JPS6030519B2 - A method to measure components in biological fluids without interference from endogenous or exogenous substances - Google Patents

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, a redox reaction using dehydrogenase is applied to the reaction process for measuring components in biological fluids, and nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (NAD (P) ↓) is finally converted into phenazine methosulfate (hereinafter referred to as PMS), which is an electron carrier.
) or its derivative, 1-methoxy-5-methylphenazine methosulfate (hereinafter referred to as m-PMS), and a chromogenic electron acceptor, tetrazoIJium compound. In the method of measuring the amount of a component from the result, if there is an interfering substance involved in the formazan coloring of the reaction in the sample, it is related to a method in which this is removed in advance by the method described below and then the component in the biological fluid is measured. The above-mentioned method of colorimetrically quantifying the color development of formazan for measuring components in serum or blood has been applied to many diagnostic reagents.
For example, in the method shown by the following reaction formula, each reaction proceeds quantitatively, so the components can be measured by colorimetrically quantifying the color development of the final formazan. '1' Component to be measured → Inducer ■ Inducer 10NAD (P) + Dehydrogenase oxidized 10NA
D(P) day 10 day 10 Inducer (3) NAD(P
)H+H++m-PMS→NAD(P)10m-PMSH
2 [4} m-PMSH20NTB → m-PMS + formazan (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 of reaction {1} above exists as an interfering substance in the sample, reactions 21 and below will proceed together with the inducer from the component to be measured, and the colorimetric determination of formazan in reaction {41 Gives a positive error. Such interfering substances include endogenous glycose in alpha-amylase measurements, exogenous maltose, L-glutamate in GOT and OPT measurements, and glycerin in triglyceride measurements. 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. The inventors are PM
Reduced form of S or m-PMS i.e. PMSH2,m-
PMSH2 is oxidized by dissolved oxygen in the solution and P
Focusing on the property of reverting to MS and m-PMS, as a result of intensive research, we developed a method for measuring components in biological fluids that is completely unaffected by endogenous and exogenous interfering substances and is easy to operate. , completed the invention.

The method of the present invention oxidizes endogenous or exogenous substances or their inducers that exhibit an interfering effect during the measurement reaction process in the presence of dehydrogenase to lose their interfering ability, and at the same time, NDA (
P)+ is reduced and the generated NAD(P) is oxidized in the presence of PMS or m-FMS and returned to NAD(P)+. At the same time, PMS and m-PMS are reduced and PMSH5, m
- This product which produces PMSH2 is directly oxidized by dissolved oxygen in the reaction solution in the presence of catalase, and the original PMS,
At the same time as it returns to m-PMS, hydrogen peroxide is generated, 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 the original PMS and m-PMS, and at the same time water is generated (hereinafter these steps are also referred to as pretreatment).

In this way, the interfering substances in the sample are extinguished without leaving behind any effects, and then the components are measured as described above, so the formazan color development in the final stage is proportional to the amount of the components, making accurate colorimetric determination possible. can.

In this case, since a tetrazolium compound, which is a color-forming electron acceptor, is present, the final stage of PMS4, m-PM
SH2 preferentially reacts with dissolved oxygen and quantitatively develops the color of formazan. As described above, the method of the present invention applies a redox reaction using a dehydrogenase, and finally generates a derivative quantitatively from the component to be measured using an electron carrier-chromogenic electron acceptor reaction system. It can be applied to the measurement of α-amylase in body fluids, the measurement of transaminases, and the measurement of triglycerides using glycerol dehydrogenase or glycerokinase-glycerophosphate dehydrogenase, etc., 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 a-amylase α-amylase in body fluids can be measured by the following reaction system: ○' Modified starch α-amylase decomposed starch ■ Degraded starch a-glucoamylase glucose (3) Glucose + ATP hexokinase Glucose-6-phosphate + ADP■ Glucose-6-phosphate 1NAD (P)
Decoglucose-6-phosphate dehydrogenase 6-phosphogluconate + NAD (P) day [51 NAD (P) day Ten HTBm-PMSNAD (P
) 10 formazan However, ATP: Adenosine triphosphate ADP: Adenosine diphosphate NTB: Nitrotetrazolium flu (tetrazolium compound).

The absorbance of formazan produced in reaction [5' is measured, and the activity value of a-amylase is calculated from the result.

If endogenous glucose or maltose, which has recently been widely used as a ring fluid, exists as an interfering substance in the biological fluid, the reaction [3] will be followed by the reaction ■, '3', then the reaction {4', [ 5) produces formazan, giving a positive error to the measured value of α-amylase. In the present invention, the following reaction {2} is carried out without adding the modified starch of reaction {1'}, and endogenous glucose and maltose are converted to 6 by reactions ■, '3', and '41.
- phosphogluconic acid and remove it.

Then the reaction {separately, does not occur because NTB is not present, and the next separate reaction proceeds. [6-NAD(P)H+H+10m-
PMS→NAD(P)+ 10m-PMS ratio'7} m-
PMS ratio 1002 → m-PMS + Buddha 02 ■ ratio 02 Takaraze day 20 Toyo 2 '9'm skin scream 2POD m-PMS ratio ○ However, 02: dissolved oxygen ratio 02: represents hydrogen peroxide.

Reactions ■～[9}' result from interfering substances.

NAD(P) day returns to NAD(P) ten and m-PM again
SH2 returns to m-PMS, and if catalase is used, it ultimately only produces water and oxygen, and POD
If used, water will be produced. Therefore, the influence of interfering substances is completely excluded in the subsequent measurement of the components. That is, after pretreatment, a-amylase can be measured accurately by performing reactions (1' to '51).1
Reagent '11ATP 5mm.

1 noku NAD 0.4 mmol
/〆m-PMS 30 French mm.

