JP2009264988A - Simplified dry mouth diagnostic kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an easy and reliable examination kit for dry mouth. <P>SOLUTION: A simple diagnostic filter in which the principle of paper chromatography that quantitatively causes subject's salivary to be soaked and develop into a filter of paper chromatography is combined with starch iodine reaction is formed, and dry mouth is diagnosed, based on the color reaction by dropping an official hydrogen peroxide solution or a novel coloring reagent thereto. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a test kit for simply measuring the degree of xerostomia.

  Xerostomia is a disease in which the amount of saliva secretion decreases due to organic disorders of the salivary glands, disorders of secretory nerves, systemic metabolic diseases, etc., resulting in dry symptoms of the oral mucosa. As a result, concomitant causes such as oral pain, burning sensation, abnormal taste, dysphagia, denture incompatibility, sleep disorder, speech disorder, halitosis, dental caries, periodontal disease, oral fistula and oral candidiasis are caused. According to a survey conducted by the Ministry of Health, Labor and Welfare's Longevity Science Research Project [Study on Xerostomia and Salivary Physical Properties of Elderly People] in 2001, 27.7% of the elderly people were always aware of dry mouth and had mild dryness. Including the conscious person, the rate was 56.7%. In addition, even among young people under the age of 65, 10.5% are always aware of dryness and 35.1% when the longitude is included, and many cases of xerostomia are recognized regardless of age (Non-patent Document 1). ). On the other hand, Field et al. (Non-Patent Document 2) report that 10% or more of the population has xerostomia.

  Early detection and diagnosis of xerostomia are important for systemic and oral health maintenance. However, although the saliva secretion amount measurement, the salivary gland imaging, etc. are performed as the diagnostic method, it is not necessarily fully utilized from the complexity of operation.

  We made a simple diagnostic filter paper that combines the principle of paper chromatography and the iodostarch reaction, and xerostomia caused by a blue or brown color reaction caused by the dripping of pharmacophoric hydrogen peroxide (oxidol) or a new coloring agent. Devised a diagnostic kit.

Yasuaki Kashiwagi, Yuki Iwaki: Research on dry mouth feeling and dryness of the mouth-Research on dry mouth and salivary physical properties of the elderly, 2002 Health and Welfare Science Research Fund Research Report, pages 22-26, 2003 Field et al., Gerontology, 2001, 18-21-24,

  The problem to be solved is that there is no simple and reliable method in the diagnosis of xerostomia.

  The present invention creates a simple diagnostic filter paper combining the principle of paper chromatography that the solution is quantitatively soaked and developed and the iodostarch reaction, and after containing it in the mouth of the subject for a certain period of time, The most important feature is to develop a blue or brown color tone by adding a pharmacopoeia hydrogen peroxide solution (oxidol) or a novel coloring agent.

  The xerostomia diagnostic kit of the present invention is a method in which a subject reacts with a dripping of a chromophoric hydrogen peroxide solution (oxide) or a novel coloring agent after the diagnostic filter paper is put in the mouth for a certain period of time. By producing a blue or brown color, there is an advantage that the result can be easily determined visually. Until now, the amount of salivary secretion could not be quantified easily by the measurement method.

Cut the chromatographic filter paper into strips, make a portion containing starch and potassium iodide at a certain position from the edge, and then develop a blue or brown color with the coloring solution, with the minimum mechanism. Quantitative measurement of saliva secretion was realized.

The composition of the reagent composition for detection and the preparation reagent composition for detection refer to reagents that are applied to filter paper in advance. The reagent composition for detection was prepared as follows using soluble starch (Kanto Chemical Co., Inc.) and potassium iodide (Kanto Chemical Co., Ltd.). Specifically, 1 g of soluble starch was prepared by dissolving 1 g of soluble starch in 99 ml of 0.1 M Tris-HCl buffer (pH 7.3). The potassium iodide solution is 0.1M of 4.97 g of potassium iodide.
A 0.3 M potassium iodide solution was prepared by dissolving in 100 ml of Tris-HCl buffer (pH 7.3). Next, 3 ml of the prepared starch solution and 1 ml of potassium iodide solution were mixed to prepare a detection reagent composition. Next, 0.1 ml of this detection reagent was added to a chromatographic filter paper having a diameter of 23 mm (thickness 0.7
mm; Advantec, No.526; Toyo Roshi Kaisha,
Ltd., Japan) and dried in the dark to prepare a detection reagent composition.

