DE2235174A1 - DIAGNOSTICS FOR DETERMINATION OF GLUTAMATE OXALATE TRANSAMINASE (GOT) AND GLUTAMATE PYRUVATE TRANSAMINASE (GPT) - Google Patents

Description

Palentcmwälie

Dr.-Ing. Wilhelm Reichel Dipl-Ing. Wolfgang Eeichel

6 Fiaiikfuri a. M. 1
Parkstrasse 13-.

TECHNICON INSTRUMENTS CORPORATION, Tarrytown, N.Y. VStA

Diagnostics for the determination of glutamate oxalate transaminase (GOT) and Glutamate Pyruvate Transaminase (GPT)

The invention relates to stable, lyophilized diagnostic agents and in particular to those used for determining activity of glutamate oxalate transaminase and glutamate pyruvate transaminase in samples containing these enzymes are.

GOT and GPT are enzymes whose increased concentration in the human and animal blood is a sign of certain dysfunctions and / or disorders. For example is an increased concentration of GOT is an important indicator of the condition of patients with severe heart disease. As a result, determining the activity of this enzyme in the body is a useful diagnostic tool.

In general, a beginning or already manifesting itself occurred heart muscle damage in an increase in GOT values. In addition, increased GOT values also provide information the possibility of severe liver dysfunction such as cirrhosis, cancer or hepatitis.

209885/0955

Similarly, the increased concentration of GPT is an indication for liver dysfunction of the type mentioned. Thus, the prognosis and the progression of liver diseases can by an exact GPT determination can be determined.

Certain distinguishing features are necessary for a substance to be an effective diagnostic agent. Among other things, this diagnostic must have good stability, it must be easy to manufacture, and the individual components must be compatible with each other. Diagnostics are generally used in a reconstituted state, i.e. as an aqueous solution, manufactured and used. It is unusual for diagnostics in this form to have significant stability.

For this reason, the reagent is generally maintained to be prepared shortly before use by combining the necessary components. The disadvantages of this Manipulation is obvious: it is subject to error, time consuming, requires adjustment of the reagent and, most importantly, the preparation of a reagent with compatible components is practically impossible.

When such a reagent as described is prepared it must be used up within a very short time, otherwise it will decompose makes the material completely unusable, so that it must be discarded.

The object of the invention is therefore to overcome the aforementioned serious drawbacks, the most important of which is the lack Stability is the production of stable, lyophilized diagnostics that are stored indefinitely can. In addition, if a certain amount of the reagent should be produced and adjusted (standardized) readjustment may not be required, so

209885/0955

that one only has what is necessary for the reconstitution Need to add amount of water. This way the possibility of error is minimized Eliminates time-consuming manufacturing steps immediately before use, as well as adjustment and, most importantly is, a stable product can be obtained that can be stored for a long time and at any time with help only of a simple reconstitution step usable can be made.

The invention relates to a stable, lyophilized diagnostic agent for the determination of glutamate oxalate transaminase in a sample that contains this enzyme, which is characterized by a content of malate dehydrogenase (M) H), a pyridine nucleotide, namely reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH), as well as a buffer with a sodium content of less than about 600 milliequivalents per liter (600 meq./l) to regulate the pH value of the diagnostic agent in the reconstituted form.

The invention also relates to a stable, lyophilized Diagnostic for the determination of glutamate pyruvate transaminase (GPT) in a sample containing this enzyme, which is characterized by a content of lactate dehydrogenase (LDH), a pyridine nucleotide, namely reduced, reduced nicotinamide adenine dinucleotide (NADH) or reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), as well as a buffer with a sodium content of less than about 600 meq./l to regulate the pH value of the diagnostic agent in the reconstituted form.

The reconstituted form of any of these diagnostic agents is preferred and is made by simply adding a predetermined amount of water, and thus to one Solution with a pH of preferably 6.0 to 8.0 is obtained. It is also possible to use a reconstituted intermediate

209885 / 09b5

Produce product with a pH outside of 6 to 8, which is then adjusted by adding more buffer can be. Although the dry reagent can be used in the analytical determination, the reconstituted Form preferred.

