JPS626700A - Measurement of biological substance using ammonia as reaction product - Google Patents

Abstract

PURPOSE:To accurately measure a biological substance using NH3 or urea as a reaction product, by terminating completely the reaction of isocitric dehydrogenase (iCDH) and measuring the formed NH3. CONSTITUTION:GlDH (glutamic dehydrogenase), alpha-KG (alpha-ketoglutaric acid), NADPH (reduced form of nicotinamide adenine dinucleotide phosphate), isocitric acid, metallic ion, e.g. Mg or Mn, and iCDH (isocitric dehydrogenase) and, as necessary, urease are added and mixed with a specimen, e.g. urine or blood serum, to consume NH3 or NH3 and urea already present in the specimen. A chelating agent, e.g. ethylenediaminetetraacetate, is added to terminate the reaction of the iCDH. An enzyme for producing NH3 or urea as a reaction product is then added to the specimen to measure the formed NH3.

Description

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

More specifically, the present invention relates to a method for accurately measuring biological substances in which ammonia or ammonia and urea already present in a specimen are consumed in advance and ammonia or urea is a reaction product.

In general, it is common practice to detect urea, creatinine, creatine, guanine, adenosine, etc. present in samples such as urine and serum, 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 glutamic acid membrane hydrogen chloride (G, O01l), and at this time, reduced nicodineamide adenine dinucleotide phosphate is converted into glutamic acid. (NADPII) ゛→Nicotine amimidodenin dinucleotide phosphate (NADPII)
The amount of NADPII decreased by the conjugation reaction of DP'' was quantified by measuring at 340°C.

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

Therefore, in the first place, the ammonia present in the sample was pretreated with F.
If 1+ reacts with α-ketogeltaric acid (α-KG) using Gjl [111 and converts it into glutamic acid, the problem will disappear. Since this ammonia → glutamic acid system involves a change from NADPII → HAOP+, it is necessary to return it to NADPH through the reverse reaction of NADP+-)NADPII. A conjugation reaction can be caused by the metal ions of. This reaction system is expressed by the following formula (% formula %). Once the ammonia in the urea has been consumed and the NADP''-→NADPII reaction has completely stopped, the amount of ammonia obtained by decomposing urea can be determined accurately.Therefore, the problem arises.The formula ( 1) NADP II in 1)
The question was how to completely stop the reaction of NADP+→N8[1PII. Conventionally, NADP-! −
→It was not known to completely stop only the NAOPH reaction.

The present inventors completely stopped the reaction of G with isocitric acid π → α- in the above formula (I) and formula (TI),
We conducted extensive research in search of a method to accurately measure biological substances that have ammonia or urea as reaction products, and found that the addition of a chelating agent completely suppressed the reaction between NADP and NADPI+. The present invention succeeded in stopping the sample with (dl DI+, α-G, NADP
H.

Isocitric acid, metal ions such as magnesium ions or manganese ions, and iCDH are added to consume the ammonia already present in the sample, and then a chelating agent is added to stop the 1cDII reaction and simultaneously or subsequently generate the reaction. This is a method for quantifying biological substances with ammonia as a reaction product, which is characterized by adding N element which causes ammonia to be produced as a substance and measuring the produced ammonia. In addition, the present invention provides 010 mouths ((,
α-KG, NADP[1, isocitrate, sq24
, Add and mix phrease and 1cDII to consume ammonia J3 and r/A'i/i already present in the sample, then add a chelating agent to stop the 1cDII reaction, and simultaneously or after this reaction. This is a method for determining biological materials with urea as a reaction product, which is characterized by adding an enzyme or enzyme group that produces urea as a product and measuring ammonia produced.

Here, metal ions include magnesium ions, manganese ions, iron ions, copper ions, zinc ions, tin ions,
It refers to calcium ions, etc., but is not limited to these ion types.

