DE2032270A1 - Diagnostic reagents for the determination of gamma-glutamyl transpeptidase in human serum - Google Patents

Description

Dlpl.-fng. Kar! Klekcbtn

Patent attorney - ', t Beriin 19 »^ iaerdanua SV

June 22, 1970 P.5103

Sankyo Company, Limited in Tokyo (Japan).

Diagnostic reagents for the determination of jf -glutamyl transpeptidase in human serum.

The invention "relates to a diagnostic reagent. It relates in particular to a diagnostic reagent for the detection and quantitative determination of Jl -glutamyltranpeptidase (hereinafter referred to as" J ^ -GrTPaSe ") in human serum which contains a specific combination of b -I-glutamic acid p-nitroanilide, a selected surfactant and a selected buffer solution.

It is known in the relevant art that Ϋ -G-TPase is mainly found in the liver and in the mere, and that a higher J'-GTPase activity is found in the serum of patients with liver diseases such as liver cancer, pathological obstruction of the liver and the like / is observed. -

■ ■ - -? -

ft ft ύ a £ "} / ι £ η-% '. ■ ■. ■. ■

More recently, A. Szewczuk et al. (Clinica Chimica Acta, 7, 755, 1962) - S. Lukasik et al. (Swiss Medical Wochenschrift, 98, 81, 1968) and M. Orlowski et al. £ Clinica Chemica Ac ta, 15, 387, 1967) reports that a jf -GIPase method is considerably more sensitive than the alkali phosphatase method (journal of Clinical Pathology, 7, 322, 1954} and the leucine aminopeptidase method ( Cancer, V \ _ _f 283, 1958 ^ which have been used for the diagnosis of liver diseases.

There is therefore an urgent need and endeavor in the relevant art to bring the above-mentioned J'-GIPase method to a practically usable level, for example for the introduction of such a method in a hospital as a routine test method. However, no favorable results have been obtained because of the poor solubility of % -L-glutamic acid p-nitroanilide (hereinafter referred to as "^ -GNA"), which is the most suitable substrate for the determination of the jf-GTPase activity, as well as because of the instability of the solution of this substrate. This determination therefore includes in particular the determination of the γ / -GTPase activity in the presence of a substrate at a concentration of 5 mM in a 0.1 M tris (tris (hydroxymethyl) aminomethane) buffer solution of pH 8 at 37 ⁰ C. However, this older relevant method has some drawbacks, namely (1), since the substrate,. y ~ GNA, tends to be decomposed into glutamic acid and p-nitroaniline in a Tris buffer solution of pH 7 to 9, i.e. the optimal pH range, the preparation of the reagent and the determination procedure should be carried out within a short period of time, and if this is not possible, the accuracy of the measured value is poor and (2) because of the solubility of the substrate

0 0 9 8 5 2/1673

in the above Tris buffer solution within the range given above pH range is at most about 2 mM at ordinary temperature, should be used for the determination of the jj'-GTPase an unstable supersaturated solution of the substrate, which 'by dissolving with heating and quenching, can be used to obtain a solution having a concentration of about 5 mM.

As a result of extensive studies on a. ver

it
improved and useful / diagnostic reagent for the determination of J '-GTPase in human. Serum has now been found that a | -G-NA solution, which is stable at high concentration and useful as a diagnostic reagent, can be obtained by selecting and applying a particular combination of % -GEA with a specific surfactant and a specific buffer solution,

It is therefore a primary object of the invention to provide a highly stable diagnostic reagent with a high substrate concentration for the determination of If-GTPase in human serum to show which is a combination of ^ -GNA and a special amphoteric or nonionic contains surfactants, as well as a special buffer solution.

Further features and advantages of the invention will become apparent to those skilled in the art from the following more specific description the invention.

As already indicated above, the inventive diagnostic reagent has been completed on the basis of the above knowledge, and it contains im special a new combination of (1) Jf-GlTA,

(2) one / or more surfactants, selected. selects from the group consisting of amphoteric surfactants and nonionic

- 4 009852/1673

surfactants, and
(3) a buffer solution selected from the group consisting of a collidine buffer solution, a boric acid buffer solution, a 5,5-diethyrbarbituric acid buffer solution and an ammonia buffer solution.

The procedure and the manner in which the The above findings are explained in more detail below.

