JPH03272699A - Reagent for measuring creatine kinase and pyruvate kinase - Google Patents

Abstract

PURPOSE:To obtain the title economic and efficient reagent having both performances of specificity to pathologic state and emergency in diagnosis of myocardial infarction, consisting of a reagent group containing creatine phosphate and a reagent group containing phosphoenolpyruvic acid. CONSTITUTION:The objective reagent consisting of at least a reagent group containing creatine phosphate and a reagent group containing phosphoenolpyruvic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to creatine kinase (hereinafter referred to as CK) in biological fluids.
It is abbreviated as ) and pyruvate kinase (hereinafter abbreviated as PK).

(Prior art) CK exists in the brain and muscle tissue, and in the field of clinical testing, CK is used for the purpose of diagnosing muscle diseases, neurological diseases, heart diseases, etc.
K activity is routinely measured.

CK is an enzyme that catalyzes both the left and right reactions of formula (1).

C lid Cr P ten ADP ≠ Cr + ATP (1) (abbreviations are CrP: creatine phosphate, Cr: creatine,
ADP: adenosine diphosphoric acid, ATP: adenosine diphosphoric acid. ) On the other hand, PK is cardiac and skeletal muscle in vivo.

It is distributed in the brain, but in recent years, research has been reported on the application of measuring PK to the diagnosis of myocardial infarction and muscle diseases, and it has attracted attention. This is another measurement item 1 for diagnosing myocardial infarction, for example.

It is believed that the aforementioned CK, lactate dehydrogenase, etc. lack specificity for the pathological condition of myocardial infarction, and PK, which is superior in this respect, has attracted attention.

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

PEP+ADP #py r+ATP (2) (abbreviation: PEP: phosphoenolpyruvate.

Pyr: pyruvic acid. ) Now, if we compare CK and PK in terms of diagnosing myocardial infarction, as mentioned above, CK activity is slightly inferior to PK in terms of specificity for pathological conditions, as it increases even with mild exercise. be. Next, when comparing the response time from a myocardial infarction attack, that is, the time from when the attack occurs until the amount of each enzyme in the serum becomes abnormal and appears, CK and PK are much earlier. It has been reported that abnormal values occur. In other words, if specificity is required when diagnosing myocardial infarction, PK
If a quick response is required, it is sufficient to measure CK, but for actual diagnosis, both of these two performances are required, so CK and PK are measured separately. It is desirable to use this as comprehensive judgment material.

Conventionally, various methods have been devised as methods for measuring CK. One of them is when measuring the activity in the right direction of equation (1),
■Method of colorimetrically measuring or measuring fluorescence by reacting the generated Cr with a dye, ■Method of using rosiferase (Japanese Patent Laid-Open No. 51-41597, Japanese Patent Laid-Open No. 55-12079)
Publication No. 6, JP-A-56-26200, and JP-A-Sho 5
7-105199), (2) a method using phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase (Japanese Patent Publication No. 59-34119).

(see JP-A-56-15500), and (2) a method using hexokinase or glucokinase and glucose-6-phosphate dehydrogenase.

Among these, glucokinase and glucose-6-phosphate dehydrogenase have been proposed to be the most preferable methods from the viewpoint of reliability, reproducibility of measurement values, stability of measurement reagents, economic efficiency, etc. (See Publication No. 62-104598)
.

In addition, as a method for measuring PK, ■Pyr produced in equation (2) is treated with pyruvate oxidase and peroxidase, and colorimetrically fixed; ■Pyr is treated with lactate dehydrogenase, and reduced at that time. Methods for measuring the rate of decrease in type nicotinamide adenine dinucleotide are known. Furthermore, “Clinical Chemistry” Volume 18, 130-13
5 (1989) proposes a method for measuring generated ATP using hexokinase and glucose-6-phosphate dehydrogenase.

On the other hand, Japanese Patent Publication No. 1-46113, Japanese Patent Publication No. 1-517
No. 86 describes a method for analyzing a plurality of enzyme activity values. They include lactate dehydrogenase (LD
It is abbreviated as H. ) and leucine aminopeptidase (hereinafter abbreviated as LAP), and 2 #i of glutamate oxaloacetate transaminase (hereinafter abbreviated as GOT) and glutamic acid pyruvate transaminase (hereinafter abbreviated as GPT). It describes the measurement of precepts.

