IL44125A - Method of assaying catalase in milk and other liquids especially of biological origin - Google Patents

Description

Method of assaying Catalase in milk and other liquids, especially of biological origin 'n T3 Tl|7»0 fl¼33- 1ΠΤ»Β31 Ο' ΠΚ 0'>ηη 37Π3 ΠΤ70|? ny*»3p7 ΠΟ'ϋ 1 ' ' > A ■ ■ ■KO NO ULT. Ι Η ΟΕΜ Ι ΟΠ noLr DAH I CLI BOX 74, iaa gl FAnOTA METHOD, OF ASSAYINC CATALASE IN MILK AND OTHER LIQUIDS , ESPECIALLY OF BIOLOGICAL ORIGIN The present invention relates to a method of assaying the enzyme, catalase in milk and other liquids, especially such of biological origin, such as blood serum. The method is character zed in particular by addition to the milk or other liquids of a substance or synergistic substances that are capable of generating hydrogen peroxide in conjunction with a substance present in the milk or other liquids, and a further substance which, while itself becoming oxidized by the generated hydrogen peroxide, displays a colour reaction, enabling the catalase to be assayed from the colour hue thus produced.

Methods of qualitative and quantitative catalase determination are known. One of these methods is based on the power of catalase to catalyze the decomposition of hydrogen peroxide, releasing free oxygen. In practice, the liquid under test is mixed with dilute hydrogen-peroxide solution in a fermentation tube, which is then generally allowed to rest for 3 h before the volume of the released oxygen is read:- 2H202 catalase ^ + ^ (l) The possibility of assaying catalase in a fast, accurate and simple way is of great importance, especially in milk as the presence of catalase in milk is an indicative of mastitis, an infectious disease of the udder of the milk- -producing animal. Being. a source of considerable loss to the farmers, it is important that the presence of mastitis can be revealed by catalase assay at an early stage through regular controls of the state of the udder.

Owing to the slow character of the above-mentioned method of assaying catalase by adding hydrogen peroxide in a fermentation tube, other methods have been devised. Some of them are based on an assessment of the degree of coagulation or- increase in the viscosity of milk to which alkaline or surface-active substances have been added. These methods do, however, also involve the disadvantage of requiring handling reagents; in addition, their accuracy is low unless very skilled personnel is used for the analytical work, especially at low degrees of mastitis.

In two. articles - Z. physiol. Chem. 3 9 ( 1 62 ) 0 and Clin. Chim. Acta Ijj ( 1967 ) 15 -163 - Johann Putter described a method of colorimetric detection of catalase by a system of reagents, ' comprising among other compounds hydrogen peroxide, peroxidase and a leuko dye. According to this method a quantity of hydrogen peroxide is added to the liquid being analyzed, the catalase that may be present in the liquid is allowed to decompose the hydrogen peroxide, and the reaction is interrupted at certain time. intervals, the quantity of the remaining hydrogen peroxide each time being determined by letting it oxidize the leuko dye, using peroxidase as catalyst, according to the formula:- Leuko dye + I^O Peroxidase > dyestuff + E^0 (2) Obviously, this method as well is too complicated and time-consuming to be used for rapid, yet reliable determinations of catalase by non-expert staff.

It is, thus, the aim of the present invention to provide a simple fast and accurate method of catalase detection.

Since the occurrence of catalase in milk is known to be closely correlated to the degree of mastitis it is also an aim of the invention to provide a simple, fast, reliable, and sensitive method of quantitative catalase determination.

It is, further, an aim of the invention to provide a method, involving the above-mentioned advantages, that makes it possible to carry, out catalase assays by a colour reaction. , Finally, it is the aim of the invention to provide a method that makes it possible to carry out the above described catalase assay by means of a test appliance, for instance in the form of a strip of paper in which the reagents necessary for the test are adsorbed.

