Method of determining the content of creatinine, creatine and sarcosine in biological fluid

专利摘要:
This invention relates to biochemistry and may find use in clinical diagnostics. The purpose of the invention is to increase the sensitivity of the method. The content of creatine, creatinine and sarcosine in a biological fluid is determined by incubating 3 samples with two reagents at a ratio by volume of sample: reagent 0.05 - 1, while reagent 1 contains a buffer with PH 7.9, 4-aminoantipyrine 0, 8 mmol / l, peroxidase 2 units / ml, in the appropriate case of sarcosine oxidide - 6.5 units / ml and additionally sodium cholate 5 mmol / l, 2,4,6-tribrom-3-hydroxybenzoic acid 8.6 mmol / l, iron-synergistic potassium 10 µmol / l, LUTENSOL @ ON 50 0.5 w / v%, lipase 2 u / ml, ascorbate oxidase 10 u / ml and in the corresponding case, creatine hydrolase 12 e / ml, and reagent II contains, in appropriate case, sarcosine oxidase, 6.5 units / ml and creatine – nogidrolase, 25 units / ml, followed by measuring the color intensity in the resulting mixture at 546 nm in comparison with the standard sample, using CHAINIA PURPUROGENA DSM - 43156 , CHAINIA OCHARACEAE DSM - 43155, STREPTOMYCES FLOCCULUS DSM - 40327. Application of the method allows to increase the sensitivity of the method for determining creatine, creatinine and sarcosine by more than three times compared to the prototype. 3 tab.
公开号:SU1582993A3
申请号:SU4027546
申请日:1986-05-28
公开日:1990-07-30
发明作者:Майр Ульрих；Меллеринг Ханс；Зидель Иоахим；Зайдель Ханс
申请人:Берингер Маннхайм Гмбх (Фирма)；
IPC主号:

专利说明:

This invention relates to biochemistry and can be found in clinical diagnostics.
The goal is to increase the sensitivity of the method.
Example 1. Cultivation of Chainia purpurogena DSM-43156.
In the shake-flask, the organism is (cultured in a complex medium of the indicated composition: 5 g yeast extract; 3 g peptone (typically digested); 2 g NaCl; 0.24 g MgS04- "7HtO; 0.014 g CaC1a x7HgO; 2 g glucose; 10 g of sarcosine; 1 l of water; pH 7.0. The culture grown at 28 ° C shows the activity of about 400 V / l in .30 h. Example 2. Isolation of sarcosine oxidase E.C.1 5.3.1 from Chainia purpurogena.

