JPH0644879B2 - Assay for glutamate oxaloacetate transaminase isozymes in human serum - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Field of Industrial Application> The present invention relates to a glutamate oxaloacetate transaminase (hereinafter referred to as GOT) isozyme in human serum,
That is, cytosolic GOT (hereinafter referred to as s-GOT) and GOT localized in mitochondria (hereinafter referred to as m-GOT)
That is, the present invention relates to a simple method for separately measuring activity.

Conventionally, blood transaminase (GOT, GPT)
Is often used for the purpose of early detection of liver and heart diseases, follow-up of healing process, and determination of prognosis. In particular, the Reitman Frankel method is regarded as the standard operating method, and several improved methods have been issued thereafter. These total GOT (total G
The measurement of (PT) is also applied to the automated measurement by an automatic analyzer.

In recent years, the need for differential quantification of GOT isozymes has increased. Fractional quantification of GOT isozymes in human serum, particularly measurement of m-GOT activity in human serum, is an important index for diagnosing diseases such as alcoholic liver injury and acute hepatitis, and for determining the degree of hepatocyte injury. The clinical and diagnostic significance is extremely high, and it is being widely used as a routine examination because it enables grasping the pathological condition before and after surgery.

<Prior Art> Conventionally, as a method for separating GOT isozymes, a chromatography method using an anion exchanger and various electrophoretic methods are known, but these methods require complicated pretreatments. Operation was necessary.

Also, the measurement results obtained by these methods do not have satisfactory reproducibility for application to routine inspection,
It didn't spread.

After that, an immunological method which is relatively excellent in operability, specificity of fractionation and reproducibility was developed, and is widely used today.

The present inventors also developed various immunological fractionation and quantification methods (for example, Japanese Examined Patent Publication Nos. Sho 56-17920 and Sho 58-30543).

<Problems to be Solved by the Invention> However, these immunological methods require a centrifugal separation operation for separating m-GOT and s-GOT in the process of operation, and therefore, the test sample The number of processing cases is limited.

That is, these conventional immunological methods that require the operation of centrifugation are a major obstacle to the application to automated measurement by automatic analyzers that have been performed for various clinical test items in recent years.

Therefore, there is no need for centrifugal operation to solve the above problems,
There is a need for a means that also allows automated measurements with automated analytical instruments.

In recent years, s obtained from strains belonging to the genus Streptomyces
A classification method was reported which utilizes the difference in the sensitivity of GOT isozymes with an enzyme having a protease activity that inhibits -GOT but not m-GOT (Japanese Patent Laid-Open No. Sho 60).
-149399).

According to comparison of various proteases in the report, s-GO
As for the inhibitory effect on T, serine protease of microbial origin, particularly serine protease obtained from a strain belonging to the genus Streptomyces is strong, and trypsin and α-chymotrypsin of animal origin are reported to have a weak inhibitory effect. Suggests that there is no possibility of.

That is, as a protease that specifically inhibits s-GOT, those of microbial origin are effective, and a method for fractionating GOT isozymes using this is disclosed.

As a result of examining the inhibitory effect of various known proteases on s-GOT activity in human serum, the present inventors surprisingly did not agree with the previously reported results.

Therefore, when the above report was examined in detail, the subject used in the comparison of the inhibitory effect on s-GOT activity of various proteases in the report was not human serum but s-GOT derived from pig heart or It was confirmed that the solution was a solution in which m-GOT was dissolved.

That is, up to now, no investigation has been conducted on a protease that enables a simple measurement for differential quantification of GOT isozymes in human serum, both inside and outside.

<Means for Solving Problems> The present inventor has found that the fact based on the above report is that GOT isozyme is a protein whose structure is obviously different between various animals, and particularly in human serum GOT isozyme and animal serum. GOT isozymes of the
-GOT, or between m-GOT) was considered to be due to the fact that a clear difference in protein structure was observed [H. Teranishi et al .: Journal of Biological Chemistry (H. Teranishi et al.,: J. Biol. Che
m.) Vol. 253, No. 24, pp. 8842-8847,
1978].

It is a well-known fact that the action of various proteases generally causes limited decomposition with respect to the amino acid sequence of the enzyme protein, which is its substrate.

From this fact, there is a possibility that the enzyme activity may be completely lost as a result of the limited decomposition of the certain protease with respect to the certain enzyme protein.

Or, conversely, even if it is limitedly decomposed, it may have the enzyme activity.

Therefore, the specificity of its action is of novel value only if it is experimentally proven.

That is, when examining s-GOT activity inhibitors for the purpose of differential quantification of GOT isozymes in human serum, it is an essential condition to target human GOT isozymes.

The object of the present invention is to enable a simple measurement of GOT isozyme in human serum.

The present inventor has found that human GO samples can be used as human GO samples.
S-GOT enabling separation of GOT isozymes in human serum targeting T-isozyme
The specific inhibitor of the above was earnestly studied.

As a result, surprisingly, human s- was among the commercially available proteases.
It was found that chymotrypsin extracted and purified from animal organs is effective as a substance that specifically inactivates GOT and hardly affects human m-GOT activity.

That is, the present invention specifically deactivates s-GOT in human serum samples by chymotrypsin of animal origin,
By measuring the remaining m-GOT by a known biochemical method, the GOT isozyme in human serum can be easily fractionated and quantified, and the series of measurement operations can be automated by an automatic analyzer. It is a thing. The chymotrypsin used in the present invention is defined to be of animal origin in the sense that it is distinguished from a protease obtained from a microorganism. There are various types of chymotrypsin such as α-form, β-form and γ-form, and any of them can be used. It is known that chymotrypsin can be obtained by reacting chymotrypsinogen with trypsin.

The present invention involves the use of chymotrypsinogen in trypsin as chymotrypsin. When chymotrypsinogen is used, trypsin is used together with chymotrypsin according to a conventional method.

The present invention will be described in more detail in the following examples and comparative examples.

The present invention is not limited to the examples below.

<Example> The m-GOT activity in human serum was measured by the method of the present invention and a conventional immunization method.

Table 1 shows the results of measurement using 20 human total GOT-positive sera as test samples, and Table 2 shows the results of measurement using 20 human total GOT-positive sera. The methods for preparing the reagents and measuring the reagents are as follows.

[Preparation of Reagents] First reagent: α-chymotrypsin (manufactured by Sigma: Type I)
20 mg of 0.1M Tris-HCl buffer (pH 8.
0) Dissolve in 20 ml.

Second reagent: The one having the following composition is used.

L-aspartic acid 200 mM α-ketoglutarate 12 mM Malate dehydrogenase 1,600 U / lactate dehydrogenase 1,000 U / NADH ₂ 0.16 mM Tris buffer 80 mM The pH of the second reagent is 7.8.

[Measurement Method] The method of the present invention was measured using a Hitachi 705 type automatic analyzer (manufactured by Hitachi, Ltd.) in which 200 μm of the first reagent and 500 μm of the second reagent were applied to 10 μm of human test sample serum.

The conventional method (immunization method) is an anti-s-GOT according to the conventional method.
The antibody and anti-s-GOT antibody sensitized to sheep red blood cells were added to the serum of the test sample, and after centrifugation, the second reagent was added to m-.
GOT activity was measured.

The measurement method uses aspartic acid and α-ketoglutaric acid as substrates, GOT produces oxaloacetic acid and glutamic acid, and when the generated oxaloacetic acid changes to malic acid in the presence of MDH, NADH ₂ becomes NAD, At this time, the decrease in the absorbance of NADH ₂ at a wavelength of 340 nm per minute was measured to determine the m-GOT activity.

All activity values in the table are in international units.

<Comparative Example> In order to confirm the difference in the protease action of chymotrypsin used in the method of the present invention on human and porcine GOT isozymes by the same method as in the example, human and porcine GOT isozymes were used as test samples, respectively. GOT
GO after reacting a protease with an isozyme
The result of measuring the T activity was the GOT activity value of 10 before the reaction.
Table 3 shows the results expressed as a change rate (%) when 0% is set.

As the protease, α-chymotrypsin was used.

<Effects of the Invention> The present invention does not require a centrifugation operation in the separation process, and thus can be directly used as a reagent for automatic analysis. That is, it is easy to operate and has an effect of enabling processing of a large amount of samples in a short time.

Claims (1)

[Claims] 1. Glutamic acid oxaloacetate transaminase isozymes present in human serum are specifically inactivated by cytoplasmic glutamate oxaloacetate transaminase, and only mitochondrial glutamate oxaloacetate transaminase is fractionated and quantified. A method for measuring glutamate oxaloacetate transaminase isoenzyme in human serum, characterized in that chymotrypsin of animal origin is used as a cytoplasmic glutamate oxaloacetate transaminase activity inhibitor in the activity measurement of acetate transaminase isozyme.