JP2015080456A - Alt activity-measuring reagent and method for measuring alanine aminotransferase activity using reagent thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a reagent which ensures storage stability and a measuring range which enable obtaining a stable result by suppressing HGD activity of LD formulated in a reagent causing an increased blank reaction in ALT reagent, and suppressing the increased reagent blank, and to provide a method for measurement using the reagent.SOLUTION: The reagent for measurement of alanine aminotransferase activity is formulated in combination with L-lactic acid dehydrogenase which reacts with an L-form of lactate dehydrogenase and daringly D-lactate dehydrogenase which reacts with a D-form of the substrate.

Description

  The present invention provides a reagent capable of obtaining a stable result by suppressing fluctuation of a reagent blank in alanine aminotransferase (hereinafter referred to as “ALT”) activity measurement, and a method for measuring alanine aminotransferase activity using the reagent. For the purpose.

  ALT is an enzyme widely distributed in the heart and liver, and is released into the blood during various diseases. Therefore, the measurement of ALT activity in biological fluids such as urine and blood can be used for diagnosis and treatment of heart disease or liver disease. It is one of the important items as a follow-up index.

  As a method for measuring ALT activity, L-alanine and 2-oxoglutarate were used as substrates, and pyruvic acid produced by ALT was changed to lactic acid by lactate dehydrogenase (hereinafter referred to as “LD”) and coexisted. A method for measuring the ALT activity in a body fluid by measuring the amount of reduced nicotinamide adenine dinucleotide (hereinafter referred to as “NADH”) in the vicinity of a wavelength of 340 nm is generally used.

In Japan, the recommended method of ALT activity measurement published in 1989 by the Japanese Society for Clinical Chemistry (JSCC) [Japan Clinical Chemistry Society: Recommended Method for Measuring Enzyme Activity in Human Serum -Alanine Aminotransferase- (1989-08-30), Clinical Chemistry, 18 (4), 250-262 (1989)], based on the principle of measuring ALT activity has been widespread (Non-patent Document 1).
The above-mentioned recommendation method published by the Japanese Society for Clinical Chemistry is a method of measuring common enzyme activity measurements that can be shared at the environmental level of clinical laboratories, etc. under optimal conditions in order to obtain compatibility of measured values. In general, it cannot be used as a daily test method. However, clinical test drug manufacturers provide reagents that can be used for compatibility with the recommended methods of the Japanese Society for Clinical Chemistry and measurements. .

  In addition, a technique for suppressing HGD activity by adding a substance having an LD inhibitory action, particularly oxamic acid or a salt thereof, to a reagent has been reported (Patent Document 1). Furthermore, LD is a commonly used L-lactate dehydrogenase that reacts only with L-lactic acid (hereinafter referred to as “L-LD”) and D-lactate dehydrogenase that reacts only with D-form lactic acid (hereinafter referred to as “D”). It is reported that a reagent that suppresses HGD activity is possible by using D-lactate dehydrogenase (D-LD) (Patent Document 2).

Japanese Patent No. 3775632 Japanese Patent No. 4884733

Clinical Chemistry Recommended Law Summary 2012 Edition

Of the above, in facilities that measure ALT activity using large-scale facilities such as clinical laboratories, in general, most of them are recommended by the Japanese Society of Clinical Chemistry as recommended methods and measured values. ALT activity measurement reagents provided by clinical test drug manufacturers that can be compatible with each other, but biological samples are a mixture of many types of components, and ALT activity measurement reagents are also a mixture of many types of components Therefore, it is very difficult to develop a reagent for measuring ALT activity that is not affected by the stabilization of the reagent and the impurities.

In particular, in the case of using an analyzer called an automatic analyzer that automatically measures by setting a measurement reagent and setting the conditions, the measurement can be performed with the lid open for several weeks. In many cases, in this case, carbon dioxide in the atmosphere is absorbed, the pH of the ALT activity measurement reagent changes, the reagent blank reaction value also fluctuates, and an error occurs in the obtained ALT activity measurement value.

It has been reported that the reaction fluctuation of this reagent blank is derived from the α-hydroxyglutarate dehydrogenase activity (hereinafter referred to as “HGD activity”) of LD, and that the pH is suppressed to the alkali side. .
On the other hand, since the oxamic acid or salt thereof used in Patent Document 1 described above is inexpensive, it is possible to supply an inexpensive reagent with suppressed HGD activity. However, only oxamic acid or a salt thereof can suppress the increase in blank value. Is insufficient, and there is a problem in the measured value stability of the reagent.

