Abstract:
The present invention is a method for the detection of hydrogen peroxide in biological fluids or an aqueous solution which involves contacting the solution with an oxidation-reduction indicator and a transition metal complex. The transition metal complex is either a creatinine coordinated with iron or a guanidine coordinated with iron.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention is a novel methodology to colorimetrically determine the concentration of hydrogen peroxide in biological fluids and in aqueous solutions by contacting a fluid or solution with an indicator and a transition metal complex which is either creatinine coordinated with iron or guanidine coordinated with iron. 
     2. Description of the Background Art 
     The detection of hydrogen peroxide in biological fluids such as blood, serum, and urine is useful in the diagnosis of various abnormalities. Hydrogen peroxide is generated as a byproduct of numerous reactions of analytes with enzymes. Tests for the detection of hydrogen peroxide in biological fluids are complicated by the presence of ascorbate, a strong reducing agent which can transfer an electron to the indicator resulting in false negative results. The inclusion of certain metal ion complexes, such as Fe-HEDTA, in the indicator reagent composition essentially eliminates ascorbate interference, however, these metal ion complexes themselves can catalyze the color-forming reaction between the peroxide and the oxidizable dye which can, under some circumstances result in false positives or erroneously high assay results due to additional dye oxidation mediated by the metal ion complex. 
     There is literature referencing the discovery of creatinine complexes of various transition metals with the exception of iron. 
     SUMMARY OF THE INVENTION 
     The objects of the present invention are provided by a method for the detection of hydrogen peroxide in biological fluids. Additionally, the present invention provides a method for the detection of an analyte by reacting the analyte with a compound to produce hydrogen peroxide. 
     This invention relates to a method for the detection of hydrogen peroxide, comprising (a) adding a transition metal complex and an oxidation-reduction indicator to a solution suspected of containing hydrogen peroxide and (b) analyzing the color transition to detect the presence or concentration of hydrogen peroxide. The transition metal complex may be creatinine coordinated with iron or guanidine coordinated with iron. The creatinine coordinated with iron has the structure: ##STR1## wherein X and Y are independently chosen from the group consisting of creatinine, chloride, fluoride, bromide, iodide, sulfate, phosphate, perchlorate, nitrate, oxalate, sulfide, ammonium, gluconate, cyanide, thiocyanate, catechol, tropolone, phenol, pyridine, buffers such as acetate, citrate, tartaric acid, malonic acid, boric acid, succinic acid, glycerol-2-phosphate, salicylic acid, oxalic acid, and malic acid. 
     The guanidine coordinated with iron has the structure: ##STR2## wherein X and Y are independently chosen from the same group as for the iron creatinine complex above and R 1  and R 2  are independently chosen from the group consisting of alkyl and aryl groups. Examples of R 1  and R 2  groups are alkyl groups such as C n  H 2n+1  and (C 2  H 4  O) n  H wherein n is an integer from 1 to 20, preferably from 1 to 12; and aryl groups such as C 5  H 4  Z, C 4  H 3  Z, and C 11  H 6  Z, wherein Z is O, S, N, or CH. 
     R 1  and R 2  can optionally be substituted with one or more functional groups which enhance water solubility such as alcohol (--OH), amine (--NH 2 ), carbonyl (--C═O), sulfonic acid (--SO 3  H), carboxylic acid (--CO 2  H), fluorine (--F), chlorine (--Cl), bromine (--Br), iodine (--I), phosphonic acid (--P(O)(OH) 2 ), or phosphate (--OP(O)(OH) 2 ). Particularly preferred iron creatinine complexes are those wherein both X and Y are chloride or both X and Y are acetate. Particularly preferred iron guanidine complexes are those wherein both X and Y are sulfate (--SO 4 ) or both X and Y are acetate, and/or wherein R 1  is H and R 2  is the sulfonic acid-substituted alkyl group --CH 2  CH 2  --SO 3  H; R 1  is H and R 2  is the amine- and carboxylic acid-substituted alkyl group --(CH 2 ) 3  CH(NH 2 )CO 2  H (arginine); or R 1  is H and R 2  is the carboxylic acid-substituted aryl group --C 6  H 3  (CO 2  H) 2 . 
     The solution containing hydrogen peroxide may be a biological fluid or an aqueous solution. The oxidation-reduction indicator and the transition metal complex may be introduced to the solution by means of a test strip to which they have been applied. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The advantages of the present invention will become apparent upon reading the following detailed description and upon reference to the accompanying FIGURE, which shows the hydrogen peroxide dose response. 
    
