Reagent for detecting occult blood

Abstract: A reagent to be used in detection of occult blood comprising a mixed solution of sodium carbonate, 3-amino phthalic acid hydrazide, and iron and sodium derivative of ethylenediaminetetraacetic acid, and hydrogen peroxide.

Description: The present invention relates to a reagent for detecting occult blood contained in human faeces or urine (hereinafter called excretion). It is hard to detect grossly the occult blood which is a trace hemorrhage in the human digestive tract or urinary organ. If occult blood is detected, however, an ulcer or tumour of the digestive tract, infection of the urinary tract, a stone, tumour, blood disease or other intractable diseases may be detected at an early stage before they are advanced too much in their symptoms, so that the progress of refractory diseases may be prevented. Therefore, the occult blood reaction for detecting whether occult blood is contained in faeces or other excretion or not has been attempted hitherto in various ways. Among such ways the method of using a catalyst was the most common, of which the principle is as follows. That is, acetic acid is caused to act with the haemoglobin in blood to form hematin acetate, and by making use of its catalytic action, a reagent such as orthotoluidine, phenolphthalein, benzidine or guaiacol acid is oxidized by hydrogen peroxide. The presence of occult blood is determined by a change in colour. Aside from such catalytic method, recently, computer-assisted diagnostic apparatus has been developed to attempt the early discovery of focus. In the catalytic method, however, the reagent includes carcinogenic substances such as benzidine. In the computer apparatus, a huge system is needed and it is very costly, and aside from the economical reason for patients, specialists were necessary to interpret the results of the computer diagnosis. It is hence a primary object of the present invention to provide a reagent for detecting occult blood that is simple, handy and inexpensive, without using the previously known catalytic method or computer system. In JP-A-63-247659 there is disclosed a reagent solution for the detection of occult blood which comprises sodium carbonate, 3-amino phthalic hydrazide, hydrogen peroxide and EDTA. In JP-A-57-071398 there is disclosed a test for the analysis of blood serum using an oxidase, a colouring hydrogen donor and hydrogen peroxide. If bilirubin is present in the blood serum it can interfere with the detection test. This document discloses the use of EDTA-iron complex to prevent or avoid that interference. In US-A-4933276 there is disclosed reagents for use in chemiluminescent assays, in particular ones using xanthine oxidase. The document teaches that the use of an iron-EDTA complex increases the chemiluminescent response linked to the xanthine oxidase induced oxidase of luminol allowing the detection threshold to be reduced. According to the present invention there is provided a reagent for use in detecting occult blood, which comprises a solution containing (a) sodium carbonate,(b)3-aminophthalicacidhydrazide,(c)an iron and sodium derivative of ethylenediaminetetraacetic acid and (d) hydrogen peroxide. By using the reagent for detection of occult blood of the present invention, if the excretion was free from occult blood, the colour of the excretion is unchanged, and foams are hardly noted, and the temperature declines. In contrast if the excretion contains occult blood, the excretion turns to a pinktinted violet colour, and foams build up and the temperature rises, so that the presence or absence of occult blood may be judged anytime, anywhere, by anyone, in hospital or at home, easily, safely and inexpensively, so that it is outstandingly effective to discover disease at an early stage before the symptoms are advanced. Besides, unlike the conventional method of sampling a trace from the excretion, the whole excretion discharged by a human is tested, and the precision of examination is improved. To achieve the above object, the invention presents a reagent for detecting occult blood which comprises a mixed solution of sodium carbonate, 3-amino phthalic acid hydrazide, and iron and sodium derivative of ethylenediaminetetraacetic acid (tradename CLEWAT, hereinafter also called iron and sodium derivative of EDTA), and hydrogen peroxide. The hydrogen peroxide may be used by mixing into the mixed solution before use. By mixing the reagent in the whole volume of the feces and urine (excretion) discharged by a human, presence or absence of occult blood (black blood) is detected. Some examples of the invention are described in detail below. Example 1 (1) A mixed solution was obtained by putting Sodium carbonate 6.5 g 3-Amino phthalic acid hydrazide 0.2 g Iron and sodium derivative of ethylenediaminetetraacetic acid (iron and sodium derivative of EDTA) 7.0 g into a container containing 185 cc of aqueous solution. (2) 31.3 g of hydrogen peroxide was separately prepared in order to put into the container filled with the mixed solution of (1) at the time of detection of occult blood. (3) Samples A and B were prepared. A: Feces and urine mixture without occult blood 388 g B: Feces and urine mixture with occult blood 488 g (4) Samples A and B were put in measuring cylinders of 5 cm in diameter and about 30 cm in height, and the whole volume of combined reagent solution including the mixed solution and hydrogen peroxide of (1) and (2) was added in each measuring cylinder. Example 2 (1) A mixed solution was obtained by putting Sodium carbonate 9.0 g 3-Amino phthalic acid hydrazide 0.1 g Iron and sodium derivative of ethylenediaminetetraacetic acid (iron and sodium derivative of EDTA) 6.0 g into a container containing 160 cc of aqueous solution. (2) 29.3 g of hydrogen peroxide was separately prepared in order to put into the container filled with the mixed solution of (1) at the time of detection of occult blood. (3) Samples A and B were prepared. A: Feces and urine mixture without occult blood 840 g B: Feces and urine mixture with occult blood 440 g (4) Samples A and B were put in measuring cylinders of 5 cm in diameter and about 30 cm in height, and the whole volume of combined reagent solution including the mixed solution and hydrogen peroxide of (1) and (2) was added in each measuring cylinder. Example 3 (1) A mixed solution was obtained by putting into a container containing 195 cc of aqueous solution. (2) 33.3 g of hydrogen peroxide was separately prepared in order to put into the container filled with the mixed solution of (1) at the time of detection of occult blood. (3) Samples A and B were prepared. A: Urine without occult blood 242.5 cc B: Urine with occult blood 420 cc (4) Samples A and B were put in measuring cylinders of 5 cm in diameter and about 30 cm in height, and the whole volume of combined reagent solution including the mixed solution and hydrogen peroxide of (1) and (2) was added in each measuring cylinder. Example 4 (1) A mixed solution was obtained by putting Sodium carbonate 5.0 g 3-Amino phthalic acid hydrazide 0.1 g Iron and sodium derivative of ethylenediaminetetraacetic acid (iron and sodium derivative of EDTA) 4.0 g into a container containing 185 cc of aqueous solution. (2) 27.3 g of hydrogen peroxide was separately prepared in order to put into the container filled with the mixed solution of (1) at the time of detection of occult blood. (3) Samples A and B were prepared. A: Urine without occult blood 291 cc B: Urine with occult blood 391 cc (4) Samples A and B were put in measuring cylinders of 5 cm in diameter and about 30 cm in height, and the whole volume of combined reagent solution including the mixed solution and hydrogen peroxide of (1) and (2) was added in each measuring cylinder. As known from the above examples, the mixing ratio of the reagent is satisfactory in the range of: 3-Amino phthalic acid hydrazide 0.2 - 0.5 % Sodium carbonate 13 - 19 % Iron and sodium derivative of ethylenediaminetetraacetic acid (iron and sodium derivative of EDTA) 10 - 17 % Hydrogen peroxide 66 - 75 % However, by using 3-amino phthalic acid hydrazide within 1%, other components may be appropiately changed. In samples A and B of the examples, the colour change, foaming, and temperature changes were as follows. 1. Colour change A: The colour change could not be recognized visually, and the excretion colour was almost unchanged. B: In 2 minutes after contact of the reagent with the excretion, a pink-tinted violet colour developed, and it returned to the original colour in about 12 minutes. 2. Foaming change A: Foams were hardly noted in any example. B: In Examples 1 and 2, at 8 minutes after the start of the test, the foaming reached a maximum height of 29 cm in the measuring cylinder. In Examples 3 and 4, at 8 minutes after the start of the test the foaming reached a maximum height of 21 cm in the measuring cylinder. 3. Temperature change A: In Examples 1 and 2, the temperature reached 31°C in 8 minutes after the start of the test. In Examples 3 and 4, the temperature reached 26°C in 8 minutes after the start of the test. B: In Examples 1 and 2, the temperature reached 37°C in 8 minutes after the start of the test. In Examples 3 and 4, the temperature reached 34°C in 8 minutes after the start of the test. The colour of excretion varies with the kind of food taken, and the mixing amount of the reagent of the invention varies with the volume of the excretion to be examined. Besides, the detected temperature varies somewhat depending on the content of the iron and sodium derivative of EDTA, and the blending ratio of hydrogen peroxide. Thus, between samples A and B, the result of experiment is obviously different. As this reason, the following point may be considered although they are not logically proved yet. The sodium carbonate in the reagent is considered to increase the pH of the reagent to turn the reagent alkaline, thereby assisting the colouring of the colouring matter (nitrogen carbonate compound). Human erythrocyte components are destroyed by hydrogen peroxide, and the iron content in the destroyed erythrocytes is chelated by EDTA. The chelate compound is destroyed by hydrogen peroxide, and the iron and reagent react to present a pink-tinted violet colour. If occult blood is present in the sample, the hydrogen peroxide in the reagent acts on the erythrocyte components and chelate compound as mentioned above. The hydrogen peroxide destroys erythrocyte components and at the same time generates oxygen to build up foams, thereby raising the temperature. The reagent of the invention should be preferably kept at 2 to 5°C by storing, for example, in a refrigerator in order to maintain the quality for a long period. Besides, in order to clarify the presence of occult blood by using the reagent of the invention, it is recommended that the person whose excretion is to be tested abstain from fish and meat considering the effects of other animal proteins for two or three days before examination. It is, meanwhile, possible to examine in a small, free atmosphere by mixing 3-amino phthalic acid hydrazide, sodium carbonate and iron and sodium derivative of EDTA, dissolving sufficiently, adding hydrogen peroxide water, and adding the mixed solution to the excretion and stirring to make the excretion odourless.

Claims: A reagent for use in detecting occult blood, which comprises a solution containing (a) sodium carbonate, (b) 3-amino phthalic acid hydrazide, (c) an iron and sodium derivative of ethylenediaminetetraacetic acid and (d) hydrogen peroxide. A reagent according to claim 1, wherein the hydrogen peroxide is added to a solution containing the other components at the time of use of the reagent. A reagent according to claim 1 or 2, wherein the solution is an aqueous solution.

Inventors: TABATA KAZUO, TABATA, KAZUO
Applicants: TABATA KAZUO, TABATA, KAZUO
Application year: 1992
Main classification: G01N33
IPC codes: G01N33