Claims
- 1. Process for the preparation of a meltoheptaose derivative of formula ##STR15## wherein R is a phenylglucoside, mononitrophenylglucoside or dinitrophenylglucoside group, comprising reacting a phenylglucoside, mononitrophenylglucoside or dinitrophenylglucoside with .alpha.-cyclodextrin, amylose or soluble starch in the presence of Bacillus macerans amylase which is free of p-nitrophenyl-alphaglucoside splitting activities, under conditions favoring formation of said maltoheptaose compound, and recovering said product therefrom.
Priority Claims (2)
| Number |
Date |
Country |
Kind |
| 2741192 |
Sep 1977 |
DEX |
|
| 2755803 |
Dec 1977 |
DEX |
|
Parent Case Info
This application is a continuation of application Ser. No. 762,579 filed Sep. 5, 1985, now abandoned, which was a divisional application of Ser. No. 311,856, filed Oct. 16, 1981, now U.S. Pat. No. 4,544,631, which was a continuation application of Ser. No. 941,834 filed Sep. 11, 1978, now abandoned.
The present invention is concerned with a process and a reagent for the determination of .alpha.-amylase.
The determination of .alpha.-amylase in serum is an important clinical parameter for the function of the pancreas. The commercially available reagents for the determination of .alpha.-amylase are preponderantly based upon a system in which starch is broken down by .alpha.-amylase and the fragments formed are determined in the visible range of light or in the UV range, depending upon whether color starch or native starch is used in the test as the substrate for the amylase. An important disadvantage of these processes and reagents is that starch, which is a macromolecule, cannot be sufficiently standardised and characterised so that the rate of reaction of individual batches can vary very greatly and, when carrying out measurements, a standard must always be included. For better results, a more uniform substrate would be necessary which provides dependable results in the case of fission.
A step forwards in the direction of a more uniform substrate occurred with the use of maltopentaose. This is split by .alpha.-amylase into maltotriose and maltose and maltotriose and maltose are converted by .alpha.-glucosidase into glucose which can then be determined by any desired method, for example by the known hexokinase method.
Besides maltopentaose, it has also already been proposed to use maltotetraose and maltohexaose as substrate (see U.S. Patent Specifications Nos. 3,879,263 and 4,000,042). However, in this case, the results obtained with the tetraose were markedly poorer than those obtained with the pentaose and with hexaose even worse results were obtained than with the tetraose. Thus, in the case of maltotetraose and -pentaose, it is still possible to obtain a stoichiometric reaction whereas in the case of the hexaose, just still tolerable deviations from the stoichiometric reaction were ascertained.
A disadvantage of maltopentaose, which was also found in the case of the tetraose, is, however, that a considerable reagent blank occurs, i.e. the measurement reaction already starts before the sample to be determined is added. Furthermore, this reagent blank is not constant in the case of comparatively high substrate concentrations but rather changes for more than 25 minutes before a constancy of this side reaction is achieved.
It has also been ascertained that the assumed different fission of maltopentaose by pancreas .alpha.-amylase and saliva .alpha.-amylase, which would have enabled a differentiation, does not actually exist (see J. BC., 1970, 245, 3917-3927; J. Biochem., 51, p. XVIII, 1952).
The present invention provides a process and a reagent for the determination of .alpha.-amylase in which a substrate is used which has a higher degree of purity and uniformity than the known substrates, is readily obtainable and satisfies the requirements with regard to the blank value without serum, the length of the lag phase and the achievable maximum activity. Furthermore, a simple measurement without expensive and complicated apparatus is to be possible and a suitability for rapid diagnostics, such as test strips, is to be provided.
The process of the invention for the determination of .alpha.-amylase by the enzymatic fission of an .alpha.-amylase substrate and measurement of a fission product essentially comprises using, as substrate, a compound of the general formula: ##STR2## in which R is a glucoside, phenylglucoside, mononitrophenylglucoside, dinitrophenylglucoside, sorbitol or gluconic acid group.
