JPH03119997A - Method for measuring ingredient - Google Patents

Abstract

PURPOSE:To enable accurate measurement of a biological ingredient without any influence of bilirubin by the presence of a berlinate or specific ferrocene compound in a reaction system for producing hydrogen peroxide utilizing an enzyme. CONSTITUTION:A biological ingredient such as cholesterol or uric acid to be measured is placed in a buffer solution such as phosphoric acid or tris-HCl and a berlinate such as potassium ferrocyanide or a ferrocene compound such as ferrocene or a ferrocenecarboxylic acid expressed by the general formula is added thereto. An enzyme (oxidase) is then reacted to generate hydrogen peroxide. The produced hydrogen peroxide is subsequently reacted with a coloring matter such as coupling coloring matter of 4-aminoantipyrine(4AA) with phenols to measure absorbance or fluorescent intensity within the visible region of the colored reaction solution and carry out colorimetry. Thereby, the influence of bilirubin causing problems in quantitative analysis of the biological ingredient utilizing enzymic reaction can be avoided to more accurately and quantitatively analyze the biological ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is used in the field of clinical testing to quantify hydrogen peroxide produced by using an enzyme reaction system such as oxidase to measure biological components such as serum and urine. This paper relates to a method for measuring biological components.

BACKGROUND ART As a method for quantifying biological components using hydrogen peroxide as a means of quantifying hydrogen peroxide, various oxidases are used, for example, in the case of cholesterol, cholesterol oxidase (C) 100. EC
, 1, 1, 3, 6>, uricase (IC) in the case of uric acid.

EC, 1, 7, 3, 3), in the case of phospholipids, phospholipase D (PLO, EC, 3, 1, 4, 4>) and choline oxidase (CLOD, EC, 1, 1, 3, 17)
Hydrogen peroxide is generated using a compound such as peroxidase (POD) and various chromogens, such as 4-aminoantipyrine (4^A) and phenols, or 4^A and Trinder's reagents to dye the hydrogen peroxide. A reaction system in which colorimetric determination is performed on the colored reaction solution is widely used. However, this reaction system is easily affected by coexisting reducing substances such as ascorbic acid, cysteine, and bilirubin, and it is known that even if hydrogen peroxide is generated quantitatively, some of these substances are consumed. [Enzymatic analysis methods and clinical tests, complete clinical enzyme analysis research, P, 43 (I983)]. In order to avoid the influence of this reducing substance, for example, ascorbic acid is treated with ascorbic acid oxitase (Allot).
) to convert ascorbic acid to dehydroascorbic acid, or to convert cysteine to N-ethylmaleimide (
This effect was avoided by reacting with NεM> to change the mercapto group to a group that is not affected by reducing substances. Regarding eliminating the interference of bilirubin, ■Method to avoid the influence by converting bilirubin to biliverdin using bilirubin oxidase (JP-A-54-151193, JP-A-5
8-61000) ■ A method of oxidizing bilirubin with various oxidizing agents, such as potassium iodate and converting it into biliverdin to avoid the effects (JP-A-56-107161) ■ Utilizing the redox reaction of potassium ferricyanide and potassium ferrocyanide. How to avoid its effects [clown,
)tsuγchi etc. Clinical Chemist cJ-(C1ini
cal Chemistry) DanNα2 227-23
1 (I980)] and a method of adding an EDT^-iron complex (Japanese Patent Publication No. 5G-22200).

Problems to be Solved by the Invention The above-mentioned ■■ has the property of oxidizing not only bilirubin but also the color sugen itself. Japanese Society of Clinical Chemistry Pension Record Vol. 22 P, 179) Operations such as reagent dispensing However, the method (2) has the drawback that the absorbance increases, giving a positive error in the measured value, and the method (2) has the drawback that it is incomplete in terms of canceling the interference of bilirubin. Therefore, there is a need to develop a method for accurately measuring biological components without being affected by bilirubin.

