508

ORIGINAL CONTRIBUTIONS

nature publishing groupsee related editorial on page 516

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CME

Gluten Causes Gastrointestinal Symptoms in Subjects
Without Celiac Disease: A Double-Blind Randomized
Placebo-Controlled Trial
Jessica R. Biesiekierski, B Appl Sci1, Evan D. Newnham, MD, FRACP1, Peter M. Irving, MD, MRCP1, Jacqueline S. Barrett,
PhD, BSc, MND1, Melissa Haines, MD1, James D. Doecke, BSc, PhD2, Susan J. Shepherd, B Appl Sci, PhD1, Jane G. Muir,
PhD, PGrad Dip(Dietetics)1 and Peter R. Gibson, MD, FRACP1OBJECTIVES:

Despite increased prescription of a gluten-free diet for gastrointestinal symptoms in individuals who
do not have celiac disease, there is minimal evidence that suggests that gluten is a trigger. The aimsof this study were to determine whether gluten ingestion can induce symptoms in non-celiac
individuals and to examine the mechanism.

METHODS:

A double-blind, randomized, placebo-controlled rechallenge trial was undertaken in patients withirritable bowel syndrome in whom celiac disease was excluded and who were symptomaticallycontrolled on a gluten-free diet. Participants received either gluten or placebo in the form of two
bread slices plus one muffin per day with a gluten-free diet for up to 6 weeks. Symptoms were
evaluated using a visual analog scale and markers of intestinal inflammation, injury, and immune
activation were monitored.

RESULTS:A total of 34 patients (aged 29–59 years, 4 men) completed the study as per protocol. Overall,
56% had human leukocyte antigen (HLA)-DQ2 and/or HLA-DQ8. Adherence to diet and supplements
was very high. Of 19 patients (68%) in the gluten group, 13 reported that symptoms were notadequately controlled compared with 6 of 15 (40%) on placebo (P = 0.0001; generalized estimating
equation). On a visual analog scale, patients were significantly worse with gluten within 1 week foroverall symptoms (P = 0.047), pain (P = 0.016), bloating (P = 0.031), satisfaction with stool
consistency (P = 0.024), and tiredness (P = 0.001). Anti-gliadin antibodies were not induced.
There were no significant changes in fecal lactoferrin, levels of celiac antibodies, highly sensitive
C-reactive protein, or intestinal permeability. There were no differences in any end point inindividuals with or without DQ2/DQ8.

CONCLUSIONS: “Non-celiac gluten intolerance” may exist, but no clues to the mechanism were elucidated.
Am J Gastroenterol 2011; 106:508–514; doi:10.1038/ajg.2010.487; published online 11 January 2011

INTRODUCTION
In clinical practice, some patients have symptoms of irritablebowel syndrome (IBS) that respond well to a gluten-free diet but
they have no markers of celiac disease. The published scientific
literature is largely devoid of the so-called “non-celiac gluten intolerance” and “wheat intolerance,” yet they are widely believed to bevery common (1–3). In the evaluation of exclusion diets, wheat
has been found to be one of the most common factors inducing
gastrointestinal symptoms (4), but it is not known whether gluten
is the responsible agent, as wheat, the major cereal removed fromthe gluten-free diet, contains other components that include otherproteins, lipids, and carbohydrates. Of particular importance are
fructans, which are poorly absorbed carbohydrates and can induce
symptoms by themselves (5,6).
The role of gluten in celiac disease is clear. The toxic peptide
sequences have been defined (7,8), the genetic susceptibility lociidentified, and the pathological processes comparatively wellknown. Deamidation of these gliadin epitopes by tissue transglutaminase enables them to be presented with a high affinity tomajor histocompatibility complex class II T cells in genetically1

