• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The invention relates to analytical chemistry, in particular, a method for the determination of f-glutamyl transpeptidase (GTP) used to diagnose liver diseases. In order to intensify the process, another more soluble substituted f-glutamyl-p-anilide — α-glutamyl-3-sulfo (or carboxy) -n-chad (GA) is used. The definition of a GTP is carried out by treating HA with glycine lysine when adding a human sigorotka. Next, the change in optical density (OD) and the rate of formation of the hydrolyzed product are determined. By the difference in OD per minute, the average values are taken and an indicator of enzyme activity is calculated. The duration of determination is halved, the maximum concentration of HA is 6-8 mmol versus 2.9. O) (2 cl С «9 4: l 1C with / 1
    公开号:SU1253439A3
    申请号:SU1974541
    申请日:1973-12-04
    公开日:1986-08-23
    发明作者:Бернт Эрих；Грубер Вольфганг；Хайд Эрих；Штэлер Фритц；Вильхельм Валефельд Аугуст；Вайманн Гюнтер
    申请人:Берингер Маннхайм Гмбх (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to an improved method for the determination of 1-glutamyltranspeptidase, which is used in clinical and chemical laboratories for the diagnosis of liver disease.
    The purpose of the invention is to intensify the process.
    This goal is achieved by using f-glutamyl-sulfo-Sily carboxy-p-anilide as a substrate.
    Example 1. Preparation of Y-GLU-tamyl-p-nitroanilide-3-carboxylic acid (GLU PA 3-carboxylic acid)
    9.1 g of 2-nitro 5-aminobenoic acid (50 mg-mol) and 14.2 phthaloylglutamic acid anhydride (55 mg-mol) are dissolved in 50 ml of absolute dioxane with the addition of 13 ml of tributylamine. The reaction mixture is boiled for 2 hours under reflux. After cooling, 100 ml of 2N ammonia is added to the reaction mixture and the extract of tributylamine with ether is extracted by agitation. The aqueous solution is concentrated to dryness, dissolved in methyl alcohol, and the pH is adjusted to tO using hydraamide hydrate. The reaction solution is held at room temperature for about 20 hours. In this case, the product is partially separated, which is then filtered and washed with methyl alcohol. The methanol filtrate is concentrated to dryness and the residual oil is dissolved in approximately 300 ml of distilled water.
    This aqueous solution was chromatographed over a column with 100 ml of Dowex (Dowex) 4 with a hydroxy form, washed with 300 MP of distilled water, graduated with 2 liters of water with 0.5 N ammonium carbonate solution. The eluate is collected by 100 ml fractions and examined by thin layer chromatography in the butyl: ice system for acetic acid 5 water in a ratio of 50j15: t5 (chromatography: blue spots under ultraviolet light or after moistening with ningin and subsequent heating are brown spots of zones containing amino acids) “Fractions containing glutamic acid-nitroanilide-3-carboxylic acid product (R value 0.25) are collected and concentrated in vacuo to dryness. The remaining powder is stirred in absolute alcohol, filtered and vacuum over x Oristà calcium

    Yield 7 g of If-glutamyl-p-nitro-anilide-3-carboxylic acid, mono-amide salt (58% of the theoretical).
    Ultraviolet spectrum: A 317 nm, e 11.7 cm / mol / at 317 nm.
    Example 2. The use of γ-glutamyl-β-nitroanilide-3-carboxylic acid (GLU-PA-carboxylic acid) for the determination of β-glutamyltranspep-Ti-schazzy (1 - GT).
    Enzymatic cleavage releases 2-nitro-5-aminobenzoic acid.
    Taking measurements
    A 1 M tris-Buffer, 100 mg-mol of glycylglycine, pH 8.25-2.8 ml, 130 mg-mol of the GLU PA-3-carboxylic acid in the specified buffer, 0.2 ml, are pipetted into the cuvette, heated to the determination begins when 0.2 ml of human serum is added, mixed, the optical density is counted at 405, and the chronometer is simultaneously turned on. After 1.2 and 3 minutes, the countdown is repeated.
    By the difference in optical densities per minute (aU / min), the average values are taken and entered into the calculation.
    The calculation is as follows.
