JP2000069997A - New method and reagent for measuring lactate dehydrogenase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and a reagent for accurately measuring lactate dehydrogenase in a wide measurement range. SOLUTION: In this method for measuring the activity of lactate dehydrogenase by using lactic acid and a coenzyme NAD as substrates, the measurement is carried out in the presence of L-glutamic acid and alanine aminotransferase so that formed pyruvic acid is converted into alanine. Consequently lactate dehydrogenase can be accurately measured in a wide measurement range.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a test method used in the field of clinical tests and food tests, and more particularly to a method for measuring lactate dehydrogenase activity in a sample. More specifically, the present invention relates to a highly quantitative lactate dehydrogenase activity measuring method and a measuring reagent.

[0002]

2. Description of the Related Art Lactate dehydrogenase is an important enzyme for the energy production of living organisms, and is contained in high concentrations in major organs and tissues such as liver and heart. Therefore, its activity is measured as a useful index for diagnosis of various diseases including myocardial infarction. One of the methods for measuring lactate dehydrogenase activity is that when the enzyme converts lactic acid to pyruvate, the coenzyme NAD
This is a method for measuring the production rate of NADH using the fact that it is reduced to ADH. The other utilizes the fact that the reaction catalyzed by this enzyme is reversible, and uses pyruvate as a substrate to convert the conversion of pyruvate to lactic acid to the coenzyme NADH.
Methods for measuring the rate at which is oxidized to NAD are widely known.

[0003] It is known that any of these methods forms a complex between the product and lactate dehydrogenase, resulting in a reverse reaction. As described above, since the reaction itself is a reversible reaction, the reverse reaction of the intended reaction proceeds as the concentration of the product increases after the start of the reaction.
Because the reaction rate decreases with time,
It was difficult to measure the activity accurately. As a method for avoiding this, an initial rate measurement, which measures the reaction rate immediately after the start of the reaction, is generally used. However, in the measurement of the initial velocity, the amount of the enzyme is proportional to the reaction rate in a region where the enzyme activity is low. Therefore, an accurate method for measuring lactate dehydrogenase having a wide measurement range has been desired. In general, a technique for converting a product into a component not involved in the reaction by a chain enzyme reaction when an inhibitory reaction by the product occurs is known. However, in the measurement of lactate dehydrogenase activity, no effective conversion by a chain enzyme reaction has been known so far.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for measuring the activity of lactate dehydrogenase accurately and over a wide measurement range.

[0005]

Means for Solving the Problems In order to provide a method for measuring the activity of lactate dehydrogenase as described above, which is accurate and has a wide measurement range, the present inventors have conducted intensive studies and found that As a method for eliminating pyruvic acid generated by the action of the present enzyme by using as a substrate by another enzyme reaction, L-glutamic acid and the enzyme alanine aminotransferase are added to convert pyruvic acid to alanine and α-ketoglutarate. As a result, they have found that it is possible to prevent the progress of the reverse reaction, and have completed an accurate method for measuring lactate dehydrogenase activity with a wide measurement range. That is, the gist of the present invention is as follows. A method for measuring lactate dehydrogenase activity, which comprises using L-glutamic acid and alanine aminotransferase in a method for measuring lactate dehydrogenase activity using lactic acid as a substrate. A reagent for measuring lactate dehydrogenase activity, which comprises using L-glutamic acid and alanine aminotransferase in a reagent for measuring lactate dehydrogenase activity using lactic acid as a substrate.

The method for measuring the activity of lactate dehydrogenase of the present invention comprises:
In a reaction in which pyruvate and reduced coenzyme NADH are converted by lactate dehydrogenase using lactic acid and coenzyme NAD as substrates, pyruvate produced is converted to alanine by adding L-glutamic acid and alanine aminotransferase. It is characterized by conversion. Alanine aminotransferase, pyruvate oxidase, pyruvate decarboxylase, and the like can be considered as enzymes that convert the generated pyruvate into other substances. However, other enzymes than alanine aminotransferase were not effective due to low substrate specificity and maximum reaction rate.

