Gastrointestinal Effects of Eating Quinoa ( Chenopodium quinoa Willd.) in Celiac Patients

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1 7 ORIGINAL CONTRIBUTIONS nature publishing group see related editorial on page x Gastrointestinal Effects of Eating Quinoa ( Chenopodium quinoa Willd.) in Celiac Patients Victor F. Zevallos, PhD 1, L. Irene Herencia, PhD, Fuju Chang, MD, PhD 3, Suzanne Donnelly, PhD 1, H. Julia Ellis, PhD 1 and Paul J. Ciclitira, MD, PhD 1 OBJECTIVES: METHODS: Celiac disease is an enteropathy triggered by dietary gluten found in wheat, rye, and barley. Treatment involves a strict gluten-free diet (GFD). Quinoa is a highly nutritive plant from the Andes that has been recommended as part of a GFD. However, in-vitro data suggested that quinoa prolamins can stimulate innate and adaptive immune responses in celiac patients. Therefore, we aimed to evaluate the in-vivo effects of eating quinoa in adult celiac patients. Nineteen treated celiac patients consumed 5 g of quinoa every day for weeks as part of their usual GFD. We evaluated diet, serology, and gastrointestinal parameters. Furthermore, we carried out detail histological assessment of 1 patients before and after eating quinoa. RESULTS: Gastrointestinal parameters were normal. The ratio of villus height to crypt depth improved from slightly below normal values (.8:1) to normal levels (3:1), surface-enterocyte cell height improved from 8.7 to 9.77 μ m and the number of intra-epithelial lymphocytes per 1 enterocytes decreased from 3.3 to 9.7. Median values for all the blood tests remained within normal ranges, although total cholesterol ( n = 19) decreased from 4. to 4.3 mmol / l, low-density lipoprotein decreased from.4 to.45 mmol / l, high-density lipoprotein decreased from 1.8 to 1.8 mmol / l and triglycerides decreased from.8 to.79 mmol / l. CONCLUSIONS: Addition of quinoa to the GFD of celiac patients was well tolerated and did not exacerbate the condition. There was a positive trend toward improved histological and serological parameters, particularly a mild hypocholesterolemic effect. Overall, this is the first clinical data suggesting that daily 5 g of quinoa for weeks can be safely tolerated by celiac patients. However, further studies are needed to determine the long-term effects of quinoa consumption. Am J Gastroenterol 14; 19:7 78; doi:1.138/ajg ; published online 1 January 14 INTRODUCTION Wheat, rye and barley are cereals, widely consumed around the world, that contain proteins capable of inducing inflammation and histological deterioration of the small intestine in individuals affected by celiac disease (CD) ( 1, ). Thus, celiac patients must follow a strict and permanent gluten-free diet (GFD) in order to avoid gastrointestinal and related symptoms. The market for gluten-free foods products has shown a compound annual growth rate of 8 % over the 8 1 period, reaching $ 4. billion in 1 in the US market ( 3 ). Maize and rice are the two main ingredients of gluten-free products, reflecting on palatability, price, and nutritional value when compared with their gluten containing counterparts. Thus, alternative gluten-free ingredients that can improve any of those qualities are a welcome addition. Guidance about toxicity of new gluten-free ingredients can be obtained from taxonomic classification (4), and in-vitro experiments but ultimately in-vivo challenge is necessary to confirm the suitability of new gluten-free products. The safety of oats as part of the GFD has been tested using in-vitro and in-vivo feeding experiments. Janatuinen et al. (5 ) suggested that 5 7 g per day can be safely added to the GFD of celiac patients. Kemppainen et al. ( ) indicated that even up to 1 g did not cause alteration of duodenal biopsies, endomysial antibody levels and general well-being in celiac patients. However, activation of CD with morphological deterioration after oats challenge was also shown in a case report ( 7 ). Recently, Cooper et al. (8 ) evaluated the 1 King s College