e-spen, the European e-journal of Clinical Nutrition and Metabolism

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1 e-spen, the European e-journal of Clinical Nutrition and Metabolism 4 (2009) e315 e320 Contents lists available at ScienceDirect e-spen, the European e-journal of Clinical Nutrition and Metabolism journal homepage: Original Article Oats in a strictly gluten-free diet is associated with decreased gluten intake and increased serum bilirubin q Astrid Løvik a, *, Anne Ulla Gjøen b, Lars Mørkrid c, Vigdis Guttormsen d, Thor Ueland e, Knut E.A. Lundin a,f a Department of Medicine, Oslo University Hospital, Rikshospitalet, Sognsvannsveien 20, N-0027 OSLO, Norway b Department of Medicine, Oslo University Hospital, Ullevaal, Norway c Department of Medical Biochemistry, Oslo University Hospital, Rikshospitalet Oslo, Norway d Akershus University College, Lillestrøm, Norway e Institute for Medical Research, Oslo University Hospital, Rikshospitalet, Oslo, Norway f Institute of Immunology, Oslo University Hospital, Rikshospitalet, Oslo, Norway article info summary Article history: Received 4 May 2009 Accepted 19 September 2009 Keywords: Bilirubin Dietary fibre intake Gluten-free diet Gluten intake Oats Nutrient intake Background and aims: To investigate clinical, nutritional, and laboratory parameters during oats challenge and to cross-sectionally examine the levels of plasma bilirubin in a cohort of coeliac disease patients. Methods: Intestinal biopsies, gastrointestinal symptom registration, standard blood tests, anthropometry, weighed food records, and calculations of nutrients and gluten were performed in 19 patients before and after oats challenge (50 g/day). To further investigate bilirubin levels, blood samples were analysed in 136 patients (82 oats consumers) and 141 healthy controls. Results: Three of 19 patients developed gastrointestinal symptoms during oats challenge. Compliance with gluten-free diet was good. Dietary fibre intake increased after oats inclusion (p < 0.024) and met recommended levels. With three exceptions, calculated gluten intake remained within the proposed safe level and decreased after oats intake. Total P-bilirubin increased after oats challenge (p < 0.003). In the cohort study a tendency of higher bilirubin levels was seen among oats consumers (17 g/day). Conclusions: Gluten-free oats were associated with recommended levels of dietary fibre, and calculated gluten intake was reduced by more than 50%. Gluten-free oats induced higher levels of the anti-oxidant bilirubin in treated coeliac disease patients. This may have positive health implications. Ó 2009 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved. 1. Introduction Coeliac disease is a chronic inflammatory condition of the small intestine caused by an inappropriate immune response to cereal proteins. The immune response in turn leads to the villous changes typical of coeliac disease with crypt hyperplasia, villous atrophy and subsequent malabsorption. 1 The disease is precipitated by Abbreviations: CD, coeliac disease; GFD, gluten-free diet; NNR, Nordic Nutrition Recommendations; BMI, body mass index; NA, not applicable. q Part of the work has been presented at the 13th International Coeliac Disease Symposium, Amsterdam, the Netherlands April 6 8, 2009 and at the 28th Congress of the European Academy of Allergy and Clinical Immunology, Warszawa, Poland June 6 10, * Corresponding author. Tel.: þ ; fax: þ addresses: astrid.lovik@rikshospitalet.no (A. Løvik), AnneUlla. Gjoen@ulleval.no (A.U. Gjøen), lars.morkrid@medisin.uio.no (L. Mørkrid), vigdis.guttormsen@hotmail.no (V. Guttormsen), thor.ueland@medisin.uio.no (T. Ueland), k.e.a.lundin@medisin.uio.no (K.E.A. Lundin). intake of wheat, rye, barley and all closely related members of the tribe Triticeae of the grass (Pooideae) subfamily. Although oats belong to the same subfamily as Triticeae, it is considered a separate tribe of Pooideae. Other species of grains like maize and rice are well tolerated. The diet excluding wheat, rye and barley, is generally referred to as a gluten-free diet. During decades, controversy has remained with respect to oats. The recommendation of excluding oats from the gluten-free diet common until the beginning of the 1990-es, was based on early feeding experiments. More recently, several studies have concluded that oats are tolerated by most coeliac patients. 