Method of enzymatically measuring hydrogen peroxide and reagent therefor

Abstract: There are described a method of enzymatically measuring hydrogen peroxide by developing a color with use of 3-methyl-2-benzothiazolinonehydrazone as a coupler and in combination of an oxidative color developing reagent with peroxidase, and a reagent therefor. In case of the color developing reaction is carried out in a neutral or weak alkali pH condition, 3-methyl-2-benzothiazolinonehydrazone reagent containing catalase is used, and the reaction is carried out in the presence of ethylenediaminetetraacetic acid or its analogue. The reagent comprises a buffer, 3-methyl-2-benzothiazolinohydrazone, and catalase.

Description: The present invention relates to a method of enzymatically measuring hydrogen peroxide with high sensitivity and a reagent therefor. The method is utilized in the field of clinical examinations to quantitatively measure various biological substances and enzymes in a body fluid as a sample, through the measurement of hydrogen peroxide. In the field of clinical examinations, at the present time, the enzymatical measurement of hydrogen peroxide has widely been carried out. For quantitatively measuring a biological substance or enzyme in a sample body fluid such as serum and urine, such an enzymatical measuring method has been developed and actually employed, which comprises steps of generating hydrogen peroxide in the sample with use of an oxidase, and leading into a color developing reaction with use of an oxidative color developing reagent and an oxidase of peroxidase, catalase or the like. Followings are relations between the biological substances to be quantitatively measured by utilizing the generation of hydrogen peroxide and exemplar oxidases to be used therefor. a) Activity of amylase : α -Glucosidase and glucose oxidase. b) Activity of guanase : xanthine oxidase and uricase. c) Glucose : Glucose oxidase. d) Creatinine : Creatininase and sarcosine oxidase. e) Activity of choline esterase : Choline oxidase. f) Cholesterol : Cholesterol oxidase. g) Cholesterol ester : Cholesterol ester hydrogenase and cholesterol oxidase. h) Neutral fat : Lipase and glycerol oxidase. i) Activity of transaminase : Pyruvate oxidase. j) Free (or non-esterified) fatty acids : Acyl-CoA synthetase and Acy-CoA oxidase. k) Lactic acid : Lactate oxidase. l) Uric acid : Uricase. m) Urea : Urease and glutaminate oxidase. n) Phospholipids : Phospholipase D and choline oxidase. o) Bilirubin : Bilirubin oxidase. Followings are color reaction utilizing the generated hydrogen peroxide. A) Methods using peroxidase (POD) : H 2 O 2 + DH 2 → D + 2H 2 O (DH 2 : Potential dye material) B) Methods using catalase : There are methods, for instance, one wherein methanol is oxidized with generated hydrogen peroxide to convert into formaldehyde, and then adding acetylacetone and an ammonium salt to cause a condensation according to Hantzsch reaction and develop a color. This method can be shown with reaction formulae, as given below. H 2 O 2 + CH 3 OH → HCHO + 2H 2 O In both types of the methods, the latter methods using catalase not only require a relatively long operation period of time but also difficult to apply the same for an automatical measuring apparatus and thus the former methods using POD have widely been employed in the clinical examination field. Among the color reactions using POD, such a method has most widely been accepted that a hydrogen doner is subjected to a condesation reaction with a coupler such as 4-aminoantipyrine (hereinafter referred to as "4-AA" ), 3-methyl-2-benzothiazolinonehydrazone (hereinafter referred to as "MBTH") or the like, in the presence of POD, and then the reaction mixture is lead into an oxidative color developing step. In this case, followings can be used as the hydrogen doner: phenol, 4-chlorophenol, 2,4-dichlorophenol and sulfonated derivatives thereof, 2,4-dibromophenol, 2,6-dichlorophenol, 2,4,6-tribromophenol, 3,5-dichloro-2-hydroroxybenzene sulfonic acid, 3-hydroxy-2,4,6-triiodobenzoic acid and the like phenols; N,N-dimethylaniline, N,N-diethylaniline, N-ethyl-N-(2-hydroxyethyl)-m-toluidine, N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N,N-dimethyl-m-anisidine, N-substituted anilines treated with alkylsulfonic acid