London, Division of Diabetes and Nutritional Sciences, Department of Gastroenterology, The Rayne Institute (KCL), Gastroenterology Laboratory, London, UK ; Departamento de Producci ó n Vegetal, Universidad Polit é cnica de Madrid, Spain ; 3 Departament of Histopathology, St Thomas Hospital, London, UK. Correspondence: Paul J. Ciclitira, MD, PhD or Victor F. Zevallos, PhD, King s College London, Division of Diabetes and Nutritional Sciences, Department of Gastroenterology, The Rayne Institute (KCL), Gastroenterology Laboratory, St Thomas Hospital, Westminster Bridge Road, London SE1 7EH, UK. paul.ciclitira@kcl.ac.uk or vfzevallos@yahoo.co.uk Received 1 August 13; accepted 5 November 13 VOLUME 19 FEBRUARY 14

2 Gastrointestinal Effects of Eating Quinoa 71 Figure 1. Typical Andean landscape, depicting the cultivation of quinoa at Kancharij Jatun Chamaca (Peruvian highlands). effect of 5 g per day for 1 year in a cohort of 4 patients with no evidence of toxicity observed despite detailed immunohistological examination. Quinoa is an Andean crop ( Figure 1 ) from the Chenopodiaceae family that is taxonomically very different from gramineae (wheat, barley, and rye). Quinoa is cultivated in a wide range of environments from latitude North in Colombia to 4 latitude South in Chile, and from sea level to an altitude of 3,8 m above sea level. The main areas of cultivation include Bolivia, Peru, Ecuador, Colombia, northern Argentina, and Chile, former territories of the Inca Empire. The achenium (dried fruit) and young leaves from the quinoa plant have been traditionally eaten for millennia by local populations because of a peculiar combination of strong adaptability to extreme environmental growing conditions (drought, frost, and soil salinity) and excellent nutritional value ( 9 ), which has motivated an increase in worldwide consumption. Recent in-vitro studies suggest that quinoa complies with the Codex Alimentarius nomenclature of gluten-free products (gluten < mg /kg), and is potentially suitable for patients with CD after examining one unknown cultivar in human T cells and organ culture explants ( 1 ). However, we have identified cultivars with putative toxic quinoa peptides that seem to elicit immunological activation of gliadin-specific CD4 + T cells, as well as to elicit secretion of cytokines when cultured with celiac duodenal biopsies ( 11 ). Furthermore, it is likely that quinoa immunogenic peptides within the prolamin and other protein fraction (11S type globulins) are involved in activation of a strong immune response (allergy and anaphylaxis), as already described in two clinical case studies in non-celiac patients ( 1,13 ). In-vivo studies in celiac patients are almost absent with the exception of a retrospective review of dietary history, indicating that quinoa consumption significantly increase the nutrient profile of the GFD ( 14 ). Therefore, the aim of the study was to examine the clinical, histological, and immunological responses of adult celiac patients before and after consuming quinoa as part of their usual GFD. METHODS Food samples Quinoa samples (flakes and grains) labeled as gluten-free were purchased from a company based in the United Kingdom and dedicated exclusively to manufacture gluten-free products. Quinoa flakes were produced and packed in the United Kingdom using various commercial quinoa cultivars harvested in Bolivia. Red quinoa grains from the cultivar Pasankalla were harvested in Peru and packed in the UK by the same company. Quinoa samples had a recommended shelf life of 1 year and were sold in 5 kg bags. Samples were carefully reweighed and repacked into 5 g transparent bags in a research dietetic kitchen using equipment that has not been in contact with gluten. Each participant received 45 transparent bags containing either quinoa grains or quinoa flakes. Purity of quinoa Gluten cross-contamination remains a concern for many glutenfree products; quinoa is less likely to be contaminated in the fields because quinoa