JPS6030696A - Method and reagent for determination of creatinine and creatine - Google Patents

Abstract

PURPOSE:To determine creatinine or creatine, in high specificity and sensitivity, free from the influence of foreign materials, by using a determination reagent comprising a combination of a specific enzyme and a coenzyme DNA. CONSTITUTION:Creatinine or creatine in the specimen is made to react with a reagent comprising a combination of creatinineamide hydrolase (except for the determination of creatine), creatineamidino hydrolase, sarcosine oxidase, formaldehyde dehydrogenase, formic acid dehydrogenase and coenzyme NAD, and the produced NADH is determined.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying creatinine and creatine in body fluids using an enzymatic method and a reagent for the assay.

Measurement of creatinine and creatine in serum and urine:
It provides useful information for diagnosing kidney diseases, muscle diseases, etc.

Conventionally, the Jaffe method using alkaline picric acid has been used for total quantification of creatinine and creatine, but this method has large measurement errors due to non-specific color development, and requires testing equipment because it uses strong alkali and picric acid. However, it has disadvantages such as contaminating or damaging equipment.

Therefore, in order to solve these drawbacks, there has been a strong demand in the jυ1 industry for a method for quantifying creatinine and creatine that uses enzymes with high specificity and mild measurement conditions, and various proposals have been made. ing. The main quantitative methods are as follows.

a) Method for completely quantifying the amount of hydrogen peroxide produced (Unexamined Japanese Patent Publication No. 1983
-83297) +Glycine The coloring body produced by the above reaction is measured.

b) Method for quantifying formaldehyde produced (Clinical Examination Vol. 22, 1331-1338 (1978))
xAHM'l": 4-7mi/-3-hydracino5-
Mercapto-1,2,4-)lyazole The reddish-purple tetrazine produced by the above reaction is measured.

The method for quantifying creatinine and creatine using these enzymes has high specificity, allowing for highly accurate quantification.
In addition, the measurement conditions are mild and it is an excellent quantitative method.
However, in measurement method a), the generated hydrogen peroxide is measured using a redox reaction under the action of peroxidase, so if reducing substances such as ascorbic acid and reduced glucthion coexist in the sample, the direct measurement will be negative. The disadvantage is that it is influenced by In addition, measurement method b) requires four steps and is complicated, and has drawbacks such as negative effects if formaldehyde dehydrogenase is mixed in with each enzyme.

Furthermore, the following method has been proposed as a quantitative method using an enzyme that does not have these drawbacks.

C) Method for quantifying the amount of NADL-I (reduced nicotinamide atenine dinucleotide) that disappears (Bergmeyer, "Merry's of Enzymatic Analysis" 2nd edition, 1787-1790 Sada (1974)) + DP + Pyruvic acid Measure the amount of NADH that disappears by the above reaction.

d) Method for quantifying the generated NADHt' (Journal of the Japan Clinical Laboratory Automation Study Group, Volume 5 Supplement, p. 119 (1980)
year)) +, H2O2 Formaltehyde Tehydrochenase Formaltehyde + NAD'.

NADH + formic acid The amount of NADH produced by the above reaction is measured.

Measurement method C) and measurement method d) have high specificity, are easy to operate, and are free from the effects of coexisting substances in the sample. However, in these measurement methods, the amount of N AD 1 that disappears from one molecule of creatine is very good.
Since only one molecule of NADH is produced, the measurement sensitivity becomes low. For this reason, when measuring a low concentration sample, highly accurate measurement cannot be performed. On the other hand, a normal creatinine in serum is 0.9 to 1.5 mg/dis, and a normal creatine in one shift is 0.3 to 0.91% l/di, so low concentrations of V atinine or creatine are accurately measured. There was a desire to establish a method for quantifying

The present inventors have conducted extensive research to resolve all the shortcomings of methods a), b), C), and d), and have found that the methods have high specificity, are easy to operate, and are susceptible to the effects of coexisting substances in the sample. not receive,
In addition, they have discovered a method for quantifying creatinine and creatine that has high measurement sensitivity and high accuracy, and have completed the present invention.

That is, the present invention achieves creatinine amide hydrolase, creatine amidinohydrolase, sarcosine oxidase, and formaldehyde tehydrogen;
This invention is based on the unique knowledge of the present inventors and has been completed with a unique configuration and has remarkable effects.

Further details of the present invention will be explained below.

The measurement principle of the present invention is as follows.