We have, surprisingly, found that maltoheptaose possesses superior properties as a substrate for .alpha.-amylase, although in the case of the oligomaltoses from maltopentaose to maltohexaose, which have already been suggested for this purpose, a decrease in suitability has been ascertained since the maltohexaose gives results which are substantially poorer than those achieved with the pentaose. Therefore, it was to have been expected that with a further lengthening of the maltose oligosaccharide chain, no longer tolerable errors would occur. Surprisingly, however, better results are achieved than with the pentaose. Thus, for example, the reagent blank value in the case of 0.02 ml. of sample with maltopentaose as substrate amounts to 73% but with maltoheptaose amounts to only 13%, referred to the end-point of the determination.
Furthermore, we have found that instead of maltoheptaose itself, certain maltoheptaose derivatives can also be employed which, by the action of .alpha.-amylase, form a derivatised fission product which can be determined especially advantageously.
The process according to the present invention is especially suitable for the determination of the fission products by means of .alpha.-glucosidase or maltose phosphorylase.
In the case of the determination with .alpha.-glucosidase and a compound of general formula (I) in which R is a glucoside group, the fission products of the maltoheptaose, i.e. maltotetraose and maltotriose, are further split to give glucose, which can then be measured in known manner. For the measurement of the glucose formed in the presence of .alpha.-glucosidase, the hexokinase process is particularly preferred. The principle of this embodiment of the process according to the present invention can be illustrated by the following equations: ##STR3##
In the above equations, HK means hexokinase, NAD means nicotinamide-adenine-dinucleotide, NADH means the reduced form thereof, G-6-PDH means glucose-6-phosphate dehydrogenase and ATP means adenosine triphosphate.
For this embodiment of the present invention, i.e. the use of .alpha.-glucosidase there are also especially useful the maltoheptaose derivatives employed according to the present invention, i.e. compounds of general formula (I) in which R is other than a glucoside group. By the action of the two enzymes .alpha.-amylase and .alpha.-glucosidase, the substituent of R, i.e. a phenolgroup, a mononitrophenyl group or a dinitrophenyl group or the terminal sorbitol or gluconic acid residue, is split off and can easily be determined. The phenyl groups can be in the .alpha.- or .beta.position. In the case of the .alpha.-position, splitting off thereof takes place by the action of .alpha.-amylase and .alpha.-glucosidase alone and the substituted or unsubstituted phenols split off can then be easily determined by known color reactions. However, the present invention can also be employed in the case of .beta.positioned substituents, in which case, in addition to .alpha.-glucosidase, .beta.-glucosidase is also employed.
In the case of the dinitrophenyl groups, the two nitro groups can be present in any desired position, for example as 2,4-, 2,6- or 3,5-substituents.
The nitrophenols or dinitrophenols liberated by the splitting off of the nitro group-containing substituents are themselves colored compounds which can easily be determined optically. If phenol itself is split off, then this can be determined by known methods, for example by reaction with a nucleophilic reagent, such as 3-methyl-6-sulphonylbenzthiazolone-hydrazone(2) (HSK), in the presence of monophenol oxidase, a red colored material being formed which can be measured.
In the case of splitting off sorbitol, this can be oxidized, for example, by sorbitol dehydrogenase to give fructose and, at the same time, NAD present is reduced to NADH. The formation of NADH can then easily be determined in known manner with a UV spectrophotometer. If this is not available, then, by reaction with a tetrazolium salt, for example 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride (INT), in the presence of diaphorase or some other electron carrier, a colored formazan is formed which can be measured in the visible spectrum.
In an analogous manner, liberated gluconic acid can be determined by known methods, for example with gluconate kinase, 6-phosphoglucate dehydrogenase and nicotinamide-adenine-dinucleotide phosphate (NADP), as well as optionally a tetrazolium salt and an electron carrier.
