JP2564295B2 - Method for measuring pyruvate kinase activity for testing vitamin E deficiency - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Field of Industrial Application The present invention relates to a method for measuring pyruvate kinase (hereinafter abbreviated as PK) activity. That is, the industrial application field of the present invention is a field where biochemical or medical significance is found in measuring PK activity, and specifically, a field for testing vitamin E deficiency can be mentioned.

(2) Prior art PK is an important glycolytic enzyme and is present in all tissues throughout the body. In the field of clinical examination, measurement of PK activity is routinely used for examination of some blood diseases such as congenital hemolytic anemia.

PK is an enzyme that catalyzes the reaction in the left and right directions of the formula (1).

(The abbreviations are PEP: phosphoenolpyruvate, ADP: adenosine diphosphate, ATP: adenosine triphosphate.) Various methods have been proposed to measure PK activity, but they are roughly classified. And a method for directly measuring the product of the above reaction (1) (direct method), and the above (1)
The product of the reaction of (1) is flown into another enzyme reaction using it as a substrate, and the change in the absorbance of another substrate involved in the enzyme reaction is measured (indirect method).

As a direct method, adenosine diphosphate (hereinafter abbreviated as ADP) or phosphoenolpyruvate (hereinafter abbreviated as PEP), which is a substrate for PK reaction, is labeled with a radioisotope, and a product therefrom, that is, adenosine triphosphate is labeled. Acid (hereinafter abbreviated as ATP) or pyrivic acid is separated and its radioactivity is measured (Anal, Biochem., 39 , 135
-140 (1971) (Anal.Biochem., 39, 135-140 (197
1)), Anal, Biochem., 134 , 495-498 (1983) (A
nal.Biochem., 134 , 495-498 (1983)).
Although these methods have high sensitivity, they have not been widely used because of the drawback of using radioactive isotopes harmful to the environment.

Another method of direct method is ATP generated by PK reaction.
Is a method of measuring luciferin with luciferin. This method is already known in principle (Method Biochem Anal., 16 , 164-168, (1986) (Methods Bichem
ochem, Anal., 16 , 164-168, (1968)), Scand. J. Denta. Lisa., 83 , 375−381 (1975) (Scand.J.Den
t.Res., 83 , 375-381 (1975), but no report has been found to date as a detailed measurement method.

As an indirect method, reduced β-in the presence of lactate dehydrogenase
There is a method for measuring the decrease in the absorbance of nicotinamide adenine dinucleotide, and the measurement principle is as follows. As a patent applying this principle, there is Japanese Patent Publication No. 55-42636.

(Of the above abbreviations, NADH is reduced β-nicotinamide adenine dinucleotide, NAD is β-nicotinamide adenine dinucleotide, and LDH is lactate dehydratase.) That is, pyruvin generated by the action of PK in the reaction of (1). Conversion of acid to lactate in the presence of reduced nicotinamide adenine dinucleotide and lactate dehydrogenase, and simultaneous spectroscopic oxidation of reduced β-nicotinamide adenine dinucleotide to β-nicotinamide adenine dinucleotide Is to be measured.

This method is currently the most widely used method for measuring PK activity. This method has the advantage that PK activity can be measured anywhere with a conventional spectrometer. However, the disadvantage is that the measurement sensitivity is low, and therefore the amount of sample (blood etc.) used for measurement is inevitably large and burdens subjects, and the sample must be used almost as the source solution for measurement. Other coexisting substances (eg ATP
It is necessary to take into account the effects of A second drawback is that it takes time to measure one sample, making simultaneous processing of multiple samples difficult.

(3) Problems to be Solved by the Invention As described above, up to now, there is no method for measuring PK activity in a biological sample simply and accurately in a short time. That is, the luciferin-luciferase method is known as a principle in the direct method, but its detailed examination has not been added. The reasons for this are firstly that it was difficult to obtain luciferin luciferase that was stable and had little noise.
ATP is always present in biological samples, and it is detected as the background of ATP measurement caused by PK reaction, which may complicate the measurement method and reduce its accuracy. . Furthermore, in biological samples
Since PK is unstable and its activity is markedly reduced especially in a dilute solution, it is important to stabilize PK to some extent in order to make the measurement easy and reproducible.

