CS254978B2 - Agent for glucose,cholesterol and haemoglobin determination - Google Patents

Description

The present invention relates to a novel reagent for the determination of glucose, cholesterol and hemoglobin, in particular in biological samples.

Numerous procedures are known for the detection and quantification of glucose, cholesterol and hemoglobin. Because of their simple feasibility and cheapness, and for reasons of technical measurement, they are the most widely used methods based on chemical color reaction.

In general, color reactions with 1-phenyl-2,3-dimethyl-3-aminopyrazolin-5-one (hereinafter referred to as 4-aminophenazone), phenol, phenol derivatives and salicylic acid derivatives are used to measure the above compounds. The reaction taking place with phenol-4-aminophenazone was first used by P. Trinder [Ann. Clin. Biochem. 8, 24 (1969)]. The medical importance of this methodology is so great that numerous similar methods have been developed for this purpose.

A general disadvantage of these methods is that the measurement characteristic is not linear and the resulting color is not sufficiently stable or light sensitive (cf. Table 1 of the above-cited article and Table 3 contained in the description of the invention later).

It is an object of the present invention to provide a new system which overcomes the disadvantages of known methods.

The invention is based on the surprising observation that when 4 aminophenazone and thymol are used to form a colored product, a photometric reaction with very good sensitivity is excellent, very well usable (stable, light insensitive and linearly characteristic) under optimal conditions.

The present invention relates to a reagent for the determination of glucose, cholesterol and hemoglobin, in particular in biological samples comprising 4-aminophenazone, a buffer solution, a stabilizer, a detergent and optionally an enzymatic catalyst, characterized in that it contains p-isopropyl -cresol (thymol).

According to a preferred embodiment of the agents according to the invention, the thymol is dissolved in a lower alcohol, in particular ethanol; the other chemicals are dissolved in distilled water and the alcoholic and aqueous solutions are mixed together in a 1: 4 ratio. The reagent thus produced contains thymol at a concentration of 2.5 ± 0.2 mmol / liter.

mol / liter of mol / liter of mol / liter of mol / liter

The concentration of other chemicals in the reagent is as follows:

phosphate buffer 0.08 + 0.02

4-aminophenazone 0.8 + 0.2 sodium azide 9.85 + 0.1 ethanol 330.0+ 20

The agent of the invention is used as follows:

Thymol and 4-aminophenazone are contacted in a buffered medium at appropriate concentrations relative to the test substances (glucose, cholesterol). As a result, hydrogen peroxide forms with specific enzymatic reactions. The resulting hydrogen peroxide releases nascent oxygen via peroxydase, which forms 4- (2'-isopropyl-5'-methyl-1 ', 4'-benzoquinone monoimino) phenazone, a colored product. In the case of substances which are themselves capable of catalyzing the decomposition of hydrogen peroxide (e.g. hemoglobin), an additional enzymatic reaction is not necessary, and only hydrogen peroxide, color-forming 4-aminophenazone and thymol are added to the reaction mixture.

The novel agent of the invention has numerous advantages, which can be summarized as follows:

a) the color reaction is carried out in a well-defined pH range (pH = 7.0 to 8.0, preferably 7.5);

(b) the maximum light absorption of the resulting color product falls within the wavelength range 465 to 480 nm; it is therefore expedient to carry out photometric measurements in this range, in particular at 475 nm;

(c) it is of particular importance that the photometrically detectable color is formed relatively quickly. It has been found that a stable color develops at room temperature within 25 minutes and at 37 ° C within 15 minutes.

(d) various buffers (such as tris-phosphate, Na2HPO4-KH2PO4, K2HPO4-KH2PO4 and sodium borate-hydrochloric acid) are suitable for adjusting the optimum pH;

e) a favorable concentration of thymol or its solvent was found. It is expedient to dissolve the thymol in methanol, ethanol or isopropanol, especially ethanol. The optimization of thymol concentration is shown in Table 1; the optimum value is mmol / liter. The measurement is performed with a glucose solution of the indicated concentration (11.10 and 27.75 mmol / liter).

Table 1

Thymol mmol / liter 1,330 2,000 2,500 3,000 4,000 5,000 extinction of 11.1 mmol / liter 0.330 0.365 0.370 0.370 0.370 0.370 extinction of 27.75 mmol / liter 0.750 0,860 0.870 0.870 0.870 0.870

Obviously, in both glucose solutions, the highest, no longer rising extinction (color reaction intensity) is obtained at a thymol concentration of 2.5 mmol / liter;

f) Optimization of the concentration of 4-aminophe nazone is given in Table 2. The measurements are again carried out with glucose solution and thymol

2.5 mmol / liter.

