DE2431779A1 - IMPROVED PROCEDURE FOR THE DETERMINATION OF GLUTAMATE AND GLUTAMINATE TRANSAMINASES - Google Patents

Description

Patent attorneys Dipl.-Ing. F. ¥ eickmann,

Dipl.-Ing. HLWeickmann, Dipl-Phys. Dr. K. Fincke H / WE / MY Dipl.-Ing. RA Weickmann, D1PL.-CHEM. B. Huber

S MUNICH 86, DEN Case 16,752-F POSTFACH 860820

MÖHLSTRASSE 22, CALL NUMBER 48 3921/22

<983921/22>

THE DOW CHEMICAL COMPANY, Midland, Michigan / USA 2030 Abbott Road

Improved procedure for the determination of glutamate and glutaminate transaminases

The invention relates to a method for determining Glutamate and glutaminate (glutamic) transaminases.

The colorimetric determinations of glutaminate transaminases in biological fluids are carried out according to various methods in many laboratories. The value of such determinations as a diagnostic aid in myocardial infarction and necrosis of liver cells is well recognized; Reitman and Frankel, Am. J. Clin. Pathol., 28, 56 (1957). The enzymes by principal Are important are glutaminate-oxalacetic acid transaminase (GOT), which is the implementation

L-aspartate + a-oxoglutarate - oxaloacetate + L-glutamate

catalyzes and the enzyme glutaminate-pyruvic acid transaminase (GPT), which is the implementation

L-alanine + α-oxoglutarate pyruvate + L-glutamate

catalyzed. The methods are based on enzyme-catalyzed transaminations of a substrate, which a-oxoglutarate and Contains a suitable amino acid, with glutamate and another acid, pyruvate in the case of GPT and oxaloacetate in the case from GOT.

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Some colorimetric methods are based on color conversions, where the acid formed is included. Recently, a method has been described in which this occurs during the transamination step formed glutamate is dehydrated, whereby the enzyme glutamate dehydrogenase (GlDH) in aqueous ammonium sulfate and the coenzyme uses oxidized nicotinamide adenine dinucleotide (NAD), where <x -oxoglutarate, ammonia and reduced Nicotinamide adenine dinucleotide (NADH) are formed,

Glutamate + H ₂ O + NAD £ i23 »cVOxoglutarate + NH ₃ + NADH

The NADH formed is treated with a colorless tetrazolium dye, 2-p-iodophenyl-3-nitrophenyl-5-phenyltetrazolium chloride (INT), with the help of an electron carrier such as N-methylphenazonium methosulfate (HfS) implemented, whereby a colored dye (INT-formazan) is formed in a reaction that is catalyzed by the electron carrier:

NADH + INT - ^ NAD + INT-formazan

The intensity of the color formed is then determined and gives a measure of the transaminase activity; Lippi and Guidi, Clin. Chim. Acta, 28, 431 (1970).

The transaminase determination is usually carried out on biological fluids such as serum in the differential diagnosis of liver and heart diseases. A predetermined volume of the sample liquid is mixed with a predetermined volume of the substrate composition, buffered to a pH of about 7 _f 4, which contains iX-oxoglutarate and either aspartic acid (for GOT) or alanine (for GPT). A color developing solution or solutions containing GlDH, INT and NAD is used. The substrate, sample and color developer are mixed in a container such as a colorimeter, color developing cell or test tube. The mixture is incubated, typically in a water bath or by heating in a block at 37 ° C for a predetermined time of about 45 to 60 minutes. The Incuba-

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tion is terminated by the addition of acid and the intensity of the resulting color is measured photometrically in a spectrophotometer or colorimeter determined at wavelengths from 490 to 550 nm and with a control serum, a comparison curve o.a. compared.

