Reaction agent for determining glycerine by photometry

专利摘要:
The invention relates to the field of medicine and can be used in clinical biochemistry for the determination of glycerol and triglycerides in blood serum. The aim of the invention is to increase the specific activity. Glycerol oxidase from ASPERGILLUS SPEC is used for this. ASM 1729.
公开号:SU1554765A3
申请号:SU813285563
申请日:1981-02-04
公开日:1990-03-30
发明作者:Гауль Хельмгард；Зайдель Ханс；Ланг Гунтер；Редер Альберт；Цигенхорн Йоахим
申请人:Берингер Маннхайм Гмбх (Фирма)；
IPC主号:

专利说明:

The invention relates to medicine and can be used in clinical biochemistry for the determination of glycerol triglycerides in serum.
The aim of the invention is a reagent comprising glycerol oxidase, characterized by a higher specific activity.
This goal is achieved by using glycerin oxidase from Aspergillus Spec. DSM 1729 (soil microorganism), which was obtained with a molecular weight of 90,000 and a specific activity between 2000 and 8000 V / mg, whose activity is reduced by SH reagents, and which is not affected by the presence of sulfuric acid copper and acetic acid lead.
In the determination of triglycerides, their enzymatic hydrolysis is first carried out under the action of lipase and
(L
esterases to glycerol and the amount of free glycerol formed is determined by glycerol oxidase, measuring either the oxygen consumption or the amount of hydrogen peroxide.
Oxygen consumption is determined polarimetrically using an oxygen electrode, less frequently using gas chromatography.
Hydrogen peroxide can be determined both by titration and by potentiometric, polarographic, and colorimetric methods. The preferred method is an enzymatic method using peroxidase. When using the latter as a chromogenic substance (dye), compounds are used which, after reaction with phenol, can be determined photometrically, for example, 2,2-aminobenethiazoline sulfonic acid, 4-aminoantipyrin, phenylene diamine sulfonic acid
SP
SL
J J O SL

