Clinical trial: gluten microchallenge with wheat-based starch hydrolysates in coeliac disease patients – a randomized, double-blind, placebo-controlled study to evaluate safety

Summary

Background  Wheat-based starch hydrolysates such as glucose syrups, dextrose and maltodextrins are found in more than 50% of European processed food. These products contain low amounts of residual gluten and it has been questioned whether they are safe for coeliac disease patients.

Aim  To investigate whether coeliac disease patients can safely consume wheat-based starch hydrolysate products.

Methods  This randomized, double-blind, placebo-controlled, prospective follow-up study involved 90 coeliac disease patients in remission randomized to consume glucose syrups, maltodextrins or placebo for 24 weeks. Small bowel mucosal morphology and inflammation, symptoms, coeliac serology and malabsorption laboratory data were evaluated at baseline and at the end of the study.

Results  Daily ingestion of wheat-based starch hydrolysates, glucose syrups and maltodextrins, had no deleterious effect on small-bowel mucosal villous architecture or inflammation in coeliac disease patients when compared to the placebo group. Neither were there any significant differences in gastrointestinal symptoms, serology or malabsorption parameters after 24 weeks.

Conclusions  Wheat-based starch hydrolysates, glucose syrups and maltodextrins did not have harmful effect on coeliac disease patients. Coeliac patients can thus safely continue to consume these products.

Introduction

Coeliac disease is a genetically linked small-intestinal disorder induced by wheat-, rye- and barley-derived gluten. It is a major public health burden in Western countries, as it occurs in approximately 1% of the population.^1, 2 A lifelong gluten-free diet is currently the only treatment for the disease and alleviation of symptoms and recovery of mucosal damage are evident on a strict diet. While it is generally agreed that a gluten-free diet should be as strict as possible, a diet completely devoid of gluten is probably impossible to maintain. The safe threshold for gluten in gluten-free products is under investigation.^3–5 Industrially purified wheat starch-based gluten-free products have been shown to contain trace amounts of gluten (0–150 mg/kg = ppm gluten).³ Although the products have proved safe in clinical practice,^6–10 some authorities recommend only cereals, which are gluten-free by nature (maize, rice, buckwheat), for the treatment of coeliac disease.^11, 12

In the above-mentioned prospective studies, wheat starch-based gluten-free products consisted solely of flours. In addition, wheat-based starch hydrolysates such as glucose syrups and maltodextrins are formulated in soft drinks, beverages, confectionaries, desserts, infant food and dietetic products and together with maize-based starch hydrolysates, these hydrolysates are found in a wide range of European processed food. Hitherto, these products have been consumed by coeliac disease patients in their daily gluten-free diet. The amount of gluten after ingesting wheat-based starch hydrolysate products is most probably lower than what has been shown in wheat starch-based gluten-free flours. With the present analytical methods for gluten and for protein, in general, technical difficulties are encountered when they are applied to hydrolysed proteins in food products.^4, 13 In wheat-based starch hydrolysates, the residual gluten can also be hydrolysed as a result of the process. The European Commission Directive 2000/13/EC (amended by Directive 2003/89/EC) demands that products derived from cereals containing gluten be labelled with an indication of their botanical origin unless they are proved not to trigger adverse reactions.¹⁴ If this issue is not properly addressed, many products would be unnecessarily labelled as gluten-containing and regarded not suitable for coeliac disease patients. This could jeopardize the treatment of coeliac disease with unnecessary and inconvenient restrictions and might increase the disease burden and reduce the quality of life. However, no clinical trials on wheat-based starch hydrolysates have so far been carried out. On the other hand, it would be unethical to allow the consumption of these products containing tiny amounts of gluten without properly investigating their safety. Clinical and histological recovery in coeliac disease patients cannot always be achieved even with a strict gluten-free diet; it is not known whether such problems may be related to gluten traces.^15–19

Our aim here was to establish whether wheat-based starch hydrolysates (glucose syrups and maltodextrins) have untoward effects on small-bowel mucosal morphology or inflammation, serology or symptoms in coeliac disease patients. This was to ensure that coeliac patients can safely continue to consume products containing wheat-based starch hydrolysates in their daily gluten-free diet.

Patients and methods

Patients and study design

This randomized, double-blind, placebo-controlled, prospective 24-week follow-up study was conducted in one centre in Finland during October 2004–December 2005. Inclusion criteria comprised biopsy-proven coeliac disease patients aged 18 years or more adhering to a strict gluten-free diet for at least 1 year and being in clinical remission as judged by an interview and clinical examination. Patients with a history of regular dietary transgressions and those suffering from a condition carrying a risk at endoscopy, from refractory sprue or malignancy were excluded.

