BIOACTIVE AND IMMUNOREACTIVE PROLACTIN VARIANTS IN HUMAN-MILK

Human milk from mothers of term (T) and preterm (PT) infants was colle cted during early (days 2-7), mature (2-16 weeks), or late (>16 weeks) lactation. PRL-like bioactivity (B) was measured by Nb2 cell prolifer ation, and PRL immunoreactivity (I) was determined by RIA. PRL activit y is reported in PRL equivalents (1 PRL equivalent = 1 ng NIDDK refere nce material). Milk from early lactation contained significantly great er 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 la ctation progressed (T mature: B, 41.74 +/- 8.9; I, 27.19 +/- 5.5; T la te: 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 wa s 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-) f raction of human milk. Four immunoreactive forms (24, 30, 32, and 40 k Da) of P-PRL were identified by immunoblot analyses. Alkaline phosphat ase 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 det ected in the Concanavalin-A-retained fraction of human milk; however, treatment of the glycosylated fraction of milk with peptide-N-glycosid ase F increased thymidine incorporation by Nb2 cells 1.67-fold compare d 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 bioa ctive PRL variants in milk throughout the course of lactation may serv e as a mechanism by which milk PRL influences neonatal development.