O4
methylbenzothiazolone and a buffer solution. In addition to these reagents, the reagent may additionally contain a solvent, a stabilizer, and / or a surfactant.
Preferred surfactants are Genaprol, alkylpolyglycol ether, triton. polyethylene octylphenol oxide
Polyethylene octylphenol oxide (fa and Bryde, as well as polyethylene glycol ether of lauryl, stearyl and oleyl alcohol.
At the specified pH value of 6.8 to 8.0, the enzyme has good resistance,
A preferred combination of reagents consists of
100-250 KV / L (333-833 mg / L)
glycerol oxidase;
5-50 KV / l (.20-200 mg / l)
peroxidase;
0.2-5 mmol / l (38-955 mg / l)
indicator (4-antipyrine
amino substituted);
5-50 mmol / l (64-6400 mg / l)
parachlorophenol;
1-5 g / l of surfactant;
pH 6.8-8.0 buffer solution
Example 1. Determination of glycerol by the formation of hydrogen peroxide. Two reagents were prepared.
Reagent 1.
0.1 mmol / l triethanolamine
(HCl buffer, pH 8.0);
2 g / l sodium cholate
(4.7 mmol / l);
1150 mg / l parachlorphenol
(10 mmol / l);
86 mg / l aminosubstituted
4-aminoantipirin (0.5 mmol / l);
40 mg / l peroxidase (10 V / ml).
Reagent 2. 1600 mg / l glyceroxydase (500 V / ml).
For the determination, 2 ml of reagent 1 and 0.2 ml of reagent 2 are pipetted into a cuvette. The extinction E is measured on a photometer at 546 nm. Immediately thereafter, initiate the reaction by adding 20 μl of the sample. For determination of glycerol, aqueous media of all types can be used as a sample, for example, food extracts, body fluids, and serum.
After reacting for 20 minutes, the extinction of Kg is measured. The holding temperature is 25 ° C.
0
five
five
0
five
0
five
The evaluation is made by the calibration straight line, where the difference in the measured extinkium & Е Е – Е1 standard glycerin solution depends on the glycerol concentration.
The final concentration in the test mixture is:
45 V / ml - 144 mg / l glycerol oxidase;
9 V / ml - 36 mg / l peroxidase;
0.45 mmol / l-86 mg / l of amino-substituted 4-antipyrine;
9 mmol / l-1156 mg / l of parachlorophenol;
4.3 mmol / l-1800 mg / l cholate
on three ;
pH 8.0 0.09 mol / l triethanolamine /
/ HC1 - buffer solution
Example 2. Kinetic definition.
Try. Aqueous standard solution (glycerin content from 10 to 100 mg / dL).
Reagent. 0.1 mol / L Pipes buffer; 1.7 mol / n H, 1U, -, PH 7.0; 3.0 g / l sodium cholate; 0.5 mmol - 90 mg / l of aminoantipyrine, amino-substituted; 10.0 mmol / l parachlorphenol (1170 mg / l) g, 10.0 V / l (40 mg / l) peroxidase; 121.0 V / l 403 mg / l glyceroxydase.
The determination is carried out on a Gemsaec Fast automatic analyzer: temperature 25 ° C; wavelength at 546 nm; sample volume 10 μl; 1 diluent () 50 μl; reagent volume 500 μl. Measurement time: the first reading is 35 seconds after the start, the second reading is 315 seconds after the start.
The following results were obtained:
Samples (aqueous standard solution).
Glycerin (VV-test), Value mg / dlglycerin-00,%
1 I105
21101
32100
43101
54100
6599
7699
8799
96102
Example 3. A reagent is used in the kinetic determination with the following quantitative ratio of components: from 0.05 to 0.5 mol / l of Pipes buffer; from 0.5 to 2.0 mol / l H3VOE-, pH from 6.8 to 8.0; from 1 to 5 g / l sodium cholate; 0.2-5 mmol / l (38-955 mg / l) of 4-aminoantipiprine, amino-substituted from 5 to 50 mmol / l (640-6400 mg / l of parachlorophenol, from 5 to 50 KV / l (20 - 200 mg / l peroxidase, from 100 to 250 KV / l (.333-833 mg / l) glycerol oxidase.
The invention can be implemented on an automated analyzer (spectrophotometer) available in biochemical laboratories.
PRI me R 4. Determination of glycerol through the formation of rOg when using various amino-substituted 4-aminoantipyrine. Carried out as in Example 1, with Reagent 1 containing various amino-substituted 4-aminoantipyrine (0.5 mmol / L).
The following results were obtained:
Try. The glycerin aqueous standard is 45 mg / dL glycerol.
Derivative Finding again, 4-aminoantipyrine Phenylenediamine sulphonic acid102
MBTH101
p-MBTH99
4-aminoantipyrin (comparison) 101
Example 5. Kinetic determination of glycerol with the use of various surfactants.
Carried out as in Example 2, with the reagent instead of 3 g / l sodium cholate containing the same amount of another surfactant.
Compiled by
five
0
The following results were obtained. Try. The glycerin aqueous standard (45 mg / dL glycerol).
Surfactant Location of substances again%
Acetyltrimethylammonium bromide101
Polyoxyethylene lauryl ether
(Brij 35) 103
20 Etocg. And sorbitan monolaurat (Tween 20) 98
10,5 Etoxn-nonylphenol (Tergntol NPX) 99
Tesit100
Sodium deoxycholate 97.5
Isotridecanol-polyglycline ether (Genapol X-80) 101

权利要求:
Claims (1)
[1]
Cholate sodium (comparison) 99 Formula of the invention
25
five
five
0
A photometric reagent for the determination of glycerol, containing glycerol oxidase from the genus Aspergillus, peroxidase, indicator, surfactant, buffer solution, characterized in that it contains glycerin oxidase from Aspergillus DSM 1729 in order to increase specific activity. as an indicator, contains 4-antipyrin amine-substituted and parachlorophenol, as a surfactant is cholate or sodium deoxynholate, and a buffer solution with a pH of 6.8-8.0 at 0 has the following ratio of components, g / l:
Glycerol acid from
genus Aspergillus
Peroxidase
4-Antipyrnum amine-substituted
Parachlorophenol
Cholat- or
deoxycholate sodium
Buffer solution
pH 6.8-8.0
Konyukhov
0.333-0.833 0.02-0.20
0.038-0.955 0.640-6,400
1.0-5.0 Up to 1 l
Editor Y. Sereda
Tehred L. Serdyukova Proofreader O. Kravtsova
Order 467Discount 478Subscription
VNIIPI State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR 113035, Moscow, Zh-35, Raushsk nab. 4/5

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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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DE3004129|1980-02-05|

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