JPH0771517B2 - Fractional quantification method of aspartate amino transphenylase isozyme - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to serum aspartate aminotransferase (strain name L-Aspartate:
2-oxoglutarate aminotransferase (EC2.6.1.1.), Synonym Gletamic Oxaloacetic Transamirase (GOT), hereinafter referred to as AST)
More specifically, it relates to a method for differentially quantifying mitochondrial AST by reacting a sample serum with a specific substrate in the presence of a specific inhibitor and measuring the obtained product.

BACKGROUND OF THE INVENTION AST is present in almost all organs of mammals, and is a cytoplasmic localized fraction of cytoplasmic AST (hereinafter, referred to as
There are two types of isozymes, s-AST) and mitochondrial AST (hereinafter referred to as m-AST), which is a fraction localized in mitochondria.
The quantitative determination of the amount, the amount of s-AST, and the amount of m-AST are all very useful for clinical diagnosis.

In particular, the differential quantification of m-AST is useful as a clinical diagnostic method for mitochondrial function, hepatitis, myocardial infarction, severity of cell damage, etc., and in recent years, there is a need for a simple and specific quantification method. It is increasing more and more.

Prior art Conventionally, measurement of AST isozymes anion exchange chromatography (clinical chemistry, 5, 163 (1979)), immunochemical methods (JP 50-19918) and electrophoresis (Robert Ray, Clinical・ Chemistry (Robert Rej, c
lin.chem.), 24 , 1971 (1978), etc., but these have various drawbacks such as time-consuming and complicated operations, and sufficient accuracy cannot be obtained. ing.

Furthermore, a method of measuring the activity at a plurality of different pHs to obtain an isozyme ratio and similarly measuring the activity at two different pHs, and further examining the influence of the s-AST inhibitor to calculate the isozyme ratio method (Robert Ray, Clinical chemistry (Robert Rej, Clin.Chem.), 27, 53
5 (1981)), but all of them are complicated in operation and lack in speed and accuracy.

In addition, a method has also been proposed in which m-AST is measured after pretreatment with an enzyme that inhibits s-AST activity (JP-A-60-149399). However, this method requires a large amount of purified enzyme, has a problem in economical efficiency, requires a relatively long time for pretreatment, and is not suitable for automatic analysis.

OBJECT OF THE INVENTION The present inventors have conducted earnest studies to find a method for fractionating and quantifying m-AST that is free of such problems, is rapid, has high accuracy, and is suitable for automatic analysis. As a result, in the presence of one or more s-AST inhibitors selected from dicarboxylic acid compounds and proteases, a new substrate different from L-aspartic acid, which is conventionally known as a substrate for AST, is allowed to act. -The present invention has been completed by finding that only AST can be specifically and rapidly fractionated and quantified with high accuracy and can be applied to automatic analysis.

DISCLOSURE OF THE INVENTION That is, according to the present invention, in the presence of one or more s-AST inhibitors selected from dicarboxylic acid compounds and proteases, L-cysteic acid and 2-oxoglutaric acid are used as substrates for m-AST in a sample. And act as the formula: The present invention provides a fractional quantification method of AST isozyme, which specifically quantifies m-AST by measuring either L-glutamic acid or β-sulfonylpyruvic acid produced according to the reaction shown in.

Using cysteic acid and 2-oxoglutarate as substrates,
Cysteinic acid transaminase is conventionally known as an enzyme that induces glutamic acid and β-sulfonylpyruvate from these (S. Darling, Nature, 170 , 749 to 750 (195
2)), which is different from AST, and there is no example of using cysteic acid and 2-oxoglutarate as substrates for the differential quantification of AST isozymes.

In the method of the present invention, examples of the dicarboxylic acid compound used as the s-AST inhibitor include glutaric acid, adipic acid, succinic acid, maleic acid and their anhydrides. Particularly preferred. Examples of the protease include actinase (produced by Kaken Chemical Co., Ltd.), papain, protease type VII, protease type VIII, protease type X VIII, promeline (all produced by Sigma) and the like. These s-AST inhibitors may be used alone or in combination of two or more.