The process according to the present invention is generally carried out at a pH value of from 5 to 9. However, it is preferable to use a pH value of from 7 to 7.5 since, in this case, the best results are obtained in the shortest reaction times. If, according to the present invention, nitrophenyl compounds are employed, then the range of the most suitable pH values is somewhat narrower and is generally from 6 to 8.5.
The buffers used can be any of those which are effective in the main activity range of the enzymes employed. Preferred buffers include phosphate buffers, HEPES (N-(2-hydroxyethyl)-piperazine-N-2-ethanesulphonic acid) and glycylglycine. The preferred buffer concentrations are from 10 to 200 mMol/litre.
The .alpha.-glucosidase is generally employed in an amount of from 0.1 to 5000 U/ml. It is a special advantage of the process according to the present invention that relatively large amounts of this enzyme can be used so that the .alpha.-amylase fission is the ratedetermining step. It is, of course, also possible to employ even larger amounts of this enzyme but this provides no further advantages.
The compounds of general formula (I) employed according to the present invention are generally used in amounts of from 0.1 to 250 mMol/litre, amounts of from 0.5 to 100 mMol/litre being preferred.
Substrate saturation of the .alpha.-amylase with maltoheptaose is present at a concentration of 8 to 10 mMol. Therefore, it is preferable to use a minimum concentration of 8 mMol of maltoheptaose if it is desired to work under the conditions of substrate saturation, which is usually the case.
Furthermore, an activation agent for the .alpha.-amylase is preferably added. Such activation agents are known, the preferred ones including sodium chloride and potassium chloride.
In the case of another preferred embodiment of the process according to the present invention, the determination of the fission products takes place by reaction with maltose phosphorylase, with the formation of glucose-1-phosphate, which is then determined in known manner. According to a specially preferred embodiment, the glucose-1-phosphate is determined by conversion into glucose-6-phosphate by means of .beta.-phosphoglucose mutase (.beta.-PGluM) and oxidation of the glucose-6-phosphate formed with NAD in the presence of glucose-6-phosphate dehydrogenase, with the formation of gluconate-6-phosphate and NADH, the formation of the latter being easily monitored photometrically. The measurement signal can be intensified by further oxidation with NAD in the presence of 6-phosphogluconate dehydrogenase with the formation of ribulose-5-phosphate and a further molecule of NADH.
The principle of this embodiment is illustrated by the following equations: ##STR4##
An advantage of this embodiment of the present invention is that maltose phosphorylase is more specific than .alpha.-glucosidase and, therefore,endogenic glucose does not disturb.
With regard to the buffer, there again applies what was stated above with regard to the embodiment of the present invention using .alpha.-glucosidase process.
Apart from the two above-mentioned embodiments of the process according to the present invention, the determination of the fragments formed from the maltoheptaose, i.e. maltotetraose, maltotriose and the maltose formed therefrom, can also take place by other methods known for this purpose.
The present invention also provides a reagent for the determination of .alpha.-amylase which comprises an .alpha.-amylase substrate and a system for the determination of a fission product formed from the .alpha.-amylase substrate by the .alpha.-amylase, the substrate used being a compound of general formula (I).
A preferred system for the determination of the fission products is the .alpha.-glucosidase system which contains .alpha.-glucosidase, an alkali metal chloride and a buffer. If the substrate consists of maltoheptaose itself, then, as enzymes, there are also necessary hexokinase (HK) and glucose-6-phosphate dehydrogenese (G6PDH), as well as NAD, ATP and magnesium ions.
A reagent based upon the .alpha.-glucosidase system preferably comprises the following components:
According to another preferred embodiment, the reagent comprises .alpha.-glucosidase, potassium chloride or sodium chloride, buffer and substrate. A reagent of this type comprises, in particular, 10.sup.2 to 5.times.10.sup.6 U/1. .alpha.-glucosidase, 1 to 100 mMol/1. sodium chloride or potassium chloride, 10 to 250 mMol/1. buffer (pH 5 to 9) and 0.1 to 250 mMol/1. of a maltoheptaose derivative of general formula (I), referred to the concentration in the test. The reagent can be present in dry and especially in lyophilised form or also in the form of a solution, as a mixture of all components or separately.
