CS263655B1 - Agent for determining diagnostic meaningfull parts of biologic liquids - Google Patents

Abstract

The solution is a diagnostic tool to determine the components of biological fluids, that generate during auxiliary reactions hydrogen peroxide. The resource by of the invention against the prior art characterized by the fact that it contains as an accelerator oxidatively coupling reaction superficially an active substance of the cationic type, for example cetyltrimethylammonium chloride, cetylpyridinium bromide, benzyldimethylhexadecylammonium chloride or p-tert-octylphenoxyethoxyethyl dimethylbenzylammonium chloride.

Description

SUMMARY OF THE INVENTION The present invention provides a diagnostic means for the determination of diagnostic components of biological fluids that generate hydrogen peroxide during appropriate auxiliary reactions and which are based on the principle of color-forming oxidative coupling.

In the determination of a number of diagnostically important components of biological fluids, such as blood, serum or urine, specific enzymatic reactions are used in clinical biochemistry, whereby the analyte to be analyzed is oxidized with airborne oxygen to form hydrogen peroxide. A typical example is the determination of glucose, which in the presence of the enzyme glucose oxidase is oxidized to gluconic acid or gluconolactone and an equivalent amount of hydrogen peroxide. However, uric acid, cholesterol or so-called triglycerides can be similarly determined when specific oxidoreductases, uricase, cholesterol oxidase or glycerophosphate oxidase are used. The analytically very difficult determination of the original complex analytes is thus converted into the determination of a relatively very simple substance - hydrogen peroxide, whose determination uses its relatively strong oxidizing properties. The most suitable and also most commonly used principle for this determination is the oxidation of a suitable colorless redox indicator to colored oxidation products, the amount of which is equivalent to the amount of hydrogen peroxide and hence the amount of the original analyte. For decades, redox indicators of the benzidine type, such as o-tolidine, o-dlanlsidine or 3,3,5,5-tetramethylbenzidine, have been used practically exclusively for this purpose. More recently, these indicators have been replaced by systems consisting of a nucleophilic nitrogen compound such as a primary aromatic or heterocyclic amine or a heterocyclic hydrazone and a coupling-capable compound such as phenols, naphthols, secondary aromatic amines or active methylene groups. These colorless components of the chromogenic system enter the oxidation-coupling reaction under the action of hydrogen peroxide in the presence of a specific peroxidase enzyme to produce intensely colored quinone imine or diazamerocyanine dyes.

These oxidative-coupling reactions are used in clinical biochemistry to determine the above-mentioned, diagnostically important body fluid components, both in solution, where the intensity of the resulting coloration is measured by absorption, and on so-called diagnostic strips, where the coloration formed on the porous carrier is evaluated. either visually or by measuring reflectance. In both these cases, however, the chromogenic system, however advantageous over previously used simple redox indicators, has the disadvantage that oxidative coupling proceeds relatively slowly even under catalytic peroxidase enzyme activity, since the reaction is carried out quantitatively and thus maximum coloration occurs. only in a long time.

This drawback is particularly pronounced in the diagnostic strips in which all reagents required for the desired assay procedure are incorporated into a porous carrier, such as filter paper. In these diagnostic devices, the oxidation-coupling reaction is slowed down especially when the carrier is provided with a semipermeable membrane on its surface to retain blood elements on the carrier surface.

This drawback removes the present solution for the determination of diagnostically important body fluid components that are converted to oxidation products by specific enzymatic reactions, while producing an equivalent amount of hydrogen peroxide, the determination of which is based on the principle of oxidative coupling. It is based on the fact that it contains a cationic-type surfactant such as, for example, ethyltrimethylammonium chloride or p-tert-octylphenoxyethoxyethyl-dimethylbenzylammonium chloride as an accelerator of the oxidation-coupling reaction.

In addition to the chromogenic system and the above-mentioned cationic-type surfactants, the composition according to the invention comprises other substances known per se and commonly used for similar purposes, such as specific oxidoreductases, peroxidase, a buffer and optionally suitable stabilizers.

