CS263028B1 - Stabilized agent to determine the catalytic concentration of alanine aminotransferase - Google Patents

Abstract

The subject of the solution is a stabilized reagent for determining the catalytic concentration of alanine aminotransferase and L-alanine, lactate dehydrogenase, albumin and nicotinamide adenine dinucleotide, which contains citric acid and albumin in a certain ratio. The reagent is used in determining the catalytic concentration in blood serum or plasma and stands out compared to those used so far, primarily due to its higher stability.

Description

The invention relates to a stabilized agent for determining the catalytic concentration of alanine aminotransferase.

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The determination of the catalytic concentration of alanine aminotransferase (L-alanine: 2-oxoglutarate aminotransferase EC 2.6.1.2.) In blood serum or plasma is a widespread analysis allowing a significant refinement of differential diagnosis of liver and heart diseases. Determination of catalytic concentration of alanine aminotransferase is one of the basic analyzes performed in clinical biochemistry.

The catalytic concentration of L-alanine aminotransferase is increasingly determined by coupled enzyme reactions. Currently, an agent for determining the catalytic concentration of alanine aminotransferase is prepared by adding the indicator enzyme lactate dehydrogenase and its substrate nicotinamide adenine dinucleotide to a buffer containing L-alanine and pyridoxal-5-phosphate. This solution is stable in the dark at +4 ° C for 2-3 days. The low stability of the stock solution is economically disadvantageous for smaller workplaces and for the use of automated analyzers that operate with very small volumes of reagents and therefore cannot process the entire volume of working solutions prepared from commercially available diagnostic kits in such a short time. Extending the stability of the prepared solutions by freezing, which is very widely used, cannot be used in this case, since the freezing of the indicator enzyme lactate dehydrogenase is impaired and thus its catalytic activity decreases. Although the stability of the alanine aminotransferase catalytic concentration reagent can be increased by the addition of albumin, which acts as a protective medium, it is only about 5 to 6 days.

The stability of the stock solutions can be prolonged several times by adding substances that protect the enzymes to the solution

263,028 and antioxidants to prevent unwanted oxidation of nicotinamide adenine dinucleotide. Many entioxidants, such as propyl gallate, ethylene glycol and ascorbic acid, widely used in the food and pharmaceutical industries, have an adverse effect on the catalytic concentration of the indicator enzyme, lactate dehydrogenase. Therefore, it is desirable to find such a novel substance that suppresses the oxidation of nicotinamide adenine dinucleotide and thereby increases the use of a catalytic concentration assay for ALT, while not adversely affecting the catalytic concentration of enzymes. The aforementioned drawbacks are significantly suppressed by the stabilized reagent for determining the catalytic concentration of alanine aminotransferases according to the invention. His - -1 1 characterized in that it comprises 0.5 to 10.0 /umol.l /umol.l citric acid and 0.5 to 10 gl ^"1 albumin.

The citric acid and albumin reagent has the following advantages. Albumin, in combination with citric acid, increases the stability of the alanine aminotransferase reagent from the original 5 to 6 days up to 10 days compared to the albumin only stabilized agent. Thus, it is a specific higher effect of albumin and citric acid, in particular on nicotinamide ad enindinucleotide, manifested by prolonging the stability of the reagent for catalytic concentration of alanine aminotransferase up to twice that of the agent stabilized by albumin alone. The range of albumin and citric acid concentrations given in the definition of the invention should be maintained since higher concentrations of citric acid will adversely affect the determination of the catalytic concentration of alanine aminotransferase. Albumin and citric acid are compatible with analytical systems most commonly used to determine the catalytic concentration of alanine aminotransferase, as opposed to other protective systems.

In a relatively very simple manner, the stability of the working solution for determining the catalytic concentration of alanine aminotransferase can be substantially increased so that it can be used for the necessary and convenient time for various types of medical laboratories.

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Example 1

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Κ 1 liter of buffer containing 0.120 mol of tris (hydroximethyl) -aminomethane and 0.480 mol of 1-alanine is added 0.5 µmol of citric acid. The pH of the buffer is adjusted to 7.6. To the solid enzyme mixture containing 0.216 mmol nicotinamide adenine dinucleotide and 36 / Ukat lactate dehydrogenase was added 10 g bovine albumin.

After mixing both components, a stabilized reagent for determining the catalytic concentration of alanine aminotransferase is obtained. The reagent is stable for at least 10 days.

Example 2

To the enzyme mixture containing 0.216 mmol nicotinamide adenine dinucleotide and 36 µl lactate dehydrogenase was added 5 µmol citric acid and 0.5 g bovine albumin. After mixing the enzyme mixture with 1 liter of 0.120 mol of tris (hydroximethyl) -aminomethane and 0.480 mol of L-alanine and 0.120 mmol of pyridoxal-5-phosphate, whose pH is adjusted to 7.6 *, a stabilized reagent for the catalytic determination is obtained. concentration of alanine aminotransferase. The reagent is stable for at least 10 days.

Example 3

To 1 ml of the stabilized reagent for the determination of the catalytic concentration of alanine aminotransferase containing 120 mmol.l ^{-1 of} tris-hydroxymethyl-D-aminomethane, 480 mmol.1 l-alanine, 0.12 mmol.1 pyridoxal-5-phosphate, 0,216 mol.l -1 * ¹ /Ukat.l* ¹ lactate dehydrogenase, 0.5 /umol.l* ¹ citric acid and 10.0 ^gl-1 bovine albumin, pH 7.6 prepared according to Example 1 was added 0.1 ml of the analyzed serum. After a 10 minute preincubation at 30 ° C, the enzyme reaction is started by adding 0.1 mol of 165 mmol / ^l 2-oxoglutaric acid. Record the change in absorbance at 340 nm per time unit at 30 ° C.

Claims (1)

Stabilized reagent for the determination of the catalytic concentration of alanine aminotransferase with L-alanine, lactate dehydrogenase, albumin and nicotinamide adenine dinucleotide, characterized in that it contains 0.5 µmol ^-1 to 10.0 µmol citric acid and 0.5 to 10 µmol. gl * albumin.