JPS6366520B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for measuring GOT activity, particularly to a method for measuring GOT activity contained in a biological sample or the like. The GOT referred to here is the enzyme aspartate amino transferase (L-aspartate:2-oxoglutarate aminotransferase, EC2.6.1.1), which is also an abbreviation for glutamate oxaloacetate transaminase. Clinically, it has long been recognized that the amount of GOT increases in serum during liver disease, heart disease, and many other diseases, and measurement of its activity is important for diagnosis. Traditionally, GOT activity has been measured using aspartate and 2-oxoglutarate as substrates.
There is a known method in which oxaloacetate and glutamate are produced by a GOT enzyme reaction, oxaloacetate is colored with dinitrophenylhydrazine, and the absorbance is measured [Reitman et al.: Amer. J. Clin. Pathol ., 28 , 56 (1957)].
However, this method has the disadvantage that it also reacts with the keto acid used as the substrate because it is not a reaction specific to a particular keto acid. There is also a method in which NADH is added to the oxaloacetate produced in the above GOT enzyme reaction, malate dehydrogenase (MDH) is allowed to act on this to oxidize NADH, and the decrease in absorbance in the ultraviolet region is measured [Carmen: J. Clin. Inveet., 34 , 131 (1955)]. However, with this method, it is difficult to process a large number of samples by manual measurement in clinical tests. Others point out that MDH often has problems with substrate specificity. Furthermore, the glutamate produced by the above GOT enzyme reaction is converted to glutamate dehydrogenase (GLDH).
There is a method of measurement using [Ritupi et al.: Clin.
Chim. Act., 28 , 431 (1970)]. But this GLDH
has the disadvantage that it is inhibited by aspartate. In view of these conventional problems, the present inventors have conducted extensive research and have discovered a method for measuring GOT activity with high specificity without being affected by substrates, and have completed the present invention. Ivy. That is, in the method of the present invention, SO ₃ ^-- produced by a GOT enzyme reaction using cysteine sulfinate and 2-oxoglutarate or cysteine sulfinate and oxaloacetate as substrates is converted into a tetrazolium salt compound via an intermediate electron carrier. GOT activity is determined by reducing , forming formazan, and measuring it. The term "cysteine sulfinate" herein means a salt of cysteine sulfinic acid, but in the present specification it also includes cysteine sulfinic acid itself. However, in relation to solubility, it is preferable to use it in the form of cysteine sulfinic acid salt. Similarly, the terms "2-oxoglutarate" and "oxaloacetate" mean salts of 2-oxoglutaric acid (α-ketoglutaric acid) and oxaloacetic acid, respectively, but in this specification, these acids themselves are also included. . In the case of these substrates, it is also preferable to use them in the form of salts in terms of solubility and the like. Here, the tetrazolium salt compound used in the present invention is not limited to a specific one, but some examples include MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) , INT (2-P-iodophenyl-3-P-nitrophenyl-5-phenyl-
2H-tetrazolium chloride), NBT (nitrotetrazolium blue) (3,3'-dimethoxy-
4,4'-biphenylylene)-bis-(2-P-nitrophenyl-5-phenyl-2H-tetrazolium chloride), TNTB (tetranitrotetrazolium blue) (3,3'-(3,3'-dimethoxy-4, 4′-biphenylylene)-bis-(2,5
-P-nitrophenyl-2H-tetrazolium chloride), TB (tetrazolium blue) (3,
Examples include 3'-(3,3'-dimethoxy-4,4'-biphenylylene)-bis-(2,5-diphenyl-2H-tetrazolium chloride). Moreover, PMS (phenazine methosulfate) is a representative example of the intermediate electron carrier used in the present invention, but the present invention is not limited thereto. In the case of the GOT enzymatic reaction using cysteine sulfinate and 2-oxoglutarate, glutamate and β-sulfinylpyruvate are produced, and β-sulfinylpyruvate is decomposed non-enzymatically to form pyruvate and SO ₃ ^{- -} to cause. Furthermore, in the case of a GOT enzyme reaction using cysteine sulfinate and oxaloacetate, aspartate and β-sulfinylpyruvate are produced, and β-sulfinylpyruvate is similarly decomposed non-enzymatically to form pyruvate. produces SO ₃ ^-- . The reaction between SO ₃ ^-- produced by the above GOT enzyme reaction and the tetrazolium salt compound is carried out as follows when, for example, PMS is used as the intermediate electron carrier. The production amount and production rate of formazan are determined by colorimetric determination. Furthermore, after adding a GOT-containing sample onto a gel or membrane and performing electrophoresis, it is incubated with a solution containing the above substrate, an intermediate electron carrier, and a tetrazolium salt compound for activity staining, and the GOT activity is determined from the staining intensity. You can also ask for it. In this case, isozymes (S-GOT, m-GOT) can be detected down to a very low activity level of 0.0003 international units (U).
It can also be applied to the fractional quantification of As described above, the method of the present invention is capable of using cysteine sulfinate and 2-oxoglutarate or cysteine sulfinate and oxaloacetate as substrates via an intermediate electron carrier using SO ₃ ^-- produced by a GOT enzyme reaction. It consists of a reaction in which a tetrazolium salt compound is reduced to form formazan, and since no enzyme is required in the indicator reaction system, problems related to the effects of substrates and products when enzymes are used, as well as the stability of enzymes, can be avoided. . As described above, the method of the present invention has excellent advantages as a method for measuring GOT activity in the field of clinical testing. Examples relating to the method of the present invention will be shown below, but these are intended to illustrate the present invention, and the scope of the present invention is not limited thereto. Example 1 An example of a method for measuring GOT activity using a colorimetric method is shown. First, prepare the following reaction solution.

