JPS60186300A - Determination of n-acetylhexosamine - Google Patents

Abstract

PURPOSE:N-acetylhexosamine oxidase is allowed to act on a sample to be assayed in the presence of water and oxygen to determine the hydrogen peroxide formed or the oxygen consumed whereby accurate, rapid and simple determination of the titled compound becomes possible for early diagnosis of nephrectopathies. CONSTITUTION:N-Acetylhexosamine oxidase, which acts on N-acetylhexosamine in the presence of water and oxygen to form hydrogen peroxide, is allowed to act on a sample such as serum. And the hydrogen peroxide formed or the oxygen consumed are determined to know the amount of N-acetylhexosamine. The amount of the hydrogen peroxide formed is determined by allowing it to react with 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium to develop the color and comparing the absorbancy of the color at 550nm wavelength with the standard.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for the enzymatic determination of N-acetylhexosamine in samples, particularly in aqueous liquids.

In recent years, in the field of clinical medicine, various chemical substances in body fluids,
Quantification of enzyme activity is gaining increasing clinical significance as it provides useful diagnostic information. In particular, enzymatic determination of N-acetylhexosamine in body fluids provides information about many sugar-related compounds and enzyme activities. These target chemicals and enzymes include, for example, N
- Acetylhexosaminidase, muramidase, β-glucuronidase, sialic acid, sialidase, mucopolysaccharide, etc.0 These clinical significances include measuring U,N-acetylhexosaminidase activity in renal diseases, especially in the proximal tubule. Measurement of muramidase activity is used for early detection of kidney failure, kidney transplant rejection, etc. Measurement of muramidase activity is used for diagnosis of acute monocytic leukemia, atypical tuberculosis, etc. Measurement of β-glucuronidase activity is used for diagnosis of urinary tract malignancy, bladder cancer, etc. The determination of sialic acid provides useful information for the diagnosis of inflammatory diseases, the measurement of sialidase activity provides useful information for the diagnosis of bacterial infections, and the measurement of mucopolysaccharide provides useful information for the diagnosis of mucopolysaccharidoses.

Hitherto, there has been no suitable method for quantifying N-acetylhexozamine, and in particular, no reports have been found regarding enzymatic methods. Conventionally, therefore, the concentration of the above-mentioned compound and the enzyme activity have been determined by measuring other reaction products, but this method has been insufficient in terms of accuracy, simplicity, and speed. In other words, N-acetylhexosaminidase activity, β-glucuronidase activity, etc. can be determined by enzymatic reaction using a chromogenic substrate bound to a coloring cap such as p-nitrophenol. Because it measures activity, it is not quantitative and there is a large variation. For sialic acid, the chemical method, thiobarbic acid method, has low specificity and requires complicated operations. The amount of sialic acid can be determined by determining the amount of pyruvin oxygen, which is a sifted reaction product. The desired enzymatic method has problems with accuracy due to endogenous pyruvate. For sialidase, the Am1noff method is known, which measures the total amount of sialic acid released using sialyllactose as a substrate, but it has problems in terms of sensitivity, accuracy, and operability.

Mucopolysaccharides have drawbacks such as the lack of suitable quantitative methods.

Taking all of these circumstances into consideration, the present inventors have developed an accurate, fast, sugar-linked compound and l! After conducting various intensive studies in order to find eight quantitative methods, we found that a method for quantifying N-acetylhexosamine using an N-acetylhexosaminine oxidase (hereinafter abbreviated as NAHOD) tube achieves the above objectives. We have arrived at the present invention.

That is, the present invention relates to N-acetylhexosamine oxidase, which generates hydrogen peroxide by reacting N-acetylhexosamine in the presence of water and oxygen. This is a method for quantifying N-acetylhexosamine, which is characterized by acting on a sample and measuring the total amount of hydrogen peroxide produced or oxygen consumed.

The measurement principle of the present invention is explained below.