In addition, in Patent Document 2, with respect to a reagent that suppresses HGD activity by using D-lactate dehydrogenase (D-LD), when this is used, the Km value for pyruvic acid is L-lactate dehydrogenation. Since it is higher than the enzyme (L-LD), there is a problem that a measurement range used in daily inspection cannot be secured.

  Therefore, the present inventors tried to suppress the increase in reagent blank reaction in a reagent for measuring alanine aminotransferase activity containing L-alanine, 2-oxoglutarate, lactate dehydrogenase, and reduced nicotinamide adenine dinucleotide. As a result, there is provided an ALT activity measuring reagent capable of obtaining a stable measurement result by reducing the influence of the HGD activity on the reagent and consequently suppressing fluctuations in the reagent blank reaction, and an ALT activity measuring method using the reagent. I succeeded in finding it. Specifically, in order to solve the above problems, not only D-lactate dehydrogenase (D-LD) but also L-lactate dehydrogenase (L-LD) having the same substrate as D-LD described above. Tried to combine.

  Thus, in the normal case, those skilled in the art react with a plurality of the same substrates, such as a combination of D-LD and L-LD, because enzyme activity measurement defines enzyme activity from Km and Vmax for the substrate. I do not think of combining enzymes. Moreover, in order to suppress the HGD activity which is the subject of the present invention, it is not usually considered to combine L-LD. The reason is that when a small amount of L-LD is combined, the reagent blank reaction may increase. Therefore, as a result of intensive studies, the present inventors can suppress HGD activity only with D-LD, but other problems also arise with D-LD alone, so as to ensure a problematic measurement range. When I tried the idea of combining L-LD, I found out that each problem could be solved.

Specifically, in a reagent for measuring alanine aminotransferase activity containing L-alanine, 2-oxoglutarate, lactate dehydrogenase, and reduced nicotinamide adenine dinucleotide (hereinafter referred to as “NADH”), a certain amount of D- By using the alanine aminotransferase activity measuring reagent in which LD and a certain amount of L-LD are combined, the above-described problems can be solved. The present invention also provides a test sample that may contain alanine aminotransferase, L-alanine, 2-oxoglutarate, a certain amount of D-LD, a certain amount of L-LD, and reduced nicotinamide adenine diene. The present invention relates to a method for measuring alanine aminotransferase activity, which comprises prescribing a certain amount of nucleotides.

  In the present invention, D-LD, which is individually known as an ALT activity measurement reagent, and L-LD of the same substrate are combined with each other so that the HGD activity exhibited by the LD itself does not affect the measurement result. As a result, the reagent blank is made smaller without losing the quantitative property, and the reagent blank is prevented from rising due to a decrease in the reagent pH after opening the reagent container, and ALT due to the apparent increase in Km of LD against pyruvic acid. We succeeded in reducing negative errors to activity measurements.

  In addition, by combining D-LD and L-LD, the problem that a sufficient measurement range cannot be ensured with only D-LD is solved, and stable measurement performance without fluctuation in measured value of HGD activity is obtained. And a method for measuring alanine aminotransferase activity using the reagent have been completed.

It is a graph which shows a time-dependent change of the blank sensitivity in an oxamic acid prescription and a comparative ALT activity measuring reagent. It is a graph which shows the measurement upper limit in the ALT activity measuring reagent for this invention and a comparison. It is a graph which shows a time-dependent change of the blank sensitivity in this invention and the ALT activity measuring reagent for a comparison. It is a graph which shows the time-dependent change of the ALT activity measured value in a pooled serum measured using this invention and the ALT activity measuring reagent for a comparison.

The ALT activity measurement reagent of the present invention includes L-alanine, 2-oxoglutarate, lactate dehydrogenase (LD), and reduced nicotinamide adenine nucleotide (NADH), which has been widely used so far. It is a revolutionary improvement. In general ALT activity measuring reagents including general-purpose L-alanine, 2-oxoglutarate, LD, and NADH, as described above, pyruvic acid produced by ALT using L-alanine and 2-oxoglutarate as substrates. ALT activity can be measured by changing the amount of NADH that had been coexisting with LD by measuring the decrease in the amount of NADH at a wavelength around 340 nm, but avoiding variations in measurement results due to fluctuations in the reagent blank reaction It has a big problem that it cannot be done.