Composition and preparation of filter paper for diagnosis of xerostomia Chromatography filter paper (67 mm in diameter) in which the detection reagent composition filter paper was cut into a diameter of 6.0 mm and holes were previously made in three places as shown in FIG. x 21.0mm; thickness 0.7mm; Advantec,
No.526; Toyo Roshi Kaisha, Ltd., Japan) to prepare a filter paper for diagnosis of xerostomia (FIG. 1). Each filter paper piece (disk) having a diameter of 6 mm contains 51 micrograms of starch and 85 micrograms of potassium iodide.

Optimization of starch concentration and iodostarch reaction To investigate the relationship between starch concentration and iodostarch reaction, 3 ml of starch solution of various concentrations (0.062% to 2%) and 1 ml of 0.3M potassium iodide solution were mixed. The following detection reagent was prepared. As shown in FIG. 2, the iodo starch reaction reacted strongly with increasing starch concentration. Therefore, a starch concentration of 1% showing sufficient iodine starch reaction was used.

Optimization of potassium iodide concentration and iodine starch reaction To investigate the optimal potassium iodide concentration for iodine starch reaction, 1 ml of potassium iodide solution and 3 ml of 1% starch solution of various concentrations (0.001M to 3M) were used. Various detection reagents were prepared by mixing. As shown in FIG. 3, the iodostarch reaction reacted strongly with increasing concentration of potassium iodide solution. However, an increase in the concentration of the potassium iodide solution resulted in poor iodine separation and tailing was observed. Therefore, 0.3M potassium iodide solution was optimized.

Hydrogen Peroxide Concentration and Iodine Starch Reaction To examine the optimal hydrogen peroxide concentration for the iodostarch reaction, various concentrations (0.0375% -3%) of hydrogen peroxide solutions were prepared. In the reagent composition for detection consisting of 3 ml of 1% starch solution and 1 ml of 0.3M potassium iodide solution, the iodostarch reaction is visually observed with 0.0375% or more hydrogen peroxide solution, and the hydrogen peroxide concentration increases. It reacted strongly (FIG. 4). Because of the safety of humans to handle and the convenience that is always available to pharmacies, we decided to use pharmacopoeia hydrogen peroxide (oxide).

Using saliva (mixed saliva, ie, mixed parotid gland, submandibular gland, sublingual gland, minor salivary gland) of healthy saliva (male and female aged 20-25, 36 men; 20 women) The relationship between the amount of saliva (200 microliters to 600 microliters) and the iodine starch reaction of the filter paper for dry mouth diagnosis was examined. As shown in FIG. 5, the relationship between the amount of saliva absorbed on the dry mouth diagnosis filter paper per 2 minutes and the iodostarch reaction showed that the iodostarch reaction disappeared as the amount of saliva increased. The iodostarch reaction was determined by dropping 0.1 ml of a local hydrogen peroxide solution (oxide) on a piece of filter paper.

The amount of saliva and the iodostarch reaction result of the filter paper for dry mouth diagnosis were investigated. After each of the mixed saliva was absorbed, the color was developed with a pharmacopoeia hydrogen peroxide solution (oxide), and then the detection reagent composition disc was removed. When the disc of the reacted reagent composition for detection was removed and arranged on another paper, it was as shown in FIG.

  In a strip of 200 microliters of absorbed saliva, all the places I, II, and III were colored blue. As the amount of saliva absorbed increases, the blue color disappears from the position of III. At 350 microliters, III disappears completely, at 500 microliters, the color of II disappears, and at 600 microliters, most of I Color lost. From these results, it was confirmed that there was quantitativeness.

Determination of xerostomia Dry xerosia diagnosis filter paper was inserted into the oral cavity (under the tongue), and resting saliva was collected for 2 minutes. Next, the dry mouth diagnosis filter paper was taken out, and 0.1 ml of oxidative hydrogen peroxide was dropped onto the filter paper piece, and after 2 seconds, the diameter was 6.0.
Xerostomia was determined from the color tone of a filter paper piece (disk) of mm. The determination was easy by observing a change in color tone on the surface opposite to the surface on which the hydrogen peroxide solution was dropped.