cultivated in the Andes is usually sowed alongside other gluten-free Andean grains. However, cross-contamination during transportation and processing is still possible. Therefore, random samples were taken from the top, mid and bottom part of each 5 kg quinoa bag. Gluten content was determined by a non-competitive enzyme-linked immunosorbent assay and western blotting using the monoclonal antibody PN3 (anti-a gliadin 31 49) and CDC5 (anti α-gliadin 57 75) as previously described (11), and no evidence of gluten cross-contamination was found. Ethical considerations Ethical approval for the study was obtained from the Ethics Committee at St Thomas National Health Service Trust. London, UK (REC number / Q7 / 83). All subjects involved in the study gave their written informed consent before the oesophagogastroduodenoscopy. All the individuals had been diagnosed with CD (positive serology, clinical symptoms, human leukocyte antigen -DQ or DQ8, and positive histology) and had an oesophagogastroduodenoscopy as part of their clinical management. Recruitment process The present study included adult celiac patients (18 years old or older) that had been diagnosed, based on the presence of subtotal or total villous atrophy of the duodenal mucosa on a diet containing gluten with subsequent improvement of the mucosa villous architecture following introduction of the GFD for at least 1 months. Exclusion criteria were drug or alcohol abuse, mental impairment, refusal to take part, inconclusive diagnosis of CD and any medical condition interfering with the study or constituting a risk to participants. Participants were recruited from a dedicated celiac clinic at St Thomas Hospital. Twenty-one potential participants meeting the inclusion / exclusion criteria were asked to eat quinoa for 14 by the American College of Gastroenterology

3 7 Zevallos et al. Table 1. Demographics of 19 celiac patients participating in the feeding study Table. Demographics of 1 celiac patients participating in the clinical challenge Celiac patients Variables Units Median Min max Participants N 19 Sex Male / female / 17 Age Years GFD Years BMI kg / m HLA DQ / DQ8 19 / BMI, body mass index; GFD, time on a gluten-free diet; HLA, human leukocyte antigen; M / F, male / female. weeks, and provide duodenal biopsies and blood samples at the beginning and at the end of the study. Nineteen patients successfully completed the study. One patient at the beginning of the study withdrew owing to difficulties in cooking. Another subject was withdrawn from the study because of insufficient criteria for a diagnosis of CD. Patient Sex Age GFD BMI HLA number M / F Years Years Kg / m DQ / DQ8 1 F DQ F DQ 3 F DQ 4 M DQ 5 F 7.8 DQ F DQ 7 F DQ 8 F DQ 9 M DQ 1 F DQ n =1 M / 8F 58 (4 7) 9. (1 4) 3 (18 3) 1 DQ BMI, body mass index; GFD, time on a gluten-free diet; HLA, human leukocyte antigen; M / F, male / female. Age, GFD and BMI are expressed as mean (s.d.). Participants Participants for this study were 19 adult celiac patients, males and 17 females, with a median age of 59 years, BMI of 3 kg / m and 9 years on a GFD. All participants had human leukocyte antigen alleles encoding for DQ and provide written informed consent before participating in the study ( Table 1 ). Gastrointestinal symptoms and serological tests were examined in all 19 participants and detailed histological assessment in 1 participants of the same group. Diet Oral and written instructions about the study diet were given to all participants. They were asked to consume 5 g of quinoa (1 pre-weighed bag) every day for weeks as part of their usual GFD and to complete a daily questionnaire indicating whether quinoa was consumed and at which meal time. Participants were free to choose the cooking method but were recommended to consume quinoa flakes for breakfast as porridge or pancakes. Quinoa grains were recommended to be prepared as rice or mixed with rice to be consumed as a side dish or added to salads. Grains could also be cooked in soups or