Biochemical analyses were done on blood samples (taken by orbital exsanguination) and spot urines collected following an overnight fast at the same time of day between 10 AM and 2 PM. Following deproteinization of heparinized plasma by filtration (NanoSep 300 K, Pall Corp., Ann Arbor, MI), plasma and urinary total pyrophosphate (PPi) concentrations were determined using a fluorometric probe (AB112155, ABCAM, Cambridge, MA). Urine PPi was corrected for urine creatinine, which was measured by LC-MS/MS or by ELISA using appropriate controls to adjust for inter-assay variability.