  As shown in FIG. 6, a subject whose resting saliva is less than 1 ml in 10 minutes can be determined to be severe xerostomia. Mucosal dryness is observed in the mouth of severe subjects. This is because the diagnosis findings of the dentist and the result of the filter paper coloring method of the present application are 3/3 in which all of the three holes are colored, and the results are in good agreement. The mild test subject was saliva with consistency, and the amount of saliva per 10 minutes was from 2 ml to less than 4 ml, and in the color development method, the color developed in only one hole was 1/3. In the case of moderate subjects, there are many cases where foamy saliva is present in the findings. The subjects observed as moderate in the findings are 1 ml or more and less than 3 ml in 10 minutes, and in color development, 1/3 to 3/3. And there was width. All normal subjects did not develop color and were determined to be 0/3.

Literature example of the determination of xerostomia According to the diagnosis standard of Sjogren's syndrome of the Ministry of Health, Labor and Welfare (Ministry of Health and Welfare Ministry of Health and Welfare Specific Disease Immune Disease Investigation Research Group) (Non-patent Document 3) Decrease, that is, (1) measurement of salivary secretion, (2) salivary gland angiography, (3) salivary gland scintigraph. The amount of saliva secretion is the amount of saliva at rest, and 1.5 ml is the reference value in 15 minutes. In the gum test, the gum is chewed for 10 minutes, and the amount of saliva during that period is 10 ml. In the Saxon test, stimulated saliva is measured, gauze is put in the mouth, gauze is chewed at a constant speed for 2 minutes, the amount of saliva is measured from weight measurement, and 2 g per 2 minutes is the reference value .

  On the other hand, some researchers have various criteria for determining xerostomia as follows (Non-Patent Documents 4 to 6). Among these values, it is reasonable to set the secretory volume per minute to 200 microliters. Therefore, in the present invention, a prototype with a reference position of 200 microliters was completed.

According to Non-Patent Document 4, a saliva secretion amount of 50 microliters +/- 90 microliters per minute is used as a reference value for xerostomia.
According to Non-Patent Document 5, the amount of saliva is less than 115 microliters +/− 20 microliters per minute, and xerostomia is determined.
According to Non-Patent Document 6, a subject with 90 microliters +/- 110 microliters in a total saliva amount at rest is considered to be dry, and a subject with 360 microliters +/- 330 microliters is determined to be normal.
In Non-Patent Document 3, 1.5 ml or less per 15 minutes is regarded as dryness. Therefore, it takes 200 microliters per 2 minutes.

ENIF Pharmaceutical News, 2006, Volume 15, pp. 20-22 Kalk WWI et al. Ann. Rheum Dis, 2001, 60, 1110-1116 Rhodus N. L. et al. J. Otolaryngol., 2000, 29, 28-34 Marton K. et al. J. Prosthet Dent. 2004 91 577-581

Non-Patent Document 7 lists the following case reports regarding the cause of xerostomia. That is, salivary secretion is reduced to 46 microliters per minute due to salivary gland damage caused by radiation irradiation, and it is determined that xerostomia is present. The amount of salivary secretion in Sjögren's syndrome due to systemic exocrine gland function decline is 60 microliters per minute, and the amount of saliva secreted due to decreased response of secretory nerves caused by drug administration is 90 microliters per minute. It is determined to be sick.

Aframian D. J. et al. Oral Disease, 2007 13: 88-92

Regarding the saliva at rest in clinical laboratory tests, according to the definition of the Dry Mouse Study Group (Tsurumi University School of Dentistry, Prof. Ichiro Saito, Representative Secretary), “Diagnosis of xerostomia in 1.5 minutes or less in 15 minutes” is stated. The test kit of the present invention is assumed to be included in the mouth for 10 minutes. Accordingly, 1.0 ml or less in 10 minutes is diagnosed as xerostomia. The test was conducted using 30 subjects.
In Example 2, it was performed with a filter paper with three holes. However, if only normal values and abnormal values are screened, one hole should be possible. A one-hole inspection kit was prototyped. The center point of the hole was set to be located 26 mm from the tip of the filter paper.
The results are shown in FIG. It is the result of having tested 30 test subjects using the 1-hole diagnostic kit. The normal person did not develop color at all and became negative (-). Many subjects with xerosis became positive (+), and some were half-positive (+/−) with half color.

