Circulating UCB levels were measured in plasma samples following a well-established protocol [30 (link), 31 (link)] using high-performance liquid chromatography (HPLC, Merck, Hitachi, LaChrom, Vienna, Austria), equipped with a Fortis C18 HPLC-column (4.6 × 150 mm, 3 μm), a Phenomenex SecurityGuard™ cartridges for C18 HPLC-columns (4 × 3 mm), and a photodiode array detector (PDA, Shimadzu). An isocratic mobile phase contained glacial acetic acid (6.01 g/L) and 0.1 M n-dioctylamine in HPLC grade methanol/water (96.5/3.5%). Before starting the procedure, all aliquots were centrifuged and 50 μL plasma/serum was mixed with 200 μL mobile phase. After a second centrifugation, 120 μL of the supernatant was injected to the HPLC at a flow of 1 ml/min.
Case-control pairs were analyzed in the same plate to minimize batch-to-batch fluctuation. Bilirubin (alpha) (purity ≥ 98%, Sigma Aldrich) acted as an external standard (3.3% IIIα, 92.8% IXα, and 3.9% XIIIα isomers, 450 nm). One reference plasma sample was assessed per analysis as internal standard. The coefficient of variation (CV) between each plate was 6%.
Case-control pairs were analyzed in the same plate to minimize batch-to-batch fluctuation. Bilirubin (alpha) (purity ≥ 98%, Sigma Aldrich) acted as an external standard (3.3% IIIα, 92.8% IXα, and 3.9% XIIIα isomers, 450 nm). One reference plasma sample was assessed per analysis as internal standard. The coefficient of variation (CV) between each plate was 6%.