JPS5934900A - Determination of component in bioliquid - Google Patents

Abstract

PURPOSE:To eliminate the interference of ascorbic acid, and to determine the components in a bioliquid specimen easily and accurately, by removing ascorbic acid from the bioliquid specimen using phenazine methosulfate or its derivative. CONSTITUTION:In the determination of the components in a bioliquid specimen by the redox reaction using a dehydrogenase or oxidase, e.g. determination of alpha-amylase activity, determination of triglyceride, determination of transaminase activity, etc., the specimen is pretreated with phenazine methosulfate or its derivative and catalase or peroxidase in the absence of a color-developing electron acceptor. The reducing property of ascorbic acid exhibiting interference action to said redox reaction can be eliminated by this pretreatment.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the removal of ascorbic acid in order to easily and accurately measure components in biological fluids without interference from ascorbic acid in biological fluids.

Colorimetric assays using oxidation and one-component reactions using dehydrogenases or oxidases have been widely used in the field of diagnostic reagents to measure components in biological fluids. However, as with any enzymatic reaction, color-forming systems are often interfered with by substances in biological fluids, and reducing substances such as ascorbic acid are particularly important.

In other words, when these reducing substances are present, the correct error in the formazan coloring reaction using dehydrogenase is
Oxidative condensation reactions using oxidases give negative errors. Therefore, for example, when ascorbic acid is present in a biological fluid, it is impossible to accurately measure the value of the component unless ascorbic acid is removed and its interference effect is eliminated prior to measuring the component. Conventionally, in order to eliminate the interference effect of ascorbic acid, it is common to use an enzyme such as ascorbic acid oxidase, but the use of an enzyme is not desirable from the viewpoint of stability and economic efficiency of the reagent.

As a result of various studies aimed at improving these drawbacks, the present inventors developed a method for removing ascorbic acid from biological fluids by utilizing the properties of phenazine methosulfate or its derivatives, and completed the present invention.

According to the present invention, in a method for measuring components in a biological fluid using a redox reaction using a dehydrogenase or an oxidase, ascorbic acid in the biological fluid that interferes with the reaction is detected in the absence of a color-forming electron acceptor. Provided is a method for measuring a component in a biological fluid, which is characterized in that the reducing property of phenazine methosulfate or a derivative thereof and catalase or peroxidase is eliminated.

In the method of the present invention, ascorbic acid is removed through the steps shown in the following schematic.

When the method of the present invention is performed as a pretreatment prior to measuring components in biological fluids, ascorbic acid is rapidly oxidized according to the above-mentioned scheme, and its interfering effect disappears. PMS here
(m-PMS) acts as a catalyst and returns to its original state, so there is no change. Therefore, there is no influence on subsequent measurement reactions of the components.

If the method of the present invention is applied as a pretreatment when dehydrogenase is used to measure components in biological fluids, the subsequent redox reaction can be performed to develop the color of formazan in the presence of newly added tetrazolium salt according to the following reaction formula. can also be quantified colorimetrically.

NAD(P)H1H++ PMS (rn-PMS)
---→NAD+(P) +PMS H2(m-PMS
H2) PMSH, (m-PMSHI)+tetrazolium salt----→PMS (m-PMS)+
formazan chromogenic) NAD+(P): nicotinamide adenoid dinucleotide phosphate or nicotinamide adenino dinucleotide phosphate NAD(P)H: reduced form of the above In this case, a tetrazolium salt which is a chromogenic electron acceptor Because of the presence of PMS (
The fact that m-PMS) can be used as it is and that there is no change in the amount after carrying out the method of the present invention, that is, after pretreatment, is also operationally advantageous compared to conventional methods that require a separate enzyme such as ascorbic acid oxygose. It is also very economically advantageous.

In addition, when using an oxidizing enzyme, after pretreatment according to the method of the present invention, the hydrogen peroxide produced by the redox reaction can be further added with an appropriate electron donor in the presence of peroxidase to carry out the next oxidative condensation reaction. This allows for colorimetric determination.

H2O2 + Electron donor 11-→ Color-forming oxidized mesh peroxidase In this case, the hydrogen peroxide produced has ascorbic acid removed, so it is not consumed by ascorbic acid, and the color of the oxidized condensate shows the exact location of the components. give.

As mentioned above, the method of the present invention is widely applicable to the measurement of components in biological fluids through redox reactions caused by dehydrogenases or oxidases.

Examples include -α-amylase activity measurement method, triglyceride measurement method, and transaminase activity measurement method.