In addition, chelating agents include EDT^ and its salts, 1.2-
Bis(0-amylaf-1noxy)ethane-N,N,N'
, N'-tetraacid and salt of I, trans-1,2 cyclo[
1-hexyldiamine-N,N,N',N'-tetraacid and its salts, dihydroxyedylglycine and its salts, 1,3-diamituprobanol-N,N,N',N
'-Tetraacic acid and its salts, diethylenetriaminepentaacetic acid and its salts, ethylenediaminediorthohydroxyphenylacetic acid and its salts, ethylenediaminediacetic acid and its salts, ethylenediaminenibropionic acid and its salts, human OJ- Shear ethylene diamine triacetic acid J3 and its salts, ethylenediaminetetrakis (methylenephosphonic acid) and its salts, glycol ether diamine diacetic acid and its salts, hydroxyethyliminodiacetic acid and its salts, iminodiacetic acid and its salts, diaminopropane tetraacetic acid and its salts, nitrilotriacetic acid and its salts,
These include nitrilotripropionic acid and its salts, nitrilotris (medylenephosphonic acid) and its salts, triethylenetetraminehexaacetic acid and its salts, etc.
: Not limited to rate agents.

α - to G α - to G ↑ Ammonia or phosphoric acid (III>
The present invention uses the above formula (II) 4 when adding a chelating agent.
This causes the change to the formula (I[[). In other words, the complete consumption of an[nia] and/or urea in the sample is expressed by the formula (II).
After complete consumption, a chelating agent is added to the reaction system to stop the reaction from NADP-π plate to NA[]Pl+.

After stopping the iCDH reaction, at the same time as or after the addition of the chelating agent, add an enzyme that generates ammonia in the sample or an enzyme that generates urea or a group of P' elements to convert ammonia to glutamic acid. 340n associated with NADPH→NADP+ reaction as military service reaction
Each substance is quantified by the decrease in absorbance of m.

The following formula (IV) is shown as a reaction between annia and the enzyme to be produced.

That is, urea is determined by urease, creatinine is determined by deimibouge, guanine is determined by guanage, and adenosine is determined by adenosine deamipase.

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 M element group that produces urea.

That is, in a system in which urea has been consumed in J, creatine is determined by creatinine, arginine is determined by full 1-bouze, and creatinine is determined by taleadipus and creatininase. .

In the present invention, the analyte in the sample is decomposed and NAOP1
Accurate quantification of the analyte is performed by consuming NΔKan 11 through the reaction of 1→N8DP+.

Termination of the 1cDH reaction with a rate agent used in the present invention can be performed using the same medium in which the reaction was terminated with NΔ0P.
It is extremely useful in that various reactions can be performed using the 11→NADP' reaction.

A chelating agent such as EDTA may be added to the reaction system in an amount of 5 mM or more.

FIG. 1 shows the influence of 111 degrees of EDTA on iC activity, and when EDTA is 5n+H, 1cDtl completely loses its activity.

Next, examples of the invention will be shown.

Example 1 G to α-! lmM NADPII 012mM Isoquene M
5 mH HaC, 1120, 2n14 (J Ro II 20u/
dicDH2u/Il! l! 0, IM Tris-HCl (pl+7.5
) Urea-containing samples adjusted to various Takimaro containing 160 mN ammonia in 2.4 d (urea state - nitrogen 0.~60
0ma/d1) 30/μλ was added. 37℃ each
After incubating for 5 minutes, add EDTA and urease so that the EDTA and urease concentrations are 5mM and 0.1u/me, respectively.
, urease mixture at 0.6ati! In addition, urea in the sample was measured using a spectrophotometer based on the decrease in absorption at 340 nm for 1 minute at 37°C.

The measurement results are shown below.

Sample number 1 234567 Urea state-N Theoretical value 600500400300200100
0 (mQ/dfJ) Measured value 585502405298201 98
01no/djl) It became possible to quantify urea in the test liquid without being affected by the high concentration of ammonia present in the sample.

[Brief explanation of drawings]

Figure 1 shows the effect of EDTA on iCDH activity? It is a diagram looking at the influence of rJ degree.

Claims (2)

[Claims] (1) Add and mix Gl DH, α-KG, NADPH, isocitrate, metal ions such as magnesium ions or manganese ions, and iCDH to the sample to consume ammonia already present in the sample, and then add a chelating agent. , a method for quantifying biological substances that uses ammonia as a reaction product, which comprises stopping the iCDH reaction, adding an enzyme that produces ammonia as a reaction product at the same time or after that, and measuring the produced ammonia. . (2) Add and mix Gl DH, α-KG, NADPH, isocitrate, metal ions such as magnesium ion or manganese ion, urease and iCDH to the sample to consume ammonia and urea already present in the sample, and then chelate urea as a reaction product, characterized in that the iCDH reaction is stopped 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. A method for quantifying biological substances.