JPS6234061A - Method for quantifying creatinine - Google Patents

Abstract

PURPOSE:To accurately quantify creatinine, by preliminarily consume ammonia already present in a specimen. CONSTITUTION:AlDH (glutamic acid dehydrogenase), alpha-KG,NAD(P)H (reducing type nicotineamide adenine dinucleotide), isocitric acid, a metal ion such as a magnesium ion or a manganese ion and iCDH are added to and mixed with a specimen to consume ammonia already present in the specimen. Subsequently, ATP or/and a chelate agent are added to the specimen to stop iCDH reaction and, at the same time or thereafter, creatininase is added to measure formed ammonia and the quantification of creatinine in the specimen is enabled without receiving the effect of high concn. ammonia present in the specimen at all.

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 accurately measuring creatinine by pre-consuming ammonia already present in a sample.

Conventionally, in order to quantify creatinine present in samples such as blood and urine derived from living organisms, substances that interfere with the alkaline picric acid reaction (Jaffe method) that are already present in the sample are removed by dialysis treatment or adsorption with resin. After removal by treatment, creatinine can be determined by quantifying it using the Jaffe method, or by converting creatinine in the sample into creatine using an enzyme that converts it to creatine, and quantifying the produced creatine using various methods. It was being quantified.

However, the dialysis treatment of the specimen is complicated and has the disadvantage that the specimen is diluted, and the adsorption treatment with a resin also has the disadvantage that the operation is complicated, both of which are not preferred. In addition, the method of converting creatinine to creatine and quantifying the generated creatine has already been used for quantifying creatine.
It is a multi-step reaction system in which more than one reaction is coupled, and the creatine in the sample must be eliminated in advance or the sample must be blind tested, which is time consuming and requires a large amount of sample. If the amount was too low, it would be impossible to measure, which was not preferable.

In addition, in the detection of creatinine, ammonia is generated and the generated ammonia is used as Gl! Converted to glutamic acid by DH (glutamic acid dehydrogenase), and at this time N
The amount of NADll decreased by the coupling reaction of ADII (ifi archetypal nicotinamide adenine dinucleotide) → cotinamide adenine dinucleotide to NADH+) 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, if the ammonia present in the sample is reacted with α-KG (α-ketoglutaric acid) and converted into glutamic acid using GΩDH in a pretreatment, the problem will disappear. Since this ammonia-glutamic acid system involves the change NAD (P)H4NAD (P), NAD(P)+ → NAD (P)
It is necessary to return it to NAD(P)H by a reverse reaction of H, and in this case, a conjugation reaction can be caused by using isocitrate as a substrate and 1cDH (isocitrate dehydrogenase) and metal ions such as magnesium ions or manganese ions. . This reaction system is shown in the following formula ([).

Creatinine ↓ Creatininase As shown in the formula (■), the measurement of ammonia obtained by consumption and decomposition of ammonia in the sample can be performed by the same coupled reaction, but the consumption of ammonia in the sample is completed. Only after the reaction of NAD(P)+→NAD(P)H is completely stopped can accurate quantification of ammonia obtained by decomposing creatinine be performed.

Therefore, the problem is that NAD(P
)H=NAD(P)+ at NAD(P)+→NA
The question was how to completely stop the D(P)H reaction. Conventionally, it was not known to completely stop the reaction of NAD(P)+→NAD(P)H.

The present inventors completely stopped the reaction of incitric acid → αKG in the above formulas (I) and (n), and iCD
We conducted extensive research to find a method for accurately measuring biological substances that use ammonia as a reaction product, and found that by adding ATP or/and a chelating agent.

NAD(P)“ NAD(P)HcDIl They succeeded in completely stopping the reaction.

The present invention provides G Q D l (, α-KG, NAD
(P)H, isocitrate, metal ions such as magnesium ions or manganese ions, and iC leaves are added and incubated to consume the ammonia already present in the sample, then ATP or/and chelating agent is added and 1 cD
This is a method for quantifying creatinine, which is characterized by stopping the II reaction, adding creatininase at the same time or after that, and measuring the ammonia produced.

Here, the metal ions include magnesila 11 ions, manganese ions, iron ions, copper ions, zinc ions, tin ions, calcium ions, etc., but are not limited to these ion types.