Another preferred embodiment of both diagnostic agents consists of the use of NADH as a pyrodine nucleotide.

The reconstituted forms of diagnostics stand out by certain defined properties and threshold values that must be adhered to in order for the diagnostic agent to be used has the desired advantages described above. For example, in the case of glutamate oxalate transaminase, the Malate dehydrogenase may be present at an activity of from about 0.5 to about 10 units / ml, with an activity of 3 units / ml is particularly preferred.

For glutamate pyruvate transaminase the corresponding is Lactate dehydrogenase activity after reconstitution 8 to about 80 units / ml, with an activity of 40 units / ml is particularly preferred.

The amount of pyridine nucleotide, preferably NADH, is in both reconstituted diagnostic agents on a value that is one optical density of 2.1 to 1.0, the optical density being spectrophotometrically at 340 mn using a 10 mm light path is determined. An optical density of 1.8 is particularly preferred.

The buffer in both reconstituted diagnostics has a sodium content of less than 600 meq./l and is used to regulate the pH value of the reconstituted product. The preferred pH range that is regulated by the buffer is between 6 and 8. Certain buffers are particularly preferred for regulation within this range. These consist of potassium phosphate, sodium phosphate and mixtures

209885/0955

from it. The terms "sodium and potassium phosphate" include all three possible salts, the dipotassium or disodium salt being particularly preferred .. In the following the disalt is always meant if the term sodium or potassium salt is not explained in more detail.

If sodium phosphate is used alone as a buffer, the amount to be used will be dictated by the sodium content in the reconstituted product. The sodium content is allowed do not exceed a concentration of 600 meq./l and is preferably around 400 meq./l ^

If potassium phosphate is used as a buffer, it is used in added in such an amount that a 0.05 to 0.4 and preferably 0.1 molar concentration is achieved.

The buffer type of choice for the purposes of the present invention found are salts of strong bases and weak acids. Typical examples are sodium and potassium phosphate. Other representatives of this type are ammonium acetate, lithium phosphate and sodium borate.

However, other buffers that do not fall into the aforementioned category, such as albumin, are in the same way and Tris buffer (trihydroxyaminomethane) can be used.

Another preferred embodiment of both diagnostic agents is the use of albumin, the preferred one Concentration after reconstitution about 0.15 g / 100 ml amounts to.

According to the invention, the named diagnostics are quantitative Determination of the appropriate enzymes used.

The usual procedure for this is to mix the sample to be analyzed with a substrate and then mix it Analyze with the help of a suitable measuring instrument

209885/0955

mentes. According to the invention, the diagnostic agent is used with the reaction product combined and the decrease in NADH concentration measured photometrically.

Used in determining glutamate oxalate transaminase a mixture of aspartic acid and α-ketoglutaric acid is preferred as the substrate, while in the determination of glutamate pyruvate transaminase the substrate is preferably a mixture of alanine and α-ketoglutaric acid.

In both methods, the unknown enzyme concentration is the photometrically determined decrease in the NADH concentration directly proportional.

The stable, lyophilized diagnostic agents are produced according to the invention by adding malate dehydrogenase or lactate dehydrogenase mixed with the pyridine nucleotide and buffer. The aqueous solution obtained is lyophilized; and the reconstituted products have a pH in the range of 6.0 to 8.0 and are by those described above Features marked.

Potassium phosphate is preferred in the process according to the invention used as a buffer and albumin as a reagent.

The essential components are in both diagnostic tools

1. an enzyme,

2. a pyridine nucleotide and

3. a buffer.

For the diagnostic means for determining glutamate oxalate transaminase the enzyme component consists of malate dehydrogenase, while the enzyme component in the diagnostic Agent for the determination of glutamate pyruvate transaminase Is lactate dehydrogenase. Both enzymes are commercially available and are usually derived from animal sources. It

209885/0955

They are enzymes, commonly called components used for reagents for the determination of glutamate oxalate transaminase and glutamate pyruvate transaminase will.

