CH650800A5 - Process for the preparation of a substrate for the determination of alpha-amylase - Google Patents

Description

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PATENT CLAIMS Process for the preparation of a substrate for the determination of a-amylase of the formula I.

OH

OH

CH, Off

HO

OH

wherein R represents a (l -> - 4) -linked phenylglucopyranosyl, mononitrophenylglucopyranosyl or dinitrophenylglucopyranosyl group which has a 1-0 (a or β) position, characterized in that the respective phenylglucopyranoside or nitrated phenylglucopyranoside reacted with a-cyclodextrin, amylose or soluble starch in the presence of Bacillus macerans amylase.

The invention relates to a method for producing a substrate for the determination of a-amylase of the formula I specified in the claim.

Serum ct amylase levels are an important clinical parameter for pancreatic function. The commercially available reagents for determining the a-amylase are predominantly based on a system in which starch is broken down by the a-amylase and the fragments formed are determined in the visible or in the UV range, depending on whether colored starch or native starch is used as an amylase substrate in uses the test. A major disadvantage of these methods or reagents is that the starch can only be insufficiently characterized and standardized as a macromolecule, so that the turnover rate of the individual batches is very different and a standard must always be carried along with the measurements. For better results, a more uniform substrate would be required, which provides reliable cleavage results.

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

In addition to maltopentaose, maltotetraose and maltohexaose have already been proposed as substrates (US Pat. No. 3,879,263,4000,042). However, the results were significantly worse with tetraose than with pentaose, and even worse results with hexaose than with tetraose. For example, a stoichiometric reaction is still given for maltotetraose and pentaose, while tolerable deviations from the stoichiometric reaction have already been found for hexaose.

However, a disadvantage of maltopentaose, which was also found in tetraose, is that a considerable reagent blank occurs, i.e. the measurement reaction is already running before the sample to be determined is added. In addition, this reagent blank value is not constant at higher substrate concentrations, but changes for more than 25 minutes before this side reaction is constant. It also turned out that the assumed different cleavage of maltopentaose by pancreatic a-amylase and saliva a-amylase, which would have made it possible to differentiate, does not actually exist (J. BC, 1970,245,3917-3927; J. Biochem. 51, p. XVIII 1952).

The invention is therefore based on the object of providing a substrate which can be used in the determination of the a-amylase and which has a better purity and uniformity than the known substrates, is easily accessible and, in terms of blank value, without serum, duration of the lag phase and achievable maximum activity meets the requirements. Furthermore, a simple measurement without expensive and complicated equipment should be possible and suitable for rapid diagnostics such as test strips.

It was found that a compound of

I.

CH_OH

Oll

HO

can be used, wherein R represents a (l - »- 4) -linked phenylglucopyranosyl *, mononitrophenylglucopyranosyl or dinitrophenylglucopyranosyl group which has a 1-0 (a or β) position.

Surprisingly, it has been shown that the compounds of the formula I have superior properties as a substrate for α-amylase, although in the case of the oligomaltoses already proposed for this purpose, a significant drop in suitability from maltopentaose to maltohexaose has been found, since they result in significantly worse results than with which pentaose can be achieved. It was therefore to be expected that with a further extension of the maltose oligosaccharide chain, errors that would no longer be tolerable would occur. Surprisingly, however, better results are achieved with maltoheptaose than with pentaose. For example, the reagent blank value for 0.02 ml sample with maltopentaose as the substrate is 73%, with maltoheptaose only 13%, based on the final value of the determination in the normal range.

In addition, it was found that instead of the maltoheptaose itself, the maltoheptaose derivatives of the formula I can also be used as the substrate, which form a derivatized cleavage product when exposed to the a-amylase, which can be determined particularly advantageously.

The a-amylase can advantageously be determined by enzymatic cleavage of a compound of formula I as the a-amylase substrate and measurement of a cleavage product. A simple process for the preparation of maltoheptaose is described in DE-OS 2 741 191.

The maltose heptaose derivatives of the formula I are prepared according to the invention by the processes specified in the patent claim.

The enzymatic preparation of the phenyl derivatives of the formula I is carried out by transglucosidation of the phenylglucoside or the corresponding nitrated phenylglucoside with a-cyclodextrin, amylose or soluble starch in the presence of an amylase from Bacillus macerans. For this transglucosidation, the known amylase from Bacillus macerans (EC 2.4.1.19. DSM 24; isolation: JA de Pinto, LL Campbell, Biochemistry 7 (1968) 114; transfer reaction: Methods in Carbohydr. Chemistry, Vol. II, 347 ) use, which in addition to their hydrolytic and cyclizing effects apparently also has a glucosyl-transferring activity.