or hydroxyalkylsulfonic acid or the like anilines. Advantages and disadvantages of the conventional methods are as follows. The methods using 4-AA as the coupler have widely been used, since a detection wave-length for dyes lies at a shorter side, it can be applied in a wide pH range, and reagents show good stability, although a sensitivity thereof is lower than that of the method using MBTH as the coupler. If a reducing substance such as reduced type nicotinamidonucleotide or the like coexists in the reaction system, however, the method is apt to be affected of the reducing action due to such substance, so that there is such a case that any color does not appear or tone of the color suppressed. While, the methods using MBTH as the coupler have been said as that suitable for increasing a sensitivity, since a sensitivity is high, detection wave-length lies at higher side, developed color shows good stability, and it is not susceptible to influence of the reducing substance, even if the substance coexists in the reaction system. However, there are disadvantages of that the MBTH reagent per se is not stable, a reagent blank value remarkably changes with the lapse of time, and it can be applied for only in acidic pH range, in practice. According to JP-A-63 246 665 catalase may be added to a coupling reagent to prevent a rise of the blank value. Therefore, the object of the invention is to provide such a method for measuring hydrogen peroxide with use of MBTH as the coupler, which has various advantages excepting the use of unstable reagent, that an increase of reagent blank value due to the change with lapse of time can be suppressed, even through the measurement shall be carried out in neutral or weak alkali pH range, and that it shows a sensitivity higher than that in any method using 4-AA as the coupler, in also such a measuring pH range. The inventors have energetically studied and investigated as to that in the method using MBTH as the coupler for the color developing system to measure hydrogen peroxide, the increase with the lapse of time of reagent blank value occurs under what condition, and what factor(s) causes such an instabilization. In the first place, they have carried out following experiment. To 10mM phosphate buffer (pH 7.0), 3.5 U/ml peroxidase, 0.5mM MBTH, 0.5mM N-ethyl-N-sulfopropyl-m-anisidine (ADPS), and 0.5mM EDTA· 2Na (as the case may be) were added, mixed at temperature of 37°C, and measured absorbance (initial absorbance) to check its value on the reagent. Each of the mixed reagents was stored in a refrigerator and the absorbance was measured with a day interval to check an increase of the value. Results are shown in following Table 1. Through the experiment, 2 kind phenomena concern to the increase of reagent blank value. One of them is an absorbance increase with lapse of time, after mixing the color reagents, and the other is that the MBTH solution used increases the initial absorbance, with the lapse of time from the preparation of the solution. The appearance of these phenomena was of remarkable in neutral to alkali pH ranges and not noticeable in acidic range (pH 5 or more less), as so hitherto reported that the method using MBTH is not suitable, when measurement is carried out in neutral or alkali pH range. Through various studies and investigations, the inventors have estimated that the former phenomena shall be caused mainly by oxidation due to a metal(s) contained in the reaction solution, although an amount thereof is very small, and that the latter shall be caused through that intrinsic or endogenous hydrogen peroxide to be formed in MBTH solution, namely dissolved oxygen in MBTH solution is made to oxygen radical by MBTH per se or foreign peroxide and others, and the oxygen radical reacts with water molecule to generate hydrogen peroxide which increase the initial absorbance, when the MBTH solution is mixed with peroxidase and oxidative color developing reagent (phenols, trinder reagent or the like). Although EP-A-0 071 730 (Jap. Pat. No. Sho 58 - 899(A)) discloses "a stabilized reagents for detecting H 2 O 2 or H 2 O 2 doner system and states that an alkali ferrocyanide, chelating agent, and alkali azide are effective as the stabilizing agent, it is not sufficient with only addition of such a