JPS6238657B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for measuring the reaction rate of creatinine and a reagent for measuring the reaction rate of creatinine.
In particular, the present invention relates to a method for measuring the reaction rate of creatinine with high accuracy, which is suitable for measuring the reaction rate of creatinine in serum without negative errors due to the presence of bilirubin in clinical biochemical tests. Traditionally, colorimetric analysis has been used for most clinical biochemical tests, but in recent years, reaction rate measurement methods (hereinafter referred to as ``rate methods'') have been used, both with the aim of more accurate measurements and advances in measurement equipment. ) are becoming increasingly important. This rate method measures the rate of a certain reaction involving the so-called test substance at the most appropriate point, that is, at the point when the endogenous error is as small as possible, and can be roughly divided into the following three types: . 1 The test substance is an enzyme, and the concentration (activity value) of the test substance is determined from the rate of an appropriate enzymatic reaction involving the enzyme. It is used to measure various transamylases, phosphatases, peptidases, etc. 2. Although the test substance is not an enzyme, the concentration of the test substance is determined from the rate of an appropriate enzymatic reaction involving the substance. Used to measure various lipids, sugars, etc. 3 The test substance is not an enzyme, and the concentration of the test substance is determined from the rate of an appropriate chemical reaction involving it. As mentioned above, the purpose of the rate method is to obtain more accurate data than the conventional colorimetric method by removing endogenous errors, and for this reason, it uses highly specific enzymatic reactions 1 and 2. The most commonly used method is the rate method, or the enzyme analysis method. However, even in the case of pure chemical reactions, it may be more advantageous to use the rate method than the endpoint colorimetric method in order to eliminate endogenous errors. This is case 3, and a typical example is the serum creatinine rate method. Creatinine is a muscle metabolite that is excreted from the kidneys, and the concentration of creatinine in serum and urine is closely related to renal dysfunction, and is one of the most important measurement items in biochemical tests. As a method for measuring creatinine, the Yathue method, which utilizes the formation of creatinine picrate under alkaline conditions as shown in the following formula (1), has been used almost exclusively. That is, the Yatsufue method determines the creatinine concentration by colorimetrically measuring the absorption of creatinine picrate produced from creatinine and picric acid under alkaline conditions at a wavelength of around 500 nm. Although this method is highly practical as it is easy to operate and is stable, it has major drawbacks such as low sensitivity and non-specific reaction. In particular, regarding non-specificity, it is said that when measuring serum from normal people, about one quarter of the measured values are due to positive substances other than creatinine. That is, proteins and acetone bodies have a color reaction similar to that of creatinine, and various sugars and ascorbic acid reduce picric acid to produce red picramine, so both of these give a positive error. In order to eliminate these various positive errors, various devices have been conventionally devised. For example, in the case of manual methods, methods have been used in which creatinine is measured after removing proteins by precipitation filtration or dialysis. However, such a protein removal operation is complicated, and automatic equipment employs a method of omitting the protein removal operation by applying a surfactant. However, even surfactants cannot completely eliminate errors caused by proteins. Moreover, no improvements have been made to prevent interfering substances other than proteins, and although it is one of the most important measurement items in clinical tests, conventional colorimetric measurements of creatinine have poor accuracy. It contains many problems in terms of. In order to eliminate positive errors in such colorimetric methods,
Recently, the creatinine rate method mentioned above has appeared. In this rate method, the production rate of creatinine picrate is determined at the optimum point after the start of the reaction.