The amount of malate dehydrogenase used in the diagnostic agent for determining glutamate oxalate transaminase is calculated by determining the activity of a reconstituted product. It has been found that for the purposes of the present invention the reconstituted product must have a malate dehydrogenase activity of from 0.5 to about 20 units / ml in order for an effective diagnostic agent to be achieved. An activity of 3 units / ml is preferred.

The amount of lactate dehydrogenase that the diagnostic Agent for the determination of glutamate pyruvate transaminase is added by determining the activity of a reconstituted Product calculated. It has been found that for the purposes of the present invention the reconstituted The product must have a lactate dehydrogenase activity of 8 to about 80 units / ml. An activity of 40 Units / ml is preferred.

The pyridine nucleotides used in the present invention are also available in the stores. For the production of the diagnostic agents according to the invention, it is imperative that that the NADH or NADPH are present in the reduced state, since the determination of the enzyme in the sample corresponds to the transition from NADH in the oxidized state, namely in NAD, directly proportional is. Similarly, when using NADPH, its Transition to the oxidized state, namely in NADP, measured.

NADH and NADPH are known cofactors and are particularly suitable for enzymatic reactions in which oxidation takes place.

209885/0955

The amount of pyridine nucleotide in the diagnostic agent for the determination of glutamate oxalate transaminase or glutamate pyruvate transaminase is determined by measuring the optical density of the reconstituted product. It was found to have an optical density in the range of 2.1 to 1.0 when using spectrophotometry at 340 nm a 10 mm light path, is required to produce effective diagnostic agents according to the invention.

The third component of diagnostic tools, namely the buffer, is the most important. The buffer seems essential To play a role in stabilizing the lyophilized material as well as the reconstituted product. How this is done in detail is, for. Not yet fully clarified.

With both diagnostic agents, the buffer must be so a satisfactory product is obtained, have a sodium content of less than 600 meq / l, which is the pH of the Agent is preferably able to regulate within the range of 6.0 to 8.0. The preferred buffer is potassium phosphate, which is added in such an amount that a 0.05 to 0.4 molar solution, based on the reconstituted Material, is obtained. Most preferred is a 0.1 molecular concentration. Other buffers that are preferred are sodium phosphate / potassium phosphate mixtures and sodium phosphate itself.

From the above it can be seen that the diagnostic agents according to the invention are characterized by properties that they have in the reconstituted product. Accordingly, the methods of making the agents are in With regard to the initial concentrations of the reagent, it is not of particular importance as long as the end product exhibits the well-defined properties clearly stated above after reconstitution ..

209885/0 9-5

- y -

The process for preparing the diagnostic agents comprises mixing malate dehydrogenase and lactate dehydrogenase, respectively with the pyridine nucleotide and the buffer material. The aqueous solution obtained is then lyophilized, -so that a dry, stable diagnostic reagent is obtained.

If the diagnostic reagent is to contain albumin, it is called Reagent introduced while mixing. Although albumin, preferably bovine albumin, is not an essential component it serves a very useful purpose. It prevents solids from settling to avoid the baseline drift during the analysis process. However, the albumin does not matter in the enzymatic conversion. If it's in the diagnostic Composition is incorporated, it must be present in an amount that the concentration after reconstitution is about 0.15 / 100 ml. This applies to both diagnostic agents.

The reconstituted diagnostic reagents as used above preferably have a pH in the range from 6.0 to 8.0. When the reconstituted product is outside this range, an appropriate setting is made prior to its analytical use.

In the process for the preparation of the reagent for glutamate oxalate transaminase or glutamate pyruvate transaminase is the use of a 0.5 molar potassium phosphate buffer solution, a 90% strength by weight aqueous solution of NADH or NADPH as well as an ammonium sulfate suspension of malate dehydrogenase or lactate dehydrogenase is preferred.