According to a further embodiment of the reagent according to the present invention of the above-described type, .alpha.-glucosidase and/or phenol oxidase and HSK can additionally be present.
According to yet another embodiment of the reagent according to the present invention, in addition to .alpha.-glucosidase, sodium chloride or potassium chloride, buffer and substrate, it also contains sorbitol dehydrogenase and NAD or gluconate kinase, ATP, 6-phosphogluconic acid dehydrogenase and NADP, and optionally also a tetrazolium salt and diaphorase or phenazinemethosulphate (PMS).
A preferred reagent of this type comprises 1.times.10.sup.2 to 3.times.10.sup.6 U/1. .alpha.-glucosidase, 2.times.20.sup.3 to 5.times.10.sup.4 U/1. sorbitol dehydrogenase, 1.times.10.sup.3 to 5.times.10.sup.4 U/1. hexokinase, 0.5 to 50 mMol/1. ATP, 10 to 500 U/1. diaphorase (Chlostridium kluyveri), 0.01 to 0.5 mMol/1. tetrazolium salt, 0.1 to 10 mMol/1. NAD, 0.2 to 5 mMol/1. magnesium chloride, 0.5 to 20 mMol/1. maltoheptaitoll to 100 mMol/1. sodium chloride or potassium chloride and 10 to 250 mMol/1. buffer (pH 5.5 to 8.5).
If desired, a non-ionic surface-active agent can also be present, for example in an amount of from 5 to mMol/1.
Another preferred embodiment of the reagent comprises 100 to 3.times.10.sup.6 U/1. .alpha.-glucosidase, 10 to 10.sup.4 U/1. 6-phosphogluconate dehydrogenase, 20 to 2.times.10.sup.4 U/1. gluconate kinase, 0.5 to 25 mMol ATP, 0.05 to 10 mMol/1. NADP, 1.0 to 20 mMol/1. maltoheptagluconic acid, 0.5 to 5 mMol/1. magnesium chloride, 1 to 100 mMol/1. sodium chloride and 10 to 250 mMol/1. buffer (pH 5.5 to 8.5).
Yet another reagent according to the present invention comprises 0.1 to 250 mMol/1. .alpha.-nitrophenylmaltoheptaoside or dinitrophenyl-maltoheptaoside, 1.times.10.sup.2 to 2.5.times.10.sup.6 U1. .alpha.-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).
Yet another embodiment of the reagent according to the present invention comprises:
Another preferred system for the determination of the fission products is the maltose phosphorylase system which consists essentially of maltose phosphorylase, .beta.-phosphoglucomutase .beta.PGluM, glucose-6-phosphate dehydrogenase (G6PDH), glucose-1,6-diphoschate (G1,6DP), NAD, buffer, maltoheptaose and optionally 6-phosphogluconate dehydrogenase (6PGDH).
An especially preferred reagent with this detection system comprises:
The reagent according to the present invention can be present in dry or dissolved form and it can also be impregnated or incorporated into a sheet-like carrier, for example a film, an absorbent paper or the like. In the latter case, it preferably consists of at least three layers or laminae, the first of which contains the substrate, the second of which serves as a barrier layer and the third of which contains the system for the determination of the fission products. If such a multi-layer reagent material, which can be used for a simple rapid test for .alpha.-amylase, is brought into contact with a liquid .alpha.-amylase-containing sample, then the .alpha.-amylase splits the substrate and the fragments diffuse through the intermediate layer into the third layer containing the determination system. As detection reaction, in this case there is preferably employed a color-formed reaction in order to make visible the concentration of the .alpha.-amylase on the basis of the resultant coloration if the fission products themselves are not already colored.
