided that we can demonstrate that the use of a variety of gluten-free products results in both clinical and histological recovery, we can assume that the gluten level in these products is acceptable, notwithstanding the fact that the sensitivity to gluten is individual. Studies on possible toxic or harmful effects of dietary transgressions or gluten challenge are summarized in Table 1. The study designs and the duration and the amount of gluten load have been highly variable. Gluten challenge studies with <10 mg have evidently not shown any effect, whereas some effect on the histology can be observed when the daily intake has been 500 mg or more. Catassi et al. 6 reported that

4 1280 P. COLLIN et al. (a) (b) Villous height /crypt depth ratio Villous height /crypt depth ratio Consumption of wheat starch-based gluten-free flours Consumption of naturally gluten-free flours (c) (d) CD3 + intraepithelial lymphocytes (cells/mm) CD3 + intraepithelial lymphocytes (cells/mm) Consumption of wheat starch-based gluten-free flours Consumption of naturally gluten-free flours Figure 2. The estimated daily ingestion of flours in 76 coeliac adults on gluten-free diet, and its correlation with small intestinal villous structure (a ¼ wheat starch-based, b ¼ naturally gluten-free diet) and intraepithelial CD3+ lymphocytosis (c ¼ wheat starch-based, d ¼ naturally gluten-free diet). No significant correlations were seen: correlation coefficient (a) )0.21, P ¼ 0.15; (b) )0.20, P ¼ 0.31; (c) )0.22, P ¼ 0.13; (d) )0.03, P ¼ Short-term treated (1 1.2 years) patients are illustrated with open and long-term (2 10 years) treated with closed squares. challenge with 100 mg of daily gliadin (corresponding to 200 mg gluten) resulted in a decrease in mean Vh/ CrD from 1.5 to 1.3 and an increase in intraepithelial lymphocytes from 11/100 enterocytes to 19/100, the respective values by 500 mg challenge being from 1.6 to 1.1, and from 10/100 to 25/100. The changes were altogether marginal and notably, the villous recovery had initially not been as good as in well-treated children in general (Vh/CrD: ). 9 Nevertheless, these earlier papers indicate that the safe threshold should

5 GLUTEN IN GLUTEN- FREE PRODUCTS 1281 Table 1. Small-bowel biopsy findings in coeliac patients taking small amounts of gluten Author n Age group Consumption of gluten Symptoms Abnormal findings in small-bowel biopsy Ciclitira et al. 4 1 Adults 10 mg gliadin (4 h) No mg gliadin (4 h) SVA and increase in IELs Ciclitira et al Adults mg gliadin daily (1 week)* No Ciclitira et al Adults mg gliadin daily (6 weeks)* Diarrhoea in four No Montgomery et al. 7 8 Adults g gluten daily, median (6 months) Increase in IELs Catassi et al Children 100 mg gliadin daily (4 weeks) Asymptomatic Dose-dependent decrease in Vh/CrD, increase in IELs mg gliadin daily (4 weeks) Anorexia and pale stools in three Sturgess et al Adults 1 g gliadin (4 h) Decrease in Vh/CrD in 3/4, increase in IELs in all Srinivasan et al Adults 500 mg gluten daily (6 weeks) Decrease in Vh/CrD in two Laurin et al Children 0.2 g gluten (39 weeks) Symptoms in 74% Increase in IELs within 4 weeks g gluten (5 weeks) No changes g gluten daily (5 51 weeks) Decrease in Vh/CrD and increase in IELs Vh/CrD, villous height and crypt depth ratio; PVA, partial villous atrophy, SVA, subtotal villous atrophy; IELs, intraepithelial lymphocytes. * Gliadin content estimated from the wheat starch-based gluten-free products. clearly be set below 500 mg of daily gluten, but could well be more than 10 mg. The current study verifies that most wheat starchbased gluten-free products contain trace amounts of gluten (Figure 1). More than 90% of Finnish coeliac disease patients consume these products. Dietary compliance, even long-term, has been good, as more than 85% are adhering to a strict diet. Complete mucosal recovery has been evident in both children and adults, 9 and even better 18 than in some other series, 19 probably because of the good compliance. The quality of life has been as good as in non-coeliac controls even after longterm treatment. 20 Further, the incidence of small 12, 21 intestinal lymphoma has been lower in patients on a long-standing strict gluten-free diet including wheat starch for a long period than in some other reports. 22,23 In earlier studies, only occasional abdominal complaints but no intestinal mucosal deterioration have been reported after taking wheat starch-based glutenfree products in an open study design (Table 2). We may therefore conclude that these products are safe in clinical practice. This was also verified in a prospective controlled study where no differences in histology, serology or quality of life were seen between wheat starch-based and naturally gluten-free products. 13 Table 3 shows how the daily amount of gluten load depends on the gluten level and the intake of flours. In the present study, we showed that coeliac disease patients have been using products containing ppm gluten or even more. The daily amount of flours has mostly been <100 g (Figure 2). Provided that the threshold for gluten content is set at 100 ppm and the daily intake is 300 g, the gluten amount does not exceed 30 mg. Such a gluten load has been shown to be safe in both clinical practice and challenge studies (Tables 1 and 2). The assay used here is new, and recognizes only one epitope found in wheat, rye and barley, but not in oats and other cereals. This is a promising method to solve problems in gluten analysis of food products. 16 The results of this study, in accord with those in previous publications, suggest that the acceptable threshold for gluten content in gluten-free products can be from the clinical point of view set at 100 ppm (¼mg/kg). This ensures a wide variety of gluten-free products and does not jeopardize the treatment of coeliac disease with inconvenient restrictions. ACKNOWLEDGEMENTS The present study and Coeliac Disease Study Group is supported by grants from the Medical Research Fund of Tampere University Hospital, the Foundation of the

