JP2937396B2 - Reagent for measuring creatine kinase and pyruvate kinase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent for simultaneous measurement of creatine kinase (hereinafter abbreviated as CK) and pyruvate kinase (hereinafter abbreviated as PK) in a biological fluid.

(Prior art) CK is present in the brain and muscle tissue, and is used for the purpose of diagnosing muscular disease, neurological disease, heart disease, etc. in the field of clinical examination.
Activity is routinely measured. CK is an enzyme that catalyzes the reaction in both the left and right directions in equation (1).

(The abbreviations are CrP: creatine phosphate, Cr: creatine, ADP: adenosine diphosphate, ATP: adenosine triphosphate.) PK is distributed to the heart muscle, skeletal muscle, and brain in vivo. However, in recent years, studies have been reported that the measurement of PK is applied to the diagnosis of myocardial infarction and muscular disease, and has attracted attention. This is because other measurement items for diagnosing myocardial infarction, such as the aforementioned CK and lactate dehydrogenase, are thought to lack the specificity for the pathological condition of myocardial infarction. It came to be.

 The reaction catalyzed by PK is as shown in equation (2).

(The abbreviations are PEP: phosphoenolpyruvate and Pyr: pyruvate.) By comparing CK and PK in the context of the diagnosis of myocardial infarction, CK activity is high in terms of specificity to the disease state as described above. Is slightly inferior to PK because it increases even with mild exercise. Next, the response time from the attack of myocardial infarction, that is, the time from the occurrence of the attack until the amount of each enzyme in the serum becomes an abnormal value, is compared between CK and PK. Outliers have been reported. In other words, PK should be measured if specificity is required in the diagnosis of myocardial infarction, and CK should be measured if a quick response is required. Therefore, it is desirable to measure CK and PK separately and use them as comprehensive judgment data.

Conventionally, various methods have been devised for measuring CK. One of them is to measure the rightward activity of equation (1).
A method in which the generated Cr is reacted with a dye for colorimetry or fluorescence measurement, and a method using rosifelase (JP-A-51-41597, JP-A-55-120796, JP-A-55-120796,
6-26200 and JP-A-57-105199),
Phosphoglycerate kinase and glyceraldehyde-3
-A method using phosphate dehydrogenase (see JP-B-59-34119 and JP-A-56-15500), and a method using hexokinase or glucokinase and glucose-6-phosphate dehydrogenase. .

Among these, glucokinase and glucose-6-phosphate dehydrogenase have been proposed as the most preferable methods from the viewpoints of reliability, reproducibility of measurement values, stability of measurement reagents, economy, and the like (Japanese Unexamined Patent Publication (KOKAI) No. 2000-163191). See JP-A-62-104598).

In addition, PK can be measured by colorimetric determination of Pyr produced in the formula (2) by the action of pyruvate oxidase and peroxidase, and by reducing lactate dehydrogenase on Pyr and reducing nicotine. Methods for measuring the reduction rate of amide adenine dinucleotide and the like are known. Further, "Clinical Chemistry", Vol. 18, pp. 130-135 (1989)
ATP produced by hexokinase and glucose-6
-A method of measuring using phosphate dehydrogenase has been proposed.

On the other hand, Japanese Patent Publication No. 1-4613 and Japanese Patent Publication No. 1-51786 disclose methods for analyzing a plurality of enzyme activity values. They include lactate dehydrogenase (hereinafter abbreviated as LDH) and leucine aminopeptidase (hereinafter abbreviated as LAP), two components, glutamate oxaloacetate transaminase (hereinafter abbreviated as GOT), and glutamate pyruvate. It describes a two-component measurement of transaminase (hereinafter abbreviated as GPT).

(Problems to be solved by the invention) Items for the purpose of diagnosing myocardial infarction are, in addition to CK and PK,
For example, there are many such as lactate dehydrogenase, oxybutyrate dehydrogenase, aldolase, myoglobin, etc. In order to make the above-mentioned comprehensive judgment, at least one item must be measured. A large amount of sample (serum) is required. However, collecting large volumes of blood from patients who have had a myocardial infarction attack is a major problem with great pain.

It takes some time to measure many items separately, but if seizures have already occurred, surgical treatment must be performed as soon as possible, and measurement of each item is extremely difficult. There is also a problem that it needs to be done quickly.

In the conventional method as described above, CK and PK are, of course, measured separately, so that a large amount of blood must be collected as described above, and some time is required before measurement results are obtained. was there.

