The latter was frozen at -20 °C for 24 hours and thawed in a microwave for 45 seconds.6 The analysis of natural human milk was performed immediately after the extraction. Of the three 10-mL aliquots, one was identified as reference (not subjected to any process), the other was assigned for administration simulation by gavage, and this website the last was assigned for administration simulation by continuous infusion. The administration by gavage was performed with a 10 mL-syringe and disposable #4 siliconized tube; the content was gravity-fed. The administration by continuous infusion was performed with a 10 mL-syringe, a disposable #4 siliconized tube, a 120 cm perfusor, and a Samtronic ST6000® infusion

pump (São Paulo, Brazil). The time set for infusion was 1 hour. All materials and techniques used followed the routine of the Neonatal Unit of the Instituto Fernandes Figueira/Fiocruz, Brazil. The amount of fat, PD-1/PD-L1 inhibition protein, and lactose in human milk was measured

by infrared spectrophotometry, using the infrared analysis equipment MilkoScan Minor (Foss, Denmark), previously validated for human milk.7 Sample size calculation was performed considering the magnitude of the difference found between measurements of fat in the two forms of administration (gavage and continuous infusion) in the study by Vieira et al.,7 power of 90%, and significance of 95%. In this study, the magnitude of the difference was 0.94 g/100 mL. Considering these parameters, the initial sample size consisted of 16 samples, which was doubled due to variability of fat content in milk

samples.8 and 9 The measurements of macronutrients and total calories in human milk samples were compared at each phase using the Wilcoxon test for paired samples. The SPSS software, version 20.0 (IBM Corp, USA), was used for the statistical analysis. This study was approved by the Research Ethics Committee of the Instituto Nacional da Saúde da Mulher, Criança e Adolescentes Fernandes Figueira and an informed consent was obtained from all participants. A total of 34 human milk samples were analyzed. There was a variation IKBKE in macronutrients between donated samples of 19% for fat, 1.9% for protein, and 1.6% for lactose. No samples of pooled human milk were analyzed. The mean content of macronutrients in g/100 mL in natural milk was 3.05 ± 1.18 for fat, 1.22 ± 0.50 for protein, and 6.09 ± 0.55 for lactose. The mean of total calories was 56.66 ± 11.76 Kcal/100 mL. Milk administration by continuous infusion significantly altered the levels of fat when compared to gavage, both during the infusion of natural and thawed milk (Table 1). A significant increase of protein in thawed milk was also observed when compared to natural milk. However, no significant difference was observed in the amounts of protein in thawed milk offered either by gavage or continuous infusion. (Table 1) The use of gavage did not result in loss of macronutrients in both natural and thawed milk (Table 1).