JPH01148198A - Determination of sarcosine - Google Patents

Abstract

PURPOSE:To determine sarcosine which is useful in clinical test and diagnosis accurately in a short time, by allowing sarcosine oxidase and a specific compound to act on a sample containing sarcosine to form a coloring substance. CONSTITUTION:A sample containing sarcosine is combined with 5-30U/test of sarcosine oxidase originating from Bacillus, and 0.5-5U/test of peroxidase originating from horseradish and 0.01W/W% of 4-aminoantypirine and N-2- hydroxy-3-sulfopropylaniline derivative of the formula (R is H, methoxy, M is alkali metal) such as sodium-N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine to obtain a reaction mixture. The mixture is kept at 5.5-9.5pH, about 37 deg.C for about 5minutes and the color developed by the reaction is measured by absorption spectroscopy to determine the content of sarcosine.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for quantifying sarcosine, and more particularly, to a method for quantifying sarcosine in a short time and with high accuracy using a small amount of enzyme.

[Conventional technology]

Quantification of creatinine and creatine in body fluids is important for diagnosis of renal diseases, muscle diseases, advanced thyroid dysfunction, etc.

The commonly used method for quantifying creatinine and creatine is the method using alkaline picric acid, but this method is affected by other substances such as acetone, pyruvic acid, and folic acid, so it is not accurate. The drawback was that it was not possible to determine the quantity.

In order to solve the drawbacks of the conventional method, sarcosine is produced by the action of creatininase and creatinase, which is then reacted with sarcosine oxidase, and the produced hydrogen peroxide is treated with 4-aminoantipyrine and phenol in the presence of peroxidase. An enzymatic method has been proposed in which the absorbance of the color is measured by reacting with an aniline derivative or an aniline derivative.

[Problem that the invention seeks to solve]

However, when this enzymatic method uses a conventional dye system, the reaction from sarcosine to the chromophore is slow, requires a large amount of enzyme, and is not affected by interfering factors in serum. It had the disadvantage of being easy.

Therefore, it has been desired to provide a method for quantifying sarcosine in a short time using a small amount of enzyme.

[Means for solving problems]

Under such circumstances, the present inventor conducted intensive research and found that 4-aminoantipyrine and the general formula (
When the N-2-hydroxy-3-sulfopropylaniline derivative (hereinafter referred to as AO3) ft represented by I) is used, AO8 significantly increases the Michaelis constant (Km value) of sarcosine oxidase for sarcosine, and even with a small amount of enzyme. The present invention has been completed by discovering that sarcosine can be quantified in a short period of time. That is, the present invention is based on the present invention. , R represents a hydrogen atom or a methoxy group, and M represents an alkali metal). This provides a method for quantifying sarcosine.

The quantitative method of the present invention can be applied to all samples containing sarcosine. Examples include samples obtained by the action of creatinase.

The sarcosine oxidase used in the present invention may be of any origin, but examples include those produced by microorganisms belonging to the genus Corynebacterium, Flavobacterium, Bacillus, etc., and the amount used is 5 to 30 U /
test.

Particularly preferred is 10-20 U/test. Also,
Peroxidase may be of any origin, such as animals, plants, microorganisms, etc., but peroxidase derived from horseradish is particularly preferred, and the amount used is 0.5 to 5 U/te.
st, especially 1 to 2 U/l@mt.

Preferred examples of AO8, which is one of the chromogens of the present invention, include -N-(2-hydroxy-3-sulfozolopyl)methanisidine (^DO8
(referred to as AlO2), N-ethyl-N-(2-hydroxy-3-sulfozologyl)aniline (referred to as AlO2), and the like. These AO8s are preferably used in a range of 001 to 1 v/v% relative to the sample.

The quantitative method of the present invention can be carried out by a known method except for using AO8ft as one of the chromogens. The pH of the measurement system may be within the stable range of color development of the color former, and for example, in the case of ADO8, pHa5 to Q5, and in the case of AlO2, p)Ie, is preferably 5 to 75. Any buffer may be used as long as it has a pH within this range; for example, phosphate buffer, Tris buffer, etc. are used.

When K is applied in this manner, AO8 and 4-aminoantipyrine undergo oxidative condensation in the presence of hydrogen peroxide to produce a chromophore, so for example, the absorbance at 542 nm for ADO8 and 565 nm for human LO8. The amount of sarcosine can be determined by measuring.

〔Effect of the invention〕

As shown above, according to the present invention, since the chromogen AO8 promotes the hydrogen peroxide production reaction from sarcosine, sarcosine can be quantified in a short time with a small amount of enzyme. In quantifying creatinine and creatine in body fluids, the hydrogen peroxide production reaction from sarcosine is the rate-limiting reaction, so the present invention, which can perform this reaction in a short time, is extremely useful in the diagnostic field of clinical tests. Meaningful.