Each of the methods described in Japanese Patent Publication No. 1-461313 and Japanese Patent Publication No. 1-51786 is intended to provide a mechanism of an apparatus for measuring a plurality of enzyme activities. Also, L
Combinations such as DH and LAP, GOT and GPT are completely incompatible with the optimal measurement conditions for enzymes, such as optimal pH, types of reagents for measurement, and conjugate enzymes. For the purpose of application, there was a problem that, of the two independent measuring reagents, only the substrate of one of the enzymes was separated as the second reagent.

An object of the present invention is to provide a reagent for simultaneous measurement of CK and PK that can be quantified with a rapid and trace amount of serum from patients with myocardial infarction attacks.

(Means for Solving the Problems) As a result of intensive studies to solve such problems, the present inventors added CrP immediately after measuring PK activity or, conversely, measured CK activity. Immediately after the addition, it was found that CK and PK activities could be measured simultaneously by adding PEP, and the present invention was completed.

That is, the present invention is a reagent for simultaneously measuring creatine kinase and pyruvate kinase in the same sample, the first reagent and the second reagent containing the following components: the first reagent: creatine phosphate; β-nicotine Creatine kinase and pyruvine, comprising: amidoadenine dinucleotide or phosphate thereof; hexokinase or glucokinase glucose 6-phosphate dehydrogenase adenosine diphosphate; and glucose second reagent: phosphoenolpyruvate. Provided is a reagent for measuring acid kinase.

The present invention also relates to a reagent for simultaneously measuring creatine kinase and pyruvate kinase in the same sample, wherein the first reagent and the second reagent include the following components: the first reagent: phosphoenolpyruvate; A creatine kinase comprising nicotinamide adenine dinucleotide or a phosphate thereof; hexokinase or glucokinase; glucose 6-phosphate dehydrogenase; adenosine diphosphate; and glucose second reagent: creatine phosphate. Provided is a reagent for measuring pyruvate kinase.

 Hereinafter, the present invention will be described in detail.

 The principle of measuring CK and PK in the present invention will be described below.

CK activity measurement principle PK activity measurement principle (The abbreviations in the reaction formulas are HK: hexokinase, GlcK: glucokinase, G6PDH: glucose 6-phosphate dehydrogenase, NAD
(P): β-nicotinamide adenine dinucleotide (phosphate), NAD (P) H: reduced β-nicotinamide adenine dinucleotide (phosphate). The reagent of the present invention is mainly composed of, for example, CrP, PEP, NAD (P), HK (or GlcK), G6PDH, ADP, glucose, etc., but may also contain other conventional activators, preservatives, stabilizers Can be used without hindrance.

The source of the HK used in the present invention is not limited, and Bakers East (Bakers Yeas) is used.
Those derived from t) can be used. GlcK, which has higher specificity for glucose than HK, can also be used. GlcK can also be used. The source of GlcK is not limited, and various sources such as those derived from microorganisms and animals can be used.
Among them, those produced by microorganisms having an optimum growth temperature of 50 ° C to 85 ° C are preferable. Such microorganisms include, for example, Bacillus stearothermophilus, Bacillus
Examples include Bacillus genus such as Thermoproteoliticus, Thermoactinomyces genus, and Thermos genus.

The source of G6PDH is not limited, but is preferably a G6PDH that acts on NAD as well as NADP as a coenzyme, such as those derived from Leuconostoc mesenteroides, Pseudomonas fluorescens and the like.

As the activator, for example, magnesium acetate,
Magnesium salts such as magnesium sulfate and potassium salts such as potassium acetate and potassium sulfate can be used. As a preservative, for example, a known salt such as sodium azide can be used. Further, as the stabilizer, for example, polysaccharides and derivatives thereof such as soluble starch and carboxymethyl cellulose, proteins such as albumin and γ-globulin, and water-soluble polymer compounds such as polyvinyl alcohol and polyethylene glycol can be used as appropriate. .

In general, the concentrations of the components of the reagent of the present invention are preferably as follows. For example, 0.1 to 40 units of HK or GlcK
/ ml, G6PDH 0.1 to 40 units / ml, CrP 2 to 70 mM, PEP
0.1-20 mM, ADP 0.1-20 mM, NAD (P) 0.05-20 mM, glucose 1-200 mM, magnesium salts 0.5-50 mM, potassium salts 0.5-50 mM. Adenosine monophosphate (AMP)
Abbreviated. ) The 0.2～30MM, diadenosine penta phosphine ate (hereinafter, abbreviated as AP ₅ A.) The not be used 1～100MyuM. More preferably, HK or GlcK 0.2-20 units / ml, G6PDH 0.2-20 units / ml, GrP 5-40 mM, PEP
0.5 to 10 mM, ADP 0.2 to 10 mM, NAD (P) 0.1 to 10 mM, glucose 2 to 100 mM, magnesium salts 2 to 15 mM, potassium salts 2 to 30 mM, AMP 0.5 mM, AP ₅ A to 2 50 μM may be used.

