DE2741192C2 - Method for the determination of alpha-amylase - Google Patents

Description

The invention relates to a method and a reagent for determining human pancreatic amylase.

TDTeBestnhmühgTIesa arnylase level in the serum an important clinical parameter for pancreatic function. The commercially available reagents for the determination of -amylase are predominantly based on a system in which starch is produced by -amyias degraded and the fragments formed are determined in the visible or in the UV range, depending on whether one colored starch or negative starch as Amylase substrate is used in the test An essential one The disadvantage of these processes and reagents is based on the fact that the starch as a macromolecule cannot be adequately characterized and standardized, see above that the turnover rate of the individual batches is very high

w turns out differently and always in the measurements a standard must be carried. For better results a more uniform substrate would be required, which would give reliable results on cleavage supplies

J5 One step forward towards a more uniform substrate was the use of maltopentaose. This becomes maltotriose and from the amylase Split maltose, maltotriose and maltose are converted into glucose by the a-glucosidase, which then can be determined by any method, for example with the known hexokinase method can.

In addition to maltopentaose, maltotetraose and maltohexaose have also been used as substrates

«Proposed (US-PS 38 79 263, 40 00 042). Included However, the results with the tetraose were significantly worse than with the pentaose, and with the hexaose even worse results than obtained with tetraose stoichiometric reaction indicated, while for the hexaose already tolerable deviations from the stoichiometric reaction were found became.

A disadvantage of the maltopentaose, which also applies to the

Tetraosis has been found, however, is that significant creep occurs; H. the measurement reaction is already running, before the sample to be determined is added. In addition, this creeping reaction at higher Substrate concentration is not constant, but rather Changed for more than 25 minutes before constancy this side reaction is achieved. Besides, they are Reagent blank values undesirably high. It also turned out that the assumed different splitting of the maltopentaose by the pancreas · «· amylase and Saliva-a-amylase. the one distinction actually not given (]. BC, 1970,245,3917 to 3927; J. Biochem. 51, p. XVIII1952). The invention is therefore based on the object

To provide a method and a reagent for matching the human Pankrcas-cj-amylase in which a Substrate is used, 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.

This object is achieved according to the invention by a method z'if determination of human pancreatic a- amylase by enzymatic cleavage of an e-Amylasesubstrats and measurement of a cleavage product, which is characterized in that is used as a substrate maltoheptaose in high purity.

Surprisingly, it has been shown that maltoheptaose has superior properties as a substrate which possesses α-amylase, although with for this purpose already proposed oligomaltoses from maltopentaose to maltohexaose a substantial drop in the Suitability was determined, as it achieved significantly worse results than with the Pentaose will. It was therefore to be expected that with a further lengthening of the maliose oligosaecharidkette no longer tolerable errors occur surprisingly, better results are achieved than with the Pentaose. So is the Reagent blank for 0.02 ml sample with maltopentaose as substrate 73%, with maltoheptaose d & against only 13%, based on the final value of the determination.

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

In the determination with α-glucosidase the cleavage products of maltoheptaose, maltotetraose and Maltotriose, further split into glucose and the latter then measured in a manner known per se. For the The hexokinase method is particularly preferred for measuring the glucose formed in the presence of α-glucosidase. The principle of this embodiment of the method according to the invention can be carried out give the following equations:

Maltoheptaose + H ₂ O maltotriose + maltoletraose maltotriose + 2 H ₂ O ₃ ^^^

Maltotetraose + 3 H ₂ O 4 glucose

7 glucose + 7 ATP

HK

7 glucose-6-P

7 glucose-6-P + 7 NAD ^{+ t} J-> 7 gluconate-6-P + 7 NADH + 7 H *

For the embodiment described above, those buffers can be used which are in the main activity range of the enzymes used, i.e. H. between pH 6.2 and 7.8 are effective. Phosphate buffer, pH 6.8 to 7.2, especially in the is particularly favorable Concentration of 50 mM as sodium phosphate buffer. Glycylglycine buffers were also very suitable, in particular in a concentration of 150 mM or Hepes buffer in a concentration of IOC mM.

