PROLACTIN HETEROGENEITY - A LIMITATION ON THE EVALUATION OF RESULTS FROM PROLACTIN ASSAYS DUE TO DIFFERENCES IN IMMUNOASSAYS AND THE DIFFERENT BIOACTIVITIES OF PROLACTIN FORMS

Prolactin exists in biological fluids in several molecular forms. This raises two questions: (1) whether the assay of prolactin by immunotec hniques is valid and reliable and (2) whether the different forms have different physiological roles, which might be exploited to improve di agnostic accuracy and data interpretation by the use of appropriate me thods. To investigate these questions, prolactin from human amniotic f luid was separated, by concanavalin A-Sepharose affinity chromatograph y, into bound, retarded and unbound fractions (bound prolactin fractio n, retarded prolactin fraction, unbound prolactin fraction), which wer e characterized by electrophoresis, immunoblotting and glycan detectio n blot. Virtually no contamination was found in the bound prolactin fr action, and the unbound prolactin fraction and retarded prolactin frac tion were 74-83% pure according to densitometry of the electrophoretic and immunoblot patterns. High variability was found among the individ ual patterns. Glycan detection in the blotted fractions revealed that the bound prolactin fraction bands corresponding to M(r) 25000 - 29000 were weakly glycolysated, whereas the bands of M(r) 60000 - 64000 wer e significantly glycan-positive. Immunoreactive bands of unbound prola ctin fraction and retarded prolactin fraction also stained positively for glycans. Using two commercial prolactin kits, the bound prolactin fraction forms were virtually undetectable. To demonstrate that the pr olactin forms may depend on the hypothalamic state, two behaviourly di fferent breeds of cattle were used as an animal model for studying hyp othalamic activities. The number of immunoreactive bands, representing the prolactin forms, and the change of the forms in response to thyro liberin differed strikingly among the groups. The bioactivity of the f orms was examined in bovine granulosa, oviductal, endometrial and sple en cells, and in murine splenocytes, the latter being activated by con canavalin A or allogeneically to create in vitro conditions that may h ave relevance for situations in vivo. The rate of incorporation of [H- 3]thymidine in murine splenocytes was dose-dependently enhanced only b y bound prolactin fraction. The increase was abolished by purified ant i-prolactin antiserum. However, the standard prolactin from the kits i nhibited the proliferation even in low dose (1.25 mu g/l) and the inhi bition was abolished in part by bound prolactin fraction. Thymidine in corporation into the bovine cells was significantly increased by low c oncentrations (2 mu g/l) of unbound prolactin fraction and retarded pr olactin fraction. Oviduct epithelial cells and splenocytes were stimul ated by unbound prolactin fraction but not by retarded prolactin fract ion in a dose of 16 mu g/l. Thymidine incorporation into granulosa cel ls was inhibited by retarded prolactin fraction (16 mu g/l) but not by unbound prolactin fraction.