JPH0143560B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to glutamate oxaloacetate transaminase (hereinafter abbreviated as GOT) in biological samples.
This invention relates to a method for storing compositions used for measuring.

More specifically, the present invention utilizes an ultraviolet absorption method (hereinafter referred to as
In the GOT measurement method based on the UV method (abbreviated as UV method),
Reduced beta-nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH) is mixed with an alkaline solution and L-aspartic acid and malate dehydrogenase (hereinafter referred to as
(abbreviated as MDH) and lactate dehydrogenase (hereinafter referred to as
The present invention relates to a method for preserving a composition containing components of LDH (abbreviated as LDH), α-ketoglutaric acid, and a PH buffer at room temperature.

The above term "microbial origin" is in contrast to the term "animal organ origin" and means that microorganisms such as yeast and bacteria are used as raw materials. Note that the origin of the above-mentioned lactate dehydrogenase does not matter. In addition, the above "PH buffer" means "PH buffer aqueous solution",
The upper limit of the above room temperature is 35°C.

The object of the present invention is to provide a useful GOT measurement composition that has a long lifespan of one day or more at room temperature of 10° C. or higher without refrigeration, together with NADH and L-aspartic acid, for GOT measurement by UV method. is to provide. Further, the effects of the present invention include not only saving the trouble of refrigerating reagents and preventing waste of reagents, but also improving the operating efficiency of a multi-channel automatic analyzer.

Quantification of GOT in biological samples such as urine and blood is important for clinical testing as an indicator for the diagnosis and treatment of various diseases, such as clinical diagnosis and follow-up, such as distinguishing between heart disease and liver disease, acute liver injury and chronic liver injury. This is one of the important items. In the UV method, oxaloacetic acid (hereinafter referred to as OAA), which is generated from L-aspartic acid and α-ketoglutaric acid, coexists with the catalytic action of GOT and MDH as a conjugate enzyme. A method of measuring GOT by reducing NADH to acid and measuring the rate of oxidation reaction of NADH to NAD (oxidized beta-nicotinamide adenine dinucleotide) (rate of decrease at 340 nm, the characteristic absorption wavelength of NADH). be. In order to understand the above description, please refer to the following first and second reaction formulas.

1st reaction formula L-aspartic acid + α-ketoglutarate GOT ――――→ Glutamic acid + OAA 2nd reaction formula OAA + NADHMDH ――――→ Malic acid + NAD The UV method is a method of measuring GOT using phenylhydrazine, which is called a color method. Accuracy, compared to
This is an excellent GOT measurement method with good accuracy. However, since the UV method uses conjugated enzymes and coenzymes for measurement, if a GOT measurement reagent containing these enzymes is stored at room temperature, it will retain its effective activity for less than a day, although it may vary slightly depending on the room temperature. Can't hold it. For this reason, the reagent solutions set in the multi-channel automatic analyzer must be replaced frequently, which is complicated and time-consuming, and the reagents that have expired are often discarded, resulting in a large amount of wasted reagents.

Therefore, there is a strong desire for a reagent composition that can be stored at room temperature for a long period of time. It is known that NADH used for measuring GOT is stable even at room temperature if it is alkaline with a pH of 9 to 11. In the GOT measurement reagent, NADH is used as a separate alkaline reagent so that it does not lose its buffering properties even when combined with other reagents containing enzymes (PH is the value at the time of measurement). There is no practical problem regarding its lifespan. Also, α-ketoglutaric acid and L-aspartic acid can be stored for a long period of time without any problem if a disinfectant is used. it contains enzymes
An object of the present invention is to stabilize a reagent for GOT measurement at room temperature.

In order to meet these demands, the present inventors developed a GOT that is highly stable, that is, can be stored for a long period of time at room temperature.
As a result of our efforts to search for a reagent composition for measurement, we have developed a reagent composition for GOT measurement that uses MDH of microbial origin in the presence of a bactericidal agent, which allows GOT to be measured when mixed with an alkaline solution of L-aspartic acid and NADH. The present invention was achieved by discovering that the composition is stable for one week or more at room temperature.

The GOT measurement composition mentioned here can be used as a GOT measurement reagent by simply combining NADH and L-aspartic acid, or it can be diluted with water or buffer or mixed with other additives. This means a composition that can be used as a GOT measurement reagent.

