JP2000270893A - Reagent for measurement of activity of gamma-glutamyl transpeptidase and its measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a reagent for measurement of the activity of γ-glutamyl transpeptidase (γ-GTP) capable of obtaining an accurate measurement scarce in error without affected from haemoglobin for samples such as blood serum, blood or the like to be measured and measuring method. SOLUTION: This reagent for measurement of the activity of γ-GTP includes the first reagent including a receptor substrate for γ-glutamyl group (GlyGly) and the second reagent including a donor substrate for γ-glutamyl group (Glu- CANA or Glu-pNA) and oxidase (peroxidase or catalase) or oxidase and a preservative (sodium azide). The γ-GTP activity of a sample is obtained from the measurement of the change of the absorption for unit time in the wavelength range of 400-450 nm of the substance produced by the reaction of reagent and the sample in the presence of the oxidase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the activity of the enzyme γ-glutamyltranspeptidase (also referred to as γ-glutamyltransferase, hereinafter abbreviated as γ-GTP) present in biological samples such as serum. And a method for measuring γ-GTP activity using the reagent. More specifically, the present invention relates to a reagent and a method for measuring γ-GTP activity, which can suppress changes over time in measured values of enzyme activity and can more accurately measure γ-GTP activity of a sample.

[0002]

2. Description of the Related Art γ-GTP is an enzyme that catalyzes a reaction of cutting off a 5-L-glitamyl group from a substrate (such as a peptide) and transferring it to an amino acid (generation of 5-L-glitamyl amino acid). This plasma membrane enzyme is widely distributed in animals and is thought to play a major role in the γ-glitamyl cycle. In particular, in a person suffering from various liver diseases such as cirrhosis, the value of γ-GTP activity in the liver and serum is higher than a healthy value. It is an essential laboratory test item for diagnosis. Generally, the value of γ-GTP activity in a clinical test is
It is determined using the measurement reagent. Conventional γ-G
The reagent for measuring TP activity includes one reagent containing glycylglycine (GlyGly) as an acceptor substrate for a γ-glutamyl group, and L-γ- as a donor substrate for a γ-glutamyl group.
Glutamyl-3-carboxy-4-nitroanilide (G
lu-CANA) or L-γ-glutamyl-4-nitroanilide (Glu-pNA). Then, these reagents are sequentially added to samples such as blood and serum to be measured, and γ-
The γ-GTP activity value of the sample is calculated by measuring a change in absorbance per unit time in a wavelength range of 400 to 450 nm of a substance produced by an enzyme reaction by GTP.

[0003]

However, in such a conventional method for measuring γ-GTP activity, when the sample to be measured is a hemolyzed sample containing an excessive amount of blood components, the sample is not allowed to coexist with a measuring reagent. There is a sign that the absorption peak around 400 nm in the wavelength range decreases with time,
As a result, the change in absorbance per unit time in the wavelength range of 400 to 450 nm, which is the measurement range of the γ-GTP activity, is apparently small, and thus the γ-G
There was a problem that the measured value of TP activity had a considerable negative error. This is because hemoglobin in the hemolyzed sample is γ
-It is speculated that the reaction is caused by inhibiting or suppressing the reaction between GTP and the reagent for measurement. The actual sample from which gamma-GTP activity is measured is often a hemolyzed sample, such as human or animal blood, serum or plasma. Therefore, in order to establish an accurate measurement with few errors in the above-mentioned method for measuring γ-GTP activity, it is absolutely necessary to remove the influence of hemoglobin in the sample.

Conventionally, for the purpose of preventing the influence of hemoglobin during the measurement of γ-GTP activity, a method of including thiourea in a reagent for measuring γ-GTP activity (Japanese Patent Publication No. 6-12998) or pyridine , Imidazoles or histamines are contained in a reagent for measuring γ-GTP activity (Japanese Patent Publication No. 3-56425) and proposed. However, these methods cannot sufficiently suppress the effects of hemoglobin, and their effects are still insufficient. In general, sodium azide may be added as a preservative to a reagent for measuring enzyme activity. In the above reagent for measuring γ-GTP activity, when sodium azide is added, γ-GTP
Our studies have shown that negative errors in activity measurements tend to be greater.

