JPH06217800A - Method of enzymatically measuring creatinine, creatine, sarcosine and glycine and measurin kit - Google Patents

Abstract

PURPOSE:To improve sensitivity of measurement by adding glycine-L- methyltransferase and S-adenosyl-L-methionine. CONSTITUTION:Glycine-L-methyltransferase and S-adenosyl-L-methionine are added to a measurement system and sarcosine is recyclably generated in the system. Hydrogen peroxide and formaldehyde are generated in larger amounts than a conventional method and sensitivity can be improved.

Description

Detailed Description of the Invention

[0001]

The present invention relates to creatine in body fluids,
The present invention relates to an improved measuring method and measuring kit for creatinine, sarcosine and glycine. More specifically, by adding glycine-N-transferase and S-adenosyl-L-methionine to the measurement system, sarcosine is recycled in the system, and hydrogen peroxide or formaldehyde, which is a direct measurement target, is usually generated. The present invention relates to an enzymatic assay method and assay kit for creatine, creatinine, sarcosine, or glycine, which is characterized in that it is generated more frequently than in the reaction of 1.

[0002]

BACKGROUND OF THE INVENTION Creatine is mostly distributed in muscle. Creatine in serum is known to increase due to diseases such as primary myopathy, myositis, muscular atrophy, and hyperthyroidism. Moreover, since creatine is reabsorbed from the renal tubule, it is usually hardly detected in urine in adults. However, it is well known that the amount of excretion in urine increases as the concentration in serum increases. On the other hand, creatinine is a dehydrated metabolite of creatine, and serum creatinine concentration has a close correlation with renal glomerular filtration rate. Creatinine is excreted in the urine because it is hardly reabsorbed by the renal tubules.

When the amount of creatinine in the serum is increased, it is suspected that the renal function is impaired, and it is used as an index thereof. In addition, creatinine in urine is known to decrease in progressive muscular dystrophy and severe renal failure,
It is used as an index. On the other hand, glycine and sarcosine, when their serum concentrations are higher than normal values, are known to cause intellectual impairment and the like as hyperglycinemia and hypersarcosinemia, respectively.
These are thought to be due to inborn errors of metabolism, but the measurement of glycine and sarcosine respectively
It is used for these diagnoses.

[0004]

2. Description of the Related Art Conventionally, chemical methods have been used to measure creatinine and creatine. This is a method called the Jaffe method, which utilizes the fact that creatinine forms a coloring compound with picric acid in an alkaline solution. However, this method has drawbacks such as lack of specificity and being greatly affected by the drug to be taken, and therefore, it is being converted to a newly developed enzyme method having higher specificity. As for this enzymatic method,
For example, creatinine is converted to creatine by creatinine amide hydrolase (CNH), the resulting creatine is converted to sarcosine using creatine amidinohydrolase (CRH), and sarcosine is further converted to glycine by sarcosine oxidase (SAO), in which case, There is a method of knowing the original amount of creatine by applying hydrogen peroxide generated at the same time to a coloring system. The enzyme method has a very high specificity and therefore has an advantage that creatinine can be accurately measured.

However, since the normal concentrations of creatine and creatinine to be measured are extremely low and the range of fluctuation due to disease is very small, there remains a problem that the range of fluctuation is difficult to capture even with the enzymatic method. In addition, for the measurement of glycine and sarcosine, an enzymatic method has not been put into practical use at present, and a method using high performance liquid chromatography is generally performed, but this method is time-consuming and It requires expensive equipment. On the other hand, the method of enzymatic recycling has been known in principle for some time, but in order to achieve recycling and obtain a good calibration curve with high sensitivity, it is necessary to devise a device. Generally, in order to achieve recycling, the ratio of enzyme,
It is said that it is difficult to set conditions such as the difference in Km with respect to the substrate. Successful examples of these include a method for amplifying NAD by alcohol dehydrogenase or malate dehydrogenase (T. Kato et al., Anal. Biochem., 53, 86.
(1973)).