Means for Solving the Problems The present inventor has proposed a solution by directly adding a berric acid salt or a ferrocene compound to a reaction system in which enzyme S (oxidase, etc.) acts on a biological component to be measured to generate hydrogen peroxide. discovered that bilirubin interference could be eliminated. That is, the present invention measures the absorption in the visible region of a colored reaction solution by reacting hydrogen peroxide, which is generated by using an enzymatic reaction with components in a sample, with a chromogen in the presence of peroxidase. The present invention relates to a method for colorimetric determination of a component, which is characterized by the presence of a berric acid salt or a ferrocene compound represented by the general formula 2N).

General formula (I1','',,ra-a・(r)8,Oku・, In the formula, R,,R, are the same or different and are hydrogen, substituted or unsubstituted lower alkyl, hydroxyl, carboxyl, ami/, -CODR (wherein R represents substituted or unsubstituted lower alkyl, aralkyl or aryl)
, (ONHR (in the formula, R has the same meaning as above) or -
COO(C2)1.0> ,,H (wherein n is 5 to 10(
D represents R).

The present invention will be explained in detail below.

As a berlinate, a compound obtained by reacting potassium or sodium ferrocyanide with a trivalent iron salt such as ferrous ammonium sulfate, iron chloride, iron acetate, etc., or a compound obtained by reacting potassium ferricyanide with a divalent iron salt such as ferrous ammonium sulfate. , a compound obtained by reacting iron chloride, iron acetate, etc., and commercially available Prussian blue.

The appropriate amount to add is 3X10-' to 3mM. Berlinerate is a blue compound, as it is called Berlin Blue, but in the present invention, the color is hardly a problem because it is used in a diluted state, and even if it is slightly colored, it will not change. Therefore, it does not affect the measured value.

In the definition of a ferrocene compound represented by the general formula [I'l,
Lower alkyl refers to straight-chain or branched alkyl having 1 to 4 carbon atoms, such as methyl, ethinoben-n-propyl, isopropyl, n-butyl, isobutyl, tert-
butyl, 5ec-butyl.

The substituents of substituted alkyl are the same or different, and the number of substituents is 1 to 1.
In the alkyl-substituted amino, the alkyl in the alkyl-substituted amino has the same meaning as the above alkyl. Aralkyl is benzyl, phenethyl, benzhydryl, etc., and aryl is phenyl, tolyl,
Naphthyl etc.

Specific ferrocene compounds include ferrocene, ferrocenecarboxylic acid, tert-aminoferrocene, α-
Hydroxyethylferrocene, hydroxymethylferrocene, dimethylaminomethylferrocene, etc. are commercially available. Further examples include compounds obtained by reacting ferrocenecarboxylic acid with various amines, alcohols, etc. using commonly used chemical reaction means. The amount added is 1, X 10-'~10m! , 1 is appropriate.

The influence of bilirubin can be avoided by allowing the berric acid salt and the ferrocene compound to be present in a reaction system that generates hydrogen peroxide by causing an enzyme (such as oxidase) to act on the component to be measured.

Moreover, as in the case of conventional ascorbic acid elimination, if the generation of hydrogen peroxide and the elimination of ascorbic acid proceeded simultaneously, the elimination of ascorbic acid would be incomplete, so the reagent was divided into two. There is no need to use a system that generates hydrogen peroxide through an enzymatic reaction after completely erasing ascorbic acid in the sample with AOD in the first reagent, and the reaction can proceed either before or simultaneously with the generation of hydrogen peroxide. Similar effects can be expected by the presence of berric acid salts or ferrocene compounds in the system. Moreover, since berric acid salts and ferrocene compounds do not react with 80D, NEM, etc., they can be allowed to coexist with these compounds to simultaneously eliminate not only bilirubin but also ascorbic acid and cysteine.

This system can be applied to any method in which hydrogen peroxide is generated from a target substance using an enzyme, this is led to the production of a dye, and the concentration of the dye is measured by absorbance or fluorescence intensity. .