The present invention has been made in consideration of the above-mentioned conventional circumstances, and an object thereof is to solve the problem that a sample such as serum to be measured is not affected by hemoglobin contained therein. Another object of the present invention is to provide a reagent for measuring γ-GTP activity which can obtain an accurate measurement value of γ-GTP activity. In addition, the present invention provides a method of reacting a sample with a reagent for measuring γ-GTP
In the method of determining the γ-GTP activity of a sample by measuring the change in absorbance per unit time at ~ 450 nm, it is possible to accurately measure the γ-GTP activity value of the sample with little error without being affected by hemoglobin. Possible γ-
It is to provide a method for measuring GTP activity. Other objects of the present invention will be understood by referring to the description of the specification including the claims.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above technical problems, and as a result, an appropriate amount of an oxidase, such as peroxidase or catalase, was previously added to a reagent for measuring γ-GTP activity. Preferably, when the glycylglycine reagent of the γ-glutamyl receptor substrate is contained, γ-GTP
When the activity is measured, the absorption peak in the vicinity of the wavelength range of 400 nm of the hemolyzed sample does not substantially decrease over time in the presence of the measurement reagent, and therefore, the substance generated by the transfer reaction by γ-GTP is not observed. Wavelength range 400-450n
In the method for determining the enzyme activity by measuring the change in absorbance per unit time at m, the fact that the γ-GTP activity value of a sample can be accurately measured without being affected by hemoglobin in the sample. I found it. further,
The present inventors presuppose that a preservative, that is, sodium azide, in addition to the oxidizing enzyme was previously contained in the reagent for measuring γ-GTP activity, and in the above-described method for measuring γ-GTP activity using the reagent, However, the present inventors have found that even a hemolyzed sample having a high hemoglobin concentration can measure the γ-GTP activity value of the sample even more accurately, and completed the present invention.

Accordingly, the present invention provides a reagent for measuring γ-GTP activity comprising a first reagent containing a γ-glutamyl group acceptor substrate and a second reagent containing a γ-glutamyl group donor substrate. An oxidase or an oxidase and a preservative are mixed with the first reagent, the second reagent or both reagents, or a third reagent containing the oxidase or the oxidase and the preservative is provided. Which relates to a reagent for measuring γ-GTP activity. More specifically, the reagent for measuring γ-GTP activity of the present invention comprises: 1) glycylglycine (Gl) as a receptor substrate for γ-glutamyl group.
yGly) and L-γ-glutamyl-3-carboxy- as a donor substrate for the γ-glutamyl group.
A second reagent containing 4-nitroanilide (Glu-CANA), L-γ-glutamyl-4-nitroanilide (Glu-pNA) or a salt thereof, and an oxidase such as peroxidase or catalase;
Alternatively, such an oxidase and a preservative (sodium azide) are blended with the first reagent, the second reagent or both reagents, or 2) the first reagent and the second reagent described above. A reagent and a third reagent comprising an oxidase such as peroxidase or catalase or an oxidase and a preservative (sodium azide) (ie, three reagent kits). Further, the present invention provides a method for measuring γ-GTP activity using the above reagent, that is,
Reacting with an activity measurement reagent in the presence of an oxidase such as peroxidase or catalase or an oxidase and a preservative;
It also relates to a method for determining the γ-GTP activity of a sample by measuring the change in absorbance per unit time at 50 nm.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The measuring reagent and the measuring method according to the present invention can be effectively used for measuring γ-GTP activity using a sample derived from a living body such as blood, serum or plasma of a human or animal as a specimen. Can be applied. In particular, the present invention is a sample susceptible to hemoglobin,
That is, it exhibits an excellent effect in measuring γ-GTP activity in serum and plasma. Γ-G according to the present invention
Except for the addition of the oxidizing enzyme and the preservative, the reagent for measuring the TP activity can be applied to the conventional preparation technique, particularly the compounding technique for the first and second reagents. That is, GlyGl is used as a receptor substrate for a γ-glutamyl group.
The first reagent containing y, and the second reagent containing Glu-CANA, Glu-pNA or a salt thereof as a donor substrate of the γ-glutamyl group, also contain other components other than the oxidase and the preservative. The composition may be the same as the conventional one, and it is also possible to add a conventional component such as a buffer, a stabilizer, an activator, a preservative and the like at the same time. Therefore, these reagents can be prepared, for example, in a composition according to the method recommended by the Japanese Society for Clinical Chemistry. As the oxidizing enzyme used in the present invention, peroxidase, catalase or a combination thereof is suitable. As peroxidase, a purified product obtained from, for example, horseradish is used, and as catalase, a purified product obtained from, for example, a microorganism or bovine organ is used. Peroxidase is usually used in the range of 1 to 10 U / mL, and its preferable amount is 2.5 to 5.0 U / mL, depending on the hemoglobin concentration in the sample. Catalase is usually used in the range of 1000 to 10000 U / mL, although it also depends on the hemoglobin concentration in the sample, and the preferable use amount is 2000 to 6000 U / mL.
mL. In addition, the method for measuring γ-GTP activity according to the present invention employs a conventionally used method for measuring γ-GTP activity, except that an oxidase or an oxidase and a preservative are added to a reagent for the measurement in advance. Techniques and procedures can be applied as well. Therefore, for example, automatic measurement of γ-GTP activity of a sample using an automatic analyzer conventionally used is also included in the scope of the present invention.