N-acetylhexosamine+H,O+ 01)"■ne4N-acetylhexosaminic acid 10H30,...
(1) In the present invention, the total amount of hydrogen peroxide produced or oxygen consumed by the above reaction is measured. Methods for measuring hydrogen peroxide include a method in which it is conjugated with peroxidase or catalase, and a method in which it is directly quantified using a hydrogen peroxide electrode. There is a method of quantifying total oxygen using an oxygen electrode.

In the present invention, N-cetylhexozamine includes N-
- Acetylglucosamine, N-acetylgalactosamine, N-acetylmuramic acid, etc.

N-acylglucosamine in the present invention may be fcN-acylglucosamine derived from a sugar linkage compound by a chemical or enzymatic method.

For example, there is N-acetylhexosamine, which is produced by subjecting N-acetylhexosaminide to the full action of N-acetylhexosaminidase. Sweet N-acetylglucosamine and N~
There are N-acetylglucosamine and N-7 cetylmuramic acid produced by muramidase and β-1,4-conjugates with ruacetylmuramin. Furthermore, the bonded sialic acid is reacted with neunyramidase to generate N-acetylneuraminic acid, and then the N-acetylneuraminic acid is reacted with neuraminic acid aldolase to produce N-acetylmannosamine and pyruvate? obtain. Next is N-acetylglucosamine, which is obtained by treating N-acetylmannosamine with acylglucosamine-2-x himerase.

The NAHOD used in the present invention may be of any origin, but for example, there is a NAHOD produced by a microorganism belonging to the genus Pseudomonas.The purification and properties of ONAI (01) were discussed at the 1988 Annual Meeting of the Japanese Society of Agricultural Chemistry. It has been announced.

All you need to do is select the optimal buffer. For example, a phosphate buffer is used in a neutral range, an acetate buffer is used in an acidic range, and a Tris-HCl buffer is used in an alkaline range. The concentration of the buffer solution is not particularly limited, but is usually about 0.01M to IM.

In addition, if necessary, a surfactant, etc. may be added in its entirety. Using the method for quantifying N-acetylhexozamine of the present invention,
Sugar linked compounds such as N-acedelhexosaminide,
The determination of sialic acid, mucopolysaccharides such as hyaluronic acid, chondroitin sulfate, dermaic acid, heparin, heparan sulfate, keratan sulfate, etc. is also OT capable. Enzyme activity preparations such as N-acetylhexozaminidase, noramidase, sialidase, etc. are also useful.

In the 1N invention, the hydrogen peroxide produced is measured by conjugating it with, for example, peroxytase or catalase, or directly by a hydrogen peroxide electrode. Vortex 1. Ascorbine in (
It can be removed by the total action of i k ascorbate oxidase (abbreviated as ASO). In this case, ASO may be removed by acting prior to other enzymatic reactions, or by being combined with and simultaneously acting with other enzymatic reactions.

A S Oi (1: It may be derived from microorganisms or plants, but those derived from plants such as %, vc capocha, cucumber, etc. are p7 size [2].

In the present invention, if necessary, the corresponding enzyme epimerase may be added in order to fully enhance the action of N-acetylhexosamine oxidase on the anomer.Next, sialic acid is quantified. This section explains how to do this.

In other words, neuraminidase' in samples containing all sialic acids.
N-acetylneuraminic acid is reacted with neuraminic acid Aldra-97 to produce N-acetylmannosamine and pyruvate t! Iru7, then TN-
7 Cetylmannosamine is subjected to the full action of acyl glue 1-samine-2-epimerase (hereinafter abbreviated as AG-epimerase) to obtain N-acetylglucosamine. Then, the sialic acid is determined by applying NAHOD'(r) of the present invention and measuring the sound of hydrogen peroxide produced or oxygen consumed.

Neuraminidase may be of any origin, but some are produced by microorganisms belonging to the genus Clostridium, Arthrobacter, Corynebacterium, Sto I/Butococcus, etc.Neuraminidase includes Enone Erysianocoli, Some are purified from rat liver, brain, etc., and may be derived from any source, but those derived from Erysian coli are particularly preferred.

The AG-epimerase may be of any origin, but for example, it is described in Ghosh and Loisemann, Journal of Biological Chemist IJ-J 1.
), Vol. 324, No. 4, p. 153, AG-epimerase purified from pig kidney. This AG-
ATPk may be added to promote the entire epimerase reaction.

