Using pyrophosphorylase crystal structure expression profile as diagnostic indicator of metabolic disorders; rational drug design
Inventors
Assignees
University of Miami • US Department of Veterans Affairs
Publication Number
US-8153436-B2
Publication Date
2012-04-10
Expiration Date
2028-02-14
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Abstract
Methods of assaying for disease-associated crystal species in biological samples are described. Such methods involve contacting a patient sample with an excess of a detectable crystal-tagging compound reactive with a plurality of crystal species under conditions that allow the detectable crystal-tagging compound to react with a plurality of crystal species, if present, to form tagged crystal species complexes. Substantially all unreacted tagging compound is then removed. If desired, chemical, enzymatic, or physical treatment can be used to selectively degrade some, but not, of the tagged crystal species. Assessment of soluble versus crystal-associated label is then performed.
Core Innovation
The invention provides patentable methods, reagents, and kits to assay for one or more disease-associated crystal species in biological samples. These assays can be qualitative, semi-quantitative, or quantitative and useful for diagnostic purposes, including monitoring disease progression and therapeutic efficacy. The general method involves contacting a biological sample with an excess of a detectable crystal-tagging compound reactive with multiple crystal species, allowing tagging compound to react and form tagged crystal species complexes. Subsequently, excess tagging compound is removed, and the tagged complexes are detected.
Preferred detectable crystal-tagging compounds comprise a crystal reactive compound such as phosphocitrate, which reacts with calcium-containing crystals, conjugated to a detectable label, including radiolabels, fluorescent labels, or members of high affinity binding pairs. The disease-associated crystal species include crystallized basic calcium phosphate species, crystallized calcium pyrophosphate dihydrate, crystallized magnesium-substituted tricalcium phosphate, crystallized calcium oxalate, or their combinations. Some embodiments include selective degradation of certain crystal species using chemical or enzymatic reagents, such as pyrophosphorylase, to release soluble detectable species for further detection and differentiation of crystal types.
Claims Coverage
The claims include one independent claim focusing on a method for assaying crystallized calcium pyrophosphate dihydrate (CPPD) in biological samples using a radiolabeled calcium crystal-tagging compound and selective enzymatic degradation.
Using radiolabeled phosphocitrate as a calcium crystal-tagging compound
The method involves contacting a biological sample with an excess of a detectable calcium crystal-tagging compound reactive with multiple crystallized calcium species, wherein the tagging compound is radiolabeled phosphocitrate.
Selective degradation of CPPD using pyrophosphorylase or pyrophosphatase
After forming detectable calcium complexes, the method includes contacting these complexes with a reagent that selectively degrades CPPD crystals, releasing a soluble detectable species containing the radiolabel. The reagent is specifically a pyrophosphorylase or pyrophosphatase enzyme.
Detection of soluble radiolabeled species for CPPD presence
The soluble detectable species containing the radiolabel is detected, thereby indicating the presence of CPPD in the biological sample.
The claims cover a method that uses radiolabeled phosphocitrate to tag calcium crystals in a biological sample, followed by selective enzymatic degradation of CPPD crystals using pyrophosphorylase or pyrophosphatase to generate and detect a soluble radiolabeled species, thus identifying CPPD presence.
Stated Advantages
Provides a rapid and specific assay to detect and quantify disease-associated calcium-containing crystal species in biological samples.
Allows differentiation of multiple calcium crystal species, including CPPD and basic calcium phosphate, through selective enzymatic degradation.
Can be performed in a clinical laboratory setting without requiring expensive and complex instrumentation used in conventional crystal identification methods.
Documented Applications
Diagnostic detection and quantification of calcium-containing crystal species in synovial fluid or other biological samples for diseases such as osteoarthritis, pseudogout, and acute calcific periarthritis.
Monitoring disease progression and therapeutic efficacy in patients with crystal-deposition diseases.
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