GB2057684A

GB2057684A - Method and Kit for Determination of Transaminases

Info

Publication number: GB2057684A
Application number: GB8023408A
Authority: GB
Original assignee: Biodata SpA
Current assignee: Biodata SpA
Priority date: 1980-07-17
Filing date: 1980-07-17
Publication date: 1981-04-01
Also published as: GB2057684B

Abstract

In a method for the determination of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in a single sample there is effected a kinetic U.V. determination on the sample using as the specific aminoacid substrates for the said transaminases L- cysteinsulfinic acid and L-alanine, respectively, and the pyruvic acid produced being determined using lactic hydrogenase so providing a measure of both transaminase activities. The sample may be of plasma, serum or other biologically-derived material. The invention also includes a kit for use in the method comprising, as enzyme substrates, L-cysteinsulfinic acid and L-alanine, optionally together with instructions.

Description

SPECIFICATION Method and Kit for Determination of Transaminases This invention reiates to a method and a kit for the determination of transaminases, particularly useful in the field of clinical diagnosis.

Transamination is the process of transferring an amino-group from an aminoacid to a keto-acid.

Enzymes which catalyze this type of reaction are named transaminases and their determination has become essential in clinical chemistry since, in 1954, La Due, Wroblewski and Karmen reported that GOT (Glutamic Oxalacetic Transaminase or Aspartate Amino Transferase or EC 2.6.1.1. according to the l.U.B. classification) increases after myocardial infarction. Variations have been noted of both GOT and GPT (Glutamic Pyruvic Transaminase or Alp nine Amino Transferase or EC 2.6.1.2. according to the l.U.B. classification) in other pathologic states, especially in epathopathies.

GOT catalyzes the following reaction:

Aspartic acid a-Ketoglutaric acid Oxalacetic acid Glutamic acid; while GPT catalyzes the following reaction:

Alanine a-Ketoglutaric acid Pyruvic acid Glutamic acid In the case of GOT, it is not easy to determine aspartic and a-ketoglutaric acids and, therefore, all methods actually in use are based upon the determination of oxalacetic acid, either directly or indirectly. Two methods are usually employed for this determination: 1) The colorimetric method based upon the reaction of oxalacetic acid with 2,4dinitrophenylhydrazine; this reaction is not kinetic, but after a pre-established period of incubation the quantity of keto-acid formed is determined by measuring at 536 nm the extinction of the thus formed phenylhydrazone.

2) The kinetic U.V. method, which is generally considered as the reference method due to its high accuracy and specificity. The oxalacetic acid produced in the transamination reaction is determined through an enzymatic reaction involving malic dehydrogenase (MDH) by measuring at 340 nm the oxidation of NADH (Nicotinamide Adenine Dinucleotide in the reduced form).

oxalacetic acid malic acid The modifications made to this method first introduced by Karmen permitted the reaction to be optimized as a function of the concentration of substrate and of the determination temperature, and the troubles due to the non-linear kinetic phases to be overcome.

In the case of GPT, similar considerations also exist in the case of the colorimetric test, while the kinetic U.V. method introduced by Henley and Pollard is based on the determination of pyruvic acid formed in the alanine transamination reaction by an enzymatic reaction involving lactic dehydrogenase (LDH), the re-oxidation of NADH being measured at 340 nm.

pyruvic acid lactic acid Again in the case of the kinetic U.V. method, the modifications introduced and described in the literature have permitted the reaction to be optimized as a function of the concentration of substrate and of the determination temperature.

As indicated above, the determination of GOT and GPT activities in the serum or in other biological samples is effected by the colorimetric or U.V. kinetic methods mentioned above. This imposes upon the analyst a requirement to carry out two different analyses for the two transaminases.

This is due to the technical impossibility of coupling the two U.V. kinetic enzymes mentioned above in a single determination as a result of cross-inhibition phenomena.

The object of the present invention is a new method which permits spectrophotometric determination, by a U.V. kinetic method involving only one assay, of the sum of the activities of the two transaminases, and thus constitutes the first screening assay as between normal and pathological values for these particular enzymatic activities. In addition, depending upon the requirements of the analyst, the new method permits, by simply modifying the order of introduction of participants and substrates in the reaction mixture, the exact determination of the activities of the two transaminases in Units per liter in the same sample of plasma or serum, thus saving time compared to methods traditionally used in the laboratory.

In fact GOT is able to catalyze transamination not only of its natural substrates (glutamic acid and aspartic acid), but also of other aminoacids. Thus, cysteinsulfinic acid is actively transaminated, in the presence of a-ketoglutaric acid, with formation of p-sulfinyl pyruvic acid, which, being unstable in aqueous solution, is quickly hydrolyzed to pyruvic acid and sulfite according to the following reaction scheme:

pyruvic acid Even in the absence of a-ketoglutaric acid, GOT is able to intereact with cysteinsulfinic acid and catalyze an a-p-elimination reaction according to the following scheme:

cysteinsulfinic acid a-a miniacrylic acid pyruvic acid In this case the final product of the reaction is again pyruvic acid.Accordingly, the activity of GOT may be determined in a spectrophotometric kinetic manner at 340 nm by measuring the re-oxidation of NADH in the presence of lactic dehydrogenase and the same enzyme may be employed for the determination of GPT activity.

