JPH0380480B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for the simultaneous determination of creatinine and creatine in body fluids, particularly serum or urine, using enzymes, and a kit therefor. Creatine is mainly synthesized in the liver and enters the bloodstream, and most of it is distributed in the muscles, where it is synthesized into creatine phosphate, a high-energy compound, by the action of creatine kinase, and plays an important role as an energy source. Creatine is excreted from the renal glomerulus, but is reabsorbed in the renal tubules, so it is hardly excreted in the urine in adult males.
Serum creatine concentration increases in primary muscle diseases, muscle atrophy, hyperthyroidism, etc., resulting in creatinuria. On the other hand, creatinine, which is an anhydrous form of creatine, is produced in muscle by irreversibly and non-enzymatically dehydrating creatine phosphate, passes through the renal glomerulus as a metabolic end product, and is excreted without being reabsorbed in the renal tubules. Therefore, in the case of renal function diseases such as uremia, chronic nephritis, and urinary tract obstruction, creatinine cannot be excreted normally and creatinine increases in serum. Measuring creatine and creatinine concentrations in serum and urine in this way serves as an important index for the diagnosis of these diseases. The number of specimens continues to increase year by year. The conventionally commonly used method for quantifying creatinine uses the Jacques reaction, which develops color by reacting with picric acid under alkaline conditions (Bonsnes and
Taussky, J. Biol. Chem. 158 581 (1945)). Since this method does not show a specific reaction to creatinine, it is not possible to obtain an accurate amount of creatinine because it is affected by other substances in body fluids, and picric acid may contaminate the reaction vessel or analyzer, causing measurement errors. Become. Furthermore, when adapted to an automatic analyzer, there were many problems in handling, such as affecting other measurement items. On the other hand, creatine is heated in acidic conditions to convert it into creatinine, and the resulting creatinine is measured using the above-mentioned Jacques reaction, and the amount of creatinine is estimated from the difference with the amount of creatinine before acidic heating.For this reason, the creatinine quantitative method eliminates errors. It has the disadvantage that it is easy to occur and the operation is complicated. It has been desired to develop a simple and accurate method for simultaneously quantifying creatinine and creatine that overcomes these conventional drawbacks. Against this background, we conducted extensive research to establish a rapid, highly sensitive, and accurate method for simultaneously quantifying creatinine and creatine.As a result, we developed four enzymes: creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, and catalase. The inventors have discovered that the intended purpose can be achieved by using a color-forming reagent and have arrived at the present invention.
The present invention converts creatine and creatinine in an aliquot of a sample into formaldehyde, hydrogen peroxide, and glycine by applying a combination of three enzymes: creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase. , a combination of two enzymes, creatine amidinohydrolase and sarcosine oxidase, was applied to another aliquot of the above sample to convert the creatine in the sample into formaldehyde, hydrogen peroxide and glycine, and the hydrogen peroxide produced in each case 2 per molecule of creatinine and creatine by reacting with methanol in the presence of catalase and converting it to formaldehyde.
Formaldehyde molecules are generated, a coloring reagent is applied to each generated formaldehyde, the resulting chromophore is measured, the total amount of creatinine and creatine, and the amount of creatine are determined, and the amount of creatine is calculated from the total amount. This is a method for simultaneously quantifying creatinine and creatine, which consists of calculating the amount of creatinine by subtracting the amount of creatinine. According to the present invention, the sample contains creatinine amidohydrolase [EC3.5.2.10; hereinafter abbreviated as CRN], creatine amidinohydrolase [EC3.5.3.3; hereinafter abbreviated as CR], and sarcosine oxidase [EC1.5.3.1]. ; hereinafter abbreviated as SOX] are activated in combination to convert creatinine and creatine in the sample into formaldehyde, hydrogen peroxide, and glycine.
Catalase generates hydrogen peroxide [EC1.11.1.6]
It is converted into one more molecule of formaldehyde by reacting with methanol in the presence of . This converts one molecule each of creatinine and creatine into two molecules of formaldehyde. Since twice the amount of formaldehyde generated here is measured by color, the sensitivity and accuracy of the measurement is extremely high, and creatinine and creatine can be measured quickly and accurately. The measurement principle of the method of the present invention is specifically shown below. [A] Enzyme reaction Creatinine + H ₂ OCRN ---→ Creatine Creatine + H ₂ OCR ---→ Sarcosine + Urea Sarcosine + H ₂ O+O ₂ SOX ----→ Formaldehyde + Glycine + Hydrogen peroxide Hydrogen peroxide + Methanol catalase --- ―――→ Formaldehyde + 2H ₂ O [B] Color reaction 2 Formaldehyde + Color reagent → Chromogen In other words, creatinine in the sample is changed to creatine with CRN, the resulting creatine is changed into sarcosine and urea with CR, and sarcosine is filtered with SOX. An enzymatic reaction that converts hydrogen oxide and glycine into formaldehyde and water by reacting the resulting hydrogen peroxide with methanol in the presence of catalase, and a coloring reaction that develops color by reacting formaldehyde generated by the enzymatic reaction with a coloring reagent. Then, measure the absorbance in the visible region of the resulting colored material,