Monash University Department of Medicine and Gastroenterology, Box Hill Hospital, Box Hill, Victoria, Australia; 2CSIRO Preventative Health Flagship, CSIRO
Mathematics and Information Sciences, Brisbane, Australia. Correspondence: Peter R. Gibson, MD, FRACP, Department of Medicine, Box Hill Hospital, Box Hill,
Victoria 3128, Australia. E-mail: peter.gibson@monash.edu
Received 16 March 2010; accepted 10 September 2010
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susceptible individuals (human leukocyte antigen (HLA)-DQ2 or
HLA-DQ8 being expressed in 99.4% of patients with celiac disease)(9). This process initiates a cascade of events, resulting in mucosal
inflammation, small intestinal villous atrophy (10), increased
intestinal permeability (11), malabsorption of macronutrients andmicronutrients (12), and resultant complications of celiac disease.
To date, the literature regarding the effect of gluten outside celiac
disease has been limited to experiments in cancer cell lines and touncontrolled clinical studies (1,2,13–17). Whether gluten itself cancontribute to gastrointestinal symptoms and/or induce injury to
the proximal small intestine in non-celiac patients has never been
directly assessed.
The aims of this study were to examine the hypotheses that gluten
can cause gastrointestinal symptoms in patients without celiac disease and to preliminary screen for potential mechanisms of whether
gluten does so by causing intestinal injury and/or inflammation insuch subjects. To do this, a randomized, double-blind, placebocontrolled, dietary rechallenge trial was conducted in subjects with
IBS who had celiac disease excluded by best practice methods andwho reported a symptom response to a gluten-free diet.

METHODS
Patients

Patients were recruited between July 2007 and December 2008
through advertisements in e-newsletters and community/state
newspapers in metropolitan Melbourne, and by referral in private dietetic practice. The inclusion criteria were age > 16 years,
symptoms of IBS fulfilling Rome III criteria that have improved
on a gluten-free diet, and adherence to the diet for at least 6 weeks
immediately before screening. Celiac disease was excluded by either(i) absence of the HLA-DQ2 and HLA-DQ8 haplotype or (ii) a normal duodenal biopsy (Marsh 0) performed at endoscopy while on agluten-containing diet in individuals expressing the HLA-DQ2 or
HLA-DQ8 haplotype. Patients with significant gastrointestinal disease (such as cirrhosis or inflammatory bowel disease), excessive
alcohol intake, intake of non-steroidal anti-inflammatory agents,
and unable to give written informed consent were excluded.
Study protocol

Patients were randomized according to a computer-generated listof random numbers held by an independent observer to either
the gluten or the placebo treatment group. Baseline symptom data
and a 7-day food diary were collected during a 2-week run-in
period. Participants continued on a gluten-free diet throughout
the study, but were asked to consume one study muffin and two
study slices of bread containing gluten (total intake of 16 g/day)or not (see below) every day for 6 weeks. Both patients and investigators evaluating patients were blinded to the study treatment.
At the end of each study week and for 3 weeks after completionof the study intervention, symptoms were evaluated, as previously
applied (6). Patients were asked to complete a symptom questionnaire containing the question for the primary outcome detailed
below, and a 100-mm visual analog scale, with 0 representing
no symptoms, assessing overall symptoms, bloating, abdominal© 2011 by the American College of Gastroenterology

pain, satisfaction with stool consistency, nausea, and tiredness.
At weeks 0 and 6, serum, urine, and stool samples were collected
for analysis, and intestinal permeability was measured. Food diaries were maintained by patients during the third and sixth study
weeks, and unused muffins and bread were returned at the endof weeks 3 and 6 of the treatment period for counting. Patientsunable to continue the study because of intolerable symptoms
were permitted to cease study food after data were collected as per
week 6 (symptom assessment, and blood, urine, and stool samples collected). The protocol was approved by the Eastern HealthResearch and Ethics Committee.
End points

The primary outcome was the proportion of patients answering“no” on more than half of the symptom assessments to the question, “Over the last week were your symptoms adequately controlled?” This question was asked at the end of each study week or atwithdrawal if premature. Secondary outcomes were the change inoverall and individual gastrointestinal symptoms as assessed by
the visual analog scale, and changes in biomarkers (see below).
Compliance with the study treatment was assessed by an unused
food count at weeks 3 and 6. Compliance with the gluten-free diet
was judged on food diary entries and on specific questioning at
the time of review.
Study food preparation