It has. been the inventors' idea to try to attain these ends by balancing the above-mentioned reaction (2), according to which a leuko dye produces a coloured dyestuff by oxidation with hydrogen peroxide, against the above-mentioned reaction according to which catalase decomposes hydrogen peroxide, the last-mentioned reaction consequently inhibiting colour reactions like the first-mentioned one.

The intention to devise a method, according to which it would be possible to use only a dry test paper, cannot be realized by adding hydrogen peroxide, which is both volatile and chemically unstable, to the paper. Hence the hydrogen peroxide has to be generated in the liquid under test or in the test paper by incorpora ing in that paper a substance that is capable of reacting with a substance present in the liquid, thereby causing hydrogen peroxide to be released. A reagent meeting these requirements is an organic or inorganic peroxide or some compound capable of generating a. peroxide.

Another possibility would be to utilize an enzymatic reaction for generating the hydrogen peroxide. The opportunities for such an approach are; particularly favourable in milk and other liquids of biological origin. So, for example, xanthine and hypoxanthine together with the en¾rme xanthine oxidase present in milk yield hydrogen peroxide.

The most favourable approach, however, would be to mix the milk orj other liquids of biological origin with an enzyme or synergistic enzymes that are capable of releasing hydrogen peroxide in the presence of. a sugar available in the liquid. Such generation of hydrogen peroxide can be obtained from the lactose in the milk in conjunction with the enzyme galactose oxidase.

However, galactose oxidase is basically specific to free galactose and reacts only slowly with lactose. It would therefore be to advantage to add also the enzyme 5 -galactosidase, which splits lactose into galactose and glucose.

Instead of galactose oxidase, or in conjunction with that enzyme, the enzyme glucose oxidase, which is specific to the glucose obtained by the splitting reaction, can be used together with S -galactosidase.

Hence a useful system for detecting catalase in milk would follow the general lines of the formula below, in which a leuko dye in the presence of peroxidase becomes oxidized catalytically by the hydrogen peroxide generated in the liquid under test, this oxidation being inhibited in the presence of catalase :- ' (J -galactosidase Ί leuko dye —> colour Lactose'— glucose leuko dye — no col (3) A 'strip of paper, in which the reagents mentioned are adsorbed should provide a convenient material for applying this system by simply immersing the paper in the liquid under test in the determination of catalase. Another possible material might involve a specific structure, such as a gel, to which the reagents are attached. Such a material can be produced in the form of a strip of gel and has been utilized for instance in pH determination for a considerable time.

It seemed improbable, however, that such a competition for the available hydrogen peroxide could be brought about between catalase and peroxidase. Following the demonstration by W. E. Knox - Biochem. Biophys. Acta 14 (1954) 117 - according to which all generated hydrogen peroxide in a system comprising glucose, glucose oxidase, catalase, peroxidase, and an oxidizable compound, i.e., a hydrogen donor, was consumed during the reaction catalyzed by peroxidase bat was not affected by the presence of catalase, it has been common belief that enzymatically generated hydrogen peroxide is generall handled by the peroxidase irrespective of catalase being prec3ent. This belief found further support from the fact that despite its power of metabolizing its substrate at a very much higher rate than other enzymes do, catalase shows a very low affinity to the substrate, whereas peroxidase has a high affinity to its substrate and has been shown most efficiently to utilize enzymatically generated hydrogen peroxide present in very low concentration.

To their surprise the inventors of the present method' found the system and reaction (3.) to work as they had hoped. The difference between their results and those published by Knox and others may be attributed at least in part to the. latters' using certain naturally occurring hydrogen donors whereas the inventors resorted' to synthetic dyes. The enzymatic generation of the hydrogen peroxide in situ leads to a comparatively constant concentration of the hydrogen peroxide, which is a prerequisite to the utilization of the competi- V tive degradation of hydrogen peroxide by peroxidase and catalase in assaying the catalase content.

