JP5633669B2 - ADP measurement method and ADP measurement kit - Google Patents

Description

  The present invention relates to a method for measuring ADP in a sample and an ADP measurement kit. Furthermore, the present invention relates to a method for measuring a specific substance in a sample that generates ADP intermediately or finally by an enzymatic reaction and a kit for measuring a specific substance.

Various methods have been developed for measuring ADP, such as a method for measuring ADP existing in a sample in advance, a method for measuring a specific substance that generates ADP by an enzymatic reaction, and the like (for example, Patent Documents). 1, Patent Document 2).
For example, phosphoenolpyruvate and pyruvate kinase are allowed to act on ADP in a sample, and then lactate dehydrogenase is allowed to act in the presence of NADH using the produced pyruvate as a coenzyme to generate NAD, and a wavelength of 340 nm. A method of measuring ADP from a decrease in NADH having absorbance is known (Non-patent Document 1). However, since this method is a measurement method in which the absorbance decreases as the amount of ADP in the sample increases, the upper limit value of ADP that can be measured may be limited by NADH in the reagent.
In addition, as a method for measuring ADP, phosphoenolpyruvate and pyruvate kinase are allowed to act on ADP in a sample, and then the generated pyruvate is allowed to act with pyruvate oxidase or the like. There is known a method for measuring the above (Patent Document 3). However, this method is easily affected by reducing substances such as uric acid and ascorbic acid in the sample, and coloring substances such as bilirubin and hemoglobin, which may affect the measured value.
Furthermore, glucose and ADP-dependent hexokinase are allowed to act on ADP in the sample in the presence of magnesium ions, and then the resulting glucose-6-phosphate is converted to glucose-6-phosphate dehydrogenase in the presence of NAD (P). A method is known in which ADP is measured from the amount of NAD (P) H produced by the action (Patent Document 2). In this method, since ADP is measured based on the amount of NAD (P) H produced, the measurement limit is high, and NAD (P) H whose molecular extinction coefficient is clear is measured. Therefore, it is said that ADP in a biological sample can be easily measured with high accuracy (Patent Document 2).

JP-A-9-285297 Japanese Patent Laid-Open No. 9-234098 Japanese Patent Laid-Open No. 5-244994

J. et al. Appl. Biochem. , 1985, 7 (4-5), 303-310.

  An object of the present invention is to provide a method for measuring ADP in a sample by measuring glucose-6-phosphate or AMP produced by allowing glucose, ADP-dependent hexokinase, and metal ions to act on ADP in the sample. Is to provide a method for accurately measuring a wide range of ADP concentrations by reducing the slope of the calibration curve with a small blank value and a measurement kit used therefor. Furthermore, another object of the present invention is to provide a method for measuring a specific substance in a sample that intermediately or finally generates ADP by an enzyme reaction using such a measurement method, and a measurement kit used therefor.

  The present inventors apply the above-mentioned method that is capable of accurately measuring ADP by subjecting a specific substance to an enzymatic reaction to generate ADP, and then allowing ADP-dependent hexokinase or the like to act on the generated ADP. The use of thio-NAD (P) as a coenzyme for the measurement of specific substances such as high concentrations of urea was examined. However, in this method, if the concentration of a specific substance in a sample is high or measurement is performed using thio-NAD (P) as a coenzyme, the slope of the calibration curve becomes extremely large, and the specific substance can be measured. It has been found that there is a problem that the concentration range becomes extremely narrow, which may cause problems in practicality. Furthermore, as a result of various studies to solve the problem, when phosphoenolpyruvate and pyruvate kinase coexist in an enzyme reaction using ADP-dependent hexokinase or the like, the blank value is unexpectedly reduced, and It has been found that the slope of the calibration curve is reduced, and as a result, the measurable concentration range of ADP and specific substances can be widened and measured accurately. The present invention has been made through this process.