    
     DETAILED DESCRIPTION OF INVENTION 
     The peroxidase and ascorbate oxidase activities of iron creatinine and iron guanidine complexes were previously unknown. This disclosure describes the first application of iron complexes for the removal of ascorbate interference through the oxidation of ascorbate to dehydroascorbate. 
     Hydrogen peroxide is produced by numerous reactions carried out in the presence of enzymes. For example: ##STR3## The determination of the presence or concentration of hydrogen peroxide is a way of determining the presence or concentration of the substrate in a biological fluid. 
     Table 1 lists examples of substrates and enzymes and the products that they produce. It is not intended to be a complete list of all reactions for which the present invention may be used. 
     
                       TABLE 1______________________________________Substrate   Enzyme         Product______________________________________Glucose     Glucose oxidase                      Gluconic acidCholesterol Cholesterol oxidase                      Cholest-4-en-3-oneD-Aspartate D-Aspartate oxidase                      Oxaloacetatean L-amino acid       L-amino acid oxidase                      a 2-oxo acida D-amino acid       D-amino acid oxidase                      a 2-oxo acidRCH.sub.2 NH.sub.2       Amine oxidase  RCHO       (flavin-containing)Pyridoxamine       Pyridoxamine-phosphate                      Pyridoxal5&#39;-phosphate       oxidase        5&#39;-phosphateRCH.sub.2 NH.sub.2       Amine oxidase  RCHO       (copper-containing)D-Glutamate D-Glutamate oxidase                      2-oxoglutarateEthanolamine       Ethanolamine oxidase                      glycolaldehydePutrescine  Putrescine oxidase                      4-aminobutanalCyclohexylamine       Cyclohexylamine oxidase                      CyclohexanonePeptidyl-L-lysyl-       Protein-lysine 6-oxidase                      Peptidyl-allysyl-peptide                    peptideL-Lysine    L-Lysine oxidase                      2-oxo-6-amino-                      hexanoateD-Glutamate D-Glutamate    2-oxoglutarate       (D-aspartate) oxidaseL-Aspartate L-Aspartate oxidase                      Oxaloacetate3-Hydroxyanthranilate       3-Hydroxyanthranilate                      6-imino-5-oxocyclo-       oxidase        hexa-1,3-diene-                      carboxylateD-alanine   D-amino acid oxidase                      pyruvategalactose   galactose oxidase                      galactonic acidL-tyrosine  polyphenyl oxidase                      4-hydroxyphenyl                      pyruvateputrescine  plasma amine oxidase                      1-diamine-4-butan-                      aldehydeL-tryptophan       1-tryptophan peroxidase                      indoleuric acid   uricase        allantoinxanthine    xanthine oxidase                      uric acid______________________________________ 
    
     The detection of hydrogen peroxide is often carried out by a reaction between hydrogen peroxide, peroxidase, and an oxidation-reduction indicator: 
     
