JPH054080B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for enzymatically quantifying N-acetylneuraminic acid. Sialic acid, which is a sugar complex of N-acetylneuraminic acid, exists in various forms in living organisms. This sialic acid is an important component of glycoproteins and resins, and in the field of clinical testing, it has been found that it changes in acute and chronic inflammation, shock, trauma, myocardial infarction, diabetes, liver disease, various cancers, etc. , sialic acid measurement has shown an important role in diagnosis. The Ehrlich method, which is a chemical method, is used to measure sialic acid.
Orcinol method, resorcinol method, periodate resorcinol method, thiobarbituric acid method, etc. are known, but they have problems with specificity, some require the use of strong acids, and some require heating operations. The disadvantage was that the operations were complicated. As an enzymatic measurement method, N-acetylneuraminic acid is released from sialic acid using neuraminidase, pyruvate is generated using neuraminic acid aldolase, and this pyruvate is reacted with lactate dehydrogenase (LDH) and NADH system to quantify pyruvate. There are methods for quantifying hydrogen peroxide produced by the action of pyruvate oxidase on acid, but these measurement methods have the drawback of being influenced by endogenous pyruvate. The present inventors have intensively investigated various methods for measuring N-acetylneuraminic acid that are easy to operate and are not affected by endogenous pyruvate and have found that neuraminic acid aldolase, acylglucosamine-2- The present invention has been achieved by discovering that the above object can be achieved by using the enzymes epimerase (hereinafter abbreviated as AG-epimerase) and N-acetylhexosamine oxidase (hereinafter abbreviated as NAHOD). That is, the present invention provides hydrogen peroxide or N-acetyl neuraminic acid produced by reacting a sample of N-acetylneuraminic acid with neuraminic acid aldolase, acylglucosamine-2-epimerase (AG-epimerase), and N-acetylhexosamine oxidase (NAHOD). This is an enzymatic method for quantifying N-acetylneuraminic acid, which is characterized by measuring glucosamic acid or consumed enzyme. The measurement principle of the present invention is as shown below. (A) Enzyme reaction N-acetylneuraminic acid neuraminic acid aldolase――――――――――――→ N-acetylmannosamine + pyruvate N-acetylmannosamine AG-epimetase―――― ――――――→ N-acetylglucosamine N-acetylglucosamine + O ₂ + H ₂ ONAHOD ――――――――→ N-acetylglucosaminic acid + H ₂ O ₂ , that is, N-acetylneuraminic acid, is converted to N-acetylglucosamine by neuraminic acid aldolase. Then, it is decomposed into N-acetylmannosamine and pyruvate, and the resulting N-acetylmannosamine is treated with AG-epimerase.
Produces N-acetylglucosamine. This N-acetylglucosamine in the coexistence of oxygen and water,
By acting with NAHOD, N-acetylglucosaminic acid and hydrogen peroxide are produced. The measuring method is characterized by measuring either hydrogen peroxide or N-acetylglucosamic acid produced at this time, or oxygen consumed. The neuraminic acid aldolase used in the present invention includes those purified from E. coli, Clostridium, rat liver, brain, etc., and may be derived from any source, but those derived from microorganisms are particularly preferred. The AG-epimerase used in the present invention may be of any origin, but for example, the AG-epimerase purified from pig kidney described in "Journal of Biological Chemistry" by Gauchiel and Roisemann, Vol. 24, No. 4, p. 153. There is an AG-epime. ATP may be added to promote this AG-epimerase reaction. The NAHOD used in the present invention may be of any origin, but for example, there is NAHOD derived from the genus Pseudomonas, which is described in Horiuchi's Proceedings of the 1981 Annual Conference of the Japanese Society of Agricultural Chemistry, page 173.