〔Example〕

Next, the present invention will be explained with reference to Examples.

Example 1 Quantification of sarcosine... Comparison of various chromogens: Reaction solution composition Phosphate buffer 40 mM, p
HEL 04-aminoantipyrine (4-^A)
0.02% peroxidase 2.5
U Chromogen 002% Sarcosine Q l pmol e Add each amount of sarcosine oxidase to 0.5 d of the above reaction solution, react for 5 minutes at 37°C, and then add 0.5% SDS solution 1
d tl'' was added to stop the reaction, and the absorbance was measured at the absorption wavelength of each chromogen. The measurement results are shown in FIG.

As is clear from this, only two chromogens, ADO8 and AlO2, showed good results as the reaction was completed in a short time with a small amount of enzyme compared to other chromogens.

The following chromogens were used.

ADO8: Sozoum N-ethyl N-(2-hydroxy-3-sulfopropyl) Meta-anisidine AlO2: Sozoum N-ethyl N-(2-hydroxy-3-sulfozolovir) Aniline TOO8: Sozoum N-ethyl N-(2-hydroxy- 3-sulfopropyl) metatoluizone ALPS; Sozoum N-ethyl-N-sulfopropyl-aniline ADPS: Sozoum N-ethyl N-sulfozolobyl-metaanisizone DAO8: Sozoum N-ethyl N-(2-hydroxy-3-sulfozolovir) - 3,5-dimethoxyaniline HDAPS: Sozoum N-sulfozolobyl-3.5
-Dimethoxyaniline PN: Phenol DE Human = Zomethylaniline Example 2 Km value of sarcosine oxidase in coloring system using ADO8'i: Reaction solution composition Phosphate buffer 40mM, pHa.

4-muA 002% chromogen
0.02% peroxidase
2.5U Sarcosine Oxidase 12.5m
Add sarcosine at various concentrations to 05m of the above reaction solution, and add 3
After reacting at 7°C for 5 minutes, 05% SO8 solution 1- was added to stop the reaction, and the absorbance of each was measured. The results are shown in Figure 2. From Figure 2, Lineveaver-Bu
The Km value determined by the rk plot is the same as that of the control 4-AA.
-The Km value of sarcosine oxidase in the phenol coloring system is 67:OmM, whereas 4-AA-
K of sarcosine oxidase in ADO8 color system
m value was 131 mM. In other words, the Km value of sarcosine oxidase for sarcosine is 4-AA-
When the ADO8 coloring system was used, it was reduced to about one-fifth compared to the 4-AA-phenol coloring system.

Example 3 Quantification of creatinine using ADO8 Time course: Reaction solution composition Phosphate buffer 40mM, pH
a.

4-AA QO2%ADO
8002% Peroxidase 25U Creatinase IOU Creatinase
20U sarcosine oxidase 20U Add creatinine to 0.5 wl of the above reaction solution Onmo 1
@ was added, and the change in absorbance at 542 am was measured at 37°C. The results are shown in Figure 3. As is obvious,
The reaction was completely completed in 5 minutes] and good results were obtained.

Example 4 Quantification of creatinine using ADO8 Calibration curve: Add each amount of creatinine to 05 ml of the reaction solution in Example 2, react at 37°C for 5 minutes, and then measure the absorbance at 542 nm. did. The results are shown in Figure 4.
It showed extremely good linearity.

[Brief explanation of the drawing]

Figure 1 shows the relationship between the absorbance and the amount of sarcosine oxidase when using various chromogens. Figure 2 shows the substrate for sarcosine oxidase in the coloring system using ADO8 and the coloring system using Fe/-F. A diagram showing a saturation curve, Figure 3 shows the use of ADO8 as a chromogen in the determination of creatinine.
Figure 4 is a diagram showing the change in absorbance over time when using ADO8 as a chromogen, and Figure 4 is a diagram showing a calibration curve when quantifying creatinine using ADO8 as a chromogen. That's all

Claims (1)

[Claims] 1. Sarcosine-containing samples include sarcosine oxidase, peroxidase, 4-aminoantipyrine, and the general formula (I) ▲ Numerical formula, chemical formula, table, etc. ▼ (I) (In the formula, R is a hydrogen atom or methoxy A method for quantifying sarcosine, which comprises reacting with an N-2-hydroxy-3-sulfopropylaniline derivative represented by the following formula (M represents an alkali metal) and measuring the resulting colored product.