Urine samples from the participants were collected in the early morning at the time of physical examination. Samples were stored at −20°C prior to use. Analysis of urinary pyrroles employed minor modifications of published methods.19–21 (link) Pyrrole adducts were measured spectrophotometrically after reaction of 0.08 mL urine with 0.08 mL guanidine hydrochloride (70%) and 0.08 mL of Ehrlich’s reagent 3% 4-dimethylaminobenzaldehyde (DMBA) in the solution of 40% vol/vol methanolic 14% boron trifluoride and 60% vol/vol ethanol.22 (link) Absorption values were measured at 526 nm with an automatic microplate reader (Infinite 1000, Tecan, Switzerland). Calculations were based on a standard curve prepared with different concentrations of 2,5-dimethylpyrrole.