DE1598828A1 - New means of determining glucose - Google Patents

Description

New means of determining glucose The quantitative determination of Glucose is used in certain technical processes, e.g. in cane sugar production and in fermentation processes. The exact one is of particular importance Determination of the glucose content in the examination of body fluids, e.g. Blood or plasma and urine in clinical laboratories. It serves above all else the safe diagnosis of diabetes mellitus and the control of insulin therapy.

In addition, abnormal blood sugar levels give clues to a number of things other metabolic disorders.

There are a number of diagnostic tools and procedures known for the determination of glucose: the glucose content is determined after the reduction process determined due to its @reducing effect. However, this method is unspecific ; fish, since other reducing substances are also included in addition to glucose. In Another known method is the determination of glucose its response with aromatic amines e.g. o-toluidine in glacial acetic acid. This method however, it has disadvantages for routine determinations, since a boiling water bath is required and the use of glacial acetic acid causes an odor nuisance. aside from that is known to use the so-called nexokinase method to determine glucose Use multiple auxiliary enzymes to perform. Although this method is gena @@, however very prone to failure and therefore only to be carried out by trained personnel. In addition the reagents for this method are technically difficult to access and only limited durable.

Most often a procedure is used in practice today (according to the consists in increasing the glucose with atmospheric oxygen in the presence of glucose oxidase oxidize and quantitatively determine the hydrogen peroxide formed (German Patent 1 075 869: Canadian Patent 745 087), which can be achieved by adding a chromogen takes place in the presence of peroxidase as an oxygen carrier and is made from the chromogen forms a colored compound, which can be determined colorimetrically can. This method works relatively precisely, but has the disadvantage that the The enzyme peroxidase used is difficult to produce and has a limited shelf life. During storage, insoluble fractions are formed, which are filtered off before use Need to become. In addition, the enzyme activity decreases.

If a partially decomposed peroxidase is used, none is obtained linear calibration curves. As a result, when the blood sugar content is high, those are according to the usual ones Calculations received values too low.

The short shelf life of the ready-to-use reagent also makes it required, the ready-made reagent mixture shortly before use from 2 as well to prepare solutions that can only be kept in the refrigerator for a limited time. This is special disadvantageous for series determinations, since it is seldom possible to see how many in advance Blood glucose tests are to be performed in a clinical laboratory in one day.

For the detection of the hydrogen peroxide, which according to the above The process discussed is released from glucose under the action of glucose oxidase a few other methods have also been proposed, but these can be found in have not proven effective in practice. For example, hydrogen peroxide has been tried to be converted into oxygen by catalase, which is measured in the Warburg apparatus will. According to another suggestion, the hydrogen peroxide should act on it on methanol or ethanol in the presence of catalase and determination of the amount thus formed Foiiu- or acetaldehydes can be detected.

It is also known that hydrogen peroxypotassium iodide is present in the presence of molybdate is converted into potassium triiodide, which is spectrometrically or potentiometrically can be measured. According to another from French Patent No. 1 256 933 known method is that formed by the action of glucose oxidase on glucose Hydrogen peroxide by adding an iodide, a molybdate and a chromogen and the detection of the dye formed from the chromogen is determined. Furthermore is also the use of copper, nickel, manganese and iron (III) salts of the Ethylenediaminetetraacetic acid or iron (II) salts together with tannic acid or Gallic acid, or pyrogallic acid, of lead sulfide, lead iodide, potassium iodide, and potassium iodide starch (Canadian patent 745 087, Belgian patent 627 415) and the use of blood, Erythrocytes and metal salt complexes with porphyrins or phthalocyanines together with amino donors (aminobenzothiazole, pyridine, nicotinic acid and bipyridylpyridine known as a redox catalyst (German patents 1,121,846 and 1,185,841). This continues Compounds proposed as catalysts have, however, due to shifting Disadvantages, e.g. B. Too little Parbau booty, compared to the current general applied perozydase method in practice is of no importance.

It has now been found that the disadvantages of the known methods for Glucose testing can largely be avoided when using a new agent that Vanadate as a redox catalyst and optionally an inert water-soluble polymer used as a protective colloid.

The present invention accordingly relates to a new reagent mixture for the determination of glucose in body fluids, especially for the determination of blood sugar, with a content of glucose oxidase, one by hydrogen peroxide to a colored one Compound oxidizable chromogen, a water-soluble iodide, a redox catalyst sator and a buffer, which is characterized by the use of potassium, sodium and / or Ammonium vanadate as a redox catalyst and optionally an additional content on an inert water-soluble polymer with protective colloid effect.