(4) Means for Solving Problems As a result of intensive studies to solve such problems, the present inventors have found that by using a stable and noise-free firefly luciferin luciferase, blood The present inventors have completed the present invention by discovering that PK activity in blood can be measured easily and with high accuracy, and that PK in a sample can be stabilized by using a buffer solution containing serum albumin to further improve reproducibility.

That is, in the present invention, the content of 10 μM to 1 mM phosphoenolpyruvate and adenosine triphosphate is 0.01% or less.
Blood for testing vitamin E deficiency, characterized by using a reagent consisting of 0.1 to 10 μM adenosine diphosphate, firefly-derived luciferin luciferase and 0.01 to 0.5% bovine serum albumin, and blood diluted with a buffer It is a method for measuring pyruvate kinase activity in the medium.

The reagent of the present invention is AD having a PEP and ATP content of 0.01% or less.
P and firefly-derived luciferin / luciferase and bovine serum albumin (hereinafter abbreviated as BSA) are the main components, and other usual buffer salts, activators, stabilizers, etc. can be freely included. Bovine serum albumin is particularly useful as a PK stabilizer. Here, as BSA, one that can be obtained as a reagent may be used. A suitable concentration of BSA is 0.01% to 0.5%, particularly 0.1 to 0.3%.

The source of ADP used in the present invention is not limited, and, for example, one derived from mouse skeletal muscle can be used. However, ADP is around 1% A
Since TP is often contained, and this ATP interferes with the measurement of PK activity, a known method (Flexible System of Enzymatic Analysis, p15
3, Academic Press, New York (1972) (A
Flexible System of Enzymatic Analysis, p153, Academi
C Press, NY (1972))), the ATP content must be 0.01% or less.

As with ADP, the source of PEP is not limited.

Luciferin luciferase is used for firefly (Photinus p
yralis) with high purity and low background luminescence is used.

Examples of the activator include potassium salts such as potassium chloride and potassium acetate, magnesium salts such as magnesium chloride and magnesium acetate, and potassium salts of ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA 2K), such as trishydroxymethyl. A known substance such as aminomethane (hereinafter abbreviated as Tris) can be used.

Known components may be applied to each component of the PK activity measuring reagent used in the present invention, but the following concentrations are generally preferable. For example, ADP may be used at 0.1 to 10 μM, PEP at 10 μM to 1 mM, potassium chloride at 0.1 mM to 10 mM, magnesium chloride at 0.1 mM to 30 mM, EDTA 2K at 0.1 mM to 10 mM, and Tris at 1 to 100 mM.
More preferably, ADP 0.5 to 5 μM, PEP 50 to 500 μM, potassium chloride 0.5 to 5 mM, magnesium chloride 0.5 to 15 mM, EDTA di-K 0.5
-5 mM and Tris 5-50 mM may be used.

Furthermore, fructose-1,6-diphosphate can be added to examine the activation of PK isozymes, but the concentration is preferably 0.1 mM to 10 mM, and particularly 0.5 to 5 mM is preferable. .

Although the reagent used in the present invention is shown as a three-reagent system in the examples, it may be used as a two-reagent system or a one-reagent system by appropriately combining known techniques depending on the convenience of an analytical instrument or the like. In this case, the final concentration of each reagent may be adjusted so that it falls within the concentration range of the above reagents.

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

Experimental Example 1 1) Reagents used for measurement The following three reagents were combined to provide a measurement reagent of the invention for measuring PK activity.

First reagent, EDTA 2K 2mM, BSA 0.1%, ADP 2μM, Tris 20mM
-Sulfate buffer (pH7.70) 2nd reagent, EDTA 2K 2mM, BSA 0.1%, potassium chloride 17.5m
M, Magnesium chloride 31.5 mM, PEP 1.4 mM, Trim 20 mM-sulfate buffer (pH 7.70) Third reagent, luciferin / luciferase solution (LKB
-Made by wallack).

 The above three reagents were kept under ice cooling after preparation.

2) Measuring method The measuring method of the present invention is performed as follows using the test used for the above-described measurement.

