JPS6058097A - Method for determination of component in body fluid free from influence with endogenous and exogenous substance - Google Patents

Abstract

PURPOSE:To eliminate the influence of the interference of an endogenous and exogenous substance, and to determine the component in a body fluid with simple operation, by oxidizing the interfering substance in the presence of a dehydrogenase during the determination process, and reducing the NAD(P)<+>. CONSTITUTION:An endogenous and exogenous substance or their derivative exhibiting interfering action during the determination process, is oxidized in the presence of a dehydrogenase to eliminate the interference activity, and at the same time, the NAD(P)<+> is reduced. The produced NAD(P)H is oxidized in the presence of a diaphorase to the original NAD(P)<+>. When the system contains a catalase, the product is immediately oxidized with the dissolved oxygen in the reaction liquid to generate H2O2, which is decomposed and eliminated by the action of the catalase. When a peroxidase is present in the system, the product is oxidized with dissolved oxygen to produce water. The color development of formazan at the final stage is proportional to the content of the component, and accordingly, the amount of the component can be determined accurately by colorimetry.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for easily and accurately measuring components in biological fluids without being influenced by endogenous or exogenous interfering substances. More specifically, we apply a redox reaction using dehydrogenase to the reaction process to measure components in biological fluids.
The produced nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NAD(P) when referring to either one) is finally combined with diaphorase, which is an electron carrier, and a tetrazolium compound, which is a chromogenic electron acceptor. In this method, in which the amount of the component is determined by colorimetrically quantifying the color development of formazan with The present invention relates to a method for measuring components in a biological fluid after removal thereof.

The method of colorimetrically quantifying the color development of formazan as described above for measuring components in serum or plasma is one example of the method shown by the following reaction formula, since each reaction proceeds quantitatively. Components can be measured by colorimetrically quantifying the color of formazan @ (1) Component to be measured → Inducer (2) Inducer + NAD (P) + 10 NAD (P) H 11 Dehydrogenase 1111 →Oxidized type inducer (3) NAD(P)H+NTB Geophorase---→NAD(P)++Formadene (color development) However, NTB: represents nitrotetrazolium blue, one of the tetrazolium compounds.

However, this reaction system is easily affected by interfering substances present in the sample, and accurate measurements cannot be made unless these are removed in advance.

If the same inducer in reaction (1) above exists as an interfering substance in the sample, reaction (2) and subsequent reactions will proceed together with the inducer from the component to be measured, and the ratio of formazan in reaction (3) will increase. Gives a positive error to color quantification. Such interfering substances include endogenous glucose in alpha-amylase measurements, exogenous nomaltose, GOT, L-glutamate in GPT measurements, glycerin in triglyceride measurements, and the like.

Conventionally, in such cases, errors have been corrected by subtracting the absorbance measured for only the interfering substance from the absorbance measured for the component to be measured and the interfering substance together. However, this method is complicated because the operation must be repeated twice, and there is also a large waste of reagents.

As a result of intensive research into diaphorase activity in the oxidation reaction of NAD(P)H, the present inventors found a method for measuring components in biological fluids that is completely unaffected by endogenous and exogenous interference substances and is easy to operate. and completed the present invention.

The method of the present invention oxidizes endogenous or exogenous substances or their inducers that exhibit an interfering effect in the course of a measurement reaction in the presence of dehydrogenase to lose their interfering ability and at the same time
The NAD(P)I produced by reducing (P) is then oxidized in the presence of diaphorase and returned to NAD(P), while at the same time being directly oxidized by dissolved oxygen in the reaction solution in the presence of catalase. At the same time, hydrogen peroxide is produced, but this hydrogen peroxide is decomposed by the action of catalase and disappears.

In addition, in the presence of peroxidase (POD), it is oxidized by dissolved oxygen and returns to its original state, at the same time producing water (hereinafter these steps are also referred to as pretreatment).

In this way, after the interfering substances in the sample have disappeared without leaving any effects, the above-mentioned general components are measured, so the final stage of formazan color development is proportional to the amount of the components, and an accurate ratio can be obtained. Capable of color quantification. In this case, since a tetrazolium compound, which is a color-forming electron acceptor, is present, diaphorase gives priority to the reaction with dissolved oxygen and quantitatively develops the color of formazan.

As mentioned above, the method of the present invention applies a redox reaction using a dehydrogenase, and finally converts the derivative to be quantitatively produced sequentially from the component to be measured into an electron carrier-chromogenic electron acceptor reaction system. It can be applied to the measurement of α-amylase in body fluids, the measurement of transaminase, and the measurement of triglyceride using glycerol dephosphate dehydrogenase, and its effects are remarkable.

