JPH05317094A - Method for determining activity of n-acetylhexosaminidase - Google Patents

Abstract

PURPOSE:To sensitively and accurately determine the subject enzyme in a simple operation and high specificity without being influenced by a coexisting substance and contribute to early discover of a renal disease by a specific method using N-acetylhexosaminin oxidase. CONSTITUTION:N-Acetylhexosaminide of the formula (R is alkyl, phenyl, etc.) is made to react with a sample (e.g. urine or serum) containing N- acetylhexosaminidase(NAG) to produce N-acetylhexosamine. Further, the N- acetylhexosamine is made to react with N-acetylhexosamineoxydase(NAHOD) and the produced hydrogen peroxide or the consumed oxygen is measured. When NAG or NAHOD reacts, a buffer capable of keeping pH to 4-10 is preferably together used and the reaction temperature is preferably kept to 20-50 deg.C. Furthermore, hydrogen peroxide is measured by e.g. hydrogen peroxide electrode.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention is directed to samples, especially N in aqueous liquids.
-A method for quantifying acetylhexosaminidase activity. BACKGROUND ART In recent years, in the field of clinical medicine, the quantification of various chemical substances and enzyme activities in body fluids has become more important in clinical significance as they provide useful diagnostic information. In particular, the enzymatic determination of N-acetylhexosamine in body fluids provides information on many sugar-related compounds and enzyme activity. Examples of these target chemical substances and enzymes include N-acetylhexosaminidase, muramidase, β-glucuronidase, sialic acid, sialidase, and mucopolysaccharide. The clinical significance of these is that the measurement of N-acetylhexosaminidase activity is for the early detection of renal diseases, especially proximal tubular injury, renal transplant rejection, and the measurement of muramidase activity is for acute monocytic leukemia. For the diagnosis of atypical tuberculosis, etc., the measurement of β-glucuronidase activity is for the diagnosis of urinary tract malignant tumor, bladder cancer, etc., the determination of sialic acid is for the diagnosis of inflammatory diseases, etc., and the measurement of sialidase activity is for bacterial infection. The measurement of mucopolysaccharides provides useful information for the diagnosis of mucopolysaccharidosis.

[0002]

Conventionally, N-acetylhexosaminidase (β-N-acetyl-D-hexosaminidase, hereinafter referred to as N-acetylhexosaminidase
The activity (abbreviated as AG) is that p-nitrophenol is released by allowing NAG to act using a chromogenic substrate in which a chromophore such as p-nitrophenol is bound to the reducing end of N-acetyl-D-glucosamine. It is common to compare the chromophores such as
Enzymol., 28, 702 (1971), Clinca Chimica et Acta 6
9 (1), 85-91 (1976)). However, in this method, the optimum pH of the target enzyme NAG (pH 4 to 5.5) and the color development pH of the chromophore p-nitrophenol (pH 9 or higher).
Therefore, in order to measure NAG activity, it is necessary to carry out the enzyme reaction and the color reaction separately. Therefore, the number of reagents and the number of operating steps are required, and it is the most suitable for obtaining the enzyme activity. There is a drawback that the so-called rate analysis method cannot be used. In addition, a method using a substrate in which phenol is bound to N-acetylglucosamine (JP-A-54-60997), a method in which a substrate in which m-cresolsulfoisophthalein is bound to N-acetylglucosamine is used (Clin. Chem. ., 29, 1713 (19
83)) also has the same drawbacks as in the case of using p-nitrophenol.

[0003]

In consideration of these circumstances, the present inventors have made various studies in order to find an accurate, simple and rapid method for quantifying sugar-related enzymes, and as a result, N-acetylhexosa The present inventors have found that a method for quantifying N-acetylhexosaminidase activity using minin oxidase (hereinafter abbreviated as NAHOD) achieves the above object, and thus arrived at the present invention.

[0004]

That is, the present invention provides N-acetylhexosaminide represented by the following formula (I) with N-acetylhexosaminide.
N-characterized by reacting a sample containing acetylhexodaminidase, reacting N-acetylhexosamine produced with N-acetylhexosamine oxidase, and measuring hydrogen peroxide produced or oxygen consumed
-A method for quantifying acetylhexosaminidase activity.

[0005]

[Chemical 2] (In the formula, R represents an alkyl group, a phenyl group, a naphthyl group, or a group having a nitro group as a substituent.)

The measurement principle of the present invention is as follows.

[Chemical 3]

Examples of the N-acetylhexosaminide represented by the above formula (Formula 1) in the present invention include methyl-
N-acetyl-β-D-glucosaminide, phenyl-N
-Acetyl-β-D-glucosaminide, p-nitrophenyl-N-acetyl-β-D-glucosaminide, methyl-N-acetyl-β-D-galactoaminide, phenyl-N-acetyl-β-D-galactosaminide, p- Examples thereof include nitrophenyl-N-acetyl-β-D-galactosaminide.