Liquids, especially in The new remedy after the invention is the previously known means for glucose determination by simpler Production of the ready-to-use reagent or by easier handling and superior results in glucose determination. Compared to the well-known The reagent containing peroxidase, which has been widely used up to now, is characterized by this novel means according to the invention in that the redox catalyst is precisely defined and is easy to dose and that its activity in storage even without cooling -im In contrast to peroxidase - does not decrease. The well-known molybdate containing reagent is the novel reagent according to the invention due to a higher maximum color yield the colorimetric determination (100 - 110% compared to 80% when using molybdate as a redox catalyst). The maximum color yield will be in the new reagent even when using a very small amount of redox catalyst (0.04 - 0.05% Vanadate), while the one known from French patent 1,256,933 Procedure 1% redox catalyst (molybdate) to achieve the maximum color yield is required. Bringing in the preparation of the ready-to-use reagent solution The small amount of catalyst in the new agent also has the advantage that the reagent is resolved after a short time. In contrast, when using molybdate as a redox catalyst, longer dissolution times are required.

It is also worth mentioning that numerous other catalysts are found in the literature for H202 redox reactions (e.g. aliphatic aldehydes, organophosphorus compounds, Nucleic acids) are known (cf. the 1964 published by Butterworths, London Monograph Perpxidase by Saunders et al., Page 176). Because of their low peroxidase activity however, these compounds are evident as catalysts for glucose determination not suitable.

The advantages of the reagent according to the invention described above, This also applies to the determination of glucose in cane sugar production and in fermentation processes is usable, were accordingly. not to be foreseen and are also for the expert surprised.

The glucose oxidase contained in the agent according to the invention is used as a dry powder. The corresponding raw preparations often contain insoluble ones Fractions which have to be separated off by filtration of the aqueous solution. That Water is then removed again by freeze-drying.

The water-soluble iodide is preferably sodium, potassium or Ammonium iodide is used. Potassium iodide is particularly suitable because it is not hygroscopic is.

As chromogens, which are colored by hydrogen peroxide Let the compound oxidize, especially aromatic amines and phenols such as Dianisidine, tolidine, anisidine, p-toluidine, o-phenylenediamine, benzidine, 2,7-diaminofluerene, also o-toluidine, α-daphthol and guaiacol are possible. Appropriately, the Chromogens in the form of their salts, e.g. B. as hydrohalides, especially as hydrochlorides and hydrobromides are used. O-dianisidine or its salts are particularly suitable such as o-dianisidine hydrochloride or hydrobromide, and especially the bis-dihydrogen phosphate, since these salts give an acidic, relatively stable solution after dissolving in water.

Mixtures of alkaline and eauer-reacting agents can generally be used as buffers Compounds with a buffer effect are used, which the reagent or the reagent solution Maintain in a pH range of about 5.5 to 9.5, preferably between 6.5 and 8.5. For example, a phosphate buffer (primary and secondary alkali, in particular Sodium or potassium phosphate) can be used. Another very suitable one Buffer is trishydroxymethylaminomethane, its application because of the additional Inhibitory effect on secondary activity of raw glucose oxidase preparations (e.g. maltase, amylase) brings special advantages. Also come as buffer substances such. B.

Sodium hydrogen carbonate, triethanolamine and barbiturates are possible.

In a preferred embodiment, a buffer mixture of a rapidly dissolving acidic compound, such as primary alkali phosphate, with a slowly dissolving alkaline compound, especially trishydroxymethylaminomethane, used. If necessary, a carbon dioxide-releasing substance can also be used in the buffer. in particular an alkali bicarbonate such as sodium carbonate may be included.

As a Redozkatalysator are in the new agent according to the invention the colorless low molecular weight (essentially monomeric to tetrameric) sodium, Potassium or ammonium vanadates, preferably the corresponding o- or m-vanadates used. The o-vanadates have the advantage that even during acid deproteinization No undesired polyvanadate formation takes place during the analysis. With neutral deproteinization Ammonium mvanadate is very suitable as a redox catalyst because it is easy to clean and not hygroscopic.

