CS236758B2 - Method of determining of alpha-amylase - Google Patents

Abstract

A substrate for the determination of alpha -amylase of the formula I is prepared by reacting the particular phenyl glucoside or nitrated phenyl glucoside with alpha -cyclodextrin, amylase or soluble starch in the presence of Bacillus macerans amylase. The radical R in the formula I has the meaning stated in the claim. <IMAGE>

Description

The present invention provides a method and an agent for determining α-amylase.

Determination of serum α-amylase is an important clinical parameter for pancreatic function. Commercial reagents for the determination of α-amylase are mainly based on a process in which starch is degraded by α-amylase and the resulting cleavage products are determined in the visible or ultraviolet range, depending on whether colored or natural starch is used as the amylase substrate . An essential disadvantage of these methods or reagents is that starch as a macromolecule can only be poorly characterized and standardized so that the degree of conversion of the individual batches is very different, and a standard must always be used in determining the measurements. To achieve better results, a more uniform substrate would be required, which yields reliable results in cleavage.

A step forward towards a more uniform substrate was brought about by the use of maltopentose. This is cleaved by α-amylase into maltotriose and maltose, maltotriose and maltose are converted by α-glucosidase into glucose, which can then be determined by any method, for example the known hexokinase method.

In addition to maltopentosis, maltotetrose and maltoheosis have already been proposed as substrates (U.S. Pat. Nos. 3,879,263, 4,000,042). However, significantly worse results were obtained with tetrose than with pentose, and with hexose even worse than with tetrose. Thus, a stoichiometric reaction is reported for maltotetrose and maltopentosis, while for hexose, just tolerable deviations from the stoichiometric reaction have already been found.

However, the disadvantage of maltopentosis, which has also been found with tetrose, is that a very blind reagent value is generated, i.e. the measurement reaction is already in progress before the sample to be determined is added. In addition, this blank value of the reagent is not constant at higher substrate concentrations, but varies for more than 25 minutes before the side reaction remains invariant. It has also been shown that the anticipated different cleavage of maltopentosis by the pancreatic α-amylase and the salivary α-amylase, which cleavage would allow differentiation, does not actually occur (J. BC., 1970, 245, pp. 3917-3927; J. Biochem., 51, XVIII (1952).

It was therefore an object of the present invention to provide a method and reagent for the determination of aamylase using a substrate having a higher purity and uniformity than known substrates, is readily available and

The 2367S8 meets the required requirements in terms of serum-free blank, lag phase duration, and achievable peak activity. Furthermore, a simple assay procedure, without expensive and complex apparatus, as well as suitability for immediate determination, such as test strips, should be allowed.

The object of the present invention is to provide a zipper for the determination of α-amylase by enzymatic cleavage of the α-amylase substrate and measurement of the cleavage product, which method is characterized in that a compound of formula I is used as the substrate.

where

R represents a glulcoside, phenylglucoside, mononitrophenylglucoside, dinitrophenylglucoside or sorbitol or gluconic acid group.

Surprisingly, it has been found that maltoheptose has more desirable properties as an α-amylase substrate, although oligomaltoses already proposed for this purpose have shown a substantial loss of suitability in switching from maltopentosis to maltohexosis, since its use results in worse results than with pentose. Thus, further extension of the maltozo-oligosaccharide chain could be expected to result in already intolerable deficiencies. Surprisingly, however, better results are obtained than with pentose. Thus, the blank value of the reagent at 0.02 ml of the sample with maltopentose as substrate 73%, with maltoheptose, on the other hand, is 13%, based on the final assay value in the normal range.

In addition, it has been found that certain maltoheptose derivatives may also be used instead of maltoheptosis, which, when treated with α-amylase, form a derived cleavage product which can be determined particularly advantageously.

The method according to the invention is particularly suitable for the determination of cleavage products by α-glucosidase or maltose phosphorylase.

