Abstract:
Enzymatic assays for determining glucose, creatine phosphokinase or plasma ammonia, wherein nicotinamide adenine dinucleotide is reduced or the reduced form of nicotinamide adenine dinucleotide is oxidized to nicotinamide adenine dinucleotide, are carried out in the presence of about 2 to 50 micromoles per liter of oxalic acid or oxamic acid, or salts thereof. The oxalic acid, oxamic acid and salts thereof inhibit lactic dehydrogenase which causes errors in the assays.

Description:
BACKGROUND OF THE INVENTION 
     In the optical method for the determination of glucose, hexokinase (HK) catalyzes the phosphorylation of glucose by adenosine triphosphate (ATP) as follows: 
     
         Glucose + ATP .sup.Hexokinase → Glucose-6-Phosphate + Adenosine Diphosphate (ADP) 
    
     glucose-6-phosphate (G-6-P) is oxidized in the presence of nicotinamide adenine dinucleotide (NAD) by glucose-6-phosphate dehydrogenase (G-6-PDH): 
     
         G-6-P + NAD.sup.+ .sup.G-6-PDH → 6-Phosphogluconate + NADH + H.sup.+ 
    
     the reduction of NAD to NADH (reduced form of NAD) at 340 nanometers (nm) is a quantitative measure of the amount of glucose present. 
     In the creatine phosphokinase (CPK) assay method of Rosalki, s. b., Journal of Laboratory and Clinical Medicine, 69; 696  1967), CPK catalyzes the reversible formation of adenosine triphosphate (ATP) and creatine from adenosine diphosphate (ADP) and creatine phosphate (CPO 4 ) according to the equation: 
     
         ADP + Creatine Phosphate .sup.CPK → ATP + Creatine 
    
     The ATP formed in the CPK mediated reaction is used to phosphorylate glucose in the presence of hexokinase (HK) producing glucose-6-phosphate. As glucose-6-phosphate is formed, ADP is generated keeping its concentration at a constant level. 
     
         Glucose + ATP .sup.Hexokinase → Glucose-6-Phosphate + ADP 
    
     the glucose-6-phosphate formed by the hexokinase reaction is then oxidized by the enzyme glucose-6-phosphate dehydrogenase (G-6-PDH) with simultaneous reduction of NAD. 
     
         G-6-P +NAD.sup.+ .sup.G-6-PDH→ 6-Phosphogluconate+ NADH + H.sup.+ 
    
     the reduction of NAD to NADH is followed spectrophotometrically by observing the resulting increase in absorbance at 340 nm. For each mole of phosphate transferred by the CPK, one mole of NADH is formed. Thus the rate of absorbance change is directly proportional to the CPK activity present in the sample. 
     Lactic dehydrogenase (LDH) (L-Lactate:NAD oxidoreductase catalyzes the following reaction: 
     
         L-(+):Lactate + NAD.sup.+ .sup.LDH ⃡ Pyruvate + NADH + H.sup.+ 
    
     in assaying for serum constituents other than LDH, any LDH present may cause interference due to the presence of either lactate or pyruvate in the sample, thus causing errors in the assay of glucose or CPK or in any assay system where NAD is reduced to NADH. Any NADH generated is subject to reaction with pyruvate in the presence of LDH, thus causing errors in the assay of glucose or CPK, for example. 
     
         H.sup.+ + NADH + Pyruvate .sup.LDH → Lactate + NAD.sup.+ 
    
     this interference may be overcome or avoided by the use of NADP as follows: 
     
         1. Creatine Phosphate (CPO.sub.4) + ADP .sup.CPK → Creatine + ATP 
    
     
         2. atp + glucose .sup.Hexokinase → Glucose-6-Phosphate (G-6-P) + ADP 
    
     
         3. g-6-p + nad phosphate (NADP).sup.+ .sup.G-6-PDH → 6-Phosphogluconate + NADPH + H.sup.+ 
    