         H.sub.2 O.sub.2 +peroxidase+indicator→color 
    
     For example, glucose activity may be measured by reacting glucose in the presence of glucose oxidase enzyme to produce hydrogen peroxide. The hydrogen peroxide then reacts with peroxidase to catalyze the oxidation of the indicator to produce a colorimetric response directly proportional to the hydrogen peroxide concentration. However, tests for the detection of hydrogen peroxide in biological fluids are complicated by the presence of ascorbate, a strong reducing agent which can transfer an electron to the indicator resulting in false negative results. The transition metal complexes of the present invention react with hydrogen peroxide to catalyze oxidation of the indicator only in the presence of hydrogen peroxide. Ascorbate causes no interference with the detection of hydrogen peroxide brought about using the transition metal complexes of the present invention. ##STR4## 
     Oxidation-reduction indicators (&#34;redox&#34;) indicators are substances which have a different color in the reduced form than in the oxidized form and which can be reversibly reduced and oxidized. Examples of oxidation-reduction indicators which can be used with the transition metal complexes of the present invention are benzidine, o-tolidine, 3,3&#39;,5,5&#39;-tetraalkylbenzidines wherein the alkyl group contains from one to six carbon atoms (e.g., 3,3&#39;,5,5&#39;-tetramethylbenzidine (&#34;TMB&#34;)), o-dianisidine, 2,7-diaminofluorene, bis-(N-ethylquinol-2-one)-azine, (N-methylbenzthiazol-2-one)-(1-ethyl-3-phenyl-5-methyltriazol-2-one)-azine, 2,2&#39;-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), 4-aminoantipyrine, potassium iodine, or combinations thereof. 
     The pH of the test sample is preferably in the range of from about 3 to about 9, most preferably in the range of from about 4.5 to about 8.2. Hydrogen peroxide may be detected in a test sample in which the concentration of hydrogen peroxide is in the range of from about 0.1 to about 100 mM (millimolar), preferably from about 1 to about 20 mM. 
     The following examples and data are listed to demonstrate the novelty of the assay as well as its practical use for the detection of hydrogen peroxide. Example 1 is of a dry reagent for the detection of hydrogen peroxide. Example 2 is of a dry reagent for the detection of glucose via the detection of hydrogen peroxide generated from the reaction of glucose with glucose oxidase. Example 3 shows a test of the peroxidase activity various transition metal complexes. 
     EXAMPLE 1 
     Reagent for the detection of hydrogen peroxide. 
     Dry reagent paper was made by sequentially impregnating reagent paper with the components of two dip solutions, drying in between dips. The reagent paper was then dried at 60° C. for approximately 7 minutes. The paper carrier used was Whatman 3 MM. Dip 1 contained iron creatinine and buffer and was prepared by the addition of 100 mL (milliliters) of water to 4.52 g (grams) of creatinine, 2.70 g ferric chloride hexahydrate, and 2.0 g malonic acid, and allowing the mixture to stir overnight; the solution was then adjusted to pH 4.6 using 1N NaOH; final concentrations were 100 mM ferric chloride, 400 mM creatinine, and 250 mM malonic acid. Dip 2 contained the redox indicator, 3,3&#39;,5,5&#39;-tetramethylbenzidine (&#34;TMB&#34;) at a concentration of 80 mM in acetonitrile. 
     Reagent strips were made from papers produced from the dips above. The strips were then dipped into urine containing various levels of hydrogen peroxide and the reflectance at 660 nm (nanometers) was measured with an Advanced Research Rapid Scanner instrument at one minute after dipping. The reflectance value was taken to represent reagent reactivity. Reagent reactivity was determined using three urine pools of differing specific gravities, 1.005, 1.015 and 1.023 g/mL. 
     Reagent color is directly proportional to hydrogen peroxide concentration. The FIGURE shows the linear dose response observed between hydrogen peroxide concentration and the K/S values obtained with the iron-creatinine reagent. The K/S is the conversion from reflectance to units which are proportional to the absorbance by the indicator. It was calculated according to the equation K/S=(1-R) 2  /2R, wherein R is the reflectance at 660 nm and 1 minute. 
     Table 2 shows that the reagent reactivity towards 3.0 mg/dL (milligrams per deciliter) hydrogen peroxide (0.88 mM) was fairly consistent in all three urine pools. The reagent remained reactive towards 3.0 mg/dL hydrogen peroxide in the presence of 100 mg/dL ascorbic acid, indicating the oxidation of ascorbate was occurring. 
     
                       TABLE 2______________________________________   Reflectance at 660 nm and 1 minute   Mean (standard deviation)     Urine      Urine        Urine     specific gravity                specific gravity                             specific gravityAdditive to urine     1.005 g/mL 1.015 g/mL   1.023 g/mL______________________________________none      72.4 (0.9) 70.4 (0.9)   69.2 (1.1)3.0 mg/dL H.sub.2 O.sub.2     29.8 (1.2) 29.9 (2.3)   36.8 (3.2)3.0 mg/dL H.sub.2 O.sub.2      37.9 (2.1)*                 40.8 (1.7)*  48.0 (1.9)*and 100 mg/dLascorbic acid______________________________________ *The reduced reactivity in the presence of ascorbate partly reflects reductive decomposition of hydrogen peroxide by ascorbate in the test solution prior to dipping of reagent. 
    
     The data in Table 2 sharply contrasts with a reagent made with horseradish peroxidase instead of iron creatinine (Table 3). 
     
                       TABLE 3______________________________________   Reflectance at 660 nm and 1 minute   Mean (standard deviation)     Urine specific                Urine specific                             Urine specific     gravity =  gravity =    gravity =Additive to urine     1.005 g/mL 1.015 g/mL   1.023 g/mL______________________________________none      71.3 (0.4) 72.3 (1.2)   74.6 (2.1)3.0 mg/dL H.sub.2 O.sub.2     32.6 (2.3) 35.3 (4.1)   39.7 (2.8)3.0 mg/dL H.sub.2 O.sub.2     77.2 (0.5) 76.1 (0.2)   78.3 (0.3)and 100 mg/dLascorbic acid______________________________________ 
    
     The peroxidase reagent was completely unreactive towards 3.0 mg/dL hydrogen peroxide in the presence of 100 mg/dL ascorbic acid. Reactivity of the iron creatinine reagent increased with decreasing pH and with increasing ratio of creatinine to iron. A ligand ratio of 4 to 1 (creatinine to iron) was selected because of lesser interference from urinary phosphate, thereby providing more consistent results in all urines. A reagent pH of 4.5 was selected because it provided adequate reactivity towards 3.0 mg/dL hydrogen peroxide, which was roughly the amount of hydrogen peroxide expected from the reaction of glucose oxidase on 100 mg/dL glucose. Malonic acid serves as a buffer to control the pH and can be substituted with other buffers such as citrate, tartaric acid, boric acid, succinic acid, glycerol-2-phosphate, salicylic acid, oxalic acid, and malic acid. 
     EXAMPLE 2 
     Hydrogen peroxide reactivity was also observed with iron chelates of guanidine. The reactivity was determined by a test done as in Example 1 except that 400 mM guanidine was used in Dip 1 instead of 400 mM creatinine. The specific gravity of the urine was 1.015 g/mL. The results appear in Table 4. 
     