First, studies on the solubility of the substrate within wide ranges of solvents were carried out in the following manner. When a conventional solvent was used, for example, an alcohol such as methanol, ethanol, propanol, butanol, glycerol and the like; a ketone such as acetone, methyl ethyl ketone and the like; a lower aliphatic acid ester such as ethyl acetate, butyl acetate and the like; a heterocyclic base such as pyridine, quinoline and the like; an aromatic hydrocarbon such as benzene, toluene and the like; Dimethylformamide; Water; and a mixture thereof, only a solution of the substrate with a concentration below 2 mM could be obtained in each of the above solvents at a temperature used for the determination in an enzyme reaction, and when a 30% aqueous solution of dimethylsulfoxide was used only a 3.5 ^m M solution of the substrate could be obtained. Briefly summarized, it therefore shows that with any of the above-mentioned solvents © in © - solution with the required concentration could not be obtained.

When different surfactants together could be used, the substrate solution with a high

009852/1673

- 5 -. ■■. ■ -. ■

Concentration above about 5 mM can be obtained. Therefore the following surfactants have been investigated: for example, cationic surfactants such as which contain quarterly ammonium ions such as benzethonium chloride, benzalkonium chloride and the like; anionic surface-active agents, such as those which contain derivatives of carboxylic, sulfonic or sulfuric acids, such as sodium laurate, sodium dodecyrbenzenesulfonate, Sodium alcohol polyathene oxysulfate; amphoter © surface-active Agents such as those containing cyclic imidinium compounds or H-ayl sarcosine, such as sodium H-lauroyl sarcosine, contain; and nonionic surfactants such as those comprising a mixed ether of one Alcohol and a phenol, such as polyoxyethylene nonylphenol ether, Polyoxyäthylenoctylalkoho.läther contain.

Besides, the reaction is what is necessary for achieving For the purposes of the present invention, an enzymatic reaction is used, and therefore one must of course select a surfactant which will perform this reaction does not inhibit.

As a result of the studies on the effectiveness of the above cationic, anionic, amphoteric and nonionic surfactants. Means was found that amphoteric and nonionic surface-active agents inhibit the / -ÖIPase activity in its way, whereas cationic and anionic surfactants significantly inhibit this activity.

Some representative examples of the above study results are summarized in the following table I, where de * state of the reaction after a lapse of 30 Minutes since the initiation of the reaction is indicated due to a change in the absorbance at 410 m / t

■. - 6 009852/1673 '.

• Table I

Classification
of the surfactant

surfactant used.

410

Cationic

Benzallronium chloride

Benzethonium chloride (1)

0.035 0.147

Anionic

ITatrite unlauryl sulfate

Sodium dodecyrbenzenesulfonate

Sodium alcohol polyethene oxysulphate ^ '

O ₉ OOO 0.053

0.053

Amphoteric

Sodium dlauroylsarcosine

0.450

Cyclic imidinium derivative «■ 0,

430

Non-ionic

Polyoxyethylene nonylphenol ether

Polyoxyethylene octyl alcohol ether

0.591

0.600

control

0.591

9852/1673

(Annotation)

(1) "Hyamine 1622", tradename, available from

Rohm & Haas.Co., U.S.A.

. (2) "Uilclrol ' ¹ NE S 202, tradename, available from. Mkko Chemicals Co., Irtd., Japan.

(3) "Hikkol Sarcosinate"'IiS, trade name, available from Mkko Chemicals Co., Ltd., Japan.

(4) "Miranol" G 2 M -SF, trade name, available by The Miranol Chemical Co., Incorporation, U.S.A.

(5) "Mkkol" HP-9, trade name, available from Mkko Chemicals Co., Ltd., Japan.

(6) "STikkol" BO 20, trade name, available from Nikko Chemicals Co., Ltd., Japan.

The method for determining the y-GTPase activity, which is used here is summarized below, the publication of the same is to be understood as a reference.

The "solution of 5 g of one of the surfactants specified above, 150 mg of J ^ -GNA and 396 mg of glycylglycine in about 90 ml of distilled water is adjusted to a pH of 8 by adding hydrochloric acid or an aqueous solution of sodium hydroxide, and it becomes that The whole is made up to 100 ml by adding distilled water. The solution obtained is used as substrate solution. The mixture of 0.5 ml of the substrate solution with 0.01 ml of serum is ^{left to stand at 37 °} C. for 30 minutes, and then 2.5 ml of a 1.3 M aqueous acetic acid solution

■ - 8 -

00985/1/167?

solution added. " The absorbance at 410 m / λ. is measured in the usual way. On the other hand, 0.5 ml of the SubstraüLösung 30 minutes at 37 ⁰ C, and then 0.01 ml of the serum and 2 * 5 ml of one. 1.3 M aqueous acetic acid solution was added. The absorbance at 410 m / t is measured as above. The difference obtained by subtracting the latter measured value from the first measured value is referred to as the activity value, expressed as Ae, ... Q.