The invention relates to an improvement in glutamate determinations through the implementation of NADH with tetrazolium indicators and includes improved glutaminate transaminase determinations by coupling the transamination by glutamate dehydrogenase and NAD with the color reaction of NADH with tetrazolium salt indicators. The invention relates to a reagent-substrate composition containing a transaminase substrate, NAD, glutamate dehydrogenase, and the tetrazolium color reagent in a single liquid composition. In the method of the present invention, the substrate-reagent composition with a sample for its transaminase activity or to be analyzed for glutamate, mixed and then incubated for relatively short times as if by about 5 or less to about 15 minutes before the acid incubation is terminated, and then the color will certainly.

The reagent-substrate composition and that of the invention Methods provide several advantages in the determination of glutamate and glutaminate transaminase enzymes. she can be produced in large quantities, with the advantage that the quantities of the ingredients can be easily measured out, the standardization is easily done and that it is for actual Determination is not required to perform mixing operations. The incubation time necessary for the determination the transaminase is required by the method according to the invention is significantly less than the time that is required in the known methods, i.e. the determination can be compared in an incubation period of 10 minutes with times of 45 minutes. The

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The method according to the invention and the agent according to the invention are highly specific and result in the desired linear relationship from color to enzyme activity. As in the present invention the transaminase activity through the glutamate produced during the incubation is formed, is determined »the invention enables the use of pure, aqueous standard solutions of glutamate, and it is not necessary to use control sera with a known enzyme activity to use.

The reagent substrate means comprises transaminase substrate components, i.e., Οί-ketoglutarate and a substrate amino acid; Color reaction components, i.e. the tetrazolium dye, and an electron carrier such as the enzyme diaphorase or FMS, and coupling reaction components GlDH and NAD in aqueous Solution, buffered to a pH at which the transamination reaction can run rapidly »In particular, the reagent substrate includes the substrate, color reaction and coupling reaction components in amounts sufficient to give measurable color necessary for transaminase activity is measurable on incubation with a glutaminate transaminase. The reagent-substrate means is further characterized by that it is essentially free of ammonia or ammonium ions.

The agent according to the invention can be produced by the ingredients are mixed in any suitable order and manner. As with diagnostic reagents high purity components should be used with quality. The agent can contain small amounts of other ingredients in addition to the ingredients mentioned above. For example, one can use surface-active agents, chelating agents (such as ethylenediamine-tetraacetic acid or EDTA) or enzyme activators such as adenosine diphosphate (ADP). Generally a small amount is one Surfactants such as polyoxyethylene (23) laurylether «1s surfactant is desirable. From un-

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about 0.01 to 0.05 moles ADP / 1 are also generally desirable. The identity of the substrate amino acid determines the specificity of the composition and the method for a special transaminase. Accordingly, the substrate amino acid can be selected to make the determination specific is for GPT in the presence of GOT (using L-alanine) or you can have all of GOT and GPT activity determine if desired (using both L-aspartate and L-alanine together).

For a GPT determination, the L-alanine can be used as L-alanine or can be used as a racemic mixture of DL-Alan in. in the in the latter case, the D-alanine is essentially inert in the determination and serves only as a diluent. It was found that in GOT determinations, D-aspartate suppressed the GOT transamination reaction inhibited. For GOT determinations, L-aspartate should be used in place of the optical isomer, D-aspartate, or racemic mixtures can be used.

The glutamate dehydrogenase is usually given as a suspension used in glycerin. Both GlDH and diaphorase should be essentially free of ammonium salts, which are usually contained in enzyme preparations.

As the tetrazole dye component, there can be any Use tetrazolium salt dye that is catalyzed by an electron barrier NADH reduction results in a colored product and which does not adversely affect the other ingredients or with other ^ materials used during the transamination reaction are present, responds. In the method according to the invention, INT, TNBT (tetranitro-blue tetrazolium), NBT (nitro-blue tetrazolium), NT (neotetrazolium) and BT (Blue tetrazolium) all measurable discoloration. However, results INT has an unexpected, more intense color than other tetrazolium salt dyes and it is also in the test mixture more soluble than most tetrazolium salts. Accordingly

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- 6- "'243 7 779

INT is the preferred tetrazolium dye.

