JPH0625767B2 - Creatinine measurement reagent - Google Patents

Description

The present invention relates to a reagent used in the Folin method for creatinine measurement using the Jaffe reaction.

"Prior Art" Creatinine is a metabolite of creatine phosphate,
It is filtered by the renal glomerulus and excreted outside the body. Insufficient renal function leads to incomplete excretion of creatinine, resulting in a high amount of creatinine in the blood. Also, if there is an abnormality in muscle contraction, the amount of creatinine produced decreases, so that the amount of creatinine in blood / urine decreases. Therefore, the measurement of the amount of creatinine in body fluids has clinically important significance as an indicator of renal diseases such as renal failure, nephritis, and uremia, or congestive heart failure, acromegaly, giant disease, muscular dystrophy, diabetes insipidus. It is a thing.

As a conventional creatinine measurement method, there is a Folin method using the Jaffe reaction (creatinine and picric acid are condensed in an alkaline solution to produce a reddish substance). However, in this method, picric acid causes a side reaction with various substances (positive substances) present in the measurement material in addition to the reaction with creatinine, thereby producing color. Therefore, in serum, albumin in particular of protein causes this side reaction and gives an error in the measurement, so that deproteinization operation is required and the convenience is impaired. Therefore, in recent years, the enzymatic method has been attracting attention with the intention of specifically measuring creatinine. However, the reagent using the enzymatic method is expensive, has low sensitivity, and is affected by an interfering substance in serum. The Folin method is inexpensive, and accurate measurement is possible if the problem with picric acid and positive substances other than creatinine (especially albumin) can be solved.

As one of the methods, in JP-A-60-38666, by adding sodium or potassium lauryl sulfate to the reagent, the binding of picric acid and the protein is blocked and the deproteinization operation is omitted, and the nonionic interface is further added. A simple measuring method has been proposed in which an activator is added to prevent the reagent from being preserved in winter and precipitation of sodium or potassium lauryl sulfate during the reaction.

By the way, in this measuring method, the addition of a nonionic surfactant is an essential condition for the solubility of sodium or potassium lauryl sulfate. However, this nonionic surfactant has a serious drawback that it partially inhibits the reaction of picric acid and creatinine, which is the main reaction, and reduces the sensitivity.

"Problems to be Solved by the Invention" In view of the above, the present inventor prevents side reactions of picric acid and protein, does not cause precipitation during low temperature conditions and reactions, and inhibits the main reaction of picric acid and creatinine. As a result of various studies on various side reaction inhibitors, Patent Publication No. 60-38
By using some specific substances among the anionic surfactants which are not preferable as an additive in Japanese Patent No. 666 (hereinafter, also referred to as a conventional method), it is possible to use the nonionic surfactant in combination. The inventors have found that the above objects can be achieved and have reached the present invention.

"Means for Solving Problems" That is, the present invention provides a Folin-type creatinine reagent containing a first reagent composed of an alkaline aqueous solution and a second reagent composed of picric acid as a main component, and using polyoxygen as a side reaction inhibitor. Ethylene alkyl ether sulfate, sodium lauroyl sarcosine, lithium lauryl sulfate,
A reagent for measuring creatinine, comprising one selected from the group consisting of fatty acid sodium tauride.

Hereinafter, the present invention will be described in detail.

Like the general Folin creatinine measuring reagent, the creatinine measuring reagent according to the present invention contains picric acid and an alkaline aqueous solution as main components, and is preferably composed of a first reagent and a second reagent which are separated from each other.

The side reaction inhibitor used in the present invention is polyoxyethylene alkyl ether sulfate, sodium lauroyl sarcosine, lithium lauryl sulfate and fatty acid tauride.

These side reaction inhibitors may be added to either the first reagent or the second reagent. However, lithium lauryl sulphate and fatty acid sodium tauride have poor solubility at low temperatures under alkaline conditions, so it is inappropriate to add them to an alkaline solution. Other than that, the above-mentioned anionic surfactants can maintain excellent solubility under both alkaline and acidic conditions.

Therefore, preferably, a combination of a first reagent consisting of an alkaline aqueous solution containing one (or two or more) of polyoxyalkyl ether sulfate and sodium lauroyl sarcosine and a second reagent containing picric acid, or
A combination of a first reagent consisting of an alkaline aqueous solution and one of polyoxyethylene alkyl ether sulfate, sodium lauroyl sarcosine, lithium lauryl sulfate, sodium fatty acid tauride and a second reagent containing picric acid is used.

The amount and concentration of each component in the creatinine measuring reagent according to the present invention, the normality of the alkaline aqueous solution, and the like are in accordance with those generally adopted in the Folin method.

In the Folin method, for example, after adding 6 m of deproteinization test solution to 2 m of serum and centrifuging for 30 minutes at 3000 rpm,
1.5m of the supernatant was sampled, 0.92% (W / V) picric acid aqueous solution was added to 0.5m and 0.75N sodium hydroxide aqueous solution, and the mixture was reacted for colorimetric determination (Clinical Examination Techniques Complete 6 Clinical Chemistry. Inspection II reference).

However, the amount of the side reaction inhibitor used in the present invention is 0.2 to 1% when the first reagent and the second reagent are used in a volume ratio of 4: 1 when added to the alkaline aqueous solution which is the first reagent. (W /
V) It is preferably 0.4 to 0.6% (W / V). If it is less than 0.2% (W / V), the effect is insufficient, and if it exceeds 1% (W / V), the effect on protein remains unchanged.