The substrate, ^ -GNA, which can be used according to the invention, shows very different stabilities in the same pH range, depending on the kinds and types of used Buffer solutions, and that this substrate is very unstable in the Tris buffer solution which was previously used for the determination the ^ -GTPase activity was used, and various Phosphate buffer solutions with a wide pH range from weakly acidic to weakly basic, which are commonly used in enzymatic reactions.

Some representative examples of the above study results are summarized in Table II below, wherein the state of decomposition of the substrate after 3 days have passed since the test solution was prepared is due to the change in the absorbance at 410 m / t

009852/1673

- - 9 -, ■ Table II

Buffer solution

Collidine - hydrochloric acid 0.5

Boric acid - sodium carbonate 0.6

Veronal ^ '- Uatrium acetate -

Hydrochloric acid '

Ammonia - hydrochloric acid 0.5

Tris (hydroxymethyl) aminomethane -

Hydrochloric acid ⁶ °

Sodium phosphate - potassium phosphate 9.0 (Note): (1) 5,5-diethylbarbituric acid.

The method for determining the stability of Jf-GIfA in the buffer solution used here is summarized below, where this publication is included as a reference.

In this method, the same substrate solution is used as in Table I above, except that "Mkkol" 3JP-9 is used as the surfactant and the indicated pH 8.0 buffer solution is used in place of distilled water and the acid or base. Immediately and 3 days after the preparation, the absorbance became ΔΈ. ^ ₀ measured. The ,

- 10 -

Difference, which results from the first measured value, deducted from the last measured value is defined as the stability value.

In an expedient embodiment of the invention, the final concentration of the surface-active agent used according to the invention is desirable in the range from about 2.5 to 15% in the reagent for the determination of V-GTPase, a value of 5 fo being particularly favorable

in
is. The ionic power of the buffer solution used in the present reagent can be varied within wide limits, mainly depending on the types and types of buffer solutions used, but the preferred strength is in the range of about 0.05 to 0.3, with about .0, 1 for the collidin or ammonia buffer solution, with about 0.2 to 0.3 for the boric acid buffer solution, and with less than 0.1 for the veronal buffer solution as particularly favorable.

It is desirable in the present invention to have a concentration of the substrate, y-GKA., From about 4 to 6 mM should be used because then precisely measured values are constantly obtained.

A considerable increase in the rate of the reaction can be achieved, although this is not essential, by further adding an appropriate amount of a peptide derivative having free terminal amino groups, such as glycylglycine and the like, to the present reagent as an acceptor for the ^ -glutamic acid which can be formed in situ as a consequence of the enzymatic reaction.

In the quantitative determination of Jf-GTPaSe im human serum using the present diagnostic reagent can be satisfactorily used the present diagnostic reagent, which either zuoror or was manufactured immediately prior to its use »

- 11 009852/1673

The procedure for the determination of Jf-GTPase im human serum can be conveniently carried out by a slight change in the "known method of determination, for example that described by D.M. Dimov et al. in Clinica Chemica Acta, 1-6, 271 (1967) has been. The details of the determination method are discussed below, this publication is included as an explanatory note.

(1) A mixture of 0.5 ml of the reagent with 0.02 ml of the human serum is allowed to stand at 37 ° C for 30 minutes, and then 2.5 ml of a 1.3 M aqueous acetic acid solution is added. The absorbance at 410 my & is measured in the usual way. On the other hand, 0.5 ml of the reagent ^{is left to stand at 37 °} C. for 30 minutes, and then 2.5 ml of a 1.3 M aqueous acetic acid solution and 0.02 ml of the serum are added. The absorbance at 410 m ^ tc is also measured in the above manner.

The difference made by subtracting the latter measured value is obtained from the first measured value is defined as the activity value.

(2) Immediately after 3.0 ml of the reagent is mixed with 0.02 ml of the human serum, the absorbance at 410 mJUL is measured in a conventional manner. Then the measured mixture ^{is left to stand at 37 0} O for 30 minutes, and then the absorbance at 410 is also measured in the above manner.

The difference made by subtracting the latter measured value is obtained from the first measured value is defined as the activity value.

The following examples, which represent representative embodiments of the composition of the diagnostic Describe reagent according to the invention, only to For the purpose of explaining the invention, given without

-12-009852 / 1673

-12-They are intended to limit the scope of the invention.

example 1

The diagnostic reagent was prepared in the following composition!