Therefore, the present invention provides a method for measuring ADP in a sample by measuring glucose-6-phosphate or AMP produced by allowing glucose, ADP-dependent hexokinase, and metal ions to act on ADP in the sample. The method for measuring ADP is characterized in that phosphoenolpyruvate and pyruvate kinase coexist at the same time.
Furthermore, the present invention provides an enzyme reaction with a specific substance in a sample to generate ADP intermediately or finally, and the ADP is subjected to the above-described measurement method. The present invention relates to a method for measuring a specific substance.
Furthermore, the present invention relates to an ADP measurement kit comprising an essential component for measuring glucose, ADP-dependent hexokinase, metal ion, glucose-6-phosphate or AMP, phosphoenolpyruvate, and pyruvate kinase.
Furthermore, the present invention measures an essential component, glucose, ADP-dependent hexokinase, metal ion, glucose-6-phosphate or AMP for subjecting a specific substance to an enzymatic reaction to generate ADP intermediately or finally. The present invention relates to a kit for measuring a specific substance, comprising essential components for the preparation, phosphoenolpyruvate, and pyruvate kinase.

  In the present invention, when measuring ADP in a sample or ADP generated by an enzymatic reaction of a specific substance in a sample using an ADP-dependent hexokinase reaction, phosphoenolpyruvate and pyruvate kinase are allowed to coexist. The slope of the calibration curve is small and the blank value is reduced, so that a wide range of ADP concentrations can be accurately measured.

FIG. 1 shows absorbance values when urea nitrogen (BUN) was measured in Comparative Example 1, Example 1 and Example 2.

The sample used in the present invention is a sample that may contain ADP or a sample that may generate ADP by an enzymatic reaction, and is preferably not particularly limited as long as it is liquid, but serum, plasma, urine, etc. Biological samples or model samples thereof are preferred.
In the present invention, ADP refers to adenosine diphosphate.

In the present specification, NAD (P) means, for example, any of NAD, NADP, thio-NAD, and thio-NADP, but as a coenzyme used for glucose-6-phosphate dehydrogenase (G6PDH) reaction A structure similar to those that function, for example, a compound having the same ring skeleton and known to have a function as a coenzyme may be used. In the present specification, NAD means nicotinamide adenine dinucleotide, NADP means nicotinamide adenine dinucleotide phosphate, thio NAD means thionicotinamide adenine dinucleotide, and thio NADP means thionicotine Refers to amidoadenine dinucleotide phosphate.
In the present specification, NAD (P) H is a corresponding reduced form of NAD (P). For example, when NAD (P) is NAD, NADP, thio-NAD, or thio-NADP, It means NADH, NADPH, thio-NADH, thio-NADPH.
In this specification, NAD (P) means NAD or NADP, NAD (P) H means the corresponding reduced form of NAD (P), and when NAD (P) is NAD or NADP Means NADH and NADPH, respectively.
In the present specification, thio NAD (P) means thio NAD or thio NADP, thio NAD (P) H means a corresponding reduced form of thio NAD (P), and thio NAD (P) When is thioNAD or thioNADP, it means thioNADH or thioNADPH, respectively.

In the present invention, glucose, ADP-dependent hexokinase, and metal ions are allowed to act on ADP as shown in the following reaction formula (this reaction may be described as an ADP-dependent hexokinase reaction in this specification). -6-phosphate (G6P) and AMP (Adenosine monophosphate) are produced, and the produced glucose-6-phosphate or AMP is measured.

As the metal ion, magnesium ion, cobalt ion, manganese ion and the like are preferable.

In the present invention, when measuring the produced glucose-6-phosphate or AMP, as shown in the following reaction formula, glucose-6-phosphate is converted to glucose-6-phosphate dehydrogenase (G6PDH) and NAD (P It is preferable to perform the reaction by generating gluconolactone-6-phosphate (6-GL) and NAD (P) H, and measuring the NAD (P) H.

In the present invention, when NAD (P) is used as the NAD (P) of the coenzyme, ADP is measured from the increase in absorbance around 340 nm, for example, at a wavelength of 330 to 350 nm, derived from the generated NAD (P) H. can do.
In the present invention, when thio-NAD (P) is used as the NAD (P) of the coenzyme, the ADP is calculated from the increase in absorbance at around 405 nm, for example, at a wavelength of 390 to 415 nm, derived from the thio-NAD (P) H to be produced. Can be measured. The use of thio-NAD (P) in the present invention is preferable in that it can be measured using a visible spectrophotometer that is inexpensive and easily available, and the ADP measurement region can be remarkably improved.
In order to further expand the ADP measurement range, thioNAD (P) and NAD (P) coexist as coenzymes to perform an ADP-dependent hexokinase reaction, and thioNAD (P) H and NAD (P) H are It is preferable to measure ADP by measuring the increase in absorbance around a wavelength of 405 nm derived from the generated thio-NAD (P) H.
In this case, the amount of NAD (P) used is not particularly limited, but is preferably 0.3 to 30 times the number of thioNAD (P) used in order to adjust the slope of the calibration curve appropriately, The mole number of -20 times is more preferable.