For example, the aforementioned cholesterol ([:HOD, PO
D), uric acid ([JC, POD),! J 7 lipid (PL
II, [:LOD, Po1l), as well as sialic acid (neuraminidase, NAN^aldolase, pyruvate oxidase, POD), pyruvate (pyruvate oxidase, POD), glucose (glucose oxidase, POD), free fatty acid (acyl Co hesynthetase, acyl-CoA oxidase, POD), lactic acid (lactate oxidase, POD), creatinine (creatininase, creatinase, sarcosine oxidase, PO[]), triglyceride (riboprotein lipase, glycerol kinase, glycerin-3-phosphate) oxidase, POD), inorganic phosphorus (purine nucleotide phosphorylase, xanthine oxidase, P
OD), magnesium (glycerol kinase, glycerol 3-phosphate oxidase, POD), calcium (PLO.

CLOD, POO), or measurement of enzyme activity in biological fluids, such as cholinesterase (substrates include various choline esters, Cl0D, F'OO), creatinine phosphokinase (substrates talleatinine phosphate, glycerol kinase, glycerol-3-phosphate) oxidase, P
OD), IJ pase (substrate glycerol ester, Anru-CoA synthetase, acyl-CoA oxidase, P
OD), monoamine oxidase (substrate allylamine,
POD) etc.

In the present invention, dyes for measuring hydrogen peroxide include the aforementioned 4A^ and phenols (phenol, P-xylenol, 3-hydroxy-2,4,6-)ribrobenzoic acid, 3-hydroxy-2,4 , 6-) ribrombenzoic acid, etc.), and 4^^ and anilines [Trinder reagent neck (Dojindo Chemical Research Institute 16th edition general catalog)
and N-ethyl-N-3-methylphenyl-N'-succinylethylenediamine, N-ethyl-N-3-methylphenyl-N'-acetylethylenediamine, NN-dimethyl-m-)luidine, SN-diethyl-m-toluidine, N
N-disulfopropyl-m-)luidine, NN-disulfobropy-35-dimethoxyaniline, etc.], as well as 3-methyl-2-benzothiazolinone hydrazone (MBTH) and the above phenols. or coupling system with anilines, or JP-A-62-
296, Japanese Patent Publication No. 59-1823 f'l, Special Publication No. 63-4
Dye sources such as those shown in 9189 can be used. The reaction is carried out using a suitable buffer such as phosphoric acid, Tris CI, succinate,
The reaction temperature is 20-50° C. in a standard buffer solution. The concentration of the buffer solution may be 10 to 500 mM, and the pH may be 5 to 9, at which the enzyme reaction is normally performed. In addition, nonionic, anionic, or cationic surfactants can be used as appropriate to solubilize turbidity caused by lipids in the sample.

Examples and reference examples of the present invention are shown below.

Example 1 Cholesterol measurement reagent A> Potassium hydrogen phthalate (pH=6.2>25m
! J diamine (E!, Isε) Potassium phosphate OD OD Cholesterol esterase HOD AA <Reagent B> Test IA1. : Ferrocenecarboxylic acid 10 m! Reagent B was prepared by adding 0.05 mM of J 511/mf1 7U/m1 2U/m1 3 and TIJ/dd (manufactured by Aldrich Chemical Company).

Reagent C> Reagent C was prepared by adding 0.05 mM of berric acid salt (BA; Prussian blue, manufactured by Aldrich Chemical Company) to the reagent.

Reagent D> Add 0 potassium ferrocyanide (manufactured by Wako Pure Chemical Industries) to the reagent.
．． 05mM was added as a reagent.

Reagent E> Add 0 potassium ferricyanide (manufactured by Wako Pure Chemical Industries) to Reagent A.
．． 025mM was added as Reagent E.

<Reagent F> Add 0.05% potassium iodate (manufactured by Wako Pure Chemical Industries) to the reagent.
The one to which n+M was added is called reagent F.

<Reagent G> Reagent A contains bilirubin oxidase (E, C, 1, 3,
Reagent G is prepared by adding 2.0 units/- of 3°5).

Reagent H> Reagent Iron ([) -EDT^ (manufactured by Isotope Chemistry Institute,
A reagent H is prepared by adding 0.05mM of Fe-EDT^).

The same operation is performed for reagents A to H.