If bovine albumin is also used, it is used in the solid state.

The lyophilized compositions described are very stable. For example, they can be for an indefinite period of time

2098 85/095 5

at a temperature of 4 ⁰ C and at least two days at a temperature of 45 ⁰ C are kept.

Even the reconstituted product is proportionate
stable. During a storage time of up to 12 hours
at 4 ^° C. it does not change the optical density, and at 25 ^° C. the optical density changes by less than 0.290 in 3 hours.

The diagnostic means described become useful
used for automated analysis of samples for glutamate oxalate transaminase and glutamate pyruvate transaminase activity. To determine these enzymes, it is known to apply a substrate to the sample under observation
mix and analyze for enzyme activity. Mixing can take place with simultaneous heating. In addition, it is generally preferred to pass the reaction mixture through a dialysis membrane. The dialysis product is then analyzed for enzyme activity.

The present invention is modified in part by the dialysis product with the diagnostic agent for the determination of glutamate oxalate or glutamate pyruvate transaminase, usually in reconstituted form, combined and photometrically measures the decrease in NADH concentration.

The enzyme concentration is directly proportional to the decrease in NADH concentration.

The sample used to determine glutamate oxalate transaminase can be a serum sample (serum glutamate oxalate transaminase), a plasma sample or an aqueous solution, suspension or dispersion. An example of the latter type is an albumin sample. When analyzing for glutamate oxalate transaminase, the preferred substrate is a mixture of
Aspartic acid and «-ketoglutaric acid, while the substrate

209885/0956

of choice for glutamate pyruvate transaminase determination Mixture of alanine and -ketoglutaric acid is.

Example I.

Manufacture of the diagnostic agent for glutamate oxalate transaminase determinations

800 ml of a 0.5 molar aqueous potassium phosphate solution were treated with 1.25 g of NADH (90% strength by weight aqueous solution) and then mixed with 15,000 units of malate dehydrogenase in the form of an ammonium sulfate suspension. The solution obtained was made by adding 0.5 molar aqueous sodium phosphate buffer solution brought to the volume of 1 liter and then filled into vials in portions of 5 ml.

The vials were then subjected to lyophilizing conditions and the dry, stable compositions obtained were stored ^{at 4 ° C.}

Reconstitution

The dry compositions prepared as described above were distilled by adding 25 ml Reconstituted water. The reconstituted diagnostic agent had the following properties:

pH 7.4

Optical density . 1.75 ± 0.05 malate dehydrogenase activity 3 units / ml Potassium phosphate concentration 0.1 molar

The dry, lyophilized product can be ^{stored indefinitely at a temperature of 4 °} C. and is ^{stable at 45 °} C. for at least 2 days. The reconstituted product is ^{stable at 4 °} C. for up to 12 hours without a change in its optical density, while at 25 ^° C. the optical density changes by less than 0.2% in 3 hours. ■ - · ■

2098-85 / 0955

Example II

The procedure according to Example I was repeated with the difference that 7.5 g of bovine albumin in the 0.5 molar potassium phosphate buffer he solution were introduced.

The properties of the reconstituted product were the same and the lyophilized material was comparable Stability.

Example III

800 ml of a 0.5 molar aqueous potassium phosphate solution were mixed with 1.25 g of NADH (90% strength by weight aqueous solution) and then mixed with 200,000 units of lactate dehydrogenase in the form of an ammonium sulfate suspension. The solution obtained was made by adding 0.5 molar aqueous potassium phosphate buffer solution added to 1 1 and then filled in 5 ml portions into vials.,

The vials were then subjected to lyophilizing conditions, and the dry, stable compositions obtained were stored ^{at 4 ° C.}

Reconstitution

The dry compositions, as described / above, were reconstituted by adding 25 ml of distilled water. The reconstituted diagnostic reagent had the following properties:

pH "7.4

Optical density 1.75 ί 0.05

Lactate dehydrogenase activity 40 units / ml

Potassium phosphate concentration 0.1 molar

The dry, lyophilized product can be ^{stored indefinitely at a temperature of 4 °} C. and is ^{stable at 45 °} C. for at least 2 days.