[0009]

The present invention will be further clarified by describing examples which are considered to be the best embodiments of the present invention.

(Sample) Based on the control serum, and
To this, a hemolytic hemoglobin (Hb) solution adjusted to an appropriate concentration by purified water is added at a ratio of 9: 1 by volume, so that the hemoglobin (Hb) concentration is 0, 100, or 20.
6 consisting of 0, 300, 400 and 500 mg / dL
Different types of samples were prepared. These six identical samples were used in all examples.

(Procedure for measuring γ-GTP activity) In each of the following examples, γ-glutamyl receptor substrate Gl
A reagent 1 containing yGly and a reagent 2 containing a donor substrate Glu-CANA for a γ-glutamyl group were prepared. Reagent 1
Is added with peroxidase (Examples 1 and 2) or catalase (Examples 3 and 4) as an oxidase, or sodium azide is added in addition to the oxidase (Examples 2 and 4). In each case, reagent 1 was used for comparison, except that oxidase and preservative were not added.
A reagent 1a having the same composition as in Example 1 was prepared. Γ-GTP of each sample
The activity was measured using a Hitachi 7170 automatic analyzer (manufactured by Hitachi, Ltd.) according to the following procedure. In each case, first add 90 μL to a 7 μL sample.
Of reagent 1 (or reagent 1a) is added and the mixture is
Reaction at 7 ° C. for 5 minutes, followed by 270 μL of reagent 2
Is added and the mixture is reacted at 37 ° C. One to four minutes after the start of the reaction,
The absorbance at 05 nm (main wavelength) and 505 nm (sub wavelength) is measured. Then, the change in absorbance per minute was obtained from the measured value, and the γ-GTP specific activity of each sample was calculated based on the K factor obtained in advance.

Example 1: Avoiding the effect of hemoglobin by peroxidase (POD) As can be seen from Table 1, γ-GTP to which POD was added
The activity measurement reagent was γ-GT to which POD was not added.
Unlike the reagent for measuring P activity, not only in the sample not containing hemoglobin (Hb) but also in the sample in which it is present at a high concentration, there is no influence of hemoglobin (Hb).
γ-GTP activity can be accurately measured.