  In the present invention, when measuring AMP to be produced, for example, myoadenylate deaminase is allowed to act on the AMP, and glutamate dehydrogenase is allowed to act on the produced ammonia in the presence of NAD (P) Hs and α-ketoglutarate. Measure the AMP by measuring the decreasing NAD (P) Hs, for example, by decreasing the absorbance at the appropriate wavelength, ie, the appropriate wavelength to measure the NAD (P) Hs described above. can do.

In the present invention, when glucose, ADP-dependent hexokinase, and metal ions are allowed to act on ADP in a sample, phosphoenolpyruvate (PEP) and pyruvate kinase (PK) are simultaneously added as shown in the following reaction formula. It is characterized by allowing it to coexist and react with ADP in the sample.

In this case, the amount of pyruvate kinase used is preferably 1/20 to 20 times KU of the ADP-dependent hexokinase used. The amount of phosphoenolpyruvate is, for example, preferably 1/20 to 20 times the number of moles of ADP supposed to be measured, more preferably 1/10 to 10 times the number of moles, and 1/5 to 5 times the number of moles. Numbers are particularly preferred.

The method for measuring ADP of the present invention comprises glucose, ADP-dependent hexokinase, and essential components for measuring metal ions, glucose-6-phosphate or AMP, phosphoenolpyruvate, and pyruvate kinase. Depending on the kit, it can be carried out, for example, at 20 to 40 ° C, preferably around 37 ° C.
The composition of this kit may be combined into one reagent, but in consideration of the stability of the reagent, two reagents such as a first reagent containing glucose and phosphoenolpyruvate, ADP-dependent hexokinase, metal It is also possible to include these components separately from the essential components for measuring ions, glucose-6-phosphate or AMP and the second reagent containing pyruvate kinase. A surfactant and a buffering agent may be appropriately added to the reagent. With this kit, ADP can be measured using an automatic analyzer or a visible part measuring device.

Furthermore, the method for measuring ADP of the present invention comprises subjecting a specific substance in a sample to an enzymatic reaction to generate ADP intermediately or finally, and subjecting the ADP to the measuring method of the present invention to determine the amount of ADP. Measurement can be applied to a method of measuring a specific substance in a sample.
In the present invention, the specific substance is not particularly limited as long as it is a substance that can generate ADP by an enzymatic reaction, and examples thereof include urea, creatine, and choline.
When measuring urea as a specific substance, as an enzyme reaction, ATP, magnesium ion, potassium ion, hydrogen carbonate ion and urea amide lyase are allowed to act on urea in a sample to generate ADP intermediately. Urea can be measured by attaching to the ADP measurement method. The reaction formula of the urea amide lyase reaction is shown below.

When measuring creatine as a specific substance, as an enzyme reaction, ADP, magnesium ion and creatine kinase are allowed to act on creatine in a sample as reagents to generate ADP in the middle, and the ADP is used in the ADP measurement method of the present invention. By attaching, creatine can be measured. The reaction formula of the creatine kinase reaction is shown below.

When measuring choline as a specific substance, as an enzymatic reaction, ADP, magnesium ion, and choline kinase derived from a reagent are allowed to act on choline in a sample to generate ADP intermediately, and the ADP is measured according to the method of ADP of the present invention. By attaching to, choline can be measured.

