Colorimetric Quantification of Serum Proteins and Urinary Metabolites

Total serum protein was determined by the Bradford method using bovine serum albumin (BSA) as the external standard. The serum samples were diluted 200-fold before analysis. The absorbance was measured at 595 nm on a Biotek Synergy HT Microplate Reader (Agilent, Santa Clara, CA, USA). The determination of total polyphenols and creatinine in urine has been described in a previous publication of our group [7 (link)], performing a standard curve with commercial gallic acid from 300 to 4.7 µg/mL in ultrapure water. To normalize the data obtained for the polyphenols, we determined the concentrations of urine creatinine by a colorimetric reaction. Urine samples were diluted 20-fold and 10 µL pipetted into a 96-well, flat-bottom plate. Next, 200 µL 0.1% picric acid and 15 µL of NaOH were added. After a 15-min incubation, the plate was read at an absorbance of 500 nm by using a Biotek Synergy HT Microplate Reader.