Optionally, the new reagent can be natural or synthetic contain inert water-soluble polymer with protective colloid effect. Are suitable those inert water-soluble polymers which, when tested according to the known methods, such as an internship in colloid chemistry by Thiele, Steinkopff-Verlag, Frankfurt / M., 1950, page 40/41, a favorable protective colloid effect and also do not react noticeably basic or acidic in aqueous solution. The so-called gold number of the polymers can be used as a measure of the protective colloid effect can be used. Particularly suitable as a protective colloid for the agent after present invention are those inert water-soluble polymers that have a gold number on the order of 0.005 or less. In particular, they can be used as protective colloids Polyvinylpyrrolidone, gum arabic, polyethylene glycol or gelatin are used with polyvinylpyrrolidone being preferred. Also other inert water-soluble ones Vinyl polymers with a good protective colloid effect, such as polyvinyl alcohol, are suitable. High gold number polymers, e.g. B. in the order of 0.25 (gold number of Strength) lie, are not suitable as protective colloids. The protective colloid prevents unwanted precipitation of the from the in the finished reagent solution Chromogenically formed dye. The addition of a protective colloid is therefore particular Recommended when measuring very high glucose concentrations. A special The advantage is that when using a protective colloid, for example from Polrinylpyrrolidone, for a reagent according to the invention, the calibration curves for the detection of glucose are linear and can be easily reproduced. If no protective colloid is added, so opacities form, which makes the measured values more difficult for the calibration curves as well become reproducible.

The protective colloid is rorzugweibe in a concentration of 0.002 Up to 0.1% by weight added. For polyvinylpyrrolidone has a concentration range of about 0.003-0.05% by weight (calculated on the ready-to-use solution) as special proved beneficial. The polymers used as protective colloid may have to be used before incorporation into the reagent of oxidizing or reducing admixtures getting cleaned. When using Polyvinylk pyrrolidon must for example & To achieve low blank values in the glucose measurement those contained in the polymer Peroxides must be removed beforehand. This can e.g. B. by treating the polyvinylpyrrolidone with a reducing agent such as zinc / salic acid in an aqueous medium or with zinc amalgam done in dichloromethane solution.

The reagent according to the invention can be in solution or in a suitable solid Shape, e.g. B. can be used as a powder or as a tablet.

It is advisable to prepare a first solution that contains the buffer, the enzyme containing iodide and vanadate, the pH of this solution being optional is set to a neutral value.

In addition, a second separate solution is made, which is the chromogenic salt contains. Optionally, the acidic components of the buffer can also be used in the second Solution, together with the chromogen salt. By putting together The ready-to-use reagent is obtained from the two solutions prepared in this way.

It is also possible from either of the two solutions or from one Mix the two solutions to remove the water by freeze drying. That The dry preparation obtained in this way is then put to use in water dissolved again.

Furthermore, the reagent can also be used to increase the shelf life dry mixing of the ingredients in solid form, e.g. as a powder will. It is recommended that vanadate is also contained in the reagent hygroscopic or hydrous acidic substances such as potassium dihydrogen phosphate, just mix loosely. If an intensive mixing, for example by Trituration of these ingredients, there is a risk that undesirable ones Form polyvanadates. Obtained by dissolving the powdered reagent in water one the ready-to-use solution.

The agent according to the invention is preferred in the form of a tablet used. In particular in this form, it is useful to have a buffer from a rapidly dissolving acidic constituent such as a dihydrogen phosphate, in particular of potassium dihydrogen phosphate, together with a carbon dioxide-releasing agent, preferably a bicarbonate such as sodium bicarbonate and a slowly dissolving one basic component, in particular trishydroxymethylaminomethane, to use.

It is expedient to produce a durable tablet assumed two granulates. The first granulate contains the acidic ones Components of the buffer, for example the primary alkali phosphate and the bicarbonate, together with the chromogenic salt.

The basic components of the buffer, for example the trishydroxyme thylaminome than, the iodide and the vanadate are processed into a second granulate. The glucose oxidase can be mixed with the first or the second granulate. Appropriately, a granulate or both granulates with an inert coated with water-soluble polymers. Such a coating can for example are made by spraying the granules with a solution of one of the obn polymers listed as protective colloid, e.g. from polyvinyl alcohol, polyethylene glycol, Gum arabic and especially polyvinylpyrrolidone, and allowing the solvent to evaporate. The two granules are then mixed well and compressed into tablets. The through the protective coating formed by the polymer separates the acid-sensitive iodides and vanadates of the acidic components of the reagent. The different speed of solving of the acidic and basic components and the content of bicarbonate enables a Effervescent tablet that ultimately creates a neutral solution. This achieves a very short dissolution times for the tablet. This advantage is particularly important because the disintegrants commonly used in tablets, e.g. Starch, for a reagent for glucose determination due to the formation of grape solutions cannot be used. When measuring glucose, the polymer prevents as Protective colloid an undesired precipitation of the colored ones formed from the chromogen Link. In addition, the polymer can act as a lubricant during tablet compression works.