6 1282 P. COLLIN et al. Table 2. Studies on the effects of wheat starch-based gluten-free diet in coeliac disease and dermatitis herpetiformis Author Study design Number of patients with wheat starch diet (all patients) Patient group Duration of wheat starch intake Outcome methods Untoward effects of wheat starch-based gluten-free diet Ciclitira et al. 24 Open challenge 7 (7) Adults 1 week Small-bowel biopsy No Ciclitira et al. 25 Open challenge 10 (10) Adults 6 weeks Small-bowel biopsy Cr-EDTA excretion Abdominal symptoms in some Ejderhamn et al. 11 Cross-sectional 11 (11) Adolescents 10 years on Small-bowel biopsy No average Chartrand et al. 29 Open challenge 17 (31) Children months AGA, EmA Symptoms in 11/17 and adults Kaukinen et al. 9 Cross-sectional 40 (52) Children 8 years on Small-bowel biopsy, No and adults average AGA, EmA, ARA Selby et al. 30 Cross-sectional 39 (89) Adults years Small-bowel biopsy, AGA, EmA No differences from naturally gluten-free diet Lohiniemi et al. 20 Cross-sectional 48 (53) Adults 9 11 years Quality of life, small-bowel biopsy to 23 No Peräaho et al. 13 Prospective, randomized 20 (40) Adults 1 year Small-bowel biopsy, AGA, EmA, BMD, quality of life No differences from naturally gluten-free diet Cr-EDTA, crept-ethylenediaminetetraacetic acid; AGA, antigliadin antibodies; EmA, antiendomysial antibodies; ARA, antireticulin antibodies; BMD, bone mineral density. Table 3. Estimated amount of daily ingested gluten from different amounts of gluten-free foods with varying gluten contamination. Six slices of bread correspond to approximately 100 g baking mix Gluten content in products, ppm (¼mg/kg) Friends of the University Children s Hospitals in Finland, the Päivikki and Sakari Sohlberg Foundation, the Finnish Medical Foundation and the Academy of Finland Research Council for Health. REFERENCES Amount of daily gluten-free foods (g) Amount of daily gluten ingestion (mg) Codex-Alimentarius-Commission. Codex Standard. Joint FAO/ WHO Food Standards Programme, Rome 118, Codex-Alimentarius-Commission. Report of the Working Groups on Prolamin Analysis an Toxicity (WGPAT). Joint FAO/WHO Food Standards Programme; CX/NFSDU03/4 Rome, Stern M, Ciclitira PJ, van Eckert R, et al. Analysis and clinical effects of gluten in coeliac disease. Eur J Gastroenterol Hepatol 2001; 13: Ciclitira PJ, Evans DJ, Fagg NL, Lennox ES, Dowling RH. Clinical testing of gliadin fractions in coeliac patients. Clin Sci 1984; 66: Ferguson A, Blackwell JN, Barnetson RS. Effects of additional dietary gluten on the small-intestinal mucosa of volunteers and of patients with dermatitis herpetiformis. Scand J Gastroenterol 1987; 22: Catassi C, Rossini M, Rätsch I-M, et al. Dose dependent effects of protracted ingestion of small amounts of gliadin in coeliac disease children: a clinical and jejunal morphometric study. Gut 1993; 34: Montgomery AM, Goka AK, Kumar PJ, Farthing MJ, Clark ML. Low gluten diet in the treatment of adult coeliac disease: effect on jejunal morphology and serum anti-gluten antibodies. Gut 1988; 29: Grefte JM, Bouman JG, Grond J, Jansen W, Kleibeuker JH. Slow and incomplete histological and functional recovery in adult gluten sensitive enteropathy. J Clin Pathol 1988; 41: Kaukinen K, Collin P, Holm K, et al. Wheat starch-containing gluten-free flour products in the treatment of coeliac disease and dermatitis herpetiformis. A long-term follow-up study. Scand J Gastroenterol 1999; 34: Högberg L, Stenhammar L, Wagermark J. Very late mucosal relapse in a girl with coeliac disease. Acta Paediatr 1993; 82: Ejderhamn J, Veress B, Strandvik B. The long-term effect of continual ingestion of wheat starch-containing gluten-free

