JP3446197B2 - Reagent for measuring γ-glutamyl transpeptidase activity - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a γ-glutamyl transpeptidase (hereinafter abbreviated as γ-GTP) activity measuring reagent. More specifically, it relates to stabilization of L-γ-glutamyl-3-carboxy-4-nitroanilide or a salt thereof used as a substrate in the measurement of γ-GTP activity.

[0002]

2. Description of the Related Art γ-GTP is an enzyme having a function of degrading a γ-glutamyl transpeptide and transferring a γ-glutamyl group, which is a by-product thereof, to another peptide or L-amino acid. γ-GTP is widely distributed in the body,
It is known that various liver diseases increase the activity in the liver and serum.

In 1995, the Japan Society for Clinical Chemistry established γ-GTP.
Published JSCC recommended method for measuring activity, L-γ
Since -glutamyl-3-carboxy-4-nitroanilide has better solubility than L-γ-glutamyl-p-nitroanilide, it was approved as a substrate for γ-GTP activity measurement. Therefore, in the measurement of γ-GTP activity, it will be mainstream to use L-γ-glutamyl-3-carboxy-4-nitroanilide as a substrate in the future.

[0004] gamma-GTP activity determination reagent is usually a first reagent comprising L-.gamma.-glutamyl-3-carboxy-4-nitroanilide as a substrate, a buffer containing an amino acid or peptide, such as glycylglycine It is composed of a two-reagent system of a certain second reagent . The problem with storing these reagents in solution is that the substrate L-γ-glutamyl-
Quality stability of 3-carboxy-4-nitroanilide over time. L-γ-glutamyl-3-carboxy-4
-Nitroanilide has a problem that it decomposes even in the absence of an enzyme during storage in a solution (hereinafter, this is referred to as non-enzymatic decomposition) to release 3-carboxy-4-nitroaniline. . As a way to solve this problem,
L-.gamma.-glutamyl-3-carboxy which is a substrate-4-
A method has been proposed in which a transition metal or a salt thereof is allowed to coexist with nitroanilide (JP-A-6-327498).

However, the transition metal is L-γ-glutamyl-
Although it has the effect of stabilizing 3-carboxy-4-nitroanilide with time, it inhibits γ-GTP activity, so that a chelating agent such as EDTA is added to a buffer solution (second reagent) containing no substrate. There is a need. In addition, when transition metals such as copper, nickel, and zinc are discharged as a waste liquid, problems such as environmental pollution may occur.

[0006]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention As a reagent for measuring γ-GTP activity which has solved these problems, the present inventors
In a L-.gamma.-glutamyl-3-on-carboxy-4-nitroanilide in the coexistence of a quaternary ammonium salt
More and inhibited non-enzymatic degradation of L-.gamma.-glutamyl-3-carboxy-4-nitroanilide or a salt thereof .gamma.
Developed a reagent for measuring GTP activity, Japanese Patent Application No. 7-325571
As for the issue, we have already applied for a patent. However , the addition of a quaternary ammonium salt increases the cost, and the carbon chain has a C
_{When 15} or more quaternary ammonium salts were added, the solubility of the quaternary ammonium salts was lowered during storage under refrigeration, and some of the quaternary ammonium salts were sometimes precipitated. The turbidity due to this precipitation may cause variations in the absorbance, or the initial absorbance may increase to narrow the measurement range and reduce the measurement sensitivity. The present invention was made in view of the above circumstances, gamma-GTP
Non-enzymatic degradation of L-γ-glutamyl-3-carboxy-4-nitroanilide used as a substrate in activity measurement is suppressed to improve stability over time, enzyme activity is not inhibited, and the cost is low. Manufacturable γ-GTP
It is intended to provide an activity measuring reagent.

[0007]

The present invention provides a γ-glutamyl transpeptidase activity measuring reagent using L-γ-glutamyl-3-carboxy-4-nitroanilide or a salt thereof as a substrate. A stabilized γ-GTP activity measuring reagent, characterized in that at least one substance selected from alkaline earth metal ions and salts thereof is allowed to coexist.