Next, the method of the present invention and its effects will be explained in more detail with reference to Examples and Test Examples.

Example 1 Measurement of α-amylase α-Amylase in body fluids can be measured using the following reaction system: α-amylase (1) modified starch decomposed starch α-glucoamylase (2) decomposed Denzo Y-1111 11. Glucose hexokinase (3) Glucose + ATP □ Glucose-6-phosphate + ADP (4) Glucose-6-phosphate + NAD (P ) +
Glucose phosphogluconate + NAD(P)H (5) NAD(P)H+ NTBL and Lnee NAD(
P) ++Formaden However, ATP: Adenosine trisunate ADP: Adenosine diphosphate NTB: Nitrotetrazolium pool (tetrazolium compound).

The absorbance of the formazan produced in reaction (5) is measured, and the α-amylase activity value is calculated from the result. If endogenous glucose or maltose, which has recently been widely used as an infusion, exists in the biological fluid as an interfering substance, reaction (3) will occur, and reactions (2) and (3) will occur, followed by reaction ( 4).

(5) Since cyformaden is produced, a positive error is given to the measured value of α-amylase.

In the present invention, reaction (2) and subsequent reactions are performed without adding the modified starch of reaction (1), and endogenous glucose and maltose are removed from reaction (2).

(3) and (4) with dirophosphogluconic acid,
Remove this. Next, reaction (5) does not occur because NTB is not present, and the next separate reaction proceeds.

(6) NAD(P)H+ H+ o2(7) NAD
(P)I(+H+10 remainder 0 except 02: represents dissolved oxygen.

NA arising from interfering substances in reactions (6) and (7)
D(P)H is returned to NAD(P), and when catalase is used, only water and oxygen are ultimately produced, and when POD is used, water is produced. Therefore, the influence of interfering substances is completely excluded in the subsequent measurement of the components. In other words, after pretreatment, reaction (1) and reaction (5) are performed.
), α-amylase can be measured accurately.

1. Reagent (1) ATP 5 mmol/A'NADP+0.
4 m mol/l soaphorase 2000 μ/1 MgC1220 m mol/l hexokinase 600 μ/l glucose-6-phosphate soo μ/ dehydrogenase glucoamylase 4800 μ/1 POD 10000 μ/100 mmol/ containing 0.1% bovine alzomine 1 pH 8,2 succinic acid buffer (2 NTB 3 m mol/l Triton 0 containing 1000, 33%
．． 6 Add 20 μL of serum sample to NHCl solution 2, procedure reagent (1) 2 ad, and add at 37°C.
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After incubating at 37°C for 5 minutes, add reagent (3) 1 d to stop the reaction, and then measure the absorbance at a wavelength of 600 nm. Separately, a sample with known α-amylase activity was operated in the same manner as above to create a standard curve, and this calibration curve was used to calculate the α-amylase activity of the serum sample.
- Measure amylase activity.

Test example 1 (Influence of glucose on re-α-amylase measurement Glucose 200.400 for serum sample.

600.800. Example 1 Absorbance was measured for a sample added at a concentration of 100 ng/dt. A method in which the same specimen is not subjected to the pretreatment of the present invention, that is, 2 μt of the specimen is added to a mixed reagent of reagent (1) 2d and reagent (2) 1 at, and after incubation at 37°C for 5 minutes, reagent (3) 1 is added. After the reaction was stopped by adding d, the absorbance was measured.

The results are shown in the following table.

As shown in the above table, if endogenous glucose is not pretreated, it will be affected by glucose, and as a result, it cannot be said that α-amylase activity is being measured. According to the present invention, α-amylase activity can be accurately measured without being affected even by glucose at a high concentration of 1000 μm.

(2) Relationship between α-amylase activity concentration and absorbance according to the method of the present invention FIG. 1 shows the results of measuring the absorbance of α-amylase activity concentration series according to the method of Example 1.

As this figure shows, there is a linear relationship between the concentration of α-amylase activity and the absorbance, indicating that α-amylase activity can be accurately measured.

Example 2 Measurement of glutamate oxidelacetate transferase (GOT) and glutamate pyruvate transferase (GPT) GOT and GPT in body fluids can be measured by the following reaction system using glutamate dehydrogenase (GLDH).