The reagent in tablet form is particularly beneficial because the ingredients the dianisidine-containing reagent in this form is precisely dosed in the tablet can be kept for a long time and the preparation of a ready-to-use solution simple dissolving of the tablet in water carried out in a very simple way can be.

A ready-to-use solution of the reagent is required for the determination of glucose recommended according to the invention with the following concentrations: iodide: about 0.006 to 0.3, preferably 0.02 to 0.03 mol / l; Vanadate: about 0.0005 to 0.04, preferably 0.002 to 0.004 mol / l; Buffers, especially phosphate buffers: about 0.02 to 0.5, preferably 0.05 to 0.2 mol / l; If necessary, addition of trishydroxymethylaminomethane: up to about 0.5, preferably 0.03 to 0.1 mol / l.

Optionally addition of bicarbonate up to about 0.2 mol / l; Protective colloid, in particular polyvinylpyrrolidone: about 0.003 to 0.1% by weight; Chromogenic, in particular o-Dianisidine: about 0.01-1, preferably 0.08 to 0.5 mmol / l.

The measurement of the glucose content with a ready-to-use solution of the reagent according to the invention is carried out in a manner known per se, such as, for example in H.-U. Bergmeyer, Methods of Enzymatic Analysis, Verlag Chemie, Weinheim; Bergstrasse (1962), page 123. For example, you give a measured Amount e.g.

0.1 or 0.2 ml of the sample to be measured, e.g. of protein-free 1:10 diluted Serum to an excess - e.g. 2 to 5 ml - of reagent solution and measure after one Incubation time of, for example, 1/2 hour, at a suitable temperature, for example Zismertemperature, the absorbance at 436 or 492 nm with a photometer against the unreacted reagent solution as a blank value. By comparison with the The colorimetric values of standard glucose solutions are obtained to be determined Glucose content.

Example 1 Reagent solution 3.45 g Na2HP04. 2H20 1.82 g NaH2PO4.2H2O 0.063 g of glucose oxidase (15 U / ml), 0.5 g of KI and 0.25 g of NH4V03 are dissolved in 250 ml of water solved. If necessary, the pH is adjusted to 7.0 (solution A).

A solution is obtained from 0.0125 g of dianisidine hydrochloride and 2.5 ml of water B manufactured.

A ready-to-use reagent solution (C) is made by mixing 100 ml of solution A obtained with 1 ml of solution B.

Carrying out the analysis a) Deproteinization: 0.1 ml of serum, plasma or Whole blood is mixed with 1 ml of a solution containing 0.16% uranyl acetate and 0.9% NaCl, mixed and centrifuged.

At the same time, a sample of a standard solution (100 mg glucose in 100 ml of water) diluted with a 10-fold volume of water. b) Color reaction: 0.2 ml protein-free supernatant obtained according to a) or the standard sample pretreated according to a) are mixed with 5 ml of ready-to-use reagent solution (C). The mixture turns brown. The mixture is left to stand for 30 minutes and the absorbances are measured against the reagent solution (C) with a photometer with a mercury-cadmium lamp 436 nm. C) Glucose content: The glucose content is calculated using the following formula: EA glucose concentration = G 5 EA = absorbance of the analysis sample ES = absorbance of the standard sample G = glucose content of the standard sample Example 2 0.892 g of K2HP04 0.945 g KH204 0.200 g trishydroxymethylaminomethane 0.04 g NH4V03 or 0.05 g Na3VO4 # xH2O 0.400 g KJ, 0.020 g glucose oxidase (25 U / ml) and 0.015 g polyvinylpyrrolidone dissolved in 100 ml of water (solution A).

0.010 g of dianisidine bis-dihydrogen phosphate are dissolved in 1 ml of water. (Solution B) For the preparation of the ready-to-use reagent solution solution A is mixed with solution B.

Carrying out the analysis with the reagent described above takes place analogously to example 1.

