FR2512960A1 - Detecting blood and peroxidisable substances in body fluid - using carrier impregnated with organic hydroperoxide, colouring, buffer, wetting and activating agents and amide or sulphonamide stabiliser - Google Patents

Abstract

Detection of occult blood and other peroxidisable substances uses agent comprising carrier impregnated with organic hydroperoxide, colouring matter (oxidn. indicator e.g. ortho-tolidine), buffer agent (e.g. citrate), wetting agent (e.g. Na lauryl sulphate), activating agent (e.g. benzo(f)-quinoline) and stabiliser comprising at least 1 cpd. (I)-(III). In the formulae, R1 is methyl, alkoxy, halogen or OH; R2 and R3 are 1-2C alkyl gp., and R4 is 1-6C alkyl. Stabiliser used is e.g. N,N'-dibenzoyl-N,N'-diethyl- ethylenediamine. The agent is useful for detecting blood and other peroxidisable substances in body fluid (e.g. urine, excrements, etc.).

Description

 Test tube for the detection of occult blood.

 The present invention relates to a test tube for the detection of occult blood. More particularly, the present invention relates to a stable test tube for the easy detection of blood and other active substances in a peroxidative manner in body fluids.

 The presence of peroxidatively active hemoglobin strap in bodily fluids such as urine, feces or vomited material may be viewed as an indication of some disease such as ulcers or inflammatory tumors of the stomach , kidneys, intestines and other urinary organs. Therefore, in this case, it is very important from the clinical point of view that the diagnosis or therapy be carried out with minimum loss of time.

For this purpose, it is necessary that the blood, or the active substance in a peroxidizing manner, present in the bodily fluid be detected accurately.

 Virtually all of the conventional methods adopted for the detection of occult blood in donated body fluids have used the peroxidative activity of occult blood. For example, a test piece was prepared impregnating a support, such as filter paper, with an organic hydroperoxide and an organic chromogenic indicator. When peroxidically active blood, or free hemoglobin, comes into contact with this test tube, they break down the hydroperoxide mentioned above by releasing oxygen, and the oxygen produced oxidizes the chromogenic indicator and causes it to take on a color.

 The organic chromogenic indicator contained in the test piece has an oxidizing power and the organic hydroperoxide also contained in the test piece is a reactive substance and therefore cannot be compatible with the indicator. Since these incompatible components co-exist in the specimen, they are essentially easily reactive with each other. Therefore, the test piece does not stand up to prolonged storage. In order to prevent this unwanted reaction of the two components during storage of the test piece, it has been proposed to incorporate a stabilizer in the test piece.

As examples of stabilizers proposed to date, mention may be made of compounds of the di- or trimorpbolidephospbate type (US Patent 3,853,471), the amine salts of organic hydroperoxide (US Patent No. 4,017,261) and boric esters ( US Pat. No. 4,071,321). Among the conventional stabilization processes, mention should be made of a process which involves the placing in microcapsules of an organic hydroperoxide with an agglutinating substance such as gelatin.

 The conventional techniques adopted for incorporating stabilizers have tended invariably to stabilize the reagents which cannot be compatible (organic hydroperoxide and organic chromogenic indicator) by preventing them from rapidly mixing with one another. Compounds of the di- or trimorpholidephosphate type are first of all insecticides (contraceptives for insects). Because of their violent actions, therefore, their manufacture is full of risks, and their handling requires a lot of precautions.

Amine salts of organic hydroperoxides are extremely volatile. Consequently, test pieces containing these compounds will easily deteriorate other test papers placed in the vicinity. In the case of boric esters, since they are used in combination with dimethyl sulfoxide, for example, test pieces containing these compounds similarly alter other test papers placed in the vicinity.

Thus, conventional stabilizers have drawbacks.