O4
2.9 kg wet weight of Chainia purpurogena DSM-43156 (obtained by treating 95 l of culture) is suspended in 15 l of phosphate buffer solution with a concentration of 20 mmol / l, pH 8.0, after which the suspension is kept for 4 h 25 ° C with 4 g of lysozyme. To the treated suspension Add 10% of a solution of polyethylenimine (Polymin-G-20 // // BASF) to complete separation of the nucleic acids and extraneous proteins of the Sarkozinoxidine, which is in the supernatant, are bound by a weakly basic anion exchange. nickname (DEAE-Sephadex), then elute with an increasing salt gradient. The addition of ammonium sulfate eluate was adjusted to a concentration of 0.6 mol / L. The enzyme is fused to phenyl-Sepharoe, followed by chromatography with an increasing ammonium sulfate gradient (indicated phosphate buffer solution). The eluate containing more than 4 V / mg protein is adjusted to a concentration of 2.4 mol / L by adding solid ammonium sulfate. The precipitate is treated with 0.1 mol / l phosphate buffer solution, after which the sarcosine oxidase is further purified by passing through molecular sieves (Sephacryl-S-200, Pharmacia). The purified enzyme has a specific activity of 5.5 V / mg.
PRI me R 3. Growing Chairiia ochraceae DSM-43155.
The organism is cultivated in a shake flask in a complex medium of the following composition: 5.00 g / l yeast extract; 3.00 g / l peptone (typically digested); 2.00 g / l Nad; 0.24 g / l MgS04 7H, 0; 0.014 g / l CaCl gx LCW; 2.00 g / l glucose; 10.00 g / l sarcosine; pH 7.0. Diluted at 28 ° C culture after 30 h gives about 200 urea / L sarcosine oxidase activity.
PRI me R 4. Selection of sarcosine oxidase from Chainia ochraceae.
2.6 kg wet weight of Chainia ochra ceae (DSM-43155) from 81 l of culture (200 urea / l, diluted in example 3) is suspended from 14 l of 20 mmol / phosphate buffer, pH 8.0. and dissolved for 4 hours at 35 ° C with 3.8 g of lysozyme. After that, as described in Example 2, negative precipitation of polyethyleneimine and chromatography of DEAE-Sephadex are carried out. The eluate is mixed with ammonium sulfate to 3.0 mol / l, pH 8.0. The precipitated sarcosine oxidase is centrifuged and transferred to phosphate buffer (20 mmol / L, pH 8.0). Partially purified enzyme has a specific activity of 2.1 urea / l.
PRI me R 5. Cultivation of Streptomyces flocculus DSM-40327.
The organism is cultivated in a shake flask in a complex medium of the following composition: 5.00 g / l yeast extract; 3.00 g / l peptone (typically digested); 2.00 g / l NaCl; 0.24 g / MgS04 7H70; 0.014 g / l CaCl2 7H20; 2.00 g / l glucose; 10.00 g / l sarcosine; pH 7.0. Cultivated at 28CC culture after 30 hours makes sarcosine oxidase activity about 30 urea / l.
Example 6. The selection of sarcosine oxidase from Streptomyces flocculus,
3.1 kg of wet mass of Streptomyces flocculus (DSM-40327) from 98 l of culture (30 urea / l, according to example 5) is dissolved by analogy with example 4 and the enzyme is isolated through precipitation with polyethylene imine, DEAE-Sephadex chromatography and precipitation with ammonium sulfate . Partially purified enzyme has a specific activity of about 0.35 urea / mg protein.
Example 7. The use of sarcosine oxidase for the determination of creatinine. Reagent I. Reagent for determining the control value of the sample: Potassium phosphate
(pH 7.9), mmol / l, 150
(or 0.1 mol / l TES / KOH, pH 7.9)
4 Aminoantipirin, mmol / l0,8
2,4,6-Tribromo-
-3-hydroxybenzoic acid,
mmol / l8,6
K4Fe (CN)
μmol / l10
Na-cholate,
mmol / l
Lutensol% N 50,
(w / v)% 0.5
Creataminohydrolase, u / ml 12
Sarkozine oxidase in example 2, u / ml
6.5
Peroxidase, -
u / ml2
Lipazch unit / mp2
Ascorbate oxidase,
u / mp10
Reagent (II) (test reagent): Reagent I plus 25 units / ml creatine-dimerolase.
Test implementation (mixture for determination): wavelength 546 nm; T 25 ° C (or 37 ° C); layer thickness 1 cm; measurement in relation to air (see tab. 1).
Incubation is carried out for 20 minutes at 25 or 37 ° C, and then the extinctions E f - E 4 are measured.
E (E4 - EE) - (E7-E,).
Calculation of the concentration of creatinine in the sample: through a co-administered standard sample of 2 mg / dd. For this, the standard sample is taken in the same mixture for the determination as the sample.
Try on Use of Sark Noxidase to Determine Sarkozy Reagent I (reagent for determining a blank):
Potassium phosphate
(pH 7.9),
mmol / l 150
(or 0.1 mol / l TES / KOH, pH 7.9)
4 Aminoantipirin, mmol / l 0.8
2,4,6-Tribrom-2-hydroxybenzoic acid,
mmol / l8,6
K4Fe (CN) t,
mmol / l10
Cholate,
µmol / Lr 5
Lutensol
ON 50,
(w / v)% 0.5
Peroxidase,
u / ml2
Ascorbate oxidase, u / ml10
Lipase, units / ml2
Reagent II (sample reagent): reagent 1 + 6.5 u / ml sarcosine oxazine.
Test implementation (mixture for determination): wavelength 546 nm; T (or 37 ° C); layer thickness
five
0
0
five
0
five
five
0
five
0
 1 cm; measurement in relation to air (see tab. 2).
Incubate for 20 minutes at 25 or 37 ° C, then extinctions Et-Et
E () - (E1-EO.
Calculation of sarcosine concentration in the sample: through the jointly administered aqueous standard (2 mg / dL). The standard must then be introduced into the determined mixture as a sample.
Limer 9. The use of sarcosine oxidase for the determination of creatine.
Reagent I (reagent for determining blank):
Potassium phosphate
(pH 7.9) mmol / l 150
(or 0.1 mol /, l TES / KOH, pH 7.9)
4 Aminoantipirin, mmol / l0,8
2,4,6-Tribrom-3-hydroxybenzoic acid,
mmol / l8,6
K4Fe (CN),
µmol / l-10
Cholate,
mmol / l 5
Lutensol
ON 50,
(weight / vol.) #, 0.5
Sarcosine oxidase according to
example 2,
u / ml6,5
Peroxidase,
u / ml2
Lipase
u / ml2
Ascorbate oxidase, u / ml10
Reagent II (sample reagent): reagent 1 + 12 units / ml of creatine and hydrolase.
Test implementation (mixture for determination): wavelength 546 nm; T 25 ° C (or 37 ° C); layer thickness 1 cm; measurement in relation to air (see tab. 3).
Incubate for 20 minutes at 25 or 37 ° C, then extinctions E, -E 4 are measured.
Е - (Е4-КЭ) - (Ё2-Е,).
Calculation of the concentration of creatine in the sample: through the jointly introduced water-j standard (2 mg / ml)
The standard must then be introduced into the determined mixture as a sample.

