JPH0710237B2 - N-acetylhexosaminidase activity assay method - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to N in a sample, especially an aqueous liquid.
-A method for quantifying acetylhexosaminidase activity. 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 providing 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, particularly 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 by 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 4-5.5) and the color development pH of p-nitrophenol (chromophore) (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 when using p-nitrophenol.

[0003]

In view 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-acetylhexosamine oxidase was found. The present inventors have found that a method for quantifying N-acetylhexosaminidase activity using (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-acetyl characterized in that a sample containing acetylhexosaminidase is allowed to act, N-acetylhexosamine produced is allowed to act with N-acetylhexosamine oxidase, and hydrogen peroxide produced or oxygen consumed is measured. This is a quantitative method of hexosaminidase 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-acetylhexosaminidase (NAG) in the present invention contains 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 reacting a sample containing NAG with 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 NAHOD to act on the produced N-acetylhexosamin 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. It suffices to select an optimum buffer solution for the pH used. 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 the 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 or 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 a sample that interferes with the determination of hydrogen peroxide is ascorbate oxidase (hereinafter referred to as ASO).
Abbreviated). In this case, ASO may be reacted prior to the other enzyme reaction, or may be conjugated with the other enzyme reaction and simultaneously acted to be removed. The ASO may be of microbial origin or plant origin, but is preferably of plant origin such as pumpkin and cucumber. 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 measured with a simple operation, with high sensitivity and without being influenced by coexisting substances.
It becomes possible to quantify acetylhexosaminidase (NAG) activity, which is extremely significant in the diagnostic field of clinical examination.

[0014]

EXAMPLES The present invention will now 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. Reagent p-nitrophenyl-N-acetyl-β-D-
Glucosaminide 1 mM sodium chloride
200 mM N-acetylhexosamine oxidase 50U / ml
Peroxidase
30U / ml TOOS
1mM 4-AA
0.5 mM 0.05
M citrate buffer (pH 5.5) 2. Measurement method To 50 μl of the NAG-containing test solution, 2 ml of the above reagent was added, reacted at 37 ° C., and the absorbance was measured at a wavelength of 550 nm to determine the color development rate. The reaction curve is shown in FIG. The calibration curve is shown in FIG. As is clear 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) to produce N-acetylhexosamine with N-acetylhexosamine oxidase. A method for quantifying N-acetylhexosaminidase activity, which comprises reacting hydrogen peroxide produced or measuring 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.)