Creatinine urinary assay kit

Urinary creatinine levels were used to adjust for variability in dilution and to determine the validity of a spot urine sample for assessing chemical exposure [23] . A creatinine (urinary) assay kit from Cayman Chemical Company (Ann Arbor, MI) was used according to the manufacturer's protocol to measure urinary creatinine levels. The creatinine levels were used to adjust the urinary concentrations of BPA measured by the HPLC-ESI-MS/MS to obtain the “creatinine-adjusted” BPA levels (BPA levels) in µg/g.

Briefly, approximately 50 ml first-void morning urine sample was collected during the day of the saline infusion test. Urine sample (10 ml of urine sample was directly used for the measurement of creatinine content using a creatinine [urinary] Assay Kit, 500701-96, Cayman, USA), and the remaining sample was treated with 3 ml PMSF (10 mM) and 50 μl leupeptin (1 mg/ml) before freezing at -80°C (16 (link), 17 (link)). Urinary EVs were isolated via ultracentrifugation, as previously described by Salih et al. (18 (link)). All centrifugations were performed in an ultracentrifuge (Optima XPN-100, Beckman Coulter, USA) with an angle rotor (Type 70 Ti Rotor, Beckman Coulter, USA) at 4°C. Briefly, frozen urine was quickly thawed at 32°C and vortexed for 90 s. The urine was first centrifuged at 17,000×g for 15 min to remove high-density particles. The supernatant obtained above was ultracentrifuged at 200,000×g for 90 min to obtain a gel-like precipitate and the new supernatant was discarded. This gel-like pellet was resuspended in 1× PBS buffer (Sangon Biotech, China), and then ultracentrifuged at 200,000 × g for 90 min to obtain a new pellet containing EVs. The pellet was resuspended in 160 μL 1× PBS buffer and froze at -80°C after aliquoting.