An experimental example is shown below.

Experimental example Oxidation reaction of ascorbic acid: Reagent (1) 2 m M m -P M 510000
v/l 0.1M phosphate buffer containing peroxidase pH 7.5 Procedure reagent (1) After incubating 2.5- at 37°C for 5 minutes, 100
Add 10 μt of my/ll ascorbic acid solution and
Determine the change in absorbance at 65 nm.

Figure 1 shows the absorption spectra of the reduced form of ascorbic acid and the oxidized form of the reaction product.

Figure 2 shows the time course of the oxidation reaction of ascorbic acid.

Under these conditions, ascorbic acid is completely oxidized within about 3 minutes from the start of the reaction.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 α-amylase activity assay reagent (1) 5mM adenosine triphosphate (ATP) 0
．． 4mM NADP 30μM m-PMS 20mM Mgα.

10000 v/L catalase 600 v/l hexokinase 500 v/
L Glycose 6-phosphate dehydrogenase 4800v/l Glycoamylase 0.1% Bovine albumin/0.1M sodium succinate buffer pH 8
,2 (2) 3mM Nitrotetrazolium Flu 1%
Triton X-1007, 2t/l
0.04M Sodium Succinate Buffer with Sodium Carboxylate/Starch pH
6,4 (3)0.33% Triton X-
Add 20μt of specimen to 0.6NHC/solution handling method reagent (1) 2 containing 100, keep warm at 37°C for 5 minutes, add reagent (2) 1-, and keep warm at 37°C for 5 minutes, then add reagent (3) 1. After stopping the reaction by adding d, the absorbance is measured at a wavelength of 600 nm. 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 sample is determined from this calibration curve.

Test Example 1 Effect of ascorbic acid on α-amylase activity measurement method: Ascorbic acid was added to the sample in advance at 12.5°25.50°C.
The absorbance was measured according to Example 1 for the sample added at a concentration of 1/dl. The results are shown in Figure 3.

It is clear that under these conditions up to 80 leu of ascorbic acid is completely eliminated and is not affected.

Example 2 Triglyceride measurement reagent (1) 20 pM m-PMS 10000v/l Peroxidase 1,6 X 1
0.02M N, N containing 011v/L lipase
'-Bis(2-hydroxyethyl)-2-aminoethanosulfonic acid buffer (BES) pH 7,7 (2) 80 v/l glycerokinase 24
00v/l glycerol triphosphate oxy 5μM
7 Rabi/adenine dinucleotide (FADl 1.5μMA, TP 15mA2 0.1mM 4-Aminoa 7tipiri 10.5mM
0.02 MBES buffer containing N-ethyl-N-sulfopropyl-m-toluidine pH 7.7 Procedure Reagent (1) Add 20 μt of specimen to 0.5 td and stir at 37°C.
After incubating for 10 minutes, remove reagent (2) 2. s tri was added, the mixture was further incubated at 37°C for 15 minutes, and the absorbance was measured at a wavelength of 550 nm. Separately, a sample whose concentration is known is operated in the same manner as above to create a calibration curve, and the triglyceride content of the sample is determined from this calibration curve.

Test Example 2 Effect of ascorbic acid on triglyceride measurement method: Ascorbic acid was added to the sample in advance at 10°17,5,2
For the specimen added at a concentration of 5,35 rrq/ltt, the absorbance was measured according to Example 2. The results are shown in Figure 4.

Even when using oxidized cicada, ascorbic acid 3
It is possible to completely eliminate the influence up to the equivalent of 0 to /dt.

As described above, the method of the present invention is useful in that it eliminates the interference effect of ascorbic acid in a sample in a simple manner and enables accurate measurement without wasting reagents.

[Brief explanation of the drawing]

FIG. 1 shows the absorption spectrum of ascorbic acid, and FIG. 2 shows the change over time in the oxidation reaction of the amount of ascorbic acid. FIG. 3 shows the influence of ascorbic acid concentration on the α-amylase activity measurement method. FIG. 4 shows the influence of ascorbic acid concentration on the triglyceride measurement method. Figure 1 558- Figure 3

Claims (1)

[Claims] In a method for measuring components in biological fluids using a redox reaction using dehydrogenase or oxidase, ascorbic acid in the biological fluid, which has an interfering effect on the reaction, is treated with phenazinemeth in the absence of a color-forming electron acceptor. 1. A method for measuring components in a biological fluid, which comprises interacting with sulfate or a derivative thereof and catalase or peroxodase to eliminate its reducing property.