Serum z

At the age of 34 weeks, mice were food deprived for 4 h, anesthetized with diethyl ether, and sacrificed. Blood was collected into microtubes (Serum Z, Sarstedt, Nümbrecht, Germany) to obtain serum. Lungs and hearts with the adjacent aorta were prepared, rinsed with 0.9% NaCl solution, weighed, and snap-frozen in liquid nitrogen. The apex of the heart was fixed in 10% paraformaldehyde neutral buffered formalin solution (Sigma-Aldrich, St. Louis, MO, United States). Tibia length was assessed for normalization of organ weights.

Blood samples of day 0, 28 and 71 (end of trial) were collected in serum tubes (Serum Z, Sarstedt AG&Co, Sarstedt, Germany), allowed clotting for 60 min at room temperature and then centrifuged at 2123 g for 15 min (15 °C), aliquoted and stored at − 80 °C until analyses. Serum was analysed for total protein, albumin, urea, aspartate-aminotransferase (AST), alanine-aminotransferase (ALT), γ-glutamyltransferase (γ-GT), total bilirubin, alkaline phosphatase (ALP), triglycerides, cholesterol and glucose using photometric methods with an automatic analyser (Eurolyser, CCA180, Eurolyser Diagnostika GmbH, Salzburg, Austria).

In both studies, feed was withdrawn 4 h prior to sacrifice. All mice were euthanized by decapitation under anesthesia with diethyl ether. Blood was collected into micro tubes (Serum Z, Sarstedt, Nümbrecht, Germany) to gain serum. Serum concentrations of ionized calcium and inorganic phosphate were measured spectrophotometrically using Fluitest® CA CPC and Fluitest® PHOS kits (Analyticon Biotechnologies AG, Lichtenfels, Germany). Serum PTH was analyzed using the mouse PTH 1-84 ELISA Kit (#60-2305) and serum iFGF23 was quantified by using the mouse/rat FGF-23 (intact) ELISA Kit (#60-6800) both purchased from Immunotopics, Inc. (San Clemente, CA, USA). To identify whether high dietary calcium concentrations may induce vascular calcification, aortic root sections were prepared and quantified via von Kossa staining (Schmidt et al., 2012 (link)). The area of calcification was related to the aortic valve tissue.