     nadp and NADPH do not react with LDH present in common biological samples; however, the use of NADP in the enzymatic determination of either CPK or glucose increases the cost of the reagent and reduces the dynamic range of the method. The use of NAD in place of NADH results in reduced costs of reagent and increased dynamic range but permits undesirable reactions and the possibility of error as hereinbefore described. 
     SUMMARY OF THE INVENTION 
     It has been found that LDH forms an inactive complex with oxalic or oxamic (oxaminic)acid and their salts, thus eliminating interferences due to the presence of LDH. The concentration of oxalic or oxamic acid or salts thereof should not be in such concentration as to inhibit or interfere with the constituents being measured but should be sufficient to inhibit the reaction of LDH. Generally a concentration of from two to nine micromoles per liter is sufficient. Up to fifty micromoles can be used for high levels of pyruvate or lactate in the presence of LDH, the appropriate concentration therefore ranging from about 2 to 50 micromoles per liter of reagent solution. The method can be employed with any assay system where NAD is reduced to NADH and where the presence of pyruvate and/or LDH would be undesirable. 
     The method of the present invention can also be employed in assay systems wherein NADH is oxidized to NAD in order to prevent interference by pyruvate or lactate in a sample which contains LDH. In such reactions, the disappearance of NADH is a quantitative measure of the constituent which is being measured. As example is the measurement of plasma ammonia using glutamic dehydrogenase. 
     In the publication by Von F. Da Fonseca-Wollheim entitled &#34;Direkte Plasmaammoniakbestimmung Ohne Enteiweissung&#34; Z. Klin. Chem. Klin. Biochem. 11, 426-431, 1973, an assay system for plasma ammonia is described and it is pointed out that unspecific changes in extinction upon initiation of the reaction are avoided by using reduced NAD phosphate (NADPH) in place of NADH as the coenzyme. This procedure uses 0.5 ml. of plasma with 1.5 ml. of reagent. Since the sample is not deproteinized, pyruvate and LDH in the sample can react with NADH and therefore be measured as apparent ammonia. Since normal ammonia levels are quite low, less than 60 micromoles per liter, equivalent to 0.1 milligrams percent, even a small interference by pyruvate or other keto acids would be highly detrimental to the assay. It is therefore apparent that in assays employing reactions in which NADH is consumed and in which interference can occur, use of the herein described method would eliminate such interferences. 
     DETAILED DESCRIPTION 
     The present invention is exemplified by use of oxalate as the free acid or potassium salt in both the CPK or glucose assays as previously described. Oxalate is effective in preventing interference by high levels of pyruvate or lactate in the presence of LDH, thus enabling one to substitute NAD for NADP. The oxalate used may be part of the buffer system or included as a separate ingredient. The advantages of using NAD rather than NADP are decreased cost and increased dynamic range. 
     CPK 
     The effect of pyruvate at a level of 100 milligram percent in the sample tested is shown in Table I. Using NADP, there is no effect because LDH does not utilize NADP. With NAD, the presence of pyruvate causes a decrease in apparent CPK activity due to the conversion of pyruvate to lactate and subsequent oxidation of NADH. When the reaction of samples containing 100 milligram percent pyruvate is run in the presence of 2.63 millimoles per liter of oxalate (Table 2), the interference due to LDH is obviated. 
     
                       TABLE I______________________________________CPK (International Units/Liter)Effect of Pyruvate on CPK ActivityNADP             NADControl    100 mg%   Control     100 mg%(No Pyruvate)      Pyruvate  (No Pyruvate                            Pyruvate                No Oxalate) (No Oxalate)______________________________________ 9         10        11          1478         80        83          80 9          9        13           9455        461       490         46965         65        70          64102        104       106         99717        726       845         83331         31        32          2819         20        25          2216         17        20          17______________________________________ 
    
     
                       TABLE 2______________________________________CPK (International Units/Liter)Effect of Oxalate on CPK Activity of SamplesContaining 100 mg% PyruvateControl         Test(No Pyruvate)   (100 mg% Pyruvate in           2.63 mmole/liter Oxalate______________________________________10              1074              7311              11422             42857              5892              93726             74029              2722              2118              18______________________________________ 
    
    
    
     EXAMPLE I 
     The following is an example of a reagent employing NAD in the presence of magnesium and activator and suitable for the enzymatic determination of creatine phosphokinase. 
     
         __________________________________________________________________________                         Quantity PerIngredient      Quantity Per 3 ml. Assay                         Milliliter of Reagent__________________________________________________________________________adenosine diphosphate (ADP)           0.5 - 5 milligrams                         0.16 - 1.6 milligramscreatine phosphate (CPO.sub.4)            10 - 30 milligrams                           3 - 10 milligramsglucose          5 - 20 milligrams                          1.6 - 6.6 milligramsnicotinamide adenine            2 - 6 milligrams                          0.6 - 2 milligramsdinucleotide (NAD)hexokinase (HK)  2 - 10 international                          0.6 - 3.3 international           units         unitsglucose-6-phosphate           5.0 - 15.0 international                          1.6 - 5.0 internationaldehydrogenase (G-6-PDH)           units         unitsmagnesium aspartate            2 - 20 milligrams                          0.6 - 6.6 milligrams__________________________________________________________________________ 2 to 9 millimoles of oxalate per liter of reagent in the form of potassium oxalate is added to prevent the noted side reaction together with a suitable buffer to maintain the pH in the range of 6.1 to 7.0. Dithioerythritol in the amount of from 2 to 25 millimoles per liter of solution is satisfactory as an activator. 
    