    The international unit U is an enzyme activity that converts 1 mmol of substrate to 91 minutes. This will be referred to as 1 ml of body fluid, for example, serum. To calculate the enzyme activity per ml (A), use the general formula
    min € - d V
    YE (mi / ml)
    ,, five
    The extinction coefficient (€) of 3-ka1-boxy 4-nitroaniline is 9.4 / mmol under conditions of the test at 405 nm. The thickness (d) of the cuvette layer is 1 cm, the volume of serum used (V) is 0.2 ml, the sum of volumes (v) is 3.2 ml. The attenuation measurement E is performed at intervals of 1 minute. Wherein
    A 4E (405 nm) / min x 1702 (mi / ml).
    Example 3. Preparation of α-glutamyl-4-nitroanilide-3-sulfonic acid.
    115 g of Annpine-t-4-nitro-3 sulfonic acid and 150 g of phthaloylglutamic acid anhydride are suspended in 2 l of absolute dimethylformand and then heated on an oil bath to. .When this temperature reaches 10
    15
    The pentium is incubated for 1 h at 145 ° C and then cooled. Sediment from-. filter and concentrate the filtrate to dryness. The oily residue is dissolved in methanol and adjusted to pH 9 with 80% hydrazine hydroxide. After 24 hours, the resulting crystals are filtered off with suction and washed with a small amount of methyl alcohol. The filtrate is dried and, when cooled, is carefully decomposed with cooled acetone. The precipitate thus formed is filtered off with suction, washed well with acetone and the residue is dissolved in water with stirring. The water-insoluble residue is filtered off and the pH of the solution is adjusted to 7.5 using ammonia. The ammonium salt of y-glutamyl-D-nitroanilide-3-sulfonic acid is crystallized by the addition of acetone.20 The product crystallizes very well into light yellow crystals. M.p. 186-187 ° C.
    The absorption maximum at 290 nm is 4.57 cm / mmol.
    A yield of about 115-120 g, and 15-20 g can still be obtained from the crystallization mother liquor.
    The total yield of about 135 g corresponds to 70% of the theoretical.
    The solubility of the compound at room temperature is good and reaches about 160% of the degree of conversion of U-glutamyl-p-nitroanilide under the same conditions.
    Example 4. The use of GLU PA-3-sulfonic acid for the determination of -c:
    Enzymatic cleavage
    25
    thirty
    35
    The international unit U is an enzyme activity, which at 25 ° C turns into 1 mmol substrate per minute. This will be referred to 1 ml of body fluid, such as serum. To calculate the enzyme activities per ml (A), use the general formula
    V.1000
     .
    The extinction coefficient (b) of the m-nor-roanilgzd-3-sulphonic acid under the conditions of the experiment at 405 nm was 5.45 cm / mol. The thickness (d) of the cuvette layer is 1 cm, the volume of serum (V) injected is 0.2 ml, the sum of the volumes (v) is 3.2. Optical density measurement 6 is performed at intervals of 1 min.
    From here half the beat
    A DE (405 nm) / min x 2936 (mi / ml)
    The proposed method for the determination of y-glutamyl transpeptidase, which provides for the use of β-glutamine-3-sulfo (or carboxy) -1-anilide as a substrate, as compared to the known method, where glutamine-nitroanchide 1Bc allows the process to be significantly intensified. The maximum reaction rate is achieved at a lis concentration in the range of 6-8 mmol, which is only feasible with the substrate according to the invention due to its good solubility. The maximum concentration achieved using a known substrate is 2.9 mmol, due to
    compounds according to the proposed method limited it. dissolve-release n-nitroanilide-3-sulphate. background acid.
    In accordance with the invention, when using a carboxyl derivative, an increase in the reaction rate by 20% is achieved, and in the case of the J derived sulfonic acid, the rate is twice as high; This means that the duration of the determination can be halved.
    Measurement
    0.1 Tris-Buffer, 150 mg-mole of shlycylglycine 45, pH 8.5, 2.8 ml of 130 mg-mole of GLU PA-3-sulfonic acid in adhesion buffer of 0.2 m are introduced into the cuvette with a pipette. n, heated to 25 ° C, determination started with 0.24 ml of human serum, over-50 was ejected, optical density was measured at 405 mi, and the chronometer was simultaneously turned on. Exactly 1, 2 and 3 minutes, the counting is repeated.