■ 20 μg of distilled water in 4 test tubes containing reagent 3.0-
■ Standard solution of cholesterol 200 mg/J, ■ 20 d of large serum containing cholesterol ester equivalent to 135 mg/a of cholesterol, and ■ ■ additionally 2 bilirubin.
204 was added to each solution to give a concentration of 0 mg/J, and the mixture was heated at 37° C. for 10 minutes. The results of measuring the absorbance at 555 nm are shown as E, E2., respectively. E3
°E4, and the cholesterol concentration was calculated from the following formula, and the results are shown in Table 1. Table 2 shows the increase in absorbance E1 when the solution (2) was left at 37° C. for 2 hours to see the increase in reagent blind test.

■Cholesterol level = 200X (E3-Snake, /E2
-8. ) ■Cholesterol level・200X (H, -E
, /E2-E,) Table 1 In the systems to which ferrocenecarboxylic acid or berric acid was added (Reagents B and C), there was very little interference from bilirubin, and there was no change in the blind test of the reagents. On the other hand,
Additive-free system (reagent A) and potassium ferrocyanide,
Potassium iodate, bilirubin oxidase and iron (
II[)-HOT^ was added to the system (reagent,
F, G, and H) could not sufficiently eliminate bilirubin interference.

Furthermore, in the systems in which potassium ferricyanide, potassium iodate, and bilirubin oxidase were added (reagents E, F, and G), the blind increase in reagents was extremely large.

Example 2 Measurement of uric acid (I) <Reagents> Gutud buffer (pH 6.5) TOO3 (manufactured by Isotope Kagaku) 4ΔA OD Ferroceneuricase 100n+M 1,2mM 0.3mM 100/d O,01mM 0.40 /d Reagent 3. Add 50 μe of distilled water to 4 test tubes containing 0-,
■Standard solution of uric acid 10mg/J, ■50 large serum containing uric acid, ■■Additionally bilirubin 20mg/c/j2
50 μQ of each solution was added to the solution to give a concentration of 100 μl, and the mixture was heated at 37° C. for 10 minutes. When the concentration was calculated from the results of measuring the absorbance at 555 nm, it was 4.
9mg, #f! , ■ But 4. ' Obtained a result of 9 mg/dR. There was no difference between the values of ■ and ■, and there was no effect of bilirubin.

(2) (Reagent) Gutud buffer (pH□6.5) 100m
MTOO5 (manufactured by Isotope Kagaku) 1.2mM4A
A 0.3mMP OD
1011/mffBA
O, 01mM uricase
Into 4 test tubes containing 0.4u/g reagent 3.0-, ■ 50μQ of distilled water, ■ Standard solution of 10+ng/y of uric acid, ■ Human serum 504 containing uric acid, ■■ and 20mg/d1 of bilirubin. 50 μQ of each solution was added to the solution to give a concentration of 100 μl, and the mixture was heated at 37° C. for 10 minutes. When the concentration was calculated from the results of measuring the absorbance at 555 nm, it was 5.2 mg/c+j for ■! , ■ But 5.
A result of 2 mg/di was obtained. There was no difference between the values of ■ and ■, and there was no effect of bilirubin.

Example 3 Measurement of triglyceride (I) <Reagent> Gud buffer (pH = 6.75) 10
mMDΔO3 (manufactured by Isotope Kagaku) 1. hM
Liton X-1000, 1% Magnesium Sulfate 3ITIM4AA
1mM glycerol kinase 0.60/mP OD
l0IJ/72 Lipoprotein Lipase 0.3+ng/m & α-Hydroxyethylferrocene 0.025mg/mc Into 4 test tubes containing 3.0ml of reagent ■ 50 heads of distilled water,
■Standard solution containing 200 mg/J of triolein, ■50 μQ of human serum containing triglycerides, and ■■ with bilirubin added to a concentration of 20 mg/J5.
0 relatives were added to each, and the mixture was heated at 37°C for 10 minutes. 5
When the concentration was calculated from the results of measuring the absorbance at 93 nm, it was 53.6 mg/dl for ■ and 53.5 m for ■.
g/J results were obtained. There is almost no difference between the values of ■ and ■.
There was no effect of bilirubin.