209885/0955

The reconstituted product is ^{stable at 4 °} C. without changing its optical density for up to 12 hours, while at 25 ° C. the optical density deviates less than 0.2% from the initial value in 3 hours.

Example IV

The procedure of Example III is followed with the exception repeats that 1.5 g of bovine albumin is placed in the 800 ml of 0.5 molar potassium phosphate buffer solution.

The properties of the reconstituted product obtained are unchanged, and the lyophilized material has a comparable stability.

Example V

The reconstituted diagnostic reagent, which had been prepared according to Example I, was used to determine the Glutamate oxalate transaminase activity of an unknown sample is used as follows:

Using an AutoAnalyzer II, SMA-12/60 and SMA-12 MICRO from Technicon Instruments Corporation, Tarrytown, N.Y. the sample (about 0.3 ml) was mixed with a substrate consisting of 75 parts by weight of aspartic acid and 1 part of α-ketoglutaric acid (about 0.4 ml) dissolved in a phosphate buffer. Dialyze the reaction products through a dialysis machine and were with the reconstituted reagent that had been prepared according to Example II (about 0.3 ml), reacted. The change in optical density was determined in a photometer at 340 nm.

The activity of glutamate oxalate transaminase is the decrease in optical! Density of the NADH directly proportional.

209885/0955

Example VI

The method according to Example V was used to determine the activity of glutamate pyruvate transaminase in a sample repeated, whereby instead of the reconstituted diagnostic agent according to Example II that according to Example IV and a mixture of alanine and α-ketoglutaric acid were used as substrate.

Example VII

The procedure of Example I was used to prepare reconstituted repeated diagnostic agents A, B and C with the following characteristics:

A 13 C

pH 6 8 7

Optical density 1.0 2.1 1.5

Malate dehydrogenase activity 0.5 10 5

Potassium phosphate concentration 0.05 mol. 0.4 mol. Q, 3 mol.

Example VIII

The procedure of Example III was used to prepare reconstituted repeated diagnostic agents A, B and C with the following characteristics:

AB C

pH 6 8 7

Optical density 1.0 2.1 1.5

Lactate dehydrogenase activity 8 80 60

Potassium phosphate concentration 0.05 mol. 0.4 mol, 0.3 mol.

Example IX

The procedure according to Example I was repeated with the difference that instead of potassium phosphate a buffer composed of a mixture of potassium phosphate and sodium phosphate was used. The reconstituted product had a sodium content of 400 meq / 1 and a comparable stability.

2 09885/0955

Example X

The procedure of Example I was followed with the exception
repeats that sodium phosphate was used as a buffer instead of potassium phosphate ·. The reconstituted product had a sodium content of 400 meq / 1 and a comparable stability. .

Example XI

The method according to Example V was used to determine the activity of glutamate pyruvate transaminase of an unknown
Sample used, with that according to Example IV would be used instead of the diagnostic reagent prepared according to Example II.

209885/0956

Claims (22)