The reagent-substrate means should be essentially free of ammonia such as ammonium ions. It was found that the presence of small amounts of ammonium salts, like them present either as impurities or as suspending agents or carriers for one or more ingredients can be or can be present in some buffer systems, strongly inhibit the entire course of the determination can. The ingredients should be selected and used so as to achieve an ammonium ion concentration below about 0.001 mol / l in the final mixture (i.e. zero or below the limit of determination of about 0.005 mol / l). The ammonium ion concentration is preferably below 0.001 mol / l. Ammonium ions from the Serum will not adversely affect the determination either at normal serum levels or at levels close to 10 or 20 times the usual.

The reagent-substrate means may contain the following ingredients in the following proportions:

Substrate

cX-ketoglutarate 0.0005 to 0.0012 moles

Substrate Amino Acid 0.01 to 0.5 moles of color reagent

Tetrazolium dye 0.0005 to 0.005 moles electron carrier, diaphorase (or equivalent, i.e. PMS 0.0001 to 0.0002 mol) 100 to 3200 international units (I.U.)

Coupling reagents

GlDH 2000 to 4000 IU
NAD 0.001 to 0.01 mole
surfactant 1 to 10g aqueous phosphate buffer (pH 7.2 to 8.5; molarity 0.01 to 1) qs to 11.

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2A3177S

A preferred agent contains the following ingredients:

(X-ketoglutarate 0.0005 to 0.0075 moles

Substrate amino acid 0.01 to 0.5 mole

INT 0.0005 to 0.005 moles

Diaphorase 250 to 2000 I.U ..

GlDH 2000 to 40 000 I.U.

NAD 0.002 to 0.004 moles

surfactant about 5 g

aqueous phosphate buffer (pH qs to 11 molarity 0.01 to 0.10), 8.0
to 8.2

For a specific application for the determination of a specific Glutaminate transaminase such as GOT or GPT will be the preferred amount of substrate amino acid depending on the one used Amino acid vary. L-alanine is used as the amino acid for GPT determinations and this is preferred in Amounts from about 0.02 to 0.4 moles / liter based on the reagent-substrate agent are present. For GOT regulations -Used as substratamine L-aspartate, and this is preferably present in concentrations of from about 0.01 to about 0.16 mol / l.

In the method according to the invention, the substrate components, the color reaction components and the coupling reaction components mixed with a small amount of a sample to be analyzed for transaminase activity. The resulting mixture is under such pH and temperature conditions that der.enzymkatalysierten transamination correspond for a predetermined short period of time incubated, then the strength or depth of the resulting color is determined. Mixing is conveniently done by • to a sample such as serum, plasma, tissue extracts, enzyme preparations or the like directly with the aqueous according to the invention Mixed composition. The relative amounts of sample and substrate-reagent agent can vary approximately, depending

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j on factors such as known or expected concentration of the enzyme in the sample, the concentration of the constituents in the composition, etc. If appropriate Procedures will be about 0.02 to about 0.5 parts by volume Sample / part by volume composition used.

The incubation pH can range from 7.2 to as high as 9.0. For best results, the pH should be in the range of 7.9 to 8.3 and is preferably controlled by adding a buffer to the substrate-reagent composition. The incubation should be carried out at a temperature of about 25 to 40 ° C and preferably at about 37 ⁰ C. In a preferred embodiment, the agent is preheated to the incubation temperature before adding the sample.

The incubation time prior to color determination is important to the successful practice of the present invention. The incubation time can be determined by known methods using known devices such as continuous flow analyzers or recording spectrophotometer. Incubation can be easily done by adding acid conveniently by diluting the mixture with aqueous hydrochloric acid to bring it to pH 0.1 to 4.5, after which the color remains stable for 30 minutes or more and during this time can the absorption can be measured. The predetermined incubation time should be sufficient for a measurable color to be formed and it should be less than about 15 minutes. For longer incubation times such as 20 or 30 minutes not only does it eliminate the benefit of rapid determination, but also the sensitivity and linearity of the determination can also be adversely affected.