In the case of the microcapsules of an organic hydroperoxide, there is a drawback due to the fact that the microcapsule, as a result of aging, becomes rigid enough to reduce the ease of the reaction and alter the uniformity of the reaction in the test tube, with the result that the color is formed irregularly. Since conventional reagents and test pieces are unstable, as described above, it has happened quite frequently that the colors developed by the test pieces prevented an accurate diagnosis from being made.

 Consequently, the object of the present invention is to provide a new test tube for the detection of occult blood in bodily fluids.

 Another object of the present invention is to provide a test tube for the detection of occult blood, which has improved color sensitivity, for easy detection.

dans laquelle, m représente un nombre entier valant 1 a 5' R1 représente des groupes méthyle, alcoxy inférieur, des atomes d'halogène, des atomes d'hydrogèneKet X représente un groupe constitué par

où R et R représentent chacun un groupe alkyle ayant un a deux atomes de carbone, un groupe

où R, R et R ont les définitions ci-dessus, R4 représente un groupe alkylène ayant 1 à 6 atomes de carbone, et n vaut 0 ou 1, un groupe

où R et R4 ont les définitions ci-dessus, et un groupe

où R et R ont les définitions ci-dessus.The object described above is achieved by a test tube for the detection of occult blood, that is to say an active substance in a peroxidative manner in body fluids, obtained by impregnating a substrate with an organic hydroperoxide, a chromogen, a buffer and a stabilizer, test piece which is characterized by the stabilizer, which is at least one compound of general formula I

in which, m represents an integer ranging from 1 to 5 'R1 represents methyl groups, lower alkoxy, halogen atoms, hydrogen atoms and X represents a group consisting of

where R and R each represents an alkyl group having one to two carbon atoms, a group

where R, R and R have the above definitions, R4 represents an alkylene group having 1 to 6 carbon atoms, and n is 0 or 1, a group

where R and R4 have the above definitions, and a group

where R and R have the above definitions.

où m représente un nombre entier valant 1 a 5, de préférence 1; RI représente les groupes méthyle, alcoxy inférieur ayant 1 à 6 atomes de carbone, des atomes d'halogène,des atomes d'hydrogène, de préférence les groupes methyle, méthoxy, un atome de brome, un atome de chlore ou un atome d'hydrogène;X représente un groupe de formule

où R2 et R3 sont chacun un groupe alkyle ayant 1 à 2 atomes de carbone', un groupe

m, R , R et R ont les définitions ci-dessus R represente un groupe alkylène ayant 1 à 6, de préférence 2 à 6, atomes de carbone, et n vaut 0 ou 1 un groupe

où m, R et R4 ont les définitions ci-dessus, et un groupe

ou R et R ont les définitions ci-dessus.Ce composé est soluble dans l'eau ou les alcools D'une façon souhaitable, il a un point de fusion d'au moins 45 C. pour acquérir une résistance thermique
Quand X est

le composé de formule générale I est un composé représenté par la formule générale II:

où m, R , R et R sont définis ci-dessus.
Quand X est

le composé de formule générale I est un composé représenté par la formule générale III

4 où m. R R2 R3 R et n sont dénis ci-dessus. The stabilizer used in the present invention is a compound of general formula I mentioned below

where m represents an integer from 1 to 5, preferably 1; RI represents methyl groups, lower alkoxy having 1 to 6 carbon atoms, halogen atoms, hydrogen atoms, preferably methyl, methoxy groups, bromine atom, chlorine atom or atom hydrogen; X represents a group of formula

where R2 and R3 are each an alkyl group having 1 to 2 carbon atoms', a group

m, R, R and R have the above definitions R represents an alkylene group having 1 to 6, preferably 2 to 6, carbon atoms, and n is 0 or 1 a group

where m, R and R4 have the above definitions, and a group

or R and R have the definitions above. This compound is soluble in water or alcohols Desirably, it has a melting point of at least 45 C. to acquire thermal resistance
When X is

the compound of general formula I is a compound represented by general formula II:

where m, R, R and R are defined above.
When X is

the compound of general formula I is a compound represented by general formula III

4 where m. R R2 R3 R and n are denoted above.

le composé de formule générale I est un composé représenté par la formule générale IV

où m, R1 et R4 sont définis ci-dessus.When X has the formula

the compound of general formula I is a compound represented by general formula IV

where m, R1 and R4 are defined above.

le composé de formule générale I est un composé représenté par la formule générale V:

où m, R , R et R sont définis ci-dessus.When X is

the compound of general formula I is a compound represented by the general formula V:

where m, R, R and R are defined above.

