Simple Silica Column-Based Method to Quantify Inorganic Polyphosphates in Cartilage and Other Tissues.

ABSTRACT

OBJECTIVE: Inorganic polyphosphates (polyP) play a multitude of roles in mammalian biology. PolyP research is hindered by the lack of a simple and sensitive quantification method. The aim of this study was to develop a robust method for quantifying the low levels of polyP in mammalian tissue such as cartilage, which is rich in macromolecules that interfere with its determination. DESIGN: Native and in vitro formed tissues were digested with proteinase K to release sequestrated polyP. The tissue digest was loaded on to silica spin columns, followed by elution of bound polyP and various treatments were assessed to minimize non-polyP fluorescence. The eluent was then quantified for polyP content using fluorometry based on DAPI (4',6-diamidino-2-phenylindole) fluorescence shift occurring with polyP. RESULTS: Proteinase K pretreatment reduced the inhibitory effect of proteins on polyP recovery. The eluent was contaminated with nucleic acids and glycosaminoglycans, which cause extraneous fluorescence signals. These were then effectively eliminated by nucleases treatment and addition of concentrated Tris buffer. PolyP levels were quantified and recovery ratio determined using samples spiked with a known amount of polyP. This silica spin column method was able to recover at least 80% of initially loaded polyP, and detect as little as 10-10 mol. CONCLUSIONS: This sensitive, reproducible, easy to do method of quantifying polyP will be a useful tool for investigation of polyP biology in mammalian cells and tissues. Although the protocol was developed for mammalian tissues, this method should be able to quantify polyP in most biological sources, including fluid samples such as blood and serum.