Detection and Characterization of Phosphorylation and other molecular modifications of Fetuin A in Human Blood

Objectives: The hepatokine Fetuin A (Fet A) has been associated with diverse pathological states like insulin resistance, type 2 diabetes, macrovascular disease and systemic ectopic and vascular calcification. Fet A may also play a role in tumor growth and metastasis. The biological activity of Fet A may be affected by various modifications, including phosphorylation, O- and N-glycosylation and fatty acid binding. Methods: We developed an antibody-based assay for the detection of Fet A phosphorylated at serine 312. Fatty acid pattern were determined by gas chromatography. Results: Using the antibody we found that the phosphorylation was stable in human plasma or serum at room temperature for 8 hours. We observed that Fet A is present in several glycosylation forms in human plasma, but the extent of Ser 312 phosphorylation was not associated with glycosylation. The phosphorylation pattern did not change during an OGTT (0–120 min). We further found that human Fet A binds preferentially saturated fatty acids (>90%) at the expense of mono- and poly- unsaturated fatty acids. Conclusions: Our results indicate that different molecular species of Fet A are present in human plasma and that these different modifications may determine the different biological effects of Fet A.