Next, the fixed stitch method using N-7 cetylhexosamindase will be explained. That is, all the samples containing the substrate KN-acetylhexorymindase gold were allowed to react, and the raw rJ! I:L
7j NAH of the present invention to N-acetylhexosamine
ODt action produces hydrogen peroxide or consumes 1
The complete determination of N-acetylhexosaminidase is carried out by measuring fluorine.

(In the formula, R represents an aruol group, a phenyl group, a kifthyl group, or a substituent group.) It may be a one-step method in which the NAHOD reaction and subsequent reactions are performed simultaneously, or a two-step method in which the N-acetylhexosaminidase reaction is performed and then the NAHOD reaction is performed.

Next, the constant method for musimidase will be explained.

That is, a sample containing all muramidase is applied to the substrate, and the N-acetylglucosamine or 1N-acetylmuramic acid produced is used as an action source for the N A H01) of the present invention,
The amount of hydrogen peroxide produced was determined by muramidase concentration by measuring the consumption and oxygen noise of UN-acetylneuraminic acid β-1,4-linked oligomers (G]uNAG) as C groups. , β-1 of N-acetylglucosamine and 1\4-acetylmuramin Nl,
4-linked oligomer ((GluNAG-NA
M)n) is appropriate.

Next, a method for quantifying β-glucuronidase will be explained.

That is, the entire sample containing β-glucuronidase was allowed to act on the substrate, and the NAHODTh of the present invention was made to act on the generated N-acylglucosamine to produce: A? β-glucuronidase is quantified by measuring the oxygen concentration or total oxygen consumed.

A suitable substrate is a glucuronide to which glucuronic acid VcN-acetylglucosamine is bound.

According to the present invention, it is possible to quantify N-acetylhexonamine in a sample with high specificity and accuracy without being influenced by coexisting substances through a scientific operation, which is useful in the diagnostic field of clinical testing. Analysis of sugar-linked compounds and enzymes in C is extremely meaningful.

Next, the present invention will be explained by examples.

The following abbreviations are used in the examples.

NAH, Of) (1-7 cetylhexosamine oxidase) 4-AA (4-aminoantipyrine) TOO8 (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium) A, G
-Epimerase (acylglucosamine-2-epimerase) ATP (adenosine triphosphate) TPP I-9 iamine birophosphate) F'AD
(Flavin adenine dinucleotide) POD (peroxidase) E D T [(coch diamine triacid i) Example 1 N-acetylguryufu, naminf & 7r in the test solution were quantified by the following method using the following reagents, QJ exam
Drug 1-J A HOD 1 unit-1-AA0.57nf' TOO82rny O,IM phosphate buffer (n7.o total 1ttiOm
62, Measuring method Solution or known concentrated N-acetyl glucose concentration 20
After reacting for 20 minutes at 1137°C in a full vise, the wavelength was set to 550 +.
Absorbance was measured at +r+. The N-acetylglucosaminic acid concentration is N-acetylglucosamic acid concentration of known ai N-acetylglucosamic acid/1. Determined by comparing with Kozamin.

Calibration curve in Figure 1? show.

Example 2 Amount of 7 Alkalic Acid in Serum Gold Using the following reagents and using the following method: To provide a detailed explanation of the present invention, all tests were carried out as follows.

Method A is a quantitative method according to the quantitative method of the present invention, in which neuraminic acid is released with sial feeding neuraminidase, pyruvate is totally generated with neuraminic acid aldolase, and pyruvate oxidase 1 is applied to this pyruvate, resulting in peroxidation. Methods for determining the total amount of hydrogen The amount of Sial # in serum was determined using the following methods. Furthermore, pyruvate oxidase is fully activated on pyruvate and birubin m2 in the serum to produce hydrogen peroxide. A method for quantifying sound was determined.