2 4 However, withdrawals among the oats consumers in randomised studies and more abdominal complaints among oats consuming coeliac patients, implied some uncertainties concerning oats in gluten-free diet. 5,6 Indeed, recent data suggest that oats are safe for the majority of coeliac patients, 7,8 but also that oats intolerance may exist among a subset of adult patients. 9,10 On this background it is appropriate to investigate clinical and nutritional aspects of gluten-free oats in a standard gluten-free /$ - see front matter Ó 2009 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved. doi: /j.eclnm

2 e316 A. Løvik et al. / e-spen, the European e-journal of Clinical Nutrition and Metabolism 4 (2009) e315 e320 diet. The content of macro- and micro-nutrients, dietary fibre and anti-oxidants in oats is abundant. 11 It is generally believed that these beneficial effects of oats are not out-weighted by low phytase activity and the frequent contamination by mycotoxins. 12 The aims of the study were to determine the possible gluten contamination of ecologically cultivated rolled oats, and to investigate clinical, biochemical and histological tolerance to oats in well-treated patients with coeliac disease. First, we performed an oats challenge study on 19 adult patients and concomitant analysis of gluten content in rolled oats. 9 The present paper reports the nutritional status of the patients and the composition of the glutenfree diet before and at 12 weeks of oats challenge. Further we examined for alterations in a range of commonly performed blood tests. This part of the study was extended by the cross-sectional determination of serum bilirubin levels in a diet treated cohort of coeliac disease patients, stratified according to oats intake, and in a cohort of healthy controls. 2. Material and methods 2.1. Patients Nineteen adults (two males) diagnosed by gastroduodenal biopsies and successfully treated with gluten-free diet without oats (Table 1), recruited from the out-patient clinics at Rikshospitalet and Ullevaal University Hospital gave informed written consent for participating in an oats challenge study (study population I). Eight participants had been treated for five years or more and the remaining patients for at least two years. Some patients were treated for additional diseases; hypothyroid disease and Sjøgrens disease (n¼1), hypothyroid disease and diabetes mellitus type I (n¼1), hypothyroid disease and constipation (n¼1) and chronic constipation (n¼1). Due to documented or self-reported food hypersensitivity, eleven patients abstained from one or several foods (milk, shrimps, citrus, eggs, rice, nuts, green apples) in addition to gluten containing foods. Exclusion criteria were dermatitis herpetiformis, diabetes mellitus with complications, vegetarian diets, inflammation modulating medications or organ transplants. To further investigate serum bilirubin levels in a larger population of oats consuming and oats non-consuming coeliac patients, frozen serum from 104 females and 32 males, recently participating in a study of anti-avenin-iga levels 13 were analysed (study population II). The patients were recruited among local support group members of the Norwegian Coeliac Association (Table 1). Inclusion criteria were: age 18 years or older, diagnosed through intestinal biopsies, gluten-free diet for the last two years and, for oats consumers, a regular intake of gluten-free oats for the last six months. Exclusion criteria were similar as for population I (oats challenge study) except for the inclusion of patients with dermatitis herpetiformis (eight patients). The participants Table 1 Population characteristics. Variables CD population I median (min max) CD population II median (min max) Controls a median (min max) Subjects Male/Female 2/17 32/104 35/104 b Age, years 44 (27 57) 50 (20 87) 50 (11 79) BMI, kg/m 2 24 (19 31) 24 (17 