Preparation of diagnostic filter paper by spot of detection reagent without using hole In Examples 1, 2, and 3, a round filter paper (disk) of the same size in which the detection reagent is permeated into the hole by making a hole in the filter paper It was inserted. However, this operation is fine and complicated. In order to improve this, a method of spotting a test reagent directly on a filter paper was devised.
The reagent used was a detection reagent composition consisting of 3 ml of 1% starch solution and 1 ml of 0.3 M potassium iodide solution using 8 micropipettes and 4 microliters (30 micrograms of starch and iodine) at 6.0 mm intervals. (Containing 50 micrograms of potassium halide) was spotted linearly at five locations. The position of the first spot was 14 mm from the end of the filter paper. After drying in the dark, it was sterilized by autoclaving at 121 ° C. for 15 minutes and used.
Using saliva (mixed saliva, ie, mixed parotid gland, submandibular gland, sublingual gland, minor salivary gland) of healthy people (20-25 year old male and female, 36 males; 20 females), the amount of saliva (0 micron Liters to 600 microliters) and the iodostarch reaction of the filter paper for dry mouth diagnosis was examined. The relationship between the amount of saliva absorbed in the filter paper for dry mouth diagnosis per 2 minutes and the iodostarch reaction showed that the iodostarch reaction disappeared as the amount of saliva increased as shown in the photograph of FIG. The iodostarch reaction was determined by dropping 0.1 ml of a local hydrogen peroxide solution (oxide) on a piece of filter paper. FIG. 9 shows the determination result in an easy-to-understand manner by plus and minus.

In FIG. 8, there are 11 sheets of diagnostic filter paper. From the left, the mixed saliva is 0 microliter, 100 microliter, 150, 200, 250, 300, 350, 400, 450, 500, 600 microliter, respectively. Was absorbed.
As a result, the result of FIG. 8 was judged in FIG. 9, and as is clear from this, the spots that developed blue as the amount of saliva decreased decreased from the bottom, and quantitativeness was recognized.

The improved color developing solution is mainly composed of hydrogen peroxide, but it has been found that the color development changes depending on the composition of the solution. That is, when color was developed using 3% hydrogen peroxide solution (pharmacopecial hydrogen peroxide solution; oxidol), the blue color on the spot spread around the filter paper. In order to improve this, a coloring reagent having the following composition was invented. The composition of the improved coloring reagent is a mixture of 1 volume of purified water and 7 volumes of ethanol to 1 volume of hydrogen peroxide (30%). The color on the spot developed by using this improved coloring reagent became clear without bleeding on the filter paper. Therefore, identification became easy by adding alcohols, such as ethanol, as a composition of a solution.

Specific examples of improvements are as follows. The conventional local hydrogen peroxide solution and the improved color reagent are shown together. The color reaction immediately after color development was clear without bleeding in the improved method (FIG. 10).
There was another good point in the improved method in which ethanol was added. In the case of color development using only hydrogen peroxide solution, the color development position changes with time after color development. A photograph after 10 minutes was shown (FIG. 11). On the other hand, with the improved ethanol addition, there was no change and stable color development was obtained. Stable color was preserved even after standing for 60 minutes (Fig. 12)
Although acetone does not bleed, the degree of color development is weak, and DEMSO (Dimethyl
Sulfoxide) could not be judged because color development was inhibited.

The degree of color development due to the difference in pH and buffer solution It was examined whether the iodostarch reaction was changed by pH. The solution of the reagent composition for detection consisting of potassium iodide and starch was prepared in pH 5 to pH 8 with 0.1 M phosphate buffer, and pH 9 to pH 10 with 0.1 M Tris-HCl buffer. As a result, blue or brown color development was detectable from pH5 to pH9, but color development was inhibited at pH10.

  Xerostomia can be diagnosed by a simple operation by performing an iodostarch reaction on a filter paper that is contained in the subject's mouth and collected saliva.