Therefore, in the present invention, as a result of repeated studies for suppressing an increase in reagent blank reaction in an alanine aminotransferase activity measuring reagent containing L-alanine, 2-oxoglutarate, LD, and NADH, an unexpected blank reaction Succeeded in suppressing the rise in In other words, the problem that the measurement range cannot be secured by D-LD alone is solved, and the influence on the reagent of the HGD activity exhibited by the LD itself is suppressed to the extent that it does not affect the measurement result, and as a result, the quantitativeness is impaired. In addition, the fluctuation range of the reagent blank reaction is extremely reduced, avoiding the increase of the reagent blank due to the decrease in the reagent pH after opening the reagent container, and the negative error in the measured ALT activity due to the increase in the apparent Km of LD against pyruvic acid. Succeeded in reducing significantly.

Specifically, the ALT activity measuring reagent of the present invention prescribes D-LD and L-LD of the same substrate in addition to the above-mentioned known constituents. Usually, those skilled in the art do not consider combining D-LD and L-LD of the same substrate in order to define enzyme activity from Km and Vmax with respect to the substrate when measuring enzyme activity. As D-LD used here, natural D-LD derived from Lactobacillus sp., Recombinant D-LD thereof, or mutant D-LD thereof can be used.

Moreover, as L-LD, for example, natural L-LD derived from chicken heart, porcine heart, porcine muscle, or Leuconostoc mesenteroides, or a recombinant L-LD thereof is used. The origin is not particularly limited. In the present invention, “LD activity” means an activity of reducing pyruvic acid to lactic acid. The unit “U” is defined as the amount of enzyme activity (standard temperature is 30 ° C.) that converts 1 μmol of substrate (pyruvic acid) into product (lactic acid) per minute.

If the amount of L-LD is less than 10 U / L, it is difficult to accurately measure the ALT activity in the sample. Conversely, if the amount exceeds 3,000 U / L, it is more than that for improving the measurement range. Since the effect cannot be expected, the prescription amount of L-LD is from 10 to 3,000 U / L, and D-LD is further prescribed thereto. If the amount of D-LD is less than 1,000 U / L, it is difficult to accurately measure the ALT activity in the sample. Conversely, even if it exceeds 30,000 U / L, it is necessary to ensure the measurement range. Since the above effects cannot be expected, the prescription amount of D-LD is preferably in the range of 1,000 to 30,000 U / L.

Furthermore, in this case, the prescription of carbonate and bicarbonate is also effective in that it suppresses the increase in reagent blank reaction. Specifically, for example, as carbonates and bicarbonates, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, calcium carbonate, barium carbonate, lithium bicarbonate, sodium bicarbonate, potassium bicarbonate, cesium bicarbonate and the like Can be used. In this case, the carbonate and bicarbonate are ineffective at less than 1.0 mmol / L, and on the contrary, even if it exceeds 150 mmol / L, no further effect can be expected in suppressing the increase in the reagent blank reaction. It is preferably within the range of 1 to 150 mmol / L.

Among the components contained in the ALT activity measuring reagent of the present invention, various components contained in the known ALT activity measuring reagent, that is, LD, NADH, L-alanine, and 2-oxoglutaric acid, are known ALT activities. It can be used in the same manner as the measurement reagent.
Furthermore, the ALT activity measurement reagent of the present invention can further contain an appropriate buffer as in the known ALT activity measurement reagent. In this case, as long as the ALT activity measurement is not adversely affected, A known buffer solution can be appropriately selected and used. Specifically, for example, tris (hydroxymethyl) aminomethane, phosphoric acid, 2- [4- (2-hydroxyethyl) -1-piperazyl] ethanesulfonic acid, bis (2-hydroxyethyl) iminotris (hydroxymethyl) Methane, 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, or n-ethylmorpholine may be used. it can.

  Furthermore, the reagent for measuring ALT activity of the present invention contains, in addition to the above-mentioned essential components and buffering agent, generally added components such as chelating agents [for example, ethylenediaminetetraacetic acid (EDTA)], antiseptics, and the like. An agent (for example, azide, etc.), a stabilizer (for example, albumin, glycerol, etc.), and / or various surfactants can be added as appropriate.