The muffins and bread were prepared and baked commerciallyin gluten-free ovens and conditions. The base mixes were gluten
free. For the gluten group, commercially available, carbohydratedepleted wheat gluten (Gemtec 1160, Manildra Group, Auburn,
NSW, Australia) was added before baking at the amount of 8 g per
muffin and 4 g per slice of bread. Analysis of the baked products
using a commercially available assay (Biokits Gluten Assay Kit;
Tepnel Biosystems, Flintshire, UK; AOAC 991·19 Method) confirmed the preservation of intact gluten and in the amount expected. The gluten used contained 91.7% protein, 1.1% crude fiber,
1.9% lipid, 1.8% starch, and 3.5% ash shown on reversed-phasehigh-performance liquid chromatography. To assess FODMAP(fermentable oligosaccharides, disaccharides, monosaccharides
and polyols) content, gluten was analyzed as described previously (17) and was shown to be free of short-chain carbohydrates,
fructans, fructose, glucose, lactose, sorbitol, mannitol, raffinose,
stachyose, nystose, and kestose. On the basis of size-exclusion
high-performance liquid chromatography, the protein content
had a distribution of 2.3% non-gluten protein (albumin/globulin),
45.7% glutenin, and 52.0% gliadin. Preliminary testing in 10
healthy individuals showed that the muffins and bread containing
gluten could not be differentiated from those that did not on thebasis of taste or texture.
Measurement of biomarkers

All markers were measured after randomization. Serum wasanalyzed for antibodies to tissue transglutaminase (i.e., tissuetransglutaminase IgA) and whole gliadin (IgA and IgG) by ELISA(enzyme-linked immunosorbent assay) using commercially
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available assays (INOVA Diagnostics, San Diego, CA). The manufacturer’s reference ranges were used to determine the classificationof the serological result. Endomysial antibodies were examinedby immunofluorescent staining of distal monkey esophagus(Chemicon Australia, Boronia, VIC, Australia).
Highly sensitive C-reactive protein was measured using an
immunoturbidimetric assay (Tina-Quant CRP Roche Diagnostics,
Basel, Switzerland). Intestinal permeability was measured using adual sugar test (18). After an overnight fast, patients emptied their
bladder and consumed a solution of lactulose (5 g) and rhamnose(1 g) dissolved in 120 ml of water. All urine over the next 5 h wascollected in containers containing boric acid as a preservative,
and samples were stored at − 80 °C until assayed by high-pressureliquid chromatography as described previously (19). The urinary
lactulose-to-rhamnose ratio was calculated. Fecal lactoferrin wasmeasured by ELISA using a commercially available kit (IBD Scan;
Techlab, Blacksburg, VA). Two dilutions of each sample wereassayed and the results expressed in units of mg/ml feces.
Statistical analyses

Power calculations were based on a placebo effect using a similar
end point and rechallenge methodology of ~20% (6) and an estimate of 60% response to gluten as there were no previous data on
which this could be judged. This indicated that 30 patients wererequired in each group to achieve a power of 80% and a P value
0.05. The study was terminated early because of difficulty withrecruitment of patients in whom celiac disease had been definitely
excluded (see above).
To determine the relationship between tolerable symptoms(“yes”/“no”) over the 6 weeks, a generalized estimating equation,
was used (primary outcome). The change in symptom severity
was calculated as the scored difference between commencement
and 1 week and was tested by the independent samples t-test forwithin-group comparisons and ANCOVA (analysis of covariance)
between groups. A linear mixed effects model assessed symptom
severity scores across the treatment period (longitudinal data). The
correlation between measured symptoms and their model residuals was assessed using Pearson’s correlation coefficient. Changes
in biomarker levels after therapy within each dietary group wereassessed by paired t-test using log-transformed data. Comparisonof change in biomarker levels between each group was assessedusing ANCOVA and that of change in the indices using an independent samples t-test. Blinding was assessed by using the κ-agreement statistic, in which a value of 1 indicated complete agreement
and 0 indicated no agreement. All statistical analyses were conducted using the R Statistical Software Package (R Development
Core Team, R: A Language and Environment for Statistical Computing, Vienna, Austria). Two-tailed P values at or below 0.05 wereconsidered statistically significant.

RESULTSLess than one-third of respondents to the advertisements weredeemed suitable for screening. Of those 103 subjects, only 39 met
the inclusion criteria and were enrolled. Subject flow is shown in
The American Journal of GASTROENTEROLOGY

Screenedn = 103
Randomised
n = 39Inadequate symptom
control during baseline (1)Celiac disease not excluded (34)
Unwillingness to participate (23)Symptomatic on gluten-free diet (7)Glutenn = 20

Placebon = 19Per protocol
Glutenn = 19Per protocol
Placebon = 15Inadequate symptom
control during baseline (3)Acute psychiatric illness (1)Figure 1. Recruitment pathway and reasons for screen failure andwithdrawals.