The inventors have carried out a great number of tests by which catalase has been assayed as described above. The mixtures of reagents applied, as stated in formula (3), in conjunction with the liquid samples, have resulted in the occurrence of dark-blue or greenish colours in the absence of catalase. With high percentages of catalase present, pale-yellow colours have developed.

The sensitivity is inversely dependent on the peroxidase content of the reagent mixture. The inventors have succeeded in adjusting the sensitivity during their tests on milk so that with milk from a completely healthy cow the colour reaction has been dark blue whereas milk from cows arranged in order of increasing severity of mastitis has resulted in colours ranging from medium blue over light bluish green to pale yellow.

The paragraphs below offer examples of reagent mixtures, containing the substances suggested above, and methods for their application in assaying catalase in milk. One drop of milk is to be mixed with one drop of a reagent solution that is made up according to one of the following alternatives:- Alternative 1 fi -galactosidase 0.1 mg/ l glucose oxidase or galactose oxidase 0.1 mg/ml peroxidase Ί.0 ig/ml o-tolidine 0.2 mg/ml Alternative 2 Galactose oxidase 0.1 mg/ml peroxidase 1 · o βε/ml o-tolidine 0.2 mg/ml Alternative 3 Hypoxanthine 0.1 mg/ml peroxidase 1.0 jHg/ l o-tolidine 0,2 mg/ml Alternative 4 Sodium perborate or urea peroxide 1.0 mg/ml peroxidase .1.0 ig/ml o-tolidine 0.2 mg/ml This technique may be replaced by using, for instance, a strip of filter pa per soaked in one of the alternative solutions mentioned, and dried. When being subsequently immersed in the milk for the purpose of assaying milk catalase, it will absorb a quantity of milk that matches the amount of reagent present in the strip.

The peroxidase employed can be of normal commercial purity (e.g., RZ = 0.6). The glucose oxidase and galactosidase preparation, on the other hand, have to be special grade with a low catalase content. To ensure reagent stability they should be dissolved in. an adequate buffer solution close to pH 7.

The sensitivity of, the single-phase reagent system described is sufficiently high to detect a developed inflammatory process of the udder, known as an acute mastitis, and its stage of development. This single-phase system has, however, proved to be insufficiently sensitive to allow a reliable ocular detection in the early stages of the inflammatory process ("subclinical mastitis") that are of particular diagnostic interest. However, these stages can be detected photometrically, using the single-phase system described.

It has been shown that the . sensitivity of the analytical method can be increased to the necessary level by developing it into a multi-phase system in that the reagents generating hydrogen peroxide and dyestuff, respectively, are separated physically from each other by means of a space through which the generated hydrogen peroxide can diffuse, and which is also capable of admitting the milk. During its passage from the hydrogen peroxide--generating phase of the system, through the separating space and! into the dye-generating phase of the system the hydrogen peroxide has enough time for total or partial decomposition, should catalase be present in the milk that has penetrated or been brought into the said space.

Thus, by arranging a diffusion path for the generated hydrogen peroxide through the liquid under test, from the hydrogen peroxide-generating enzyme to the dye-generating system, containing mainly a catalyzing enzyme, it is nov; possible to detect even very small amounts of catalase and hence mastitis at an initial . stage of development.

The following paragraph offers a number of examples of practical applications of the method according to the present invention:- Example 1 A test cell fitted with two compartments one of which is permanently closed by a semipermeable membrane and contains 1 - 10 U of galactose oxidase (as counted on lactose as the substrate). A small amount, 0.5 - 1 ml, of the liquid to be analyzed for catalase, is added in the open compartment of the Lest cel.. A test paper, containing peroxidase and a leuko dye as described in Example 2 (below), is arranged in the liquid in such a way that its nearest part remains at a fixed distance of a few mm from the semipermeable membrane. Various sensitivities to catalase can be attained, depending on the amount of galactose oxidase, the sample volume, and the fixed distance mentioned. With an arrangement as described it has been possible to carry out semiquantitative determinations of catalase concentrations of approximately 2 - 20 U/ml by observing the development of colour in the test paper, which is maximum after a few minutes in the absence of catalase but attains gradually weaker intensity maxima the higher the catalase concentration.

