CS269205B1 - Stabilized solution of chromogene for urea's enzymatic determination by means of berthelot reaction - Google Patents

Abstract

The stabilized chromogen solution consists from an aqueous solution of 3-ethyl-6-isopropylphenol at a concentration of 1 to 5 mmol / l containing complexing agents capable of mask heavy metals at a concentration of 0.05 up to 5 mmol / l, such as acid ethylenediaminetetraacetic or sodium azide, Trace amount of reducing substance at concentration 10 to 500 pmol / l and aliphatic alcohol of 1 to 3 atoms 0.1 to 2 mol / l carbon. The stabilized chromogen solution may be prepared with β buffer at pH 5 to 9, which is compatible with β enzyme urease, as well as Berthelot reaction. Due to the new type of chromogen and specific the composition of the chromogen solution is dosa synergistic effect manifested with high stability of the chromogen solution i complete solution. Absorbance is suppressed control solution and chromogen solution of said composition does not inhibit the enzyme urea, the content of which in complete solution can be reduced several times. The solution is very stable and simplified thus the analytical determination of urea by one step while increasing accuracy and accuracy and price reduction due to savings enzyme urease.

Description

CS 269205 B1

BACKGROUND OF THE INVENTION The present invention relates to a stabilized solution of a chromogen for the enzymatic determination of urea by Berthelot reaction.

Urea determination is widely used in feed, biochemistry, human and veterinary diagnostics. Its determination in biological material, especially in serum, belongs to the basic analysis, because urea together with creatinine is an indicator of kidney function. Its level is increased In acute glomerulonephritis, pyelonephritis, obstruction, uremia, but also in liver diseases, gastrointestinal disorders and inadequate heart.

A variety of methods have been used to determine urea in biochemistry. Of the photometric methods, which are still the most prominent, urea and some alpha-diketones and their oximes, such as diacetyl, acetylbenzoyl and diacetylmonoxime, react with ureas at elevated temperatures of 80 to 100 ° C under strongly acidic conditions. it is difficult to automate the determination. Another group is a method suitable for a cine-based assay, for example, a chemical assay with o-phthaldehyde and N- (1-naphthylethylenediamine or an enzyme assay with urease, glutamate dehydrogenase, and reduced nicotinamide adenine dinucleotide in the ultraviolet region of the spectrum).

The main method of urea determination is still in biochemistry and clinical chemistry is the enzyme determination with urease, which catalyzes the decomposition of urea to ammonia and carbon dioxide. Typically, ammonia is determined photometrically after its conversion to indophenol according to the Berthelot method. However, this determination, which is highly specific and sensitive, has several drawbacks, since it is a combination of two separate and at the same time contradictory reactions. The contradictory requirements of the reaction conditions for urea enzyme decomposition and for the determination of ammonia by Berthelot is a major source of difficulty. Because the urease enzyme contains thiol groups, urease-converted urea catalyzed ammonia is inhibited by heavy metals. Urease also inactivates substances with oxidizing effects that oxidize its thiol groups. In urea decomposition, urease can be prolonged by the presence of heavy metal masking agents such as complex-forming agents such as ethylenediaminetetraacetic acid, citric acid, and substances and thiol groups such as glut athlon (W Schneider et al .: Ger. Offen, 2,612,726, 1977, Chem Abstr., 7, 196, 469 (1977). The enzymatic hydrolysis rate increases with the presence of some amino acids which, however, interfere with the determination of ammonia by the oxidation-coupled Berthelot. In particular, the Berthelot-leaching agents include amines, thiols, reducing agents, complexing agents, and generally quinone-nucleophilic agents with which they preferably form monoalkyl to bis (alkylamino) hydroquinones, thus preventing the formation of indophenol (TT NGo et al .: Anal., Chem., 54, 46, 1932). According to Pyma Milham (Anal. Chem. 48, 1413, 1976), the Berthelot reaction yield reduced by 20 to 90% fluorides, citrates, oxalates and ethylenediaminetetraacetic acid. The condition of the Bert helot reaction is that the phenol is present in the solution in the form of a phenolate anion. Its actual concentration in the solution depends not only on the pH but also on the dissociation constant of the phenol used. The sensitivity of the reaction and the rate of indophenol formation will depend on the phenol type and the type of sub-substituents on the phenolic core. The determination of ammonia by Bert-Helot with phenol is sensitive to serum analysis, with a sample volume of 0.02 ml, the resulting solution volume is high and reaches up to 10 ml. The urea urease enzyme assay in combination with the Berthelot reaction is performed as a multi-stage, elaborately complicated analysis. Most of the time, the sample is incubated with the urease solution, then a solution containing phenol and nltroprusside is added to serve as a catalyst, and an oxidizing solution containing mostly alkali hypochlorite or other substances is added as active chlorine sources. The resulting blue color is measured photometrically. Combining the enzyme mixture with phenol and the catalyst in a single incubation solution is difficult because phenol inhibits urease. CS 269205 Bl