If necessary, mutarotase, which promotes conversion of anomers, may be added to promote this NAHOD reaction. Furthermore, the neuraminidase used to measure sialic acid by the action of neuraminidase may be of any origin, but is produced from microorganisms belonging to the genus Clostridium, Arthrobacter, Corynebacterium, Streptococcus, etc. There is. In the present invention, a sample refers to a sample containing N-acetylneuraminic acid produced by reacting neuraminidase to a sample containing serum, blood, or sialic acid. Any method may be used to quantify the hydrogen peroxide produced in the present invention, such as a colorimetric assay using an enzymatic method using peroxidase or catalase, and a method using a direct electrode. In the method using peroxidase, 4-aminoantipyrine and phenol or a derivative thereof, 4-aminoantipyrine and aniline or a derivative thereof, 3-methyl-2-benzothiazolinone hydrazone and aniline or a derivative thereof, etc. are used as a chromogen. There is a way. Examples of phenol derivatives include p-chlorophenol, 2,4-dichlorophenol, p-promophenol, o-chlorophenol, m-chlorophenol, 2,6
-dichlorophenol, 2,3-dichlorophenol, etc. Also, examples of aniline derivatives include N,N-dimethylaniline, N,N-diethylaniline, N,N-diethyl-m-toluidine,
N,N-dimethyl-m-anisidine, N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(β-hydroxyethyl)-m-toluidine, N-ethyl-N
-(2-hydroxy-3-sulfopropyl)-m-
Toluidine (hereinafter abbreviated as EHSPT), N-ethyl-N-sulfopropyl-m-toluidine, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy-
Examples include 3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-m-anisidine, and the like. As a method using catalase, there is a method in which catalase is allowed to act on hydrogen peroxide and methanol, the resulting formaldehyde is condensed with acetylacetone in the presence of ammonia, and the absorbance at 412 nm is measured. This method allows accurate concentration measurements by taking a sample blank. As a method of quantifying oxygen consumption, there is a method of quantifying oxygen using an oxygen electrode. Furthermore, in the present invention, ascorbic acid in the specimen, which interferes with the color development of hydrogen peroxide, can be removed by the action of ascorbic acid oxidase (hereinafter abbreviated as ASO). In this case, ASO
may be removed by acting prior to other enzymatic reactions, or by conjugating with other enzymatic reactions and acting simultaneously. ASO may be derived from microorganisms or plants, preferably from pumpkin or cucumber. The reagent according to the present invention is a composition buffered at pH 3 to 10, and the reaction temperature when measuring N-acetylneuraminic acid using the reagent according to the present invention is about 4 to 50.
It is ℃. The composition for measuring N-acetylneuraminic acid according to the present invention is an enzyme for quantifying neuraminic acid aldolase, AG-epimerase, NAHOD enzymes, hydrogen peroxide produced or N-acetylglucosaminic acid, or oxygen consumed. or reagents by appropriately combining them. These reagents and enzymes are prepared as a liquid, solid, or lyophilized agent, and if necessary, a buffer solution is added before use to prepare a measurement reagent. The measurement method is to first treat the sample with neuraminic acid aldolase and AG-epimerase, converting N-acetylneuraminic acid to N-acetylmannosamine.
Furthermore, it is set as N-acetylglucosamine. next
Hydrogen peroxide is measured by reacting with NAHOD and a reagent that measures the hydrogen peroxide produced. In the present invention, the measurement method may be a one-reagent system or a two-reagent system, and may also be performed using any number of reagent systems. In the present invention, by allowing neuraminic acid aldolase, AG-epimerase, and NAHOD to act on the sample, it is possible to quantify N-acetylneuraminic acid with simple operation and high accuracy without being affected by endogenous pyruvate. It is also extremely meaningful in the diagnostic field of clinical testing for sialic acid. Next, the present invention will be explained by examples. Example 1 The concentration of N-acetylneuraminic acid in a solution was determined using the following reagent and the following method. 1 Reagent Neuramic acid aldolase 40 units AG-epimerase 300μ NAHOD 4 units 4-AA 0.5mg EHSPT 2mg ATP 60.53mg 0.1M phosphate buffer (PH7.0) Total volume 10ml 2 Measurement method Place 20μ of the sample in a test tube and add the above After adding 3 ml of reagent and reacting for 20 minutes at 37°C,