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As already mentioned above, the compounds of the formula I are used as substrates in the determination of a-amylase. This determination method is particularly suitable for the determination of the cleavage products using a-glucosidase or maltosephosphorylase.

When determining with a-glucosidase and a compound of formula I, the cleavage products of the maltohep

Taose derivatives of the formula I, maltotetraose and maltotriose, further split into glucose and the latter then measured in a manner known per se. For the measurement of the glucose formed, the hexokinase method is particularly preferred in the presence of the a-glucosidase. The principle of this process can be represented by the following equations:

Maltoheptaose + HbO "-A" 1-'la »e Maltotriose + Maltotetraose Maltotriose + 2 H? 0" - ° ll "- '0>" iuse ^ 3 Glucose

Maltotetraose + 3 H2P "-Cil" c0; '' djie ► 4 Gl ucose

7 glucose + 7ATP-tü ^^ 7 glucose-6-P

7 glucose-6-P + 7NAD + I * £ 2ö- ^ 7 gluconate-6-P + 7NADH + 7H +

The compounds of the formula I are particularly suitable for this process. When the two enzymes a-amylase and a-glucosidase act, the substituent, that is to say a phenyl group, mononitrophenyl group or dinitrophenyl group, is split off and can be easily determined. The phenyl groups can be in the a or β position. If the a position is involved, it is split off by the action of a-amylase and a-glucosidase alone, and the split, substituted or unsubstituted phenols can then be easily determined by known color reactions. This method can also be used for ß-positioned substituents. In the latter case, β-glucosidase is also used in addition to the a-glucosidase.

In the case of the dinitrophenyl radicals, the two nitro groups can be in any position, for example as 2,4, 2,6 or 3,5 substituents.

The nitrophenols or dinitrophenols released during the cleavage of the substituents containing nitro groups are themselves colored compounds which can be readily determined optically. If phenol itself is split off, this can be determined by known methods, for example by reaction with a nucleophilic agent such as 3-methyl-6-sulfonyl-benzothiazolone hydrazone- (2) (HSK) in the presence of monophenol oxidase. This reaction forms a red dye that can be measured.

PH values between 5 and 9 are generally suitable for carrying out the process mentioned. It is preferred to work at pH values between 7 and 7.5, since this gives the best results and the shortest reaction time. If nitrophenyl compounds are used, the range of the suitable pH values is somewhat narrower and is generally between pH 6 and pH 8.5.

Particularly suitable buffers are those which are active in the main activity range of the enzymes used. Phosphate, HEPES (N- (2-hydroxyethyl) piperazine-N-2-ethanesulfonic acid) and glycylglycine are preferred. Before-

20 concentrations are between 10 and 200 mmol / l.

The a-glucosidase is generally used in amounts between 0.1 and 5000 U / ml. It is a particular advantage of the process that relatively large amounts of this enzyme can be used, so that the a-amylase-25 cleavage is the rate-determining step. It is of course also possible to use even higher amounts of this enzyme, but this does not result in any further advantages.

The compounds of the formula I are generally used in the test in amounts between 0.1 and 250 mmol / l. Amounts between 0.5 and 100 mmol / l are preferred.

Substrate saturation of a-amylase with maltoheptaose is at a concentration of 8 to 10 mmol. A minimum concentration of 8 mmol 35 of maltoheptaose is therefore expediently used if work is to be carried out under conditions of substrate saturation, which is usually the case.

In addition, an activating agent for the α-amylase is expediently added. Such activation agents are known. Sodium chloride or potassium chloride is preferred.

The cleavage products can also be determined by reaction with maltose phosphorylase to form glucose-1-phosphate, which is then determined in a manner known per se. The glucose-45 1 -phosphate is preferably determined by conversion into glucose-6-phosphate by means of β-phosphoglucose mutase, oxidation of the glucose-6-phosphate formed with NAD in the presence of glucose-6-phosphate dehydrogenase to form gluconate -6-phosphate and NADH, the formation of the latter 50 being easy to follow photometrically. The measurement signal can be amplified by further oxidation with NAD in the presence of 6-phosphogluconate dehydrogenase to form ribulose-5-phosphate and another molecule of NADH.

