JPS6225360B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to γ-glutamyl transpeptidase (hereinafter referred to as γ-GTP) which uses a novel compound in which a substituent is introduced into γ-glutamylanilide as a substrate.
(abbreviated as )) relates to quantitative reagents. [Prior art] γ-GTP is converted into glutathione and other γ-GTP in vivo.
It is an enzyme that transfers the γ-glutamyl group from glutamyl peptide to receptors for amino acids, peptides, etc., and is present in large amounts in various organs such as the liver, kidney, and pancreas. Clinically, increased γ-GTP activity is observed in liver diseases such as obstructive jaundice, chronic active hepatitis, liver cancer, and alcoholic hepatitis. It is said that there is a relationship between the transition to the liver and increased production in liver tissues damaged by chronic active lesions. Therefore, gamma-GTP activity measurements are now increasingly being performed in clinical laboratories for the diagnosis of liver diseases. Conventionally, L-γ-glutamyl-p-nitroanilide has been generally used as a substrate for quantitative measurement of γ-GTP, but it is well known that this has a serious drawback in substrate solubility. Therefore, this drawback is usually solved by adding dilute hydrochloric acid, a surfactant, and an organic solvent to dissolve the substrate. [JP-A-52-29799] [Problems to be Solved by the Invention] However, even in this case, the stability is extremely poor due to self-decomposition after dissolving the substrate and bubbles generated due to the addition of a surfactant. Therefore, L-γ-glutamyl-
In order to overcome the above-mentioned drawbacks, it is desired to produce a new substrate that is more soluble in water than p-nitroanilide. The substrate for γ-GTP measurement described in JP-A No. 49-86338 is L-γ-glutamyl-3-carboxy-4-nitroanilide obtained by introducing a carboxyl group into the 3-position of p-nitroaniline. It is stated that solubility could be improved by introducing sulfonic acid into the 3-position of p-nitroaniline to obtain L-γ-glutamyl-3-sulfonic acid-4-nitroanilide. However, although this substrate is suitable for automatic analysis using the initial velocity analysis method, commonly performed colorimetric measurements require the use of a measurement wavelength of around 400 nm, which may be affected by bilirubin, etc., present in living organisms. Not suitable for receiving. Therefore, it is still desired to develop a substrate with good solubility that is suitable for general use for quantitative measurement of γ-GTP activity. [Means for Solving the Problems] As a result of research to solve the problems, the present inventor selected γ with excellent solubility from various compounds in which substituents were introduced into L-γ-glutamylanilide.
-A new compound for quantitative measurement of GTP was discovered. That is, the present invention relates to the following structural formula [] or [] (In the formula, R is L-γ-glutamyl group:

Represents [formula]. ) This is a reagent for quantifying γ-GTP, which is characterized by using a compound represented by the following as a substrate. The substrate according to the present invention is characterized in that a substituent is introduced into L-γ-glutamylanilide. Specifically, they are L-γ-glutamylanilide with a carboxyl group and a hydroxyl group introduced, and L-γ-glutamyl-4-carboxyanilide with glycine amide bonded. The novel compound thus obtained is L-γ-glutamyl-3-hydroxy-4-carboxyanilide or L-γ-glutamyl-4-carboxyglycinamide anilide. The reagent for quantifying γ-GTP of the present invention comprises the above-mentioned substrate and p-GTP generated from the substrate through the enzymatic action of γ-GTP.
It is basically composed of a color former of p-dimethylaminocinnamaldehyde which gives color to aminosalicylic acid or p-aminohippuric acid. The substrate according to the invention is a conventional γ-glutamyl-
The drawbacks of p-nitroanilide can be successfully overcome.
That is, the optimal substrate concentration required for γ-GTP quantification is about 6-7mM, whereas conventionally it is about 3-4mM.
The solubility was 15 mM, but in the present invention, it can be dissolved in water at a concentration of 15 mM or more without a solubilizing agent. Below, the solubility of the substrate according to the present invention and γ-GTP
Table 1 shows the decomposition rates of the substrates. The decomposition rate was expressed as a relative value when L-γ-glutamyl-p-nitroanilide was used as 100. (Measurement conditions are 37℃ for 15 minutes.)

