DEVELOPMENT AND VALIDATION OF A RADIOIMMUNOASSAY FOR FOLLISTATIN IN HUMAN SERUM

OBJECTIVE Follistatin (FS/FSH-suppressing protein/activin-binding prot ein) is a single-chain glycoprotein, structurally distinct from inhibi n, that has been shown to have inhibin-like activity in suppressing FS H secretion both in vivo and in vitro. The aim of these studies was to develop and validate a radioimmunoassay (RIA) for FS in human serum, and to describe the physiological variations of serum FS in humans. PA TIENTS Serum was collected from normal men, and normal women in the fo llicular and luteal phases of the menstrual cycle. Clinical samples we re also collected from male patients with hypogonadism, post-menopausa l women and pregnant women in the first, second and third trimesters. MEASUREMENTS A RIA for FS in human serum was developed using antisera raised against purified bovine FS and using bovine FS as tracer and st andard. Serial dilutions of serum were non-parallel to purified bovine FS standard in the RIA. The addition of sodium dodecyl sulphate (SDS 0.05%) to the assay buffer resolved the non-parallelism suggesting tha t the interference of serum in the RIA was an assay matrix effect. To characterize the serum FS immunoactivity further, serum was fractionat ed by gel filtration on Sephadex G-100. At neutral pH, FS immunoactivi ty eluted as a major peak in the molecular weight range > 200 kDa. In 0.1 M HCI, a second peak of FS immunoactivity eluted in the molecular weight range 30-60 kDa consistent with the known sizes of FS. This sug gested that FS was dissociating from a larger complex. Fractions taken from the low molecular weight region diluted in parallel with bovine FS in contrast with fractions from the high molecular weight region wh ich were non-parallel. It is concluded that the non-parallelism of hum an serum in the FS RIA is due to the binding of serum FS to an unknown high molecular weight factor. This interference is eliminated by the inclusion of 0.05% SDS in the assay buffer. The RIA in 0.05% SDS has b een applied to the description of the normal and pathophysiological va riations of FS in human serum. RESULTS There were no significant diffe rences between FS levels in serum from hypogonadal men, normal women i n the follicular phase of the menstrual cycle, post-menopausal women a nd pregnant women from the first, second and third trimesters. FS leve ls in serum of women in the luteal phase of the menstrual cycle were s ignificantly lower than all other groups. FS levels in normal men were significantly higher than those in both luteal phase women and women in the first trimester of pregnancy (P < 0.05). CONCLUSIONS These find ings argue against a role for circulating follistatin in the control o f gonadotrophin secretion and suggest that the gonads and/or conceptus are not the primary source of follistatin immunoactivity in serum. Th e lower follistatin levels in the luteal phase of the menstrual cycle remain unexplained.