Example 3 A reagent solution containing an easily soluble, red dye instead of the brown dye is obtained analogously to the procedure in Example 2 produced, however, an additional 200 mg of polyvinylpyrrolidone in solution A are given.

The analysis is carried out as in Example 1, but with the difference that the absorbances run at 492. be measured.

Example 4 reagent tablets for one glucose determination each: 124 g KH2P04, 76 Q NaHCO3 and 0.35 g of dianisidine hydrochloride are triturated together and sifted. The powder is stirred vigorously. with a solution of 10 g of polyethylene glycol sprayed in 100 ml of CH2Cl2. When the solvent has completely evaporated, be 29.5 g trishydroxymethylaminomethane, 2.5 g glucose oxidase, 5.0 g ammonium vanadate, 30 g of potassium iodide and 1 g of polyvinylpyrrolidone mixed in.

It is sieved again, dried in vacuo at 300C, again sieved and pressed into tablets weighing 70 mg each.

Carrying out the analysis: (Only one standard sample is used for each analysis series a) Deproteinization 0.04 ml of whole blood (or standard solution) are mixed with 0.5 ml of a solution of 5 g of trichloroacetic acid mixed in 100 ml of 1.0% sodium hydroxide solution and centrifuged. b) color reaction; 1 tablet is in 3 ml of water solved. Add 0.1 ml of protein-free supernatant or standard mixture and leave Stand for half an hour at room temperature. Then the absorbance measured at 436 run against a blank value (solution VO11 1 tablet in 3 ml H2O).

The calculation is carried out as in example 1.

Example 5 500 mg sodium o-vanadate (50% Na3VO4), 2.5 g potassium iodide, 0.25 g of glucose oxidase (30,000 U / g) are dissolved in 50 ml of 0.4 M phosphate buffer (p = 7.4) solved.

If necessary, the solution is filtered. Before use, this Solution added a solution of 7 mg of o-dianisidine dihydrochloride in 7 ml of water and then the resulting reagent solution is shaken. With this solution you can spray paper or thin-layer chromatograms, with a large excess of reagent, which washes out the stains should be avoided. The spots (violet-gray, at higher concentration dark brown) develop in the presence of glucose (3γ / spot) immediately and are evaluated roughly quantitatively after 2 to 3 minutes.

Claims (7)

Claims 1. Means for determining glucose in liquids, in particular in body fluids with a content of glucose oxidase, a through Hydrogen peroxide can be oxidized to a colored chromogen, a water-soluble one Iodide, a redox catalyst and a buffer, characterized in that as Redox catalyst potassium, sodium and / or ammonium vanadate is used and that optionally an inert water-soluble polymer with protective colloid effect is included. 2. Composition according to claim 1, characterized in that it is used as a chromogen o-Dianisidine, as a water-soluble iodide potassium iodide and as a redox catalyst ammonium vanadate contains. 3. Means according to claim 1 and 2, characterized in that it is powder form and as a buffer a mixture of a primary and a secondary alkali phosphate, preferably from primary and secondary sodium or potassium phosphate, if appropriate mixed with trishydroxymethylaminomethane. 4. Means according to claim 1 and 2, characterized in that it is in tablet form has and as a buffer a mixture of a rapidly dissolving in an aqueous medium acidic compound, preferably primary alkali phosphate, such as primary sodium or Potassium phosphate, and a basic compound that dissolves slowly in an aqueous medium, preferably trishydroxymethylaminomethane, along with an aqueous acidic Solution carbon dioxide-releasing compound, preferably an alkali bicarbonate such as Sodium or potassium bicarbonate. 5. Means according to claim 4, characterized in that it consists of a consists of two granules compressed tablet, the acidic components of the Buffer and a chromogenic salt in the first granulate, a water-soluble iodide, galium, Sodium and / or ammonium vanadate and the slowly dissolving basic compound in the second granulate, and the glacose oxidase in the first or second granulate are, and one of the granules, preferably the first granules, with a protective coating is encased from an inert water-soluble polymer. 6. Means according to claim 1 to 5, characterized in that it is as inert water-soluble polymer with protective colloid effect polyvinylpyrrolidone, Contains gum arabic, gelatin or polyethylene glycol. 7. Method for the determination of glucose in liquids, especially in body fluids, especially in the blood, characterized in that the to determining sample brought together with an agent according to claim 1 to 6 and the Content of the colored compound formed is measured colorimetrically.