Ninety eligible patients were randomized either to glucose syrups (A), maltodextrins (B) or to the placebo group (C) in a ratio of 1:1:1 using a double dummy technique. Laboratory analysis, gastroscopy and duodenal biopsy and clinical evaluation including gastrointestinal symptoms were carried out at baseline and after 24 weeks.

The primary outcome measurements were changes in small-bowel mucosal morphology or inflammation. Safety assessments included monitoring of adverse events, vital signs, physical examinations, laboratory evaluations (haematology, malabsorption parameters and coeliac disease serology) and a symptom questionnaire. A dietary interview with a 4-day record of food intake was employed.

Study products and dietary assessment

The study products were provided by The Association des Amidonniers et Féculiers (AAF, Brussels, Belgium). The dietary exposure to gluten through the use of wheat-based starch hydrolysates was assessed based on food consumption data from three European countries, the Netherlands, Ireland and Italy and the compositions of the study products were chosen to cover the high end of the realistic mean exposure. The products were powder packed in single-use sachets to be diluted in 300 mL water. The subjects were advised to use one sachet daily or seven weekly during the study period. Each glucose syrup sachet contained 27.7 g glucose syrups (DE 41) and the maltodextrin products 2.7 g maltodextrins (DE 19). The protein/gluten content of one sachet measured by high-performance liquid chromatography was 4.3 ppm and 1.7 ppm respectively. In both products, the gluten contents established by sandwich and competitive R5-enzyme-linked immunosorbent assays (ELISAs) were under detection limits (<3.1 ppm and <2.4 ppm respectively). Placebo sachets contained no gluten.

The subjects were told to continue on a strict gluten-free diet. If the diet contained oats, wheat starch-based flours, products containing wheat-based starch hydrolysates, or only products gluten-free by nature, it continued as it was. Consumption of the study products, changes in dietary habits and possible dietary transgressions were inquired at each visit and by regular telephone contacts. At baseline and at the 24-week follow-up visit, a dietitian undertook a detailed dietary analysis and a history of occasional or regular consumption of gluten-containing products by means of an interview and a 4-day record of food intake.

Small-bowel mucosal morphology and inflammation

Six small-bowel biopsy specimens were taken from the distal part of the duodenum upon oesophago-gastroduodenoscopy; the specimens were evaluated by the same investigator without prior knowledge of the history or findings. Three biopsies were processed and stained with haematoxylin-eosin and studied under light microscopy. Poorly oriented sections were discarded; when necessary, the samples were dissected repeatedly until they were of good quality. Morphometric analysis measuring villous height and crypt depth ratio (Vh/CrD) was made in well-oriented biopsy samples as previously described.²⁰

Three biopsies were snap-frozen for immunohistochemical stainings. The specimens were freshly embedded in optimal temperature compound (OCT, Tissue-Tec; Miles Inc., Elkhart, IN, USA), snap-frozen in liquid nitrogen and stored at −70 °C. Immunohistochemical studies were carried out on 5-μm-thick frozen sections. CD3+ intraepithelial lymphocytes (IELs) were stained with monoclonal antibody Leu-4 (Becton Dickinson, San Jose, CA, USA), αβ+ IELs with monoclonal βF1 antibody (Endogen, Woburn, MA, USA) and γδ+ IELs with TCRγ antibody (Endogen).²¹ IELs were counted with a ×100 flat-field light microscope objective in randomly selected surface epithelium and the density of IELs expressed as cells/millimetre of epithelium as previously described.²¹ The reference values were set at 37 cells/mm for CD3+, at 25 cells/mm for αβ+ and at 4.3 for γδ+ IELs.²¹

Serology and chemical analysis

Serum IgA-class anti-tissue transglutaminase antibodies (tTG-ab) were determined by ELISA (Celikey; Phadia, GmbH, Freiburg, Germany: reference values <5.0 U/L) and anti-endomysial antibodies (EmA) by an indirect immunofluorescence method using human umbilical cord as antigen (reference values titres 1:<5).²² None of the study subjects suffered from selective IgA deficiency. Blood haemoglobin (reference values for women 115–152 g/L and for men 132–164 g/L), serum iron (reference values 9–34 μmol/L), serum-ionized calcium (reference values 1.18–1.30 mmol/L) and erythrocyte folic acid levels (reference values 212–1453 nmol/L) were measured using routine laboratory methods.