7 GLUTEN IN GLUTEN- FREE PRODUCTS 1283 products in coeliac patients. In: Kumar, PJ, ed. Coeliac Disease: One Hundred Years. Leeds: Leeds University Press, 1988: Collin P, Reunala T, Pukkala E, Laippala P, Keyriläinen O, Pasternack A. Coeliac disease-associated disorders and survival. Gut 1994; 35: Peräaho M, Kaukinen K, Paasikivi K, et al. Wheat-starchbased gluten-free products in the treatment of newly detected coeliac disease. Prospective and randomized study. Aliment Pharmacol ther 2003; 17: Denery-Papini S, Nicolas Y, Popineau Y. Efficiency and limitations of immuno-chemical assays for the testing of glutenfree foods. J Cereal Sci 1999; 30: Valdes I, Carcia E, Llorente M, Mendez E. Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol. Eur J Gastoenterol Hepatol 2003; 15: Immer U, Vela C, Mendez EFJ. PWG collaborative trial on gluten in gluten-free food through Coctail Elisa. In: Stern, M, ed. Proceedings of the 17th Meeting Working Group on Prolamin Analysis and Toxicity. Zwickau: Verla Wissenschaftliche Scripten, 2002: Ladinser B, Rossipal E, Pittschieler K. Endomysium antibodies in coeliac disease: an improved method. Gut 1994; 35: Collin P, Mäki M, Kaukinen K. Complete small intestinal mucosal recovery is obtainable in the treatment of celiac disease. Gastrointest Endosc 2003; 59: Lee SK, Lo W, Memeo L, Rotterdam H, Green PHR. Duodenal histology in patients with celiac disease after treatment with a gluten-free diet. Gastrointest Endosc 2003; 57: Lohiniemi S, Mäki M, Kaukinen K, Laippala P, Collin P. Gastrointestinal symptoms rating scale in coeliac disease patients on wheat starch-based gluten-free diet. Scand J Gastroenterol 2000; 35: Collin P, Pukkala E, Reunala T. Malignancy and survival in dermatitis herpetiformis: a comparison with coeliac disease. Gut 1996; 38: Cooper BT, Read AE. Coeliac disease and lymphoma. Q J Med 1987; 63: Green PH, Fleischauer AT, Bhagat G, Goyal R, Jabri B, Neugut AI. Risk of malignancy in patients with celiac disease. Am J Med 2003; 115: Ciclitira PJ, Ellis HJ, Fagg NL. Evaluation of a gluten free product containing wheat gliadin in patients with coeliac disease. BMJ 1984; 289: Ciclitira PJ, Cerio R, Ellis HJ, Maxton D, Nelufer JM, Macartney JM. Evaluation of a gliadin-containing gluten-free product in coeliac patients. Hum Nutr Clin Nutr 1985; 39C: Sturgess R, Day P, Ellis HJ, et al. Wheat peptide challenge in coeliac disease. Lancet 1994; 343: Srinivasan U, Leonard N, Jones E, et al. Absence of oats toxicity in adult coeliac disease. BMJ 1996; 313: Laurin P, Wolving M, Fälth-Magnusson K. Even small amounts of gluten cause relapse in children with celiac disease. J Pediatr Gastroenterol Nutr 2002; 34: Chartrand LJ, Russo PA, Duhaime AG, Seidman EG. Wheat starch intolerance in patients with celiac disease. J Am Diet Assoc 1997; 97: Selby WS, Painter D, Collins A, Faulkner-Hogg KB, Loblay RH. Persistent mucosal abnormalities in coeliac disease are not related to the ingestion of trace amounts of gluten. Scand J Gastroenterol 1999; 34:

RIDASCREEN Gliadin. Validation Report. R-Biopharm AG. Art.No. R7001

RIDASCREEN Gliadin. Validation Report. R-Biopharm AG. Art.No. R7001 RIDASCREEN Gliadin Art.No. R7001 AOAC-Official Method New of Analysis (2012.01) AOAC-RI certified (120601) Codex Alimentarius Method (Type I) Validation Report Test validation RIDASCREEN Gliadin is a sandwich