Creatinine amide hydrolase (1) Creatinine + H20 Creatine + Urea + Glycine + H2O2 Formaldehyde dehydrogenase (4) Formaldehyde + NAD 1-NADH + Awakening By measuring the ultraviolet absorbance of NADH generated in the above reactions (4) and (5), creatinine Or creatine can be quantified. According to the present invention, N produced from one molecule of frettinin or one molecule of creatine
Since ADH has two molecules, it has high measurement sensitivity and can be quantified with high accuracy even when measuring low concentration specimens such as serum.

Furthermore, the excellent effects of the present invention are listed below: (1) The reaction is fast, so quantification can be carried out in a short time. (2) It is easy to operate because five steps of reaction can proceed simultaneously;
(3) It does not use a redox reaction such as using a tetrazolium salt or acting peroxidase on hydrogen peroxide, so it can be quantified without being affected by reducing substances such as ascorbic acid in the sample.

In addition, when measuring the amount of creatine, it can be measured by allowing the entire reaction to proceed without using creatinine amide hydrolase. In addition, if both creatinine and creatine are present in the tumor body, from the total amount of creatinine and creatine obtained by proceeding the reaction with creatinine amide hydrolase, creatinine obtained by proceeding the reaction without using creatinine amide hydrolase. The true amount of creatinine can be determined by subtracting the amount and, conversely, the amount of creatinine amidohydrolase first? After quantitating the amount of creatine by measuring the absorbance of NAD and H produced by allowing the reaction to proceed without using creatine, the same test solution was treated with creatinine amide hydrolase and the absorbance of NAD14 was measured again. NA obtained by the reaction starting from
The amount of creatinine can also be determined by a one-liquid method by subtracting the absorbance of DH.

Regarding the formate dehydrogenase used in the present invention, see Berbmeyer, Mary's of Enzymatic Analysis, 2nd edition, pp. 1551-1554 (1974).
A method for quantifying formic acid using formate dehydrogenase was disclosed in 2010). However, as shown below, the method of the present invention cannot be easily inferred from this disclosure.

That is, in the method of the present invention, formic acid, which is one of the products produced by the action of formaldehyde, NADH, and formic acid is made to act on formic acid, which is one of the products, but at this time, the other product, NADH 1 European Journal of Biochemistry by H6PNER et al.
, Vol. 83, 485-498 Sada (1978), NA
It has been specified that DH is an inhibitor of formate dehydrogenase. Therefore, even if formate dehydrogenase is allowed to act on formic acid in a state in which NADH, an inhibitor of formate dehydrogenase, coexists with formic acid in a sufficient amount, it is unlikely that the reaction will proceed rapidly; Since NADH is also generated by the reaction of It was inconceivable that this would proceed. Surprisingly, however, when creatinine and creatine were measured using a combination of enzymatic reactions according to the method of the present invention, the reaction of formate dehydrogenase also proceeded rapidly and was completed in a short time.
It was confirmed that DH was produced, and it was discovered that creatinine and creatine could be quantified with high sensitivity and accuracy, and the present invention was completed.

In quantifying creatinine in a test sample, the present invention produces NADH' by acting in combination with creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and coenzyme NADt.
Is it easy to implement by e-measurement? In addition, in quantifying nocreanan in a test sample, by acting in combination with creatine amidinohydrolase, sarcosine oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and coenzyme NADk, and measuring the generated N and ADH'. It can be easily implemented.

Further, to describe the embodiments of the present invention in detail, the present invention provides a method for quantifying creatinine in a test specimen by using, for example, a solution containing creatinine amidinohydrase as the first solution, and creatine amidinohydrolase and sarcosine oxidase. A solution containing formaldehyde dehydrogenase, formate dehydrogenase and NADk is used as the second solution. First, the Al solution and the second solution are added to the test sample and incubated. The resulting color is measured and the absorption coefficient is defined as 1rES.

), separate from this, add the second solution to the test sample and incubate it, measure the resulting coloration, and measure the absorbance value of E.
Let it be B. ), (ES-EB') can be easily carried out by calculating the amount of creatinine.

Furthermore, in quantifying creatine in a test sample, the present invention uses, for example, a solution containing creatine amidinohydrolase as the first solution, sarcosine oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and N.
Using the liquid contained in AD' as the second liquid, first add the first and second liquids to the test sample and incubate to check the resulting color 'k.
ill! This absorption straightness is defined as f:Es.)
, Separately, a second solution was added to the test sample and incubated, and the color development was measured (this absorbance 1[i'
k EB. ), (ES-EB) is the amount of creatine? It can be easily implemented by asking for it.