stews. Patients received at least three telephone calls during the study period and were free to contact the investigators at any time. Gastrointestinal symptoms Subjects were given a diary card to record any symptoms of diarrhea, abdominal pain, increased bowel sounds, or vomiting. The severity of their symptoms was graded daily ( for none, 1 for mild, for moderate and 3 for severe). The score for each symptom was recorded each week throughout the study period. Serological tests Celiac antibody screen serology included (immunoglobulin (Ig)A AGA, IgA ttg, IgA, and IgG EMA) were used to monitor compliance with a GFD. Immunoglobulins (IgA and IgG) were used to determine deficiency. Full blood count, liver, and renal profile were used to follow the health status of all the subjects. Iron, vitamin B1, serum folate, and lipid profile were also used to determine any effects of quinoa on their GFD. All the tests were analyzed at St Thomas Hospital hematology and clinical chemistry laboratory before and after the study. Clinical challenge Ten individuals with CD underwent upper gastrointestinal endoscopy at the beginning and end of the study, using an Olympus GIF-T14 scope (Olympus GIF Q end-viewing gastroscope, Tokyo, Japan). Biopsies were obtained from the duodenal bulb to the second part of the duodenum randomly from four quadrants using Jumbo endoscopy forceps (Olympus JR4). The first four biopsies were used for histological analysis; the subsequent three were snap frozen and stored on liquid nitrogen for future use ( Table ). Histological assessment Morphometric measurements were performed on duodenal biopsies and include: ratio of villus height to crypt depth (VH:CD), surface-enterocyte cell height (SECH), and number of intra-epithelial lymphocytes (IEL) per 1 enterocytes. Th e normal range for these parameters are VH:CD between 5:1 and 3:1, SECH between 9 and 34 nm, and IEL < IEL / 1 surface cell enterocytes. CD is normally diagnosed when the VH:CD is < 3:1, SECH is < 9 nm, and IEL > IEL / 1 VOLUME 19 FEBRUARY 14

4 Gastrointestinal Effects of Eating Quinoa 73 enterocytes. Negative alterations of a combination of these parameters can be used as an indicator of exacerbation of the condition. Biopsies of each patient were cut and stained for histological comparison before and after the quinoa challenge. Each morphometric measurement was assessed as follows: the VH:CD ratio in 1 different areas with at least 1 measurements of this ratio per biopsy on hematoxylin and eosin (H & E)-stained slides, the SECH in at least 3 randomly selected enterocytes in the mid-third of villi per biopsy on H & E-stained slides and the IEL in 1 different areas per biopsy stained for CD3 + cells. Microscopical appearance were also assessed on H & E slides and included degree of villous atrophy (DVA) ( 15 ) and mononuclear cell infiltration (MCI) ( 5 ). A score was assigned to the DVA as follows: normal, 1 partial,, subtotal, and 3 total specimens. In partial atrophy, villi were broadened and shortened. In subtotal villous atrophy, villi were more damaged and almost completely absent. In total villous atrophy, no villous projections from the surface mucosa were present. Mononuclear cell infiltration was graded as: none, 1 mild, moderate, and 3 severe. The morphometric measurements were assessed randomly and blindly by an experienced gastrointestinal pathologist (F.C.) at St Thomas Hospital and also by V.F.Z. and H.J.E. Gastrointestinal symptoms Table 3. Blood tests results from 19 celiac patients 5 Figure. Self-reported gastrointestinal symptoms (diarrhea, abdominal pain, increased bowel sounds, and vomiting) were expressed combined per cohort / week during weeks with a hypothetical maximum of 8 per week and a minimum of. Pre Post Normal Blood tests Median (Q1, Q3) Median (Q1, Q3) Range Vitamin B1 (ng / l) 3 (38, 38) 3 (18, 355) Statistical analysis PASW statistics 18, release 18.., and Microsoft Office Excel 7 were used to analyze the data. Paired tests were used to compare