Since it is determined from the rate of change in absorbance at a wavelength around 500 nm, it is possible to reduce errors caused by the various interfering substances mentioned above. The optimal point is the point at which the production of picramine by the reducing agent is completed and the reaction between proteins, acetone bodies, etc. and picric acid does not proceed. At such an optimal point, the change in absorbance is primarily due to the formation of creatinine picrate, and the positive error due to interfering substances is significantly reduced compared to conventional colorimetric methods. However, it has recently been found that bilirubin coexisting in serum gives a negative error in the creatinine rate method. Moreover, since this interference caused by bilirubin appears in the entire region after the start of the reaction, it was found that errors caused by this cannot be eliminated no matter how the above-mentioned starting point of rate measurement is selected. Recent experiments have shown that the negative error due to bilirubin in creatinine rate measurements is extremely large; for example, when measuring jaundice serum containing 20 mg/dl of bilirubin, the measured creatinine rate is 1.0 mg/dl lower than the true value. was also found to be low. Considering that the normal value of creatinine is 0.8 to 1.2 mg/dl, this negative error is extremely large and significantly impairs the reliability of the biochemical test. In order to solve the above-mentioned problems, an object of the present invention is to provide a method for measuring the reaction rate of creatinine, which is free from negative errors due to the coexistence of bilirubin and can be measured with high accuracy. In order to achieve the above object, the present invention adds picric acid to a biological sample under alkaline conditions, and calculates the rate of reaction of creatinine picrate formed between creatinine in the biological sample and the picric acid. In the creatinine reaction rate measurement method to determine the concentration of creatinine in a sample,
The biological sample is placed in a reagent solution containing any one of the group consisting of dichloroisocyanuric acid, chloramine B, chloramine T, and sodium hypochlorite until bilirubin contained in the biological sample is oxidized. This is a method for measuring the reaction rate of creatinine, which comprises forming creatinine picrate after incubation, and determining the concentration of creatinine in the biological sample from the rate of production of the creatinine picrate. The principle of the present invention will be explained below. In order to investigate the cause of the negative error due to bilirubin as described above, the present inventor added a high bilirubin control serum to a creatinine measurement reagent and investigated temporal changes in the bilirubin spectrum. As a result, it was found that such negative errors were not due to the reaction between bilirubin and picric acid, but were due to oxidative denaturation of bilirubin under alkaline conditions. The results are shown in FIG. That is, spectrum 1 in Figure 1 is the absorption curve immediately after diluting the high bilirubin control serum containing 10 mg/dl bilirubin with a reagent for measuring the rate of creatinine, and spectrum 2 is the absorption curve after diluting the same solution at 37°C for 20 minutes. This is an absorption curve after being left to stand. The dilution ratio is similar to that for creatinine rate determination.
That's 13 times more. Spectrum 3 is an absorption curve of creatinine picrate obtained by reacting a creatinine standard solution containing 1 mg/dl of creatinine at 37° C. for 20 minutes. Note that all spectra 1 to 3 are reagent blank controls. That is, as is clear from the figure, bilirubin has an absorption maximum at 415 nm under alkaline conditions, but this absorption decreases with the passage of time, and conversely, broad absorption with a maximum around 600 nm increases. The reason for this is thought to be that bilirubin is oxidized in an alkaline environment and converted to biliverdin, as shown in the following formula (2). On the other hand, creatinine picrate, which is a combination of creatinine and picric acid, has an absorption maximum at 490 nm, but its color development sensitivity is low, and even if 1 mg/dl of creatinine is reacted at 37°C for 20 minutes, its absorption is as shown in spectrum 3. It is. In other words, the three spectra in Figure 1 show that when jaundice serum containing a high concentration of bilirubin is measured using the rate method described above, an increase in absorption (around 500 nm) due to the production of creatinine picrate and a decrease in absorption due to oxidation of bilirubin. This shows that the error is significantly negative because the error is canceled out by the There are three possible ways to solve this problem: 1. Method for preventing oxidation of bilirubin 2. Method for prolonging pre-incubation time 3. Method for promoting oxidation of bilirubin As a method for preventing oxidation of bilirubin, there is a method of coexisting a reducing agent in the reagent, or a method of combining with bilirubin. A possible method is to coexist a protective agent that increases the stability. However, as mentioned above, many reducing agents themselves react with picric acid;
Since bicramine is produced, there is a possibility that a positive error may be given, and therefore, their addition is considered undesirable. In addition, bilirubin originally exists in serum in a stabilized state bound to proteins, but it is difficult to find a more powerful protective agent in an alkaline aqueous solution, and even if such a protective agent Even if there is a chemical agent, there is a high possibility that it will react with picric acid. In addition, the method of measuring creatinine by adding picric acid after the pre-incubation time under alkaline conditions is sufficiently long and most of the bilirubin has been oxidized is correct in principle, but is not effective in the case of manual methods. be. However, as shown in Figure 1, even after being left at 37°C for 20 minutes, the amount of bilirubin remaining is about 50%.