As described above, in the method for measuring a specific substance of the present invention, ATP is generally converted to ADP as an essential component for subjecting the specific substance to an enzymatic reaction to generate ADP intermediately or finally. In general, it contains an enzyme, a substrate for the enzyme and ATP. In this case, for example, the amount of phosphoenolpyruvate is preferably 1/20 to 20 times the number of moles of ATP, more preferably 1/10 to 10 times the number of moles, and particularly preferably 1/5 to 5 times the number of moles. In the method for measuring a specific substance of the present invention, the amount of pyruvate kinase used is preferably 1/20 to 20 times KU of the ADP-dependent hexokinase used.
The method for measuring a specific substance according to the present invention includes an essential component for generating ADP intermediately or finally by enzymatic reaction, glucose, ADP-dependent hexokinase, and metal ion, and glucose-6-phosphate produced Alternatively, it can be carried out, for example, at 20 to 40 ° C., preferably around 37 ° C., using a specific substance measurement kit containing an essential component for measuring AMP, phosphoenolpyruvate, and pyruvate kinase.
The composition of this kit may be combined into one reagent, but it is also possible to include these components in two reagents in consideration of the stability of the reagent. When measuring urea as a specific substance, for example, glucose, potassium ion, ATP, phosphoenolpyruvate is included in the first reagent, and magnesium ion, NAD (P), ATP-dependent hexokinase, ureaamide is included in the second reagent. Lyase, glucose-6-phosphate dehydrogenase, and pyruvate kinase can be included. A surfactant and a buffering agent may be appropriately added to the reagent. A specific substance can be measured by this kit using an automatic analyzer or a visible part measuring device.

EXAMPLES Hereinafter, although an Example and a comparative example demonstrate this invention further in detail, this invention is not limited to these at all.
Examples 1 and 2 and Comparative Example 1
Method for measuring urea nitrogen using ADP-dependent hexokinase and pyruvate kinase

1. In a urea nitrogen measurement system using urea amidolyase, ADP-dependent hexokinase, and glucose-6-phosphate dehydrogenase, measurement comprising pyruvate kinase and phosphoenolpyruvate as enzymes acting on ADP in the composition The system is shown as an example.
In the measurement method using urea amide lyase, urea in the sample is decomposed by urea amide lyase in the presence of ATP, hydrogen carbonate ions, and potassium ions to generate ADP and ammonium ions. Subsequently, the generated ADP produces glucose-6-phosphate and AMP by the action of ADP-dependent hexokinase in the presence of glucose. The generated glucose-6-phosphate uses NAD (P) as a coenzyme, and 6-phosphogluconolactone and NAD (P) H are generated by the action of glucose-6-phosphate dehydrogenase. The amount of increase in the generated NAD (P) Hs is measured, for example, as the amount of increase in absorbance near the wavelength of 340 nm or near 405 nm, and urea in the sample is measured as the urea nitrogen concentration.
In the above reaction, ADP-dependent hexokinase is reacted with ADP derived from the generated sample, and at the same time, excessive ADP is converted to ATP by competitive reaction with pyruvate kinase in the presence of phosphoenolpyruvate, Measure the appropriate amount of ADP.
As Comparative Example 1, a composition containing no phosphoenolpyruvate and pyruvate kinase was used as a control. Furthermore, as a substrate for glucose-6-phosphate dehydrogenase, a reagent prepared as NAD alone was used in Example 1, and NAD and thio-NAD were mixed together.
A urea solution was prepared so that the calibration curve was prepared with urea nitrogen at 200 mg / dL, and each concentration was appropriately diluted with physiological saline.

The reagents and samples used are as follows.
Reagent composition of Example 1
First reagent (pH 7.5) :
20 mM Tris (Tris (hydroxymethyl) aminomethane)
10 mM D-glucose 10 mM potassium bicarbonate 10 mM ATP · 2Na (adenosine triphosphate · disodium)
10 mM phosphoenolpyruvate 0.1% surfactant
Second reagent (pH 7.0) :
200 mM Tris
10 mM magnesium acetate tetrahydrate 4 mM NAD
0.1% Surfactant 2KU / L ADP-dependent hexokinase 2KU / L Urea amide lyase 2KU / L Glucose-6-phosphate dehydrogenase 20KU / L Pyruvate kinase

Reagent composition of Example 2
First reagent (pH 7.5) :
20 mM Tris
10 mM D-glucose 10 mM potassium bicarbonate 10 mM ATP · 2Na
10 mM phosphoenolpyruvate 0.1% surfactant
Second reagent (pH 7.0) :
200 mM Tris
10 mM magnesium acetate tetrahydrate 2 mM NAD
2 mM Thio NAD
0.1% Surfactant 2KU / L ADP-dependent hexokinase 2KU / L Urea amide lyase 2KU / L Glucose-6-phosphate dehydrogenase 20KU / L Pyruvate kinase