(2) Reagent> Gutsud buffer (pH=6.75) 10m!
JDAO3 (Isotope Kagaku) 1.8mM
Liton X-1000, 1% Magnesium Sulfate 3+nA14AA
B A
0.025mg/m & reagent 3
．． 4 test tubes containing 0 ml of ■ 50 μQ1 of distilled water ■ 200 mg/dl standard solution of triolein, ■ 50 μg of large serum containing triglyceride, and ■■ to which bilirubin was added to a concentration of 20 mg/dl. 5
0 cows were added to each, and the mixture was heated at 37° C. for 10 minutes. 5
When the concentration was calculated from the results of measuring the absorbance at 93 nm, it was 41. , 6II+g/a, ■ but 41
．． A result of 2 mg/Lil was obtained. There was almost no difference between the values of ■ and ■, and there was no effect of bilirubin.

Example 4 Measurement of creatinine (I) <Reagents> Gud buffer dipotassium phosphate Triton mf+ 601J/d dimethylaminomethylferrocene 0.002mM reagent 3
．． 4 test tubes containing 0 ml of ■50 μa1 of distilled water ■Am solution with 200 mg/Li1 of creatinine, ■50 d of human blood containing creatinine, and ■■ to which bilirubin was added to a concentration of 20 mg/d1. 5
0 μg of each was added and heated at 37° C. for 10 minutes.

When the concentration was calculated from the results of measuring the absorbance at 555 nm, it was 86 mg/a of O in ■ and 0.86 mg in ■.
/a result was obtained. There was no difference between the values of ■ and ■, and there was no effect of bilirubin.

(2) <Reagents> Gutud buffer 10 mM
Dipotassium phosphate 50mM Triton X-1000, 1% EMSE 0.25mg
/mffP OD 5U
/ml sarcosine oxidase 401J/m
R creatinase 90U/rd.

Creatininase 6011/m1 Ascorbic acid oxidase 10 [1/mQ (for ascorbic acid elimination) BA 0025mM Reagent 3.0- in 4 test tubes, ■ 50 distilled water, ■ Standard solution of creatinine 10 mg/d1, ■Human blood containing creatinine/Iv50JdI, ■■ plus 20mg/c+ of bilirubin! l! 50 μg of each solution was added to the solution to give a concentration of 100 μg, and the mixture was heated at 37° C. for 10 minutes.

When the concentration was calculated from the results of measuring the absorbance at 555 nm, it was 0.9 mg/dl for ■ and 0.9 mg/dl for ■.
I got the result of d1. If the difference between the values of ■ and ■ is fL<, IJ
Ruhi.

There was no impact on the results.

Example 5 The same operation as in Example 4(I) was carried out using the ferrocene compound shown in Table 4 obtained by the method shown in the reference example below in place of dimethylaminomethylferrocene in Example 4(I). However, I got the results shown in Table 3.

Table 3 Reference Example: Synthesis of ferrocenecarboxylic acid derivatives To 1m+nol of ferrocenecarboxylic acid, 1.5mmol+ of various amines and alcohols and 2.5+nmol of dicyclohexylcarbodiimide were added, and the mixture was reacted in acetonitrile under the conditions shown in Table 4. The compounds shown in Table 3 were obtained.

Table 4 Effects of the Invention According to the present invention, when quantifying biological components using an enzyme reaction system, the influence of bilibin, which is an interstitial substance, can be avoided, and biological components can be determined more accurately.

Claims (1)

[Scope of Claims] Hydrogen peroxide produced from components in a sample using an enzymatic reaction is reacted with a chromogen in the presence of peroxidase, and the absorption in the visible region of the colored reaction solution is measured. A method for measuring a component by colorimetric determination, characterized in that a berric acid salt or a ferrocene compound represented by the following general formula (I) is present. General formula (I) ▲There are numerical formulas, chemical formulas, tables, etc.▼(I) In the formula, R_1 and R_2 are the same or different and are hydrogen, substituted or unsubstituted lower alkyl, hydroxyl, carboxyl, amino, -COOR (in the formula represents a substituted or unsubstituted lower alkyl, aralkyl or aryl), -CONHR (in the formula, R has the same meaning as above) or -COO(C_2H_4O)_nH (in the formula, n is 5 to 10)
represents an integer number).