Gastrointestinal symptoms and body mass index

Gastrointestinal symptoms were evaluated by the Gastrointestinal Symptom Rating Scale (GSRS).^23, 24 This comprises altogether 15 items within five subdimensions describing: diarrhoea, indigestion syndrome, constipation, abdominal pain and gastro-oesophageal reflux. Each item is graded from one to seven; a higher score indicates more gastrointestinal symptoms. This questionnaire has previously been applied in the assessment of gastrointestinal symptoms in coeliac disease.^9, 25, 26 The body mass index (BMI) was calculated as weight in kilograms divided by the square of height in metres (normal range: 18–25 kg/m²).

Adverse events

Subjects were asked at each visit and by monthly telephone contacts about changes in their medical condition and whether they had experienced any adverse events since the last visit. They were also encouraged to contact the investigator at any time in the case of questions or adverse events. An adverse event was defined as follows: adverse event is any untoward medical occurrence in a patient or clinical trial subject administered an investigational product and not necessarily having a causal relationship with this treatment. Each adverse event was evaluated to determine severity, relationship to the study product, duration, action taken and whether the adverse event was serious or not.

Statistics

The required number of participants was determined to achieve the statistical power of 0.80 at a significance level of 0.05; a difference of eight or more [standard deviation (s.d.) = 11)] in αβ+ IELs and >0.5 in Vh/CrD (s.d. = 0.65) was considered clinically relevant. It was estimated that 30 subjects were needed for each group. All analyses were made on the intention-to-treat principle. Study groups A (glucose syrups) and B (maltodextrins) were compared with the placebo (C) group.

Differences between baseline and end values (Δ-values) were compared with those in the placebo group by Mann–Whitney U-test or t-test, when appropriate. Data were given mainly as medians, lower and upper quartiles (Q1, Q3) and range. In addition, means with 95% confidence intervals were given, when appropriate. A P-value <0.05 was considered statistically significant.

Ethical considerations

The study protocol was approved by the Ethical Committee of Tampere University Hospital. All subjects gave written informed consent.

Results

Characteristics of patients

Altogether 106 treated coeliac disease patients volunteered preliminarily; six were found to have contraindications for endoscopy. Seven changed their decision shortly afterwards and eventually did not wish to participate. Of the remaining patients, 90 were enrolled and randomized. Baseline characteristics were well balanced between the study groups (Table 1).

Table 1.   Demographic data on coeliac disease patients classified in different intervention groups at baseline

	Glucose syrups (n = 30)	Maltodextrins (n = 30)	Placebo (n = 30)
CD, coeliac disease. * Osteoporosis and disturbances in bone metabolism, neurological and psychiatric disorders, arthritis, fatigue, enamel defects in permanent teeth, elevated liver enzymes and fibromyalgia. † Family history of CD and autoimmune thyroid disease.
Female (%)	19 (63)	16 (53)	22 (73)
Median age (range), years	55 (24–81)	57 (25–75)	61 (25–75)
Median duration of gluten-free diet (range), years	8 (1–42)	9 (1–30)	11 (2–34)
Symptoms and signs leading to the diagnosis of CD, n (%)
Abdominal symptoms	24 (80)	27 (90)	25 (83)
Malabsorption, anaemia, loss of weight	20 (67)	16 (53)	22 (73)
Dermatitis herpetiformis	4 (13)	3 (10)	5 (17)
Extraintestinal symptoms*	5 (16)	5 (16)	5 (16)
Screening of risk groups†	2 (7)	1 (3)	2 (7)
Diagnosis made by chance	1 (3)	3 (10)	2 (7)
Family history of CD, n (%)	18 (60)	16 (53)	17 (57)

Gluten-free diet and consumption of the study product

At baseline and during the follow-up, all subjects told the investigator and the study nurse that they had adhered to a strict gluten-free diet and did not make any changes in their dietary habits. The dietitian afterwards estimated that altogether five patients had committed minor dietary transgressions less than once a month during the study: four in the glucose syrup and one in the maltodextrin group. One patient in the placebo group reported slight dietary transgressions one to four times a month during the study.

Those patients who completed the study as planned consumed in the glucose syrups group a median of 174 (range 153–178) product sachets, in the maltodextrins group a median of 172 (range 148–180) sachets and in the placebo group a median of 173 (160–176) sachets. The planned number of sachets during the study period was 168.