The sample is typically serum, but is not limited to this, and may be any sample that can be used for measurement in which quantification of m-AST is desired.

L-Cysteinic acid and 2-oxoglutaric acid used as substrates may be of commonly available quantitative reagent grade.

L-glutamic acid or β-produced according to formula [I]
The method for measuring sulfonylpyruvic acid is not particularly limited, and any known method for quantifying these compounds can be adopted.

For example, L-glutamic acid causes L-glutamic acid dehydrogenase (GlDH) to act in the presence of NAD, an electron carrier such as diaphorase (DI) and phenazine methosulfate (PMS), and nitrotetrazolium blue (NT).
B), 3- (4,5-dimethyl-2-thiazolyl) -2,5-
A method in which a tetrazolium salt such as diphenyl-2H-tetrazolium bromide (MTT) is added to develop a color, and measurement is carried out at a wavelength in the visible region can be suitably adopted.

Surprisingly, according to the inventors, the formula: In the presence of NADH, MDH, which is conventionally thought to use oxaloacetate as a substrate, is allowed to act on β-sulfonylpyruvate according to the reaction shown by
Quantification of β-sulfonylpyruvate by measuring its change to NAD (not showing absorption in the ultraviolet region), that is, decrease in optical density of NADH in the ultraviolet region, by utilizing the specific absorption at m). It was found that the fractional quantification of m-AST isozymes can be performed with extremely high accuracy and speed. Therefore, in the method of the present invention, when β-sulfonylpyruvic acid is measured, after the reaction of the formula [I], the formula [II]
It is preferable to carry out the quantification operation by carrying out the reaction described above, and thus, the present invention also includes the presence of one or more s-AST inhibitors selected from the group consisting of dicarboxylic acid compounds and proteases in a sample. A method for fractionating and quantifying m-AST isoenzyme, which comprises reacting m-AST with L-cysteic acid and 2-oxoglutarate as substrates and allowing the produced β-sulfonylpyruvate to act with MDH in the presence of NADH. It is a thing.

In the present invention, the reaction of the formula [I] and the reaction of the formula [II]
The L-glutamic acid and β-sulfonylpyruvic acid produced, including the reaction of, can be measured in the same system.

The method of the present invention includes, for example, appropriately diluting a sample,
At the same time, an s-AST inhibitor, L-cysteic acid, 2-oxoglutarate, and a reagent for measuring L-glutamic acid or β-sulfonylpyruvate in an aqueous solution state,
Or added sequentially, after holding at a predetermined temperature for a predetermined time,
Using purified water and blind test as controls, the optical density at a given wavelength and its change per unit time were measured, and m-AST
It can be carried out by quantifying the amount of m-AST in the sample from the measured value using a standard and the calibration curve created thereby. In practice, in the case of the reaction system of MDH, it is desirable to store at the optimum pH of m-AST, that is, pH 6.0 to 8.0, preferably pH 7.0 to 7.5. Bis-Tris or Tris is usually preferably used in a concentration of about 10 to 100 mM. Usually, s-AS is used in the reaction system.
T inhibitor 100 to 300 mM (for dicarboxylic acid compounds) or 1000 to 3000 tyrosine units / ml (for actinase), L-cysteic acid at 100 to 250 mM, 2-oxoglutarate at 5 to 20 mM. The reaction system at 37 ° C.
After holding for about 1 to 30 minutes before and after, the optical density is measured. In the case of a GlDH reaction system, preferably pH7.
In order to maintain the concentration at 5 to 8.5, 10 to 200 mM of the same buffer is used, and the concentration of 2-oxoglutaric acid is 0.5 to 5 mM. For example, GlDH, NAD, P
When measuring L-glutamic acid using MS and NTB, the optical density at 560 nm is measured using a blind control. When β-sulfonylpyruvate is measured using NADH and MDH, purified water is used as a control and the change in optical density at 340 nm per unit time is measured.