The target creatinine can be quantified. Creatine can be quantitatively determined by allowing the enzymatic reaction to proceed without using CRN. In addition, when you want to know the amount of creatinine present in a sample, you can use the total amount of creatine and creatinine obtained by advancing the enzyme reaction using CRN in the above enzyme reaction.
It is obtained by subtracting the amount of creatine obtained by proceeding with the enzymatic reaction without using CRN. The CRN used in the present invention may be of any origin, but examples thereof include CRN produced by microorganisms belonging to the genus Alcaligenes, Pseudomonas, Flavobacterium, and the like. The CR used in the present invention may be of any origin, but examples thereof include CR produced by microorganisms belonging to the genus Pseudomonas, the genus Flavobacterium, and the like. The SOX used in the present invention may be of any origin, but examples thereof include SOX produced by microorganisms belonging to the genus Pseudomonas, Corynebacterium, Micrococcus, and the like. The catalase used in the present invention may be of any origin, such as animals, plants, microorganisms, etc. As the coloring reagent for quantifying aldehydes of the present invention, any coloring reagent may be used as long as it reacts with aldehyde and quantitatively develops color. One example is 4-amino-3-hydrazino-1.2.4-triazole (hereinafter referred to as AHMT). There is a compound called (abbreviated). This compound is reacted with formaldehyde under alkaline conditions and then oxidized with sodium metaperiodate to develop color. According to the method of the present invention, quantification of creatinine and creatine using the four enzymes CRN, CR, SOX, and catalase is more sensitive than the method using the Jathue reaction, is not affected by coexisting substances, and is highly specific and accurate. It is possible to quantify creatinine and creatine in body fluids, and it is extremely meaningful in the diagnostic field of clinical tests. Since hydrogen peroxide is converted to formaldehyde using catalase, one molecule each of creatinine and creatine is converted to two molecules of formaldehyde each, and twice the amount of formaldehyde produced here is being quantified. This method is more sensitive than methods that quantify hydrogen peroxide by color. Furthermore, the present invention provides a kit for simultaneously quantifying creatinine and creatine in a liquid, which will be specifically explained below. The kit for simultaneous determination of creatinine and creatine according to the present invention includes a reagent A containing four enzymes: CRN, CR, SOX, and catalase, a reagent B containing three enzymes, CR, SOX, and catalase, and a reagent containing two enzymes, SOX and catalase. C and a formaldehyde coloring reagent. Reagent A, Reagent B, and Reagent C in the kit may be in either powder or liquid form, and in the case of powder, the optimum pH6 for carrying out the enzymatic action of the four enzymes CRN, CR, SOX, and catalase. .0
A buffer containing methanol, such as a 50 mM phosphate buffer (PH 7.8) containing 4% methanol, is used as the enzyme lysing solution. If it is in liquid form, dissolve Reagent A, Reagent B, and Reagent C containing the enzymes CRN, CR, SOX, catalase, etc. in a buffer such as 50mM phosphate buffer containing 4% methanol to a pH of 6.0 to 9.0. preferably PH7.8)
All you have to do is adjust it. Any formaldehyde coloring reagent may be used as long as it is a reagent that quantitatively colors formaldehyde, but examples include 3N potassium hydroxide solution, 1.0% AHMT solution, 0.04% sodium metaperiodate solution, etc. can be mentioned. It is preferable to store the kit in a cool, dark place, especially below 5°C. Next, the present invention will be explained by examples. Example Creatinine and creatine in a reagent were quantified using the following reagent and the following method. 1 Reagent Reagent A CRN 280 units CR 360 units SOX 240 units Catalase 20000 units Methanol 0.4ml50mM phosphate buffer (PH7.8) 10ml Reagent B CR 360 units SOX 240 units Catalase 20000 units Methanol 0.4ml 50mM phosphate buffer (PH7.8) PH7.8) 10ml Reagent C SOX 240 units Catalase 20000 units Methanol 0.4ml 50mM phosphate buffer (PH7.8) 10ml Reagent D 3N-Potassium hydroxide solution Reagent E 1% AHMT solution Reagent F 0.04% Sodium metaperiodate Solution 2 Measurement method Take 20μ of the sample and creatinine standard solution,
Thereafter, perform the operations as shown in Table 1,
After absorbing light at a wavelength of 550 nm, measure the amount of creatinine and creatine in the sample using the formula shown below.

