*4.6. Study 1 Urine Analysis*

Urine was collected by participants for a 24-h period prior to the clinic appointment in large plastic bottles provided to participants. These samples were also analyzed by the NATA-accredited pathology laboratory for creatinine, sodium, and potassium excretion. Samples were analyzed fresh in singular; any abnormal results were flagged and rerun to verify results. Samples were analyzed on a either a Roche Cobas 8000 or a Roche Cobas Pro using the creatininase method for creatinine and the ion-specific electrode-indirect method for potassium and sodium.

From this urine bottle, 1.5 mL was retained and aliquoted in cryovials. These cryovials were stored at −80 ◦C with no preservative prior to F2-isoprostane assessment using previously described methods [50]. Briefly, urine samples were thawed, acidified to a pH of 3, and internal standard was added. Separation of F2-isoprostanes was achieved by using silica and reverse-phase cartridges and high-performance liquid chromatography. Samples were analyzed in singular, using gas chromatography/electron capture negative-ionization mass spectrometry and the peaks were identified through the comparison of retention times with known standards. The within- and between-assay reproducibility was 6.7% and 3.7%, respectively.