In particular, in the method of the present invention, the s-AST inhibitor, L-
Cysteinic acid and L-glutamic acid or β-sulfonylpyruvic acid measuring reagents are added to the reaction system, and, for example,
After holding at 37 ° C. for 1 to 10 minutes, 2-oxoglutaric acid was added, and again holding at 37 ° C. for 1 to 10 minutes revealed that the fractional quantification of m-AST isozymes could be performed with high accuracy.

The present invention also provides a reagent kit for m-AST isozyme fractionation and quantification, which is a simple and rapid method for carrying out the method of the present invention.

Such a reagent kit is composed of at least one reagent selected from the group consisting of the s-AST inhibitor, L-cysteic acid, 2-oxoglutaric acid and L-glutamic acid or a reagent for measuring β-sulfonylpyruvic acid, If desired, other agents such as m-AST standards, buffers and the like may be combined. Each reagent may be a lyophilized product, a solid such as powder or granules, a liquid such as a concentrated liquid, a dilute liquid, etc., according to a usual formulation technique, if necessary, appropriately mixed at a predetermined ratio, and appropriately packaged. Can be manufactured.

As described above, in the present invention, the formulas [I] and [I
According to the reaction [I], β-sulfonylpyruvic acid is preferably measured, and 2-oxoglutarate is preferably added to the reaction system separately from other reagents. Therefore, in the present invention, the reagent kit is a combination of a first reagent containing s-AST inhibitor, L-cysteic acid, NADH and MDH, and a second reagent containing 2-oxoglutarate. Is preferred.

For example, the first reagent is dissolved in purified water at the time of use to give L-
Cysteinic acid 100-300 mM, preferably about 200 mM, glutaric acid 100-400 mM, MDH 5-50 units, preferably 20 units
/ mL, NADH 0.08 to 0.23 mg / ml can be prepared as a powder or solution in which each reagent is mixed. In order to maintain the reaction system at pH 6.5 to 8.0 during use, a buffering agent such as bis-Tris or Tris can be added to the first reagent to a concentration of 10 to 100 mM.

In addition, the second reagent is dissolved in purified water at the time of use and has a concentration of 150 to 630 mM in the case of the manual method and a concentration of 20 to 630 in the case of the automatic analysis method.
It can be a powder or a solution containing 2-oxoglutaric acid at 110 mM.

To separate and quantify the m-AST isozyme in serum using this kit, the sample serum was treated with the first reagent to give about 37
After preheating at 1 ° C for 1 to 10 minutes, preferably 2 to 6 minutes, the second reagent is added and reacted at the same temperature for 1 to 10 minutes, preferably 2 to 6 minutes, using purified water as a control. The decrease in absorbance at 340 nm per minute is measured, and the m-AST activity value (U /) in the sample serum is calculated by the following formula based on these data.

In the formula, ΔEsa: difference in absorbance of sample serum per minute ΔEstd: difference in absorbance of standard m-AST per minute x: activity value of standard m-AST (U /) As described above, according to the present invention. For example, since only m-AST in serum can be specifically quantified and can be carried out extremely simply and quickly without any special device, the fractional quantification method and its reagent of the present invention -Very useful for clinical examination of AST.

Next, the following experiment was conducted in order to investigate the m-AST fractionation quantitative effect according to the present invention.

Hereinafter, the method of the present invention for measuring β-sulfonylpyruvic acid is referred to as the present invention method (1), and the method for measuring L-glutamic acid is referred to as the present invention method (2).

Experiment Experiment 1 Various s- in the conventional method using L-aspartic acid and the method of the present invention using L-cysteic acid as a substrate
Effect of AST inhibitor (1) Preparation of sample s-AST sample: m-AST by immunoprecipitation method, electrophoresis method, etc.
S-AST derived from pig heart confirmed to contain no
(Boehringer) was diluted with 1 mg / ml bovine serum albumin-containing bis-Tris buffer (pH 7.5) to give an activity value of 130.6 U /
S-AST sample is prepared.

m-AST sample: s-AST by immunoprecipitation method, electrophoresis method, etc.
Standard m-AS derived from pig heart confirmed to contain no
T (manufactured by Eiken Chemical Co., Ltd.) was diluted with the above buffer solution to give an activity value of 154.8
Prepare U / m-AST samples.