【table】

[Table] The calibration curve for creatinine is shown in Figure 1, and the calibration curve for creatine is shown in Figure 2. Furthermore, the measured values of creatinine when control serum was used as a sample were as shown in Table 2.

[Table] Using the above reagent, the concentration of creatinine in serum was determined and compared with the conventional Jacques reaction method. The results were as shown in Table 3.

【table】

[Table] In the Yatsufue reaction method, a solution of picric acid and sodium hydroxide is added to a sample, and the absorbance of the red compound produced by the reaction of active methylene is measured. The standard solution was operated in the same way to 515n.
This method calculates the concentration in the sample by comparing the colors with m. The method of the present invention uses a specific enzymatic reaction compared to the conventional Yatsufue method and can perform accurate measurements, and can simultaneously quantify creatinine and creatine in a sample. As explained in detail above, the present invention provides a method and a kit for simultaneously quantifying creatinine and creatine in a liquid accurately and rapidly, and is particularly useful for diagnosing kidney and muscle diseases in clinical medicine. It is something that can be done.

[Brief explanation of drawings]

FIG. 1 is a calibration curve for creatinine, and FIG. 2 is a calibration curve for creatine.

Claims (1)

[Claims] 1. A combination of three enzymes, creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase, is applied to one aliquot of a sample to convert creatine and creatinine in the sample into formaldehyde, hydrogen peroxide, and glycine. creatine amidinohydrase and sarcosine oxidase are applied to another aliquot of the above sample in combination to convert the creatine in the sample into formaldehyde, hydrogen peroxide and glycine, producing in each case Hydrogen peroxide is reacted with methanol in the presence of catalase to convert it into formaldehyde, producing two molecules of formaldehyde for each molecule of creatinine and creatine, and a coloring reagent is applied to each of the resulting formaldehyde to produce the resulting color. A method for simultaneously quantifying creatinine and creatine, which comprises measuring the body, determining the total amount of creatinine and creatine, and calculating the amount of creatinine by subtracting the amount of creatine from the total amount. 2 Coloring reagent is 3N potassium hydroxide solution, 1%
The method of claim 1, wherein the 4-amino-3-hydrazino-1,2,4-triazole solution and the 0.04% sodium metaperiodate solution. 3 Reagent A containing four enzymes: creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, and catalase;
A kit for the simultaneous determination of creatinine and creatine, consisting of Reagent B containing three enzymes, creatine amidinohydrolase, sarcosine oxidase, and catalase; Reagent C containing two enzymes, sarcosine oxidase and catalase; and a formaldehyde coloring reagent. 4 Coloring reagent is 3N potassium hydroxide solution, 1%
The kit according to claim 3, which is a 4-amino-3-hydrazino-1,2,4-triazole solution and a 0.04% sodium metaperiodate solution.