title: Bioactive and immunoreactive prolactin variants in human milk.
abstract: Human milk from mothers of term (T) and preterm (PT) infants was collected during early (days 2-7), mature (2-16 weeks), or late (> 16 weeks) lactation. PRL-like bioactivity (B) was measured by Nb2 cell proliferation, and PRL immunoreactivity (I) was determined by RIA. PRL activity is reported in PRL equivalents (1 PRL equivalent = 1 ng NIDDK reference material). Milk from early lactation contained significantly greater PRL-like B compared to I (T:B, 132.5 +/- 13.0; I, 83.43 +/- 12; PT:B, 195.8 +/- 56; I, 74.45 +/- 13.7). PRL-like B and I declined as lactation progressed (T mature: B, 41.74 +/- 8.9; I, 27.19 +/- 5.5; T late: B, 17.84 +/- 5.5; I, 27.33 +/- 1.8; PT mature: B, 59.85 +/- 16; I, 45.16 +/- 4.3). Milk PRL B to I ratios were consistently greater than serum B to I ratios during early lactation (milk: T, 1.4 +/- 0.3; PT, 3.6 +/- 1.3; serum: T, 1.0 +/- 0.2; PT, 0.58 +/- 0.12). During early lactation, high PRL-like B was widely distributed among several (n = 4-6) bioactive forms differing in molecular mass [8 to > 66 kilodaltons (kDa)] in T milk, but the majority of B in PT milk was detected in two or three forms. During mature and late lactation, lower PRL-like B was associated with two or three peaks (20 to > 66 kDa). A large fraction of PRL-like B (67%-84%) was associated with the phosphorylated (P-) fraction of human milk. Four immunoreactive forms (24, 30, 32, and 40 kDa) of P-PRL were identified by immunoblot analyses. Alkaline phosphatase treatment converted the 40-kDa immunoreactive P-PRL to 24-kDa PRL, increased the B of the P-fraction by 2-fold, but did not change total PRL I detected. PRL in the Concanavalin-A-retained fraction accounted for 59-69% of PRL in milk based on RIA results. No PRL-like B was detected in the Concanavalin-A-retained fraction of human milk; however, treatment of the glycosylated fraction of milk with peptide-N-glycosidase F increased thymidine incorporation by Nb2 cells 1.67-fold compared to that in controls. The results of this study show that human milk contains considerably greater PRL-like activity than previous reports based on RIA detection. The appearance and regulation of multiple bioactive PRL variants in milk throughout the course of lactation may serve as a mechanism by which milk PRL influences neonatal development.
authors.affiliation: Department of Nutrition, Pennsylvania State University, University Park 16802, USA.
authors.forename: L A
authors.initials: LA
authors.lastname: Ellis
authors.forename: M F
authors.initials: MF
authors.lastname: Picciano
mesh_terms: D005260:Female; D014644:Genetic Variation; D006031:Glycosylation; D006801:Humans; D007120:Immunochemistry; D007231:Infant, Newborn; D007234:Infant, Premature; D007774:Lactation / Q000235:genetics / Q000378:metabolism; D008895:Milk, Human / Q000737:chemistry*; D008970:Molecular Weight; D010766:Phosphorylation; D011388:Prolactin / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology
chemical_list: D011388:Prolactin