Combining the incubation solution with the oxidizing agent eliminates the nature of their components. Therefore, other phenol derivatives were sought which would be at least partially compatible with urease. One of these is sodium salicylate (Tabacco et al., Clin. Chem. 25: 336, 1979), which can be added directly to the incubation solution but at the expense of a substantial increase in urease concentration in the solution of the usual 15 to 20 U /. ml up to 50 U / ml because sodium salicylate reduces the urease catalytic activity 1. Thereby, the determination of urea can be simplified by one stage, but at the same time its cost is greatly increased. Another disadvantage associated with the increase in urease content of the incubation solution is the increase in absorbance of the blank. Urease contains traces of traces of ammonium salts, which increase the absorbance of the blank by proportional to the increase in enzyme load.

These disadvantages, due to the differing and contradictory requirements for urea enzyme hydrolysis reagents for urease and for ammonia determination by the iBerthe-Lot reaction, together with a complicated multi-step analysis procedure, are the reason for finding other, more advantageous solutions that would these disadvantages have been eliminated or at least suppressed. In particular, it is desirable to find a more suitable phenol type as a chromogen for oxidative coupling in a Berthelot reaction that does not inhibit urease, and indenophenol would have a lower absorption molar coefficient in order to avoid large volumes of photometric measurements. To overcome these drawbacks, the aqueous stabilized solution of chromogen is directed to the urea enzyme determination of the invention by a Berthelot reaction consisting of an aqueous solution of 3-methyl-6-isopropylphenol having a concentration of 1 to 5 mmol / l, a complexing agent capable of masking heavy metals such as ethylenediaminetetraacetic acid or sodium azide at a concentration of 0.05 to 5 mmol / l, a trace amount of a reducing agent, preferably ascorbic acid, sulfite or thiobarbituric acid at a concentration of 10 to 500 µmol / l; an aliphatic alcohol having a concentration of 0.1 to 2 mol / l of a total of 1 to 3 carbon atoms and a buffer of pH 5 to 9 compatible with the enzyme urease and Berthelot reaction, preferably barbituran, phosphate or alkali metal citrate .

The use of 3-methyl-6-isopropylphenol as a chromogen for the enzymatic determination of urea in conjunction with the Berthelot reaction has a number of advantages over the chromogens used, which are phenol and sodium salicylate. Phenol inhibits the enzyme and the chromogen solution does not bond with the enzyme. Sodium salicylate having a dissociation constant of pK & about 13.4 must be added to the reaction mixture in an amount of 62 mmol / l to achieve the optimum analytical concentration in the reaction mixture, where it has to be added in a dissociated, reactive form, while 3-methyl-6-isopropylphenol is sufficient in a concentration of more than orders of magnitude less than 1-5 mmol / L. The amount of urease can be reduced by up to four times with the chromogen of the present invention, thereby substantially reducing the absorbance of the control solution and refining the entire analysis. Heavy metal masking agents together with a trace amount of reducing agent protect the chromogen solution from its spontaneous oxidation, causing gradual staining of the chromogen, which is an undesirable property accompanying the oxidation of virtually all phenols. The concentration of both substances that interfere with the Berthelot reaction is freely specific so that these substances do not interfere with the oxidative coupling of ammonia with 3-methyl-6-isopropylphenol and with alkaline hypochlorite.