(Problems to be Solved by the Invention) There are many items other than CK and PK for the purpose of diagnosing myocardial infarction, such as lactate dehydrogenase, oxypate dehydrogenase, aldolase, myoglobin, etc. In order to make a comprehensive judgment, it is necessary to measure as many items as possible, and for this purpose a relatively large amount of specimen (
serum) is required. However, collecting a large amount of blood from a patient suffering from a myocardial infarction is extremely painful.

This is a serious problem.

In addition, it takes some time to measure many items separately, but if a seizure has already occurred, surgical treatment must be performed as soon as possible, and it is extremely difficult to measure each item. Another problem is that it needs to be done quickly.

In the conventional method described above, CK and PK are of course measured separately, so there are the problems that a large amount of blood is required to be collected as mentioned above, and it takes some time to obtain the measurement results. was there.

The methods described in Japanese Patent Publication No. 1-46113 and Japanese Patent Publication No. 1-51786 are both intended to provide an apparatus mechanism for measuring a plurality of enzyme activities. Also, LDH and LAP.

Combinations such as GOT and GPT have very little in common in terms of optimal enzyme measurement conditions 1, for example, types of reagents for optimal pH 1 measurement, conjugated enzymes, etc., and are simply used for the purpose of application to the mechanism of the device.

There is a problem in that the substrate of one of the two enzymes is simply separated as a second reagent out of two independent measurement reagents.

The main object of the present invention is to provide a reagent and a drug for simultaneous measurement of CK and PK, which can be quantified quickly and with a small amount of serum from patients suffering from myocardial infarction.

(Means for Solving the Problems) As a result of extensive research to solve these problems, the present inventors added CrP immediately after measuring PK activity. Alternatively, the inventors discovered that it is possible to simultaneously measure CK and PK activities by adding PEP immediately after measuring GK activity, thereby completing the present invention.

That is, the gist of the present invention is a reagent for simultaneous measurement of CK and PK, which is characterized by being composed of a reagent group containing at least CrP and a reagent group containing at least PEP.

The present invention will be explained in detail below.

The principle of measuring GK and PK in the present invention is shown below.

■ CK activity measurement principle CrP + ADP Cr + ATP (3) Glucose +^TP ■ PK activity measurement principle PEP + ADP - pyruvic acid + ATP (6) Glucose +^TP I summer tV + is GLCM) Glucose 6-phosphate + ADP (7) (in reaction formula The abbreviation is HK: hexokinase.

GlcK: glucokinase, 08PDH nigercose 6-phosphate dehydrogenase, NA(D)P: β-nicotinamide adenine dinucleotide (phosphate), NA(D)
) PH: Represents reduced β-niconamide adenine dinucleotide (phosphoric acid). ) The reagents of the present invention are 9 e.g. Cry, PEP, NAD (
P), HK (or GJcK), G6PDH, ADP
, glucose, etc. are the main components, but other usual activators, preservatives, and stabilizers can be used without any problem.

The source of HK used in the present invention is not limited, and Baker's yeast
Yeast, etc. can be used.

In addition, GA has higher specificity for glucose than HK.
cK 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 which GlcK is produced by microorganisms whose optimal growth temperature is 50°C to 85°C. is preferred. Examples of such microorganisms include 9.

Examples include Bacillus genus such as Bacillus stearothermophilus and Bacillus thermoproteolyticus, Thermo to Actinomyces genus, Thermus genus, and the like.

Although the source of G6FDH is not limited, it is preferable to use not only NADP but also N as a coenzyme.
06PDH that also acts on AD, such as those derived from Leuconostoc mesenteroides and Pseudomonas fluorescens, are preferred.

Examples of activators include magnesium salts such as magnesium acetate and magnesium sulfate.

Potassium salts such as potassium acetate and potassium sulfate.

As the preservative, known preservatives such as sodium azide can be used. Furthermore, examples of stabilizers include soluble starch, polysaccharides such as carboxymethyl cellulose, and derivatives thereof.