[Table] Take 3.0ml of this reaction solution and heat it to 37℃ for about 5 minutes.
Human serum containing GOT 0.56U/ml15, 30, 45,
60 μl was added, and the change in absorbance at a wavelength of 570 nm was recorded for 3 minutes while heating to 37° C., and the change in absorbance per minute was determined. At that time, when the amount of change in absorbance per minute was expressed in a graph, a linear relationship as shown in FIG. 1 was obtained. Example 2 An example of a method for measuring GOT activity by a colorimetric method using oxaloacetate instead of 2-oxoglutarate in the reaction solution of Example 1 will be described. First, prepare the following reaction solution.

【table】

[Table] As in Example 1, take 3.0 ml of this reaction solution and add 37
After heating to ℃ for about 5 minutes, add 15, 30, 45, 60 μl of human serum containing 0.56 U/ml of GOT, and record the change in absorbance at a wavelength of 570 nm for 3 minutes while heating to 37℃.
The change in absorbance per minute was determined. When the amount of change in absorbance per minute at each time was represented in a graph, a linear relationship as shown in FIG. 2 was obtained. Example 3 An example of quantification by activity staining of GOT enzyme using electrophoresis is shown. First, prepare the following reaction solution.

[Table] An enzyme solution sample containing 0.45 U/ml of GOT extracted from E. coli was prepared. This GOT enzyme solution 3, 6,
10, 15, and 20μl by Davis method [Davis:
Ann. NYAcad. Sci. 121 404 (1964)], and this was placed between electrodes of 150 volts and 2 milliamps for 2 hours of electrophoresis. Next, the gel was taken out and immersed in the reaction solution having the above composition for 10 minutes while being heated to 37°C in the dark. The gel was taken out and immersed in a 1.0% acetic acid solution, and then scanned with a densitometer at a wavelength of 530 nm. As a result, a good linear relationship was obtained between the amount of GOT enzyme solution used for the sample and the scanning area as shown in Figure 3.

[Brief explanation of drawings]

Figures 1 and 2 are GOTs measured by colorimetric method.
As the activity, the vertical axis shows the change in absorbance per minute at a wavelength of 570 nm, and the horizontal axis shows the dilution rate of human serum containing 0.56 U/ml of GOT. FIG. 3 shows the scanning area on the vertical axis and the amount of GOT enzyme solution (0.45 U/ml) on the horizontal axis as quantitative determination by activity staining of GOT enzyme by electrophoresis.

Claims (1)

[Claims] 1. Using cysteine sulfinate and 2-oxoglutarate or cysteine sulfinate and oxaloacetate as substrates,
A method for measuring GOT activity characterized by using a tetrazolium salt compound as a coloring agent together with an intermediate electron carrier. 2. In the method according to claim 1, adding the sample to a reaction solution containing the substrate, intermediate electron carrier, and tetrazolium salt compound,
A method to determine sample GOT activity by measuring its absorbance. 3. In the method according to claim 1, the sample is subjected to electrophoresis, and then incubated with a reaction solution containing the substrate, intermediate electron carrier, and tetrazolium salt compound, and the staining intensity is determined. How to determine GOT activity.