In a preferred method, the incubation. at pH 8.0 to 8.2 for about 5 to about 15 minutes

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carried out at 35 to 40 ⁰ C and terminated by adding an aqueous hydrochloric acid. The strength of the color that is formed is determined by measuring in a colorimeter or spectrophotometer the absorption of light having a wavelength of about 450 to 55 Ω nm. The enzyme activity is determined by comparing the absorption measurements that are obtained with control sera, standard solutions containing known amounts of glutamate, standard curves or the like.

The following examples illustrate the invention without restricting it.

i
example 1

A reagent-substrate composition containing the following ingredients in aqueous, ammonia-free solution in the following amounts is prepared:

INT 8 χ 10 " ⁴ Minor NAD 21.4 χ 10 Minor L-alanine 0.1 Minor OC-ketoglutarate .1 χ 10 " ³ Minor EDTA 0.2 χ 10 Minor surface active
Medium (Brij - 35) 5 6/1 GlDH (as glycerine
suspension) 2.7 I.U./ml Diaphorase 0.5 I.U./ml Phosphate buffer pH 8.1
(KH ₂ PO. And NaOH) 0.8 Minor

1 ml of the agent is placed in a series of ampoules and mixed well. The ampoules are used for the determination of GPT in a sample control serum or dilutions of the identical control serum with aqueous 0.85 # saline solution used. Each ampoule is heated in a heating block at 37 ° C for 5 minutes before adding the sample.

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100 / ul of the appropriate liquid (control serum or dilution of which) are added to every five ampoules and the contents are mixed. 100 / μl of an aqueous 0.85? Saline solution is mixed with the contents of another ampoule and serves as a blank sample. After 10 minutes of incubation at a temperature of about 37 ° C, the incubation is terminated by adding the contents of each ampoule with 2 ml of aqueous hydrochloric acid (approx. 0.1N) diluted.

The intensity of the color in each ampoule is then determined by measuring the absorbance at 500 nm using a photoelectric colorimeter determined. (The instrument is previously distilled to zero absorption using Water adjusted.) An identical set of reference serum and serum dilutions is also analyzed, with a commercially available ultraviolet enzyme determination method is used. A comparison of the graphs the results against concentrations of serum in the samples show excellent linearity and a good agreement with the colorimetric determination corresponding to the ultraviolet determination.

Example 2

A reagent-substrate composition is prepared containing 1.6 χ 10 ~ ⁴ Hol / l PMS, 8 χ 10 ~ ⁴ mol / l INT, 21.4 χ 10 ~ ⁴ mol / l NAD 1000 χ 10 ~ ⁴ mol / l L-alanine, 10 χ 10 "* ⁴ mol / l CX-ketoglutarate, 0.2 χ 10 ~ ⁴ mol / l EDTA and 2,700 IU / L GLDH in phosphate buffer solution containing also containing a surfactant and 1 g Bovine Serum Albumin / L The agent is tested according to a similar procedure to that described in Example 1, using various known amounts of standard aqueous glutamate solution to test the linearity of the substrate and the process in the presence of added protein the absorption values versus the concentration of added glutamate (ten determinations of

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0.4 χ 10 to 4 χ 10 "moles of glutamate in the test mixture) shows excellent linearity.

Example 3

A GOT reagent-substrate composition is prepared which contains the following components in the concentrations given below:

OUKetoglutarate 0.001 mol / l

L-aspartic acid 0.020 mol / l

INT 0.0008 mol / l

NAD 0.003 mol / l

Diaphorase 500 I.U./l

GlDH 8000 IU / l phosphate buffer (pH 8.1) 0.05 mol / l

A GPT reagent substrate composition is prepared containing the same ingredients in the same concentrations, except that the GPT composition contains 0.05 mol / L L-alanine and no aspartic acid.

The compositions are placed separately in different ampoules, 1 ml / ampoule. They are used in terms of GOT and GPT. In each method, the compositions in a 37 ^O C heating block for 10 minutes to be preheated and then with 0.1 ml of a serum sample, control serum, distilled water as a blank or aqueous mixed glutamic acid standard solution. The mixtures are incubated for 10 minutes at 37 ° C., then mixed with 2.0 ml of 0.1N hydrochloric acid in order to terminate the enzymatic reactions. The resulting color is determined in a colorimeter with light with a wavelength of approximately 500 nm.