When adding to the second reagent, picric acid aqueous solution,
It is 0.5 to 1.5% (W / V), preferably 0.9 to 1.1% (W / V). If it is less than 0.5 (W / V), the effect is insufficient, and if it exceeds 1.5% (W / V), the effect remains unchanged.

"Examples" Hereinafter, the present invention will be described more specifically with reference to Examples.

Example 1 Comparison of Solubility of Anionic Surfactants in Acidic and Alkaline Table 1 shows the solubility of 21 kinds of anionic surfactants as acidic,
This is a comparison under alkaline conditions of 4 ° C and 25 ° C. The alkaline solution is 0.1 N sodium hydroxide, the acidic aqueous solution is 1.2% (W / V) picric acid aqueous solution, and the anionic surfactant is added at a concentration of 3% (W / V). is there. The anionic surfactant used in the present invention exhibits good solubility for 11 months.

Example 2 Effect on Albumin Table 2 compares the effect of addition of the anionic surfactant of the present invention on albumin to the case of no addition.

1 of each anionic surfactant used in the present invention is added to an aqueous solution.
% (W / V) is added, ((lithium lauryl sulfate,
Fatty acid tauride sodium is excluded), the second reagent is 1.2%
(W / V) picric acid solution. In the case of B, the first reagent is
0.1N sodium hydroxide aqueous solution, the second reagent is 1.
1% of each anionic surfactant in 2% (W / V) picric acid solution
% (W / V) is added. For comparison, both A and B also use a reagent to which an anionic surfactant is not added. The measurement method is as follows: 1.6% (W / V) albumin solution 0.07 m in the first reagent 2.
After adding 8 m and 0.7 m of the second reagent and heating at 37 ° C. for 10 minutes, the absorbance at 505 nm is measured using the reagent blank as a control. At the same time, a substance with a known creatinine value is similarly measured as a standard substance, and the error caused by albumin is converted into a creatinine value to obtain the value.

The effect of the anionic surfactant of the present invention on albumin is very excellent and does not require deproteinization.

Example 3 Comparison of measured values and reaction sensitivities of the present invention and a nonionic surfactant-containing reagent (conventional method) in serum creatinine measurement Table 3 shows measured values and reaction sensitivities of this method and conventional methods. . As is clear from Table 3, this method has higher reaction sensitivity than the conventional method in which sodium lauryl sulfate is used in combination with a nonionic surfactant. The correlation between the creatinine conversion values of both measurement methods is good.

It should be noted that the improvement in the value of the present invention with respect to the value of the comparative example of the reaction absorbance shown here is around 10%, but this difference is a decisively significant difference in this technical field.

<Measurement method> The conventional method and the present measurement method are the same, and 2m of the first reagent and 2m of the second reagent (and 2m of the blind reagent instead of the second reagent for serum blind test) are added to 50μ of serum. Add 1 at 25 ° C
After standing for 0 minutes, the reaction absorbance at 520 nm is determined. Further, the same operation is performed for the standard substance, and the serum creatinine value is calculated from the obtained absorbance.

<Reagent> 1. Conventional method: The reagent preparation example of Japanese Patent Publication No. 60-38666 was used as it was.

The first reagent is 0.5N sodium hydroxide solution. The second reagent is 0.2% (W / V) picric acid solution with sodium lauryl sulfate 0.8% (W / V), polyoxyethylene lauryl ether 0.4% (W / V), sodium benzoate. 0.1% (W /
What was added so that it might become the density | concentration of V) was used. For serum blind test, sodium lauryl sulfate 0.8% (W / V), polyoxyethylene lauryl ether 0.4% (W / V), sodium benzoate 0.1% (W / V). An aqueous solution added to the concentration was prepared.

2. This method: The first reagent is 0.5N sodium hydroxide solution. As a second reagent, 0.2% (W / V) picric acid solution, lithium lauryl sulfate, sarcosinate LN, NES-202, NE
Five kinds of S-303 and Surf Royal KTW were added to each to have a concentration of 0.8% (W / V). Also, 0.8% (W / V) aqueous solution of each anionic surfactant was prepared for serum blind test.

"Effect of the Invention" As is apparent from the above, according to the present invention, by using a specific anionic surfactant, the side reaction of picric acid and protein can be effectively prevented, and a nonionic surfactant is used in combination. It is possible to provide a reagent that enables simple, accurate and highly sensitive creatinine measurement without the need.

Claims (3)

[Claims] 1. A Folin creatinine reagent containing a first reagent composed of an alkaline aqueous solution and a second reagent composed of picric acid as a main component, wherein polyoxyethylene alkyl ether sulfate, lauroyl sarcosine sodium, and lauryl are used as side reaction inhibitors. A reagent for measuring creatinine containing one selected from the group consisting of lithium sulfate and fatty acid sodium tauride. 2. The first reagent comprising an alkaline aqueous solution containing one of polyoxyethylene alkyl ether sulfate and sodium lauroyl sarcosine, and the second reagent comprising picric acid. The creatinine measurement reagent according to the item. 3. The first reagent, wherein the reagent comprises an alkaline aqueous solution.
The second reagent comprising the reagent and picric acid containing one of polyoxyethylene alkyl ether sulfate, sodium lauroyl sarcosine, lithium lauryl sulfate, and sodium fatty acid tauride, according to claim 1. Creatinine assay reagent.