[0006]

Under the above circumstances, as a result of earnest studies on improvement of the creatinine enzyme method capable of highly sensitive measurement, the present inventors have found that the glycine-N-methyltransferase and S-adenosyl-L-methionine. It was found that the sarcosine recycling reaction was established by adding the above-mentioned to the conventional enzyme method reagent, and as a result, the measurement sensitivity was linearly improved, and the present invention was completed. The recycling reaction between glycine and sarcosine has not been known so far, and it was found that the reaction was established for the first time by the present invention, and it was successfully put into practical use.

That is, the present invention provides a method for enzymatically measuring creatine, creatinine, sarcosine or glycine, characterized by using glycine-N-methyltransferase and S-adenosyl-L-methionine, and glycine-N-methyltransferase and S-
The present invention relates to an enzymatic assay kit for creatine, creatinine, sarcosine or glycine, which contains adenosyl-L-methionine as an essential component. The following reaction formula A shows the principle of the present invention.

[0008]

[Chemical 1]

In the formula, CNH represents creatinine amidohydrolase, CRH represents creatine amidinohydrolase, SAO represents sarcosine oxidase, SAM represents S-adenosyl-L-methionine, and GMT represents glycine-glycine-. Represents N-methyl transferase.

The above reaction formula will be briefly described. First, creatinine to be measured is hydrolyzed to creatine by creatinine amide hydrolase (CNH). Creatine is hydrolyzed to sarcosine by creatine amidinohydrolase (CRH). And sarcosine is sarcosine oxidase (SAO)
Is decomposed into glycine and formaldehyde,
At that time, hydrogen peroxide is generated. In the conventional enzymatic method, the creatinine is quantified by measuring the hydrogen peroxide generated here with a chromogenic reagent.

To this system, a sufficient amount of S-adenosyl-L-
When methionine (SAM) and an appropriate amount of glycine-N-methyltransferase (GMT) are added, glycine in the system is methylated and converted into sarcosine. The sarcosine produced here is once again oxidized by sarcosine oxidase in the measurement system and converted into glycine, formaldehyde and hydrogen peroxide. In this way, hydrogen peroxide and formaldehyde accumulate in the measurement system. The method of the present invention quantifies creatinine by quantifying the accumulated hydrogen peroxide using a peroxidase, a chromogen, or the like. Similarly, the creatinine can be quantified by measuring the NADH produced from formaldehyde with formaldehyde dehydrogenase.

In the actual quantification, in order to eliminate the influence of sarcosine and glycine in the measurement sample, D-
Treatment with an amino acid oxidase, glycine dehydrogenase or the like is preferable. Also, in advance C
The effect can be eliminated by measuring the amount of hydrogen peroxide generated from endogenous creatine or glycine using RH and SAO and subtracting it as a sample blank.

Further, according to the method of the present invention, not only creatinine but also creatine, sarcosine and glycine can be measured with high sensitivity. That is, creatine can be measured by removing CNH shown in the above principle of the present invention from the measurement system, and sarcosine and glycine can be measured by removing CNH and CRH. Glycine can be measured by removing sarcosine in the sample by pretreatment, and sarcosine can be measured by removing glycine in the sample by pretreatment.

The enzyme used in the present invention may be of any origin, but the following may be used, for example. Glycine-N-methyltransferase is used in animal organs,
For example, those derived from rabbit liver and rat liver can be used.
Furthermore, those produced by incorporating these production genes into bacteria, yeast, etc. can also be used. As the sarcosine oxidase, a bacterium, for example, one derived from the genus Arthrobactor can be used. Creatine amidinohydrolase is a bacterium, for example, Actinobatillus.
us) genus origin is available. Creatinine amide hydrolase is used in fungi, for example Pseudomonas.
nas) can be used. Glycine-N-methyltransferase (GMT) used in the present invention
Is used at a final concentration of 0.01 to 1000 U / ml.