Example 2 The hydrogen peroxide-generating system is applied to one side of a porous structure, the colour-generating system to its other side. The porous structure will thus form the space separating the two systems. It can absorb liquid to be analyzed and constitutes the diffusion path for the hydrogen peroxide. The separating space thus formed may be reinforced by a semipermeable membrane, isolating the systems from each other. The porous structure may consist of one or more layers of porous paper. j The two reagent systems may be applied by being fixed in layers of gel. The two following solutions are prepared at about 40 °C, preferably in 20 - 40 per cent gelatin or some other gel-forming preparation :- Solution 1 Peroxidase (EC 1.11.1.7, RZ 0.6) 0.5 mg/ml o-tolidine 0.5 mg/ml buffer salt yielding an almost neutral pH, such as phosphate dase (EC 1.1.3-9» about 20 u/rng, as' substrate, npn-catalase) buffer salt as above Example 3 · Instead of arranging the two enzyme systems' on each side of a porous layer by enclosing thejn in separate gel layers it is also possible to arrange two porous layers, for instance of paper, following adsorption of one system in each, on both sides or in some other manner in different parts of a third porous layer, which may also be of paper. The two layers carrying the reagents' are preferably isolated from the intermediate layer, by means of semipermeable membranes.

Example 4 Instead of being enclosed in gel layers the reagents according to Example 2 may be encapsulated in so-called microcapsules according to any of the methods described in the literature. Cf., for example, Chang, T. M. S., Macintosh, P. C, and Mason, S. G. - Semipermeable Aqueous Microcapsules - Can. J. Physiol. Pharmacol. 44 ( 1 66) 115· Prepare two solitinns similar to those described in Example 2. The solutions are enclosed separately in individual microcapsules, which are then placed in different layers on each side of a porous diffusion layer or mixed with an inert diffusion medium, such as cellulose fibres, the mixture finally being formed into a porous structure.

Example 5 The semipermeable membranes of Example 3 ma be omitted if the enzymes used are fixed (insolubilized) on each side or in various parts of a porous structure in such a way as to be without immediate contact with each other. Any of the methods for attaching enzymes for instance to paper as described in the literature, may be applied. Cf., for example, Stasiw, R. 0., Patel, A. B. , and Brown, H. D. - Utilization of Bound Lactase in Clinical Chemistry -Biotechnol. Bioeng. (1 72) 62 .

When stated in the Examples that the various enzyme systems are to be arranged in some way in different parts of a porous structure this must not be construed as implying only an arrangement for instance at each end of a strip i of paper but also an attachment to various component parts of the porous structure or various elements which can be used in forming such structure. The latter case is referred to in Example 4· There, the microcapsules containing the reagents are components of the structure created when, according-

Claims (1)

