A 2-SITE CHEMILUMINESCENT ASSAY FOR ACTIVIN-FREE FOLLISTATIN REVEALS THAT MOST FOLLISTATIN CIRCULATING IN MEN AND NORMAL CYCLING WOMEN IS IN AN ACTIVIN-BOUND STATE

Follistatin (FS) is a monomeric protein that binds and regulates the b ioavailability of activin. Previously, we found circulating levels of total FS to be similar in men and cycling women. Because relative amou nts of activin-bound and free FS are important considerations in deter mining activin bioavailability, we asked here whether the relative pro portions of these two changed during different physiologic states. For this, we developed a two-site, solid-phase, immunochemiluminescent as say for free FS. The assay recognizes the 288 or 315 amino acid varian ts of human FS and has a detectable limit of 1 ng/mL. Inhibin, transfo rming growth factor-beta, or alpha-2-macroglobulin do not cross-react or interfere in this assay. Preincubation of FS with activin results i n dose-dependent loss of immunoreactivity, confirming specificity of t he assay for free FS. Human follicular fluid, pituitary extract, and s erum with added FS dilute parallel with the recombinant human FS-288 s tandard. Recovery of recombinant human FS-288 from serum is quantitati ve. Using this assay, we found circulating concentrations of free FS t o be at or below the detection limit of the assay throughout the menst rual cycle. Comparison of circulating total and free FS levels in post menopausal or cycling women and normal men suggested that at least 90% is activin-bound. In contrast, measurable quantities of free FS were found in follicular fluid and pituitary extracts. The results of this study, showing that most; circulating FS is normally activin-bound, ar gue against an endocrine role for FS and suggest that a major role of circulating FS is to bind and neutralize the bioactivity of circulatin g activin. The roles of FS as a local autocrine or paracrine regulator of activin in target tissues, where FS exists in free form, or as an endocrine regulator in human pathophysiology, warrants further investi gation.