As mentioned above, the specific oxidoreductases serve to enzymatically convert the original analyte or its transformed product into an oxidation product and an equivalent amount of hydrogen peroxide. In the case of glucose assays, it is the enzyme glucose oxidase (EC 1.1.3.4), and in the determination of uric acid the enzyme uricase (EC 1.7.3,3). Cholesterol oxidase (EC 1.1.3.6) or L-glycerophosphate oxidase (EC 1.1.3) may be used in cholesterol and triglyceride preparations. However, the cholesterol and triglyceride formulations also contain other auxiliary enzymes such as cholesterol esterase (EC 3.1.1.13), and a number of specific enzymes catalysing the conversion of the original analyte, triglycerides, to glycerol-3-phosphate, accessible to the coupling oxidase reaction. These more complex enzyme systems have been known per se and have been used in classical biochemistry for many years.

As mentioned above, hydrogen peroxide formed during the oxidase reaction described oxidizes the colorless chromogenic system to produce intensely colored coupling products.

However, this reaction proceeds very slowly; therefore, the enzyme peroxidase (EC 1.11.1.7), which is a catalyst for the oxidative effect of hydrogen peroxide, is a common component of all similar means for the determination of the above analytes - and hence the means of the invention.

A further essential component of the composition according to the invention is a chromogenic system consisting of colorless components capable of undergoing an oxidation-coupling reaction under the action of hydrogen peroxide, resulting in an intensely colored quinone imine or diazamerocyanine dye Aromatic or heterocyclic amines are the most commonly used active ingredients. especially 4-aminoantipyrine and / or heterocyclic hydrazones such as N-methyl-2-benzthiazolinone hydrazone. Various phenols or naphthols, secondary or tertiary aromatic or heterocyclic amines, and finally some compounds with an active methylene group, can be used as passive components.

The composition according to the invention further comprises a buffer, the purpose of which is not only to produce an optimum pH for the final oxidation-coupling reaction, but also for all of the above-described enzyme reactions necessary for the transformation of the original analyte into its oxidation product and hydrogen peroxide. For this purpose, the composition of the invention may comprise any of the commonly known and commonly used buffers at pH 4.0 to 9.0, such as citrate, phosphate, tris- (hydroxymethyl) -aminomethane and the like.

Thickening agents, which are natural or synthetic polymeric substances, such as gelatin, caraginan, alginate, polyvinylpyrrolidone, polyvinyl alcohol, etc., may be further active ingredients of the composition of the invention. These thickeners stabilize the entire reaction system of the composition and have a positive effect on enhancing the coloration.

Another essential and essential component of the present invention is the cationic surfactant, such as cetyltrimethylammonium chlorides or bromides, cetylpyridinium, benzyldimethylhexadecylammonium, dimethyloctylbenzylammonium, and the like. Although it has been known in principle for many years, to date only substances of the non-ionic or anionic type have always been used for this purpose. On the contrary, surfactants of the cationic type have been thought to be either completely ineffective or, on the contrary, even slightly slowing the course of the oxidation-coupling reaction. The reason for this conclusion was the fact that the cationic substances quantitatively precipitate proteins of the examined biological fluids and thus could negatively influence the course of all necessary reactions. The finding that the cationic substances, on the other hand, greatly accelerate the course of the oxidation-coupling reaction is surprising and is also applied in the composition of the invention. This new and unexpected effect is particularly potent in dg. If the reaction system contains a suitable cationic surfactant, as shown in Fig. 1, which shows the difference in color development rate of the strips, the surface of which is covered by a semipermeable membrane for separating blood elements can be reduced by half. for the determination of blood glucose containing a non-ionic or anionic surfactant (curves A, B) and a cationic type surfactant according to the invention (curve C) hitherto used.

The importance of the present invention is therefore that for all strips produced so far, after removing the blood elements from the stripe surface, which is usually 30 to 60 seconds after the blood has been applied, it is necessary to wait for an additional 1 to 2 minutes before coloration can be evaluated. This time must be kept very precisely, because otherwise there are distorted findings of glucose content, which differ from the correct value by up to - 50% relative. In contrast, in a composition comprising a cationic surfactant according to the invention, the result can be evaluated either immediately or at most within 30 seconds after removal of the blood elements; moreover, the resulting coloration is stabilized by the presence of a cationic surfactant to such an extent that it no longer changes for up to several hours or even days.