-GNA 150 mg

1/10 K hydrochloric acid 15 ml

Glycylglycine 395 mg

"Nikkol" NP-9 5 g

1/7 M "Yeronal sodium" (sodium

5,5-diethyl barbiturate} - 22 ml

Sodium Acetate (pH 8.0) ■ Distilled water (q.s.) up to 100 ml

The reagent was prepared as follows: The j> -GNA was dissolved in the hydrochloric acid (referred to as solution A). On the other hand, the glycylglycine and "Nikkol" were in a) share τοη about 55 ml of distilled water dissolved, and then the resulting solution to a pH 8 ₅ 0 is set by the addition of dilute aqueous Natriumhydr-oxide ₅ is an alkaline solution to form · (referred to as solution B) ₀ Su the Lösung.B were the solution A and added to the Veronalpufferlösung, and then sufficient distilled water was added until the whole volume - ^'! uf 100 wl was filled.

The diagnostic reagent was made with; the

1 ^

"BAD ORIGINAL-

same composition as in Example 1 above with the Exception that 15 g instead of 5 g "ITikkol" NP-9 is used and that 30 ml of 1/5 M collidine hydrochloric acid (pH 8.0) buffer solution instead of the "veronal" buffer solution were used.

Example 3

The diagnostic reagent was prepared with the same composition as in Example 1 above, except that 2.5 g instead of 5 g of "Nikkol" ΪΤΡ-9 was used, and that 15.5 ml of 1/10 Ή aqueous ammonia was used in place of "Veronal" buffer solution was used.

Example 4

The diagnostic reagent was prepared with the same composition as in Example 1 above with the Exception that 18.3 ml of 1/5 M boric acid - sodium carbonate buffer solution (pH 8.0) were used in place of the "Veronal" buffer solution.

Example 5

The diagnostic reagent was prepared with the same composition as in Example 1 above, except that 5 g of "Nikkol" BO 20 was used in place of "Nikkol" KB-9 .

00985271873

Example 6

The diagnostic reagent was prepared with the same composition as in Example 1 above with the Exception that 5 g "Miranol ™ C2M-SF" instead of "Fikkol" WP-9 were used.

Example 7

The diagnostic reagent was prepared with the same composition "as in Example 1 above, except that 5 g of" Miranol "C2M-SI ^{1 were used in} place of" Fikkol "1ΪΡ-9, and that 15-5 ml 1/10 Έ aqueous ammonia were used in place of the "Yeronal" buffer solution.

Patent claims:

- 15

S Ο if% ITtJ £%

Claims (5)

- 15 claims: 1. A diagnostic reagent for the determination of y -glutaroyl transpeptidase in human serum by a combination of (1) Y- L-glutamic acid p-nitroanilide, (2) one or more surfactants, selected from the group consisting of amphoteric surfactants and nonionic surfactants, and (3) a buffer solution selected from the group consisting of an olidin buffer solution, a Boric acid buffer solution, a 5.5 ~ diethylbarbituric acid buffer solution and an ammonia buffer solution. ■ 2. Reagent according to claim 1 _3, characterized in that the surface-active agent is one which Matriurnlauroylaarkosin, a cyclic iiaidinium derivative, Polyoxyäthyleniionylphenoläther or Polyoxyäthylenoctyl- · alcohol ether .. contains. ■ 3. ReageiiE according to claim 1, characterized geke'mizeichnet that the buffer solution is a Gollidin-Chlorviasserstoffäureimfferlösungj a boric acid e-iiatrium carbonate buffer solutionj a sodium-5, S-cliätliylbarbj-turat - iTatritisacetatbuffer ¹ - = - suspension or 'aqueous ammonia » 4 »Res · -öus according to claim 1, άβαΐίΓοΙι characterized, 'that the fon;: ·; - ^ p; ätion des ol) ^ i? _{Surface-activeii means 2?} 5 to 15 $ at ·· » ¹ -; / t, ντιδ. that the buffer solution has an ion power of 0.05 "bi.r-- ·""', .... 98S2 / .1BT3 2 O 3? ? 7th 5. Reagent according to claim 1, characterized by a content of 0.15 J ^ Jf -L-glutamic acid-p-nitroanilide, 5 # polyoxyethylene nonylphenol ether and $ 22 a 1/7 M sodium 5,5 diethyl barbiturate - sodium acetate buffer solution. 009852/1673