In the α-glucosidase assay and the compound of formula (I) wherein R is a glucoside group, the cleavage products of maltoheptose, maltotetrose and maltotriose are further cleaved to glucose, and this is then determined by a known method. The hexokinase method is then particularly suitable for the determination of the glucose formed in the presence of [alpha] -glucosidase. The principle of this embodiment of the method according to the invention can be illustrated by the following equations:

α-amylase maltoheptose + H2O ------> maltotriose + maltotetrose α-glucosidase maltotriose + 2 H ₂ O --------> 3 glucose α-glucosidase maltotetrose + 3 H2O ------- -> 4 glucose glucose + 7 aidenosine triphosphate (ATP) ->

hexokinase (HK) ---------- ► 7 glucose-6-phosphate.

glucose-6-phosphate + 7 oxidized nicotinamide adenine dinucleotide 4glucose-16-phosphate dehydrogenase + ---------------->

-> 7 gluconate-6-phosphate + 7 reduced nicotinamide adenine dinucleotide (NADH) -h + 7 H +.

Maltoheptose derivatives used according to the invention, i.e. compounds of formula I, in which R is not a glucoside group, are particularly suitable for carrying out the process according to the invention. By the action of both the .alpha.-amylase and .alpha.-glucosidase enzymes, this substituent, that is, the phenyl, moinonitrophenyl or dinitrophenyl group, or the end-bound sorbitol or gluconic acid residue, is cleaved off and can be readily determined. Said phenyl groups may be linked from an α- or β-position; in the α-position, they are cleaved by α-amylase and α-glucosidase alone and the cleaved, substituted or unsubstituted phenols can then be readily determined by known color reactions. However, the invention can also be used with substituents bonded in the β position. In this case, besides N-glucosidase, β-glucosidase is also used.

For dimitrophenyl residues, both nitro groups can also be bonded at any positions, for example as 2,4-, 2,6- or 3,5-substituents.

The nitrophenols, or dinitrophenols, which are released upon the cleavage of the nitro-containing substituents, are themselves colored compounds which can be readily determined optically. When only phenol is cleaved, it can be determined by known methods, for example by reaction with a nucleophilic reagent such as 3-methyl-6-sulfonylbenzthiazolohydrazone (2) - (HSK) in the presence of monophenol oxidase. This reaction produces a red dye which can be measured.

When sorbitol is cleaved, it can be oxidized, for example, by sorbitol dehydrogenase to fructose; simultaneously, the oxidized nicotinamide adenine dinucleotide (NAD) present is reduced to a reduced nicotinamide adenine dinucleotide (NADH). The formation of NADH can be determined in a known manner by a UV-spectrophotometer. If this device is not available, color reaction with tetrazolium salt, such as 2- (p-iodophenyl) -3- (p-nitrophenyl) -5-phenyltetrazolium chloride [INT], can also form a colored electron transfer agent formazan, which can be determined by measurement in the visible range.

Analogously, the liberated gluconic acid can be determined by appropriate known methods, for example by gluconate kinase, 6-phosphogluconic acid dehydrogenase and oxidized nicotinamide adenine dinucleotide phosphate - (NADP), optionally with tetrazolium salt and an electron transfer agent.

PH values in the range of 5 to 9 are generally suitable for carrying out the process of the invention. Preferably, pH values in the range of 7 to 7.5 are used, since the best results are obtained in the shortest reaction time. When nitrophenyl compounds are used in the process of the invention, the range of suitable pH values is somewhat narrower and is usually in the range of 6 to 8.5.

Suitable buffers are those which are effective over the main activity of the enzymes used. Phosphate, N- (2-hydroxyethyl) piperazine-N-2-ethanesulfonic acid (HEPES) and glycylglycine are preferably used.

Preferred concentrations are in the range of 10 to 200 mmol / l.

α-glucosidase is usually used in an amount of 0.1-5000 J / ml. It is a particular advantage of the process according to the invention that a relatively large amount of this enzyme can be used, so that the rate-determining factor of an even higher amount of the enzyme can be used, but no other advantages are achieved.

The compounds of formula (I) used in the process of the invention are generally used in an amount of 0.1 to 250 mmol / l. Amounts in the range of 0.5 to 100 mmol / l are preferred.

The saturation of the? -Amylase substrate with maltoheptose is in a concentration range of 8 to 10 mmol. Therefore, a minimum concentration of 8 mmol maltoheptose is expediently used when operating under substrate saturation conditions, as is usually the case.

In addition, an activating agent for α-amylase is expediently added. Such activation means are known. Sodium chloride or potassium chloride is preferably used.