On the other hand, methods for measuring lactate dehydrogenase activity using pyruvic acid and NADH as substrates have been widely used, but no suitable enzyme for preventing the reverse reaction in this case is known. Therefore, in the method for measuring lactate dehydrogenase activity using lactic acid and coenzyme NAD as substrates, the present invention is characterized by coexistence of L-glutamic acid and alanine aminotransferase.

The origin and purification degree of alanine aminotransferase are not particularly limited as long as there is no contamination with lactate dehydrogenase, but those derived from animals such as humans and pigs are preferred. The amount of the enzyme used may be 1 to 10 units per measurement. L-glutamic acid is preferably 5 to 50 mM. These are usually suitable buffers, such as Tris-
It is used after being dissolved in a hydrochloric acid buffer or a glycine-sodium hydroxide buffer. On the other hand, lactate which is a substrate of lactate dehydrogenase to be measured is 50 to 300 mM, and NAD is 1 to 10 mM.
It is preferred to use it in mM. These substrates are also used by dissolving them in the same Tris-HCl buffer or glycine-sodium hydroxide buffer as described above. Lactic acid, NAD, L-glutamate and alanine aminotransferase, which are constituents of the above-mentioned measuring reagents, may be all prepared in the same solution or may be prepared in two or more solutions. In the case of preparing two or more liquids separately, the combination is not particularly limited unless L-glutamic acid and alanine aminotransferase are added after lactate dehydrogenase in the measurement sample comes into contact with lactic acid and NAD.

[0009]

EXAMPLES The present invention will be described in more detail with reference to Examples, but it should not be construed that the invention is limited thereto.

Example 1 The following two reagents were prepared.

Reagent A Tris-HCl buffer (pH = 8.8) 200 mM Lactic acid 150 mM NAD 5 mM

Reagent B Tris-HCl buffer (pH = 8.8) 200 mM Lactate 150 mM NAD 5 mM L-glutamic acid 20 mM Alanine aminotransferase 10 IU / ml

300 μl each of reagent A and reagent B
10 μl of three kinds of serum specimens were added thereto, and reacted at 37 ° C., and the absorbance at a wavelength of 340 nm was measured. The change in absorbance per minute from 1 minute to 2 minutes after the start of the reaction and 3 minutes from 2 minutes later The change in absorbance per minute up to the minute was compared. The results are shown in Table 1. As a result, in the sample A having a high lactate dehydrogenase activity in the reagent A containing no L-glutamic acid and alanine aminotransferase,
Comparing the absorbance difference (ΔA1) after 2 minutes and 1 minute and the absorbance difference (ΔA2) after 3 minutes and 2 minutes, ΔA2 is significantly lower, indicating that the reaction is inhibited. . On the other hand, in reagent B containing L-glutamic acid and alanine aminotransferase, ΔA2 and ΔA1 showed almost the same value, indicating that there was no inhibition of the reaction.

[0014]

[Table 1]

[Example 2] Using the reagents A and B shown in Example 1, the specimen 1 measured in Example 1 was subjected to 5/5, 4/5, 3/5, 2 / The sample was serially diluted to 5, 1/5 and measured. The change in absorbance (ΔA2) per 2 to 3 minutes after the start of the reaction was measured, and the dilution linearity of the sample was compared between the reagent A and the reagent B. As shown in FIG. 1, the results were as follows.
Above / 5, the slope clearly decreased. On the other hand, in the case of the reagent B according to the present invention, the dilution linearity was observed over the entire dilution range, indicating that the quantitativeness was high.

[Brief description of the drawings]

FIG. 1 is a diagram showing the dilution linearity of a high-value sample.

[Explanation of symbols]

 ○ indicates the measurement result of the reagent A, and □ indicates the measurement result of the reagent B.

Claims (2)

[Claims] 1. A method for measuring lactate dehydrogenase activity, which comprises using L-lactic acid as a substrate in the presence of L-glutamic acid and alanine aminotransferase. 2. A reagent for measuring lactate dehydrogenase activity, wherein L-glutamic acid and alanine aminotransferase coexist in a reagent for measuring lactate dehydrogenase activity using lactic acid as a substrate.