Close to the Cthrc1 expressing paraventricular cells we observed Cthrc1 immunoreactivity inside the lumen of small vessels (Fig. 6N), which provides indirect evidence that Cthrc1 may be secreted into the circulation. In young animals many tissues are still growing and undergoing remodeling, including the skeletal system, which constantly remodels. We did indeed find Cthrc1 expression in some osteocytes and osteoblasts (Fig. 6O) raising the possibility that Cthrc1 derived from bone may contribute to hormonal effects on liver and muscle. In the anterior pituitary immunoreactive Cthrc1 was observed in close association with chromophobe cells (Fig. 7A–D) suggesting that these cells express Cthrc1. Until now, no specific hormone has been reported to be expressed by chromophobe cells of the anterior pituitary lobe. Expression of Cthrc1 in the pituitary gland of pigs was confirmed at the mRNA level by RT-PCR (Fig. 5). Pharmacokinetic Properties of Cthrc1 Immunoblotting of plasma from Cthrc1 transgenic mice [1] revealed that the pharmacokinetic properties of Chtrc1 are compatible with a role of a circulating factor (Fig. 2A). To determine the half-life of Cthrc1 in circulation, we injected 125I- labeled Cthrc1 into the carotid artery of Cthrc1 mice and obtained the clearance curve shown in Fig. 8A. The half-life was calculated to be 146 minutes. The integrity of the injected radiolabeled Cthrc1 after 30 minutes in circulation was examined Hormonal Functions of Cthrc1 by subjecting plasma to SDS-PAGE under non-reducing condi- tions followed by autoradiography. Apparent monomeric and dimeric forms of the protein were dominant with higher molecular weight oligomers also detectable (Fig. 8B). Six hours after the Cthrc1 injection the amount of Cthrc1 bound in various organs was determined. Per mg tissue the highest levels of bound Cthrc1 were found in the liver (Fig. 8C). Smaller radiolabeled Cthrc1 fragments were detected in the urine suggesting that at least some of the radioactivity detected in the kidney represents degraded, excreted Cthrc1 and not bound Cthrc1 (data not shown). Cthrc1 in Human Plasma We generated mouse monoclonal antibodies suitable for detection of Cthrc1 by ELISA. These antibodies conjugated to magnetic beads were used to isolate Cthrc1 from 15 ml of plasma. For one sample (#1) (Fig. 9A) the eluate from the beads was examined for the presence of Cthrc1 by Western blotting with HRP-conjugated anti-Cthrc1 antibodies showing a single band of Cthrc1 with a similar molecular mass as Cthrc1 expressed in CHO-K1 cells (Fig. 9A). Three more plasma samples from different donors (#2, #3, #4) were analyzed in the same manner with the exception that the eluates from the magnetic beads were immunoblotted with biotin-conjugated anti-Cthrc1 followed by HRP-conjugated streptavidin (Fig. 9B). Similar results were obtained as shown in Fig. 9A. Figure 7. Cthrc1 accumulation in areas surrounded by chromophobe cells of the anterior pig pituitary. Adjacent sections were stained for Cthrc1 (A) and morphology (C, H&E). (B) and (D) are enlarged images of the boxed area shown in (A). Arrows indicate areas of Cthrc1 accumulation. Scale bar = 50 mm. doi:10.1371/journal.pone.0047142.g007 PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e47142