The sarcosine oxidase (SAO) used in the present invention is used at a final concentration of 0.01 to 1000 U / ml. S-adenosyl-L-methionine (SAM) used in the present invention is used at a final concentration of 0.1 to 500 mM, preferably 1.0 to 200 mM. Creatine amidinohydrolase (C used in the present invention
RH) is used at a final concentration of 0.1 to 2000 U / ml. The creatinine amide hydrolase (CNH) used in the present invention is used at a final concentration of 0.1 to 2000 U / ml.

In the present invention, in order to carry out the measurement with high sensitivity, the recycling rate is preferably 1.5 or more in the reaction time of about 10 minutes, and for that purpose, the GMT used is at a final concentration of 0.3 to 5 U / ml. , SAO is 4-10 at the final concentration.
It is preferably 0 U / ml. SAM in this case
Is preferably 1 to 50 mM at the final concentration. A more preferable recycling rate is 3 in the same reaction for 10 minutes.
Above, for that, GMT is 0.8-5 at the final concentration.
The final concentration of U / ml and SAO is preferably 15 to 100 U / ml. For the measurement of creatine, it is preferable to add CRH to the above components at a final concentration of 50 to 300 U / ml, and for the measurement of creatinine,
CRH and CNH are preferably added to the above components in a final concentration range of 50-300 U / ml. As the buffer, those having a pH of 5 to 9 can be used, and for example, Good's buffer (TAPS etc.), Tris-hydrochloric acid buffer etc. can be used.

[0017]

The present invention will be described in detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

Example 1: Amplification measurement of creatinine The following reagents 1 and 2 were prepared, and a solution of creatinine of known concentration (0, 0.1, 0.2, 0.3 and 0.4) was prepared as a sample.
mM; 50 μl). First, add the sample to Reagent 1 (500 μl), leave at 37 ° C. for 5 minutes, and then
Reagent 2 (500 μl) was added and allowed to stand at 37 ° C. for 10 minutes, after which the production of quinone dye in the solution was measured at 570 nm. The results are shown by ◇ in Fig. 1.

Reagent 1-creatine amidinohydrolase (Actinobacillus genus) ... 200 U / ml-peroxidase (horseradish-derived) ...... 10 U / ml sarcosine oxidase (Arthrobactor genus) ... 20 U / ml D-amino acid oxidase ( From pig kidney) ...... 800 U / ml ・ N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine ・ ・ ・ 6 mM ・ TAPS buffer (pH 8.3) ・ ・ ・ 100 mM

Reagent 2 • Creatinine amidohydrolase (from Pseudomonas sp.) ...... 200 U / ml · Glycine-N-methyltransferase (GMT, from rabbit liver) …… 3.6 U / ml S-adenosyl-L-methionine (SAM) …… 10 mM ・ 4-aminoantipyrine (4-AA) …… 0.6 mM ・ 2-hydroxybutyric acid ・ ・ ・ 50 mM ・ TAPS buffer (pH 8.3) …… 100 mM

Comparative Example 1: Measurement of creatinine by a conventional enzyme method A reagent 3 was prepared by removing the D-amino acid oxidase from the reagent 1 of Example 1, and the glycine methyltransferase, 2-hydroxybutyric acid and S were prepared from the reagent 2. -Reagent 4 was prepared by removing adenosyl-L-methionine, and reagent 3
A creatinine solution of the known concentration (the same as above) was measured by the same operation as in Example 1 using the reagent 4 and the reagent 4.
The results are shown by □ in FIG.

Reagent 3 ・ Creatine amidinohydrolase (from Actinobacillus genus) ・ ・ ・ 200 U / ml ・ Peroxidase (from horseradish) ・ ・ ・ 10 U / ml sarcosine oxidase (from Arthrobactor genus) ・ ・ ・ 20 U / ml ・ N-ethyl-N -(2-Hydroxy-3-sulfopropyl) -m-toluidine: 6 mM TAPS buffer (pH 8.3): 100 mM

Reagent 4 ・ Creatinine amidohydrolase (from Pseudomonas sp.) ...... 200 U / ml ・ 4-Aminoantipyrine (4-AA) ・ ・ ・ 0.6 mM ・ TAPS buffer (pH 8.3) ・ ・ ・ 100 mM (Discussion) From FIG. As is apparent, in Example 1, the sensitivity was about four times that of Comparative Example 1, and the calibration curve was a straight line.

