Direct detection and quantitative determination of bovine lactoferricin and lactoferrin fragments in human gastric contents by affinity mass spectrometry.

Lactoferricin (Lfcin) is a bioactive fragment of lactoferrin derived from the bactericidal and putative lymphocyte receptor binding domain(s) located within the N-lobe of lactoferrin. Although known to be liberated from at least three species of lactoferrin, conditions leading to Lfcin generation in vivo and factors affecting its distribution are still not known. Recently, we have developed a method of surface-enhanced laser desorption/ionization (SELDI) affinity mass spectrometry using n-butyl terminal groups for surface-enhanced affinity capture (SEAC) to quantify not only Lfcin generated in vivo but also other lactoferrin fragments. Unlike previous efforts to detect lactoferrin and Lfcin with specific antibodies, the SELDI affinity assay distinguished lactoferrin, lactoferrin fragments, Lfcin and unrelated peptides without their interference with each other. To evaluate Lfcin generation in vivo, the experimental design involved feeding 200 mL of 10 mg/mL (1.22 x 10(-4) mol/L) bovine lactoferrin to an adult. Gastric contents were recovered 10 min after ingestion. Lfcin produced in vivo was directly captured by the SEAC device. The amount of Lfcin in the gastric contents was 16.91 +/- 2.65 micrograms/mL (5.350 +/- 0.838 x 10(-6) mol/L). However, a large proportion of the ingested lactoferrin was not completely digested. Lactoferrin fragments containing the Lfcin region were analyzed by in situ hydrolysis with pepsin after being captured by the SEAC device. As much as 5.740 +/- 0.702 x 10(-5) mol/L of the partially degraded lactoferrin fragments were found to contain the Lfcin region, including peptide domains 17-43, 17-44, 12-44, 9-58, and 16-76 of bovine lactoferrin. These results show that bovine Lfcin can be produced in the human stomach after ingestion of an infant formula supplemented with bovine lactoferrin. It is now important to determine whether Lfcin is generated in the intestinal tract of formula-fed and breast-fed infants, and geriatric patients consuming foods enriched with lactoferrin.