JPH0218077B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying creatinine.

More specifically, the present invention relates to a method for directly quantifying creatinine present in a sample containing ammonia.

Conventionally, creatinine present in samples such as blood and urine derived from living organisms has been quantified using the alkaline picric acid reaction (Jaffe method), which is already present in the sample.
After removing substances that interfere with creatinine through dialysis treatment or adsorption treatment with a resin, quantify using the Jaffe method, or convert creatinine in the sample into creatine using an enzyme that converts creatinine to creatine. Creatinine was determined by various methods.

However, the dialysis treatment of the specimen is complicated and the specimen is diluted, and the adsorption treatment with the resin is complicated, both of which are undesirable. In addition, the method of converting creatinine to creatine and quantifying the produced creatinine is a multi-step reaction system that couples three or more reaction steps to quantify creatine.
In addition, it is not preferable that creatine in the sample must be eliminated in advance or that the sample must be blind tested, which is time-consuming and makes it impossible to measure if the amount of sample is small.

The present invention has been made in order to improve such conventional drawbacks in quantifying creatinine.

The present invention uses glutamate dehydrogenase (hereinafter referred to as GlDH), α-oxoglutaric acid (hereinafter referred to as α-OG), and reduced nicotinamide adenine dinucleotide (hereinafter referred to as α-OG) in a sample containing ammonia.
By adding an enzyme that reduces nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide (hereinafter referred to as NAD ⁺ ), and a substrate for the enzyme that reduces NAD ⁺ , the ammonia already present in the sample is converted into water and glutamic acid, and the ammonia produced at this time is The resulting nicotinamide adenine dinucleotide is converted to reduced nicotinamide adenine dinucleotide again using an enzyme that reduces nicotinamide adenine dinucleotide, and then an excess amount of creatinine deiminase and reduced nicotinamide adenine dinucleotide are added to the sample. Phosphate (NADPH)
This is a method in which creatinine is quantified by adding creatinine (called 340 nm) to cause a reaction, and determining the amount of decrease in NADPH by determining the decrease in absorbance at 340 nm.

The feature of the present invention is that ammonia already present in the sample is converted to glutamic acid by GlDH, α-OG and NADH, and then ammonia generated from creatinine by the reaction of creatine deiminase is converted to glutamic acid by the same GlDH and NADPH. The point is that it reacts and changes into glutamic acid and water.

According to the method for quantifying creatinine of the present invention, the ammonia already present in the sample is consumed in advance, so ammonia is directly generated from creatinine in the sample using creatinine deiminase added directly to the same sample. Since the total amount of ammonia produced can be measured as ammonia decomposed by creatine deiminase, the creatinine content can be measured accurately.

An example of the reaction of the ammonia consuming system of the present invention shown here is as follows when expressed by formula (1).

The method for quantifying creatinine of the present invention is used for quantifying creatinine in ammonia-containing specimens, such as serum and urine. Since large amounts of ammonia are constantly present in these samples, if a direct glutamate production reaction is used for measurement, a considerable amount of ammonia will be added to the creatinine amount, resulting in accurate quantitative values. It was something that could not have been done. In the present invention, after the ammonia in the sample is consumed by NADH, creatinine deiminase is made to act on creatinine in the sample, so that the ammonia generated therein is
The amount of NADP ⁺ is precisely measured as the amount of creatinine.

The method for quantifying creatine of the present invention can quantify creatinine in a sample containing ammonia by simply using an endpoint method, and can also quantify creatinine in a sample containing ammonia by selecting an appropriate amount of creatine deiminase.
Creatinine can be quantified using Rate Assay.

In the present invention, glutamate dehydrogenase (GlDH) (EC1.4.1.3), which generates water and glutamic acid from ammonia and α-ketoglutaric acid, is first required to consume the ammonia that already exists.

This reaction requires the presence of NADH as a coenzyme, but in order to reduce the amount of NADH added ^{, glucose}
6-phosphate dehydrogenase (G-6-PDH)
An enzyme that reduces NAD ⁺ such as (EC1.1.1.49) is added together with an excess of an enzyme reaction substrate that reduces NAD ⁺ such as glucose-6-phosphate (G-6-P). -While producing phosphogluconic acid (6-PG), ammonia is completely converted into water and glutamic acid by α-ketoglutaric acid.

In the reaction of formula (1), when NADH becomes NAD ⁺ due to the change from α-OG to glutamic acid, the absorbance at 340 nm decreases once, but because it changes back to NADH by G-6-PDH, the absorbance at 340 nm decreases. increases, and when there is no change in absorbance, it means that all the ammonia has been consumed.