39) 23 (17 33) c Years since diagnosis 3 (2 30) 10 (2 50) NA Years on GFD 3 (2 30) 10 (2 50) NA a Controls for population II. b Excluded: unrecognized CD (female), uncertainty concerning oats intake (male). c N ¼ 103. documented dietary compliance and gluten-free oats intake by food frequency questionnaire, and recorded oats intake (household measures) during three weeks preceding blood sample collection. An age-matched control group of 141 individuals (oats consuming and oats non-consuming) were recruited from the hospital staff and their healthy family members (Table 1). The amount of regular oats intake was not asked for. However, the average whole sale data on oats (rolled oats and oats flour) in the healthy population was 7.8 g per day in Norway at the time the blood samples were drawn. 14 For technical reasons, only 136 of 139 control samples collected could be analysed for bilirubin content. Further, three oats non-consuming female controls were excluded because of the small number Gluten-free oats Rolled oats were purchased from Norsk Bokhvetedyrkerlag, Arendal, Norway, and examined by ELISA for gluten (Ridascreen gluten test, Food Diagnostics, Oslo, Norway) as described previously Symptom registration, serological tests and blood tests In the challenge study, recordings of gastrointestinal symptom scores were made at six and twelve weeks into the test (intensity of abdominal pain, distension, flatulence and number of bowel movements). Intestinal biopsies were taken before and at 12 weeks of oats inclusion as reported elsewhere. 9 Investigations included quantitative testing for IFN-g. 15 Serological tests (AGA, EMA, ttg) and 13 C-D-xylose breath test were performed before and at 12 weeks of oats challenge. 16 Standard blood tests (B-haemoglobin, P-sodium, P-potassium, P-calcium, P-phosphorous, P-creatinine, P-total protein, P-albumin, P-total bilirubin, P-ASAT, P-ALAT, P-phosphatase alkaline, P-total cholesterol, P-HDL-cholesterol, S-folate, erytrocytefolate, S-vitamin B 12,P-iron, P-ferritin, P-transferrin, S-FT4, S-TSH, P-PTH) were analysed at Rikshospitalet before and at 12 weeks of oats inclusion. Serum levels of selenium and FT3 analysis in population I and total serum bilirubin analysis in population II were done at a commercial laboratory Anthropometric assessments Anthropometric assessments were made by body weight recordings (Seca , electronic scale, Vogel & Halke Gmbh & Co) without shoes and with light clothing, by height recordings (Seca 242, fixed electronic measuring rod, Vogel & Halke Gmbh & Co), by triceps skinfold measurements (Harpenden skin fold calliper, Baty international. RH15 9LB, England), and by arm, waist and hip circumference recordings. Mid-arm-muscle circumference was calculated using the formula: Arm-muscle circumference (cm) ¼ Arm circumference (cm) (0.314 triceps skin fold (mm)) Dietary records, nutrient calculations and estimation of gluten levels Instructions on weighed food records were given and patients were advised to maintain their usual gluten-free diet. Patients recorded what they ate and drank by means of record forms and digital scales (Tefal electronic scale/philips electronic kitchen scale, automatic switch-off, HR2383) for four consecutive days, including one Saturday or Sunday, before and during the oats containing period. Patients were told to record the recipes and the names of products that they used. Control of dietary compliance was performed by a 4-day weighed food record at week six of the study. Calculations of nutrients and dietary fibre were accomplished by