Absorbance was measured at 550 nm. The concentration of N-acetylneuraminic acid in this sample was determined by comparing it with a known concentration of N-acetylneuraminic acid. Figure 1 shows the calibration curve. Example 2 The amount of sialic acid in serum was determined using the following reagent and the following method. In order to explain the effects of the present invention, tests were conducted as follows. Neuraminidase acts on sialic acid in serum to convert it to N-acetylneuraminic acid,
The method of measurement using the quantitative method of the present invention is referred to as method A. Method B is a method in which sialic acid in serum is converted to neuraminic acid by neuraminidase, pyruvic acid is produced by neuraminic acid aldolase, pyruvate oxidase is allowed to act on this pyruvic acid, and the resulting hydrogen peroxide is quantified. The amount of sialic acid in serum was determined by methods A and B. Furthermore, the amount of pyruvate present in the same serum was quantified using a method in which pyruvate is reacted with pyruvate oxidase and the hydrogen peroxide produced is measured. A Method 1 Reagent neuraminidase (manufactured by Hanui Chemical) 10 units neuraminic acid aldolase (manufactured by Hanui Chemical)
40 units AG-epimerase 0.3ml NAHOD 1 unit 4-AA 0.5mg EHSPT 2mg ATP 60.53mg 0.1M phosphate buffer (PH7.0) Total volume 10ml 2 Measurement method Place serum in a 20μ test tube and add 3ml of the above reagent. , after reacting at 37℃ for 20 minutes, the wavelength was 550nm.
Absorbance was measured. The amount of cyanic acid in serum was determined by comparing known concentrations of sialic acid. B Method It was measured by the method described in "Clinica Himika Acta", Vol. 108, pp. 493-498 by Sugawara et al. 1 Reagent Reagent B Neuraminidase (manufactured by Hanui Chemical) 2 units neuraminic acid aldolase (manufactured by Hanui Chemical)
10 units 6mM phosphate buffer (PH6.8) Total volume 10ml Reagent B Mix the following reagent B-B- in a volume ratio of 5:2. B- Pyribate oxidase 100 units TPP 14μmole FAD 0.36μmole p-chlorophenol 57μmole MgCl ₂ 150μmole 20mM phosphate buffer (PH7.4) Total volume 15ml B- POD 550 units 4-AA 89μmole NaN ₃ 1.55mmole 4mM phosphate buffer (PH7.0) 50ml Reagent B EDTA 50mmole Na ₂ HPO ₄ 0.1mole Sodium citrate 0.1mole Triton
After adding 0.5 ml and incubating at 45° C. for 30 minutes, adding 1 ml of reagent B and incubating for an additional 15 minutes, adding 3.5 ml of reagent B and standing at room temperature for 10 minutes, the absorbance was measured at a wavelength of 505 nm. The amount of sialic acid in serum was determined by comparing with known concentrations of sialic acid. Pyruvate concentration measurement 1 Reagent Reagent C Mix the following reagents C-C- at a volume ratio of 5:2. Reagent C- Neuraminidase (manufactured by Hanui Chemical) 2 units neuraminic acid aldolase (manufactured by Hanui Chemical)
10 units 6mM phosphate buffer (PH6.8) Total volume 10ml Reagent C- POD 550 units 4-AA 89μmole NaN ₃ 1.5mmole 4mM phosphate buffer (PH7.0) 50ml Reagent C EDTA 50mM NaN ₂ HPO ₄ 0.1mmole Cigar Sodium chloride 0.1 mole Triton
After adding 1 ml and incubating at 37°C for 15 minutes, 3.5 ml of Reagent C was added and left at room temperature for 10 minutes, and then the absorbance was measured at a wavelength of 505 nm. Pyruvate concentration in serum was determined by comparing with known concentration of pyruvate. Measured values of sialic acid concentration and pyruvate concentration in serum are shown in Table 1. Table 1 also shows values obtained by converting the amount of pyruvate in serum into sialic acid concentration.

【table】

[Table] As is clear from Table 1, in the conventional method (Method B), sialic acid is excessively determined due to the influence of pyruvic acid present in serum, but in the present invention, sialic acid is determined in excess due to the influence of pyruvic acid present in serum. The concentration of sialic acid can be determined accurately.

[Brief explanation of drawings]

FIG. 1 shows the calibration curve of Example 1.

Claims (1)

[Claims] 1. Hydrogen peroxide or N produced by reacting neuraminic acid aldolase, acylglucosamine-2-epimerase and N-acetylhexosamine oxidase with N-acetylneuraminic acid sample
- An enzymatic method for quantifying N-acetylneuraminic acid, which comprises measuring acetylglucosaminic acid or consumed oxygen.