After the first and second part of this triple test subjects performed for 15 minutes with 60 W at 80 rpm. After the third part subjects continued exercise for three minutes with 60 W and 80 rmp and stopped then. The whole test procedure lasted between 80 and 90 minutes, depending on duration of each step test/part. Blood pressure was controlled after each 100 W

and after the last step of each ergometry. Gas exchange variables were monitored continuously throughout the step tests as described above. During the 15 minutes intervals between the ergometry step tests the facemask was removed to consume 750 mL of plain water, in total over the whole test procedure. Fourteen CHIR-99021 purchase weeks later this procedure was repeated on the same cycle ergometer, with the same investigator, standardized room temperature (20°C) and humidity (60%). Blood and feces collection We conducted blood collections in supine position from a medial cubital vein at each triple ergometry test: before exercise (Pre) and within 10 min post exercise (Post). Venous blood was

collected to determine carbonyl proteins (CP), malondialdehyde (MDA), total oxidation status of lipids (TOS), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). After centrifugation for 10 minutes plasma was removed and samples were frozen at AZD8931 −70°C until analysis. For zonulin and α1-antitrypsin from

Gemcitabine manufacturer feces the subjects collected samples at baseline and after 14 weeks with standardized stool tubules within 24 hours prior to bringing the sample in a cool bag to the laboratory. All samples were analyzed within 72 hours after dispensing. Throughout the 14 weeks treatment the subjects recorded a stool protocol to monitor stool appearance with help of the Bristol stool scale/chart [28]. Stool analyses Zonulin and α1-antitrypsin were analyzed with commercially available ELISA kits (Immundiagnostik AG, Bensheim, Germany). The zonulin analysis is based on a competition between the free antigen in the samples or standards and the antigen coated on the wells of the microplate. Standards, samples and the primary anti-zonulin antibody are transferred directly into the precoated microplate wells. The antigen in the samples competes with the antigen immobilized on the wells of the microplate for the binding sites of the specific anti-zonulin antibody. A peroxidase-conjugated antibody is used for detection, and tetramethylbenzidine as a peroxidase substrate. The this website enzymatic reaction is terminated by acidic stop solution. The quantification is based on the optical density at 450 nm. Data are expressed in ng/mL.