Ka1652

As each BAL sample consisted of ELF in saline, the dilution of ELF was determined to convert ciprofloxacin concentrations in BAL fluid to ciprofloxacin concentrations in ELF. The urea dilution method was used to assess ELF dilution [31 (link)]. Urea freely diffuses through several body compartments, including ELF, as supported by experiments in the isolated perfused dog lung [32 (link)]. If the concentrations of urea in plasma (C_urea,plasma) and BAL fluid (C_urea,BAL) are known, concentrations of ciprofloxacin in BAL samples (C_{ciprofloxacin,BAL}) can be corrected for dilution of ELF by saline to obtain ciprofloxacin concentrations in ELF (C_{ciprofloxacin,ELF}) according to the following equation:
C_{ciprofloxacin,ELF} = C_{ciprofloxacin,BAL}/(C_urea,BAL/C_urea,plasma)
To assess plasma concentrations of urea, one plasma sample was drawn near to the timepoint of BAL. The concentration of urea in both plasma and BAL fluid was determined using a specific urea assay kit (Abnova KA1652) according to the manufacturer’s protocol. Urea reactions were performed in 96-well plates and concentrations were assessed using an EnSpire^® 2300 Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) at an absorbance of 520 nm. All standards and samples were run in duplicate and the mean values were used for analysis. The limit of quantification for plasma and BAL was 0.8 µg/mL.

Blood samples were collected at defined times (see Figure 1a). After centrifugation at 4 °C and 10,000× g for 15 min, the supernatant was obtained and stored at −30 °C. Quantitative analysis of urea in blood plasma was performed using an urea assay kit (KA1652, Abnova GmbH, Heidelberg, Germany). The marker was normalized to a kidney weight of 100 g.