The aliphatic alcohol facilitates the preparation of the chromogen solution and simultaneously and simultaneously suppresses bacterial contamination. Thus, both substances simultaneously preserve the solution.

For the urea enzyme assay, a limited range of buffers can be used in conjunction with the Berthelot reaction since many of them, for example, all aminoalcohols, interfere with the coupling. For example, barbituran and alkali metal phosphates or citrates, which have a beneficial effect not only on the stability of the chromogen solution, but also on the stability of the complete solution containing both the urease enzyme and the coupling catalyst, meet the requirement of buffer compatibility with both reaction systems. Stabilized

Claims (3)

CS 269205 B1 The chromogen solution of the invention is stable for at least one year. The complete solution of the chromogen-urease is stable in the dark and cold at +10 ° C for at least 14 days. The stabilized solution of the chromogen according to the invention, containing a specific combination of preservatives, which themselves interfere with the enzymatic determination of urea by the urea-bonding with the Berthelot reaction, stabilize the chromogen with its synergistic effect and also support the good stability of the combined chromogen-urease solution. The use of a stabilized chromogen solution for the determination of urea is exemplified by this example. In a stabilized chromogen solution, an amount of urease dissolves, corresponding to its catalytic concentration of 15 to 12 kU / l, together with nitroprusside sodium at a concentration of 1 to 10 mmol / l. Add 1.5 ml of the complete solution to a urea sample of 0.01 to 0.02 ml and incubate for 10 minutes at 20 ° C, add 0.5 ml of hypochlorite solution and measure the absorbance in the absorbent for a further 10 minutes. band of 600 to 700 nm. With the chromogen solution of the invention, the analysis procedure can be minimized and the resulting reaction volume reduced up to five times. EXAMPLE 1 A phosphate buffer solution of pH 7.0 containing 2 mmol / l 3-methyl-6-isopropylphenol dissolved in ethanol 0.3 mol / l of ethylenediaminetetraacetic acid disodium salt in an amount of 1 mmol / l and 100 µmol / l was prepared. 1 ascorbic acid. Example 2 Prepare a 5 mM citrate buffer solution containing 5 mmol / l 3-methyl-6-isopropylphenol dissolved in 0.1 mol / l methanol, sodium azide at 0.05 mmol / l and 10 µmol / l. Thiobarbituric acid. Example 3 Prepare a solution of barbiturane buffer pH 8.0 containing 3-methyl-6-i-propylphenol at a concentration of 1 mmol / l dissolved in 2 mol / l propanol, 5 mmol / l and 500 µmol / l ethylenediamine tetraacetic acid. 1 sodium sulfite. OBJECT OF THE INVENTION A stabilized chromogen solution for urea enzyme determination by a Berthelot reaction, comprising an aqueous solution of 3-methyl-6-isopropylphenol at a concentration of 1 to 5 mmol / l, a complexing agent capable of masking heavy metals such as ethylenediaminetetraacetic acid or azide 0.05 to 5 mmol / l, a trace amount of a reducing agent, preferably ascorbic acid, sulfite or thiobarbituric acid, at a concentration of 10 to 500 µmol / l, and an aliphatic alcohol at 0.1 to 2 mol / l 1 to 3 carbon atoms and a buffer of pH 5 to 9 compatible with urease and Berthelot reaction, preferably barbitan, phosphate or alkali metal citrate.