3 A. Løvik et al. / e-spen, the European e-journal of Clinical Nutrition and Metabolism 4 (2009) e315 e320 e317 program Mat på data, 3.0c 1997 and Reorg, 3.0c Intake of vitamin B 12 was calculated by means of the Norwegian food composition table. No corrections were made for preparation loss of nutrients. In order to estimate the maximum level of gluten exposure before and after oats inclusion, wheat starch containing flours and wheat starch-based pre-made foods labelled as glutenfree, were taken into consideration. The calculated values were based on analyses reported by Collin and collaborators. 17 Underreporting of energy intake (EI) was assessed by calculating basal metabolic rate (BMR) 18 and BMR-factor (EI/BMR). EI/BMR-ratio before and during oats intake (week 12) was compared with the relevant group reference value Ethical considerations The studies were approved by the Regional Ethics Committee for medical research, Helseregion I. Signed informed consent was obtained from each patient Statistical analysis SPSS-PC, version 11.0 (Statistical Package for the Social Sciences) and Excel software for Windows Xp were used for the analysis of data in the challenge study. Version 16.0 of SPSS was applied for analysis of the bilirubin data. Paired tests were used to compare data before and after oats challenge (t-test for normally distributed data, Wilcoxon signed ranks sum test for non-parametric data). Correlations were assessed by Spearman s correlation coefficient. Bilirubin data were log-transformed to obtain an approximately normal distribution. Multiple regression analysis was applied for testing the variations of bilirubin levels between oats consuming and oats non-consuming patients in the cohort study. P values (2-tailed) <0.05 were considered significant. 3. Results 3.1. Purity of oats Pre-challenge analysis of gluten in 145 samples of rolled oats showed no gluten contaminated samples, with one exception Challenge study clinical events, symptom scores and compliance Nineteen adult coeliac disease patients agreed to ingest oats in the open-label study over 12 weeks. Eighteen of nineteen patients completed the challenge and kept a strict gluten-free diet which included adequate amounts of gluten-free rolled oats median (Q1, Q3) at week six and week 12 was 50 (50, 59) and 50 (50, 55) grams respectively, p < 0.78). Five patients did not use wheat starch containing gluten-free foods at all in their diet. Three patients failed to complete dietary records by week 12 and two patients failed by week six. Still these patients reported to have consumed oats as instructed. Rolled oats were used as cold or hot cereal for breakfast by 14 patients. Four patients split the daily oats intake between breakfast cereals and oats containing bread. One patient had oats partly from bread and partly as dry snacks. Most of the patients (12 of 19), had positive opinions on glutenfree oats, and total symptom scores for the study group did not change during the study period. Three patients developed gastrointestinal symptoms when eating oats. One patient (no 19) left the study after two weeks and recorded no dietary data or specific symptoms. The second patient (no 15) managed to complete the challenge. The third patient (no 5) developed an unspecific rash, mild gastrointestinal symptoms and partial villous atrophy (Marsh 3a). Withdrawal of oats for 12 weeks normalised the villous architecture. After re-introduction of oats, the patient developed a severe rash and abdominal pain. A repeat biopsy at this stage showed subtotal villous atrophy. 9 Although the number of bowel movements for this patient more than doubled by week 12, her subjective experience at this point was abdominal wellness and increased satiety Anthropometric and nutritional data in study population I There were no changes in anthropometric data when genders were assessed separately. Based on BMR-factor, the patients underreported energy intake median (Q1, Q3) before 9.9 MJ (7.2, 11.1) and after challenge 7.7 MJ (7.1, 9.3) by 4 27%, in average 9% during oats challenge. In women s data there appeared a trend of increased dietary fibre content after oats inclusion (2.2 vs 3.2 g/mj per day, p < 0.087). Further there was a trend for reduced energy intake with accompanying reductions in calculated selenium (p < 0.005), vitamin A (p < 0.023) and niacin (p < 0.028) levels during oats challenge compared to pre-challenge levels (Table 2). The intake of vitamin D and folate was below recommendations in 11 of 15 patients before and by 12 weeks of oats challenge (Table 2). The level of vitamin D was below the lower recommendation (2.5 mg/day) in five patients before and in three patients during the challenge. The