data before and after quinoa challenge for each patient, T-tests were used for normally distributed data, and Wilcoxon signed rank tests were used for non-parametric data. P values.5 were considered significant. RESULTS Gastrointestinal symptom scores Nineteen celiac patients were graded daily ( none, 1 mild, moderate, and 3 severe) of gastrointestinal symptoms (diarrhea, abdominal pain, increased bowel sounds, and vomiting). Ten patients did not report any symptoms. Nine patients reported symptoms ranging from mild to moderate during the first weeks of the study and one patient reported mild vomiting ( Figure ). Blood tests Blood samples were taken from 19 participants at the beginning and at the end of the study. All measurements were within the appropriate normal range, except total cholesterol (TC) and lowdensity lipoprotein (LDL), which were slightly higher than the recommended levels ( 1 ) throughout the study ( Table 3 ). Serological celiac antibody screen Celiac antibody screen (IgA AGA, IgA TTG, and IgA and IgG EMA) before and after the study ( Table 4 ) were used to determine compliance. Patient had a high IgA anti-gliadin (AGA) measurement (9 U / ml) before the study that was normalized after the study (7 U / ml), high titers of AGA are an indication of Serum folate ( μ g / l) 9.4 (4.1, 13.5) 9.3 (3.9, 19).5 1 Ferritin ( μ g / l) 4 (5, 79) 38 (3, 7) Triglycerides (mmol / l) Total cholesterol (mmol / l).8 (.,.97).79 (.4, 1.4) < 4. (4.1, 5.4) 4.3 (3.7, 4.8) < 4 HDL (mmol / l) a 1.8 (1.51,.1) 1.8 (1.35, 1.8) > 1 LDL (mmol / l).4 (1.78, 3.8).45 (1.9,.8) < Albumin (g / l) a 4 (44, 48) 44 (43, 47) 4 5 Alkaline phosphate (U / l) 1 (48, 77) 4 (45, 78) Bilirubin ( μ mol / ml) 7 (, 11) 8 (, 14) 1 Alanine transaminase (IU / l) (14, 34) 19 (14, 7) 4 45 Sodium (mmol / l) 14 (14, 14) 14 (14, 14) Potassium (mmol / l) 4.3 (3.8, 4.5) 4.3 (3.9, 4.4) Urea (mmol / l) 4.5 (3.8, 5.3) 5 (3.7, 5.5) Creatinine ( μ mol / l) 58 (5, 71) 58 (44, 7) Iron ( μ mol / l) 1 (15.3,.7) 19. (15., 1.5) 11 9 Hemoglobin (g / dl) 13.1 (1.7, 14.1) 13.5 (1, 13.9) 1 15 HDL, high-density lipoprotein; LDL, low-density lipoprotein. Values are presented in median and quartiles. a P.5 using the Wilcoxon signed rank test. dietary transgression in celiac patients. However, the initial IgA anti-ttg, reliable marker of compliance, was within normal levels and the patient s mucosa improved after quinoa consumption. 14 by the American College of Gastroenterology

5 74 Zevallos et al. Table 4. Normal values of IgA anti-ttg and IgA anti-gliadin range between and U / ml and EMA (IgG and IgA) in treated celiac patients are expected to be negative IgA anti-gliadin IgA anti-ttg EMA (IgG and IgA) Immunoglobulins (U/ ml) (U/ ml) Pos / Neg IgA (g/ l) IgG (g/ l) Patient code Pre Post Pre Post Pre Post Pre Pre Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Mean (s.e.) 11.9 (.3) 9.4 (5.4) 3.8 (.7) 9.7 (.) Neg Neg 1.7 (.) 8.1 (.1) Ig, immunoglobulin; Neg, negative; Pos, positive. Most of the participant in the study had normal values before and after the study. Table 5. Morphometric measurements of 1 patients before and after quinoa challenge Ratio of villous height to crypt depth Surface enterocyte cell height Intraepithelial lymphocyte count VH:CD SECH ( μ m) IEL (%) Patient number Pre Post Pre Post Pre Post Mean (s.d.).8 (.98) 3. (.89) 8.7 (4.5) 9.77 (.45) 3.3 (14.49) 9.7 (13.5) VH:CD, villus height to crypt depth ratio; SECH, surface-enterocyte cell height measurements; IEL, intra-epithelial lymphocytes. Patient 9 also showed an increment of AGA from 7 to 5 U / ml, but IgA anti-ttg was reduced from 3 to U / ml and the mucosa remained within normal levels after the study. Immunoglobulins (IgA and IgG) ( Table 4 ) were used to determine deficiency. Values were within normal levels in all patients but one. Patient 7 had slightly lower values (IgA.5 and IgG 5. g /l) that could indicate deficiency, and as a result measurements from celiac serology screening become unreliable. IgA anti-gliadin antibody has good sensitivity but poor specificity for CD, as it can be positive in healthy individuals or could be an indication of other conditions such as rheumatoid arthritis ( 17 ). Morphometric measurements Duodenal biopsies from 1 patients were assessed randomly and blindly before and after eating quinoa using three morphometric parameters (VH:CD, SECH, and IEL; Table 5 ). Five patients had VOLUME 19 FEBRUARY 14