In a further preferred embodiment of the process according to the invention, the determination of the cleavage products is carried out by reacting with maltose phosphorylase to form glucose-1-phosphate, which is then determined in a manner known per se. In a particularly preferred embodiment, the determination of glucose-1-phosphate is performed by conversion to glucose-6-phosphate by β-phospho-glucosomutase (β-PGluM), by oxidation of the resulting glucose-6-phosphate by NAD in the presence of glucose-6-phosphate dehydrogenase (G- 6-PDH) to form gluconate-6-phosphate and NADH, the formation of the latter being readily monitored photometrically. The measured signal can be further amplified by further oxidation with NAD in the presence of 6-phosphogluconate dehydrogenase (6-PGDH) to form ribulose-5-phosphate and another NADH molecule.

The principle of this embodiment is expressed by the following equations:

a-amylase maltoheptose ψ Η? Ο -------> maltotriose -h maltotetrose -> maltose maltose phosphorylase maltose + PO4 ³ '-----------> glucose + β-glucose-1-phosphate β-PGluM 'β-glucose-T-phosphate ----> glucose-6-phosphate

G-6-PDH glucose-6-phosphate + NAD + ---- · »gluconate-6-phosphate-1-NADH + H +

6-PGDH gluconate-6-phosphate + NAD + -----> ribulose-5-phosphate + NADH + II +.

An advantage of this embodiment of the method of the invention is that maltose phosphorylase is more specific than α-glucosidase, and therefore endogenous glucose is not an obstacle.

As far as buffers are concerned, the remarks made when describing embodiments of the method of the invention using α-glucosidase apply accordingly.

In addition to the two above-described embodiments of the process of the invention, the resulting maltoheptose cleavage products, i.e. maltotetrose, maltotriose - and maltose resulting therefrom, can be determined by other known methods.

As already mentioned, the invention not only provides a quick and specific method for the determination of [alpha] -amylase, but also completely or largely eliminates the lag phases, which is of particular importance. using the method of the invention on automatic analyzers. In addition, the process according to the invention can be carried out in many variations without complicated evaluation apparatus; the method is therefore particularly suitable for rapid diagnosis and for visual determination in the visible range. At the same time, however, it is possible to determine various embodiments of the method according to the invention even with measuring instruments in the UV range. Further advantages are the strict proportionality and no disturbance of the chemically related substances contained in the blood.

The substrates used according to the invention are readily available and in high purity. A simple process for the production of maltoheptosis is disclosed in German published patent application DOS 27 41 191. This method is suitable for the determination of α-amylase in biological fluids such as serum heparin plasma, urine and the like, or in other liquid or solid substances.

The invention is illustrated by the following examples.

Example 1

Maltogenic method (α-glucoside system)

A mixture of reagents containing α-glucosidase, glucose-6-phosphate dehydrogenase, hexokinase, oxidized nicotinamide adenine dinucleotide, adenosine diphosphate, maltoheptose, magnesium Mg + +, sodium chloride and phosphate buffer is dissolved in 2.0 ml of distilled water. The shelf life of the reagent at room temperature is about 1 hour, at temperatures below 8 ° C for 6 hours.

The resulting solution is characterized by the following concentrations of each reagent:

phosphate buffer

NaCl

Mg2 + maltoheptose deenssiotrifosfate oxidized oikstinamide amine eiodinukiestide hexokinase glucose-6-phosphatehydrogeoase α-glucosidase mmol / l; pH 7 mmol / 1 mmol / 1 mmol / l

1.2 mmol / 1 mmol / 1 w 2j / i x 2j / i w 10j / i

0.02 ml of the serum sample is added to the solution at 25 ° C, the mixture is transferred to a cuvette thickness of 1 cm and then the extraction is determined in a photometer at Hg 365 nm, 340 nm or Hg 334 nm. After 10 minutes, the extinction is read and then repeated five times at 1 minute intervals.

From this determined difference per minute (ΔΕ / min), the average value is generated, the reagent blank is subtracted and the corrected value is used for the calculation.

The calculation is performed as follows:

j / 1 25 cc = 4244 χ 365 E 365 nm / min = 2290- χ Δ E 340 nm / min = 2335 χ Δ E 334 nm / min.

