P0111 PP QUANTITATIVE DETERMINATION OF TOTAL GLUTAMINE CONTENT IN BREAST MILK

Abstract

Introduction: Glutamine (Gln) may be a conditionally indispensable amino acid in pre-term infants with low birth weight. Moreover, in term infants an adequate Gln supply is necessary for an optimal development of gut integrity and function. Knowledge of total Gln content (free and protein bound) of breast milk is thus of utmost importance. Respective data are, however, lacking due to methodological problems (instability of Gln during acid hydrolysis). The aim of the present study was, thus, to develop a new methodology enabling quantitative analysis of total Gln in human breast milk. Methods: Breast milk was harvested in 16 healthy women at days 4–7 (immature milk, IM) and 29–34 (mature milk, MM) after term delivery (>37 gestational weeks). Samples (2–3 mL gained 5 min after beginning of feeding) were immediately stored at −70 °C until analysis. Protein bound Gln: After removal of interfering fat and carbohydrates (extraction/dialysis) bound Gln was converted to acid stable diaminobutyric acid (DABA) by treatment with excess bis(1,1-trifluoroacetoxy)iodobenzene (BTI) for 4h at 50 °C. After evaporation, samples were hydrolyzed using microwave technology using boiling HCl. Liberated DABA was determined by HPLC with precolumn derivatization and fluorescence detection. Free Gln: After protein precipitation, Gln was determined in the supernatant by HPLC. Protein content was measured using a photometric assay. Precision and accuracy of the BTI method was confirmed by analysis of a standard oligopeptide with known Gln content. Results are presented as means ± SD. Results: The new BTI method enabled reliable analyses of bound Gln (CV: <5%) in breast milk proteins (IM: 6.1 ± 1.1 g; MM: 6.4 ± 1.3 g/100 g protein; NS, t-test). Free Gln concentrations were higher in MM (296 ± 205 μmol/L) compared with IM (143 ± 108 μmol/L, p=0.002). Considering the average protein content of IM (1.4 g/100 mL) and MM (1.0 g/100 mL), total Gln content in IM was 6.2 ± 1.4 mmol/L (0.9 ± 0.2 g/L) and in MM 4.6 ± 1.1 mmol/L (0.7 ± 0.2 g/L; p<0.001). Conclusion: The new method developed allowed for the first time a reliable quantification of total Gln content of breast milk. These data now allow calculation of Gln intake during breastfeeding (ca. 0.2 g/kg and day) and can be used to optimize nutrition strategies in infants requiring complementary feeding.