Method A Reagent Neuraminic acid enzyme (manufactured by Gyudon Kagaku) 10 units Neuramic acid aldolase (manufactured by Gyudon Kagaku) 40 units AG
-Epimerase 0.3- N A I (OD 1 unit 4-AA O,5Tq T 00 S 2my ATP 2.52f 001M phosphate buffer (vH7,0) Total jilorn
I! 2 Measurement method 20 ml of serum or known concentrated sialic acid f was taken, and the above three reagents were added thereto. After reacting at 37°C for 20 minutes, the absorbance was measured at a wavelength of 550°C.

Sialic acid AtG in serum was compared to known concentrations of sialic acid.

Figure 2 shows the calibration curve.

13 Methods [Clinica Himika Acta] No. 10 by Sugawara et al.
It was measured by the method described in Vol. 8, pp. 493-498.

1 Reagent Reagent Bl Neuraminidase (manufactured by Gyudon Kagaku) 2 units Reagent Bn Mix the following reagents BH-■ and Bff-■ at a total volume ratio of 5°2.

B11-■ Pyruvate oxydase 100 units T
P P 14 B mole F'AI) 0.36 // mole full presence 15m1 Bll-■ I' (month) 550 units 4-AA 891tmole NaN, 1.55 m mole 4mM phosphoric acid mild; ) so-reagent B
m EDTA 50 mmole Na+HPO+ 0.1 mole Sodium citrate 0-1 mole Triton 'i and incubate at 45°C for 30 minutes, then add 1 ml of reagent Bn and then add 1 ml of gold.
Incubate for 5 minutes, reagent B1113.5mf! After adding and leaving it at room temperature for 10 minutes, absorbance at a wavelength of 505 nm? It was measured. Sialic acid concentration in serum was determined by comparing with known concentrations of sialic acid.

Pyruvate concentration measurement 1 Reagent Reagent CI The following reagents CI-■, CI-■ Imayo Ryuhi 5:
Mix in 2 parts.

Reagent C [~■ Neuraminidase (manufactured by Gyudon Kasho) 2-position neuraminic acid aldolase (Gyudon Kagaku) 10 6.M Acid buffer a)i 6.8) Total volume 10rn
/! Reagent C1-■ POD 55 (I unit 4-AA 891rnole NaNs 1.5 m mole Reagent CII EDTA 50mM NaN*, 1↓Pot 0.1 m rool
e Sodium citrate 0, 1 mole trit: /X -40539 Ion exchange water Total amount 1t 2 11i17 Determination method
Add 1 mg of reagent Cl to this and heat at 37°C.
After incubating for 5 minutes, all of the reagent C113,5 was added, and after being left at room temperature for 10 minutes, the absorbance was determined at a wavelength of 505 nm. By comparing the pyruvate concentration in the serum with the pyruvate at a known level, the measured values of the sialic acid concentration and the pyruvate concentration in the serum can be determined as follows.
Shown in the table.

Table 1 As is clear from Table 1, the conventional method (Method B) measures the total excess amount of pyruvic acid in the sample to quantify the total amount of sialic acid.
The present invention (Method A) allows accurate total quantification of sialic acid.

Example 3 The amount of N-acetylmannosamine in a sample was determined by the method described below using all of the reagents listed below.

1. Reagent NAHOD 1 unit AG-epimerase 0.3me 4-AA O,5ki TOOS 2ki 2 Measurement method Take a 20ml sample, add the above reagent 3 to it, and react at 37°C for 20 minutes. After that, the absorbance is measured at a wavelength of 550 nm.

3 Inspection-■″m is shown in Figure 3.

[Brief explanation of drawings]

FIG. 1 shows a calibration curve for flN-acetylglucosamine. FIG. 2 shows a calibration curve for N-acetylneuraminic acid. FIG. 3 shows a calibration curve for N-acetylmannosamine. Patent Applicant: Toyobo Co., Ltd. Figure 1 5O N-7 謬Chiru 2) Reco length min (mγdffi) $2 [!
l

Claims (1)

[Claims] N-acetylhexosamine is reacted with N-acetylhexosamine in the presence of water and oxygen to produce hydrogen peroxide.N-acetylhexosamine oxidase is reacted with all samples to measure the hydrogen peroxide produced or the oxygen consumed. Featured N-
Determination method for acetylhexosamine.