Small-bowel mucosal morphology and inflammation

At baseline, small-bowel mucosal villous morphology and densities of CD3+, αβ+, γδ+ IELs were comparable in all groups (Table 2, data on CD3+ and γδ+ IELs not shown). During the study, there were no statistically significant changes in these parameters between study subjects in the glucose syrup and placebo groups (Figures 1 and 2). In the maltodextrins group, the density of γδ+ IELs decreased significantly when compared with the placebo group (Figure 2b).

Table 2.   Small-bowel mucosal findings of coeliac disease patients classified in different intervention groups at the baseline of the study

	Glucose syrups (n = 30)	Maltodextrins (n = 30)	Placebo (n = 30)
IELs, intraepithelial lymphocytes; Q1, lower quartile; Q3, upper quartile.
Villous height and crypt depth ratio
Median	3.2	3.2	3.3
Range	2.3–4.3	1.2–4.2	2.6–4.3
Q1, Q3	2.8, 3.5	2.9, 3.6	2.9, 3.4
Density of αβ+ IELs (cells/mm)
Median	33	35	29
Range	10–63	8–67	3–64
Q1, Q3	21, 40	20, 46	23, 36

Figure 1.  Median and upper and lower quartiles of the change (Δ) in villous height crypt depth ratio in different study groups. Differences between glucose syrups and placebo group and between maltodextrins and placebo group were not statistically significant.

Figure 2.  Median and upper and lower quartiles of the changes (Δ) in the densities of (A) αβ+ and (B) γδ+ intraepithelial lymphocytes (IELs) in different study groups. Only the difference in the Δ density of γδ+ IELs between the maltodextrins and the placebo groups was statistically significant. There were no statistically significant changes in the densities of CD3+IELs (data not shown).

Clinical outcome

During the study, there were no statistically significant changes in GSRS total (P = 0.367, ΔGSRS total score in glucose syrups vs. placebo; P = 0.161, ΔGSRS total score in maltodextrins vs. placebo group) and subdimension scores (data not shown) between the glucose syrups and placebo groups and the maltodextrins and placebo groups. None of the twelve patients suffering from dermatitis herpetiformis developed a rash during the study. BMI also remained unchanged (P = 0.308, ΔBMI in the glucose syrups group vs. placebo; P = 0.578, ΔBMI in the maltodextrins vs. placebo group, data not shown).

Coeliac serology and laboratory values

Two patients in the maltodextrins group were EmA-positive at baseline and at the end of the study; both were initially tTG-ab-negative, but one of them became positive. One additional patient in this group became tTG-ab-positive during the trial. The rest remained both EmA- and tTG-ab-negative throughout the study.

Changes (Δ-values) in blood haemoglobin, erythrocyte folic acid, serum iron and ionized calcium levels were not statistically significant between the glucose syrups or maltodextrins and placebo groups (data not shown).

Adverse events

Altogether, 68 adverse events were reported in 43 (48%) patients during the study; two of these events, unrelated to study products, were classified as serious adverse events (hospitalizations because of gastrointestinal bleeding after endoscopy and suspicion of temporal arthritis). The 68 reported adverse events were distributed practically equally over all three study groups; there were 27 adverse events in the glucose syrups group, 21 in the maltodextrins group and 20 in the placebo group. This was also the case in those 21 adverse events which were regarded to be possibly related to the study products (data not shown).

Premature withdrawals from the study

Seven patients withdrew from the study for reasons of symptoms: one in the glucose syrups, three in the maltodextrins and three in the placebo group; the intended end-point examinations were carried out in four cases. In addition, one patient was noncompliant and dropped out (Table 3). The consumption of sachets by those who prematurely withdrew from the study was as follows: glucose syrups group 22 sachets; maltodextrins group 33, 90, 90 and 120; and placebo group 9, 36 and 71 sachets. As shown in Table 3, the consumption of glucose syrups or maltodextrins products was not the reason for premature withdrawal more often than the consumption of placebo products. None of the patients withdrawing seroconverted positively or developed small-bowel villous atrophy.

Table 3.   Data from eight coeliac disease patients withdrawing prematurely from the trial