Method (1) of the present invention (1) Preparation of reagent First reagent: L-cysteic acid (Tokyo Kasei Co., Ltd.) 4.40 g, malate dehydrogenase (Amano Pharmaceutical Co., Ltd.) 2500 units, β-NADH
(Oriental Yeast Co., Ltd.) 16.0 mg, s-AST inhibitor (see Tables 1 and 2 below) and Bis-Tris (Dojindo Co., Ltd.) 0.54 g were dissolved in purified water to make 100 ml in total (pH 7). .Five).

Second reagent: 2-oxoglutaric acid (Tokyo Kasei Co., Ltd.) 0.46 g
Is dissolved in purified water to bring the total volume to 50 ml (pH 6.5).

(2) Method 0.40 ml of the first reagent was added to 0.020 ml of the sample and pre-heated at 37 ° C for 5 minutes, and then 0.10 ml of the second reagent was added and reacted at the same temperature, while using purified water as a control at 340 nm. The decrease in absorbance per minute was measured using a Hitachi 705 automatic analyzer, and from these data, m-
The activity value and relative activity of AST were calculated.

Conventional method (1) Preparation of reagent First reagent: L-aspartic acid (manufactured by Hanai Chemical Co., Ltd.) 3.46 g,
Malate dehydrogenase 100 units, β-NADH 16.0mg, s-AST
Inhibitors (see Tables 1 and 2 below) and Tris 0.
Dissolve 31 g in purified water to bring the total volume to 100 ml (pH 8.0).

Second reagent: 0.46 g of 2-oxoglutarate is dissolved in purified water to bring the total volume to 50 ml (pH 6.5).

(2) Method The method is performed in the same manner as in the method (1) of the present invention.

The results are shown in Tables 1 and 2.

As is clear from the results in Tables 1 and 2, addition of a dicarboxylic acid compound or a protease to the conventional method using L-aspartic acid as a substrate did not inhibit the activity of s-AST, but L-cysteine According to the method of the present invention using an acid, a particularly remarkable s-AST activity inhibitory effect was obtained when glutaric acid, maleic anhydride and actinase were used.

Experiment 2 Activity of AST isozyme to L-cysteic acid substrate (1) Preparation of sample s-AST sample: An s-AST sample with an activity value of 352 U / is prepared in the same manner as in Experiment 1 above.

m-AST sample: An m-AST sample having an activity value of 155 U / is prepared in the same manner as in Experiment 1 above.

Method (1) of the present invention (1) Preparation of reagent First reagent: L-cysteic acid 3.63 g, malate dehydrogenase (manufactured by Amano Pharmaceutical Co., Ltd.) 2000 units, β-NADH 13.0 mg, glutaric acid 2.84 g and bis- Dissolve 0.45 g of Tris in purified water to bring the total volume to 100 ml (pH 7.5).

Second reagent: 2.74 g of 2-oxoglutarate is dissolved in purified water to bring the total volume to 50 ml (pH 6.5).

(2) Method 2.9 ml of the first reagent was added to 0.12 ml of the sample and pre-heated at 37 ° C for 5 minutes, and then 0.10 ml of the second reagent was added and reacted at the same temperature, while using purified water as a control at 340 nm. The decrease in optical density per minute was measured using a Shimadzu UV210A spectrophotometer, and from the data, m was calculated according to the above formula (A).
-The AST activity value and relative activity were calculated.

Inventive Method (2) (1) Preparation of Reagent Preparation of Substrate Detection Enzyme Solution: L-Cysteinic Acid 1.69 g, 2-
Oxoglutarate 7.3 mg, L-glutamate dehydrogenase (Oriental Yeast Co., Ltd.) 1500 units, NAD + (Oriental Yeast Co., Ltd.) 0.15 g, oxaloacetate decarboxylase (Toyo Brewing Co., Ltd.) 500 Units, glutaric acid 1.32 g, m- 5.0 mg of phenazine methosulfate, 30 mg of nitrotetrazolium blue and 0.61 g of tris (hydroxymethyl) aminomethane are dissolved in purified water to a total volume of 50 ml (pH
7.8).

