JPS6167500A - Method and reagent for determination of substance in body fluid - Google Patents

Abstract

PURPOSE:To enable the accurate determination of a substance in body fluid, by adding specific first and second reagents to the specimen body fluid, and determining the produced H2O2. CONSTITUTION:The specimen fluid is added with the second reagent consisting of a buffer solution of 6.0-8.0pH containing 20-100U/l of peroxidase, 20-1,000U/l of keto-acid dehydrogenase, 10-900mg/l of nicotinamide adenine dinucleotide, 1-400mg/l of a keto-acid, 0.3-30mg/l of 4-aminoantipyrine, 5-1,000mg/l of sodium-N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, and 8-400mg/l of NaN3. The intrinsic lactic acid acting as an interference substance can be eliminated by this treatment. The treated specimen is mixed with the first reagent consisting of a buffer solution of 6.0-8.0pH containing 30-360U/l of alpha-hydroxy-acid oxidase and 200-2,000U/l of catalase, and the produced H2O2 is determined by colorimetry.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method and reagent for quantifying component concentrations and enzyme activities in body fluids.

[Prior art and problems]

Conventionally, in the field of clinical testing, reduced nicotinamide adenine dinucleotide (N
A method in which ADH) is produced and the target component concentration and enzyme activity are determined from the amount produced is frequently used in daily life. The quantitative method includes a method of calculating the concentration of the target component and enzyme activity value from the change in absorbance at 340 nm, which is the characteristic absorption band of the generated NADH. There is a method of conjugating an electron transport chain to generate a tetrazolium salt, converting this into colored formazan (reduced tetrazolium), and determining the concentration of the target component and enzyme activity value using a colorimetric method.

However, these methods are not good methods for measuring trace amounts of NADH. In other words, the molecular extinction coefficient of NADH at 340 nm is small and the sensitivity is low, and it is also affected by endogenous lactic acid, which causes a large error when quantifying it, so correction with a sample blank is necessary. Therefore, it cannot be applied to general automatic analyzers. On the other hand, in a system in which formazan is generated and measured using a colorimetric method, although some improvement is observed in terms of sensitivity, it is still not sufficient, and the above-mentioned N
Similar to the ADH measurement system, correction using a sample blank is required, and the formazan produced is poorly soluble, so there are problems such as extremely shortening the lifespan of the cell and reaction system of the automatic analyzer. This method also cannot be performed with automatic analyzers.

[Purpose of the invention]

The present invention provides a method and reagent for quantifying substances in body fluids that have higher sensitivity than conventional NADH measurement systems and can be easily applied to automatic analyzers.

[Technical means and effects]

In the quantitative method of the present invention, reduced nicotinamide adenine dinucleotide (NADH), which is produced by coupling an enzyme reaction to a substance to be determined in body fluids, is combined with a dehydrogenase reaction of a keto acid such as pyruvic acid and α-hydroxy This method is characterized by conjugating the oxidase reaction of acid to generate hydrogen peroxide, and measuring the amount of hydrogen peroxide produced.

The quantitative method of the present invention uses keto acid dehydrogenases such as lactate dehydrase (LD'H) and α-hydroxy acid oxidase, such as lactate oxidase (
LOX').

Since the method of the present invention uses a hydrogen peroxide measuring system, the sensitivity is very good, 2 to 5 times that of the conventional NAD) f measuring system.

In addition, in quantifying components or enzyme activities in body fluids by the method of the present invention, as a pretreatment, endogenous lactic acid, which is an interfering substance, is converted to water under the conjugation of an enzymatic reaction between α-hydroxy acid oxidase and catalase. By doing so, the influence as an interfering substance can be eliminated. Lactate oxidase (L OX
) is shown in formulas (r) and (II).

Note that the remaining catalase is dissolved in sodium nitride (NaN:
l).

LOX lactic acid → pyruvate + H2O2... CI) catalase Regarding the reaction principle of the measurement method of the present invention, bile acids in serum are the measurement target, and LDH1α- is used as a keto acid dehydrogenase.
Taking as an example a case where LOX is used as hydroxy acid oxidase to convert bile acids to NADH and finally produce a water-soluble pigment, first, as shown in formulas (III) to (V) below, Bile acids and nicotinamide adenine dinucleotide (NAD) are reacted under the enzymatic reaction coupling of 3α-hydroxysteroid dehydrogenase (3α-H3D) to generate ketosteroids and NADH, and the generated NADH is converted into LDH. The lactic acid is reacted with pyruvate in the presence of pyruvic acid to convert it into lactic acid and NAD, and then the lactic acid is conjugated with LOX oxidase to convert it into pyruvate and hydrogen peroxide.
4-aminoantipyrine (4-A
A) and sodium-N-ethyl-N-(2-hydroxy-3-sulfopropyl)-metatoluidine (TO
O5) to form a dye.