Example 2: Avoiding the effect of hemoglobin with peroxidase (POD) and sodium azide From the comparison between Table 1 and Table 2, it can be seen that when sodium azide coexists in the reagent for measuring γ-GTP activity without POD, the effect of hemoglobin (Hb) on the γ-GTP activity value is considerably increased. Nevertheless, quite surprisingly, in the reagent for measuring γ-GTP activity to which POD was added together with sodium azide, the effect of hemoglobin (Hb) was eliminated at all, and hemoglobin (Hb) was concentrated at a high concentration. Γ-
It can be seen that accurate measurement of GTP activity becomes possible.

Example 3: Avoiding the effect of hemoglobin by catalase (CAT) As can be seen from Table 3, γ-GTP to which CAT was added
The activity measurement reagent was γ-GT to which CAT was not added.
Unlike the reagent for measuring P activity, not only in the sample not containing hemoglobin (Hb) but also in the sample in which it is present at a high concentration, there is no influence of hemoglobin (Hb).
γ-GTP activity can be accurately measured.

Example 4: Avoiding the effect of hemoglobin by catalase (CAT) and sodium azide From the comparison between Table 3 and Table 4, it can be seen that the effect of hemoglobin (Hb) on the γ-GTP activity value is significantly increased when sodium azide is used in the CAT-free γ-GTP activity measurement reagent. Nevertheless, surprisingly, in the reagent for measuring γ-GTP activity to which CAT was added together with sodium azide, the effect of hemoglobin (Hb) was almost eliminated, and the hemoglobin (Hb) was concentrated at a high concentration. Γ-
It can be seen that accurate measurement of GTP activity becomes possible.

[0016]

As described above, according to the present invention,
That is, by using the reagent for measuring γ-GTP activity of the present invention, an accurate measurement value of γ-GTP activity can be obtained for a sample such as serum to be measured without being affected by hemoglobin contained therein. Is obtained. In another aspect, according to the measurement method of the present invention, that is, a sample is reacted with the reagent for measuring γ-GTP activity of the present invention, and a change in absorbance per unit time in a wavelength range of 400 to 450 nm of a substance to be generated is obtained. Γ-GTP by measurement
In the method for determining the activity, the effect is obtained that the γ-GTP activity value of the sample can be accurately measured with little error without being affected by hemoglobin. Therefore, the measuring reagent and the measuring method according to the present invention have an advantage that they can contribute to accurate diagnosis of liver disease in clinical tests on humans and animals.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Tamotsu Goda 3-5 Yarokunai Inada, Kasama City, Ibaraki Pref.

Claims (5)

[Claims] 1. A method for measuring γ-glutamyl transpeptidase activity comprising a first reagent containing a γ-glutamyl group acceptor substrate and a second reagent containing a γ-glutamyl group donor substrate. An enzyme or an oxidase and a preservative are mixed with the first reagent, the second reagent or both of them, or a third reagent containing the oxidase or the oxidase and the preservative is provided. A reagent for measuring γ-glutamyl transpeptidase activity, characterized in that: 2. The acceptor substrate is glycylglycine, and the donor substrate is L-γ-glutamyl-3-carboxy-4-nitroanilide, L-γ-glutamyl-
The reagent for measuring γ-glutamyl transpeptidase activity according to claim 1, which is 4-nitroanilide or a salt thereof. 3. The method according to claim 2, wherein the oxidase is peroxidase, catalase or a combination thereof.
The reagent for measuring γ-glutamyl transpeptidase activity according to claim 1 or 2. 4. The reagent for measuring γ-glutamyl transpeptidase activity according to claim 1, wherein the preservative is sodium azide. 5. A method for reacting a sample with a reagent for measuring γ-glutamyl transpeptidase activity and producing a substance having a wavelength range of 4.
In a method for determining the γ-glutamyl transpeptidase activity of a sample by measuring the change in absorbance per unit time at 00 to 450 nm, the reaction between the sample and the reagent is carried out in the presence of an oxidase or an oxidase and a preservative. A method for measuring γ-glutamyl transpeptidase activity, characterized in that: