Prolactin bioassay by induction of N-acetyllactosamine synthetase in mouse mammary gland explants

Abstract

Prolactin stimulates a number of metabolic activities in mammary gland explants. The bioassay described here uses the induction of an enzyme, N-acetyllactosamine (NAL) synthetase, as an end-point. Explants from midpregnant nulliparous mice are incubated at 37 C in medium TC 199 containing insulin (10 µg/ml) and cortisol (20 µg/ml). The material to be assayed or ovine prolactin standard is added at the start of incubation. After 48 hr the explants are homogenized and NAL synthetase activity is measured. This enzyme, which catalyzes the reaction N-acetylglucosamine (NAG)+UDP-galactose→NAL+UDP, can be conveniently measured radiochemically after incubation of homogenates with NAG+UDP-galactose-14C. The assay easily detects 0.1 mU prolactin/ml; increments in response are observed with increasing concentrations of prolactin up to 10 mU/ml. HGH stimulates NAL synthetase activity, but this effect can be abolished by preincubation with specific antibodies against HGH. Human plasma may be assayed directly. Prolactin is undetectable in the plasma of normal males; plasma levels are elevated during post-partum lactation and in various pathologic states associated with galactorrhea. © 1971 by The Endocrine Society.