Method for determining glutamatoxalacetattransaminaze or glutamatpyruvattransaminaze

专利摘要:
This invention relates to clinical biochemistry and is intended to determine glutamate oxalacetate transaminase and glutamate pyruvate transaminase and a reagent for their determination. The purpose of the invention is the acceleration of the method. Pyruvate and α-aminobutyrate are added to glutamate. In this case, the buffer solution has a pH of 7.0-9.0. Aminobutyratetransaminase and semi-aldehyde dehydrogenase are added to the reaction mixture. Perform photometry. The glutamate pyruvate transaminase activity is then calculated. 4 tabl.
公开号:SU1276269A3
申请号:SU792793851
申请日:1979-08-06
公开日:1986-12-07
发明作者:Денеке Ульферт；Шталь Петер；Шнайдер Вальтер
申请人:Берингер Маннхайм Гмбх (Фирма)；
IPC主号:

专利说明:


sn
The invention relates to methods for determining the activity of the enzyme glutamate oxalacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT), as well as reagents suitable for carrying out this method and can be used to determine the enzymes mentioned in biological fluids such as serum, urine or other substances.
The purpose of the invention is to accelerate the analysis.
Determination of glutamate oxalacetate transaminamine or glutamate bypassing the enzyme under investigation with glutamate in a buffer solution followed by photometry, for which 50 mmol / l glutamate is previously reacted with 0.150 mmol / l oxanate or in place at a time in a gymnast or in a gymnitral gymnast by an athlete. 9.0, then 10-300 mmol / l of Jf-aminobutyrate with 500-2000 U / l of aminobutyrate transaminase are added to the reaction mixture, then semi-aldehyde dehydrogenase is added. The resulting reconstituted NADP is photometric.
The method is carried out as follows.
Combine the reactions according to the equations
PAV-GT
about -KS-aminobutyrate
bough
cinatsemaldehyde + od-glutamate, where GAB-GT-j is aminobutyrate transaminase; , succinate semialdehyde + NADP --- Sqq d) iji
-S- succinate + NADPH + H, where
SS -AB-DH succinate semialdehyde dehydrogenase,
and carrying out a reaction which is carried out with a tetrazole salt in the presence of an electron carrier to re-oxidize NADPH while simultaneously forming colored formazan, which can be easily measured in the visible part of the spectrum. This corresponds to the equation:
NADPIl 4-MTT + K DiaForaz |
+ NADP / where MTT 3- (4,5-dimethylthiazol-2) -2,5-diphenyltetrazolyl bromide.
The method is carried out at pH 7-9.5. At higher and lower pH values, the reaction is significantly slowed down with a corresponding lengthening of the time required to carry out a single test. The best results are obtained with pH values in the range of 8-8.6.
A suitable buffer concentration is in the range of 5-0.5 mol / l, which corresponds to a concentration of a buffer salt in the test of 0.05-6%. Preferably the use of a buffer concentration of 10-100 mmol / l.
MTT is used as a tetrazole salt.
Good results have been obtained with nitrogolubic tetrazole (NBT) chloride and 2- (p-iodophenyl) -3- (p-nitrophenyl) -5-phenyltetrazole (INV) chloride. When measuring the dye formazan, it is advisable to add surface-active substances that improve the solubility of the dye.
Abbreviations are used in the examples: GPT - glutamate pyruvate transaminase;
GAB-GT - - aminobutyrate transaminase:
SS-A1-DH - sukusnatsemitialdehyde dehydrogenase;
GOT - glutamate oxalacetate transaminase,
NADP - nicotinamide adenine dinucleotide phosphate;
NADPH - nicotinamide adenine dinucleotide phosphate reduced;
MTT - 3- (4,5-dimethylthiazolyl-2) -2, 5-diphenyltetrazolyl bromide;
Tris - Tris (hydroxymethyl) -aminomethane; Triton RX100 - alkylaryl polyethylene glycol ether.
Example 1. GPT definition. The formation of NADPH is a measurement signal. The temperature is 25 ° С, the wavelength is 365 nm, the test volume is 3 ml, 1 centimeter cuvette.
The data are given in table.1.
The original solution is stirred. Start the reaction with 0.5 ml of serum (GPT-enriched), mix, incubate for 2 min, take readings.
after 1, 2, 3 and 4 minutes of exact time
Meni, then removed. Shows E
and E,
9
Based on the data obtained, the value of E / min is calculated
GPT activity in the sample is calculated by the formula
TG E. 1.714.
U / ml

