2.2. Study Design (Secondary Analysis)
breast milk choline, ±2, breast milk and blood sample, breast milk
SECONDARY
During the period of intervention, breast milk samples were collected twice weekly to measure choline-containing compounds. All infants received at least 100 mL/kg/d of enteral nutrition at the time of inclusion into the underlying RCT (median, day 7), and the first blood samples evaluated in this secondary analysis were taken on day 17 in median. Venous blood samples to measure plasma choline, betaine and PC concentrations were scheduled on days 14 (±2) and 28 (±4) after randomization. To avoid study-driven blood sampling, the latter was re-scheduled until a clinical indication arose. Choline content of the closest breast milk sample (collected at median 1 day (p25–p75: −1–+1 day) before blood sampling) was used to calculate actual enteral choline intake (comprising breast milk choline content; choline content of the administered standard—or study fortifier; protein supplement and formula) on the day before blood sampling. Only one breast milk–plasma pair per patient was included, which was always the first available breast milk–plasma pair after birth, for which the time difference between breast milk and blood sample was less than 5 days.