CS262317B1 - Reagent to determine the catalytic concentration of aminotransferase containing pyridoxal-S-phosphoric acid - Google Patents

Abstract

The pyridoxal-5-phosphoric acid reagent for determining the catalytic concentration of aminotransferases contains pyridoxal-5-phosphoric acid, tris-(hydroxymethyl)aminomethane, neutral surfactants, e.g. polyethylene glycol or ethoxylated oleyl alcohol, neutral filler, e.g. sodium or potassium chloride, lactose, maltose, dextran. The solution prepared from this reagent is stable for several months at a temperature of +2 to +8 °C.

Description

The present invention relates to an agent for determining the catalytic concentration of aminotransferases containing pyridoxal-5-phosphoric acid.

Determination of the catalytic concentration of aminotransferases in blood serum or plasma is a very valuable analysis allowing a considerable refinement of differential diagnosis of liver and ischemic heart diseases. Therefore, since its introduction into clinical practice, it has become one of the most commonly performed analyzes in clinical biochemistry.

Papers published over the past decade clearly confirm that pyrodoxal -5-phosphoric acid is a coenzyme of aminotransferases. The level of endogenous pyridoxal-5-phosphoric acid is quite variable and is often below physiological concentration. Therefore, it is necessary to saturate the reaction mixture by adding pyridoxal-5-phosphoric acid. This method of determining the catalytic concentration of aminotransferases is also recommended in the reference method of the International Federation of Clinical Biochemistry.

Conversely, in many cases, the catalytic concentration of aminotranferases determined in the presence of an excess of exogenous pyridoxal-5-phosphoric acid also suggests a deficit of endogenous pyridoxal-5-phosphoric acid. This value is also of increasing clinical importance.

Currently, the working solution is prepared by adding pyridoxal-5-phosphoric acid to the buffered substrate solution. This significantly reduces the stability of the buffer stock solution to about one third of the original time. This drawback can be overcome by preparing a multiple concentrated solution of pyridoxal-5-phosphoric acid, which is added to the reaction mixture just prior to use. However, pyridoxal-5-phosphoric acid is not only poorly soluble in aqueous solutions, but also limited in stability. The solubility of pyridoxal-5-phosphoric acid can be easily increased by adjusting the pH of the alkali to above 6.5. Conventional alkali such as sodium hydroxide and sodium carbonate have been shown to catalytically catalyze the decomposition of pyridoxal-5-phosphoric acid. Therefore, it is necessary to select a substance which adjusts the pH to the desired value, which will cause catalytic decomposition and which is also compatible with the biochemical analysis system.

The aforementioned drawbacks are substantially reduced by the pyridoxal-5-phosphoric acid aminotransferase-containing reagent for the determination of the catalytic concentration, which comprises, per 1 mmol of pyridoxal-5-phosphoric acid, 0.5 to 10 mmol of tris (hydroxymethyl) aminomethane, 0.01 to 10 mmol. 1.5 g of a neutral surfactant such as polyethylene glycol or ethoxylated oleyl alcohol and 0.35 to 60 g of a neutral filler such as sodium or potassium chloride, lactose, maltose or dextran. The reagent can also be prepared as an aqueous solution with 0.55 to 111 moles of water.

The pyridoxal-5-phosphoric acid reagent of the invention has the following advantages.

Tris (hydroxymethyl) aminomethane simultaneously acts as an alkali converting sparingly soluble pyridoxal-5-phosphoric acid into a well-soluble salt and has the specific feature that, unlike other common alkalis, it increases the stability of the reagent solution in pyridoxal-5-phosphoric acid solutions. prepared by neutralization with conventional alkali, e.g. sodium carbonate, after 4 weeks only 10% of the original active ingredient content was found. In contrast, 95% of pyridoxal-5-phosphoric acid was found in a solution containing tris (hydroxymethyl) aminomethane.

Tris (hydroxymethyl (aminomethane) is also compatible with analytical systems most commonly used to determine the catalytic activity of aminotransferases.

Example 1

A homogeneous mixture is prepared from 1 mmol of pyridoxal-5-phosphoric acid or a salt thereof, 0.5 mmol of tris (hydroxymethyl) aminomethane, 0.01 g of polyethylene glycol and 0.35 g of sodium chloride.

Example 2

0.1 mmol of pyridoxal-5-phosphoric acid, mmol of tris (hydroxymethyl) aminomethane, 6 g of potassium chloride, 1 g of ethoxylated oleyl alcohol are dissolved in 20 ml of distilled water.

Example 3

To 1 ml reaction mixture containing 122 mmol. 1 ¹ tris. (hydroxymethyl) aminomethane, 244 111,101.1 ^- ^

-1-1 L-aspartic acid, 0.20 mmol-1 nicotinamide adenine dinucleotide, 7.6 µl malate dehydrogenase, 11 µl lactate dehydrogenase were added 20 µl pyridoxal-5-phosphoric acid reagent solution prepared according to Example 2 and 100 The serum was analyzed. After 10 minutes of preincubation at 30 ° C, the enzyme reaction is started with 100 µl of 144 mmol of 2-oxoglutaric acid. ^-1 . The change in absorbance at 340 nm per time unit at 30 ° C is recorded.

Claims (2)

SUBJECT OF THE INVENTION A reagent for determining the catalytic concentration of aminotransferases containing pyridoxal-5-phosphoric acid, characterized in that it contains, per 1 mmol of pyridoxal-5-phosphoric acid, 0.5 to 10 mmol of tris (hydroxymethyl) aminomethane, 0.01 to 1.5 g a neutral surfactant such as polyethylene glycol or ethoxylated oleyl alcohol; and 0.35 to 60 g of a neutral filler such as sodium or potassium chloride, lactose, maltose or dextran. 2. A reagent for determining the catalytic concentration of aminotransferases according to claim 1, characterized in that it contains 0.55 to 111 moles of water.