Purification and characterization of intact lactoferrin found in the urine of human milk-fed preterm infants

Abstract

Little is known about the metabolic fate of ingested lactoferrin in human-milk-fed term or preterm infants. Enzyme-linked immunosorbent assays and sodium dodecyl sulfate/polyacrylamide gradient gel electrophoresis (SDS-PAGE) with immunoblots have demonstrated that the urinary excretion of lactoferrin by preterm infants fed exclusively human milk exceeded that by formula-fed infants. The origin and molecular integrity of the excreted lactoferrin, however, are unclear. We have developed extraction and separation procedures involving a stationary phase of immobilized single-stranded DNA (ssDNA) that allows the efficient (>80%) and rapid isolation of pure, intact lactoferrin from infants urine. We purified lactoferrin to apparent homogeneity from infants' urine in one step, using the immobilized ssDNA with mobile phases containing up to 6 mol of urea per liter. The purified lactoferrin was evaluated by SDS-PAGGE; silver-staining revealed one protein band at 78 kDa; immunoblots confirmed the presence of intact lactoferrin. High-performance affinity chromatography with use of immobilized metal ion (Cu2+) suggested the presence of intact, iron-saturated lactoferrin. Subsequent chromatography on high-performance reversed-phase (C18) columns independently verified sample identity and purity.