  The reagent configuration of the ALT activity measurement reagent of the present invention is not particularly limited, as is the case with known ALT activity measurement reagents. For example, the reagents can be combined into one reagent, or two or more reagent configurations. In general, it is preferable to have a reagent configuration in which each constituent component is divided into stable conditions and can react under optimum conditions for activity measurement. As such a reagent structure, for example, it is divided into a first reagent containing alkaline and stable NADH and LD, and a second reagent containing 2-oxoglutaric acid and the like. Each of these reagents is configured to have a pH, and L-alanine can be added to either the first reagent or the second reagent, or both, but is not necessarily limited to this. is not.

  In addition, when measuring a test sample that may contain pyruvic acid (for example, body fluid such as blood, serum, plasma, or urine, or cell tissue) as a test sample, it is derived from the test sample. In order to eliminate the influence of pyruvic acid, it is preferable to use a two-reagent system containing at least LD as the first reagent and 2-oxoglutaric acid as the second reagent. In this case, the test sample that may contain pyruvic acid and the first reagent containing LD are mixed, and after removing pyruvic acid derived from the test sample, the second reagent is added, thereby the test sample. The ALT activity can be measured without being affected by pyruvic acid derived therefrom.

  Even in the case of a two-reagent system containing at least LD and 2-oxoglutaric acid as the second reagent, pyruvate derived from the test sample was erased in about several tens of seconds to one minute after the addition of the second reagent, for example. The ALT activity can be measured later, or the same purpose can be achieved even with one reagent system. Therefore, the ALT activity measuring reagent of the present invention is not limited to a specific reagent configuration.

The ALT activity measuring reagent of the present invention can be used in the ALT activity measuring method of the present invention. In the ALT activity measurement method of the present invention, the test sample is brought into contact with L-alanine, 2-oxoglutarate, LD, and NADH, and the decrease in NADH amount is measured at a wavelength of around 340 nm, whereby the ALT activity is measured. Can be measured stably.
The test sample is not particularly limited as long as it is a test sample that may contain ALT activity. For example, a biological fluid commonly used for clinical diagnosis, for example, blood, serum, Plasma, urine, cell tissue, experimental sample, etc. can be mentioned.

  The contents of the present invention will be described below based on the description of examples, but the present invention is not limited to these examples.

  As reagents for measuring ALT activity of the present invention, a first reagent having the composition shown in Table 1 and a second reagent having the composition shown in Table 2 were prepared (hereinafter referred to as “reagent A”). In addition, as a comparative example, a first reagent not containing oxamic acid was prepared and combined with a second reagent having a composition shown in Table 2 (hereinafter referred to as “reagent B”).

The above-described reagent A and reagent B were subjected to an open storage stability test.
Reagent A and Reagent B prepared as described above are set in an automatic analyzer (H7180; manufactured by Hitachi, Ltd.) with the lid of the container opened, and allowed to stand for 21 days. Each blank value was measured on the 14th and 21st days.

  Specifically, after adding 4.5 μL of physiological saline and 120 μL of the first reagent as a sample to the reaction cell of the above automatic analyzer, the mixture was stirred for 5 minutes in a 37 ° C. environment, 40 μL of the second reagent was added and stirred, and then the change in absorbance at a wavelength of 340 nm from about 1 minute to 5 minutes was calculated by an automatic analyzer. The results are as shown in FIG. 1. Reagent A in which oxamic acid is formulated according to FIG. 1 has a lower blank value than that of reagent B as a comparative example, and the variation in blank over time is 0.6 mAbs. / Min, which is very small, it is clear that blank fluctuations over time are suppressed.

  As a reagent for ALT activity measurement of the present invention, a first reagent having the composition shown in Table 3 and a second reagent having the composition shown in Table 2 of Example 1 were prepared (hereinafter, this two-reagent reagent was used). Referred to as “Reagent C”). As a comparative example, a first reagent in which only L-LD was excluded from the composition shown in Table 3 was prepared, and a two-reagent reagent combined with the second reagent in Table 2 was prepared (hereinafter, this two-reagent reagent was referred to as Referred to as “Reagent D”).

As a result of examining the upper limit of the measurement range, the following became clear.
That is, the reagent C and the reagent D prepared in Example 2 were set in the same automatic analyzer as used in Examples 1 and 2 described above, and measurement was performed by the same method as in Example 1. In this case, the specimen used was High Level Check E (manufactured by Sysmex Corporation) diluted in five stages with physiological saline. For the measurement of the ALT activity of the sample, an enzyme calibrator (manufactured by Kainos Co., Ltd.) was used instead of the sample, and the calculation was performed based on the obtained measurement results.