55 The principle of this embodiment is represented by the following equations:

Maltoheptaose + H; 0 "-Amvljse ►Maltotriose + Maltotetraose -" - Maltose Maltose + POr ► Glucose + ß-Glucose-1 -P

ß-Glucose-l-P iipoluM ► Glucose-6-P

Glucose-6-P + N AD + «£!>" _ ► Gluconate-6-P + NADH + H + Gluconate-6-P + NAD + 6t'GDH ► Ribulose-5-P + NADH + H +.

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An advantage of this method is that maltose phosphorylase is more specific than a-glucosidase and therefore does not interfere with endogenous glucose.

With regard to the buffers, the same applies to the a-glucosidase process.

In addition to the two methods listed above, the maltoheptaose fragments formed, ie maltotetraose, maltotriose and maltose formed therefrom, can also be determined by other known methods.

A reagent for the determination of the a-amylase contains an a-amylase substrate and a system for the determination of a cleavage product formed from the a-amylase substrate by the a-amylase, the substrate being a compound of the formula I.

A preferred system for the determination of the cleavage products is the a-glucosidase system, which contains a-glucosidase, alkali chloride and buffer. If the substrate is maltoheptaose itself, hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) as well as NAD, ATP and magnesium are required as enzymes.

A reagent based on the a-glucosidase system preferably consists of

5 x 103 to 3 x 104 U / 1 a-glucosidase,

103 to 5 x 104 U / 1 HK,

103 to 5 x 104 U / 1 G6PDH,

0.5 to 8 mmol / 1 NAD,

0.5 to 5 mmol / 1 ATP,

1 to 3 mmol / 1 Mg2 +,

25 to 100 mmol / 1 NaC! or KCl,

25 to 200 mmol / 1 buffer, pH 6.2 to 7.8 and

5 to 100 mmol / 1 maltoheptaose or a multiple or fraction thereof, in dry or dissolved form.

Another reagent contains α-glucosidase, KCl or NaCl, buffer and substrate. Such a reagent contains in particular 102 to 5 × 106 U / 1 a-glucosidase, 1 to 100 mmol / 1 NaCl or KCl, 10 to 250 mmol / 1 buffer, pH 5 to 9 and 0.1 to 250 mmol / 1 maltoheptaose derivative of the formula I, based on the concentration in the test. The reagent can be in dry, in particular lyophilized form, or in the form of a solution, as a mixture of all components or separately.

A reagent of the above type preferably additionally contains β-glucosidase and / or phenol oxidase and 3-methyl-6-sulfonyl-benzothiazolone hydrazone- (2) (HSK).

Another reagent contains a-glucosidase, NaCl or KCl, buffer and substrate, sorbitol dehydrogenase or gluconate kinase and 6-phosphogluconic acid dehydrogenase as well as NAD and optionally a tetrazolium salt and diaphorase or phenazine methosulfate (PMS).

A preferred reagent of this type contains 1 x 102 to 3 x 106 U / 1 a-glucosidase, 2 x 103 to 5 x 10 "U / 1 sorbitol dehydrogenase, 1 x 103 to 5 x 104 U / 1 hexokinase, 0.5 to 50 mmol / 1 ATP, 10 to 500 U / 1 diaphorase (Chlostridium Kluy-veri), 0.01 to 0.5 mmol / 1 tetrazolium salt, 0.1 to 10 mmol / 1 NAD, 0.2 to 5 mmol / 1 MgCh , 0.5 to 20 mmol / 1 Maltohep-tit, 1 to 100 mmol / 1 NaCl or KCl and 10 to 250 mmol / 1 buffer, pH 5.5 to 8.5.

If necessary, a nonionic surfactant is still present in an amount between 5 and 50 mmol / l.

Another preferred reagent contains 100 to 3 x 106 U / 1 a-glucosidase, 10 to 104 U / 1 6-phosphogluconate dehydrogenase, 20 to 2 x 104 U / 1 gluconate kinase, 0.5 to 25 mmol / 1 ATP , 0.05 to 1.0 mmol / 1 NADP, 1.0 to 20 mmol / 1 maltoheptagluconic acid, 0.5 to 5 mmol / 1 MgCh, 1 to 100 mmol / 1 NaCl and 10 to 50 mmol / 1 buffer, pH 5.5 to 8.5.

