Gluten challenge and urine collection

In initial experiments, participants followed a gluten-free diet for one day, and then continued the gluten-free diet and collected a pooled urine sample for 8 h on the second day, beginning with the second morning void. On the third day, volunteers underwent a dietary gluten challenge consisting of two bagels made with wheat flour (approximately 9 g gluten each). After the gluten challenge, participants were permitted to eat ad libitum, and a pooled urine sample was collected for 8 h. For studies that required a barley or rye enriched diet, pancakes prepared with approximately 1.5 cups of barley (Arrowhead Mills), rye (Bob’s Red Mill), or wheat (Bob’s Red Mill) flour were substituted for bagels prepared with wheat flour, and participants refrained from eating any wheat, rye, or barley products until the end of the 8 h urine collection. In later experiments, volunteers were not prescribed a gluten-free diet the day prior to the gluten challenge but were instructed to fast overnight. After fasting, participants collected a spot urine sample, and underwent the dietary gluten challenge as previously described. Subsequent to this gluten challenge, a pooled urine sample was collected for 8 h in a 4 L urine container (Simport Scientific #B3504L), whereas spot urine samples were collected in 120 mL urine collection cups (Thermo Scientific #010001). Urine was stored at 4 °C throughout the collection day and was preprocessed and frozen on the same day it was collected. To do so, urine samples were centrifuged in 50-mL polypropylene tubes for 3000 × g for 20 min and transferred to 15 mL polypropylene tubes in 10–15 mL aliquots. Samples were stored at −80 °C until further analysis.