Biochemical analysis of tissue and serum samples from fro/fro;Col1a1-Smpd3 mice. (A) Schematic representation of the Col1a1-Smpd3 transgene construct. (B, top) Semiquantitative PCR analysis confirming bone-specific expression of the transgene (TG). (bottom) Hprt expression analysis has been shown as a control for the cDNA amount. Amplicon sizes in base pairs are indicated on the left. (C) Enzymatic assays using ¹⁴C-labled methyl-sphingomyelin shows no change in nSMase activity in fro/fro;Col1a1-Smpd3 brain samples. As expected, in fro/fro;Col1a1-Smpd3 bone samples, the nSMase activity is increased in comparison with the fro/fro bone samples. (D–G) Lipid analysis using liquid chromatography/mass spectrometry of sphingomyelin (D), total ceramide (E), individual ceramide species (F), and dihydroceramide (G). A significant increase of total ceramide levels is caused by the increase of several long-chain ceramide species in fro/fro bones (n = 4). (H–J) Serum calcium (H), P_i (I), and alkaline phosphatase (J) activities are comparable in +/fro, fro/fro, and fro/fro;Col1a1-Smpd3 mice (n = 5). Error bars represent standard deviations. *, P < 0.05; **, P < 0.01.