It is explanatory drawing which showed the filter paper for xerostomia diagnosis. (Example 1) The size of the filter paper was 67 mm long and 21 mm wide, and three holes were made along the center line of the strip-shaped filter paper. The right end (insertion part) of this figure is inserted into the mouth and applied to the saliva surface to absorb saliva. The position of the center point of the hole III is 26 mm from the right end, the center point of the hole II is 38 mm, and the center point of the hole I is 42 mm. The diameter of the hole is 6 mm. It is explanatory drawing which showed optimization of a starch density | concentration, and iodo starch reaction. Example 1 It is explanatory drawing which showed optimization of a starch density | concentration, and iodo starch reaction. Example 1 It is explanatory drawing which showed hydrogen peroxide concentration and iodo starch reaction. Example 1 It is the figure which showed the iodostarch reaction of the saliva amount and the filter paper for xerostomia diagnosis. (Example 2) The number described on the horizontal axis of FIG. 5 indicates the amount of mixed saliva absorbed (microliter), and the amount absorbed is 200 to 600 microliters. Roman numerals I, II, and III indicate the positions of the holes into which the filter paper infiltrated with the detection reagent composition is inserted as shown in FIG. After each of the mixed saliva was absorbed, the color was developed with a pharmacopoeia hydrogen peroxide solution (oxide), and then the detection reagent composition disc was removed. When the disc of the reacted reagent composition for detection was removed and arranged on another paper, it was as shown in FIG. It is explanatory drawing which showed determination of xerostomia. (Example 1) A normal person did not develop color at all and became negative (-). Many subjects with xerosis became positive (+), and some were half-positive (+/−) with half color. It is explanatory drawing which showed the determination (1 hole type) of dry mouth. (Example 2) It is the result of having tested 30 test subjects using the 1-hole diagnostic kit. The normal person did not develop color at all and became negative (-). Many subjects with xerosis became positive (+), and some were half-positive (+/−) with half color. It is explanatory drawing which showed determination (spot type | formula) of xerostomia. (Example 3) There are eleven sheets of diagnostic filter paper, and mixed saliva from the left side has a capacity of 0 microliter, 100 microliter, 150, 200, 250, 300, 350, 400, 450, 500, 600 microliter, respectively. Was absorbed. As a result, as clearly shown in FIGS. 8 and 9, the spots that developed blue as the amount of saliva increased decreased from the bottom, and quantitativeness was recognized. It is explanatory drawing which showed determination of the amount of saliva and a color reaction. (Example 3) The result of FIG. 8 is shown as a judgment. It is clearly shown by plus and minus It is explanatory drawing which showed the color development comparison (just after color development) of the color developing solution of a conventional method, and the color developing solution of an improved method. (Example 4) The left side of the photograph is an improved color developing solution to which ethanol is added. On the right is a color-developing solution that contains only pharmacopeia hydrogen peroxide. It is explanatory drawing which showed the coloring comparison of the coloring liquid of a conventional method and the coloring liquid of an improved method (it is left for 10 minutes after coloring). (Example 4) The left side of the photograph is an improved color developing solution to which ethanol is added. On the right is a color-developing solution that contains only pharmacopeia hydrogen peroxide. It is explanatory drawing which showed the coloring comparison of the coloring solution of a conventional method and the coloring solution of an improved method (60 minutes after coloring). (Example 4) The left side of the photograph is an improved color developing solution to which ethanol is added. On the right is a color-developing solution that contains only pharmacopeia hydrogen peroxide.

Claims (5)

In order to diagnose dry mouth, filter paper pre-impregnated with potassium iodide and starch is manufactured, and one end of the filter paper is put into the mouth to absorb saliva. A method for measuring the amount of saliva secreted by applying blue hydrogen peroxide water (oxidol) and coloring blue, and a dry mouth diagnosis kit.
2. The method of measuring the amount of saliva secreted by blue color development by applying a chloride ion solution (perchloric acid solution or sodium hypochlorite solution) as a composition of the coloring reagent (detection reagent composition) in claim 1 And xerostomia diagnostic kit.
The method for measuring salivary secretion by brown color development and a kit for diagnosing xerostomia according to claim 1, wherein the composition of the detection reagent composition is a solution in which alcohol is added to hydrogen peroxide water. .
In claim 1, the composition of the detection reagent composition of claim 3 is applied by applying a solution in which 1 volume of hydrogen peroxide (30%) is mixed with 1 volume of purified water and 7 volumes of ethanol. Of measuring salivary secretion amount by coloration of lipase and kit for diagnosing xerostomia.
The kit for diagnosing xerostomia according to claim 1, wherein the filter paper for diagnosing xerostomia is spotted at a specific position or applied on a line, rather than permeating the position where potassium iodide and starch are preliminarily permeated.