The typical compounds of the general formula I are p-bromobenzoyl-dimethylamide, p-chlorobenzoyl-dimethylamide, benzoyl-dimethylamide, anisoyl-dimethylamide, p-bromobenzoyldiethylamide, p-chlorobenzoyl-ethylamide, N, N'-dibenzyl-N, N'dimethylhydrazine , N, N'-dibenzyl-N, N'-diethylhydrazine, N, N'ditoluoyl-N, N'-dimethylhydrazine, N, N1-ditoluoyl-N, N'-diethylhydrazine, N, N'-dibromobenzoyl- N, N'-dimethylhydrazine, N, N'dichlorobenzoyl-N, N '-dimethylhydrazine, N, N'-dibromobenzoyl-
N, N'-diethylhydrazine, N, N'-dianisoyî-N, N '-dîméthylb.ydrazine,
N, N'-dianisoyl-N, N '-diethylhydrazine, N, N'-dibenzoyl-N, N' diethylethylene diamine, N, '- dibenzoyl-N, N'-dimethylethylene diamine, N, N'-dibenzoyl- N, N'-diethylhexamethylene-diamine,
N, N '-dibenzoyl-N, N'-dimethylhexamethylene-diamine, N, N' -dichlo-
robenzoyl-N, N'-diethylhexamethhlene-diamine, N, N'-dibromo
benzoyl-N, N'-diethylhexamethylene diamine, N, N '-dianisoyl-N, N'-
diethyl-hexamethylen <liamine, N, N'-ditoluoyl-N, N '-diethyl-
hexamethylene diamine, N, N "-dibenzoyl-N, N'-diethylene diamie,
N, N1-dibromobenzoyl-N, N'-diethylene diamine, N, N'-ditoluoyl
N, N'-diethylene diamine, N, N'-dianisoyl-N, N'-diethylene-
diamine, p-toluene-sulfonyl-N-diethylamide, p-bromobenzene
sulfonyl-N-dimethylamide, p-methoxybenzene sulfonyl-N-diethyl
amide, and N, N'-dimethylbenzene-sulfonamide.

 We will now describe the process for manufacturing N, N'-dibenzoyl-N, N'-diethylethylenediamine which is one of the compounds which can be used as stabilizers in the test tube of the present invention.

 A chloroform of benzoyl chloride solution is obtained by adding 13.5 ml of benzoyl chloride to 50 ml of chloroform. Separately, a chloroform solution of N, N 'diethylethylene diamine is obtained by adding 7.1 ml of N, N'-diethylethylene-di2mine to 50 ml of chloroform. The two solutions are mixed gently while cooling. To this mixture is added 25 ml of a 20% aqueous solution of sodium carbonate. The resulting solution is heated to 600C for one hour. The reaction solution thus obtained is dried under vacuum with an evaporator. To remove the salt, the reaction product is dissolved in chloroform and the resulting solution is dried with an evaporator. As a result, 14.2 g of N, N'-dibenzoyl-N, N'-diethylethylene diamine are obtained.

 As examples of organic hydroperoxides advantageously usable for the present invention, mention should be made of: 2,5-dimethylhexane-2,5-dihydroperoxide, cumene hydroperoxide, 2,5-dimethylhexanone-2,5-dihydroperoxide, di-isopropylbenzenehydroperoxide, 6-butylhydroperoxide, p-methanehydroperoxide, and 4-methylphenylisopropylhydroperoxide.