权利要求:
Claims (6)
[1]
Example 2. GPT definition. The formation of formazan is chosen as the measurement signal. The temperature is 25 ° C, the wavelength is 578 nm, the test volume is 3 ml, and the cuvette is 1 cm thick.
[2]
2. The initial solution is stirred. Begin the reaction with 0.1 ml of serum (GPT-enriched), mix, incubate for 2 minutes, and show 1, 2, 3, and 4 minutes for exactly the same time. Further, E, E, E and E are taken, on the basis of which the CPT-E / min value is calculated. The activity in the sample is calculated using the following formula: LE 1,796 U / ml Example 3. GPT determination The formation of NADPH is accepted as a measurement measure. The measurement temperature is 25 ° C, the measurement wavelength is 365 nm, the test volume is ml, the cuvette is 1 cm thick. The substances presented in Table 2 are pipetted into the cuvette.
[3]
3. The original solution is stirred. The reaction is started with 0.5 ml of serum (GOT-enriched), stirred, incubated for 2 minutes, readout E, after 1, 2, 3 and 4 minutes of the exact time, then take readings of Ej, Е 3 4 5 based on the obtained data calculate the LU / min. GOT activity in the sample is calculated according to the formula U / ml LU 1, 717 Example
[4]
4. Definition of GOT Formazan formation is taken as a measurement signal. The temperature is 25 ° C, the wavelength is 578 nm, the test volume is 3 ml, the cell is 1 cm thick. The initial solution is stirred. The reaction starts with 0.05 ml of serum (GOT-enriched), stirred, incubated for 2 minutes, EJ readings reduced, after 1, 2, 3 and 4 minutes of exact time, then her and her EC readings ZV 694 GOT ACTIVITY in the sample is calculated according to the formula U / ML 3,593. PRIOR
[5]
5. Definition on the test strip. A suitable paper is impregnated with a solution containing all of the reagents shown in example 2, carefully dried, fixed on the IGODRUM carrier, sealed and cut into strips. After dipping, a red-blue coloration appears, the intensity of which is proportional to the ORT activity in the sample. The evaluation can be carried out by comparison with a suitable standard staining scale, as well as with a reflective photometer. Example
[6]
6. Determination of GOT on the test strip. A suitable paper is impregnated with a solution of the reagents prepared according to Example 4, carefully dried, fixed on a suitable carrier, sealed if necessary, and cut into strips. After. Serum kpacHo staining appears in the serum, the intensity of which is proportional to the GOT activity in the sample. Assessment can be made by comparing with a suitable standard color scale, as well as using a reflective photometer. Claim method for determining glutamate pyruvate transaminamine glutamate oxalacetate transaminase by incubating the enzyme under investigation with glutamate in a buffer solution followed by photometry, characterized in that, in order to speed up the analysis, 50 mmol / l glutamate is preliminarily introduced into the mutant: mmole / l oxanate or pyruvate, respectively, in a buffer solution with a pH of 7.0-9.0, then 10,300 mmol / I J-aminobutyrate and 5002000 U / l amine butyrate transammones are added to the reaction mixture, then semi-aldehyde dehydrate is added antigenase; the resulting reconstituted NADP photomet.
Tris, HC1, pH 8;
3.50 m) ol / l
(0.6055%)
NADP, 27 mmol / l
(2%)
- Aminobutyrate, 9 mmol / l, pH 8.3 (9%) 0.2
Glutamate, 0.6, mol / l
pH 8.3 (9.8%)
Pyruvate sodium,
0.55 mol / l (6%)
GAB-GT, 86 U / ml (11% protein)
, 02
25 (0.30275) 0.9 (0.067)
60 (0.06) 0.1 (1.47) 5.4 (0.06)
I, 43 U / ML (1,183)
Tris, HC1, pH 8.3; 50 mmol / l (O, 6055%) i containing 2% Triton RX100
NADP, 27 mmol / L (2%)
J-Aminobutyrate, 0.9 mol / L (9%)
Glutamate, 0.6 mol / L (8.8%)
Pyruvate sodium, 0.55 mol / L (6%)
table 2
Triton KHGOO
(one%)
Tris, 25 (0.30275)
0.9 (0.067)
0.2 60 (0.06)
0.5 0.1 (1.47)
0.03 5.4 (0.06)
12762698
jZIZiIZZI
0.2 0.1 (0.00414)
1, 4: 3 U / ml
0.04 (0.183%)
2.2 U / l
0.05 (0.183%)
 0.077 U / ml 0.01 (0.0017%)
0.26
". ....... ".." | ... ".
Quantity- Concentration
Imidazole, HC1 pH 7.6
100 mmol / l
(6,808%)
NADP, 27 mmol / L (2%)
y-Aminobutyrate,
0.9 mol / l pH 8.3 (9%)
Gluzamat, 0.6 mmol / l
pH 8.3 (8.8%)
Oxalacetate
20.8 mmol / l (0.275%)
GAB-GT, 86 U / l
(11% protein)
SS-AI-DH, 133 U / ml
(11% protein)
HjO0,1
The continuation of the table.2.
I x
Table 3
in, mm in the dough,
mmol / l (%)
50 (3.404) 0.9 (0.067)
90 (0.9) 60 (0.88)
0.65 (0.00917)
1.43 U / ml (0.183%)
2.22 U / ml (0.183%)

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引用文献:

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US4575488A|1982-06-25|1986-03-11|Technicon Instruments Corp.|Interference free transaminase assay|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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DE19782834706|DE2834706A1|1978-08-08|1978-08-08|METHOD AND REAGENT FOR DETERMINING GLUTAMATE-OXALACETATE TRANSAMINASE AND GLUTAMATE-PYRUVATE TRANSAMINASE|

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