The reagent for measuring γ-GTP activity of the present invention comprises a first reagent containing L-γ-glutamyl-3-carboxy-4-nitroanilide as a substrate and a buffer containing an amino acid or peptide such as glycylglycine. is composed of two-reagent system of the second reagent is. Then, an alkali metal ion, an alkaline earth metal ion or a salt thereof is allowed to coexist in a liquid containing L-γ-glutamyl-3-carboxy-4-nitroanilide or a salt thereof used as a substrate. Them into coexist substrate solution include lithium, sodium, potassium, rubidium, cesium, alkali metals and beryllium francium such as magnesium, calcium, strontium, barium, alkaline earth metal odor acids such as radium, hydrochloric acid , Carbonic acid, phosphoric acid,
It is reacted with an acid such as fluoric acid , hypochlorous acid, nitric acid, lactic acid, sulfuric acid, acetic acid, oxalic acid, citric acid, iodine, formic acid, phthalic acid, etc. , and added to the substrate solution in the form of an appropriate salt. As a preferable metal, lithium or magnesium is used, and for example, lithium bromide, lithium chloride, lithium carbonate, lithium phosphate, lithium oxide, lithium fluoride, lithium hypochlorite, lithium nitrate, lithium lactate, lithium sulfate, Lithium acetate, lithium oxalate, lithium citrate, lithium iodide, lithium formate, lithium phthalate, magnesium bromide, magnesium chloride, magnesium carbonate, magnesium phosphate, magnesium oxide, magnesium fluoride, magnesium hypochlorite, magnesium nitrate , Magnesium lactate, magnesium sulfate, magnesium acetate, magnesium oxalate, magnesium citrate, magnesium iodide, magnesium formate,
Examples thereof include magnesium phthalate. These alkali metals, alkaline earth metal ions may be used alone, or in combination of two or more.

[0009] alkali metal ions used in the present invention, the amount of alkaline earth metal ions or their salts are preferably added per substrate 1mM 0.01mM~1M. This can not be sufficiently suppressed non-enzymatic degradation and an amount less than 0.01 mM, tend not stability is obtained, the positive charge in the reaction that the amount in excess of 1M are It tends to increase and affects the γ-GTP activity value.

The buffer solution of the present invention has a buffer concentration range of 10 to 1000 mM and a pH of 7.5 to 9.0.
Those in the range of are adopted. As such a buffer,
A Good's buffer is preferably used. For example, 2-morpholinoethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-T
ris), N- (2-acetamido) iminodiacetic acid (ADA)
, Piperazine-N, N-bis (2-ethanesulfonic acid) (PIP
ES), 1,3-bis tris (hydroxymethyl) -methylaminopropane (Bistrispropan), N-2- (acetamido)-
2-aminoethanosulfonic acid (ACES), 3- (monophorino) propanesulfonic acid (MOPS), bis (2-hydroxyethyl)-
2-aminoethanesulfonic acid (BES), tris (hydroxymethyl) methyl-2-aminomethanesulfonic acid (TES), hydroxyethylpiperazine-2-ethanesulfonic acid (HEPE
S), N-tris (hydroxymethyl) methyl-methylglycine (Tricine), tris (hydroxymethyl) aminomethane (Tris), N, N-bis- (2-hydroxyethyl) glycine (Bicine), glycylglycine ( Glycylglycine), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPAS), glycine (Glycine), cyclohexylaminopropanesulfonic acid (CAPAS) and the like.

[0011]

[Example 1]

To investigate the inhibitory effect of non-enzymatic degradation of L-.gamma.-glutamyl-3-carboxy-4-nitroanilide using the additives shown below, we were simulated test by acceleration. The values for the following additives are the substrate preparation liquid 6
Indicates the concentration added to mM.・ Choline chloride 0.358 M ・ Lithium chloride 0.125 to 0.5 M ・ Magnesium chloride 0.125 to 0.5 M Test method L-γ-glutamyl-3-carboxy-4-nitroanilide was dissolved in purified water to a solution adjusted to 6 mM, The above-mentioned three kinds of additives were added in the respective concentrations described above. Further, after the adjustment with purified water as a comparison, it was absorbance measurements for those additives not added. The pH of each substrate adjustment solution was adjusted. Evaluation method These adjusted four kinds of solutions were enclosed in a vial bottle shielded from light and the absorbance at 405 nm of a spectrophotometer was measured. Thereafter, as a simulation by acceleration, the sample was stored in a thermostat at a temperature of 25 ° C. for 15 days, and the absorbance at 405 nm of each substrate preparation solution was measured with time to obtain L-γ-glutamyl-3-carboxy-. It was examined the inhibitory effects of non-enzymatic degradation of 4-nitroanilide. The results of this measurement are shown in Table 1.

