ORIGIN OF INTACT LACTOFERRIN AND ITS DNA-BINDING FRAGMENTS FOUND IN THE URINE OF HUMAN MILK-FED PRETERM INFANTS - EVALUATION BY STABLE ISOTOPIC ENRICHMENT

The origin of intact (78-kD) lactoferrin found in the urine of human milk-fed preterm infants was investigated using human milk containing proteins enriched with [C-13]leucine and [N-15(2)lysine or [H-2(4)lysine. Mothers of infants selected for the study were infused i.v. with [C-13] leucine and [N-2(15)]lysine or [H-4(2)]lysine to label milk proteins. The labeled milk was collected from each mother, pooled, fortified with a lyophilized human milk fraction, and fed to her preterm infant by continuous orogastric infusion for a period of 48 h. Urine was collected from each infant for 96 h. Intact lactoferrin (78 kD) and DNA-binding lactoferrin fragments (51 and 39 kD) were purified from the urine by affinity chromatography on columns of immobilized single-stranded DNA-agarose. The concentration and isotopic enrichment of the intact lactoferrin and DNA-binding fragments were determined separately after their isolation by high-performance reverse-phase (phenyl) chromatography. Mass spectral analyses indicated that the isotopic enrichment of the purified urinary lactoferrin was 87 to 100% of that in the labeled human milk lactoferrin. Similar results were obtained for the isolated DNA-binding lactoferrin fragments. The ratios of isotopically labeled leucine to lysine in the purified milk lactoferrins and urinary lactoferrins were similar for each mother/infant pair. Isotopically labeled lysine, added to the milk as free amino acid, was not incorporated into the purified urinary lactoferrin. These results demonstrate that undegraded (78-kD) lactoferrin of maternal origin is absorbed by the gut and excreted intact in the urine of preterm infants; nearly all of the urinary lactoferrin was of maternal origin. The possible immunoregulatory functions of the absorbed intact, maternal lactoferrin are discussed.