Another reagent contains 0.1 to 250 mmol / 1 a-nitro-

phenyl-maltoheptaoside or dinitrophenyl-maltoheptaoside, 1 x 102 to 2.5 x 106 U / 1 a-glucosidase, 1 to 100 mmol / 1 sodium chloride or potassium chloride and 10 to 250 mmol / 1 phosphate buffer, pH 7.0 to 8.0.

Another reagent contains:

0.1 to 250 mmol / 1 a-phenylmaltoheptaoside,

1 x 102 to 1.5 x 106 U / 1 a-glucosidase,

10 to 105 U / 1 monophenol oxidase,

0.1 to 10 mmol / 1 HSK,

1 to 100 mmol / 1 NaCl or. KCl and 10 to 250 mmol / 1 buffer.

Another preferred system for determining the cleavage products is the maltose phosphorylase system, which essentially consists of maltose phosphorylase, ß-phosphogluco-mutase (ßPGluM), glucose-6-phosphate dehydrogenase (G6PDH), glucose-1,6-diphosphate (G1,6DP) , NAD, buffer, maltoheptaose and optionally 6-phosphogluconate dehydrogenase (6PGDH).

A particularly preferred reagent with this detection system consists of

0.5 x 103 to 20 x 103 U / 1 maltose phosphorylase,

lxlO2 to 1 x 104ß-PGluM,

2 x 102 to 3 x 104 U / 1 giucose-6-phosphate dehydrogenase, 0.5 to 10mM / l NAD,

0.001 to 1 mM / 1 glucose-1,6-diphosphate,

0.5 to 100 mM / 1 buffer, pH 6.0 to 7.5,

5 to 50 mM / 1 maltoheptaose and optionally

1 x IO2 to 1 x 104 U / 1 6-phosphogluconate dehydrogenase.

The reagent can be in dry or dissolved form, it can also be on a sheet support, e.g. a film, an absorbent paper or the like, impregnated. In the latter case, it expediently consists of at least three layers or layers, a first layer containing the substrate, the second layer serving as a barrier layer and the third layer containing the system for determining the fission products. If such a multilayered reagent material, which is suitable for a simple rapid test for a-amylase, is brought into contact with a liquid sample containing a-amy-laser, the a-amylase cleaves the substrate and the fragments diffuse through the intermediate layer into the third layer containing the determination system. In this case, a color-forming reaction is expediently used as the detection reaction in order to make the concentration of the a-amylase visually visible on the basis of the discoloration that occurs, if the cleavage products are not themselves already colored.

As already mentioned, the use of the compounds of the formula I as substrate for the determination of a-amylase not only enables a rapid and specific method for determining the a-amylase, but in particular the lag phase is completely or largely eliminated, which by is of particular importance when applying the method to automatic analyzers. In addition, the determination of the a-amylase can be carried out in many embodiments without complicated apparatus for the evaluation and is therefore particularly suitable for rapid diagnosis and for optical determination in the visible range. At the same time, however, the various embodiments of the determination method can also be carried out using UV measuring devices. Further advantages are strict proportionality and no interference from chemically related blood components.

According to the invention, the substrates of formula I are readily accessible and in high purity.

The determination of a-amylase can be carried out in biological liquids, e.g. Serum, heparin plasma, urine and the like, or in other liquid or solid materials.

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The following example illustrates the invention.

Example p-nitrophenyl-a-maltooligosaccharide by enzymatic synthesis with Bacillus macerans amylase (E.C.2.4.1.19 from Bac. Mac. DSM 24).

In addition to hydrolytic and cyclizing effects, Bacillus macerans amylase also has glycosyl-transferring properties which can be used for the synthesis of oligosaccharides and derivatives (Methods in Carbohydrate Chemistry II, 347). For the synthesis of p-nitrophenyl oligosaccharides, this process was optimized as follows:

680 mg Bacillus macerans-amylase (EC2.4.1.19 from Bac. Mac. DSM 24) (lyophilisate) (0.46 U / mg weight, protein content of the weight 28.5%, free of p-nitrophenyl-aD-glu-coside -cleaving activities), 400 mg «-Dp-nitrophenylglucoside,

3.5 g of a-cyclodextrin, and

70 ml Soerensen phosphate buffer, pH 6.2; 0.01 M are mixed. The mixture is incubated at 37 ° C for 24 hours. For purification, a- and β-cyciodextrin formed are first separated off via the tetrachlorethylene inclusion compound. After chromatography on cross-linked dextran (Sephadex LH20), 50 mg of lyophilisate are obtained from p-nitrophenyl maltoheptaoside, which is highly active in the amylase test.