That is, first, the sample used for measurement is dissolved or diluted with the second reagent (this can reduce the influence of ATP and the like derived from the sample). Next, add 1 volume of the second sample prepared by dissolving or diluting the sample to 6 volumes of the first reagent,
After shaking and stirring well, leave it in a constant temperature bath at 37 ° C or on a constant temperature plate. After 30 minutes, add 1.5 volumes of the third reagent, and measure the amount of luminescence per minute with an appropriate luminescence detector (Luminescence Reader (Aloka Co., Ltd., Tokyo)). Inclusion in sample
The number of PK units is calculated from the calibration curve.

Experimental Example 2 A calibration curve for purified PK (in the following Experimental Examples 4 and Examples 1 to 3, purified PK or rat plasma was used as a target), the rabbit skeleton was prepared using the reagents obtained in Experimental Example 1). A PK standard purified and purified from muscle was used as a measurement test, dissolved and diluted with the second reagent, and the PK activity was measured by the measurement method described in 2) of Experimental Example 1. The result is shown in FIG. In FIG. 1, the horizontal axis represents the PK concentration in the second reagent, and the vertical axis represents the amount of luminescence per minute ([counts / min.]: The same applies to FIGS. 2, 3, and 4 below). This measurement was carried out once every 5 days on different days including preparation of reagents and dissolution / dilution of PK (every 2 weeks), but the variation was small.
For comparison, a test was also conducted when 1 mM of fructose 1,6-diphosphate, which activates the PK isozyme, was added to the second reagent. As already known, the activation did not occur in skeletal muscle PK, so that the results were almost the same as the results of the above-mentioned measurement without addition of fructose 1,6-diphosphate (Fig. 2).

The above test was repeated within the same day, but PK 1 mU /
No decrease in activity was observed within 10 hours even under the highly diluted condition of ml.

Experimental Example 3 Reaction Time Dependence of Purified PK PK purified and purified from rabbit skeletal muscle in the same manner as in Experimental Example 2
Was used to examine the reaction time dependence of PK activity. The measuring method was based on the solid measuring method described in 2) of Experimental Example 1. That is, in the same manner as in Experimental Example 1, the PK solution dissolved / diluted in the second reagent was mixed / stirred with the first reagent according to the measurement method described in 2) of Experimental Example 1 and left in a 37 ° C. constant temperature bath. A certain amount of the mixed solution was taken out at appropriate time intervals, the third reagent was added, and the amount of luminescence for 1 minute was measured. The results are shown in FIG.
In the figure, a, b, and c show the results of the purified PK 1 mU / ml, 3 mU / ml, 10 mU / ml, and 30 mU / ml solutions, respectively. The luminescence amount increased linearly until the reaction time of 30 minutes at any concentration of PK.

Experimental Example 4 Reaction Time Dependence of PK in Rat Plasma Rat plasma was used instead of the purified and purified PK in Experimental Example 2, and rat plasma was diluted with the second reagent in 1) of Experimental Example 1 PK in rat plasma under the same conditions as in Experimental Example 3 except that it was diluted 400-fold with
The reaction time dependence of activity was examined. The results are shown in Fig. 4. Four rats (rat numbers a, b, c, d) were used for the measurement, and their respective plasmas were used as targets. A, b, c, d in the figure
The straight line indicates the rat plasma P corresponding to rat numbers a, b, c, and d.
The measurement results of K are shown.

Even in this case, the amount of luminescence increased linearly up to the reaction time of 30 minutes.

Example 1 Additive Recovery Experiment It is said that PK is leaked from skeletal muscle into plasma when a rat is fed with a vitamin E-deficient diet. An addition recovery experiment was conducted to examine whether the present method is useful for the measurement in this case. The plasma of a normal control rat and a vitamin E deficient diet-fed rat was diluted 400-fold with the second reagent of Experimental Example 1 1), and an approximately equivalent amount of purified and purified PK was added, and Experimental Example 1 2) was added. The PK activity was measured by the measuring method described above, and the recovery rate of the added purified PK was calculated. The results are shown in FIG. In the figure, the results of the normal control rat, the vitamin E-deficient diet-fed rats at 7 weeks, 10 weeks, and 14 weeks, are shown in order from the left in the figure. To make it easier to see the fluctuations, the results for each week were connected by a solid line or a dotted line. In the figure, ● or − ● − indicates the result of this Example, and ○ or… ○ indicates the fructose 1, which activates the PK isozyme (isozyme) for comparison.
6 shows the results when 1 mM of diphosphoric acid was added to the second reagent. The results are shown as the average value of 6 rats.

