JPS61247963A - Quantitatively determining method for vital material with ammonia as resulted product of reaction - Google Patents

Abstract

PURPOSE:To measure exactly a vital material with ammonia or urea as the resulted product of reaction by consuming the ammonia which exists already in a specimen then adding ATP or/and chelate agent to stop iCDH reaction, adding enzyme to form ammonia to the specimen and measuring the formed ammonia. CONSTITUTION:GlDH, alpha-KG, NADH, isocitric acid, metallic ions such as magnesium ions or manganese ions and iCDH are added and mixed to and with the specimen to consume the ammonia which exists already in the specimen. The ATP or/and chelate agent are then added to the specimen to stop the iCD reaction. The enzyme to form ammonia as the resulted product of reaction is added simultaneously with or after said stop and the formed ammonia is measured. The change from the formula I the formula II is made by the addition of the ATP or/and chelate agent. The reaction of the enzyme to form ammonia is exemplified by the formula III.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying biological substances using ammonia or urea as a reaction product.

More specifically, the present invention relates to a method for accurately measuring a biological substance that uses ammonia or urea as a reaction product by consuming ammonia or ammonia and urea already present in a sample in advance.

In general, it is common practice to detect urea, creatinine, creatine, guanine, adenosine, etc. present in samples such as urine and blood, and to measure the activities of various enzymes involved in these substances.

In detection of these substances and enzymatic reactions, ammonia is generated, and the generated ammonia is converted to glutamic acid by GQOH (glutamic acid dehydrogenase), and at this time, NADH (fi archetypal nicotinamide adenine dinucleotide) → NAD. The amount of NADH decreased by the conjugation reaction of cotinamide adenine dinucleotide) was quantified by measuring at 340 nm.

However, since ammonia is always produced in this reaction system, the ammonia present in the sample is included in the measured value, making accurate quantification difficult.

Therefore, in the first place, the ammonia present in the sample is converted into α-KG (α-ketoglutaric acid) by GΩDH in pretreatment.
The problem disappears if it is converted to glutamic acid by reacting with glutamic acid. Since this ammonia → glutamic acid system involves a change from NADH → NAD+, it is necessary to reverse the reaction of NAD+ → NADH to return it to NAD)I, using isocitrate as a substrate.
isocitrate dehydrogenase) and metal ions such as magnesium ions or manganese ions. This reaction system is shown in the following formula (1).

As shown in equation (1), the consumption of ammonia in the sample and the measurement of ammonia obtained by decomposing urea can be performed by the same coupled reaction, but once the consumption of ammonia in the sample is completed, the NAD ”→Accurate quantification of ammonia obtained by decomposing urea is only possible when the NADH reaction is completely stopped.

Therefore, the problem is that NADHd in equation (1)
The challenge was how to completely stop the reaction of NAD"→NADH in NAD+. Conventionally, NAD"→NADH reaction was completely stopped.
It has not been known to completely stop the DH reaction.

The present inventors sought a method to completely stop the reaction of incitric acid → αKG in the above-mentioned formulas (1) and (If), and to accurately measure biological substances whose reaction products are CDH, ammonia, or urea. After intensive research, I found that ATP
Or/and by adding a chelating agent, they succeeded in completely stopping the CDH reaction.

In the present invention, the specimen contains GQDH1α-KG, NA[lH,
Isocitric acid, metal ions such as magnesium ions or manganese ions, and 1cDll are added and mixed to consume the ammonia already present in the sample, then ATP or/and chelating agent is added to stop the 1cDH reaction, and at the same time Alternatively, this is a method for quantifying a biological substance using ammonia as a reaction product, which is characterized by adding an enzyme that produces ammonia as a reaction product after that and measuring the produced ammonia.