No.	Study group	Reason for withdrawal	Small-bowel biopsy findings				GSRS total score
			Vh/CrD		αβ+ IELs (cell/mm)
			I	II	I	II	I	II
Vh/CrD, villous height and crypt depth ratio; abnormal ratio compatible to active CD <2.0. IELs, intraepithelial lymphocytes; reference values for αβ+ IELs 25 cells/mm. GSRS, Gastrointestinal Symptom Rating Scale; 95% confidence intervals of noncoeliac controls 1.8–2.2.^26 NT, not taken; I, baseline of the study; II, end of the study; G, glucose syrups group, M, maltodextrins group; P, placebo group.
Withdrawal due to abdominal symptoms
1	G	Flatulence	3.4	3.7	31	38	1.9	2.3
2	M	Abdominal pain	1.2	NT	8	NT	1.7	NT
3	M	Abdominal pain	3.8	NT	23	NT	1.5	NT
4	M	Abdominal pain	3.0	3.5	20	6	1.9	2.3
5	P	Flatulence, nausea	3.2	3.0	50	81	1.7	5.3
6	P	Heartburn	3.4	3.6	16	22	2.1	1.8
7	P	Diarrhoea	3.8	NT	16	NT	2.5	NT
Withdrawal due to noncompliance
8	M	Noncompliance	3.4	NT	67	NT	2.4	NT

Discussion

We found in this prospective, placebo-controlled, double-blind intervention study that daily ingestion of wheat-based starch hydrolysates had no deleterious effect on small-bowel mucosal villous architecture or inflammation, serology, gastrointestinal symptoms or malabsorption parameters in coeliac disease patients. On the contrary, we noted an improvement in many histological items during the study period (Figures 1 and 2). The most likely explanation is that strict surveillance even further improved dietary compliance. Nevertheless, the improvement was equal in the study groups and the placebo group. It is also important to note that none of the patients suffering from dermatitis herpetiformis experienced any flare-ups of their skin disorder during the intervention study, again showing that the wheat-based starch hydrolysed products in question are not harmful to coeliac disease and dermatitis herpetiformis patients.

The overall compliance in the study was good, even though the protocol comprised two endoscopies and daily usage of study products for 24 weeks. Adverse events and premature withdrawals were distributed equally over all three different intervention groups. Only four of the 90 patients did not consent to complete the last study visit and follow-up small-bowel biopsy. However, when we hypothesized that if these four patients would have developed abnormal small-bowel mucosal morphology with total villous atrophy and massive inflammation, the main results of the study did not change (data not shown). Some of the patients prematurely withdrawing consumed the study product for less than 1 month. Even such a short period can be regarded as long enough to uncover harmful effects of gluten; there are studies showing that in coeliac disease patients, even a span of 4 h in vivo gluten challenge may suffice to damage the small-bowel mucosa.^27, 28 Furthermore, it is important to note that when the patients with minor dietary transgressions or positive EmA were excluded from the analysis, the results did not differ from those in the whole series (data not shown).

After randomization, the baseline characteristics of all three intervention groups were equal (Tables 1 and 2). Two EmA-positive and two patients with initially abnormal small-bowel mucosal biopsy findings were all randomized to the maltodextrins group and the baseline situation was therefore slightly different. Regardless of this, maltodextrins proved not to have any deleterious effect on coeliac disease patients when compared to the subjects in the placebo group.

Even in the placebo group there were clear changes in small-bowel mucosal morphology and inflammation during the study (Figures 1 and 2, Table 3). According to the literature, some patients having long-term treated coeliac disease may have abnormal small-bowel mucosa or positive coeliac serology, despite a strict gluten-free diet.^{5, 10, 18, 29} A recent study from the US even showed that only 20% of long-term treated patients consuming a strict naturally gluten-free diet had normal small-bowel mucosal villous architecture.¹⁶ Compared to these reports, patients in this study had evinced an excellent response to a strict gluten-free diet, even though they had consumed wheat starch-based flours and hydrolysates for years before the present intervention study, and they remained in good remission after 6 months additional daily challenge with wheat starch-based hydrolysates.

To conclude, wheat-based starch hydrolysates, glucose syrups and maltodextrins had no harmful effect on coeliac disease patients. In fact, on the basis of finding of the present study the European Food Safety Authority (EFSA) has issued an opinion that such wheat-based starch hydrolysates are unlikely to cause adverse reactions in coeliac disease patients, provided that the provisional value of gluten (100 ppm) considered by the Codex Alimentarius for foods rendered gluten-free is not exceeded.^30, 31 On the basis of the EFSA opinion, the European Commission decided on 27 November 2007 that these wheat-based starch hydrolysates are permanently excluded from allergen labelling (Directive 2007/68/EC).³² Altogether this means that also in the future, coeliac disease patients can safely continue to consume these products.

Acknowledgements

Declaration of personal interests: The authors have no conflict of interest. Declaration of funding interests: The study was financed by the Association des Amidonniers et Féculiers (AAF, Brussels, Belgium), and they commissioned the study to the Tampere University Hospital and University of Tampere owned company Finn-Medi Research Ltd, Tampere, Finland. Authors’ work was independent of the funding institutions.