Example 2 Measurement of Serum Creatinine by the Method of the Present Invention Using Reagent 1 and Reagent 2, the same procedure as in Example 1 was carried out using the sample prepared by adding creatinine to serum as a sample to measure creatinine. Was done. The creatinine added was adjusted to a final concentration of 0, 0.1, 0.2, 0.3 and 0.4 mM, respectively, and the mixing ratio was 9: 1 by volume.
And The result is shown by + in FIG. (Discussion) As is clear from FIG. 1, the result is a line parallel to the calibration curve of Example 1, and it can be seen that creatinine in serum can also be measured accurately.

Example 3 Sarcosine Amplification Assay Reagent 5 was prepared by removing creatine amidinohydrolase and sarcosine oxidase from reagent 1, and reagent 6 was prepared by removing creatinine amidohydrolase from reagent 2 and adding sarcosine oxidase. Using Reagents 4 and 5, using a solution of sarcosine of known concentration (0.1, 0.2, 0.3 and 0.4 mM; 50 μl) as a sample, the concentration of the sarcosine solution of known concentration was measured by the same operation as in Example 1. did. The result is shown by + in FIG.

Reagent 5 ・ Peroxidase (derived from horseradish) ・ ・ ・ 10 U / ml ・ D-amino acid oxidase (derived from pig kidney) ・ ・ ・ 800 U / ml ・ N-ethyl-N- (2-hydroxy-3-sulfopropyl)- m-Toluidine: 6 mM TAPS buffer (pH 8.3): 100 mM

Reagent 6 ・ Sarcosine oxidase ・ ・ ・ 20 U / ml ・ Glycine methyltransferase (GMT, derived from rabbit liver) ・ ・ ・ 3.6 U / ml ・ S-adenosyl-L-methionine (SAM) ・ ・ ・ 10 mM ・ 4-aminoantipyrine ( 4-AA) ... 0.6 mM 2-hydroxybutyric acid ... 50 mM TAPS buffer (pH 8.3) ... 100 mM

Comparative Example 2: Measurement of sarcosine by conventional enzymatic method Reagent 7 was prepared by removing D-amino acid oxidase from Reagent 5, and glycine methyltransferase, 2-hydroxybutyric acid and S-adenosyl-L were prepared from Reagent 6. −
Reagent 8 excluding methionine was prepared, and a sarcosine solution having a known concentration (the same as in Example 3) was used as a sample by using Reagent 7 and Reagent 8 and the sarcosine concentration was determined by the same procedure as in Example 1. It was measured. The results are shown in Fig. 2
Indicate.

Reagent 7-Peroxidase (derived from horseradish) ... 10 U / ml-N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine ... 6 mM-TAPS buffer (pH 8.3) ... … 100 mM

Reagent 8 ・ Sarcosine oxidase ・ ・ ・ 20 U / ml ・ 4-Aminoantipyrine (4-AA) ・ ・ ・ 0.6 mM ・ TAPS buffer (pH 8.3) ・ ・ ・ 100 mM (Discussion) As shown in FIG. In Example 3, the sensitivity is about four times that of Comparative Example 2, and the calibration curve is linear.

Example 4 Glycine Amplification Measurement Reagent 9 was prepared by removing D-amino acid oxidase from Reagent 5, and reagent 10 was prepared by removing 2-hydroxybutyric acid from Reagent 6, and Reagent 9 and Reagent 10 were prepared. Using a solution of glycine of known concentration (0, 0.1, 0.2, 0.3 and 0.4 mM; 50 μl) as a sample, the concentration of the glycine solution of known concentration was measured by the same operation as in Example 1. The results are shown by □ in FIG.