1. α-ketoglutarate + L-asunoyaraginoic acid --- → L-glutamate + oxalacetic acid 2. α
-ketogeltare-) + L-alanine PT -1 → L-glutamate + pyruvate 3, L-glutamate + NAD(P) + H20GL
D) I NAD (P) H + α-ketoglutarate + NH3 Soaphorase 4, NAD (P) H + NTB NAD (P) + Formazan The absorbance of the formazan produced in the above reaction was measured, and from the results, the activities of GOT and GPT were determined. Calculate the value.

L-glutamate, which is present as an interfering substance in serum or plasma, reacts with GLDH and produces NAD(P)H, giving a positive error to the measured value of GOT or GPT.

In the present invention, endogenous L-glutamate is first converted into GL
The reaction between DH and NAD(P) converts it into α-ketoglutarate, which is then removed. Then, the generated NA
Removal of D(P)H is carried out in exactly the same manner as described in Example 1, and the interfering substance L-glutamate is eliminated.

Then L-aspartic acid or L-alanine, α-
Reagents containing ketoglutarate and NTB can be added to accurately measure transaminase (GOT, GPT) activity.Reagent (1) Catalase 10000 μ/l glutame)
40,000μ/l of dehydrogenase diaphorase 4,000μ/l of 100 mmol/l pH7,8 phosphate buffer (2) DL-alanine for GPT measurement 800 mmol/Lα-ketoglutarate 11 mmol/1NTB 1. Om
100 mmol/l PH7,8 phosphate buffer containing mol/L L-aspartic acid for GOT measurement 400 mmol/l α-ketoglutarate 1.6 mmol/zNTB 1. Om
100 mmol/l pH 7,8 phosphate buffer containing mol/L (3) Triton X-1000, containing 1%. IN-H
Add 20μt of serum sample to C1 solution, procedure reagent (250μt),
℃, 10 horses + 1 JI mata wet diameter K, trial product (21250μ
After further incubating at 37°C for 30 minutes, add the reagent (3
) After stopping the reaction by adding 3 d, the wavelength was 560 n.
Measure the absorbance at m.

Separately, a sample with known GOT, GPT activity is operated in the same manner as above to create a calibration curve, and the GOT and GPT activities of the serum sample are determined from this calibration curve.

Test Example 2 (1) Effect of L-glutamate on GO'l', GPT measurement L-glutamate was added to the serum sample at 20.40°so,xo
The absorbance of the sample added at the concentration of omp/a was measured according to Example 2.

Separately, there is a method in which the same specimen is not subjected to the pretreatment of the present invention, that is, 250 μt of reagent (1) and 250 μt of reagent (2).
Add 20μt of sample to μt mixed reagent and heat at 37°C for 30 minutes.
After incubating for a minute, 3 d of reagent (3) was added to stop the reaction, and the absorbance was measured.

The results are shown in the following table.

As the table above shows, if no pretreatment is performed for endogenous L-glutamate, L-glutamate
Under the influence of Kanashi, as a result, GOT, GPT
It cannot be said that activity is being measured. According to the invention, 1
GOT and GPT activities can be accurately measured without being affected by L-glutamate at a high concentration of 0.00 mg/lie.

(2) Relationship between GOT and GPT activity concentration and absorbance according to the method of the present invention The absorbance was measured for a concentration series of GOT and GPT activities using the method of Example 2. The results are shown in FIG.

As this figure shows, there is a linear relationship between the concentration of GOT and GPT activity and the absorbance, which indicates that GOT and GPT activities are correctly related.
It turns out that T activity can be measured.

Overall, the method of the present invention as described above is extremely useful in that it removes interfering substances from a sample in a simple manner and enables accurate measurements without wasting reagents.

[Brief explanation of drawings]

The vertical axes in FIGS. 1 and 2 indicate absorbance, and the horizontal axes indicate serum dilution. In addition, the O mark in Figure 1 indicates α-amylase activity in serum.
In FIG. 2, ○ marks indicate GOT activity, and Δ marks indicate GPT activity. Figure 1 iEl, series command

Claims (1)

[Claims] To measure components in biological fluids, we apply a redox reaction using dehydrogenase to generate reduced nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate in an electron carrier-chromogenic electron acceptor reaction. In the method of colorimetrically measuring the content of components in a biological fluid using a colorimetric system, endogenous or exogenous substances or their inducers in the biological fluid that interfere with the reaction are removed in advance from the presence of dehydrogenase. oxidation, and the reduced nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate produced at that time is removed by reacting with diaphorase and catalase or peroxidase in the absence of a chromogenic electron acceptor.