1/So MgC12 2nd hm.

1/〆Hexokinase 600 mountains/〆Glucose-6-phosphate 500 French/g Dehydrogenase glucoamylase 4800〆/〆P〇D
IOOOO Buddha/Sogyu Alpmin 10cm containing 0.1%
ol/so PH8.2 succinate buffer [2'NTB 3hmol/Z
Triton
．． 4 Concinate buffer {31 Triton X-100
0.0-33% included. State HCI Solution 2 Procedure Add 20 pieces of serum sample to reagent '112' and add 37q0,
After incubating for 5 minutes, add the reagent [2'1 secretion,
After keeping warm for 5 minutes, reagent '3'1 skin is added to stop the reaction, and the absorbance is measured at a wavelength of 60 m.

Separately, a sample with known α-amylase activity is operated in the same manner as above to create a calibration curve, and the α-amylase activity of the serum sample is determined from this calibration curve. Test example 1 ○) Effect of glucose on a-amylase measurement
The absorbance was measured according to Example 1 for the sample added at a concentration of 1000 females/d'.

Separately, there is a method in which the same specimen is not subjected to the pretreatment of the present invention, that is, 20 ml of specimen is added to the mixed reagent on reagent '1' and reagent leg 1, and after incubation for 5 minutes, reagent '311' is added. was added to stop the reaction, and then the absorbance was measured. The results are shown in the following table. As shown in the above table, if no pretreatment is performed on endogenous glucose, the results are significantly 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 by glucose at a high concentration of 1000 9/d'. '21 Relationship between α-amylase activity concentration and absorbance according to the method of the present invention The absorbance was measured for a concentration series of α-amylase activity using the method of Example 1. The results are shown in FIG.

As shown in this figure, 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 Glutamate oxal acetate transferase (
GOT) and glutamate pyruvate transferase (GP
Measurement of T) GOT and OPT in body fluids can be measured by the following reaction system using glutamate dehydrogenase (GLDH).

l α-ketoglutarate + L-aspartic acid GOT
L-glutamate 10 oxalacetic acid 2 a-ketoglutarate 10 L-alanine GPTL-glutamate + pyruvate 3 L-glutamate + NAD (P) 10 10 days 20
GLDHNAD (P) Niten-a-ketoglutarate + NH
34 NAD(P) day 10NTBm-PMSNAD(P)
+Formazan The absorbance of formazan produced in the above reaction is measured, and the activity values of GOT and OPT are calculated from the results.

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

In the present invention, endogenous L-glutamate is first
The reaction between D and NAD(P) converts it into a-ketoglutarate, which is then removed.

Next, the removal of the generated NAD(P)H+ was carried out in exactly the same manner as described in Example 1, and the interfering substance L-glutamate was eliminated. Reagents containing L-aspartic acid or L-alanine, a-ketoglutarate and NTB are then added and transaminase (G
OT, GPT) activity can be accurately measured. 1
Reagent'1) Catalase 1000 skin/Soglutamate 4000 skin/Dehydrogenase NA
D14 valent mol/〆m-PMS
100 mm.

1/10 hmol including 〆/〆 PH
7.8 Phosphate buffer ■ DL-alanine for OPT measurement 80 nmol/soa-
Ketoglutarate 11 mmol/NTB
I-ratiohm.

1/10 dishes including the final product mol/10 dishes PH7
．． 8 Phosphate buffer for GOT measurement L-aspartic acid 40 hmol/A-ketoglutarate 18 hmol/N field
1. Skin mol/1 breast hmol including evening/〆
PH7.8 phosphate buffer {3} 0.1N daily CI solution 2 containing 0.1% Tritoso X-100
Procedure Reagent '1} Add 20 〆〆〆 of serum specimen to 250A, 37
After incubating at 10° C. for 10 minutes, add 250 ml of reagent (37q0), and after incubating for 3 minutes at 37q0, add reagent {313 secretion to stop the reaction, and then measure the absorbance at wavelength 56 skin blood.

Separately, a sample with known GOT and OPT activity was operated in the same manner as above to create a calibration curve, and from this calibration curve, GO of the serum sample was
Determine T,OPT activity.

Test example 2'11 Effect of L-glutamate on GOT and GPT measurements.
The absorbance was measured according to Example 2 for the samples added at concentrations of 9/d' of 40, 80, and 100.

Separately, for the same specimen, there is a method of not performing the pretreatment of the present invention, i.e., add 20 meth of the specimen to the mixed reagent, add 20 meth of the specimen to the mixed reagent, and incubate for 30 minutes, then add the reagent ``3'' to the mixed reagent. After terminating the reaction by adding 3 chlorides, the absorbance was measured. The results are shown in the following table. As shown in the above table, when no pretreatment is performed against endogenous L-glutamate, the results are considerably affected by L-glutamate, and as a result, it cannot be said that GOT and GPT activities are being measured. According to the present invention, G
OT and GPT activities can be measured accurately. (2) Relationship between GOT and OPT 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 shown in this figure, there is a linear relationship between the concentration of GOT and GPT activities and the absorbance, indicating that GOT and OPT activities can be measured correctly.

As described above, the method of the present invention 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 circle mark in Figure 1 indicates the a-amylase activity in the serum.
In FIG. 2, ○ marks indicate GOT activity, and △ marks indicate GPT activity. Figure 1 Figure 2

Claims (1)

[Claims] 1. When measuring components in biological fluids, a redox reaction using dehydrogenase is applied and the generated reduced nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate is subjected to 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 reacted with phenazine methosulfate or its derivatives and catalase or peroxidase in the absence of a chromogenic electron acceptor. A method for measuring a component in a biological fluid, the method comprising: removing a component in a biological fluid;