In a further preferred embodiment of the method of the invention, the determination takes place Cleavage products by reaction with maltose phosphorylase to form glucose-1-phosphate, which is then determined in a manner known per se. According to In a particularly preferred embodiment, the glucose-1-phosphate is determined by converting it into glucose-6-phosphate by means of ^ -phosphoglucose mutase, oxidation of the glucose-6-phosphate formed with NAD in the presence of glucose-6-phosphate dehydrogenase Formation of gluconate-6-phosphate and NADH _1, whereby the formation of the latter can easily be followed photometrii-: h. The measurement signal can be further increased by further oxidation with NAD in the presence of e-phosphogluconate dehydrogenase with the formation of ribulose-5-phosphate and another molecule of NADH.

The following equations show the principle of this embodiment:

Maltohcntaose + H ₂ O ^ -> maltotriose + maltotetraose maltose phosphorylase

Maltose

j-

Maltose + PO4

A-PGIuM ",, _D

jJ-glucose-IP ~ »G! ucose-6-P

G 6 PDH

Glucose + ^ -Glucose-1-P Glucose-6-P + NAD * Gluconate-6-P + NADH- ¹ -H ⁺ Gluconate-6-P + NAD ⁺ ribulose-5-P + NADH + H ⁺

An advantage of this embodiment of the invention is that maltose phosphorylase is more specific as α-glucosidase and therefore does not interfere with endogenous glucose

With regard to the buffers, the same applies to the embodiment of the method according to the invention with the α-Glucosidase procedure carried out in the same way.

In addition to the two embodiments of the method according to the invention listed above, the Determination of the formed fragments of the maltohep taose, so Mai.otetraose, maltotriose and from it The maltose formed can also be carried out by other methods known for this purpose.

Substrate saturation of α-amylase with maltoheptaose is at a concentration of 8 to 10 mM. Appropriately, therefore, within the framework of the Method according to the invention a minimum concentration of 8 mM of maltoheptaose is used, if under Conditions of substrate saturation should be worked, which is usually the case.

Another object of the invention is a reagent for the determination of the human pancreas ff amylase containing an oligomaltodextrin as a-amylase substrate and a system for determining one formed from the ff-amylase substrate by the α-amylase Fission product, which is characterized in that the substrate consists of Maitoheptaose.

Preferred systems for the determination of the cleavage products are the oc-glucosidase system, bei which as enzymes still hexokinase (HK) and glucose-ö-phosphate dehydrogenase (G6PDH) as well NAD, ATP, magnesium, alkali chloride and buffers are needed.

Particularly preferably, a reagent based on the -glucosidase system consists of r>

5 10J to 3 χ 10 "U / l-glucosidase.

Wbis5xl0 <HK,

If »his S χ 10 <Ci6PDH.

0.5 to 8mM / lNAD,

0.5 to 5 mM / l ATP,> »

Ibis 3 mM / L Mg ²⁺

25 to 100 mM / l NaCl or KCl.

25 to 100 mM / l buffer, pH 6.2 to 7.8 and

8 to 100 mM / l maltoseheptaose

or a multiple or fraction thereof, in 2 ~> dry or dissolved form.

Another preferred system for determining the breakdown products is the maltose phosphorylase system, which essentially consists of maltose phosphorylase, / 3-phosphoglucomutase (0PMG), glucose-6-phosphate jo dehydrogenase (G6PDH), glucose-1,6-diphosphate (GI, 6DP), NAD, buffer, maitoheptaose and optionally 6-phosphogluconate dehydrogenase (6PGDH).

A particularly preferred reagent with this detection system consists of 0.5 χ ICP to 20 χ 10 ³ U / l maltose phosphorylase,

1 xl0 ² bisl XlO ⁴ P-PGIuM,

2 χ 10 ³ to 3 χ WU / IGlucose-ö-phosphate dehydrogenase,

0.5 to 10 mM / l NAD, 0.001 to 1 mM / l glucose-1,6-diphosphate,

0.5 to 50 mM / l buffer, pH 6.0 to 7.5,

5 to 50 mM / L maitoheptaose

and optionally 1 χ Ιΰ ² to 1 χ ΙΟ ⁴ U / l 6-phosphogluconate dehydrogenase.