    The difference between optical density and SS tei in 1 min (LE / min) is found to be the average value and enter it into the calculations.
    The calculation is as follows.
    ten
    15
    , 20
    25
    0
    five
    The international unit U is an enzyme activity that, when; 25 C is converted to 1 mmol of substrate per minute. This will be referred to 1 ml of body fluid, such as serum. To calculate the enzyme activities per ml (A), use the general formula
    V.1000
     .
    The extinction coefficient (b) of m-nitroanilgzd-3-sulfonic acid under the conditions of the experiment at 405 nm is 5.45 cm / mol. The thickness (d) of the cuvette layer is 1 cm, the volume of injected serum (V) is 0.2 ml, the sum of the volumes (v) is 3.2. Optical density measurement 6 is carried out at intervals of 1 minute.
    From here half the beat
    A DE (405 nm) / min x 2936 (mi / ml).
    The proposed method for the determination of y-glutamyl transpeptidase, which involves the use of glutamine-3-sulfo (or carboxy) -1-anilide as a substrate, compared to the known method, where glutamine-nitroanchivine VB is used as a substrate, which significantly intensifies the process. The maximum reaction rate is achieved when the concentration of lisch is in the range of 6-8 mmol, which is feasible only with the substrate according to the invention due to its good solubility. The maximum concentration achieved using a known substrate is 2.9 mmol, due to
     by the bridge
    In accordance with the invention, when using a carboxyl derivative, an increase in the reaction rate by 20% is achieved, and in the case of the J derived sulfonic acid, the rate is twice as high; This means that the duration of the determination can be halved.
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    Method for determining f-glutamyl-transpeptidae by treating the substituted-glutamyl-p-anilide with glycylglycine with the addition of human serum and measuring the rate of formation of hydrolyzed products S12) 14CH96
    one with different themes. Priority n g) featured:
    that, in order to intensify the process. O). 12.71 of the starting product — t-gluta-h-amino-acid is used as the substituted y-glutamyl-glutamyl-3-carboxy-and-anilide.
    mil-3-sulfo (or carboxy) -VI-an-03.07.73 The starting product is glutalide, min-3-sulfo-1 -anilide.
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    同族专利:
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    引用文献:
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    US2951090A|1958-04-17|1960-08-30|Ogilvie Flour Mills Co Ltd|Preparation of dl-glutamine|
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    US3803223A|1970-07-20|1974-04-09|Searle & Co|3-amino-n-substituted succinamic acids and intermediates thereto|JPS584560B2|1976-06-01|1983-01-26|Sankyo Kk|
    JPS5635661B2|1976-06-01|1981-08-19|
    CA1087075A|1977-08-05|1980-10-07|Robert J. Gargiulo|Composition and method for determining transferase and protease activity|
    US4167449A|1976-07-29|1979-09-11|American Hospital Supply Corporation|Composition and method for determining transferase and protease activity|
    JPS6111219B2|1977-05-30|1986-04-01|Wako Pure Chem Ind Ltd|
    JPS5649904B2|1978-11-16|1981-11-25|
    US4511651A|1982-07-30|1985-04-16|American Monitor Corporation|Reagent composition and assay for the determination of γ-glutamyltransferase activity|
    US4567138A|1982-08-30|1986-01-28|Beckman Instruments, Inc.|Method for determining γ-glutamyltransferase activity and kits containing a novel substrate solution for use therein|
    US4751178A|1985-09-26|1988-06-14|Eastman Kodak Company|Substrates, compositions, elements and methods for the determination of gamma-glutamyltransferase|
    US4851572A|1987-07-03|1989-07-25|Wako Pure Chemical Industries, Ltd.|γ-L-glutamyl-4-nitroanilide derivatives and process for determining γ-GTP activity using the same|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    DE19722259512|DE2259512C3|1972-12-05|gamma-glutamyl-4-nitranilid-3carboxylic acid, its salts, process for the preparation of these compounds and their use|
    DE19732333798|DE2333798C3|1973-07-03|gamma-glutamyl-4-nitranilide-3sulfonic acid, its salts, process for the preparation of these compounds and their use|
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