[0012]

[Table 1]

According to Table 1, in the case of no addition to the substrate solution, the absorbance change ΔABS was 0.6568, whereas that of lithium chloride
Alternatively, the one to which magnesium chloride is added has a lower ΔABS value than that of the one without magnesium chloride, and it is clear that non-enzymatic degradation is suppressed. In particular, the addition of 0.05 M magnesium chloride showed ΔABS of 0.3817, indicating that the decomposition of the substrate was effectively suppressed. It should be noted that the addition of choline chloride (reference example)
Matrix degradation has been effectively suppression.

Example 2 Next, in order to examine the inhibitory effect on non-enzymatic decomposition in the actual reagent composition, 3 lots of each of the reagents shown below were prepared and stored in a refrigerator at 4 ° C. It examined the inhibitory effect of non-enzymatic degradation of when I went to. Reagent (1) (1st reagent) 230 mM Glycylglycine buffer pH 7.9 (2nd reagent) 18 mM γ-glutamyl-3-carboxy-4-nitroanilide 0.125M to 0.5M lithium chloride reagent (2) (1st reagent) ) 230 mM glycylglycine buffer pH 7.9 (second reagent) 18 mM γ-glutamyl-3-carboxy-4-nitroanilide 0.716M choline chloride test method Add 266 μl of the first reagent to 8 μl of physiological saline, and add 5 μl at 37 ° C.
After preheating for 1 minute, 133 μl of the second reagent was added and stirred, and the initial absorbance at a main wavelength of 405 nm and a sub wavelength of 700 nm was measured. An automatic analyzer (Hitachi 7150) is used for measuring the absorbance.
It was used. The measurement results are shown in Table 2.

[0015]

[Table 2]

From Table 2, in the long-term refrigeration storage, the reagent (1) containing lithium chloride was the reagent containing choline chloride.
Compared to (2), the increase in absorbance is suppressed and the substrate γ
-The non-enzymatic degradation of glutamyl-3-carboxy-4-nitroanilide was suppressed.

Example 3 A reagent having the following composition was prepared,
The γ-GTP activity of the control serum was measured. (1st reagent) 230 mM glycylglycine buffer pH 7.9 (2nd reagent) 18 mM L-γ-glutamyl-3-carboxy-4-nitroanilide 0.125 to 0.5M lithium chloride Test method Two types of control serum (Monitrol IX, Monitrol IIX: manufactured by Kokusai Reagent), add 266 μl of the first reagent to 8 μl, and add 5
After pre-warming for 1 minute, add 133 μl of the second reagent and stir.
The amount of change in absorbance per unit time at 5 nm (main wavelength) and 700 nm (sub wavelength) was measured. The enzyme activity value of each serum was determined based on the amount of change in the absorbance of the standard serum that was obtained at the same time. The measurement was performed with an automatic analyzer (Hitachi 7150). The results are shown in Table 3.

[0018]

[Table 3]

From Table 3, the addition of 0.5 M lithium chloride did not affect the measured γ-GTP activity.

[0020]

INDUSTRIAL APPLICABILITY As in the present invention, at least one substance selected from alkali metal ions, alkaline earth metal ions and salts thereof is added to L-γ-glutamyl-3-carboxy-4-nitroanilide which is a substrate. By making them coexist, it is possible to effectively suppress the non-enzymatic decomposition of the substrate during storage in a solution state, and moreover, it is possible to perform accurate measurement with little influence on the measured value. Also,
Alkali metals and alkaline earth metals are economically cheaper than conventional additives and have relatively low toxicity, so they are also advantageous when treating waste liquids.

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Claims (3)

(57) [Claims] 1. L-γ-glutamyl-3-carboxy-
Γ using 4-nitroanilide or its salt as a substrate
-A reagent for measuring γ-glutamyl transpeptidase activity, which is characterized in that in a reagent for measuring glutamyl transpeptidase activity, at least one substance selected from lithium ion, magnesium ion and salts thereof coexists as a substrate. 2. A substrate 1mM per, lithium ion, Ma
The reagent for measuring γ-glutamyl transpeptidase activity according to claim 1, which comprises 0.01 mM to 1 M of at least one substance selected from gnesium ions and salts thereof. 3. Lithium ion, magnesium ion and
And at least one substance selected from salts thereof,
The γ-group according to claim 1 or 2, which contains lithium ions.
Retamyl transpeptidase activity measurement reagent.