     GLUCOSE 
     The effect of pyruvate on glucose determinations is shown in Table 3, giving lower values due to the presence of pyruvate and LDH causing oxidation of the NADH formed. When oxalate is included in the formulation, pyruvate shows no interference in the presence of LDH (Tables 4 and 5). 
     
                       TABLE 3______________________________________Glucose (mg%)Effect of Pyruvate on GlucoseDeterminations (Native Sera)Control           Test(No Pyruvate)     (100 mg% Pyruvate)______________________________________64                5786                7795                8378                6296                8075                6180                6793                81152               13976                65______________________________________ 
    
     
                       TABLE 4______________________________________Glucose (mg%)Effect of Oxalate on Samples ContainingPyruvate (200 mg%)______________________________________Control            Test(No Pyruvate)      (200 mg% Pyruvate in(39.6 mmole/liter Oxalate)              39.6 mmole/liter Oxalate______________________________________86                 87105                10592                 9224                 24227                22881                 8080                 81286                28376                 7673                 7486                 86______________________________________ 
    
     
                       TABLE 5______________________________________Glucose (mg%)Effect of Oxalate on Determination of Glucosein Presence and Absence of Pyruvate______________________________________Control(No Oxalate     100 mg% Pyruvate                    100 mg% PyruvateNo Pyruvate     No Oxalate     9 mmole/liter Oxalate______________________________________123       71             12467        24             6752        12             5281        34             8250         9             4926         0             2776        30             7568        23             6982        31             8265        22             66______________________________________ 
    
     EXAMPLE II 
     The following is an example of a reagent employing NAD in the presence of magnesium and suitable for the enzymatic determination of glucose. 
     
         __________________________________________________________________________                           Quantity PerIngredient      Quantity Per 3 ml. Assay                         Milliliter of Reagent__________________________________________________________________________adenosine triphosphate (ATP)           0.5 - 5.0 milligrams                         0.16 - 1.6 milligramsnicotinamide adenine           0.5 - 5.0 milligrams                         0.16 - 1.6 milligramsdinucleotide (NAD)hexokinase (HK) 0.5 - 5.0 international                         0.16 - 1.6 international                units           unitsglucose-6-phosphate           0.5 - 5.0 international                         0.16 - 1.6 internationaldehydrogenase (G-6-PDH)                units           unitsmagnesium aspartate            2 - 10 milligrams                          0.6 - 3.3 milligrams__________________________________________________________________________ 
    
     2 to 9 millimoles of oxalate per liter of reagent in the form of potassium oxalate is added to prevent the noted side reaction. A suitable buffer is added to maintain the pH at about 7.5. 
     The ingredients of the reagents of either Examples I and II may be mixed to provide a product. Accordingly, a single, dry, stable reagent for the enzymatic assay of glucose or creatine phosphokinase is provided. 
     The studies reported in Tables 1 through 5 were carried out at a temperature of 37° C, using a total volume of reagent and sample of 3.02 milliliters, including 0.02 milliliters of sample. A recording spectrophotometer, such as the Perkin-Elmer model 124, can be used for making the determination. 
     The reagents of Examples I and II can be reconstituted by adding a quantity of distilled water and gently mixing to dissolve the contents. The reagents are stable at room temperature for 6 months and in solution for at least 8 hours at room temperature or 24 hours at 4° C. 
     In use, the reconstituted reagent is separated into 3.0 ml. aliquot portions, to each of which is added 0.02 ml. of sample. The mixture is incubated at 37°C. for at least 10 minutes and the absorbance is recorded versus a reagent blank to determine the concentration of the ingredient being measured. 
     While 3.0 milliliter portions of reagent were employed in the studies reported herein, other quantities can be used as desired. The concentrations indicated in Examples I and II refer to the reagent in the form of a solution or to a dry powder mixture which, upon reconstituting in the desired amount of water or other solvent, will provide the indicated concentrations.