1. 9 to one technique, they are mixed with an inert diffusion medium after which the mixture is formed into a porous structure. Patent Claims WHAT IS CLAIMED IS : 1. A method for qualitative and quantitative determination of catalase in milk and other liquids, mainly of biological origin, characterized by the addition of a reagent comprising a substance or synergistic substances that in the presence of a substance available in the milk or other liquids, can , , o i n the p resence of the enzyme peroxi dase re.lease hydrogen peroxide,/and another reagent that upon oxidation by hydrogen peroxide yields a colour reaction, the catalase content being determined with the aid of the hue of the colour produced. 2. . A method according to Claim 1 , characterized by the reagent oapable of releasing hydrogen peroxide in the presence of a substance available in the milk or other liquids, being an organic or inorganic peroxide or a compound capable of generating a peroxide. 3. . A method according to Claim 1 , characterized by the reagent being a compound capable of releasing hydrogen peroxide in the presence of. xanthine oxidase available in the milk, . e .g. , xanthine or hypoxanthine. 4. A method according to Claim 1 , characterized by the reagent or the synergistic substances capable of releasing hydrogen peroxide in the presence of a substance available in the milk or other liquids, being one or more enzymes, respectively. J 5 . A method according to Claim 1 , characterized by the reagent capable of releasing hydrogen peroxide in the presence of a substance available in the milk or other liquids, being the enzyme galactose oxidase. 6. A method according to Claim 1 , characterized by the synergistic substance capable of releasing hydrogen peroxide in the presence of a substance available in the milk or other liquids, being the enzymes S-galactosidase and glucose oxidase. 7 . A method according to Claim 1 , characterized by the synergistic substance capable of releasing hydrogen peroxide in the presence of a substance available in the milk or other liquids, being the enzymes {h-galactosidase and galactose oxidase. 8 . A method according to any of the previou Claims, characterized by the reagent yielding a colour reaction in the presence of hydrogen peroxide, be i ng a leuko.dye. ' ' · ' 9. A method according to any of the previous Claims, characterized by the two reagents being separated physically from each other by means of a space through which the generated hydrogen peroxide can diffuse and which is cap-acje of admitting the liquid. ' · ,;. 10 · A method according to Claim 9 , ' characterized by the reagents being isolated from each other by' at least one semipermeable membrane through' which the hydrogen peroxide but not the reagents can pass..— 1 1 . A method according to any of Claims 9. and 1 (X, characterized by the two reagents being attached to separate sides or otherwise to different parts of a porous structure. ' 12. A method according to Claim 1 1. characterized by the two reagents being enclosed in one gel layer each, the layers then being attached to the porous structure . * * . ■■· - 1 3. . A method according to Claim 1 1 > characterized by the two reagents being adsorbed in- one porous layer each, the layers then being attached to. the porous structure. , A method according to Claim 9 , £haracterized by the two reagents being enclosed in separate microcapsules, the capsules then being mixed with an inert diffusion medium, which is subsequently formed into a porous structure, or by attaching the capsules to separate sides or otherwise to different part of a porous structure. 1 ^ · A method according to Claim 9 , characterized by the two reagents being fixed (insolubilized) on separate sides or otherwise on different parts of a porous structure. , ' , ■·. ·, ■ „ " '-» 16. A porous structure for use in the qualitative or, quantitative determination of catalase in jnll k. and other liqutds., 'according to claim 1, which /has impregnated therein a first reagent which when ontacted with the liquid or a constituent thereof generates hydrogen peroxide, and a second reagent which on oxidati by hydrogen peroxide undergoes a change in colour. 17. A porous structure according to Claim 1 , wherein the '< ■" first and second reagents are physically separate from each other. 18.- A porous structure according to Claim 17,. which has one or more semipermeable membranes located between the · first and second reagents, , 19. A porous structure according to any one of Claimsi :; j... 16-18, which is formed of porous paper. ! : 20. A porous structure according to any one of Claims < \-' 16-19, wherein the first and second reagents are separately encapsulated in microcapsules dispersed in the porous ;; ' r s tructure, 21. A porous structure according to any one of Claims · 'K;' 16-18, wherein the first and second reagents are separately incorporated in layers of gel. 22. A porous structure according to Claim17* wherein the : first and second reagents are insolubilised on the porous , * structure.. · ' . ". - '. ·■'¾ ' 23.. A porous structure according to Claim 6, substantially ae herein described in the Examples. v! - 24·. A composition for use in theBetermina tion of catalase \ * in milk by the method of Claim 1, which comprises from , : ■ ■ ■ ■ · ' ■ ' J . . ■ ·'." 1 ■'·'·· -H< 0.09 to 0.11 mg/mlp-galactosidase, from 0.09 to 0.11 mg/nl'/' ·< ' · - ' ' glucose oxidase or galactose oxidase, from 0.9 to 1.1 ,·'/< JL e/ml peroxidase and from 0.19 to 0.21 tag/ml p- tolidine. · .