Fig. 1 of the accompanying drawing shows the results using a human serum dilution series with saline obtained by this method.

If the reagent is divided into two batches, one containing maltose and the other containing a mixture of all the remaining reagents, the shelf life of the solutions produced can be increased. It is 30 hours at room temperature for temperatures up to 8 cg for about 8 hours at room temperature. The shelf life of the maltoheptose solution is analogously 6 weeks, or approximately 1 week.

Example 2

Determination of maltose phosphorylase system

Two reagents are prepared, consisting of an amylase substrate and a maltose phosphorylase system. One agent - contains a maltoheptose substrate, the other agent contains soluble starch according to the prior art. The concentration of reagents after dissolution in water is as follows:

According to the invention

Comparison phosphate buffered oxidized nicotinamide adenine dinucleotide maltoheptose soluble starch glucose-1,6-diphosphate maltose phosphorylase (microorg.) O-phosphoglucomutase (microorg.) Glucose-6-phosphate dehydrogenase (Leuconcstoc mesent)

6-phosphogluconate dehydrogenase (Leuconostoc mesent)

20 May mmol; pH 6.5 20 May mmol; pH 6.5 2 mmoly 2 mmoly 10 mmol - - 5 mg / ml tracks tracks 3 j / ml 3 j / ml 1 j / ml 1 j / ml 9 j / ml 9 j / ml 1 j / ml 1 j / ml

The resulting solution was incubated at 30 ^° C, sample solution was added and the extinction difference at HCl 334 nm was determined by photometer. After a 10-million pre-incubation, the extinction difference is measured over time. 10 minutes. For 2.0 ml of reagent and 0.10 ml of sample, the following relation is obtained:

Δ E / min x

2.1 x 1000

6.18 x 0.1 x 0.2

- Δ E / min x 1699 J / L

(b) phenyl-α-D-tri- (tetra) -glucopyranoside - β-α-glucosidase

4- 3 (4) НЮ ---------> phenol +

4- (4) glucose monolenol oxidase

c) phenol-1-HSK-1? ---------------->

-> dye 4- 2 H2O.

Using five different human sera, the following values were found using the above reagents:

Serum According to the invention starch 1 37.4 J / L 15.3 j / 1 2 61.2 J / L 27.3 J / L 3 95.1 J / L 39.1 j / 1 4 114 j / 1 44.2 J / L 5 374 j / 1 161 j / 1

Example 3

Detection of α-amylase with phenyl-α-maltoheptoside as substrate

Phenyl-α-maltoheptoside is cleaved by α-amylase into phenyl-α-maltotriide or phenyl-ω-maltetraraid, which is converted to phenol and glucose by α-glucosidase. The liberated phenol is oxidatively coupled with the monophenol oxidase to the nucleophilic reagent 3-methyl-6 sulfonylbenzthiazolone hydrazone- (2) - (HSK) to form a red dye whose rate of formation is proportional to the amylase activity in the sample and can be monitored photometrically.

Test principle:

1.

α-amylase

a] phenyl-cis-maltoheptoside IDO ---------->

-> phenyl-α-D-tri-phthetra-1-glucopyranoside -h maltotetra- (tri) -ose

Measurement conditions:

^C C. measured wave length 492 nm, 1 cm cuvettes; the composition of the reagent is shown in the following table.

TABLE

Reagent · Test concentration

phosphate buffer, pH 7.4 0.05 mol / 1 sodium chloride 0.05 mol / l phenyl-N-D-maltoheptoside 10 mmol / l HSK 1 mrnol / l monophenol oxidase 1 j / ml α-glucosidase 10 j / ml Volume at. test: 2.0 ml.

Other buffers, such as glycine, glycylglycine, Hepes, tris, tra and others, may be used instead of phosphate buffer.

Example 4

Determination of α-amylase by p-nitrophenylmaltoheptoside

Test principle:

α-amylase p-nitrophenyl-α-maltoheptoside —------>

-> (glucose) _n 4-p-nitrophenyl-

3G α-glucosidase

-α- (glucose) _m ------- ► n glucose +

4- p-nitropheno]

After cleavage of the maltoheptoside, the cleavage products are degraded to glucose and p-nitrophenol. The p-nitrophenolate anion is yellow in the alkaline solution and can therefore be detected directly by optical measurement.