The spike recovery was between 90% and 110% in both normal control and vitamin E deficient diet-fed rats.

Example 2 Inter-measurement variation For the same purpose as in Example 1, the inter-measurement variation (measured three times on the same day) was determined for the plasma of normal control rats and vitamin E-deficient diet-fed rats. The same procedure as in Experimental Example 2 was performed except that the collected rat plasma was diluted 400-fold with the second reagent of Experimental Example 1 1), and the same specimen was used on the same day in the morning.
PK activity was measured three times in the afternoon and in the evening (results are shown in Table 1). In one case, the inter-measurement variability of the three measurements exceeded 10%, but good results were obtained in other cases. In this case as well, it was shown that there was almost no activity decrease within 10 hours on the same day even under the high dilution condition of 400-fold dilution of plasma as in Experimental Example 2.

Example 3 Change in plasma PK activity of vitamin E-deficient diet-fed rats Since the skeleton of the measurement method has been examined up to Example 2, changes of plasma PK activity of vitamin E-deficient diet-fed rats were actually determined by the method of the present invention. I looked it up. Plasma of normal control rats and rats fed a vitamin E deficient diet was diluted 400-fold with the second reagent, and the PK activity in plasma was measured in the same manner as in Example 2 except for the above. The results are shown in Fig. 6. In the figure,
Normal control rats and vitamin E, which were measured in the same manner as
The results of the rats at 7-week, 10-week, and 14-week of deficient diet feeding are shown in order from the left. The stippling column in the figure shows the results of this example,
For comparison, the white column shows the results when 1 mM of fructoque 1,6-diphosphate that activates the PK isozyme is added to the second reagent. The results are shown as the average value ± standard error of 6 rats. Plasma PK activity of rats fed a vitamin E deficient diet started to increase from the 7th week of feeding, and increased to 8 to 10 times that of the normal control rats at 14th week. This is in good agreement with the data reported so far, including the PK content in plasma.

Although BSA was added in order to stabilize PK in the above-mentioned experimental examples up to 2 to 4 and the examples up to 1 to 3, in order to improve the stabilization further, dissolution / dilution of the PK sample was performed by using a substance other than glass. It is preferable to use a container (known one such as plastic).

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the PK concentration in the second reagent and the amount of luminescence per minute. FIG. 2 is a graph showing the relationship between the PK concentration in the second reagent added with 1 ml of fructose 1,6-diphosphate and the amount of luminescence per minute. FIG. 3 is a graph showing the relationship between the reaction time and the light emission amount per minute. FIG. 4 is a graph showing the relationship between the reaction time and the light emission amount per minute. FIG. 5 is a graph showing the relationship between the week of the normal control rat and the vitamin E-deficient diet and the average recovery rate. FIG. 6 is a graph showing the relationship between the week of normal control rats and the diet fed with vitamin E-deficient diet and pyruvate kinase activity.

Continuation of the front page (56) References JP-A-57-54599 (JP, A) JP-A-57-91198 (JP, A) J. Nutr, 112 (7), 1982, P. 1437-1440 J. Nutr, 108 (12), 1978, P. 1963-1968 J. Clin Chem Clin Biochem, 20 (2), 1982, P. 91-94 Anal Biochem, 148 (2), 1985, P. 282-287

Claims (1)

(57) [Claims] 1. A phosphoenolpyrvic acid of 10 μM to 1 mM,
Adenosine triphosphate content 0.01% or less 0.1-10
μM adenosine diphosphate, luciferin derived from firefly
Method for measuring pyruvate kinase activity in blood for examination of vitamin E deficiency, characterized by using a reagent comprising luciferase and 0.01-0.5% bovine serum albumin and blood diluted with a buffer