The enzymes used in the present invention may be of any origin and origin, such as creatinine amidohydrolase, creatine amidinohydrolase, sarcosinolase,
Regarding formaldehyde dehydrogenase, the enzymes used in normal creatinine and creatine measurements are used without exception, but what are some examples of them? Flare, for example.

Examples of tininamide hydrolase include creatinine amidohydrolase produced by alkaline earth metals, microorganisms such as Pseudomonas and Pseudomonas, and creatinine amidinohydrolase produced by microorganisms such as Pseudomonas and Bacillus. There is creatine amidinohydrolase produced by microorganisms such as alkaline earth metals.

In addition, examples of sarcosine oxidase include sarcosine oxidase produced by microorganisms such as Corynebacterium, Bacillus, and Arthrobacter.
For polardehyde dehydrogenase, for example,
There are formaldehyde dehydrogenases produced by microorganisms such as Pseudomonas, Arthrobakucus, and Cyclococcus. On the other hand, there are no restrictions on the origin of formate dehydrogenase; for example, formate dehydrogenase τ produced by microorganisms such as Pseudomonas, Candida, and Chloesocera
Examples include, but needless to say, the present invention is not limited to these.

The pH during the measurement of creatinine and/or creatine in the present invention is generally in the range of 6.5 to 8.5, preferably around pH 8, and the buffer for this purpose is a buffer known per se. , such as phosphate buffer, Tris buffer,
Although phosphoric acid buffers and the like are used without exception, phosphate buffers are commonly used.

Thus, the present invention provides a highly accurate method for quantifying creatinine that is simple, sensitive, and unaffected by coexisting substances, and will greatly contribute to the diagnostic field of clinical testing. There is something called a dog.

Examples will be shown below and explained in more detail, but the present invention is not limited thereto.

Example 1. Reagent for quantitative determination of creatinine in serum Enzyme reagent A: 550 units of creatinine amidohydrolase. Make the total volume to 10rnl with the following buffer.

Enzyme test solution B: creatinamidinohydrolase 125 units, sarcosine oxogose 50 units, formaldehyde dehydrogenase 3 units, oxate dehydrogenase 5 units, -NAD 5 mg. The total volume is IO- with the following buffer solution.
Make it.

buffer nitriton Adjust the total volume to 40 rnl with acid buffer (pH 8.0).

Assay serum or creatinine standard solution 100μt? : and add the above enzyme test solution A to this. 5- and enzyme test solution 81, 5m
1" and react at 37°C for 10 minutes. Separately, prepare a test solution blank layer solution by performing the same procedure using purified water instead of the serum or creatinine standard solution.

Using this test solution blank M solution as a control, wavelength 3 40
Measure the absorbance in nm.

Let the absorption directly be kEs and Estd. Also, serum 1
00 μt, 0.5 ml of the above buffer solution and enzyme reagent solution B1,
In addition to 5-, after heating at 37° C. for 10 minutes, the absorbance at a wavelength of 340 nm is measured using the test solution blank solution as a control. Let its absorption directly from the farm be kEB. The amount of creatinine in serum is determined by comparing <Es-EB) and Estd. Figure 1 shows the calibration curve.

Example 1. The amount of creatinine in serum was measured using the method described above, and compared with the value measured using the conventionally used Jatsufue method. The Janoffe method is a method of determining the amount of creatinine in a sample by measuring absorption-IIV at a wavelength of 520 nm of an orange-red additive compound formed by picric acid and creatinine in an alkali. The results are shown in Table 1.

Since the method of the present invention uses a specific reaction sound compared to the conventional Yasofue method, it is possible to perform highly accurate measurements.

Table 1 Example 2. Reagent for quantitative determination of creatine in serum Enzyme reagent A: Creatine amidinohydrolase 350 units. Make the total volume 10- with the following buffer.

Enzyme test solution B: Sarcosine oxidase 50 units, formaldehyde dehydrogenase 3 units, formate dehydrogenase 5 units, NAD 5zv0. Make the total volume 10- with the following buffer.

Buffer Nitriton X-10040■. Make the total volume 40 ml with 0.1 MIJ acid buffer (pH 8,0).