权利要求:
Claims (1)
[1]
Invention Formula
The method for determining the content of creatinine, creatine and cap cosine in a biological fluid by incubating the sample with a reagent containing a buffer substance, 4-aminoantipyrine, peroxidase and sarcosine oxidase, followed by measuring the color intensity of the mixture in comparison with a standard sample, differs in 20 u and so; that, in order to increase the sensitivity of the method, two reagents are used, the incubation of three
15829938
Samples are carried out at a ratio (by volume) of the sample: reagent 0.05-1, reagent I contains a buffer with a pH of 7.9, 4-aminoantipyrin 0.8 mmol / l, peroxidase 2 units / ml, in appropriate case sarcosine oxidase , 6.5 units / ml, and additionally sodium bathrobe, 5 mmol / l, 2,4,6-tribrobrom-3-hydroxybenzoic acid, 10 8.6 mmol / l, iron-synergistic potassium, 10 µmol / l, Lutensol® ON 50 0.5% (May / vol), lipase 2 units / ml, ascorbate oxidase 10 units / ml and, in the corresponding case, creatine hydrolase, 12 units / ml, and reagent II contains, in the corresponding case, sarcosine oxidase 6, 5 units / ml and creatinogenoid - roll 25 units / Ml the color intensity measurement is carried out at 546 nm, the use of sarkozinoksidazu Chainia purpurogena DSM-43156, Chainia ochraceae DSM-43155, Strepto- myces flocculus DSM-40327.
Reagent 11.00
Reagent II
H200.05
Try
Table 1
table 2
1.00
1.00 0.05
1.00 0.05
I ii
1.00 0.05
1582993
Table 3
ten
1.00 0.05
1.00 0.05

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

JPS6029473B2|1977-10-04|1985-07-10|Toyo Jozo Kk|

---

JPS5748956B2|1978-08-28|1982-10-19|

---

JPS6243670B2|1979-12-26|1987-09-16|Hideaki Yamada|

---

DE3326888A1|1983-07-26|1985-02-14|Boehringer Mannheim Gmbh, 6800 Mannheim|SARCOSINE OXIDASE|

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JPH0134035B2|1985-01-11|1989-07-17|Noda Sangyo Kagaku Kenkyusho|

---

DE3519218A1|1985-05-29|1986-12-04|Boehringer Mannheim Gmbh, 6800 Mannheim|H 2 O 2 SARCOSINOXIDASE, ITS PRODUCTION AND USE|

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IS1550B|1986-08-28|1994-08-10|Union Carbide Canada Limited|Mix for the defense and de-icing of aviation|

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TW257792B|1992-10-01|1995-09-21|Lilly Co Eli|

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法律状态:
2005-01-10| REG| Reference to a code of a succession state|Ref country code: RU Ref legal event code: MM4A Effective date: 20020529 |

优先权:

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