 Among the other organic hydroperoxides listed above, 2-5-dimethylhexanone-2,5-dihydroperoxide, cumene hydroperoxide and 2,5-dimethylhexane-2,5-dihydroperoxide are preferred, because their rate of decomposition is relatively low.

 The chromogens used for the present invention are those which form dyes by oxidation and which are called "oxidation indicators". Examples of these chromogens include orthotoluidine, benzidine, 3,3 ', 5,5'-tetramethyl-benzidine, 2,7-diaminofluorene, mixtures containing these compounds in various proportions, guaiacols, various substituted phenylenediamines, pyridine derivatives, substituted azines and leuco-malachite green.

 When the test tube is immersed in the body fluid, the pH value of the latter must be maintained in the range of 4 to 8, preferably from 5 to 7. Therefore, a buffer is used to maintain the pH value of the composition contained in the test piece. Examples of buffers which are advantageously usable for the present invention include: citric acid-sodium citrate, tartaric acid-sodium tartrate, malic acid-borax, potassium acid phthalate-dipotassium phthalate and sodium acid succinate disodium succinate.

 In order to ensure uniform penetration of the body fluid into the test piece, it is desirable to incorporate a wetting agent into the composition used in the test piece. Typical wetting agents are surfactants including alkyl sulfates such as sodium lauryl sulfate, sodium dodecyl sulfate, and sodium tetradecyl sulfate; alkylbenzene sulfonates such as sodium dodecylbenzene sulfonate, and dialkylsulfosuccinates such as sodium dioctylsulfosuccinate and sodium diheptylsulfosuccinate.

 Optionally, the test tube of the present invention may include a reagent capable of accelerating the activity of hemoglobin peroxidase. Typical activators that can be used for this purpose are quinoline and its derivatives, including quinine, cinchonine, o-methoxy-quinoline, quinaldine, 8-amino-6-methoxy-quinoline, 2-quinolinol, isoquinoline , benzo (f) -quinoline and 3-amino-quinoline. The presence of such an activator in the test piece generally serves to accelerate the speed of the oxidation reaction, and to increase the sensitivity of the test piece itself.

 An adhesiphore can be used to prevent the composition from being removed from the test piece. Typical adhesiphores suitable for this include polymers such as polyvinyl alcohol, polyvinylpyrolidone, polyethylene glycol, polyacrylates, polyacrylamide, poly (hydroxyethyl methacrylate), poly (hydroxyetbyl acrylate) and carboxymethylcellulose , gelatin and gum arabic.

 The amounts of the reagents to be used in the test tube of the present invention are 1 to 10,000 parts by weight, preferably 100 to 1000 parts by weight, of chromogen; 10 to 10,000 parts by weight, preferably 100 to 1000 parts by weight of buffer; 10 to 10,000 parts by weight, preferably 100 to 1000 parts by weight stabilizer; 0 to 1000 parts by weight, preferably 10 to 10 parts by weight of the wetting agent; 0 to 10,000 parts by weight, preferably 190 to 1000 parts by weight1 of product increasing the activity of peroxidase (sensitizer), and O to 10,000 parts by weight, preferably 100 to 1000 parts by weight, adhesiphora, respectively based on 100 parts by weight of the organic hydro peroxide. The combined amount of these reagents to be produced is from 0 to 50% by weight, preferably 0 to 10% by weight, calculated on the substrate.

 These reagents are first dissolved in a suitable solvent such as benzene, toluene, methanol, ethanol, chloroform or water, and the resulting solution is used to impregnate the substrate. For example, the organic hydroperoxide and the stabilizer are dissolved in a solvent, and the substrate is widely wetted in the resulting solution. The wet substrate is then dried. Then, the adliésiphore and a buffer are dissolved in a solvent. The above-mentioned substrate is largely wetted in this solution, then dried. Finally, the chromogen and the product increasing the peroxidase activity are dissolved in a solvent, and the same substrate is largely wetted with the resulting solution and dried.

Thus, a test tube is obtained for 1. detection of occult blood.