Example 4

A reagent-substrate composition, which is 0.05 mol / l L-aspartate, 0.0075 mol / l CX-ketoglutarate, 0.0028 mol / l NAD, 0.002 mol / l INT, 800 IU diaphorase and 4000 IU / l GlDH in

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0.05 mol / l phosphate buffer (pH approximately 8.1), the 0.5 $ one . contains surfactant (Brij-35) 'manufactured. The composition is used to measure GOT activity on aliquots of 42 human serum samples using essentially the same procedure as described in Examples 1 and 3 is. The GOT activity of an aliquot of the same serum samples is also determined according to the procedure by Henry, Chiamori, Golub and Berkman, Am.J.Clin.Path. 34, 381 (1960) determined. A statistical analysis of the results shows a good correlation between the two methods.

According to a similar procedure, a GPT composition by a method similar to Example 3 for correlation with the above comparative method using 34 serum samples and an excellent correlation between the two methods notices.

In a particularly useful embodiment, the reagent-substrate composition is in the form of separate compositions and one of them contains the substrate amino acid and the other contains the color reagent and the Coupling components. The (X-ketoglutarate can be used in any Composition or it can be added separately. This separate reagent composition that the color reagent and the coupling components in the proportions given above can be used directly for quantitative Glutamate determinations are used.

The enzymatic activity is determined separately Using the components appropriate to use the biological fluid samples as individual blanks to be able to. In such a procedure, the color reagent and coupling reagents are with the samples during

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incubated for a time sufficient for the absorption of the resulting mixture reaches a stable, constant value. This time is generally short, from 1 to 3 minutes. During this incubation, small amounts of glutamate or substrate amines will be present in the samples are used up, and the resulting stable absorption value can be used as a blank value or as a baseline for the Can be used for comparison. The remaining ingredients are then added to make the finished substrate-reagent composition and then the incubation is continued, the absorbance determined and compared to the blank and the enzyme activity is determined as described above. This embodiment is illustrated in the following example explained.

Example 5

Reagent-substrate compositions are prepared for the separate determinations of GOT and GPT. The color reagent coupling ingredient composition is made in concentrated form, so that the dilution of 2 ml of it with 1 ml of the suitable substrate a final composition of 0.006 mol / l NAD, 0.0008 mol / l INT, 0.00002 mol / l ethylenediamine-tetraacetic acid, 0.010 mol / l ADP, 50 IU / l Diaphorase and 8000 IU / l GIdH in 0.1 mol A aqueous phosphate buffer, pH 8.0. The GOT and GPT substrate compositions are produced separately in concentrated form, to give ready-made mixtures containing: 4 mmol C * -ketoglutarate in pH 8, 0.1 mol / l phosphate buffer and 0.060 mol / l L-aspartic acid (in the case of the GOT substrate) or 0.1 mol / l D-L-alanine (in the case of the GPT substrate).

The compositions are used to determine both GOT and GPT in 43 human serum samples, and the Results are compared to the results obtained by using aliquots of the same 43 sera accordingly the method of Henry et al, which is in Example 4

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! was written, analyzed. .At the determinations will be Heat 2 ml of the color reagent-coupling reagent concentrate to 37 ° C and mix with 100 µl of the serum samples. The mixture it is then incubated for 2 minutes at 37 ° C., then the absorption (at approximately 500 nm) is determined and recorded as the serum blank value. 1 ml of the appropriate GOT or GPT substrate concentrate is then added and then mixed. The resulting finished mixture is at 37 ° C for 10 minutes incubated., After this 10 minute period, the absorption measured and recorded, the time being controlled so that the incubation is terminated Acid is not required. The enzyme activity is determined from the difference in absorbance between that incubated Mixture and the blank sample by comparing the absorption measurements obtained with the glutamate standard solution, calculated.