In addition, the present invention includes GI20H1α-KG, NAD
H, isocitrate, metal ions such as magnesium or manganese ions, urease and 1CDH are added and mixed to consume the ammonia and urea already present in the sample, then ATP or/and chelating agent is added to initiate the 1cDll reaction. A method for quantifying biological substances with urea as a reaction product, characterized by adding an enzyme or a group of enzymes that produce urea as a reaction product at the same time or after that, and measuring the ammonia produced. be.

Here, the metal ion is a magnesium ion.

Examples include manganese ions, iron ions, copper ions, zinc ions, tin ions, calcium ions, etc., but the ion species are not limited to these.

In addition, chelating agents include EDTA and its salts, 1,2-
Bis(0-aminophenoxy)ethane-N, N, N
', N'-tetraacetic acid and its salts, trans-1,2-
Cyclohexanediamine-N, N, N', N'-
Tetraacetic acid and its salts, dihydroxyethylglycine and its salts, 1,3-diaminopropanol-N, N,
N', N-tetraacetic acid and its salts, diethylenetriaminepentaacetic acid and its salts, ethylenediaminediorthohydroxyphenylacetic acid and its salts, ethylenediaminediacetic acid and its salts, ethylenediaminenipropionic acid and its salts, hydroxyethylethylenediaminetriacetic acid and its salts salt, ethylenediaminetetrakis (methylenephosphonic acid) and its salts, glycol ether diaminetetraacetic acid and its salts, hydroxyethyliminodiacetic acid ° and its salts, iminodiacetic acid and its salts, diaminopropanetetraacetic acid and its salts, nitrilotriacetic acid and its salts, nitrilotripropionic acid and its salts, nitrilotris (methylenephosphonic acid) and its salts, triethylenetetraminehexaacetic acid and its salts, etc.

There is no limitation to these chelating agents.

Ammonia or ammonia or the present invention changes the formula (II) to formula (m) by adding ATP or/and a chelating agent. That is, complete consumption of ammonia and/or urea in the sample is performed according to formula (II), and after complete consumption, ATP is added to the reaction system.
Or, after adding ATP and/or a chelating agent to stop the LCDH reaction, an enzyme that generates ammonia in the sample or an enzyme that generates urea at the same time or after the addition of ATP and/or a chelating agent. The substance is quantified based on the decrease in absorbance at 340 nm accompanying the NADH→NAD+ reaction as a conjugate reaction from ammonia to glutamic acid.

The enzyme reaction that produces ammonia is expressed by the following formula (
IV) is shown.

That is, urea is quantified by urease, creatinine is quantified by deaminase, guanine is quantified by guanase, and adenosine is quantified by adenosine deaminase. These reactions of the present invention also include measurement of the activities of these enzymes.

Further, the following formula (V) is shown as a reaction of an enzyme or enzyme group that produces urea.

That is, in a system in which urea has already been consumed, creatine is determined by creatinase, arginine is determined by arginase, and creatinine is determined by creatinase and creatininase. Furthermore, these reactions of the present invention also include measurement of the activities of these enzymes.

In the present invention, the analyte in the specimen is decomposed.

NADH is consumed through the reaction of "NADH→NAD" to accurately quantify the analyte.

Termination of the LCDH reaction using ATP or/and a chelating agent used in the present invention is extremely useful in that various reactions can be carried out using the NADH-+NAD+ reaction in the same medium in which one reaction was terminated.

The amount of ATP added to the reaction system may be 15d or more. Figure 1 shows the effect of ATP concentration on 1CDH activity.
It can be seen that DH has completely lost its activity.

Further, the amount of the chelating agent, such as EDTA, added to the reaction system may be 10 mM or more. Figure 2 shows the influence of EDTA concentration on 1cDH activity.

It can be seen that 1CDH completely loses its activity when the EDTA concentration is 10 mM or higher.

Next, examples of the present invention will be shown.

Example 1 α-KG 10mM NADHO, 16m isocitrate 5mM ADP 0 , 5mM MgCQ2 1mM GQDH 100u/m 41 iCDH 2u/m 0 or more containing 0, 1M phosphate buffer (pH 7,5) 2
．． Urea-containing samples adjusted to various concentrations containing 16On+M ammonia in 4mQ (θ ~ 100 as urinary nitrogen)
0 mg/dρ) 30 μg was added. 5 at 37℃ each
After incubating for minutes, the ATP and urease concentrations were each 2.
Add 0.6 taQ of ATP and arease mixture to 0mM and 0.1u/mM, and measure the urea layer-nitrogen in the sample from the decrease in absorption at 340nm for 1 minute at 25°C using a spectrophotometer. Shown below.

Sample number 1 2 3 4 5 6 7
8 9 10 11 Example 2 α-KG 10dNAD)I
O, 16mM inc citric acid 5m
M ADP 0.5 shoes M
gCO21mM (JDH100u/mQ iCDH2u/an 0, 1M phosphate buffer (pH 7,5) 2
．． Urea-containing specimens adjusted to various concentrations including 1601 ammonia at 4 mΩ (urea layer - O ~ 1000 m as nitrogen)
g/dfl) 30 μQ was added. After incubating each at 37°C for 5 minutes, the EDTA and urease concentrations were adjusted to 1.
Add 0.6+o12 EDTA and urease mixture to 0mM and 0.1u/mQ and measure at 25℃ using a spectrophotometer.
The measurement results of the urea layer-nitrogen in the sample were measured from the decrease in absorption at 340 nm for 1 minute are shown below.

Sample number 1 2 3 4 5 6 7
8 9 10 11 Example 3 α-G 10mM NADH0, 2mM Isocitric acid 10IIIM ADP 1++MMg(j
h O,4raMGlDH,00
u/+Q iCDH4u/mf1 containing 0,1M Tris-HCl (pH7,5)2
．． Creatinine-containing specimens (A = 12 mg/d) adjusted to various concentrations containing 100 mM ammonia in 88 m12
L B =24mg/dQ, C=48mg/dfl, D
= 96IIg/dQ) 20μQ was added. After incubating each at 37°C for 10 minutes, add a mixture of ATP and creatininase to 100μΩ so that the ATP and creatinine deiminase concentrations are 20mM and 3u/mfl, respectively.
Addition.

The absorbance change at 340 nm at 37° C. was measured using a spectrophotometer.

ΔE; A=0.044, B=0.087, C=0.
175, D=0.352.

As a result of calculating this using the following formula, the ammonia 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.

Creatinine amount ΔE = Absorbance change due to decrease in NADH 6.2 = N
Absorbance of 1mM of ADH 3, OO = total reaction volume (an) o, o! = Sample amount (mfl) l! 3 = Molecular weight of creatinine

[Brief explanation of the drawing]

FIG. 1 shows the effect of ATP'a concentration on 1CDH activity, and FIG. 2 shows the effect of EDTA concentration on 1cDH activity. Agent Patent Attorney 1) Parent Male EDTAJJI mm

Claims (2)

[Claims] (1) GlDH, α-KG, NADH, isocitrate, metal ions such as magnesium ion or manganese ion, and iCDH are added and mixed to the sample to consume ammonia already present in the sample, and then ATP or/and chelating agent A biological material containing ammonia as a reaction product, characterized in that the iCDH reaction is stopped, and at the same time or after that, an enzyme that produces ammonia is added as a reaction product, and the produced ammonia is measured. Quantification method. (2) Adding and mixing GlDH, α-KG, NADH, isocitrate, metal ions such as magnesium ions or manganese ions, urease and iCDH to the sample,
Ammonia and urea already present in the sample are consumed, then ATP or/and chelating agent is added and iC
A biological material that uses urea as a reaction product, which is characterized by stopping the DH reaction, adding an enzyme or enzyme group that produces urea as a reaction product at the same time or after that, and measuring the produced ammonia. Quantification method.