The maltoheptaose derivatives employed according to the present invention can be prepared in various ways. In the case of the phenylated derivatives, there can be used not only chemical but also enzymatic methods. The chemical syntheses are, in principle, based upon the reaction of peracetylated maltoheptaose with the appropriate phenol in the presence of a Friedel-Crafts catalyst. This method can be used not only for phenol itself but also for mononitrophenol and dinitrophenol. On the other hand, it is also possible first to prepare the phenyl derivative and subsequently to nitrate it, for example with the use of the process described in Bull Chem. Soc. Japan, 34, 718/1961. This method is particularly suitable for the mononitro derivative. Under certain circumstances, a separation of the resultant o and p-nitrophenyl derivatives can also be carried out.
This reaction is preferably carried out by melting or boiling under reflux in non-polar solvents with zinc chloride, stannic chloride or titanium tetrachloride as Friedel-Crafts catalyst. After the introduction of the phenol or nitrophenol, the protective groups are split off in known manner, for example with sodium methylate, ammonia, potassium hydroxide or barium methoxide, in each case in methanolic solution,or with aqueous barium hydroxide solution or the like.
The enzymatic preparation of the phenyl derivatives can take place by transglucosidation of the phenyl glucoside or of the corresponding nitrated phenyl glucoside with .alpha.-cyclodextrin, amylose or soluble starch in the presence of a specific microbial transferase. For this purpose, it is preferred to use a transferase from Bacillus macerans. In this case, there can be used for this transglucosidation the known amylase from Bacillus macerans (E.C. 2.4.1.19.DSM 24; isolation see J.A. de Pinto, L.L. Campbell, Biochemistry, 7, 114,/1968; transfer reaction see Methods in Carbohydrate Chemistry, Vol. II, 347 (1963)) which besides itshydrolytic and cyclising action, clearly also displays a glucosyl-transferring effectiveness.
Those compounds of general formula (I) in which R is a sorbitol residue can be obtained from maltoheptaose by reduction with sodium borohydride under mild conditions.
Finally, the compounds of general formula (I) in which R is a gluconic acid group can be prepared chemically or enzymatically by methods known for the preparation of gluconic acid from maltoheptaose, for example by oxidation with bromine (see Methods in Carbohydrate Chemistry, Vol. II, 13 (1963)).
As already mentioned, the present invention not only provides a rapid and specific process for the determination of .alpha.-amylase but also completely or substantially removes the lag phase, which is of particular importance when the process is used in automatic analysers. Furthermore, the process according to the present invention can be carried out in many embodimental forms without the use of complicated apparatus for the evaluation and is, therefore, especially useful for rapid diagnostics and for optical determination in the visible range of light. However, at the same time, the various embodiments of the process according to the present invention can also be determined with UV measurement devices. Further advantages are the strict proportionality and the absence of disturbance by chemically related components of the blood.
The substrates employed according to the present invention are readily obtainable in a high state of purity. A simple process for the preparation of maltoheptaose is described in German Patent Specification No. P 27 41 191.2. The process can be used for the determination of .alpha.-amylase in biological fluids, such as serum, heparin plasma, urine and the like, as well as in other liquid and solid materials.
The following Examples are given for the purpose of illustrating the present invention:
US Referenced Citations (4)
Non-Patent Literature Citations (7)
| Entry |
| French et al-JACS vol.76 (1954) pp. 2387-2390. |
| Tilden et al-JACS vol. 64 (1942) pp. 1432-1433. |
| De Pinto et al-Biochemistry vol. 7 No. 1 Jan. 1968 pp. 114-120. |
| Foldi Dissertation, University of Freiburg, pp. 11, 12, 109-111, 124-128 and 158 (1977). |
| Pazur et al, "Methyl-Maltotetraoside", Methods In Carbohydrate Chemistry, vol. II, Academic Press Inc., New York, 347-349 (1963). |
| Barman, Enzyme Handbook, vol. I, Springer-Verlag New York Inc., 320 (1969). |
| Wallenfils et al, Carbohydrate Research, 61: 359-368, May 1978. |
Patent Grant
5108913