Table 2

4-aminophenazone 0.200 0.400 0,600 0,800 1,000 mmol / liter extinction 0.350 0.370 0.370 0.370 0.370 11.1 mmol / liter extinction 0,800 0,860 0.870 0.870 0,871

27.75 mmol / liter

According to this table, the maximum extinction, which is no longer changed, is reached at a concentration of 0.600 mmol / liter. For safety reasons, a concentration of 0,800 mmol / liter is considered optimal.

g) The peroxide concentration required for glucose and cholesterol measurements is also optimized (see Table 3). A concentration of 15 U / ml (1 U = 1 international unit; i.e., an amount that replaces 1 mmol of substrate per minute) has been shown to be optimal. The peroxidase used must not contain other enzymatic impurities in interfering concentrations.

Table 3

Peroxidase activity extinction 27.75 mmol / liter 8 U / ml 0.820 10 U / ml 0.850 12 U / ml 0,865 14 U / ml 0.870 peroxidase activity 15 U / ml 17 j./ml extinction of 27.75 mmol / liter 0.870 0.870

From the above table it can be seen that the maximum intensity (extinction) of the color produced by glucose is already achieved with an enzyme activity of 14 U / ml. For safety, an activity of 15 U / ml is used.

h) Detergent is used in the cholesterol assay to increase solubility. 9-lauryl ether polydecanol (Sigma, (JSA), Triton X-100 (octylphenol polyethylethylene glycol ether, LOBA, Austria) or Brij 35 (polyoxyethylene lauryl ether, BDH, UK) can be used for this purpose.

The optimum concentration range of the above composition is 0.4 to 1.2%.

The practical applicability of the agents of the invention is illustrated by the following:

1. Experience gained with 'calibration'

The detectability and quantitative measurability of glucose is determined over a concentration range of 2.775 to 33.300 mmol / liter. The data are partly included in Table 4 and> partly are shown in Fig. 1 (after mathematical evaluation). It can be seen from the above data that the measurement characteristic is - within the stated range - linear. The color reaction is complete and glucose is perfectly measurable.

о со гЧ Ю со сл сч Y-Ч CZ СО

СО гН т — Г СЧ ~ +

со со ю со со гЧ г) СЧ СТ) СП о θ 'θ' θ '

SS5

LO UO Ю in Ó 00 θ 'θ' CD +

Table 4

The following abbreviations have been used in Table 4:

x = concentration of weighed glucose n = number of parallel measurements у = mean value of the measured extinctions

Y = extinction calculated from the calibration line equation d% = percentage difference between measured and calculated extinction (calculated = 100%)

F _r = regression test

F _l = 'F ^: ' linearity test FG = degree of freedom of linearity

P = linearity probability; since this value is not more than 10%, the curve does not deviate statistically significantly from the straight line.

2. Experience gained in comparison with the reference method

The internationally recognized hexokinase UV test (Boehringer) is used as a reference method. In this case, 45 human blood samples (not selected) are used as the sugar solution. Figure 2 shows that there is no difference between the results obtained by these two methods, since the correlation coefficient (r = + 0.9971) is close to the theoretical value of 1,000 and the slope of the regression line deviates from the theoretical value 1,000 and only 0.4%.

3. Sensitivity, reproducibility and process bias shall be determined on the basis of the measurement results obtained. These statistical calculations provide the following results:

sensitivity:

+ 0.17 mmol / liter (based on glucose)

+ 0.09 iminol / liter (based on glucose) bias:

+ 0.35% (based on glucose)

In addition to the above advantages, the agent of the invention also has the following essential advantages:

The reagent is suitable on the one hand for the quantitative determination of glucose, cholesterol or hemoglobin, which are present in biological samples of human and animal origin (such as blood, urine, body fluids) and, on the other hand, for determining the glucose content of beverages intended for human consumption (for example in alcoholic beverages, juices, refreshing beverages), or for measuring other solutions containing glucose or cholesterol.

It should be noted that the agent of the invention clearly outperforms the prior art tests. The numerical comparison shows that the color achieved with the method of the invention remains completely unchanged for at least 240 minutes, whereas in the known methods a continuous decrease in color intensity can be observed after a short maximum phase (after 45 minutes) (cf. FIG. 3).