Example l

Diagnostic strips for determination of glucose in capillary blood

The 180 g / m 2 filter paper is impregnated successively with the solutions of the following composition until saturation by thoroughly drying between 50 and 55 ° C between each impregnation. The paper thus prepared is then cut into 6x6 mm squares, which are then glued with a 6x85 mm felt pad.

Impregnating solution I;

Phosphate buffer of concentration

0.2 mol / l, pH = 6.0 30.0 ml gelatine; 1.5% aqueous solution 20.0 ml glucose oxidase 40 ku peroxidase 5 kU Impregnation solution II: methanol 25 ml distilled water 12 ml

1- (4'-sulfophenyl) -3-methyl-5-pyrazolone 1.10 g 4-aminoantipyrine 0.31 g benzyl-dimethyl-hexadecyl-ammonium chloride, 2% ethanol solution 17.0 ml

Impregnation solution III:

methylhydroxymethylcellulose, 3% solution in methanol-dichloromethane 1: 1 20.0 ml ethylcellulose, 4% solution in toluene 15.0 ml polyvinyl acetate B 20 H, 5% solution in ethanol 17.5 ml

The glucose determination with these strips is performed by applying a drop of capillary blood to the indicator zone of the strip. After 1 min, the surface drop 8 is removed by rinsing with water or wiping with a damp cellulose wadding. The resulting red coloring of the strip is semi-quantitatively evaluated by visual comparison with an experimental color comparison scale. These. bands which reliably indicate and through which capillary blood glucose content in the concentration range of 6 to 44 mmol / l glucose can be used either as such or in combination of said indicator zone with a zone containing, for example, 3,3 ', 5' as chromogen, 5'-tetramethylbenzidine and which is suitable for the determination of glucose concentrations in the range of 2 to 10 mmol / l; a preferred combination of both of these zones on a single strip is described, for example, in U.S. Patent No. 4,427,770.

Example 2

Strips for determination of uric acid in serum

The strips are prepared as follows: uvedeným impregnating solutions:

Impregnation solution I:

methanol distilled water

4-aminoantipyrine primaquine diphosphate p-tert-octylphenoxy-ethoxy-ethyl-dimethyl-benzylammonium chloride

Impregnation solution II:

phosphate silencer with conc. 0.2 mol / l and pH 7.0 urikasa hydroxypropylcellulose, 28 aqueous peroxidase solution

Example 3

Urine glucose test strips

The strips are prepared as indicated in the solutions:

Impregnation solution I:

phosphate buffer pH 6.3 with conc. 0.2 mol / l gelatin; 1% aqueous solution of glucose oxidase peroxidase

Impregnation solution II:

methanol water distilled 1-phenyl-3-methyl-5-pyrazolone

4-aminoantipyrine tartrazine o-dianisidine

1- (4'-sulfophenyl) 3,4-dimethyl-5-pyrazolone cetyltrimethylammonium bromide of Example 1, but using the following

10.0 ml 10.0 ml 0.6 g 1.3 g

0.15 g

20.0 ml 1.6 U 10.0 ml 3.0 0 of Example 1, but using these impregnants36.0 ml 20.0 ml 39.6 kU

6.0 kU

30.0 ml 16.0 ml 0.9 g 0.2 g

0.02 g 0.80 g

0.40 g 0.40 g

These strips indicate the formation of a light red to dark reddish-brown color, with urine glucose in the range of 2 to 170 mmol / l, and the glucose concentration in the test sample can be determined with relatively high reliability by visually comparing the color of the strip with an experimental scale.

Claims (1)

P ft E D Μ 6 T OF THE INVENTION Means for the determination of diagnostically important components of biological fluids which are converted to their oxidation products by specific enzymatic reactions, producing an equivalent amount of hydrogen peroxide, the determination of which is based on the oxidation-coupling reaction principle, characterized in that it contains an oxidation-coupling reaction a cationic type surfactant such as cetyltrimethylammonium chloride, cetylpyridinium bromide, benzyldimethylhexadecylammonium chloride or p-tert-octylphenoxyethoxyethyl-dimethylbenzylammonium chloride.