6 Gastrointestinal Effects of Eating Quinoa 75 VH:CD measurements within normal limits before and after the study. Five patients had VH:CD measurements between 1.9:1 and.3:1 before the study and between 1.5:5 and.8:1 after the study. Four patients had SECH measurements within normal range before and after eating quinoa. Six patients had SECH between 3. and 7.9 μ m before eating quinoa and between 7.3 and 34. μm after eating quinoa. Five patients had normal counts of IEL, five patients had counts ranging from 34 to before quinoa and between 5 and 45 IEL after quinoa challenge. The worst case was patient with reduced VH:CD (from 1.9:1 to 1.5:1), increased SECH (from 3. to 7.3 μ m), and increased IEL (34 41). The best case was patient 1 with increased VH:CD (from.3:1 to.4:1), increased SECH (from 7.91 to 9.44), and decreased IEL (from 4 to 5 % ). Microscopical appearance Microscopical appearance was scored according to the DVA and mononuclear-cell infiltration in 1 patients before and after the study ( Table ). Two patients had normal villi before and after quinoa consumption. Three patients had partial villous atrophy before eating quinoa and normal villi after eating quinoa. Five patients had partial villous atrophy before and after eating quinoa. MCI was normal in four patients before and after the feeding study. Six patients had mild MCI before and after the study and one patient had mild MCI before the study and none after the study. DISCUSSION The addition of quinoa to the GFD of 19 celiac patients was well tolerated and did not exacerbate their CD. We evaluated Table. Degree of villous atrophy ( normal, 1 partial,, subtotal, and 3 total) and mononuclear cell infiltration ( none, 1 mild, moderate, and 3 severe) in 1 patients before and after eating quinoa Degree of villous atrophy Mononuclear cell infiltration Patient Pre Post Pre Post Mean gastrointestinal symptoms, diet, serology, and histological parameters before and after eating 5 g of quinoa for weeks; we found no evidence of disease alteration. Most of the patients reported either no or mild gastrointestinal symptoms during the -week study period, mainly mild abdominal pain during weeks 1 and that disappeared by week 3 and remained absent until week. This might be due to an increase in dietary fiber ( 14 ), as reported in other feeding studies that involved testing the safety of oats, which is also known to have high levels of fiber ( 19 ). Adherence of quinoa consumption reached 9 % according to a self-monitoring questionnaire, subjects mostly consumed quinoa as porridge for breakfast, felt fuller than when eating their regular breakfast, and have continued eating quinoa after the study. This could be interpreted as an early indication that quinoa is well tolerated among individuals with CD. All median values for blood tests were within normal levels and no significant difference was observed after eating quinoa, except for albumin that decreased significantly from 4 to 44 g /l but remain within normal parameters (4 5), and TC and LDL levels that were higher than the recommended 4 and mmol / l, respectively, ( 1 ) before and after the study. According to two recent studies, untreated CD is associated with low serum cholesterol. West et al. ( ) compared EMA-positive undiagnosed CD patients with EMA-negative controls and found that individuals with undiagnosed CD had.5 mmol / l lower serum cholesterol than their counterparts. Furthermore, Lewis et al. (1 ) found that newly diagnosed CD patients had lower TC when compared with the general population. The cholesterol profile of CD