The use of cysteinsulfinic acid as the substrate for GOT thus permits the employment of only one revealing enzyme, lactic hydrogenase (LDH), in coupled determination of the two transaminases, according to the following scheme:

cysteinsulfinic acid pyruvic acid lactic acid Thus, the invention provides a method for the determination of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in a single sample which comprises effecting a kinetic U.V. determination on the sample using as the specific aminoacid substrates for the said transaminases L-cysteinsulfinic acid and L-alanine, respectively, and lactic dehydrogenase as the revealing enzyme for both transaminase activities.

Preferably, the sample is biologically-derived, e.g. a serum or plasma sample.

The decrease of extinction at 340 nm provides a measure of the quantity of pyruvic acid produced and, as a consequence, of the activities of both transaminases. It is important to note that the simultaneous determination of the transaminases is possible in the method of the invention thanks to the absence of cross-inhibition between the substrates employed.

The particular kinetic properties of the reaction between GOT and cysteinsulfinic acid permit two different applications of the invention, as illustrated in the following specific Examples which are given further to illustrate the invention.

Example 1 Method of Determination of GOT and GPT-absolute Activites To a reaction mixture of voiume 2.5 ml containing L-alanine (200 mM), a-ketoglutaric acid (2 mM), p-NADH (0.24 mM) and 3.6 units of lactic dehydrogenase in Tris-HCI buffer (80 mM), pH 8.5, 0.5 ml plasma or serum are added and then the decrease of extinction is measured at 340 nm (or at other wavelengths e.g. 334 or 365 nm, according to the instrument employed). The decrease is in direct relationship to the amount of transformation of alanine to pyruvic acid caused by GPT.

After a few minutes of reaction, 0.1 ml of a solution of cysteinsulfinic acid are added, so that the final concentration in the reaction mixture is 66 mM. The increase, hE/min, is due, in this case, to the transformation of cysteinsulfinic acid to pyruvic acid as caused by GOT.

AE/min observed before and after the addition of cysteinsulfinic acid, the exact values of GOT and GPT enzymatic units present in the serum by a single determination process.

Example 2 Method of Screening for Determination of Normal and Pathological Values of GOT and GPT Activities in Plasma and Serum To a reaction mixture of volume 2.5 ml containing L-alanine (200 mM), a-ketoglutaric acid (2 mM), p-NADH (0.24 mM), cysteinsulfinic acid (7 mM) and 3.6 Units of lactic dehydrogenase in Tris-HCI buffer (50 mM), pH 8.5, 0.5 ml of serum or plasma are added and the extinction decrease is measured at 340 nm (or at other wavelengths, e.g. 334 or 365 nm, according to the instrument employed). In this case the decrease of extinction is in direct relationship to the amount of transformation of both alanine and cysteinsulfinic acid to pyruvic acid, caused respectively by GPT and GOT, and is therefore a measure of the sum of the two enzymatic activities.

The kinetic characteristics of the assay permit the establishment of a limit value of AE/min between normal and pathological values.

Through opportune transformation coefficients it is possible, in the case of normal samples to calculate exactly the actual enzymatic units of both GOT and GPT, while in the case of pathogenic samples the method involves an error of about 10--15%.

The present invention includes a kit for use in the determination of GOT and GPT comprising, as enzyme substrates L-cysteinsulfinic acid and L-alanine, optionally together with instructions. Such a kit may also comprise one or more of lactic dehydrogenase, NADH and a-ketoglutaric acid.

The invention further provides L-alanine or L-cysteinsulfinic acid for use in the method of the invention (i.e. when in a suitable "get up" for use in the method, when intended for use in such method or when manufactured, sold, disposed of or otherwise dealt with for use in such method).

Claims

1. A method for the determination of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in a single sample which comprises effecting a kinetic U.V. determination on the sample using as the specific aminoacid substrates for the said transaminases L-cysteinsulfinic acid and L-alanine, respectively, and lactic hydrogenase as the revealing enzyme for both transaminase activities.

2. A method as claimed in claim 1, wherein the sample is biologically-derived.

3. A method as claimed in claim 2, wherein the sample is a serum or plasma sample.

4. A method as claimed in any one of claims 1 to 3, wherein the total of GOT and GPT transaminase activities is determined by measuring the total hE/min with the said two aminoacid substrates both being present from the start of enzymatic reaction.

5. A method as claimed in any one of claims 1 to 3, wherein hE/min is first measured in the presence of L-alanine as a substrate and the hE/min after the addition of L-cysteinsulfinic acid is measured, the latter hE/min increase (if any) being a measure of GOT transaminase activity.

6. A method as claimed in claim 1 and substantially as hereinbefore described either in Example 1 or in Example 2.

7. A kit for use in the determination of GOT and GPT comprising, as enzyme substrates, Lcysteinsulfinic acid and L-alanine, optionally together with instructions.

8. A kit as claimed in claim 7 also comprising one or more lactic hydrogenase, NADH and - ketoglutaric acid.

9. L-alanine for use in a method as claimed in claim 1.

10. L-cysteinsulfinic acid for use in a method as claimed in claim 1.