Figure 1. After randomization, five patients had to be withdrawn.
Thus, 34 patients completed the study as per protocol: 19 received
gluten and 15 received placebo.
The details of those patients are shown in Table 1. All patientswere negative for tissue transglutaminase and endomysial antibodies and there were no differences for whole gliadin antibodies
between the gluten or placebo groups, including those within the
DQ2/8-positive group.
All patients adhered to the gluten-free diet during the study.
Alcohol intake did not differ during the treatment period, and did
not differ between groups. Nearly all food supplements (95 and96%) were consumed in the placebo and gluten groups, respectively. The blinding technique was successful, supported by a
κ score of 0.24 (low agreement between actual treatment andparticipant guessing).
Nine patients ceased the study diet prematurely because of intolerable symptoms. Six patients were in the gluten arm and they
withdrew after a median of 7 (range 2–18) days, whereas three inthe placebo arm withdrew after 16 (range 11–21) days. There wereno statistical differences between the groups in frequency and timing of withdrawal. Serum, urine, and stool samples were collectedfrom all of these patients upon cessation of the diet as per week 6.
Significantly more patients in the gluten group (68%; n = 13/19)
reported the answer, “no” (the primary outcome question) compared with those on placebo (40%; n = 6/15) for more than half ofthe study therapy duration (P = 0.001; generalized estimating equation). As shown in Figure 2, changes in symptoms from baseline
to end of week 1 as scored on the visual analog scale after 1 week’s
therapy were significantly greater in those patients who consumed
the gluten diet for overall symptoms, pain, bloating, satisfaction
with stool consistency, and tiredness, but not for wind or nausea.
Over the entire study period, the severity scores of pain, satisfaction with stool consistency, and tiredness were significantly higher
for those consuming the gluten diet (Figure 2). The correlation
between model residuals to estimate symptom score redundancy
was assessed. Correlation coefficients ranged between 0.3 and 0.9,
with the highest correlation between overall score and pain.
As shown in Table 2, neither treatment group had significant
changes from baseline for any of the biomarkers measured. Similarly, no significant differences were observed in the magnitudeof changes between the groups, whether assessed using raw data
(ANCOVA) or by comparing changes in the indices (t-test). Fecal
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Table 1. Patient characteristics according to the dietary treatment
group
Patient characteristic
Number of patientsMedian age (range) in years
Men
Median body mass index range (range)

GlutenPlacebo19

15

40 (29 – 55)

49 (33 – 51)

16%

7%

23 (18 – 41)

22 (18 – 33)

16

20

Number with predominant bowel habit
Constipation in percentage
Diarrhea in percentage

58

33

Alternating percentage

26

47

HLA type
DQ2 or DQ8 positive in percentage

53

60

DQ negative in percentage

47

40

Elevated serum celiac antibodies (percentage of patients (mean (s.e.m.)Units/ml))
Tissue transglutaminase (IgA)

00

Tissue transglutaminase (IgG)

00Endomysium (IgA)

00

Whole gliadin (IgA)

27 (39 (9))

30 (33 (9))

DQ2/8 positive

5 (33 (0))

7 (24 (0))

Whole gliadin (IgG)

25 (25 (1))

0

DQ2/8 positive

5 (23 (0))

0HLA, human leukocyte antigen.
There were no significant differences between dietary groups for any index(independent samples t-test and χ2 test).

lactoferrin was below the detectable level before and after treatment in all but one patient in the placebo arm whose level was
36 mg/ml at both weeks 0 and 6. Removal of this patient’s datafrom the analysis had no effect on the results (data not shown).
Symptomatic responses to gluten did not significantly differ inthose expressing HLA-DQ2 and/or HLA-DQ8 (n = 10) comparedwith those who did not (n = 9; data not shown). Similarly, no differences in the response of biomarkers to gluten exposure were notedaccording to HLA-D status (data not shown).