(2) Method Add 0.5 ml of substrate detection enzyme solution to 0.2 ml of the above sample, and at 37 ℃
After warming for 20 minutes, 5.0 ml of 0.1N hydrochloric acid was added to stop the reaction, and the absorbance at 560 nm was measured using a Shimadzu UV210A spectrophotometer with a blind consisting of purified water as a control. The activity value and relative activity of m-AST were calculated using the calibration curve of.

Conventional method (1) Method Nescort GOTV ₃ (S), which is a reagent that has been commonly used for the measurement of AST activity by the Carmen method using the above sample
SCC) (manufactured by Nippon Shoji Co., Ltd.)
The activity value and relative activity of ST isozyme were calculated, and the results are shown in Table 3.

As is clear from Table 3, regarding the m-AST activity,
Both of the methods (1) and (2) of the present invention show the same values as those of the conventional method, while regarding the s-AST activity, the method (1) of the present invention has a relative activity of 0.6%, and the method (2) of the present invention has a relative activity. Activity 0.0
%, Showing that the AST isozyme can be fractionated and quantified by the method of the present invention.

Experiment 3 Effect of glutaric acid on s-AST activity (1) Preparation of sample Sample I: s-AST derived from pig heart confirmed by immunoprecipitation method, electrophoresis method and the like (manufactured by Boehringer) ) Was diluted with 1 mg / ml bovine serum albumin-containing bis-Tris buffer (pH 7.5) to give an activity value of 251 U / s-AST.
Prepare the sample.

Sample II: An s-AST sample with an activity value of 507 U / is prepared as described above.

Sample III: An s-AST sample with an activity value of 760 U / is prepared as described above.

(2) Preparation of Reagent Prepare in the same manner as in Experiment 1 above.

(3) Method The activity value and relative activity of s-AST were measured in the same manner as in Experiments 1 and 2 above. The results are shown in Table 4.

As is clear from Table 4, the activity of s-AST was completely inhibited by the methods (1) and (2) of the present invention using glutaric acid.

Experiment 4 m-AST activity in the coexistence of s-AST The activity value was 0, and m-AST sample with activity value of 155 U /, respectively.
151, 316 and 456 U / s-AST samples were added and s-AS
The activity value of m-AST was calculated in the same manner as in T and the method (1) of the present invention in Experiment 1. The results are shown in Table 5.

As is clear from Table 5, the m-AST activity value is hardly affected even in the presence of s-AST.

Therefore, the method (1) of the present invention can specifically separate and quantify m-AST.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 Preparation of a calibration curve by the method (1) of the present invention (1) Preparation of reagent First reagent: 3.63 g of L-cysteic acid, malate dehydrogenase 2
000 units, β-NADH 13.0 mg, glutaric acid 2.84 g and bis-Tris 0.45 g are dissolved in purified water to bring the total volume to 100 ml (pH
7.5).

Second reagent: 2.74 g of 2-oxoglutarate is dissolved in purified water to bring the total volume to 50 ml (pH 6.5).

(2) Preparation of standard serum Serum having an m-AST activity value of about 340 U / l was diluted with physiological saline to 1/5, 2
Prepare by diluting to / 5, 3/5, 4/5, 5/5.

(3) Measurement operation and results Take 0.12 ml of each standard serum of each concentration described above in 5 test tubes, add 2.9 ml of the first reagent thereto, preheat at 37 ° C. for 5 minutes, and then add the second reagent 0.1. Add ml and allow to react at 37 ℃,
Using purified water as a control, the decrease in absorbance at 340 nm per minute was measured, and the activity value of m-AST was determined from the data by the above formula (1) to prepare a calibration curve. This is shown in FIG. As is clear from the figure, a straight line passing through the origin was obtained, confirming the quantitativeness of the method (1) of the present invention.

Example 2 (1) Preparation of Reagent Prepared in the same manner as in Example 1 above.