Reagent composition of Comparative Example 1
First reagent (pH 7.5) :
20 mM Tris
10 mM D-glucose 10 mM potassium bicarbonate 10 mM ATP · 2Na
0.1% surfactant
Second reagent (pH 7.0) :
200 mM Tris
10 mM magnesium acetate tetrahydrate 4 mM NAD
0.1% surfactant 2KU / L hexokinase 2KU / L ureaamide lyase 2KU / L glucose-6-phosphate dehydrogenase

  The measurement was performed as follows. Using an automatic analyzer, 3.0 μL of the sample and 100 μL of the first reagent are mixed at 37 ° C. for 5 minutes, and then 100 μL of the second reagent is added and reacted at the same temperature for 5 minutes. In Example 1 and Comparative Example 1, the absorbance after the color development reaction was measured at a main wavelength of 340 nm and a sub wavelength of 405 nm by the one-point end method of the model. In Example 2, the absorbance after the color reaction was measured at a main wavelength of 405 nm and a sub wavelength of 546 nm by the one-point end method of the model.

2. The results are shown in FIG. From the results of FIG. 1, it can be seen that in Comparative Example 1, when the urea nitrogen concentration is 25 mg / dL or more, the absorbance is the upper limit of the measuring instrument and measurement is impossible. On the other hand, in Example 1, it can be seen that the urea nitrogen concentration does not reach the upper limit of absorbance up to around 125 mg / dL and can be measured. Furthermore, it can be seen that the reagent composition of Example 2 does not reach the upper limit of absorbance even when the urea nitrogen concentration becomes 200 mg / dL, and the measurable range is wider.

  As described in detail above, in the present invention, when measuring ADP in a sample or ADP generated by an enzyme reaction of a specific substance in a sample using an ADP-dependent hexokinase reaction, phosphoenolpyruvate and pyruvin are measured. By coexisting with acid kinase, the slope of the calibration curve is small and the blank value is reduced, so that a wide range of ADP concentrations can be accurately measured.

Claims (7)

In the sample, glucose, ADP-dependent hexokinase, metallic ions, NAD (P) compound, glucose-6-phosphate dehydrogenase (G6PDH), phosphoenolpyruvate and pyruvate kinase were added,
Reactions represented by the following chemical formulas 1 and 2


Simultaneously perform the reaction represented by the following chemical formula 3,

A method for measuring ADP in a sample, which is carried out by measuring produced NAD (P) Hs . NAD (P) and thio-NAD (P) are used together as NAD (P), and NAD (P) H is NAD (P) H and thio-NAD (P) H The method for measuring ADP according to claim 1, which is carried out by measuring an increase in absorbance around a wavelength of 405 nm derived from thio-NAD (P) H. A specific substance in the sample is subjected to an enzymatic reaction to generate ADP intermediately or finally, and the ADP is subjected to the measurement method according to claim 1 or 2 , and the ADP is derived from the amount of ADP in the sample. A method for measuring specific substances. A particular substance urea, enzyme reaction, ATP urea in a sample, magnesium ion, potassium ion, bicarbonate ion, and urea amide-lyase is allowed to act intermediate or final generate the ADP to claim 3 A method for measuring the specific substance described in 1. The method for measuring ADP according to claim 1, comprising glucose, ADP-dependent hexokinase, metal ion , NAD (P), glucose-6-phosphate dehydrogenase (G6PDH) , phosphoenolpyruvate, and pyruvate kinase. kit for. Essential components for subjecting specific substances to enzymatic reaction to generate ADP intermediately or finally, glucose, ADP-dependent hexokinase, metal ions , NAD (P) s, glucose-6-phosphate dehydrogenase (G6PDH ), phosphoenolpyruvate, and pyruvate kinase, kit for a method for measuring a specific substance according to claim 3. A first reagent comprising glucose and phosphoenolpyruvate, and
Second reagent comprising ADP-dependent hexokinase, metal ions, NAD (P) s, glucose-6-phosphate dehydrogenase (G6PDH) and pyruvate kinase
The kit according to claim 5, comprising: