Leptin identification and quantification in human and bovine milk and infant formulas

Title (20) Leptin identification and quantification in human and bovine milk and infant formulas. Introduction (120) Leptin is a small protein (14 KDa) present in plasma and milk. It plays a key role in the regulation of body weight and its concentration is affected by many physio-pathological parameters especially body weight variation and lipodystrophic conditions. Leptin is encoded by the obesity gene on human chromosome 7 and it is mainly secreted by adipocytes. It is possible that serum leptin concentration in breastfed infants is associated to early adipose tissue production and to the leptin levels of milk. In order to understand the role of different newborn feeding (breastmilk, formulas, bovine milk) on infant leptin production and risk of obesity, we aimed to develop a selective LC-MS method to evaluate leptin content in different milk matrices. Methods (120) Human milk and infant formulas were provided from local pediatric hospital. In order to detect leptin concentrations < 0.1 fM, immunoaffinity extraction was performed. A Thermo Ultimate 3000 nanoLC system Hyphenated to an Orbitrap Fusion high resolution MS and ETD source was utilized. The developed method was compared to a classical SRM quantitation by LC-Q trap analysis (using a Sciex 5500 Qtrap coupled with Shimadzu Nexera UFLC). C4-RP columns were used with a linear gradient from 5% mobile phase (acetonitrile with 0.1% formic acid) to 60%. Preliminary data (300) At first human and bovine leptin were extensively characterized by high resolution mass spectrometry in order to evaluate interferences. Full MS and CID/ETD MS/MS spectra of fifteenth fold charged positive ions of standard proteins were acquired. A top-down approach was used with the aim of reduce to a minimum sample manipulation. Accuracy, precision, LLOQ, linearity, extraction recovery and stability were then assessed. The comparison between nanoLC-Orbitrap HRMS analyzer and UFLC-Qtrap analyzer is at present under evaluation. The method was then applied to investigate about the presence of leptins in about twenty different commercial infant formulas. Finally mean concentration in a small series of plasma and milk samples was determined in order to establish correlations between mother and infant Body Mass Index. Data acquisition of new samples for statistical purposes is being implemented. Novel aspects (20) Development of novel methods to evaluate quality of infant formulas by top-down protein analysis.