(2) Measurement operation and results Using the first and second reagents and the standard serum used in Example 1, the same operation as in Example 1 was performed on 16 serum samples to determine the m-AST activity value. On the other hand, for the same serum sample,
M-AST reagent "Eiken" (manufactured by Eiken Chemical Co., Ltd.), which is generally used for measuring m-AST activity by a known immunochemical method.
Was used to determine the m-AST activity value by a conventional method. From these, a correlation diagram between the method of the present invention (1) and the conventional method was prepared.
This is shown in FIG.

From this, the correlation coefficient is r = 0.989, and when the measured value of the conventional method is x and the measured value of the method (1) of the present invention is y, the equation of the regression line shows a good correlation of y = 1.058x + 2.1. The accuracy of invention method (1) was confirmed.

Example 3 Preparation of calibration curve by method (2) of the present invention (1) Preparation of reagent Substrate detection enzyme solution: 1.69 g of L-cysteic acid, 7.3 mg of 2-oxoglutarate, 1500 units of L-glutamate dehydrogenase, NAD + 0. 15 g, oxaloacetate decarboxylase 500
The unit, 1.32 g of glutaric acid, 5.0 mg of m-phenazine methosulfate, 30 mg of nitrotetrazolium blue and 0.61 g of tris (hydroxymethyl) aminomethane are dissolved in purified water to a total volume of 50 ml (pH 7.8).

(2) Preparation of standard serum Prepared in the same manner as in Example 1 above.

(3) Measurement operation and results Take 0.12 ml of each standard serum of each concentration described above in 5 test tubes, add 0.5 ml of the substrate detection enzyme solution to it, and heat at 37 ° C for 30 minutes, and then add 0.1N hydrochloric acid 5.0 ml. Was added to stop the reaction, and the absorbance at 560 nm was measured using a blind test consisting of purified water as a control. From these data, m-AS was calculated by the above formula (2).
The activity value of T was determined and a calibration curve was prepared. This is shown in FIG. As is clear from the figure, a straight line passing through the origin is obtained,
The quantitative property of the method (2) of the present invention was confirmed.

[Brief description of drawings]

FIG. 1 is a graph showing a calibration curve in the method (1) of the present invention,
FIG. 2 is a graph showing a correlation between the method of the present invention (1) and an immunochemical method, and FIG. 3 is a graph showing a calibration curve in the method of the present invention (2).

Claims (5)

[Claims] 1. A mitochondrial aspartate aminotransferase in a sample in the presence of one or more cytoplasmic aspartate aminotransferase inhibitors selected from the group consisting of dicarboxylic acid compounds and proteases, and L-cysteic acid Aspartate aminotransferase characterized by specifically quantifying mitochondrial aspartate aminotransferase by allowing 2-oxoglutarate to act as a substrate and measuring either L-glutamic acid or β-sulfonylpyruvate produced. Fractional quantification method of isozymes. 2. The method of fractionation and quantification according to item (1), wherein the dicarboxylic acid compound is glutaric acid. 3. Mitochondrial aspartate aminotransferase in a sample in the presence of one or more cytoplasmic aspartate aminotransferase inhibitors selected from the group consisting of dicarboxylic acid compounds and proteases, and L-cysteic acid and It is characterized by specifically quantifying mitochondrial aspartate aminotransferase by allowing 2-oxoglutarate to act as a substrate and allowing the produced β-sulfonylpyruvate to act as a malate dehydrogenase in the presence of NADH. Fractional quantification method of aspartate aminotransferase isozyme. 4. One or more cytoplasmic aspartate aminotransferase inhibitors selected from the group consisting of dicarboxylic acid compounds and proteases, L-cysteic acid, 2-oxoglutarate, and L-glutamic acid or β-sulfonyl. A reagent kit for fractional quantification of mitochondrial aspartate aminotransferase isozyme, which comprises at least one reagent selected from the group consisting of reagents for measuring pyruvic acid. 5. A first reagent and a second reagent containing one or more cytoplasmic aspartate aminotransferase inhibitors selected from a dicarboxylic acid compound and a protease, L-cysteic acid, NADH and malate dehydrogenase. A reagent kit for fractionation and quantification of mitochondrial aspartate aminotransferase isozyme according to the above (4), which comprises combining a second reagent containing oxoglutarate.