folate intake exceeded the lower recommended level (100 mg/day) in all patients. On a regular basis three patients used cod liver oil (included in the calculations) or multi-vitamin supplements (not included in the calculations). Three other patients had casual intakes of supplements (not included in the calculations) Intake of grain products and gluten in study population I The main gluten contributor was bread, 80% containing wheat starch before and 76% after oats challenge (Table 3). Females Table 2 Nutrients per MJ before and after oats challenge, CD population I (male/ female ¼ 2/13). Variable Baseline median (Q1, Q3) Week 12 median (Q1, Q3) NNR 2004 MJ/day 9.9 (7.2, 11.1) 7.7 (7.1, 9.3) Protein, g 8.2 (7.5, 10.8) 9.2 (8.3, 9.4) E% 14 (13, 18) 15 (14, 16) Fat, g 9.3 (7.9, 10.4) 9.2 (8.0, 10.3) E% 36 (30, 39) 35 (30, 40) Saturated fat, g 3.6 (3.1, 4.1) 3.6 (2.8, 3.9) Monounsaturated fat, g 3.0 (2.4, 3.7) 3.1 (2.4, 3.8) Polyunsaturated fat, g 1.5 (1.0, 1.8) 1.7 (1.4, 1.8) Carbohydrate, g 27.0 (24.0, 31.2) 28.2 (23.3, 31.5) E% 47 (41, 53) 48 (41, 54) Dietary fibre, g 2.7 (1.9, 3.3) 3.2 (2.7, 3.8) a Alcohol, g 1.1 (0, 1.3) 0.5 (0, 1.3) E% 3 (0, 4) 2 (0, 4) max 5 Calcium, mg 80 (70, 108) 101 (81, 122) 800 Iron, mg 1.8 (1.6, 2.1) 1.8 (1.5, 2.1) F/M:15/9 Zink, mg 1.2 (0.9, 1.4) 1.2 (1.1, 1.3) F/M: 7/9 Selenium, mg 6 (5, 9) 6 (4, 7) F/M:40/50 Vitamin A (RE), mg 116 (87, 268) 114 (63, 140) F/M:700/900 Vitamin D, mg 0.53 (0.21, 0.7) 0.63 (0.34, 1.04) 7.5 Vitamin E (TE), mg 1.2 (0.9, 1.6) 1.3 (1.0, 1.7) F/M: 8/10 Vitamin C, mg 14.2 (10.3, 16.8) 17.1 (11.5, 28.5) 75 Thiamin, mg 0.22 (0.15, 0.29) 0.21 (0.17, 0.35) F/M:1.1/1.4 Riboflavin, mg 0.20 (0.17, 0.37) 0.21 (0.16, 0.41) F/M:1.3/1.7 Niacin, mg 3.88 (3.2, 4.5) 3.60 (3.28, 4.33) F/M:15/19 Vitamin B 6, mg 0.2 (0.1, 0.2) 0.2 (0, 0.2) F/M:1.2/1.6 Folate, mg 25 (23, 34) 26 (23, 33) 300 b Vitamin B 12, mg 0.6 (0.4, 0.9) 0.7 (0.5, 0.9) 2.0 a p < (Wilcoxon signed ranks test). b Women of reproductive age: 400 mg.

4 e318 A. Løvik et al. / e-spen, the European e-journal of Clinical Nutrition and Metabolism 4 (2009) e315 e320 consumed 17 g of bread/mj before and 18 g/mj at week 12 of oats introduction. Estimated gluten intake was reduced by more than 50% after oats inclusion (Table 4). Although the individual gluten intake was within recommended safe level (30 mg/day) 17 in most patients, three patients consumed higher amounts (38 45 mg/day) before oats challenge. There was no detectable association between gluten intake and functional mucosal area (modified xylose test), levels of gluten antibodies, interferon-g, bilirubin level or symptom scores Blood tests Compared to the pre-challenge level, we observed a reduction of median, Q1, Q3 P-total proteins (73, 70, 74 vs 75, 72, 77 g/l) (p < 0.012), P-albumin (42, 40, 43 vs 45, 43, 45 g/l) (p < ), P-total cholesterol (4.9, 4.2, 5.6 vs 5.3, 4.4, 6.2 mmol/l) (p < 0.011), S-folate (10.3, 7.9, 15.3 vs 13.6, 10.7, 22.1 nmol/l) (p < 0.002), P-ALAT (18, 13, 25 vs 24, 15, 37 U/L) (p < 0.009), P- phosphorous (1.0, 0.8, 11 vs 1.1, 1.0, 1.2 mmol/l) (p < 0.012) and an increase in total P-bilirubin (11, 8, 15 vs 7, 6, 9 mmol/l) (p < 0.003) (Fig. 1) and P-ASAT (18, 13, 25 vs 10, 7, 15 U/L) (p < 0.006). The differences were strongly significant also in women s data analysed separately (data not shown) Cross-sectional study We next analysed the serum levels of bilirubin in oats consumers (n ¼ 82) and oats non-consumers (n ¼ 54) recruited from local coeliac support groups and in 133 oats consuming healthy controls. The bilirubin values among the controls were not different from the Nordic reference interval for healthy individuals (NORIP 2000), indicating a proper storage of blood samples before analysis. 