【table】

[Table] [Effect] The present invention can fully replace conventional substrates. The advantage obtained is that substrate solutions of 100 mmol or more can be prepared. It is highly soluble in water and therefore does not require the addition of solubilizers. The rate of substrate decomposition by enzymes is high. Suitable for commonly used colorimetric measurements, etc. can be provided. Hereinafter, the preparation of a reagent for quantifying γ-GTP activity using the substrate according to the present invention will be explained with reference to Examples. Example 1 Process for producing L-γ-glutamyl-3-hydroxy-4-carboxyanilide p-aminosalicylic acid 8.1g (57.9 mmol)
and phthaloyl-L-glutamic anhydride 15.0g
(58.0 mmol) and add 150 ml of anhydrous dioxane.
Stir at 65°C for 1 hour. Then, it was distilled off under reduced pressure, dissolved in methanol (250 ml), and adjusted to pH 9.0 with 80% hydrazine hydrate. Leave at room temperature for 24 hours. The crystals were collected by filtration and suspended in 1N HCl (100 ml) to remove insoluble materials, and the filtrate was adjusted to pH 7.0 with 2N NH ₄ OH and evaporated under reduced pressure. The residue is crystallized by adding acetone, washed with ethanol, and dried under reduced pressure to obtain 10.2 g (62.4% of theory) of the desired product. Melting point 212-214℃ (decomposition), [α] ¹⁸⁰ = _+17.2 (C
= 0.5, 1N-HCl) Example 2 Process for producing L-γ-glutamyl-4-carboxyglycinamide anilide 10.0 g (51.5 mmol) of p-aminohippuric acid and 14.6 g (56.7 mmol) of phthaloyl-L-glutamic anhydride
Add 150 ml of anhydrous dioxane to 60℃
Stir for 1 hour. Then, the residue was distilled off under reduced pressure to obtain an oily product of phthaloyl-L-γ-glutamyl-4-carboxyglycinamide anilide. Dissolve this oil in methanol (250 ml) and adjust the pH to 9.0 with 80% hydrazine hydrate. Leave at room temperature for 2 days. The crystals are collected by filtration, washed with cold water and ethanol, and dried. Add 0.5N-HCl (200ml), stir vigorously at room temperature for 30 minutes, remove insoluble matter, and dilute the filtrate with 1N-Na ₂ CO ₃
Set the pH to 7.0. This is concentrated under reduced pressure and acetone is added to the residue to obtain 9.0 g (54% of theory) of the target compound. Melting point 244-246℃ (decomposition), [α] ²⁰ ₀ = +18.6 (C
= 1.0, 1N-HCl) Example 3 Reagent for quantifying γ-GTP (1) Reagent preparation L-γ-glutamyl-3-hydroxy-4
-Dissolve 2.27g of carboxyanilide, 6.05g of tris(hydroxymethyl)aminomethane and 11.88g of glycylglycine in purified water to reach a pH of 8.3.
~ After preparing in 8.4, the total volume was made to 1000 ml, and this was used as the substrate solution. p-dimethylaminocinnamaldehyde
After dissolving 900mg in 600ml of ethanol,
Add purified water to make a total volume of 1000ml and use it as the coloring solution. Use 0.3N hydrochloric acid as the reaction stop solution. A mixed solution is prepared by mixing a color developing solution and a reaction stop solution at a ratio of 2:1. A standard solution (400 mU/ml) is prepared by dissolving 0.918 g of 5-aminosalicylic acid in a 15 mM sodium hydroxide solution to make a total volume of 1000 ml. (2) Quantification method Dilute serum to 1/5 to 5/5 with normal saline, and dilute 20μ
After mixing 1 ml of the substrate solution and incubating at 37°C for 15 minutes, add 3 ml of a mixture of coloring solution and reaction stop solution. After standing for 5 minutes, absorbance is measured at a wavelength of 540 nm using a reagent blank as a control. The results are shown in Table 2. Example 4 Reagent for quantifying γ-GTP (1) Reagent preparation 1.97 g of L-γ-glutamyl-4-carboxyglycinamide anilide, 6.05 g of tris(hydroxymethyl)aminomethane, and 11.88 g of glycylglycine were dissolved in purified water. PH8.3～
After preparing in 8.4, make the total volume 1000ml and use it as the substrate solution. p-dimethylaminocinnamaldehyde
After dissolving 900mg in 600ml of ethanol,
Add purified water to make a total volume of 1000ml and use it as the coloring solution. Use 0.3N hydrochloric acid as the reaction stop solution. A mixed solution is prepared by mixing a color developing solution and a reaction stop solution at a ratio of 2:1. A standard solution (400 mU/ml) is prepared by dissolving 1.164 g of p-aminohippuric acid in 15 mM sodium hydroxide solution to a total volume of 1000 ml. (2) Quantification method Operate in the same manner as in Example 3-(2). The results are shown in Table 3.

【table】

【table】

Claims (1)

[Scope of Claims] 1. A γ-glutamyl trans characterized by comprising a substrate obtained by introducing a substituent into L-γ-glutamylanilide represented by the following structural formula [] or [], and p-dimethylaminocinnamaldehyde. Reagent for quantifying peptidase. Structural formula (In the formula, R is L-γ-glutamyl group:
Represents [formula]. )