When measuring CK and PK using the reagent of the present invention,
The two reagent system must be used separately. Specifically, PEP must be the second reagent and all reagents other than PEP must be the first reagent, or conversely, CrP must be the second reagent and all reagents other than CrP must be the first reagent. First, a certain amount of the first reagent was placed in a cell kept at 30 ° C or 37 ° C, and then a sample such as serum was added.
The rate of change of absorbance at nm is measured and CK (or P
K) Obtain activity. Next, a fixed amount of the second reagent is added to the reaction solution, and the rate of change in absorbance at 340 nm is also measured. Activity can be obtained.

(Examples) Next, the present invention will be specifically described with reference to examples.

Example 1 107.5 mM imidazole-acetate buffer (p
H6.8), 25mM glucose, 7.5mMADP, 2.5mM NADP, 6.25mMAM
P, 12.5 μMAP ₅ A, 25 mM N-acetyl-L-cysteine, 20 mM
Magnesium sulfate, 40 mM potassium sulfate, 5 u / ml GlcK (Seikagaku Corporation), 5 u / ml G6PDH (Boehringer Mannheim Yamanouchi Co., Ltd., derived from Leuconostok messenteroides), and 35 mM CrP were prepared. As a second reagent, 20 mM imidazole-acetate buffer (pH 6.8) and 10 mM PEP were prepared. As a sample, a commercially available standard serum was used.

For the measurement, first, 2.4 ml of the first reagent was placed in a cell having an optical path length of 1 cm, and the cell holder was incubated for 3 minutes in a spectrophotometer maintained at 37 ° C. 20 μ of standard serum was added, and the change in absorbance per minute at 340 nm was measured. The CK activity value was obtained from the change in absorbance at this stage and the dilution ratio of the sample with the reagent. Further, 0.6 ml of the second reagent was added, and the change in absorbance per minute at 340 nm was measured. From the absorbance change amount in the second stage, the first
The amount of change in absorbance at each stage was subtracted, and the PK activity value was calculated from the dilution ratio of the sample with the reagent and the like. This procedure was repeated 10 times, and the reproducibility of simultaneous measurement of CK and PK activities was examined.

As a result, CK: average value of 98.2u /, standard deviation of 2.96, coefficient of variation 3.01%, PK: average value of 62.3u /, standard deviation of 1.57, coefficient of variation of 2.52%, showed that both activities were extremely reproducible. .

(Effects of the Invention) The reagent for simultaneous measurement of CK and PK of the present invention is not limited to the difference in substrate, but also the optimum pH, the type and amount of the reagent for measurement, the purpose of the measurement, and the urgency. , So that simultaneous and economical measurement is possible.

When diagnosing myocardial infarction, it has both specificity and urgency for the pathological condition of myocardial infarction and enables rapid measurement.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazuhiko Nagata 23 Uji Kozakura, Uji-city, Kyoto Unitika, Central Research Institute, Inc. (72) Inventor Hiroyuki Tsubota, 1144 Owada Nitta, Yachiyo-shi, Chiba Prefecture, Japan ) Inventor Koji Sakamoto 1144 Owada Nitta, Yachiyo-shi, Chiba Pref. Yatron, Inc. (56) References JP-A-62-104598 (JP, A) Clinical Chemistry, Vol. 18, No. 3 (1989) 130-135 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12Q 1/00-3/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A reagent for simultaneously measuring creatine kinase and pyruvate kinase in the same sample, comprising: a first reagent and a second reagent comprising the following components: first reagent: creatine phosphate; β-nicotinamide Creatine kinase and pyruvine, comprising: adenine dinucleotide or its phosphate; hexokinase or glucokinase; glucose 6-phosphate dehydrogenase; adenosine diphosphate; and glucose second reagent: phosphoenolpyruvate. Reagent for measuring acid kinase. 2. A reagent for simultaneously measuring creatine kinase and pyruvate kinase in the same sample, the first and second reagents comprising the following components: first reagent: phosphoenolpyruvate; β-nicotine Amide adenine dinucleotide or its phosphate; hexokinase or glucokinase; glucose 6-phosphate dehydrogenase; adenosine diphosphate; and glucose second reagent: creatine phosphate; Reagent for measuring acid kinase.