If MDH derived from animal organs is used instead of the MDH derived from microorganisms, its instability is significantly increased. Therefore, animal organ origin
When using MDH, it is not possible to measure GOT in the sample after it has been stored at room temperature for several days.

It has been known for some time that MDH derived from microorganisms (mostly MDH derived from baker's yeast) can be used as a reagent for measuring GOT. However, it was completely unknown that this enzyme, unlike those derived from animal organs, was stable at room temperatures above 10°C. Moreover, it was completely out of consideration that the activity would not decrease even if the GOT measuring reagent was stored at room temperature for more than one day, especially for more than three days.

Therefore, although it is known that NADH is stable in an alkaline solution, there are almost no reagent kits on the market that use NADH as a separate reagent. As a result, when a reagent kit mixed with NADH is stored in a measurement state, it is only stable for about 3 days at most even if stored in a refrigerator. Also
For example, the difference in the effect on stability between low temperatures below 10°C and room temperature is significant.
TA-E kit (GOT measurement reagent manufactured by Yatron)
is stable for 7 days at low temperatures (6-8°C), but only for 5 hours at room temperature (25-30°C).

Indeed, since it is known that microbial-derived MDH is used as a reagent for GOT measurement, it is possible to take out the reagent from the refrigerator and charge it to the measurement device.
If it is left on a desk or the like for some time, it is kept at room temperature, but these cases are during the preparation period for measurement and do not fall under the category of preservation as described in the present invention. However, if the device is left charged at room temperature for one day or more, this is a case covered by the present invention.

GOT can be measured using the reagents stored using the method of the present invention, but the principle of the measurement is based on the first method described above.
Based on the reaction formula and the second reaction formula, GOT is
It is proportional to the rate of decrease in NADH. The activity of GOT can be determined by measuring the decrease in absorbance at 340 nm at a constant temperature using a spectrophotometer.

The MDH used in the present invention may be of any type as long as it is of microbial origin, including those of baker's yeast (Saccharomyces cerevisiae), heat-resistant bacteria (Thermus thermophilus, Thermus
flavus, Bucillus stearo thermophilus, etc.), Brevibacterium flavum
Those originating from bacteria belonging to the genus Brevibacterium, such as Brevibacterium flavum, are preferred.

As mentioned above, it is necessary to use MDH of microbial origin, but as already mentioned, LDH of any origin can be used, including those of animal organ origin and microbial origin. It is preferable to add a sulfhydryl compound and a chelating agent to this LDH. These sulfhydryl compounds and chelating agents are capable of stabilizing LDH, and have little effect on the stabilization of microbial-derived MDH. Therefore, even if the reagent that has been stored at room temperature for one week or more in the measurement state does not contain sulfhydryl compounds or chelating agents, GOT can be measured without any problems as long as the sample contains a large amount of pyruvic acid.

Bactericidal agents that can be used in the present invention include, for example, alkali metal azides, cationic surfactants, benzoic acid derivatives, methyleneglutaronitrile bromide, various antibiotics, and the like.

The preferred concentration of microbially derived MDH in the composition according to the invention is 0.1-5 u/ml. Further, preferred concentrations of other components of the composition are as follows.

LDH 0.1-5u/ml α-Ketoglutar 1.0-50mM Buffer 0.02-0.25M Bactericide 0.01-1.0% by weight NADH and L-aspartic acid which are added to the composition of the present invention and used for measuring GOT The preferred concentration after addition to the composition is 0.1~
0.5mM concentration and 0.05-1.5M concentration. In addition, in order to improve the distribution and drainage of automatic analyzers,
~0.1% by weight of nonionic surfactants may be added.

The buffering agent in the above composition adjusts the pH of the composition to 5.5 to 9.0.
Of those that exhibit a buffering ability in part or all of the range of 340 nm, any material may be used as long as it has little light absorption at 340 nm and does not significantly interfere with GOT measurement by UV method. Specifically, for example, Tris buffer 0.025
A concentration of ~0.25M is preferred.

Methods for measuring the activity of MDH and LDH above
of Enzymatic Analysis (2nd.Ed.) Volume 1 485
(Method for measuring MDH), Volume 2, 574 (Method for measuring LDH),
It was carried out according to the method described in Academic Press 1974. However, the measurement temperature was 30°C.