DISCUSSION
Gluten intolerance in individuals without celiac disease is a controversial issue and has recently been described as the “no man’sland of gluten sensitivity” (20). The evidence base for such claims
is unfortunately very thin with no randomized controlled trials
demonstrating that the entity does actually exist. Most publisheddescriptions involve patients with positive serology associated withceliac disease or with intraepithelial lymphocytosis in the duodenum. In other words, evidence of immunological responses seen
in celiac disease has been present (21) and this may just represent
celiac disease not fulfilling the ESPGHAN (European Society for© 2011 by the American College of Gastroenterology

Paediatric Gastroenterology, Hepatology and Nutrition) criteria
for diagnosis. This double-blind, randomized, placebo-controlled rechallenge trial in patients who claim considerable improvement in gut symptoms with the institution of a gluten-free dietdoes indeed support the existence of non-celiac gluten sensitivity.
Gluten specifically induced symptoms including bloating, dissatisfaction with stool consistency, abdominal pain, and tiredness.
Recruitment of patients for this study was not easy mainly
because of the failure of most to have celiac disease effectively ruledout. Although the final number (n = 34) of participants recruited
was less than the a priori power calculations suggested and relatively small, our interim power analyses confirmed that the reduced
number was adequate to infer a statistically robust result, withunequivocal significant separation of the two groups. All patientsdeveloped exacerbation of symptoms in response to gluten and did
so within the first week of rechallenge in contrast to the placebogroup in which symptom induction occurred more slowly and thelevel of symptoms reached was less severe. This occurred acrossthe relevant abdominal symptoms of bloating, pain, and satisfaction with stool form, whereas no differences between the treatmentgroups were shown for the less relevant symptom of nausea. Interestingly, the symptom that quantitatively differentiated the treatment groups to the greatest extent was tiredness, mainly due toplacebo having no apparent effect on this end point. Tiredness is
a common symptom of IBS (21), and its induction by gluten may
provide insights into a mechanism of action.
A key question is by what mechanism symptoms were inducedby the ingestion of gluten. It might be anticipated that somepatients reporting symptomatic improvement from the gluten-freediet have undiagnosed celiac disease. Celiac disease can be patchy(22) and, although unlikely, it is therefore possible that somepatients with undiagnosed celiac disease were included. However,there were no significant changes in celiac antibodies seen in eithergroup. About one-half did not carry HLA-D genes believed to be
essential for the development of celiac disease. Although the study
was not powered to determine differences in responses between
genotypes, no clear differences were noted.
Simple non-invasive studies were performed to look for a signalthat inflammation and/or intestinal damage was being induced.
This was particularly suspected as evidence of an immune basis
for at least a proportion of patients with functional gastrointestinal disorders has already been shown (23,24), and gluten had aprominent effect on tiredness in this population, suggesting a more
systemic process. The change in highly sensitive C-reactive protein is considered a marker for systemic circulation of cytokines
from a localized site, but no effect on this was observed. Fecal lactoferrin levels increase in the presence of intestinal inflammation
due to transepithelial migration of neutrophils to the lumen andthe inability of gut bacteria to degrade lactoferrin (25). However,levels were not increased by the interventions. Finally, intestinal
permeability, as examined using a dual sugar absorption test, isbelieved to be a sensitive marker of intestinal injury, but this also
did not change overall, and there were no differences between thegluten and placebo groups. Clearly, these markers may not havethe required sensitivity to detect subtle inflammation and/or
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Overall symptoms

VAS (0-100 mm)

40

GlutenPlacebo30

VAS Score: 0 = None 100 = Worst
20
10

k6

k5

W
ee

W
ee

k4

k3

W
ee

W
ee

k2

k1

0

W
ee

COLON/SMALL BOWEL

50

W
ee

512

Pain

50

50

40

40

30

30

20

20

10

10

0

0

50

Satisfaction withstool consistency

Bloating

Tiredness
50
40

40

30

30

20
20
10
10
0
0

50

–10

Wind

50

40

40

30

30

20

20

10

10

0

0

NauseaFigure 2. Change in symptom severity from baseline in the gluten and placebo-treated groups over 6 weeks of the study. Data shown represent the meanchange for subjects remaining on study therapy at each time point. The differences were compared at week 1 by an independent samples t-test, in which
overall symptoms (P = 0.047), abdominal pain (P = 0.016), bloating (P = 0.031), satisfaction with stool consistency (P = 0.024), and tiredness (P = 0.001)
were statistically significant, but wind (P = 0.053) and nausea (P = 0.120) were not. The differences were also compared over the entire study period using
a linear mixed effects model, in which abdominal pain (P = 0.02), satisfaction with stool consistency (P = 0.03), and tiredness (P = 0.001) were statisticallysignificant, but overall symptoms (P = 0.15), wind (P = 0.08), and nausea (P = 0.69) were not. VAS, visual analog scale.