Proteins such as albumin and γ-globulin, and water-soluble polymer compounds such as polyvinyl alcohol and polyethylene glycol can be used as appropriate.

The following concentrations are generally preferred as concentrations of each component of the reagent of the present invention. For example, HK or GlcK is 0.1-4
0L=t/m1. G6PDH at 0.1-40 units/m1. CrP 2-70mM, PEP 0.1-20
mM, ADP 0.1-20 mM.

NAD (P) 0.05-20mM, glucose 1
~200nM, magnesium salts 0.5-50M
M, potassium salts at 0.5-5QmM, adenosine-phosphate (hereinafter abbreviated as AMP) at 0.2-30mM
, diadenosine pentaphosphate (hereinafter abbreviated as APsA) may be used at 1 to 100 μM. More preferably, HK or GlcK is 0.2-20 units/d, 06PDH is 0.2-2OL=t/-, CrP
5-40mM, PEP 0.5-10mM, ADP 0.2-10mM, NAD (P) 0.1-10mM
M, glucose 2-100mM, magnesium salts 2-15mM, potassium salts 2-30mM, AM
Po, 5-15mM, APsA 2-50μM
Just use it.

When measuring CK and PK using the reagent of the present invention, it must be used separately into two reagent systems. Specifically, PEP must be used as the second reagent and all reagents other than PEP must be used as the first reagent, or conversely, CrP must be used as the second reagent and all reagents other than CrP must be used as the first reagent. In the measurement, first, a certain amount of the first reagent was added to
9C or 37°C.Next, a sample such as serum is added, and the rate of change in absorbance at 340 nm is measured to obtain CK (or PK) activity.

Next, a certain amount of the second reagent is added to this reaction solution, and the rate of change in absorbance at the same wavelength <340 nm is measured, and the change in absorbance due to the reaction of the first reagent is subtracted and calculated. ) activity can be obtained.

(Example) Next, the present invention will be specifically explained with reference to Examples.

Example 1 First reagent: 107.5mM imidazole-acetate buffer (pH 6,8), 25mM glucose, 7.5mMA
DP, 2.5mMNADP, 6.25mMAMP, 12
．． 5pMAPsA, 25mM N-acetyl-L-cysteine, 20mM magnesium sulfate, 40mM potassium sulfate, 5u/mff1Gj! cK-(Ihigaku Kogyo Button,
5 u/rrdtG 6 P DH (Boehringer Mannheim Yamanouchi ■, derived from Leuconostoc mesenteroides) and 35 mM CrP were prepared. 2 as the second reagent
0mM imidazole-acetate buffer (pH 6,8), 10
mMPEP was prepared. A commercially available standard serum was used as the sample.

For the measurement, first, the first reagent of 2.4- was placed in a cell with an optical path length of 1 cm, and a portion of the cell holder was incubated for 3 minutes in a spectrophotometer kept at 37°C.

20 μl of standard serum was added, and the change in absorbance per minute was measured by incubating 340 nm. A CK activity value was obtained from the amount of change in absorbance at this stage and the dilution ratio of the sample with the reagent. Furthermore, a second reagent of 0.6- was added, and the change in absorbance per minute at the same wavelength of <340 nm was measured. The amount of change in absorbance in the first step was subtracted from the amount of change in absorbance in the second step, and the PK activity value was calculated from the dilution ratio of the sample with the reagent, etc. This operation was repeated 10 times to examine reproducibility in simultaneous measurement of CK and PK activities.

As a result, ■CK: average value 9B, 2u/j! , standard deviation 2.96. Coefficient of variation 3.01%, ■PK: Average value 62.
3u/j! , standard deviation 1,57. The coefficient of variation was 2.52%, and both activity measurements had extremely good reproducibility.

(Effects of the Invention) The reagent for simultaneous measurement of CK and PK of the present invention is compatible with the purpose and urgency of the measurement, as well as the type and amount of the reagent for optimal pH1 measurement, in addition to the difference in substrate. Since it is common throughout, it enables economical and efficient simultaneous measurements.

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

Claims (1)

[Claims] (1) A reagent for measuring creatine kinase and pyruvate kinase, comprising a reagent group containing at least creatine phosphate and a reagent group containing at least phosphoenolpyruvate.