Claims Stable, lyophilized diagnostic agent for the determination of glutamate oxalate transaminase (GOT) in one Sample that contains this enzyme, consisting of malate dehydrogenase (MDH), a pyridine nucleotide, namely reduced Nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) and one Buffers with a sodium content of less than about 600 ml equivalent per liter to regulate the pH value of the product in reconstituted form. 2. Means for the determination of glu'tamate pyruvate transaminase (GPT) in a sample that contains this enzyme, consisting of lactate dehydrogenase (LDH), a pyridine nucleotide »viz reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) ■ and a buffer with a sodium content of less than about 600 milliequivalents per liter to regulate the pH value of the product in reconstituted form. 3. Means according to claim 1 or 2,
characterized in that it contains a buffer which keeps the reconstituted product within a pH range of 6 to 8. 4. Means according to claim 3,
characterized in that it contains potassium phosphate, a mixture of potassium and sodium phosphate or sodium phosphate as a buffer. 5. Means according to claim 1 or 2, characterized in that that it contains NADH as a pyridine nucleotide. 209885 / Ü95S - ■ 17 - 6. Means according to claim 1, ■ '._. characterized, that it contains malate dehydrogenase in such an amount that after reconstitution an activity of about 0.5 units / ml to about 10 units / ml and especially about 3 units / ml is achieved. . 7 · Means according to claim 2, characterized marked, that it contains lactate dehydrogenase in an amount such that, after reconstitution, an activity of 8 to about 80 units / ml and in particular about 40 units / ml is achieved. . ,. 8. Means according to claim 1 or 2, characterized in that that it contains the pyridine nucleotide in such an amount that, after reconstitution, an optical density of 2.1 up to 1.0 and in particular from 1.8, determined spectrophotometrically at 340 nanometers using a 10 mm light path, is achieved. 9. Means according to claim 3 »
characterized in that it contains potassium phosphate in such an amount that an approximately 0.05 to 0.4 and in particular 0.1 molar concentration is obtained after reconstitution. 10. Means according to claim 1. Or 2, characterized in that. . . that it contains albumin in such an amount that after reconstitution a concentration of about 0.15 g / 100 ml is achieved. 20988 57 * 0 955 11. Stable aqueous solution with a pH of 6.0 to 8.0, containing the agent according to claim 1 or 2, dissolved in water. 12. Method for the quantitative determination of the activity of glutamate oxalate transaminase (GOT) in a sample by mixing the sample with a substrate and analyzing it, characterized in that the reaction product formed by the mixing combined with an agent according to one or more of claims 1, 3 to 6 and 8 to 11 and the decrease the NADH concentration is measured photometrically. 13. Method for the quantitative determination of the activity of glutamate pyruvate transaminase (GPT) in a sample by mixing the sample with a substrate and analyzing it, characterized in that the reaction product formed by the mixing is used combined with an agent according to one or more of claims 2 to 5 and 7 to 11 and the decrease the NADH concentration is measured photometrically. 14. The method according to claim 12 or 13, characterized in that the reaction product of sample and substrate by passes a dialysis membrane before combining it with the diagnostic agent. 15. The method according to claim 12 or 13, characterized in that the reaction mixture from the sample and the substrate heated. 20988 5/0955 - 19 - ·. ■ 2235: 74 16. The method according to claim 12, characterized., that a mixture of aspartic and -ketoglutaric acid is used as the substrate. 17. The method according to claim 13, characterized in that that the substrate is a mixture of alanine and α-ketoglutaric acid used. 18. The method according to claim 12, characterized in that ' that the diagnostic agent contains malate dehydrogenase, NADH and potassium phosphate. 19. The method according to claim 13, characterized in that that the diagnostic agent contains lactate dehydrogenase, NADH and potassium phosphate. 20. A method for producing a stable, lyophilized diagnostic agent according to one or more of Claims 1, 3 to 6 and 8 to 11, characterized. i suspect that one malate dehydrogenase, a pyridine nucleotide and a buffer with a sodium content of less than about 600 ml-1! equivalent / 1 to regulate the pH of the agent mixed together in reconstituted form and the obtained aqueous product lyophilized, 21. Method of making a stable, lyophilized diagnostic agent according to one or more of claims 2 to 5 and 7 to 11, characterized in that lactate dehydrogenase, a pyridine nucleotide and a Buffers with a sodium content of less than about 600 ml equivalent / l to regulate the pH value of the agent mixed with each other in reconstituted form and the 209885 / na-KK -20- 2z35174 keep aqueous product lyophilized. 22. The method according to claim 20 or 21, characterized. that a 0.5 molar aqueous solution of potassium phosphate is used as a buffer. 209885 / 0.9 55