                       TABLE 4______________________________________Reflectance at 660 nm and 1 minuteMean (standard deviation)            Additive to urineIron Complex      none       3.0 mg/dL H.sub.2 O.sub.2______________________________________Guanidine 2-ethyl sulfonic acid             73.4 (3.1) 43.4 (3.6)Guanidine benzene-3,5-             71.7 (0.9) 51.1 (2.7)dicarboxylic acidL-arginine        72.9 (1.5) 63.5 (3.0)______________________________________ 
    
     EXAMPLE 3 
     Reagent for the detection of glucose. 
     Dry reagent paper was prepared as in Example 1. The first and second dips were prepared as in Example 1 except that 500 units/mL glucose oxidase was also added to the first dip before the pH adjustment. 
     The reflectance at 660 nm obtained at one minute after dipping the strip in urine containing various levels of glucose was taken to represent reagent reactivity. 
     Reagent color is directly proportional to glucose concentration. The reagent reactivity towards 100 mg/dL glucose was fairly consistent in all three urine pools (Table 5). The reagent remained reactive towards 100 mg/dL glucose in the presence of 100 mg/dL ascorbic acid, indicating that oxidation of ascorbate was occurring. 
     
                       TABLE 5______________________________________     Reflectance at 660 nm and 1 minute     Mean (standard deviation)       Urine specific                  Urine specific                             Urine specific       gravity =  gravity =  gravity =Additive to urine       1.005 g/mL 1.015 g/mL 1.023 g/mL______________________________________none        68.4 (3.3) 74.1 (3.1) 69.7 (2.7)100 mg/dL glucose       41.5 (1.2) 49.1 (2.6) 47.8 (4.1)100 mg/dL glucose       49.4 (1.6) 54.5 (3.5) 57.6 (3.1)and 100 mg/dLascorbic acid______________________________________ 
    
     EXAMPLE 4 
     Reagent for the detection of hydrogen peroxide using various iron complexes. 
     Various transition metal complexes were tested for peroxidase activity at pH 5.8 by measuring the oxidation ram of the redox indicator, TMB, for a 3-part water to 2-part acetonitrile solution containing 3.1 mM metal chelate, 44 mM malonic acid, 14 mM diisopropyl dihydroperoxide, and 11.6 mM TMB at pH 5.8. The results shown in Table 6 show iron (Fe 2+  and Fe 3+ ) complexes with poly-L-arginine and creatinine to be the most active. While copper shows some reactivity, it is to a much lower extent (1/10). No other metal (in the list given) showed peroxidase activity under these conditions. Results appear in Table 6. 
     
                       TABLE 6______________________________________       Peroxidase Rate       (Abs @ 660 nm/min)Chelate       Fe.sup.2+   Fe.sup.3+                            Cu.sup.2+______________________________________Fe ammonium   0.00        0.00Fe sulfate    0.00        0.00Fe EDTA       0.02        0.03Fe HEDTA      0.06        0.09Fe DTPA       0.00        0.19Fe EGTA       0.00        0.00Fe CDTA       0.00        0.01Fe dipyridine 0.00        0.07Fe albumin    0.03        0.00Fe poly orithine         0.03        0.05Fe poly-D-lysine         0.03        0.04Fe poly-L-lysine         0.04        0.06Fe poly-L-arginine         0.12        0.19Fe poly-L-histidine         0.08        0.12Fe poly-L-glycine         0.01        0.02Fe L-histidine         0.03        0.05Fe creatinine 0.40        0.63Cu creatinine                    0.05______________________________________ 
    
     The full chemical names for the abbreviations used in Table 6 are: EDTA: ethylenediaminetriacetic acid; HEDTA: N-(2-hydroxyethyl)ethylenediamine triacetic acid; DTPA: diethylenetriamine pentaacetic acid; EGTA: ethylene bis(oxyethylenenitrilo tetraacetic acid); and CDTA: 1,2-diaminocyclohexane-N,N,N&#39;,N&#39;-tetraacetic acid.