Next, the present invention will be specifically explained with reference to Examples of the composition of the present invention.

Example 1 α-ketoglutarate 15mM concentration, sodium azide 0.02% by weight, Triton X-100 0.01% by weight,
Yeast-derived MDH1500u/, pig heart-derived
Tris (hydroxymethylamine) methane-hydrochloric acid buffer (PH
7.8) (Liquid to be preserved according to the present invention) 1 was prepared and designated as Reagent A. In addition, a solution 1 was prepared in which MDH derived from animal organs was used instead of MDH derived from yeast as a component of reagent A, and this was designated as reagent A'.

Reagent A and Reagent A' were stored at 27°C for 10 days, and the activity of MDH during that period was measured using the Methods of
Enzymatic Analysis (2nd.Ed.) Volume 1 485,
It was determined according to the measurement method described in Academic Press 1974 (measurement temperature was 30°C). This measurement method is based on the reaction shown in the second reaction equation above.
MDH is quantified based on the rate at which NADH changes to NAD. The results are shown in Figure 1. As shown in the No. 1 line in Figure 1, yeast origin
Whereas MDH is stable for more than 10 days
The product derived from animal organs, shown by line No. 2, had 50% residual activity after 2 days and had almost lost its activity after 7 days.

Example 2 Prepare the GOT measurement reagent A used in Example 1, then add 4mM concentration of NADH and sodium azide.
A 1% by weight aqueous solution of NaHCO ₃ containing 0.02% by weight, 0.01% by weight of Triton Next, L-aspartic acid 1M concentration, sodium azide 0.02% by weight, Triton X-100
A 0.1M Tris buffer with a pH of 7.8 containing 0.01% by weight was prepared. This is called reagent C. These reagents A, B, and C were stored at room temperature of 27°C. This reagent A...
The GOT of the control serum was measured using samples B and C immediately after preparation and those stored for 7 days (27°C) after preparation, respectively. The results are as shown in FIG. 2, and a pattern consistent with the use of the reagent immediately after preparation and the use of the reagent stored after 7 days was obtained. This pattern was straight up to 1400u/.

The amounts of each composition of the above reagents are 0.4ml of reagent A,
There were 0.03 ml of reagent B, 0.12 ml of reagent C, and 0.05 ml of serum.

Example 3 Similar to Example 2, GOT measurement reagents A, B, and C
10 days after preparation, these reagents were mixed and added to the sample control serum, and its GOT was measured. On the other hand, a commercially available kit for GOT measurement (Fujisawa Kit) was used for the same control serum.
As a result of measuring the GOT using the GOT and plotting the correlation between the two results on a graph, as shown in FIG. 3, a good correlation was obtained showing that the two results almost coincided.

Example 4 Similar to Example 2, GOT measurement reagents A, B, and C
was prepared. In addition, A' was prepared by replacing MDH in reagent A with one derived from animal organs, and each was stored at 27°C. These reagents were mixed in the same manner as in Example 3, and GOT in the control serum was mixed with reagent A.
Comparative measurements were made using the +B+C combination and the reagent A'+B+C combination. As shown in Figure 4, microbial origin
The method using MDH could be stably measured for one week, but the method using MDH derived from animal organs had 80% of the GOT activity of the control serum after being stored for 3 days.
After 7 days of storage, only 10% activity could be measured.

[Brief explanation of drawings]

Figure 1 shows the composition of the GOT measurement composition according to the present invention.
The stability of MDH at 27°C is shown. FIG. 2 shows the linearity of GOT measurements of control serum using reagents after implementing the preservation method of the present invention. FIG. 3 shows the results of measuring the GOT of control serum using reagents and commercially available kits subjected to the preservation method of the present invention. Figure 4 shows MDH of microbial origin and animal organ origin.
FIG. 3 is a diagram showing a comparison of GOT measurement results using a reagent using MDH.

Claims (1)

[Claims] 1 Yeast-derived malate dehydrogenase and lactate dehydrogenase, α-ketoglutarate, bactericide, and PH buffer used for measuring glutamate oxaloacetate transaminase together with reduced beta-nicotinamide adenine dinucleotide and L-aspartate. A method of storing a composition containing each component of the liquid at room temperature of 10°C or higher for one day or more.