The sample containing N-acetylhexodaminidase (NAG) in the present invention is a sample containing body fluids such as urine and serum, and N-acetylhexosamine in the present invention is a substrate represented by the above formula, It is N-acetylhexosamine produced by allowing a sample containing NAG to act on N-acetylhexosaminide.

The NAHOD used in the present invention may be of any origin, for example, NAHOD produced by a microorganism belonging to the genus Pseudomonas. NAHOD
The refining and properties of sucrose have been announced at the Japan Society for Agricultural Chemistry 1983 conference.

The conditions for causing the N-acetylhexosaminide to act on the sample containing NAG and allowing the resulting N-acetylhexosamine to act on NAHOD are not particularly limited, but pH 4.0 to 10.0 is used. A reaction temperature of 20 to 50 ° C. is suitable, using a suitable buffer to maintain. The type of buffer solution is not particularly limited. The optimum buffer solution for the pH used may be selected. For example, there are a phosphate buffer solution in the neutral range, an acetate buffer solution in the acidic range, and a Tris-HCl buffer solution in the alkaline range. The concentration of the buffer solution is not particularly limited, but is usually about 0.01M to 1M. The reaction method may be a one-step method in which the N-acetylhexosaminidase reaction and NAHOD reaction are carried out simultaneously, or a two-step method in which the N-acetylhexosaminidase reaction is carried out first and then the NAHOD reaction is carried out. If necessary, a surfactant and the like may be added to the reaction system.

In the present invention, the produced hydrogen peroxide is measured by coupling it with, for example, peroxidase or catalase, or directly by a hydrogen peroxide electrode. In the measurement system coupled with perosidase, a system using 4-aminoantipyrine and a phenol derivative or an aniline derivative is generally preferable.

Ascorbic acid in the sample that interferes with the determination of hydrogen peroxide is ascorbic acid oxidase (hereinafter referred to as ASO).
Abbreviated). In this case, ASO may be allowed to act prior to the other enzymatic reaction, or may be conjugated with the other enzymatic reaction to act simultaneously and removed. The ASO may be of microbial origin or plant origin, with plant origin such as pumpkin and cucumber being particularly preferred. The amount of oxygen consumed in the HAHOD reaction system can be measured by, for example, an oxygen electrode.

[0013]

INDUSTRIAL APPLICABILITY According to the present invention, N-type in a sample can be accurately and accurately obtained by a simple operation with high sensitivity and without being influenced by coexisting substances.
It becomes possible to quantify the acetylhexosaminidase (NAG) activity, which is extremely significant in the diagnostic field of clinical examination.

[0014]

EXAMPLES Next, the present invention will be described with reference to examples. The abbreviations in the examples indicate the following. NAHOD (N-acetylhexosamine oxidase) 4-AA (4-aminoantipyrine) TOOS (N-ethyl-N- (2-hydroxy-3-)
Sulfopropyl) -m-toluidine sodium)

[0015]

Example The amount of N-acetylhexosaminidase activity in a test solution was measured by the following method using the following reagents. 1. Reagents p-nitrophenyl-N-acetyl-β-D-glucosaminide 1 mM sodium chloride 200 mM N-acetylhexosamine oxidase 50 U / ml peroxidase 30 U / ml TOOS 1 mM 4-AA 0.5 mM 0.05M citrate buffer (pH 5.5). ) 2. Measurement method 2 ml of the above reagent was added to 50 μl of NAG-containing test solution, and 3
The reaction was carried out at 7 ° C., and the absorbance was measured at a wavelength of 550 nm to obtain the color development rate. The reaction curve is shown in FIG. The calibration curve is shown in FIG. As is apparent from FIGS. 1 and 2, the method of the present invention can accurately and easily perform NAG rate assay in a short time.

[Brief description of drawings]

1 shows a reaction curve in Example 1. FIG.

FIG. 2 shows a calibration curve in Example 1.

Claims (1)

[Claims] 1. A sample containing N-acetylhexosaminidase is allowed to act on N-acetylhexosaminide represented by the following formula (I), and the N-acetylhexosamine thus produced is subjected to N-acetylhexosamine oxidase. A method for quantifying N-acetylhexosaminidase activity, which comprises: reacting with hydrogen peroxide to measure hydrogen peroxide produced or oxygen consumed. [Chemical 1] (In the formula, R represents an alkyl group, a phenyl group, a naphthyl group, or a group having a nitro group as a substituent.)