intestinal damage. Examination at the tissue level is warranted tobetter address this issue.
Other potential mechanisms by which a dietary product can
induce functional gut symptoms include induction of intestinal
distension through the fermentation of poorly absorbed gluten
peptides. However, passage of excessive flatus was not a prominent feature (as it is for carbohydrate sources; see the study by
Gibson and Shepherd (26) and malodorous flatus might be
The American Journal of GASTROENTEROLOGY

anticipated because of sulfide production, but was not reported
by patients in the study. Indeed, if hydrogen sulfide production was increased, this might potentially alter visceral sensitivity (27). Alternatively, gluten may mediate cholinergic activationas has been shown in murine models of gluten sensitivity (28).
This may lead to increased smooth muscle contractility and indirectly have effects on luminal water content. Other functional gut
symptoms might also be induced by stimulation of the enteric
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Non-Celiac Gluten Intolerance

513

Biomarker

Gluten (n =19)

Placebo (n =15)

Baseline

With therapy

ChangeBaseline

With therapy

Change3.0 (2.0 – 7.0)

4.5 (2.0 – 7.0)

0.6 (0.3)

3.0 (2.0 – 10.0)

3.5 (2.0 – 10.0)

0.4 (0.5)

Whole gliadin (IgA)

10.8 (3.5 – 241.5)

4.6 (0.1 – 51.3)− 29.7 (19.2)

6.6 (0.1 – 36.6)

5.9 (0.1 – 36.6)− 4.3 (2.5)

Whole gliadin (IgG)

Celiac serology (Units/ml)
Tissue transglutaminase (IgA)

14.6 (12.1 – 31.5)

15.5 (11.4 – 50.6)

2.5 (2.0)

11.9 (10.9 – 14.6)

11.9 (10.6 – 15.7)

0.2 (0.3)

Intestinal permeability (L:R ratio)

0.02 (0.01 – 0.6)

0.01 (0.01 – 2.4)

0.09 (0.1)

0.04 (0.01 – 0.15)

0.02 (0.01 – 0.18)− 0.01 (0.02)

Highly sensitive C-reactive protein (mg/l)

1.4 (0.3 – 5.3)

0.3 (0.4 – 19.8)

2.1 (1.4)

1.1 (0.2 – 8.2)

1.2 (0.3 – 13.1)

0.5 (0.9)

ANCOVA, analysis of covariance; L:R ratio, lactulose-to-rhamnose ratio
There were no statistically significant differences within each dietary group (paired t-test on log-transformed data) or between treatment groups whether evaluated using
baseline and treatment data (ANCOVA) or the changes in indices (independent samples t-test; all P ≥ 0.1).

nervous system either directly by the supply of neuroactive
molecules or by indirect release of neurotransmitters from,e.g., mast cell activation. Neurally active peptides from gluten
digestion might potentially gain access to enteric nerve endings,
but these are not known to occur and their absorption might seemless likely given normal intestinal permeability. Newer techniquessuch as examining basophil activation in response to the gluten
used might be instructive in this manner (29).
The other key issue is whether symptoms are being inducedby peptide(s) derived from gliadin proteins or non-gliadin partsof gluten, or by a contaminant of the gluten. There is ample evidence in vitro that suggest that gluten can induce injury andchanges in epithelial cells by non-DQ2-restricted mechanisms.
For example, gliadin is able to increase epithelial permeability
and alter protein expression of components of the tight junction(16), induce apoptosis (14,30), and increase oxidative stress (15)in Caco-2 (human colon adenocarcinoma) monolayers, a surrogate model for the human gut epithelium. In addition, gliadin
may inhibit RNA and DNA synthesis (17). Of non-gliadin components, carbohydrates would be considered a likely candidate,especially as fructans are present in wheat, are poorly absorbed
in the small intestine, and do induce functional gut symptoms(6). However, the gluten used was devoid of FODMAPs. Wheat
proteins are commonly implicated in food hypersensitivity andit must be considered that the induction of symptoms by gluten
in this study might be a wheat-specific phenomenon, and not
gluten specific. Such a finding would have implications for the
dietary restriction that would be necessary in such patients toattain good symptomatic control.
The prevalence of non-celiac gluten intolerance among
patients with functional gut disorders is unknown. Patients inthis study were highly selected because of the frequent failure ofinvestigative work-up by health professionals for celiac diseaseor from self-administered therapy without any investigations atall. Methods to identify these patients are required. At present,
they are restricted to ruling out celiac disease, followed bytrial of a gluten-free diet, followed by rechallenge. Better diagnostics will only derive from understanding the mechanism of© 2011 by the American College of Gastroenterology