Reagent 9-Peroxidase (derived from horseradish) ... 10 U / ml-N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine ... 6 mM-TAPS buffer (pH 8.3) ... … 100 mM

Reagent 10 sarcosine oxidase ...... 20 U / ml glycine methyltransferase (GMT, derived from rabbit liver) ...... 3.6 U / ml S-adenosyl-L-methionine (SAM) ...... 10 mM 4-aminoantipyrine ( 4-AA) ... 0.6 mM TAPS buffer (pH 8.3) ... 100 mM (Discussion) As is clear from Fig. 3, a linear and highly sensitive calibration curve was obtained.

Example 5: Amplification measurement of creatinine Reagent 1 and reagent 2 were prepared in the same manner as in Example 1. However, various concentrations of glycine transmethyl ferrase and sarcosine oxidase in the reagent were changed to measure the recycling rate of each. TAPS (pH = 8.0) was used as the buffer.

[0035]

[Table 1]

(Discussion) As is clear from Table 1, at each concentration, a recycling rate of 2 to 4 times was obtained,
It was confirmed that highly sensitive measurement was possible.

[0037]

EFFECTS OF THE INVENTION Since the method of the present invention can measure creatine, creatinine, sarcosine and glycine with higher sensitivity than conventional enzyme methods, accurate diagnosis and pathological analysis of muscle diseases, renal diseases and the like are possible. In addition, it is possible to quickly and accurately diagnose inborn errors of metabolism such as hyperglycinosis and high sarcosine.

[Brief description of drawings]

FIG. 1 shows the measurement results of creatine concentration. □ indicates the conventional method (Comparative Example 1), ◇ indicates the method of the present invention (Example 1),
+ Indicates the method of the present invention (Example 2), respectively.

FIG. 2 shows the measurement results of sarcosine concentration. □ indicates the conventional method (Comparative Example 2), and + indicates the method of the present invention (Example 3).

FIG. 3 shows the measurement results of glycine concentration. □ indicates the method of the present invention (Example 4).

Claims (10)

[Claims] 1. A method for enzymatically measuring creatine, creatinine, sarcosine or glycine, which comprises using glycine-N-methyltransferase and S-adenosyl-L-methionine. 2. The method for enzymatically measuring glycine according to claim 1, wherein glycine-N-methyltransferase, S-adenosyl-L-methionine and sarcosine oxidase are used. 3. The method for enzymatically measuring sarcosine according to claim 1, wherein glycine-N-methyltransferase, S-adenosyl-L-methionine and sarcosine oxidase are used. 4. The method for enzymatically measuring creatine according to claim 1, wherein glycine-N-methyltransferase, S-adenosyl-L-methionine, sarcosine oxidase and creatine amidinohydrolase are used. 5. The method for enzymatically measuring creatinine according to claim 1, wherein glycine-N-methyltransferase, S-adenosyl-L-methionine, sarcosine oxidase, creatine amidinohydrolase and creatinine amide hydrolase are used. 6. A kit for enzymatically measuring creatine, creatinine, sarcosine, or glycine, which contains glycine-N-methyltransferase and S-adenosyl-L-methionine as essential components. 7. The kit for enzymatically measuring glycine according to claim 6, wherein glycine-N-methyltransferase, S-adenosyl-L-methionine and sarcosine oxidase are essential. 8. The kit for enzymatically measuring sarcosine according to claim 6, which essentially comprises glycine-N-methyltransferase, S-adenosyl-L-methionine and sarcosine oxidase. 9. The kit for enzymatically measuring creatine according to claim 6, which essentially comprises glycine-N-methyltransferase, S-adenosyl-L-methionine, sarcosine oxidase and creatine amidinohydrolase. 10. The kit for enzymatically measuring creatinine according to claim 6, which essentially comprises glycine-N-methyltransferase, S-adenosyl-L-methionine, sarcosine oxidase, creatine amidinohydrolase and creatinine amide hydrolase.