compound. Namely, for increasing the sensitivity and stability, it necessary to exclude or suppress both of said 2 instabilizing factors. The inventors have continued investigation under assumption that if the metal(s) contained in reaction solution can be masked with a chelating agent such as ethylenediaminetetraacetic acid or its analogue and a method for disappearing or suppressing an influence of hydrogen peroxide to be formed from dissolved oxygen in MBTH solution can be established, the increase of reagent blank value may be suppressed to make possible a high sensitive measurement, even though the measurement is carried out in neutral or alkali pH range. As a result, they have finally found that both of the abnormal increase of reagent blank value, namely absorbance increase with the lapse of time, after mixing of MBTH solution and the coloring reagents and increase of initial absorbance, after the mixing can remarkably be suppressed, by coexisting ethylenediaminetetraacetic acid or its analogue, during the reaction and by adding catalase in MBTH solution, and that the addition of the ethylenediaminetetraacetic acid or its analogue and catalase and the coexistence thereof in reaction system do not give any bad influence on the color developing system to establish the invention. Therefore, the enzymatical method according to the invention for measurement of hydrogen peroxide is characterized in that in case of using 3-methyl-2-benzothiazolinonehydrazone (MBTH) as a coupler to develop a color in combination with an oxidative color developing reagent and peroxidase, and carrying out a color developing reaction due to oxidational condensation in a pH range of 5.5 - 9.5, the reagent containing 3-methyl-2-benzothiazolinonehydrazone (MBTH) further contains catalase, and an ethylenediaminetetraacetic acid or its analogue as specified in claim 1 coexists during the reaction, to provide a high sensitive measurement and attain the object of the invention. For carrying out the method according to the invention, a phenol and trinder reagent known per se can be used as the oxidative color developing reagent. As shown below, a quinone dye will be formed, if the former is used and in the other case, an indamine dye will be formed. Although the method according to the invention can be applied for from a weak acidity pH range to an alkali pH range of about 10, but it is preferable in pH range of 5.5 - 9.5. An amount of catalase in the reagent containing MBTH is at least 10 units per 1ml of the reagent. As the catalase which is one of enzymes to decompose hydrogen peroxide, those obtained from an animal, plant and microorganism can be employed for the method according to the invention, if it has been sufficiently purified. As the ethylenediaminetetraacetic acid and its analogues, ethylenediaminetetraacetic acid per se , its sodium, potassium, ammonium, lithium or the like salt, and cyclohexanediamine tetraacetic acid and the like can be used. The invention will now be further explained in more detail with reference to Examples, which will refer to a drawing, wherein Fig. 1 is a graph showing a reaction time course, when glucose was measured by a hydrogen peroxide color developing system using glucose oxidase and 3-methyl-2-benzothiazolinonehydrazone, to show a difference between a case of that catalase coexists in the measuring system and another case of that no catalase therein. Example 1 1. Reagents (a) MBTH reagent This reagent was prepared by dissolving 2.2mg of MBTH and 0 - 20000 units of catalase in 10ml of 50mM phosphate buffer (pH 8.0, containing 0.5mM EDTA· 2Na). (b) ADPS reagent This reagent was prepared by dissolving 3.0mg of ADPS and 70 units of peroxidase in 10ml of 50mM phosphate buffer (pH 8.0, containing 0.5mM EDTA· 2Na). 