action and what component of gluten is actually inducing thesymptoms.
In conclusion, this double-blind, randomized, placebo-controlled rechallenge study of patients with IBS without celiac disease
who have reached satisfactory levels of symptom control with agluten-free diet shows that gluten is indeed a trigger of gut symptoms and tiredness. No evidence for intestinal inflammation ordamage, or for latent celiac disease was found to offer a mechanistic explanation for symptom deterioration caused by gluten. How
common non-celiac gluten intolerance is, how it can be reliably
identified, and what its underlying mechanisms are, warrant further evaluation.
ACKNOWLEDGMENTS

We acknowledge the sponsorship of Well and Good P/LClassic Bread Mix and Muffin Mix by Sam Barak (Noble Park,
VIC, Australia); Gemtec Wheat Gluten by Manildra Group Milling(Manildra, NSW, Australia); Evan Perrett (Bakers Delight Centreof Excellence, Box Hill Institute of TAFE, Box Hill, VIC, Australia),
and Lynette McCarthy (Gluten Free Gourmet, Mornington,
VIC, Australia).
CONFLICT OF INTEREST

Guarantor of the article: Peter R. Gibson, MD, FRACP.
Specific author contributions: Enrolment of patients and assessmentof recruited patients: Jessica R. Biesiekierski, Evan D. Newnham,Peter M. Irving, Jacqueline S. Barrett, and Melissa Haines; analysisof data: Jessica R. Biesiekierski, Evan D. Newnham, and Peter M.
Irving; writing of paper: Jessica R. Biesiekierski, Evan D. Newnham,Peter M. Irving, Jacqueline S. Barrett, Melissa Haines, James D.
Doecke, Susan J. Shepherd, and Jane G. Muir; approval of the final
draft: all authors; design of study: Peter M. Irving, Susan J. Shepherd,
and Peter R. Gibson; statistical analysis: James D. Doecke; recruitment of patients: Susan J. Shepherd; supervision of J.R.B.: Jane G.
Muir; oversight of study: Jane G. Muir and Peter R. Gibson;
mentoring and writing of study: Peter R. Gibson.
Financial support: This study was supported by the Helen
Macpherson Smith Trust, the National Health and Medical
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Table 2. Celiac serology, intestinal permeability, and C-reactive protein results before and during therapy with gluten or placebo, shown as
median (range), and changes in those indices, shown as mean (s.e.m.)

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Research Council (NHMRC) of Australia, and the Vera and Les
Erdi Foundation. Susan J. Shepherd was supported by a Dora Lush
Scholarship from the NHMRC of Australia. Jacqueline S. Barrett
was supported by Sir Robert Menzies Memorial Research Scholarship. Evan D. Newnham was supported by a scholarship from the
Gastroenterological Society of Australia. Jessica R. Biesiekierski was
supported by a scholarship from the Eastern Health Clinical School,
Box Hill Hospital, the Faculty of Medicine, Nursing and Health
Sciences, Monash University.
Potential competing interests: Susan J. Shepherd has publishedcookbooks directed toward issues of dietary fructan restrictions,fructose malabsorption, and celiac disease. She has also publishedshopping guides for low FODMAPs and low fructose and fructan
foods.

Study Highlights
WHAT IS CURRENT KNOWLEDGE

3The existence or non-existence of non-celiac gluten
intolerance is contentious.
3Although wheat is one of the most common factors inducing
gut symptoms and an existing public perception that gluten
intolerance in individuals without celiac disease iscommon, the published scientific literature is devoid ofwell-designed studies that directly investigate this area.

WHAT IS NEW HERE

3This study is significant as it provides for the first time,
high-quality evidence that gluten itself may trigger gut
symptoms and fatigue in individuals who do not have celiac
disease.

3The findings will stimulate a large body of subsequent workto, e.g., confirm the effect, determine mechanisms, and to
define the prevalence of this condition in the community.

3The impact on clinical practice will be in the use of dietary
gluten restriction in the management of patients withfunctional gut symptoms.

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