Fig. 1.
Butyric acid-induced blood cytosolic oxidative stress is correlated to heme accumulation. Jugular blood was collected after BA injection as previously published (Cueno et al. 2013), and animal studies performed were in accordance with the guidelines set by the Kyoto Institute of Nutrition and Pathology Inc. a Jugular blood cytosol purity. Cytosol/Particulate Rapid Separation Kit (BioVision) was used to isolate blood cytosol. Pierce® Detergent Removal Spin Columns (Thermo Scientific) was used to purify samples from traces of detergents. Pierce® Microplate BCA Protein Assay Kit-Reducing Agent Compatible Kit (Thermo Scientific) was used to standardize the protein concentration in all samples used. All kits used were according to manufacturer's recommendation. Western blotting was performed to establish blood cytosol purity. Briefly, blood cytosolic proteins were separated by SDS-PAGE and transferred to Hybond-C nitrocellulose membrane (Amersham Biosciences). Membranes were subsequently blocked with DifcoTM Skim Milk (BD Company) probed with antibodies, and immunoreactive proteins were visualized using SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Anti-GAPDH (GeneTex) was used to detect the glyceraldehydes-3-phosphate in the blood cytosol to serve as control. Anti-HSP60 (StressMarq Biosciences Inc., Canada) was used to determine cytosolic heat-shock protein 60 in the blood cytosol extract to verify the purity of the cytosolic samples. Anti-MTC02 (Novus Biologicals) is a mitochondria-specific antibody used to confirm the absence of mitochondrial components, anti-PCNA (Thermo Scientific) was used to detect the presence of the proliferating cell nuclear antigen, and anti-PMCA (Thermo Scientific) which recognizes the plasma membrane calcium ATPase were used to further establish the purity of the isolated blood cytosol. b Total heme levels in blood cytosolic samples. QuantiChromTM Heme Assay Kit was used to measure blood cytosolic heme levels (free heme and heme–proteins) according to manufacturer's recommendation. c Catalase activity in blood cytosolic samples. EnzyChromTM Catalase Assay Kit (BioAssay Systems) was used to measure cytosolic catalase (CAT) activity according to manufacturer's recommendation. d Hydrogen peroxide amounts in blood cytosolic samples. Red Hydrogen Peroxide Assay Kit (Enzo Life Sciences) was used to measure blood cytosolic hydrogen peroxide (H2O2) amounts according to manufacturer's recommendation. Results shown correspond to n = 5 replicates of six independent samples. Jugular blood cytosol from 0, 60, and 180 min after butyric acid injection are indicated. Statistical analyses were performed using Student's t test (** p < 0.01)