2. Operations The MBTH reagent and ADPS reagent were taken in equiamount and mixed. After lapsed 30 seconds at 37°C , an absorbance (initial absorbance) of the mixture was measured at measuring wave-length of 540nm. Then the mixture has been stored at 4°C under shielding from the light. The mixture was taken out at 2nd, 3rd and 6th day from the storing, and absorbance (initial absorbance) after lapsed 30 seconds at 37°C was measured to check the initial absorbance changing with the lapse of time. 3. Results and consideration Results are shown in following Table 2. In the MBTH reagent according to the invention, which also contains the catalase, as apparently seen therefrom, an increase of the initial absorbance can significantly be suppressed. The suppressing effect increases, as an amount of the catalase increases, but desired effect can be attained, when the catalase is added 10 units or more per 1ml of the MBTH containing reagent. When the catalase was added 10 units per 1ml of the MBTH containing reagent, the increase of initial absorbance after lapsed 6 days was about 1/6, in comparison with that in a MBTH containing reagent with no catalase. Day elasped Containing amount of catalase (Units/ml) 0 10 20 200 1000 2000 0 34 30 27 21 14 15 2 131 38 37 20 18 17 3 160 41 40 18 16 14 6 235 42 41 20 15 14 (In Table 2, the values are given in mAbs) Example 2 1. Reagents (a) First reagent (MBTH reagent containing catalase) This reagent was prepared by dissolving 1.1mg of MBTH and 8500 units of catalase in 10ml of 50mM phosphate buffer (pH 7.0, containing 0.5mM EDTA· 2Na). (b) Control reagent (MBTH reagent containing no catalase) This reagent was prepared by dissolving 1.1mg of MBTH in 10ml of 50mM phosphate buffer (pH 7.0, containing 0.5mM EDTA· 2Na). (c) Second reagent This reagent was prepared by dissolving 3800 units of glucoseoxidase, 700 units of peroxidase and 20mg of TOOS in 10ml of 50mM phosphate buffer (pH 7.0, containing 0.5mM EDTA · 2Na). TOOS : N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (d) Glucose solution This solution was prepared by dissolving 40mg of glucose in 100ml of refined water. 2. Operations 20µl of the glucose solution or refined water was mixed with First reagent at 37°C , left to stand for 5 minutes, added 0.2ml of Second reagent, left to stand for further 5 minutes, and then change of absorbance (reaction time course) was measured at measuring wave-length of 590nm. As a control, the change of absorbance was measured as described above, but with use of Control reagent instead of First reagent. 3. Results and consideration Results are shown in Fig. 1. When the control reagent containing no catalase was employed, as apparently seen from the Figure, a sudden increase in absorbance had been recognized on the reagent blank, just after the addition of the second reagent (Please note the term of "Initial Absorbance" given in this specification designates the absorbance at this increased period of time). It has been estimated that the sudden increase of absorbance was caused by endogenous hydrogen peroxide to be formed in the reaction solution, as referred to in the preamble part of this specification. While, according to the method of the invention, which uses First reagent containing catalase, a phenomenon of sudden increase of absorbance on the reagent blank did not appear to show good reaction time course. It shall be so concluded from the above that the reduction of accuracy due to increase of reagent blank value which is caused by instability of the reagent can be effectively avoided by the method according to the invention.

Claims: A highly sensitive method of enzymatically measuring hydrogen peroxide in a color developing reaction using an oxidative color developing reagent, an oxidative coupling reagent of 3-methyl-2-benzothiazolinonehydrazone, and peroxidase, and being carried out in a pH range of 5.5 - 9.5, characterized in that catalase is added to the oxidative coupling reagent, and that the reaction is carried out in the presence of a compound selected from the group consisting of ethylenediaminetetraacetic acid, its sodium, potassium, ammonium and lithium salts as well as cyclohexanediaminetetraacetic acid. A method as claimed in Claim 1, characterized in that said oxidative coupling reagent contains at least 10 units of catalase per 1ml of the reagent.

Inventors: KURONO MASAYASU, SAWAI KIICHI, TAKEHIRO OSAMU, UNO SHIZUO, KURONO, MASAYASU, SAWAI, KIICHI, TAKEHIRO, OSAMU, UNO, SHIZUO, KURONO, MASAYASU, SANWA KAGAKU KENKYUSHO CO.,LTD., SAWAI, KIICHI, SANWA KAGAKU KENKYUSHO CO.,LTD., TAKEHIRO, OSAMU, SANWA KAGAKU KENKYUSHO CO.,LTD., UNO, SHIZUO, SANWA KAGAKU KENKYUSHO CO.,LTD.
Applicants: SANWA KAGAKU KENKYUSHO CO, SANWA KAGAKU KENKYUSHO CO., LTD.
Application year: 1991
Main classification: C12Q1
IPC codes: C12Q1