Method and reagent for determination of cholesterol in remant-like particles

Abstract

The present invention provides a method and a reagent for the simple and sensitive determination of cholesterol in remnant-like particles in a biological sample without the separation of components of the sample. The method comprises contacting the sample with (i) cholesterol esterase, (ii) cholesterol oxidase or cholesterol dehydrogenase, and (iii) phospholipase in the presence of oxygen or an oxidized coenzyme, and measuring the formed hydrogen peroxide or reduced coenzyme. The reagent comprises (i) cholesterol esterase, (ii) cholesterol oxidase or cholesterol dehydrogenase, and (iii) phospholipase.

What is claimed is:  
     
         1 . A method for the quantitative determination of cholesterol in remnant-like particles in a biological sample, which comprises contacting the biological sample with (i) cholesterol esterase, (ii) cholesterol oxidase or cholesterol dehydrogenase, and (iii) phospholipase in an aqueous medium in the presence of oxygen or an oxidized coenzyme, and measuring the formed hydrogen peroxide or reduced coenzyme.  
     
     
         2 . The method according to    claim 1    wherein a surfactant exists in the aqueous medium when the biological sample is contacted with (i) cholesterol esterase, (ii) cholesterol oxidase or cholesterol dehydrogenase, and (iii) phospholipase.  
     
     
         3 . The method according to    claim 1    wherein a surfactant is added to the biological sample prior to the contact with (i) cholesterol esterase, (ii) cholesterol oxidase or cholesterol dehydrogenase, and (iii) phospholipase.  
     
     
         4 . The method according to    claim 2    or    3    wherein said surfactant is a surfactant which inhibits the action of cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase on cholesterol in lipoproteins other than remnant-like particles.  
     
     
         5 . The method according to any of    claims 2    to    4    wherein said surfactant is a polyoxyalkylene derivative or a polyoxyethylene-polyoxypropylene copolymer or its derivative.  
     
     
         6 . The method according to    claim 1    wherein a polyoxyethylene-polyoxypropylene copolymer or its derivative is added to the biological sample prior to the contact with (i) cholesterol esterase, (ii) cholesterol oxidase or cholesterol dehydrogenase, and (iii) phospholipase, and a polyoxyalkylene derivative is added when the biological sample is contacted with (i) cholesterol esterase, (ii) cholesterol oxidase or cholesterol dehydrogenase, and (iii) phospholipase.  
     
     
         7 . The method according to    claim 5    or    6    wherein said polyoxyalkylene derivative is polyoxyethylene alkyl ether, polyoxyethylenestyrenated phenyl ether or polyoxyethylene long-chain branched alkyl ether.  
     
     
         8 . The method according to    claim 5    or    6    wherein said polyoxyethylene-polyoxypropylene copolymer or its derivative is a compound represented by general formula (I):  
       RO—(C 2 H 4 O) a —(C 3 H 6 O) b —(C 2 H 4 O) c —H  (I)  
       (wherein a, b and c, which may be the same or different, each represents an integer of 1 to 200, and R represents straight-chain or branched alkyl).  
     
     
         9 . The method according to any of    claims 1    to    8    wherein said phospholipase is phospholipase D, phospholipase C or phospholipase A2.  
     
     
         10 . A reagent for the quantitative determination of cholesterol in remnant-like particles, comprising (i) cholesterol esterase, (ii) cholesterol oxidase or cholesterol dehydrogenase, and (iii) phospholipase.  
     
     
         11 . The reagent according to    claim 10   , further comprising a surfactant.  
     
     
         12 . The reagent according to    claim 11    which comprises a first reagent comprising the surfactant and a second reagent comprising (i) cholesterol esterase, (ii) cholesterol oxidase or cholesterol dehydrogenase, and (iii) phospholipase.  
     
     
         13 . The reagent according to    claim 11    or    12    wherein said surfactant is a surfactant which inhibits the action of cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase on cholesterol in lipoproteins other than remnant-like particles.  
     
     
         14 . The reagent according to    claim 11    or    12    wherein said surfactant is a polyoxyalkylene derivative or a polyoxyethylene-polyoxypropylene copolymer or its derivative.  
     
     
         15 . The reagent according to    claim 12    wherein said surfactant in the first reagent is a polyoxyethylene-polyoxypropylene copolymer and the second reagent further comprises a polyoxyalkylene derivative.  
     
     
         16 . The reagent according to    claim 14    or    15    wherein said polyoxyalkylene derivative is polyoxyethylene alkyl ether, polyoxyethylenestyrenated phenyl ether or polyoxyethylene long-chain branched alkyl ether.  
     
     
         17 . The reagent according to    claim 14    or    15    wherein said polyoxyethylene-polyoxypropylene copolymer or its derivative is a compound represented by general formula (I):  
       RO—(C 2 H 4 O) a —(C 3 H 6 O) b —(C 2 H 4 O) c —H  (I)  
       (wherein a, b and c, which may be the same or different, each represents an integer of 1 to 200, and R represents straight-chain or branched alkyl).  
     
     
         18 . The reagent according to any of    claims 10    to    17    wherein said phospholipase is phospholipase D, phospholipase C or phospholipase A2.