The reagent according to the invention can be in dry or dissolved form, it can also be on a sheet-shaped carrier, e.g. B. a film, an absorbent paper or the like, impregnated. in the in the latter case it is expedient at least three layers, a first layer containing the maitoheptaose, the second layer as a barrier layer and the third layer contains the system for determining the fission products multi-layer reagent material of this type, which is suitable for a simple rapid test for α-amyiasis suitable, with a liquid, -amylase-containing sample in When brought into contact, the α-amylase splits the substrate and the fragments diffuse through it Intermediate layer in the third layer containing the determination system In this case, a color-forming reaction is used to determine the coloration that occurs Make the concentration of α-amylase visually visible.

The method and reagent according to the invention enable a rapid and reliable determination the Λ-amylase. The procedure is characterized by short lag phase, lower reagent underflow than with previously known substrates, good sensitivity with regard to the measurement signal, so that small amounts of sample can be used and the possibility of use different detection systems for the fragments of the substrate with equally good results. That The substrate used according to the invention is readily available and in high purity. A method to his Manufacture is in J. Am. Chem. Soc. 71, 356 (1949). The method is suitable for determination of human pancreatic e-amylase in fluids, such as Serum, heparin plasma, urine or in solid materials. The following examples illustrate the invention.

Example I.
Maltogenic method («-glucosidase system)

A reagent mixture containing α-glucosidase, G6PDH, HK, NAD, ATP, maitoheptaose, Mg ²⁺ NaCl 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. 6 hours.

The solution obtained has the following concentration of the reagents:

Phosphate buffer 50mmol / L; pH7.0 NaCl 50 m mol / l Mg ²⁺ 2 mmol / l Maitoheptaose lOmmol / 1 ATP 1.2 mmol / l NAD 2 mmol / l HK > 2 U / l G6PDH > 2U / 1 Λ-glucosidase > 10 U / l

0.02 ml of serum sample is added to the solution at 25 ° C. in a cuvette with a path length of 1 cm measured and then the absorbance at Hg 365 nm, 340 nm or Hg 334 nm determined in a photometer. After 10 minutes the absorbance is read and then five times that at one minute intervals Reading repeated

The mean value is calculated from the absorbance differences per minute (ΔΕ / mm) determined in this way, the reagent release is subtracted and the corrected value is used in the calculation

The calculation is made as follows:

U / l 25 ° C - 4244χζ1 £ 365ηΓη / ηιίη
- 2290xd £ 340nm / min
»2335xzl £ 334nm / min

F i g. 1 of the drawing shows the results of a dilution series of human serum with physiological Saline solution obtained by this method.

If the reagent is divided into two batches, one of which contains the maitoheptaose and the other the mixture of all other reagents, the shelf life of the solutions produced with it can be increased. For the mixing of the reagents at temperatures up to 8 ^° C. it is 30 hours, and at room temperature it is about 8 hours. The shelf life of the maltoheptaose solution is 6 weeks or approx. 1 week.

Betspiel 2 Determination with the maltose phosphorylase system

Two reagents were prepared consisting of the amylase substrate and the maltose phosphorylase-s; One reagent contained maltoheptaose substrate, the other, according to the state of the art, soluble starch. The reagent concentration after dissolving in water was as follows:

invention

comparison

Phosphate buffer 20 mM NAD 2 mM Maltohcptaosc in mM soluble &quot; strong Glucose - !. 6- diphosphate traces Maltose Phosphory I- 3 U / ml ase (microorg.)

pH 6.5

2OmM; pH 6.5 2 mM

5 mg ml

traces

3 U ml

Invention comparison

I U'ml

1 L ^: ml

β-phosphoglucomitase

(Micro org.)