Use of the maltohep-taose derivatives produced according to the invention

I. Detection of a-amylase with phenyl-a-maltoheptaoside as substrate

Phenyl-a-maltoheptaoside is cleaved by the a-amylase into phenyl-a-maltotriid or tetraid, which are converted to phenol and glucose by a-glucosidase. The released phenol is oxidatively coupled by monophenol oxidase with the nucleophilic reagent 3-methyl-6-sulfonyI-benzthiazo-ion-hydrazone- (2) (HSK) to a red dye, the rate of which is proportional to the amylase activity in the sample and photometrically can be tracked.

Test principle:

la) phenyl-a-maltoheptaoside + H2O "Am'lase ► phenyl-aD-tri- (tetra) -glucopyranoside + maltotetra- (tri) -ose b) phenyl-aD-tri- (tetra) -gIucopyranoside + 3 (4th ) HiO «chiucosidase phénol + 3 (4) glucose c) phenol + HSK + O? ► dye + 2H2O

Measurement conditions:

25 ° C, measuring wavelength 492 nm, 1 cm cuvettes; the reagent composition is shown in the following table.

table

Reagent concentration in the test

Phosphate buffer, pH 7.4 0.05 mol / 1

Sodium chloride 0.05 mol / l

Phenyl-a-D-maltoheptaoside 10 mmol / 1

HSK I mmol / 1

Monophenol oxidase 1 U / ml a-glucosidase 10 U / ml

Test volume: 2.0 ml

Instead of phosphate buffers, glycine, glycylglycine, Hepes, Tris, Tra and other buffers also proved to be useful.

2. Determination of a-amylase with p-nitrophenyl malto-heptaoside.

Test principle:

p-Nitrophenyl-a-maltoheptaoside "-Am-'lase ►

(Glucose) 0 + p-nitrophenyl-a- (glucose) m f'- ° llu: oslda> e ► n glucose + p-nitrophenol.

After cleavage of the maltoheptaoside, the cleavage products are broken down into glucose and p-nitrophenol. The p-nitrophenolate anion is colored yellow in alkaline solution and can therefore be measured directly optically.

Test system:

Reaction mixture:

50 mM / 1 phosphate buffer, pH 7.4

50 mM / 1 sodium chloride

5 to 50 U / ml a-glucosidase

5 or 10 mM / 1 p-nitrophenyl-a-maltoheptaoside

Test approach:

1 ml reaction mixture + 50 ^ 1 sample (serum)

Temperature: 30 ° C wavelength: 405 nm

Serum start after reaching the measurement temperature (10 minutes preincubation).

3. Determination of a-amylase with maltoheptite

Test principle:

Maltoheptait + H2O "• Amyhlse ► Maltotriit + Maltotetraose Maltotriit + H2O Sorbit + Maltose

Sorbitol + NAD + -512ÜK fructose + NADH + H + NADH-t-INT + H + -! I! ÜE! HHiL ^ NAD + SDH = sorbitol dehydrogenase

Test system:

Reaction batch ml

Concentration in the test

Na / K-PO4 buffer 0.02 mol / 1

1.92

12.8 mmol / 1 P04

+ Triton XI00 2 ml / 100 ml

+ NaCl 10 mmol / 1, pH 6.9

6.4

mmol / 1

MgCh 0.3 mol / 1

0.01

0.01

1.0 mmol / 1

Maltoheptite 100 mmol / 1

0.10

3.3 mmol / 1

NAD c = 40 mg / ml

0.05

1.0 mmol / 1

INT c = 0.6 mg / ml

0.20

0.09 mmol / 1

Diaphorase (Clostr. Kluyveri)

0.05

167 rev / 1

5 mg / ml +

ATP c = 50 mg / ml

0.10

3.29 mmol / 1

Hexokinase 280 U / ml +

0.05

4666 U / 1

SDH 180 U / ml *

0.30

18000 U / 1

a-glucosidase 1120 U / ml *

0.20

74670 U / 1

Sample (serum)

0.02

in test buffer

Start by adding a sample: measurement takes place at 92 nm; Temperature: 25 ° C

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