patients after treatment (GFD) was evaluated by Brar et al. ( ), who concluded that the GFD contributed to an 11 % increase in their TC. However, another study ( 1 ) observed no increase in TC after 1 year on a GFD. The TC in the present study population fell from 4. to 4.3 mmol / l, LDL from.4 to.45 mmol / l, high-density lipoprotein (HDL) from 1.8 to 1.8 mmol / l ( P.5), and triglycerides from.8 to.79 mmol / l after eating quinoa for weeks ( Figure 3 ). Epidemiological data suggest that lowering TC and LDL would reduce the risk of cardiovascular events by % for each 1 mmol / l of LDL reduction ( 3,4 ). Furthermore, these results are in agreement with an earlier in vivo study that showed a reduction in cholesterol after feeding mice with quinoa (5), and more recently Foucault et al. ( ) found that quinoa extracts given to mice on a high-fat diet decreased the expression of several genes involved in lipid storage. The active compound of quinoa (saponins, ecdysteroids, and other proteins) remains to be determined, although our results confirm previous animal experimental data, suggesting that consumption of quinoa has a mild hypocholesterolemic effect but the benefit / detriment of this reduction remains to be elucidated. The histological assessment of duodenal biopsies of 1 celiac patients ( Figure 4 ) indicate that the mean values of VH:CD (3:1) and SECH (9.77 μ m) at the end of the study were at the lower end of normal range (3:1 5:1 and 9 34 μ m, respectively), which is relatively expected in a group of individuals with CD with a wide range of time on a GFD (1 33 years). This is potentially due to the 14 by the American College of Gastroenterology

7 7 Zevallos et al. Total cholesterol (mmol/l) HDL (mmol/l) * LDL (mmol/l) Triglycerides (mmol/l) Figure 3. Total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and triglycerides levels were reduced in 19 celiac patients after eating quinoa. ( a ) TC from 4. to 4.3 mmol / l, ( b ) LDL from.45 to.4 mmol / l, ( c ) HDL from 1.8 to 1.8 mmol / l, * represents signifi cant reduction ( P.5), and ( d ) Triglycerides from.8 to.79 mmol / l. 4 VH:CD (ratio) 4 SECH (μm) IEL (%) 4 Figure 4. Villus height to crypt depth (VH:CD) ratio, surface-enterocyte cell height (SECH) measurements, and intra-epithelial lymphocytes (IEL) count of 1 patients at week and at week of the quinoa challenge. ( a ) The mean values of VH:CD went from slightly below normal levels (.8:1) to normal levels (3:1), the same occurred in panel b. SECH from 8.7 to 9.77 μ m. ( c ) IEL decreased from slightly abnormal (3.3) to just below normal (9.7). Although a positive trend was observed (increased VH:CD and SECH, and decreased IEL), no signifi cant difference was achieved in any of the measurements when statistically analyzed using the Wilcoxon sign rank test with a 95 % confi dence level. VOLUME 19 FEBRUARY 14

8 Gastrointestinal Effects of Eating Quinoa 77 Degree of villous atrophy Mononuclear cell infiltration Figure 5. Histological status of intestinal biopsies was maintained or improved in 1 celiac patients after quinoa consumption. Hematoxylin and eosin (H & E)-stained slides (original magnifi cation) 1 ( a ) before and ( b ) after quinoa challenge (patient 9). Microscopical appearance was determined by degree of ( c ) villous atrophy (DVA) and ( d ) mononuclear cell infiltration (MCI) before and after the study with a maximum hypothetical score of 3. No signifi - cant difference was observed. fact that it can take more than years to achieve either normal or nearly normal morphometric parameters following initiation of treatment with GFD ( 7 ). In some subjects, full recovery is never achieved for various reasons such as hidden sources of gluten in their diets, development of refractory CD, or other unexplained reason (8 ). The other morphometric parameter IEL count was conversely on the higher end of normality ( 3), after quinoa consumption, at 9.7. Nasseri-Moghaddam et al. ( 9 ) in a review to determine the range of normality of IEL