Of the ammonia consuming group of the present invention, GlDH is essential, but the enzyme that reduces the coenzyme NAD ⁺ is G-6.
-Not limited to PDH, any enzyme can be selected as long as it reduces NAD ⁺ as a coenzyme. For example, G-6-PDH (EC1.1.1.49) (glucose-6-
6-P-GDH (EC1.1.1.44) (6-phosphogluconate dehydrogenase) iC-DH (EC1.1.1.42) (isocitrate dehydrogenase), etc. When used, G-6-P, 6-PG, and isocitric acid may be selected and added as excess substrates, respectively.

In this way, the ammonia already present in the sample was consumed, and the ammonia produced by the action of creatine deiminase became ready for direct measurement.

Next, the reaction system for quantifying creatinine in the present invention can be expressed by formula (2) as follows.

The thick line is related to the reaction of ammonia consumption system, and the thin line is related to the reaction of creatinine deiminase.

That is, by adding a substrate/enzyme and filtered NADPH according to the amount of creatinine to be determined to the sample and causing a reaction, the amount of creatinine can be adjusted to NADPH.
The amount of decrease can be determined by measurement at 340 nm. Here, α-
It is also possible to use OG and beef liver GlDH that were added to the initial ammonia consumption reaction system. If the initial amount added is small, additional amounts can be added here. It is also essential to add creatinine deiminase. Also
Addition of NADPH is essential;
The decrease at 340 nm caused by the change to NADP ⁺ allows the creatinine content to be determined.

The reaction is carried out at 25°C in a pH 7.5 buffer.
In the reaction of formula (2), creatinine is decomposed by creatinine deiminase and converted from ammonia to α
-When NADPH becomes glutamic acid with GlDH together with -OG, it becomes NADP ⁺ and accumulates.
At this time, it is feared that NADH, which was added to consume ammonia in the sample, will be oxidized in the same way as NADPH, and then returned to NADPH by iCDH. However, since NADH exists in a very small amount, for example, less than one-tenth of that of NADPH, the iCDH cycle can be ignored.

There is also a concern that iCDH, which is contained in the sample to consume ammonia, may reduce NADP ⁺ , but this is a concern because the enzyme contained here has been selected to have high specificity for coenzymes. is not required.

The creatinine content of the generated NADP ⁺ can be directly measured by the decrease in absorbance at 340 nm.

Further, urea can be determined in the same manner as creatinine. In other ammonia production reaction systems, substances that produce ammonia can be quantified in exactly the same way as creatinine.

As described above, the present invention enables direct quantification of creatinine in the same sample by consuming ammonia in the sample in advance in quantifying creatinine, and is an extremely suitable method for automatic analysis of creatinine.

Next, examples of the present invention will be shown.

Example For quantification of creatinine α-OG 4.2mM NADH 0.013mM Isocitric acid 3.2mM iCDH 3.2u/ml GlDH (derived from bovine liver) 0.1M Tris-HCl buffer (PH
7.5) Creatinine-containing samples adjusted to various concentrations containing 2mM ammonia in 3ml (A = 0.12mg/
ml, B=0.24mg/ml, C=0.48mg/ml, D=0.96
mg/ml) was added. After incubating at 25°C for 5 minutes, the absorbance at 340 nm was measured, and when the change in absorbance stopped, 5mM NADPH 72 μ was added, and after tracking the increase in absorbance at 340 nm for about 2 minutes, 50 μ of creatine deiminase was added.
The decrease in absorbance at 340 nm was measured.

ΔE; A=0.042 B=0.084 C=0.168 D=0.335.

As a result of calculating this using the following formula, the 2mM of ammonia that was already present in the sample was completely erased, and the creatinine content in the sample was determined without affecting the quantification of creatinine that was subsequently measured.

Creatinine amount mg/ml=ΔE/6.2×3.142/0.02×113/1000 ΔE = decrease in absorbance due to decrease in NADPH 6.2 = 1mM absorbance of NADPH 3.11=Total reaction volume 0.02=sample amount 113 = molecular weight of creatinine

Claims (1)

[Claims] 1. Add and mix glutamate dehydrogenase, α-oxoglutaric acid, reduced nicotinamide adenine dinucleotide, and an enzyme and substrate that reduce nicotinamide adenine dinucleotide to the sample,
The ammonia already present in the mixture is consumed, and the nicotinamide adenine dinucleotide produced at this time is converted again into reduced nicotinamide adenine dinucleotide using an enzyme that reduces nicotinamide adenine dinucleotide, and then A method for quantifying creatinine, which comprises adding and reacting reduced nicotinamide adenine dinucleotide phosphate and creatinine deiminase.