 As a substrate, filter paper, fiberglass or plastic nonwoven fabric can be used. Any other substrate can be adopted provided that the substrate does not react with the impregnation products or the solvents contained in these products and has an absorbent capacity.

 The test tube of the present invention for the detection of occult blood is immersed in the body fluid for approximately one second, then removed from the body fluid, and allowed to stand in air for 60 seconds to be prepared for examination . The examination is carried out by comparing the color formed by the test piece with a standard color table, finding the comparison color in the table, and noting the symbol indicated accordingly. The concentration of occult blood in the body fluid can be determined based on the degree of color formation which is found from the standard color table mentioned above.

The present invention is illustrated by the following descriptive and nonlimiting examples
Example 1
Solution I
2,5-dimethylhexane-2,5-dihydroperoxide 1.5 g
Sodium dioctylsulfosuccinate 1.0 g
p-bromobenzoyldimethylamine 10 g
Methanol 100 ml
Solution I is prepared by mixing the compounds mentioned above. Filter paper is completely immersed in solution I, then it is removed from the solution and dried in an oven at 400C for 20 minutes.

Solution II Gelatin 2.0 g
Citric acid 2.5 g
Sodium citrate 7.5 g
Water 100 ml
Solution II is prepared by mixing the above mentioned compounds. The filter paper mentioned above5 which has been impregnated with solution I and dried, is completely wetted with solution II, removed from the solution, then dried in an oven at 400C for 50 minutes.

Solution III
Ortho-toluidine 0.3 g
3-amino-quinoline 0.3 g
Benzene 100 ml
Solution III is prepared by mixing the compounds indicated above. The above-mentioned filter paper, which has been impregnated with solution II and dried, is completely wetted with solution III, removed from the solution, and dried in an oven at 500C for 10 minutes. This gives a test tube for the detection of occult blood.

Example 2
The process of Example 1 is followed, except that N, N'-dibenzoyl-N, N'-dimethyl-hydrazine is used as the stabilizer in place of p-bromobenzoyl-dimethylamide. This gives a test tube for the detection of occult blood.

Example 3
The procedure of Example 1 is followed, except that N, N'-dibenzoyl-N, N'-diethylethylenediamine is used as the stabilizer in place of p-bromobenzoyl-dimethylamide.

Example 4
The procedure of Example 1 is followed, except that p-toluene-sulfonyl-N-diethylamine is used as stabilizer in the p-bromobenzoyl-dimethylamide plate.

Example 5
The procedure of Example 1 is followed, except that N, N'-ditoluoyl-N, N'-diethylene diamine is used as the stabilizer in place of p-bromobenzoyl-dimethylamide.

Example 6
In a mortar, 100 ml of gum arabic mucilage
(prepared by adding 200 g of gum arabic to 500 ml of boiling water) and 2 ml of sodium dioctyl sulfo-succinate (5% weight / volume) are added in the order indicated. Then 5 ml of cumene hydroperoxide are added. The resulting mixture is ground in the mortar for five minutes to obtain a crude emulsion. Separately, a citrate buffer is prepared by adding a mixture consisting of 163 g of sodium citrate and 37 g of citric acid in 1500 ml of boiling water.

The citrate buffer is mixed with 0.7 g of gelatin. Then.

50 ml of the resulting buffer-gelatin mixture and the crude emulsion are stirred vigorously. Then 1 ml of a 1% dialdehyde-starch dispersion is added while stirring vigorously. Finally, 12 ml of a 5% aqueous solution of sodium laurate are added to obtain a composition.

This composition is homogenized in a manual homogenizer.

While continuing to agitate, 50 ml of the buffer which was prepared using 500 ml of water, 163 g of sodium citrate and 37 g of citric acid, as described above, are added to the homogenized composition. In the resulting emulsion, a piece of fine paper is immersed to be impregnated. The wet piece of paper is dried in an oven at 750-800C. for 24 hours. Finally, a solution of 320 mg of ortho-toluidine in 16 ml of chloroform is applied by immersion on the piece of fine paper. The wet paper is gently heated to remove excess chloroform. Thus, a test tube for the detection of occult blood is obtained.