When determining GOT, the results correlate well with the results obtained with the comparison method, with a correlation coefficient of 0.9932 and a linear correlation graph with a slope of 0.9547. The relationship of the absorption results indicates that GOT activity is linear to about 300 IU GOT / ml Serum is. Excellent correlation is obtained in the GPT determination with a correlation coefficient of 0.9937 and a graph with a slope of 1.034. The results obtained show excellent Linearity up to approximately 200 IU GPT / ml serum.

For the person skilled in the art, it is familiar that the above-described Process can be varied many times, for example one can o ^ -Ketoglutarate to the color reagent coupling ingredient concentrate or you can add it separately or you can adjust the concentration of the ingredients in the concentrates change while maintaining the proportions of the components relative to one another.

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Claims (10)

Claims Λ A method for the determination of glutamate, characterized in that a sample that is to be analyzed is mixed with a solution of oxidized nicotinamide adenine dinucleotide., Glutamate dehydrogenase, a tetrazolium dye and an electron carrier, the mixture under pH conditions and temperature conditions that are suitable for the Formation of a measurable color therein are capable of being incubated and the resulting color is measured after a predetermined incubation time, the solution being essentially free of ammonium ions. 2. The method according to claim 1, characterized in that the color is measured after a predetermined incubation time sufficient to give a measurable color and less than about 15 minutes. 3. The method according to claim 1, characterized in that a mixture of a biological liquid is used as the sample with a glutaminate transaminase substrate containing <* - oxoglutarate and a substrate amino acid. 4. The method according to claim 3, characterized in that the mixing is carried out by a biological Liquid sample with a solution of a substrate amino acid, CX-oxoglutarate, nicotinamide adenine dinucleotide, glutamate dehydrogenase, a tetrazolium dye and an electron carrier mixed and that the color is measured after a Incubation of the resulting mixture for a predetermined time sufficient to give a measurable color, and is less than about 15 minutes. 5. The method according to claim 1, characterized in that one additionally has a biological fluid sample mixed with a solution containing an oxidized nicotinamide adenine dinucleotide, glutamate dehydrogenase, a tetrazolium 409886/1236 dye and an electron carrier in the absence of the Substrate amino acid contains, the resulting mixture, up to a stable color is formed, incubated, the resulting stable color is measured and then a substrate amino acid adds, the resulting mixture is incubated in the presence of CX-oxoglutarate for a predetermined time, which is sufficient to form a measurable color therein and which corresponds to the glutamate which is produced after the transamination reaction of the OC-ketoglutarate and the substrate amino acid was formed, and the resulting color is measured. 6. The method according to claim 5, characterized in that the tetrazolium dye is 2-p-iodophenyl-3-nitrophenyl-5-phenyltetrazolium chloride and used as electron carrier diaphorase. 7. The method according to claim 6, characterized in that the substrate amino acid is L-alanine or L-aspartic acid used. 8. Aqueous composition valuable for the determination of glutamate and glutaminate transaminases containing a solution of a tetrazolium salt dye which is suitable for producing a colored reaction product with reduced nicotinamide adenine dinucleotide, to give oxidized nicotinamide adenine dinucleotide and glutamate dehydrogenase, in proportions from about 0.0005 to about 0.005 moles of tetrazolium salt, from about 0.001 to about 0.01 moles of oxidized nicotinamide adenine dinucleotide to about 2000 to about 40,000 I.U. glutamate dehydrogenase, with the solution additionally contains an electron carrier in a concentration sufficient to cause the tetrazolium salt dye to react with reduced nicotinamide adenine dinucleotide, and containing a buffer that has a pH of approximately 7.9 to about 8.3 therein and wherein the composition is essentially free of ammonium ions. 409886/1236 9. The composition according to claim 8, characterized in that there is used as an electron carrier and diaphorase iTetrazolium Salt Dye 2-p-Iodophenyl-3-nitrophenyl-5-phenyltetrazolium salt used. 10. Composition according to claim 9 »characterized in that it additionally (λ-Ke toglutarate and a substrate amino acid such as L-alanine and / or L-aspartic acid
contains. 409886/1236