in celiac patients indicated that < 35, using slides stained by immunohistochemistry, could be considered normal, 3 39 borderline and more than 4 abnormal. Moreover, normal IEL counts should take into account environmental exposures, ethnic background, and regional differences. Furthermore, scores of DVA and MCI in biopsies before and after the study indicated that all the subjects experience either maintenance or improvement in the histological status of their intestine ( Figure 5 ). We previously analyzed the effects of quinoa prolamins in celiac patients using in-vitro methods ( 11 ) and found that a minority of celiac patients could have an immune response to some quinoa cultivars. However, these potential effects were not replicated in-vivo, suggesting that quinoa as a whole food behaves in a different way compared with quinoa prolamin extracts, possibly due to a matrix effect or other compounds within quinoa grains acting as a buffer, which will require further investigation. To conclude, this study provides in vivo evidence that shortterm consumption of quinoa is well tolerated among individuals with CD, it does not exacerbate the condition, and could have a mild hypocholesterolemic effect in CD patients and potentially non-cd subjects. The limitations of this study include the lack of a run-in gastrointestinal assessment to determine a baseline score of the participants and the uncertainty about the long-term effects of quinoa consumption for which further studies are needed; in the meantime, we can suggest that quinoa can be safely included within a GFD for individuals with CD. ACKNOWLEDGMENTS We are very grateful to all celiac patients participating in the study. CONFLICT OF INTEREST Guarantors of the article: Victor F. Zevallos, PhD and Paul J. Ciclitira, MD, PhD. Specific author contributions: V.F.Z., H.J.E., and P.J.C. conceived and designed the experiments. V.F.Z. performed the experiments. V.F.Z. analyzed the data. L.I.H., F.C., and S.D. contributed reagents / materials. V.F.Z. wrote the manuscript. V.F.Z., L.I.H., H.J.E., and P.J.C. made a critical revision of the manuscript. Financial support: This work was supported by the Food Standards Agency PG117. Potential competing interests: None. 14 by the American College of Gastroenterology

9 78 Zevallos et al. Study Highlights WHAT IS CURRENT KNOWLEDGE 3 Celiac disease is an enteropathy triggered by dietary gluten found in wheat, rye, and barley, the current treatment involves strict avoidance of those cereals, affecting the palatability, price and nutritional value of the patient s diet. 3 Quinoa is an Andean crop with exceptional nutritional applications that offers a clear advantage over cereals, as it contains higher levels of protein, vitamins, minerals, fiber, as well as good culinary properties. 3 In vitro studies have shown that quinoa could stimulate innate and adaptive immune responses in celiac patients but there are limited clinical data describing the effects of eating quinoa in celiac patients. WHAT IS NEW HERE 3 Celiac patients can tolerate 5 g of quinoa per day for weeks. 3 There was a positive trend involving improvement in small intestinal morphology following weeks ingestion of 5 g quinoa per day. 3 Quinoa consumption has a mild hypocholesterolemic effect that warrants further evaluation not only in individuals with celiac disease but also non-celiac subjects. REFERENCES 1. Jun ker Y, Z eissi g S, Kim SJ et al. Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4. J Exp Med 1 ;9 : Bai JC, Fried M, Corazza GR et al. World gastroenterology organisation global guidelines on celiac disease. J Clin Gastroenterol 13 ;47 : Packaged Facts. Gluten-Free Foods and Beverages in the U.S., 4th edn. Packaged Facts: LA, Kasarda DD. Grains in relation to celiac disease. Cereal Foods World 1 ;4 : Janatu i ne n E K, Pi k kar aine n PH, Kempp ai ne n TA et al. A comparison of diets with and without oats in adults with celiac disease. 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