This dye exhibits absorption at 546 nm.

(pancreatic acid
(77)-sub)ko4]ko)POX HzO2+4AA+TOO3□→dye...CV)(5
46nm) The above measurement was performed for α-hydroxy acid oxidase with LOX of 30 to 360 U/N, and catalase of 200 to 200 U/N.
a first reagent which is a buffer solution of pH 6,0-8.0 containing 0 U/N, and POX 20-100 U/l;
Keto acid dehydrogenase such as LDH 20-1000 U
/ f, 3α-H3D3~35U/l NADIO~
900 n; x/l, keto acids such as pyruvic acid 1-400
mg/l, 4-AAo, 3-30nv/l, T
OOS 5-1000 mg/II, and sodium nitride (N a N :l) 8-400 mg/I!
The first reagent is a buffer solution containing pH 6.0 to 8.0. A second reagent is added to the sample body fluid as a pretreatment to eliminate endogenous lactic acid, which is an interfering substance, by the reactions of formulas (1) and (II) above, and then the second reagent is used to perform the reactions of formulas (III to (V)). The concentration of the target component or enzyme activity in the body fluid can be determined by spectrophotometrically analyzing the resulting pigment.This quantitative procedure is
Of course, it can be carried out manually using a conventional automatic analyzer.

〔Example〕

(Automatic analysis of serum bile acids) (1) Reagent prescription (preparation of 20m6 scale) (1)
First reagent (R+): pH containing LOX 72U, catalase 400U
7,0 phosphate buffer solution (2) Second reagent (Rz): Bar oxidase (POX) 20USLDH20U
, 3 cx-HS D 7 U, NAD 1
0+ng, pyruvic acid 10mg, 4-AA 0.1
+ng, TOO34呵, NaN, pH 7 containing 3■
,0 phosphate buffer solution.

(II) Measurement operation A Hitachi 705 automatic analyzer was used as the automatic analyzer.

1st reagent (RI) 350 μm 2 sample serum 1o117
! was added, and pretreatment (reaction formula (1), [■
]) After that, 50 μl of the second reagent (Rz) is added and treated, and the bile acids are quantified by the end point method using reaction formulas (III) to [■]).

CIII) Measurement Results (11 Linearity) As a result of measuring diluted serum containing 120 μM bile acids, a good linear relationship was observed as shown in FIG.

(2) Effect of endogenous lactic acid Add lactic acid to serum at 10m+r/d1.20a++r/a, 4
As a result of adding and measuring lactic acid at a rate of 0 mg/a and 80 μ/dl, no effect of lactic acid was observed as shown in FIG.

(3) Correlation with conventional methods (manual methods) Commercially available bile acid measurement kit (for manual methods) [Dai Pharmaceutical Co., Ltd.]
FIG. 3 shows the correlation between the serum bile acid measurement results obtained using the method and the measurement results obtained by the above-mentioned automatic analysis. However, Y=
0.973 X -0,711, r = 0.988,
n=60.

It can be seen that the remeasurement method has good correlation.

〔Effect of the invention〕

According to the present invention, it is possible to quantify substances in body fluids more accurately than conventional methods, and it is also possible to perform measurements using an automatic analyzer, which was previously impossible, and is suitable for clinical testing fields that require processing of multiple samples. It contributes to

[Brief explanation of drawings]

Figure 1 is a graph showing the linearity of the measured values according to the present invention, Figure 2 is a graph showing the influence of interfering substances on the measured values according to the present invention, and Figure 3 is the measured values of the present invention and the conventional method. It is a graph showing the correlation.

Claims (2)

[Claims] (1) A keto acid dehydrogenase reaction and an α-hydroxy acid oxidase reaction are coupled to reduced nicotinamide adenine dinucleotide (NADH), which is produced by coupling an enzyme reaction to a substance to be quantified in body fluids. 1. A method for quantifying a substance in a body fluid, the method comprising: producing hydrogen peroxide and measuring the amount of hydrogen peroxide produced. (2) α-Hydroxyacidase 30-360U/l
, pH 6 containing 200-2000 U/l of catalase
．． A first reagent consisting of a buffer solution of 0 to 8.0, peroxidase 20 to 100 U/l, and keto acid dehydrogenase 20 to 1
000U/l, nicotinamide adenine dinucleotide 10-900mg/l, keto acid 1-400mg/l, 4
- Contains 0.3-30 mg/l of aminoantipyrine, 5-1000 mg/l of sodium-N-ethyl-N-(2-hydroxy-3-sulfopropyl)-metatoluidine, and 8-400 mg/l of sodium trinitride. pH6
．． A reagent for quantifying substances in body fluids in combination with a second reagent comprising a buffer solution of 0 to 8.0.