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 (methiI)
nonphosphonic acid) and its salts, glycol ether diamine tetraacetic 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, nitrilotripropion Examples include acids and salts thereof, nitrilotris (methylenephosphonic acid) and salts thereof, triethylenetetraminehexaacetic acid and salts thereof, but are not limited to these chelating agents.

In the present invention, the above formula (n) is transformed into formula (III) by adding ATP or/and a chelating agent. That is, complete consumption of ammonia in the sample is performed according to formula (n), and after complete consumption, ATP or/and a chelating agent is added to the reaction system, and NAD(P)+→NAD(P)Hi
It stops the reaction of cDl (.

After stopping the iC leaf reaction, creatininase is added to the sample at the same time or after the addition of ATP and/or chelating agent, and NAD(P)II→NAD( P
) Each substance is quantified by the decrease in absorbance at 340 nm accompanying the reaction of +.

The reaction is shown by the following formula (■).

In the present invention, creatinine in the sample is decomposed and N
NAD by the reaction of AD(P)H→NAD(P)+
(P)H is consumed to accurately quantify the analyte.

Termination of the iCDH reaction using ATP or/and a chelating agent used in the present invention is extremely useful in that various reactions can be performed using the reaction of NAD(P)H → NAD(P)'' in the same medium in which the reaction has been terminated. be.

The amount of ATP added to the reaction system may be 151 or more.

Further, the amount of the chelating agent 2, such as EDTA, added to the reaction system may be 1 mM or more.

Next, examples of the present invention will be shown.

Example 1 α-KG 10mM NADH0, 16mM Isocitric acid 5mM ADP 0.5mM MgCf12 0.5mMC+QDH50
u/mQ icDfl 2 0, 1M triethanolamine hydrochloric acid (pH 7,5) containing more than 2 u/mQ
Creatinine-containing specimens (0 to 100 mg/
dQ) 30 μQ was added. After incubating each at 37°C for 5 minutes, the ATP and creatininase concentrations were 20% each.
Add 0.6 mu of ATP and creatininase mixture to give a concentration of 0.3 u/mρ at 37°C using a spectrophotometer.
Creatinine in the sample was measured from the decrease in absorption at 340 nm for 1 minute. The measurement results are shown below.

Sample number 1 2 3 4 5 6 7
9 10 11 Example 2 α-KG10mM NADII 0.2mM incitric acid
5mM ADP 0.5mM MgCQ2 0.5mM GQDII 50 u/muiCDH2
0,1M triethanolamine hydrochloric acid (p
)17.5) Creatinine-containing samples adjusted to various concentrations (0 to 1) containing 150mM ammonia in 2.4mQ
000mg/d+2) 30μQ was added. 37 each
After incubating at ℃ for 5 minutes, 0.6 mQ of EDTA and creatininase mixture was added so that the EDTA and creatininase concentrations were 5IIIM and 0.3 u/mfl, respectively. Creatinine in the sample was measured from the decrease in absorption. The measurement results are shown below.

Sample number 1 2 3 4 5 6 7
9 10 11 Example 3 α-KG 10mM NADPHO, 2mM Incitric acid 10mM MgCI22 0.5mM GflDll 20 u/mQiCDH
0,1M triethanolamine hydrochloric acid (p
H7,5) Creatinine-containing samples adjusted to various concentrations including 2.4mM and 150mM ammonia (0 to 10
0mg/dQ) 30μQ was added. 5 at 37℃ each
After incubating for minutes, add IEDTA so that the EDTA and creatininase concentrations are 5mM and 0.3u/mQ, respectively.
0.6 creatininase mixture was added, and the creatinine in the sample was measured using a spectrophotometer from the decrease in absorption at 340 nm for 1 minute at 37°C. The measurement results are shown below.

Sample number 1 2 3 4 5 6 7
9 10 11 It became possible to quantify creatinine in the test liquid without being affected by the high concentration of ammonia present in the sample.

Claims (1)

[Claims] (1) GlDH, NADH (or NADPH) in the sample,
Isocitric acid, a metal ion such as magnesium or manganese ion, and iCDH are added and mixed to consume the ammonia already present in the sample, then ATP or/and a chelating agent is added to stop the iCDH reaction and A method for quantifying creatinine, which comprises adding creatininase at the same time or afterward and measuring the ammonia produced.