  The result is as shown in FIG. 2. Compared with the reagent D of D-LD alone, the reagent C, which is a mixture of L-LD and D-LD, ensures a wider measurement range. I understand that. Specifically, for both the reagent C and the reagent D, the sample measurement value of 1 / 5-fold dilution is 500 U / L, whereas in the sample that is not diluted, the measurement value of the reagent D is 2316 U / L. The measured value of Reagent C was 2424 U / L, and the measurement range was improved when Reagent C was used.

  As a reagent for ALT activity measurement of the present invention, a first reagent prepared by excluding only potassium hydrogencarbonate from the compositions shown in Table 3, and a second reagent having the composition shown in Table 2 of Example 1 above. (Hereinafter, this two-reagent reagent is referred to as “reagent C”). Further, as Comparative Example 1, a first reagent having the composition shown in Table 4 was prepared, and a two-reagent reagent in which this was combined with the second reagent in Table 2 was prepared (hereinafter, this two-reagent reagent is referred to as “reagent E”). Called). Further, as Comparative Example 2, a first reagent having the same composition as the known ALT activity measurement reagent shown in Table 5 was prepared, and a two-reagent reagent combined with the second reagent having the composition shown in Table 2 was prepared (hereinafter referred to as “reagent”). This two-reagent reagent is referred to as “reagent F”).

As a result of examining the open storage stability, the following became clear.
That is, Reagent C, Reagent E and Reagent F prepared in Example 1 and Example 3 are set in an automatic analyzer (H7180; manufactured by Hitachi, Ltd.) with the container lid opened, and left for 35 days. At the same time, measurements were carried out on the 7th day, 14th day, 21st day, 28th day, and 35th day by the same method as in Example 1. In this case, the sample was measured using physiological saline and pooled serum.

  The ALT activity measurement result of the physiological saline (blank) in that case is shown in FIG. In this case, when the lid is left open, the reagent blank value (absorbance mAbs / min) fluctuates (decreases) with time in the comparative reagent E and reagent F, whereas the present invention In reagent C, the reagent blank value hardly changed. Specifically, with respect to the change of reagent E: 1.2 mAbs / min and reagent F: 1.7 mAbs / min, the blank value of the reagent C of the present invention was not changed. Moreover, it has been confirmed that the reagent blank can be significantly reduced from -2.2 mAbs / min to -0.3 mAbs / min by dare combining L-LD and D-LD which are the same substrates. did.

Moreover, the measurement result of the ALT activity when pooled serum is used as a specimen is shown in FIG. Also in this case, the ALT activity was measured by the same method as in Example 2. As for the measured value of the specimen, if the reagent is left in a state where the lid is opened, the measured ALT activity (U / L) increases with time for the reagent E and F for comparison. On the other hand, with the reagent C of the present invention, there was almost no change in the measured value of the ALT activity.

Claims (6)

  In a reagent for measuring alanine aminotransferase activity including L-alanine, 2-oxoglutarate, lactate dehydrogenase, and reduced nicotinamide adenine dinucleotide, D-LD is combined with L-LD of the same substrate. A reagent for measuring alanine aminotransferase activity, characterized in that   The reagent for measuring alanine aminotransferase activity according to claim 1, wherein L-LD is formulated at 10 to 3,000 U / L and D-LD at 1,000 to 30,000 U / L. The reagent for measuring alanine aminotransferase activity according to claim 1 or 2, wherein 1 to 150 mol / L of carbonate and bicarbonate is formulated.   Formulating a test sample possibly containing alanine aminotransferase, L-alanine, 2-oxoglutarate, D-LD, L-LD of the same substrate as D-LD, and reduced nicotinamide adenine dinucleotide A method for measuring alanine aminotransferase activity, comprising:   The alanine aminotransferase activity according to claim 4, wherein L-LD is formulated to be 10 to 3,000 U / L and D-LD of the same substrate as that of D-LD is 1,000 to 30,000 U / L. Measuring method. The method for measuring alanine aminotransferase activity according to claim 4 or 5, wherein 1 to 150 mmol / L of carbonate and bicarbonate is formulated.

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