Example 7:
The test tubes for the detection of occult blood obtained in Examples 1 to 6 are stored at 37 "C. and kept under observation to verify the change in appearance during aging. The results are shown in Table 1. These results allow '' evaluate the test pieces on their stability to resist degradation by aging.

Table 1
Change in appearance due to aging
Test tube One week Two weeks Three weeks Four weeks
Example 1 No change - No change - No change - Slight turn to
gement gement brown gement
Example 2 """No change
Example 3 ""
Example 4 """Slight turn to
brown Example 5 """No change
Example 6 Turning to Turning to Turning Black
black black
From Table 1, it can be seen that the test pieces of Examples 1 to 5 according to the present invention do not vary at all up to three weeks of storage. On the contrary, the test piece of Example 6, according to the conventional method, turns black after only two weeks of storage. This shows that the test pieces of Examples 1 to S according to the present invention have excellent stability.

 When the test tubes of the present invention are used in fluids to be exanuated, such as for example urine containing occult blood, they show a stable property of color formation.

 According to the present invention, since the test piece is impregnated with at least one compound of general formula I, mentioned above, in combination with an organic hydroperoxide, a chromogen and a buffer, the stability of the test piece to resist degradation by aging, is notably improved and the sensitivity of the test piece in the detection of occult blood in the body fluid is increased. Therefore. the test piece gives an exact stable color formation on contact with occult blood. It provides sensitive and accurate detection of occult blood.

Claims (11)

 1. Test tube for the detection of occult blood, that is to say a peroxidant active substance, in body fluids, obtained by impregnating a support with an organic hydroperoxide1 a chromogen, a buffer and a stabilizer, this test tube being characterized by the fact that the stabilizer is at least one compound having the general formula I:

 wherein m represents an integer from 1 to 5; R1 represents methyl, lower alkoxy, halogen atoms or hydrogen atoms. and X is a group with the formula

 where R and R each represent an alkyl radical having 1 to 2 carbon atoms, a group

 or m, R, R, R are defined above, R is an alkylene group before 1 to 6 carbon atoms, and n is O or 1 is a group

 where m, R1 and R4 are defined above, and a group

 where R2 and R3 are defined above.  2. A test piece according to claim 1, characterized in that m is 1; R is a methyl group, methoxy. a bromine atom> of chlorine or a hydrogen atom, and R4 represents an alkylene group having 2 to 6 carbon atoms, respectively, in the general formula I.  3. A test piece according to claim 1, characterized in that X represents a group

 where R and R3 are defined above, in general formula I.  4. Test tube according to claim 1, characterized in that "e X represents the group

 in which mr. R e R2 R3 and R4 are defined above, in the general formula I.  5. A test tube according to claim 1, characterized in that X represents a grouse

 where m, R1 and R4 are defined above, in the general formula I.  6. A test piece according to claim 1, characterized in that X represents a group

 where R and R are defined above. in general formula I.  7. A test piece according to claim 1, characterized in that a wetting agent and a product increasing the peroxidase activity are additionally incorporated.  8. A test piece according to claim 7, characterized in that an adhesiphora is incorporated in addition.  9. A test piece according to claim 2, characterized in that the stabilizer is a compound chosen from p-bromobenzoyl-dimethylamide. N, N'dibenzoyl-N, N'-diethylethylenediamine, p-toluenesulfonyl-N-diethylamide, and N, N'-ditoluoyl-N.N'-diethylenediamine.  10. A test piece according to claim 9, characterized in that the organic hydroperoxide is at least one compound chosen from 2.5-dimethylhexane-2,5-dihydroperoxide, cumene hydroperoxide, and 2,5-dimethylhexanone-2 , 5dihydroperoxide.  11. A test piece according to claim 10, characterized in that the organic hydroperoxide is 2,5-dimethylhexane-2,5-dihydroperoxide.