JPH0218073B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for preprocessing a specimen. More specifically, the present invention relates to a method of eliminating ammonia present in a sample such as urine or blood in advance when quantifying a target substance present in the sample using an ammonia production system.

Conventionally, substances that produce ammonia as a reaction product present in samples such as urine and blood derived from living organisms,
For example, to quantify creatinine, urea, etc., use a reagent that specifically reacts with creatinine or urea, such as alkaline pyrophosphate (for creatinine:
There was a method of adding diacetyl monooxime (in the case of urea: Fearon reaction) and measuring the absorption maximum of the substance produced by the chemical reaction. However, this chemical coloring method is undesirable because it has the disadvantage that there are many substances in the specimen that exhibit the same coloration as the target substance.

Another method is to convert creatinine or urea in a sample to ammonia using an enzyme that converts creatinine or urea into ammonia, and then determine the amount of creatinine or urea by quantifying the ammonia produced. However, although this method is simple, it has the disadvantage that it cannot be accurately quantified because a large amount of ammonia is already mixed in the sample.

The present inventors have arrived at the present invention as a result of research into the quantitative determination of a substance that generates ammonia as a reaction product in a sample containing ammonia.

That is, in quantifying a target substance in a sample, the present invention injects glutamate dehydrogenase (hereinafter referred to as
GlDH), 2-oxoglutaric acid (hereinafter referred to as α-KG), reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADPH),
Then, ^an enzyme and a substrate that reduce nicotinamide adenine ^dinucleotide phosphate (hereinafter referred to as ^NADP It is characterized in that it is converted back into NADPH using a reducing enzyme. Furthermore, a method for pretreatment of a specimen is provided, which is characterized by adding and mixing urease.

The feature of the present invention is that ammonia already present in the sample can be converted into GlDH, α-KG,
It is converted into glutamic acid and water by NADPH, and the NADP ⁺ generated at that time is converted back to NADPH using an enzyme that reduces NADP ⁺ .Furthermore, urea is changed to ammonia by urease. It is converted into glutamic acid and water, and the NADP ⁺ generated at that time is converted into NADPH again.
The point is to convert it into

Here, an example of the reaction system for ammonia elimination of the present invention is represented by formula (1).

In the present invention, in order to eliminate ammonia present in a sample in advance, GlDH is first required in the existing system that generates water and glutamic acid from ammonia and α-KG. This reaction system requires the presence of NADPH as a coenzyme. However, since this NADPH also affects ammonia, which is a reaction product that will be quantified later, it is not possible to add a sufficient amount necessary to eliminate the ammonia already present in the sample in advance.

Therefore, in the present invention, in order to add NADPH to an extent that does not affect the quantitative determination of the target substance, NADP ⁺
By coexisting in the reaction system an enzyme that reduces NADP + and a substrate, NADP ⁺ is converted to NADPH,
The above problem was solved by minimizing the amount of NADPH added. In other words, in order to reduce the amount of NADPH added, an enzyme that reduces NADP ⁺ such as glucose-6-phosphate dehydrogenase (hereinafter referred to as G-6-PDH), which reduces NADP ⁺ produced in the reaction, is added to excess glucose -. 6-phosphoric acid (hereinafter referred to as G-
6-P) is added together with an enzyme reaction substrate that reduces ^NADP It converts it into water and glutamic acid.

In the reaction of formula (1), when NADPH becomes NADP ⁺ due to the change of glutamic acid from α-KG,
Although the absorbance at 340nm decreases once, G-6
−To be changed back to NADPH by PDH
The absorbance at 340 nm 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 NADP ⁺ is GlDH.
The enzyme is not limited to -6-PDH, and any enzyme can be selected as long as it reduces NADP ⁺ as a coenzyme.

For example, for NADP ⁺ , G-6-PDH
[EC1.1.1.49], 6-phosphogluconate dehydrogenase (hereinafter referred to as 6-PGDH) [EC1.1.1.44], isocitrate dehydrogenase (hereinafter referred to as iCDH)
[EC1.1.1.42], etc., and when using these, G-6-P, 6PG, and isocitric acid may be selected and added as excess substrates, respectively.
Furthermore, urea produced as a reaction product of ammonia, which is present in large amounts in the specimen, can be eliminated by adding urease as a pretreatment of the specimen. This is effective when the enzyme used to quantify the target substance is a crude enzyme containing urease.

In this way, the ammonia already present in the sample was eliminated, and the ammonia produced by the action of the enzyme that produces ammonia from the target substance could now be directly measured.

In this way, the present invention makes it possible to directly quantify the substance that generates ammonia in the same reaction system by eliminating the ammonia present in the sample in advance in quantifying the substance that generates ammonia in an ammonia-containing sample. This method is extremely suitable for automatic analysis of substances that produce ammonia.

Next, examples of the present invention will be shown.

Example 1 (For quantification of creatinine) α-KG 4.2mM NADPH 0.013mM G-6-P 3.2mM G-6-PDH (derived from yeast) 3.2u/ml GlDH (derived from beef liver) Contains 38u/ml or more 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℃ for 5 minutes, the absorbance at 340 nm was measured, and when the absorbance stopped changing, 72μ of 5mM NADH was added, and after tracking the increase in absorbance at 340nm for about 2 minutes, 50μ of creatinine deiminase was added. and the decrease in absorbance at 340 nm was measured.

ΔE; A=0.042 B=0.084 C=0.168 D=0.335 It was hot.

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

Amount of creatinine mg/ml = ΔE / 6.2 × 3.142 / 0.02 × 113 / 1000 ΔE = Decrease in absorbance due to decrease in NADH 6.2 = Absorbance of 1mM of NADH 3.11 = Total reaction volume 0.02 = Sample amount 113 = Molecular weight of creatinine Example 2 (For quantification of creatinine using crude enzyme solution of creatinine deiminase) α-KG 4.2mM NADPH 0.013mM G-6-P 3.2mM G-6-PDH (derived from yeast) 3.2u/ml GlDH (cow) 0.1M Tris-HCl buffer (derived from liver) containing 38u/ml or more (PH
7.5) Creatinine-containing sample adjusted to contain 2mM ammonia and 2mM urea in 3ml (0.48ml)
mg/ml) was added. After incubating this at 25℃ for 5 minutes, the absorbance at 340 nm was measured. When the change in absorbance stopped, 5mM NADH 72μ was added, the increase in absorbance at 340nm was followed for about 2 minutes, and then 20u/ml creatinine deiminase was added. crude enzyme solution
50μ was added and the decrease in absorbance at 340nm was measured. The change in absorbance at this time was 0.326. This change in absorbance showed a value nearly twice the creatinine content.

On the other hand, when a creatinine-containing sample was added and 10 u of urease was added per sample, the same operation was performed and the decrease in absorbance at 340 nm was measured to be 0.168. This absorbance change was consistent with the creatinine content.

Claims (1)

[Scope of Claims] 1. In quantifying a target substance in a sample, glutamate dehydrogenase, 2-oxoglutaric acid, reduced nicotinamide adenine dinucleotide phosphate, and nicotinamide adenine dinucleotide phosphate are added to the sample. A reducing enzyme and a substrate are added and mixed to eliminate ammonia already present in the sample, and the nicotinamide adenine dinucleotide phosphate produced at this time is reduced again using an enzyme that reduces nicotinamide adenine dinucleotide phosphate. 1. A method for pretreatment of a specimen, characterized by converting it into type nicotinamide adenine dinucleotide phosphate. 2. The method for pretreatment of a specimen according to claim 1, which further comprises adding and mixing urease.