Legend to pictures:

Giant. 1:

the ordinate shows the extinction values, the abscissa axis shows the mmol / liter concentration;

Giant. 2:

the ordinate shows the mmol / liter values achieved by the method of the invention; the abscissa axis shows the mmol / liter values obtained using the known method (Boehringer);

Giant. 3:

the ordinate shows the extinction values at a sugar concentration of 11.1 mmol per liter, the abscissa axis shows the values in minutes, curve 1 is for the method of the invention, curve 2 is for the known method (Sclavo), curve 3 is for the known method (Galenopharm).

The agent of the invention and its use are illustrated by the following examples. However, these examples do not limit the scope of the invention in any way.

Example 1

Determination of blood glucose

Solution A:

Dissolve the following substances in 0,1 mol / liter phosphate buffer solution (pH = 7,5; Na2HPOd. 2 H2O and KH2PO4):

glucosidase peroxidase 4-aminophenazone sodium azide 12.5 U / ml 18.0 U / ml 1.0 mmol / liter 12.3 mmol / liter Solution B:

12.5 mmol / liter thymol in 96% ethanol (with distilled water)

The reagent is prepared by mixing solution A and solution V in a 4: 1 ratio.

Standardization is carried out with a glucose solution (11.1 mmol / liter) (dissolved in cold saturated benzoic acid).

Determination:

Weigh 3.0 ml of reagent, 0.02 ml of standard and 0.02 ml of blood serum into tubes.

The substances were mixed and allowed to stand (at room temperature for 30 minutes or at 37 ° C for 15 minutes). In the latter case, the color produced after 10 minutes of cooling is compared photometrically to the blank (cuvette thickness 1 cm, wavelength 475 nm).

The blood glucose concentration (in mmol glucose per liter) is calculated according to the following equation:

sample extinction__ χ 111 standard extinction ’

The process sequence is linear over a concentration range of 0 to 30 mmol / liter.

Example 2

Determination of total cholesterol in blood serum

Solution A:

The following substances are dissolved in phosphate buffer (0.1 mol / liter; pH = 7.5):

peroxidase cholesterol esterase cholesterol oxidase

4-aminophenazone sodium azide 9-lauryl ether Triton X-100

Brij-35

Solution B:

18.0 U / ml

12.5 U / ml

6.0 U / ml

1.0 mmol / liter

12.3 mmol / liter

5.0 g / liter; or

5.0 g / liter; or

5.0 g / liter

12.5 mmol / liter of thymol (in 96% freshly distilled ethanol)

The reagent is prepared by mixing solution A and solution V in a 4: 1 ratio before use.

Standardization is performed using a cholesterol standard (5.2 mmol / liter) dissolved in ethanol.

Determination:

0.02 ml of standard solution or serum is added to 2.0 ml of reagent in each case. The mixture was stirred and allowed to stand either for 30 minutes at room temperature or for 15 minutes at 37 ° C. The color produced after 10 minutes of cooling is compared photometrically to the blank (cuvette thickness 1 cm; wavelength: 475 nm).

Calculation:

serum cholesterol concentration (mmol / liter) = extinction of standard extinction sample

X 5.2

In a concentration range of 0 to 13 mmol per liter, the process is linear.

Example 3

Determination of uric acid in blood serum

Solution A:

The following substances are dissolved in phosphate buffer (0.1 mol / liter; pH 7.5):

peroxidase uricase sodium 4-aminophenazone azide

18.0 U / ml

6.0 U / ml

1.0 mmol / liter

12.3 mmol / liter

Solution B:

12.5 mmol / liter thymol (in 96% ethanol)

The reagent is prepared by mixing the solutions A and V in a 4: 1 ratio.

Standardization was performed using a uric acid standard (595 µmol / liter).

Determination:

0.2 ml of the standard or serum sample is added to each 0.2 ml of reagent. The mixture is mixed and allowed to stand either for 60 minutes at room temperature or for 30 minutes at 37 ° C. The resulting color is compared photometrically to a blank (cuvette thickness 1 cm; wavelength 475 nm);

Calculation:

uric acid concentration in blood serum (/ jmol / liter) = standard extinction sample extinction

The process ranges from 0 to 900 µmol per liter linear.

Claims (2)

A reagent for the determination of glucose, cholesterol and hemoglobin, in particular in biological samples, comprising 4-aminophenazone, a buffer solution, a stabilizer, a detergent and optionally an enzymatic catalyst, characterized in that it contains p-isopropyl-m-cresol as an indicator. 2. The reagent of claim 1 comprising p-isopropyl-m-cresol dissolved in a lower alkanol such as ethanol at a concentration of 2.30 to 2.70 mmol per liter.