20 The bilirubin level in study population II, was higher in patients vs controls, men and women combined (p < ) and in women separately (p < ) (Fig. 2). According to food frequency questionnaire, the patients adhered to a strict gluten-free diet, 94% using wheat starch containing products. The median (range) recorded intake of gluten-free rolled oats during the previous three weeks, was 17 (123) g/day. There was an age difference (52 vs 46 years) between the oats consuming and the oats non-consuming female patients (p ¼ 0.036). Oats consumers were diagnosed at a higher age (44 vs 34 years) (p < 0.002) and had less years of dietary treatment at the time of the study (8 vs 14 years) (p < 0.002) as compared to the oats non-consumers. There was no association between body mass index and bilirubin level. The intake of soy proteins in the diet was assessed as negligible. Oral contraceptives and menstrual cycle among the females was not investigated. Table 4 Calculated a daily gluten intake in CD population I. Gluten intake Before median (Q1, Q3) N ¼ 18 Week 6 median (Q1, Q3) N ¼ 16 Week 12 median (Q1, Q3) N ¼ 15 Gluten, mg/day 15 (0, 26) 6 (0, 26) 0 (0, 17) a Theoretical values based on data reported by Collin et al. 17. P value ¼ 0.012, before and week 12, Wilcoxon signed ranks test. P value ¼ 0.012, week 6 and week 12, Wilcoxon signed ranks test. Here we show that introduction of gluten-free oats in a group of nutrition motivated patients, resulted in a reduction of gluten exposure, increased dietary fibre intake, and, intriguingly, increasing levels of serum bilirubin. The intake of gluten-free products with bread as the dominating component, was at the same level as documented in a questionnaire based consumption survey among coeliac disease patients. 21 The pre-challenge bread consumption among females (data not shown) in our study was 65% higher than recorded in a Swedish gluten-free diet without oats. 22 The E% from fat remained higher (37 E% vs 34 E%) compared to the Swedish study and might reflect the regular use of bread spread and toppings in the Norwegian bread meals. The difference in fat content was even more distinct compared to Størsrud s oats challenge study (32 E% from fat). Further we found the same trends towards decreased fat and increased carbohydrate intake after oats inclusion. 23 Although the patients reached the recommended level of dietary fibre after oats introduction, 24 the variation in fibre intake was wide. The patient who got serious gastrointestinal symptoms and developed oats intolerance, had very low fibre intake before challenge (0.8 g/mj), an observation also detected among drop-outs in Størsrud s study. 4 The other patient (no 15) complaining of symptoms after oats inclusion, ingested 2.7 g fibre/mj. However, the retained amount of fibre was less due to occasional vomiting. Coeliac disease patients considering oats inclusion should have dietary fibre levels checked, should be informed of possible side effects, and advised to a gradual introduction of oats. Total gluten exposure from gluten-free products and from accidental gluten contamination or other regular gluten sources (medication, cosmetic products, soluble wheat proteins, inhalation of instant wheat proteins, etc) is not known. The Norwegian Food Control Authority has no running control of gluten-free products. The long term safe limit of gluten intake is assessed to be between 10 and 100 mg/day. 25 A gluten intake of 30 mg/day has been shown to be safe when correlated to histology. 17 Regardless of the level of 4. Discussion The role of oats in the gluten-free diet is debated. Less attention has been paid to the impact of oats on nutritional status, dietary intake and possible effects on risk-factors for concomitant diseases. Table 3 Intake of grain products before and after oats challenge (week 12). Grain products Before median (Q1, Q3) N ¼ 18 Week 12 median (Q1, Q3) N ¼ 15 P-value a Gluten-free bread, breakfast 169 (128, 226) 145 (90, 200) cereals, g/day Gluten-free cookies, waffles, 33 (0, 79) 0 (0, 30) pastries, sweet rolls, g/day Gluten-free pizza, pasta (dry), 31 (9, 78) 25 (15, 28) n.s. g/day Rice, corn, gluten-free oats, 42 (0, 76) 66 (50, 125) g/day Total grain products, g/day 303 (194, 361) 231 (181, 354) n.s. a Wilcoxon signed ranks test. Fig. 1. Increase in total P-bilirubin values after oats challenge (n ¼ 18), p < 0.003, Wilcoxon signed ranks sum test. One patient (no 15) who showed lower value after challenge, had occational vomiting. Another patient (no 10) had unchanged value after challenge.

