Stabilized coenzyme solutions and their use for determining dehydrogenases or substrates thereof

Abstract

Stabilized aqueous solution of a coenzyme for hydrogen-transferring enzymes characterized in that the solution contains NAD, NADP or a derivative thereof in an oxidized or reduced form and one or several organic compounds or salts thereof having a pKa value between 1.5 and 6.0 and/or a nitrogen compound of the general formula (I) in which the residues R 1 , R 2 and R 3 are the same or different and denote hydrogen, or a saturated or unsaturated alkyl or aryl group as well as the use of the solution to determine dehydrogenases in particular lactate dehydrogenase or substrates thereof.

1 . Stabilized aqueous solution of a coenzyme for hydrogen-transferring enzymes characterized in that the solution contains NAD, NADP or a derivative thereof in an oxidized or reduced form and one or several organic compounds or salts thereof having a pKa value between 1.5 and 6.0 and/or a nitrogen compound of the general formula (I)  
       
         
           
           
               
               
           
         
       
       in which the residues R 1 , R 2  and R 3  are the same or different and denote hydrogen, or a saturated or unsaturated alkyl or aryl group.  
     
     
         2 . Stabilized solution as claimed in    claim 1   , characterized in that it contains an acid or a salt thereof with a buffering action in the pH range of 1.0 to 7.0 as the organic compound.  
     
     
         3 . Stabilized solution as claimed in    claim 1    or    2   , characterized in that it contains citric acid or a citrate salt.  
     
     
         4 . Stabilized solution as claimed in    claim 3   , characterized in that it contains ca. 5 to 500 mM citric acid or a citrate salt.  
     
     
         5 . Stabilized solution as claimed in one of the    claims 1    to    4   , characterized in that the pH value of the solution is between 1.0 and 7.0.  
     
     
         6 . Stabilized solution as claimed in one of the    claims 1    to    5    containing a hydroxyl, O- or N-alkyl-hydroxyl, O-benzylhydroxylamine and/or boric acid derivative.  
     
     
         7 . Stabilized solution as claimed in    claim 6   , characterized in that it contains a hydroxylamine derivative at a concentration between 2 and 300 mM.  
     
     
         8 . Method for determining a hydrogen-transferring analyte or a corresponding dehydrogenase in the presence of a hydrogen-accepting coenzyme characterized in that the coenzyme is contained in a stabilized aqueous solution as claimed in    claims 1    to    7   .  
     
     
         9 . Method for the determination as claimed in    claim 8   , characterized in that the analyte lactate, glutamate, ammonia, alcohol, glyceraldehyde-3-phosphate or glucose is determined in the presence of a lactate, glutamate, alcohol, glycerol-3-phosphate or glucose dehydrogenase.  
     
     
         10 . Method as claimed in one of the claims  8  or  9 , wherein the pH value is in a range of 8.5 to 10.0 and the final concentration of citrate salt, boric acid and/or hydroxylamine derivative is in each case between 2 and 50 mM.  
     
     
         11 . Kit for determining a hydrogen-transferring analyte in a sample comprising the following components: 
 a first reagent which contains a dehydrogenase in a suitable system buffering between pH 8.5 and 10.0    and    a second reagent which contains a coenzyme for hydrogen-transferring enzymes and an organic compound having a pKa value between 1.5 and 6.0 and/or a nitrogen compound of the general formula (I)                          in which the residues R 1 , R 2  and R 3  are the same or different and denote hydrogen, or a saturated or unsaturated alkyl or aryl group.    
     
     
         12 . Kit for determining the enzyme activity of a dehydrogenase in a sample comprising the following components: 
 a first reagent which contains a hydrogen-transferring analyte in a suitable system buffering between pH 8.5 and 10.0    and    a second reagent which contains a coenzyme for hydrogen-transferring enzymes and an organic compound having a pKa value between 2.0 and 4.0 and/or a hydroxylamine derivative of the general formula (I) as claimed in    claim 11   .    
     
     
         13 . Kit as claimed in    claim 11    or    12   , characterized in that the second reagent contains citric acid, a citrate salt, a boric acid and/or hydroxylamine derivative.  
     
     
         14 . Kit as claimed in    claim 11    or    12   , characterized in that the first reagent contains a boric acid derivative and the second reagent contains citric acid, a citrate salt and/or a hydroxylamine derivative.  
     
     
         15 . Kit as claimed in    claim 11    to  14 , characterized in that the second reagent has a pH value between 1.0 and 7.0.  
     
     
         16 . Kit as claimed in one of the    claims 11    to    15   , characterized in that the second reagent has a pH value of about 3.0.  
     
     
         17 . Kit as claimed in one of the    claims 11    to    16   , characterized in that the first or second reagent contains approximately 5 to 200 mM of a citrate salt, about 2 to 300 mM of a boric acid derivative and/or 2 to 300 mM of a hydroxylamine derivative.