Assay method Take 100ml of serum or creatine standard solution and add the above enzyme test solution A 005- and enzyme test solution 81.5+n to it.
Add l'e and react at 37°C for 10 minutes. Separately, a test solution blank solution is prepared by performing the same operation using purified water instead of serum or creatine standard solution. Using this test solution≠book≠Shishikui blank solution as a control, wavelength 3
Measure the absorbance at 40 nm. Its absorbance 'k ES
and Estd. Further, 0.5 me of the above buffer solution and 1.5 ml'5 of enzyme reagent solution 8 are added to 100 μt of serum, and after heating at 37°C for 10 minutes, the absorbance at a wavelength of 340 nrn is measured using the reagent blank solution as a control. Its absorbance is 1 straight 7! -EB. The amount of creatine in serum is (
Determine by comparing ES - EB ) with Estd and t. Figure 2 shows the calibration curve.

Comparative example 1. Determination of creatine in serum [Conventional method] Determination reagent Enzyme reagent A: Same as Example 2.

Enzyme test solution B: Example 2. The composition excluding formate dehydrogenase.

Buffer: Example 2. Same as .

Quantitative method Example 2. Same as .

FIG. 3 shows the calibration curve of Comparative Example 1.

As is clear from FIGS. 2 and 3, the sensitivity of the present invention is higher than when formate dehydrogenase is not used.

Example 3. Determination of Creatinine in Serum Example 1. Creatinine in the same serum was measured repeatedly using the method 1.0 and the method using a test solution from which formate dehydrogenase was removed. The results are shown in Table 2.

As is clear from the results shown in Table 2 above, the method of the present invention has high measurement sensitivity, better reproducibility, and enables highly accurate measurements compared to the measurement method that excludes formate dehydrogenase gold. .

[Brief explanation of the drawing]

FIG. 1 shows the calibration curve in Example 1, where the horizontal axis is the creatinine concentration CIIl&/dg) and the vertical axis is C340n.
It represents the absorbance at m. FIGS. 2 and 3 show Example 2, respectively. The calibration curve for Comparative Example 1° is shown, and in both cases, the horizontal axis is the creatine concentration CT.
n9/dl) The vertical axis represents absorbance at 340 nm. Patent applicant: Wako Pure Chemical Industries, Ltd. No. 11! I Creatinine concentration (■/dL) Figure 2 Creatine concentration (mL1/dt) Figure 3 Creatine concentration (mg/dz) Procedural amendment 1. 2. Title of the invention 3. Relationship with the case of the person making the amendment Patent applicant 4. Date of the amendment order 5. Number of inventions reduced by the amendment 26. A column for the title of the invention, a column for the scope of claims, and a column for the detailed description of the invention in the specification to be amended. 7. Contents of amendment (1) "Method for quantifying creatinine and creatine and reagent for quantifying" stated in the column of title of the invention is amended to read 1. "Method for quantifying creatinine and creatine." (2. The scope of claims is amended as shown in the attached sheet. (3) "Genus Ncrococcus" stated in line 16 of page 14 of the specification is amended to "genus Micrococcus". (4) Line 6 of page 19 of the specification "Serum or creatine standard?&100ml serum or creatine standard solution 100 μt" as described in Appendix 2, Claims (1) In quantifying creatinine in a test sample, creatinine amidohydrolase, creatine It is characterized by measuring NADH (reduced nicotinamide adenine dinucleotide) produced by reacting amidinohydrolase, sarcosine oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and coenzyme NAD in combination with cotinamide adenine dinucleotide). , a method for quantifying creatinine. (2) For the determination of creatine in a test sample, the NADH'i produced by the combined action of creatine amidinohydrolase, sarcosine oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and coenzyme NAD'i is measured. A method for quantifying creatine.

Claims (4)

[Claims] (1) When quantifying creatinine in a test sample,
A method for quantifying creatinine based on creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, formaldehyde dehydrogenase, formate dehydrogenase and coenzyme NAD. (2) Creatine is characterized in that in determining the total amount of creatine in a test sample, creatine amidinohydrolase, sarcosine oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and coenzyme NAD' are used in combination to measure the generated NADHt. Quantification method. (3) A reagent for talleatinine regularization, which is a combination of creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and coenzyme NAD'. (4) A creatine quantitative reagent comprising a combination of creatine amidinohydrolase, sarcosine oxidase, formaldehyde dehydrogenase, formatetecht'logenase and coenzyme NAD'.