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Simple Silica Column–Based Method to Quantify Inorganic Polyphosphates in Cartilage and Other Tissues
- Source :
- CARTILAGE. 9:417-427
- Publication Year :
- 2017
- Publisher :
- SAGE Publications, 2017.
-
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.
- Subjects :
- 0301 basic medicine
Biomedical Engineering
Physical Therapy, Sports Therapy and Rehabilitation
Sensitivity and Specificity
Chemistry Techniques, Analytical
Fluorescence
Phosphates
03 medical and health sciences
Mammalian tissue
Basic Science
Polyphosphates
otorhinolaryngologic diseases
medicine
Animals
Humans
Immunology and Allergy
Fluorometry
neoplasms
Simple (philosophy)
Mammals
Cartilage
Reproducibility of Results
Silica column
pathological conditions, signs and symptoms
Anatomy
Silicon Dioxide
digestive system diseases
surgical procedures, operative
030104 developmental biology
medicine.anatomical_structure
Biophysics
Subjects
Details
- ISSN :
- 19476043 and 19476035
- Volume :
- 9
- Database :
- OpenAIRE
- Journal :
- CARTILAGE
- Accession number :
- edsair.doi.dedup.....e1bfe15ba28cd2a2373bc80809470209