G6PDH (Leticonostoc meseni) 9 U ml 9 U ml 6PGDH (Leuconostoc mesent) IU ml 1 L ^- ml The solution obtained was incubated at 30 (, mixed with the sample solution and the hyperlinklion difference at Hg 334 nni was determined in a photometer The exclusion differences are measured over 10 minutes before incubation. For 2.0 ml reagent and 0.10 ml sample, the following calculation formula results:

. 2.1 χ 1000

M. min χ = at min χ 1699 V I

6.IX χ 0.1 χ 0.2

At tinsat? of five different lliimansera were based on the above considerations, the following values were found:

invention

Sliirke

37.4 L I

61.2 U I

95.1 U 1

114 Ll

374 L I

15th 3 l: 3 L. 39 I. U 44.2 L ¹ ΛI L.

For this purpose I sheet drawings

Claims (10)

Patent claims: 1. Method for the determination of human pancreas ff amylase by enzymatic cleavage of an oligomaltodextrin as a-amylase substrate and measurement of a cleavage product, characterized in that the substrate is maltoheptaose in high purity is used. 2. The method according to claim I _1, characterized in that the cleavage products are converted into glucose by α-glucosidase and determined in a manner known per se. 3. The method according to claim 1, characterized in that the cleavage products are converted by maltose phosphorylase to form Glucose-1-phosphate and the latter is determined. 4. The method according to claim 3, characterized in that <Hß to determine the formed Glucose-1-phosphate this is converted into glucose-6-phosphate with phosphoglucomutase and with the latter NAD in the presence of glucose-ö ^ phosphate dehydrogerase is reduced to NADH, which is measured. 5. Reagent for the determination of human pancreatic α-amylase, containing sin oligomaltodextrin ff-amylase substrate and a system for determination a cleavage product formed from the amylase substrate by the ff-amylase, characterized in that the substrate is made from maltoheptaose consists. 6. Reagent according to Auspruo. 5, characterized in that the system. to determine a cleavage product formed from the amylase substrate by <-AmyIase consists essentially of α-glucosidase, hexokinase, glucose-6-phosphate dehydrogenase, NAD, ATP, Mg ⁷⁺ , NaCl and phosphate buffer. 7. Reagent according to claim 6, characterized in that it consists of 5 χ 10 ³ to 3 χ 10 ⁴ U / liter «-glucosidase, 10 ¹ to 5 χ 1 0 * U / liter hexokinase, 10 ^{3 to} 5x 10 ⁴ U / liter of glucose-6-phosphate dehydrogenase, 0.5 to 8 mmol / liter NAD, 0.5 to 5 mmol / liter ATP, 1 to 3 mmol / liter Mg ²⁺ , 25 to 100 mmol / liter NaCI or KCI, 25 to 100 mmol / liter buffer, pH 6.2 to 7.8 and 8 to 100 mmol / liter of maltoheptaose or a multiple or fraction thereof in dry or dissolved form 8. Reagent according to claim 5, characterized in that that system for determining one from the amylase substrate formed by the "-amylase cleavage product essentially from maltose phosphorylase, p-phospho-glucomutase, glucose-6-phosphate dehydrogenase, glucose-1,6-diphosphate, NAD, buffer and optionally 6-phosphogluconate dehydrogenase. 9. Reagent according to claim 8, characterized in that it consists of 0.5 χ 10 'to 2 χ lOHJ / l.itcrmaltose- phosphorylase, I χ IO ² bisl χ K ^ U / liter ^ -phospho- gluco-mutase, 2x10 ^{J to} 3x l0 ⁴ U / liter glucose-6- phosphate dehydrogenase, 0.5 to 10 mmol / liter NAD, 0.001 to 1 mmol / liter glucose-1 ₁ 6-diphosphate, 0.5 to 50 mmol / liter buffer, pH 6.0 to 7.5 5 to 50 mmol / liter maltoheptaose and possibly 1 χ IO ² to 1x10 * U / Iiter δ-phospho-gluconate dehydrogenase 10. Reagent according to one of claims 5 to 9, characterized in that it is impregnated or incorporated into a Bbo-shaped carrier material is present