Using this method is not very effective. Therefore, in the present invention, a method of promoting the oxidation of bilirubin was attempted so as to obtain a sufficient effect within the total incubation time of the automatic analyzer. In this case, the necessary conditions are of course to oxidize bilirubin as quickly as possible, but it is important not to interfere with the production of creatinine picrate. Therefore, we investigated the oxidation rate of bilirubin and the production status of creatinine piclade in creatinine measurement reagents containing various oxidizing agents. As a result, it was found that bilirubin is easily and rapidly oxidized in strong oxidizing agents such as hydrogen peroxide, permanganate, and dichromate. Furthermore, in this case, no increase in absorption in the long wavelength region as shown in FIG. 1 is observed, and bilirubin is thought to be in a more oxidized state than biliverdin. However, on the other hand, it was found that creatinine picrate was also not produced at all in the presence of such a strong oxidizing agent. As a result of further investigation, it was found that dichloroisocyanuric acid, chloramine B, chloramine T, and sodium hypochlorite, which are oxidizing agents with relatively weak oxidizing power, are optimal for the above purpose. FIG. 2 shows the temporal change in the absorption curve of a high bilirubin control serum (containing 10 mg/dl bilirubin) in a creatinine rate measurement reagent containing 1% chloramine B. The dilution rate was 13 times, Spectrum 4 is the absorption curve immediately after mixing, and Spectrum 5 is the absorption curve after the same solution was left at 37° C. for 10 minutes. That is, it is shown that almost all bilirubin is oxidized within 10 minutes in a reagent for measuring creatinine to which 1% chloramine B is added. In addition, Figure 3 shows the absorption curve when 1 mg/dl creatinine standard solution was reacted with both the above-mentioned reagent with and without chloramine B added under the same conditions (15 minutes at 37°C). . The dilution rate is 13 times, and spectrum 7 shows the case where chloramine B is added, and spectrum 6 shows the case where it is not added. Both spectra agree within experimental error, indicating that the presence of 1% chloramine B does not interfere with the production of creatinine viclate at all. Similar results were obtained for chloramine T and dichloroisocyanuric acid. In other words, the results shown in Figures 2 and 3 show that in the rate method for creatinine, an appropriate oxidizing agent is allowed to coexist in the alkali during pre-incubation, and after bilirubin is almost completely oxidized, bicrinic acid is added to produce creatinine. This shows that it is possible to eliminate the negative error mentioned above by measuring . The concentration of the chlorine-based oxidizing agent in the reagent for measuring creatinine is preferably about 1 to 10%. 1
%, it is difficult to completely oxidize bilirubin. Moreover, if the amount exceeds 10%, the chlorine-based oxidizing agent will not be completely dissolved, and therefore, even if a larger amount is added, the reagent will be wasted. Hereinafter, embodiments of the present invention applied to an automatic analyzer will be described in detail. In this example, 100μ of sample serum is first sampled diluted into a reaction vessel together with 0.6ml of the first reagent. This first test solution uses a conventional alkaline buffer. Specifically 1
Each bottle contains 5.2g of disodium phosphate, 15.0g of sodium borate, 15.0g of sodium hydroxide, and 6.0g of sodium chloride. Then, 10.0 g/chloramine B is added to this first reagent. The reaction vessel is pre-incubated for 12 minutes at 37°C. Next, 0.6 ml of the second test solution is added, but this second test solution is a conventional one (per 1
Contains 3.2g picric acid and 1.0g sodium lauryl sulfate). Two minutes after adding the second test solution, this test solution was sucked into a constant temperature bath (37°C) flow cell of a photometer for rate measurement.
The absorbance at a wavelength of 505 nm was measured for 1 minute, and the relationship between the rate of increase in absorbance and creatinine concentration was determined using a high bilirubin control serum with a known concentration (20 mg/min).
dl) and creatinine standard solution (10 mg/dl) at three different ratios were measured using both the above method and the conventional rate method.

[Table] As is clear from Table 1, with the conventional method, as the concentration of bilirubin increases, the actual value of creatinine becomes lower than the theoretical value, but it can be seen that there is no such error in the actual value with the method of the present invention. . In the above example, the present invention was applied to the measurement of the reaction rate of creatinine in serum in a clinical biochemical test, but the scope of application of the present invention is not limited to this, and it can be applied to other general biological samples such as urine. It is clear that it can be similarly applied to measuring the reaction rate of creatinine in a sample. As explained above, the present invention provides a method for measuring the reaction rate of creatinine in which the concentration of creatinine is determined from the production rate of creatinine picrate by adding picric acid to a biological sample under alkaline conditions, and a reagent for the measurement. After incubating a biological sample in a test solution containing an oxidizing agent that promotes the oxidation of bilirubin and does not interfere with the production of creatinine picrate, the rate of production of creatinine picrate can be measured. Therefore, the accuracy of creatinine rate measurement can be significantly increased simply by adding an appropriate oxidizing agent to the reagent of the conventional rate method, which has the excellent effect of significantly improving the reliability of clinical tests.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the spectrum variation due to oxidation of bilirubin under alkaline conditions, Figure 2 is a diagram showing the acceleration of bilirubin oxidation due to the coexistence of an oxidizing agent, and Figure 3 is a diagram showing the creatinine peak of the oxidizing agent. FIG. 3 is a diagram showing the influence on rat production.

Claims (1)

[Claims] 1 Adding picric acid to a biological sample under alkaline conditions and determining the concentration of creatinine in the biological sample from the reaction rate of creatinine picrate formed between creatinine in the biological sample and the picric acid. In the reaction rate measuring method, the biological sample is placed in a reagent solution containing any one of the group consisting of dichloroisocyanuric acid, chloramine B, chloramine T, and sodium hypochlorite. A method for measuring the reaction rate of creatinine, which comprises incubating until bilirubin is oxidized, then forming creatinine picrate, and determining the concentration of creatinine in the biological sample from the rate of production of the creatinine picrate.