Abstract
Although free amino acids (FAA) account for a small fraction of total nitrogen in mammalian milk, they are more abundant in human milk than in most formulas, and may serve as a readily available source of amino acids for protein synthesis, as well as fulfill specific physiologic roles. We used reversed phase Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS) technique for FAA profiling in milks from three species (human, rat and cow) with a simple and rapid sample preparation. The derivatization procedure chosen, combined with UPLC-ESI-MS/MS allowed the quantitation of 21 FAA using labeled amino acids (Internal Standards) over a 10 min run time in micro-samples of mammalian milk (50 μL). The low limit of quantitation was 0.05 pmol/μL for most FAA with good repeatability and reproducibility (mean CV of 5.1%). Higher levels of total FAA were found in human (3032 μM) and rat milk (3460 μM) than in bovine milk (240 μM), with wide differences in the abundances of specific FAA between species. This robust analytical method could be applied to monitor FAA profile in human breast milk, and open the way to individualized adjustment of FAA content for the nutritional management of infants.
Highlights
Due to its many health benefits, breastfeeding is considered the gold standard for feeding infants in the first 6 months of life (Horta et al 2007)
Ultrapure water was obtained from a Milli-Q purifier (Millipore, Eschborn, Germany), UPLCgrade solvents, derivation reagents, and Ultra Performance Liquid Chromatography (UPLC) column were purchased from Waters (Milford, USA), acetonitrile was from Biosolve (Valkenswaard, Netherlands), hydrochloric acid (HCl) from CARLO ERBA (Val de Rueil, France), and sulfosalicylic acid from Sigma-Aldrich
7.5 aassay concentration of aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) amino acid derivatives, 1 μL injected. bcoefficient variation of reproducibility, calculated on 6 independent milk samples tested on 2 days with 2 operators
Summary
Due to its many health benefits, breastfeeding is considered the gold standard for feeding infants in the first 6 months of life (Horta et al 2007). FAA account for only 2% of total nitrogen in human milk (Carratù et al 2003), they may serve as a source of readily available nitrogen, in the small intestine of preterm infants with limited proteolytic enzymatic activity (Schanler and Garza 1987), and impact early postnatal growth. Besides their role as building blocks for protein synthesis, several amino acids have a specific physiologic role, and their availability during the first few weeks of life may be of crucial importance. Other free amino acids may be conditionally essential in the first few weeks of life: in a porcine model, maternal arginine supplementation was shown to increase sows’ milk arginine concentration, and enhance piglet growth (Mateo et al 2008)
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