5 A. Løvik et al. / e-spen, the European e-journal of Clinical Nutrition and Metabolism 4 (2009) e315 e320 e319 P -bilirubin ( µ mol/l) patient with oats patient without oats controls Fig. 2. P-bilirubin among CD patients (population II) and healthy controls (individual P-bilirubin levels and retransformed group medians from lg values). Estimates: median values (Q1, Q3) were 10 (8,13) mmol/l in female patients with oats (C), 9 (6,12) mmol/l in female patients without oats (B), 13 (9,18) mmol/l in male patients with oats (-) and 11 (8,16) mmol/l in male patients without oats (,). The values among female controls (C) were 7 (5,10) mmol/l and in male controls (-) values were 8 (6,12). There was no statistically significant difference in bilirubin values between patients with oats vs patients without oats (p ¼ 0.133, ANOVA & post-hoc LSD). However, when corrections were made for gender and age (multiple regression analysis) there was a tendency for higher bilirubin values among patients with oats (p ¼ 0.08). gluten contamination, a more than 50% reduction of gluten intake after introduction of gluten-free oats, as estimated in our calculations, may represent an improved treatment quality for many patients. Changes in plasma bilirubin levels in coeliac disease patients after oats challenge have not been investigated so far. Increased levels of plasma bilirubin were first seen in a small cohort eating 50 g of gluten-free oats per day. Based on the data from the oats challenge study, the developed hypothesis was that oats consumption increased serum bilirubin independently of gender. Multiple regression analysis of the data and adjustments for gender and age confirmed a trend of higher bilirubin values among oats consuming patients in the large cohort (population II), even if the intake of oats was one third of the intake in the small cohort. This difference might indicate an oats induced change. The mechanisms behind the observation could be multiple (prolonged intestinal transit time, enzyme inductions, changes in entero-hepatic passage, increased permeability, etc). Most likely longer transit time with increased bowel reabsorption of bilirubin could be the reason for higher levels. Peraaho et al. 6 found more gastrointestinal symptoms in well-treated patients consuming oats compared to patients on a regular gluten-free diet. Whether these patients also had prolonged transit time, was not investigated. Further, the delayed small bowel transit in untreated coeliac disease patients is documented. 26 Contributing factors to increased or delayed bowel transit could be alcohol intake and oral contraceptives. 27 In healthy subjects, lower serum bilirubin levels are associated with endothelial dysfunction and increased carotid intima-media thickness, while moderately increased serum bilirubin levels are connected to reduced risk for atherosclerosis. 28 Whether serum bilirubin in oats consuming coeliac disease patients is an independent and inversely related predictor of atherosclerosis has yet to be studied. So far there are no documented clear nutritional advantages of oats in gluten-free diet, except for increased dietary fibre intake. Results from our pilot study of nineteen patients and supportive evidence from a large cohort of patients, suggest increased levels of bilirubin after oats intake. To confirm this finding, controlled and randomised studies in oats consuming and oats non-consuming coeliac disease patients have to be performed. 5. Statement of authorship All authors have made substantial contributions to the study design, acquisition, analysis and interpretation of data, or drafting and revising the article and the final approval of the submitted version. Conflict of interest No author has any conflict of interest with this study, either financial or personal. Acknowledgements We wish to thank the following persons for invaluable support during the study: Jorunn Bratlie, Institute for Medical Research, Oslo University Hospital, Rikshospitalet, Elin Bjørge Løken, Department of Nutrition, University of Oslo, Kerstin Trygg, Department of Nutrition, University of Oslo, Stig Tollefsen, Institute of Immunology, Oslo University Hospital, Rikshospitalet, and Thor Willy Ruud Hansen, Department of Paediatric, Oslo University Hospital, Rikshospitalet, Oslo. References 1. 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