Inorganic pyrophosphate (diphosphate, pyrophosphoric acid or PPi) is a small molecule formed during, or required as a substrate for, a number of biochemical reactions. Enzymatic reactions that generate PPi include ATP hydrolysis, DNA and RNA polymerization, cyclic AMP formation, and enzymatic activation of fatty acids to form their CoA esters. Conversely, enzymes that utilize PPi may include cyclases, hydrolases and ligases. Measurement of inorganic pyrophosphate (PPi) in plasma, serum and other biologic fluids is important in studies of bone metabolism, renal stone disease, and certain types of arthritis. Assay Genie's Pyrophosphate (PPi) Assay Kit provides a fast, convenient and ultrasensitive method for determination of free inorganic pyrophosphate levels in biological material. PPi produced during biotic processes is detected through a series of reactions which utilize a proprietary enzyme mix and probe, generating a stable product that can be quantified by either colorimetric or fluorometric readout. Generated fluorescence (Ex/Em = 535/587 nm) or color (OD: 570 nm) intensities are directly proportional to the concentrations of pyrophosphate, enabling precise measurements. Monomeric inorganic phosphate (Pi) does not interfere with the assay. Our assay delivers an easy and robust method suitable for use in a variety of biological samples and can be performed in a convenient microtiter-plate format. The kit provides sufficient reagents for 100 fluorometric or 50 colorimetric assays, respectively. This kit can detect as low as 1.8 PPi in plasma and serum samples.

Figure: Pyrophosphate (PPi) Standard Curves: Fluorometric (A) and Colorimetric (B). Quantification of pyrophosphate in 20 µl de- proteinized undiluted human serum and pooled plasma (C). Spike and recovery in 20 µl of normal human plasma. Plasma samples were spiked with 0.06 nmol of PPi Standard and assayed according to kit protocol yielding 96% PPi recovery (D). Pyrophosphate measured in HepG2 and HeLa cell lysates according to kit protocol. Cells were extracted directly in the Assay Buffer (E).