Test system

Reaction mixture:

mM phosphate buffer, pH 7.4

5Ό mmol / l sodium chloride up to 50 u / ml crglucosidase or 10 mmol / l of p-nitrophenyl-α-maltoheptoside.

Test Handle:

ml of reaction mixture 50 μΐ of sample (serum), temperature: 30 ° C, wavelength: 405 nm, start by adding serum after reaching the measuring temperature (pre-incubation 10 minutes).

Test system:

Reaction batch

Na- (K-PO4-buffer 0.02 mol / l + triton x 100 2 ml / 100 ml 4- NaCl 10 mmol / l, pH 6.9

MgCl2 0.3 mol / l maltoheptitol 100 mmol / l

NAD c = 40 mg / ml

INT c = 0.6 mg / ml diaphorase (Clostr. Kluyveri) 5 mg / ml *) ATP c = 50 mg / ml hexokinase 280 j / ml *)

SDH 180 j / ml *) α-glucosidase 1120 j / ml *) sample (serum)

8

Example 5

Determination of α-amylase by maltoheptitol

Test principle:

α-amylase maltoheptitol + H2O ------> maltotritol 4- maltotetrose α-glucosidase maltotriitol 4- H2O ------—->

-> sorbitol 4-maltose

SDH sorbitol + NAD ⁺ ---> fructose 4+ NADH + H ⁺ diaphorase

NADH 4 INT 4 H ⁺ ---—> formazan 4+ NAD ⁺ .

SDH = sorbitol dehydrogenase

ml Test concentration 1.92 12.8 mmol / 1 POi 6.4 mmol / l 0.01 1.0 mmol / l 0.10 3.3 mmol / l 0.05 1.0 mmol / l 10Q 0.09 mmol / l 0.05 167 171 0.10 3.29 mmol / l 0.05 4 666 171 0.30 18 000 171 0.20 74 670 Her 0.02

*) in assay buffer

Start by addition of sample: measurement is performed at 492 nm; temperature: 2'5 ^° C

Example 6

Determination of α-amylase by maltoheptagluconic acid

Test principle:

maltoheptagluconic acid 4α-amylase

4- H2O ---—> maltotriogluconic acid 4- maltotetrose maltotriogluconic acid 4α-glucosidase

4- H ₂ O ------- ► Gluconic acid 4- “l · Maltose Gluconic acid 4gluconate kinase

4- ATP -------->

6-phosphate acid

230758 Glucone + adenosine diphosphate ribulose-5-phosphate + NADPH +

Gluconic acid 6-phosphate + + CO2 + H ⁺ dehydrogenase acid

6-phosphoglucone + NADP '------------- Testing system:

Reaction batch m ¹ Test concentration Na- / K-PO4-buffer 0.02 mol / l 2.19 14.6 mmol / l phosphate, + NaCl 10 handles / l, pH 6.9 7.3 mmol / l NaCl MgCl2 1 mol / l 0.01 3.3 mmol / l maltoheptagluconic acid 150 mmol / 0.10 5 mmol / l NADP c = 10 mg / ml 0.10 0.38 mmol / l ATP c = 50 mg / ml 0.10 2.7 mmol / l gluconate kinase 40 J / ml 0.03 1066 J / L 6-phosphogluconic acid dehydrogenase 24 J / ml 0.10 800 J / L α-glucosidase 1120 j / ml 0.30 11.2 x 10 ⁴ J / L sample (serum) 0.02 '-

Start by adding a sample; measurement is performed at 340 nm or 365 nm, temperature: 25 ° C, test volume: 3.00 ml.

Claims (1)

OBJECT OF THE INVENTION Method for the determination of α-amylase by enzymatic cleavage of an α-amylase substrate and measurement of a cleavage product, characterized in that a compound of formula (III) is used as the substrate wherein the phenylglucoside or sorbitol group R is a glucoside, phenylglucosidone or gluconic acid residue. vou, mononitrophenylglucoside, dinitro1 sheet drawings Severografla, n. P., Plant 7, Most Price 2,40 Kčs