JP6480278B2 - Simplified determination method for gingivitis and periodontitis - Google Patents

Description

  The present invention relates to a simple determination method of gingivitis and periodontitis and a simple determination kit thereof, and particularly, it is possible to quickly and easily determine gingivitis and periodontitis in a non-invasive manner (not damaging a living body) by color tone. It relates to a method and a kit.

  At present, periodontal disease is positioned as a lifestyle-related disease in Japan, and its prevalence is increasing due to the westernization of lifestyle and increased mental stress. Periodontal disease progresses before subjective symptoms such as pain appear, and if left untreated, it results in tooth loss. Furthermore, the relationship between periodontal disease and pneumonia, osteoporosis, diabetes, myocardial infarction, premature birth, metabolic syndrome, etc. has been reported in recent years, and interest in prevention and treatment of periodontal disease is rapidly increasing.

  Conventionally, the periodontal disease-affected state has been diagnosed by CPI (CommunityPeriodialIndex), which measures the depth of the periodontal pocket. However, it has been pointed out that the diagnosis requires expert technical skills, the diagnosis takes time, and uncomfortable feeling when inserting the probe into the periodontal pocket.

  Saliva contains many substances that can be useful markers of periodontal disease, and in recent years, it has been proposed to use a saliva test for early detection and diagnosis of periodontal disease. To date, aspartate aminotransferase, alanine aminotransferase, dipeptidyl peptidase, lactate dehydrogenase (hereinafter referred to as LDH), alkaline phosphatase, acid phosphatase, γ-glutamyltransferase, matrix metalloprotease, creatinine, urea, free hemoglobin, etc. Relevance to periodontal disease has been pointed out.

  In particular, it has long been reported that LDH is useful as a marker for periodontal disease. LDH is an enzyme present in the cytoplasm of almost all human cells, but leaks out of cells due to cell damage or necrosis. It is known that LDH activity of saliva is also significantly high in periodontal disease patients, and salivary LDH activity can be used as an index of the diseased state of periodontal disease (Patent Documents 1 to 4, Non-patent Documents). 1 to Non-Patent Document 9).

  However, conventional methods for measuring LDH activity require laboratory instruments such as dispensers and measuring instruments such as spectrophotometers, and in particular, in terms of technology and equipment, it is actually the case that periodontal patients themselves measure. Have difficulty.

  On the other hand, a periodontal disease diagnostic test paper, in which an aqueous solution of pH 7-11 containing an LDH substrate, LDH coenzyme, diaphorase, tetrazolium salt, enzyme stabilizer and pH buffer is permeated into the water-absorbing base material and dried. Alternatively, a periodontal disease diagnostic test paper is used in which an aqueous solution having a pH of 7 to 11 containing an LDH substrate, an LDH coenzyme, a phenazinium methylsulfate derivative, a tetrazolium salt, an enzyme stabilizer and a pH buffer is permeated into a water-absorbing base material and dried. Proposed a method to create a color sample by dividing the color tone of colors from 1 to 10, and to determine between period 7 and 8 as a boundary value, and to determine periodontal disease when the boundary value is exceeded (Patent Document 5).

JP 2004-229537 A JP 2004-329141 A JP 2002-181815 A JP 2005-201768 A JP 2010-130924 A

Clin. Chim. Acta298, 187-191, 2000 Med. OralPatrol. OralCir. Bucal 11, 115-119, 2006 J. et al. OralSci. 48,117-183,2006 J. et al. OralSci. 50, 53-56, 2008 Chir. Dent. Fr. 56, 63-69, 1986 J. et al. Periodontol. 58,614-621,1987 J. et al. Clin. Periodontol. 16,164-169, 1989 Arch. OralBiol. 52,911-915,2007 Clin. Chim. Acta 101, 321-326, 1980

  However, although the periodontal disease diagnosis test paper described in Patent Document 5 is excellent in that it can be quickly and easily determined periodontal disease, it cannot be used for accurate determination of the state of the oral cavity.

  In view of such problems, an object of the present invention is to provide a simple determination method for gingivitis and periodontitis that can easily and accurately determine the state in the oral cavity.

  Therefore, a simple determination method for gingivitis and periodontitis according to the present invention is a method of pH 7 to 11 including lactate dehydrogenase substrate, lactate dehydrogenase coenzyme, diaphorase, tetrazolium salt, enzyme stabilizer and pH buffer. A test paper in which an aqueous solution is permeated into a water-absorbing substrate and dried, or a pH 7-11 containing a lactate dehydrogenase substrate, a lactate dehydrogenase coenzyme, a phenazinium methylsulfate derivative, a tetrazolium salt, an enzyme stabilizer and a pH buffer. Using test paper in which an aqueous solution is permeated into a water-absorbing substrate and drying it, the color tone of the test paper when a certain period of time elapses after the saliva of normal subjects, gingivitis patients and periodontitis patients are immersed in the test paper The distribution of the number of normal persons, the distribution of the number of gingivitis patients and periodontitis patients, and between the maximum number of normal colors and the maximum number of gingivitis patients The intermediate color tone is the first boundary value, the intermediate color tone between the maximum number of gingivitis patients and the maximum number of periodontitis patients is the second boundary value, and the patient's saliva is immersed in the test paper. A patient whose color tone exceeds a first boundary value after a certain period of time is simply determined as gingivitis, and a patient whose color tone exceeds a second boundary value is determined as periodontitis.

  Moreover, the simple determination method of gingivitis / periodontitis according to the present invention has a pH of 7 to 11 including a lactate dehydrogenase substrate, lactate dehydrogenase coenzyme, diaphorase, tetrazolium salt, an enzyme stabilizer, and a pH buffer. A test paper in which an aqueous solution is permeated into a water-absorbing substrate and dried, or a pH 7-11 containing a lactate dehydrogenase substrate, a lactate dehydrogenase coenzyme, a phenazinium methylsulfate derivative, a tetrazolium salt, an enzyme stabilizer and a pH buffer. Using test paper in which an aqueous solution is permeated into a water-absorbing substrate and drying it, the color tone of the test paper when a certain period of time elapses after the saliva of normal subjects, gingivitis patients and periodontitis patients are immersed in the test paper Between the maximum number of normal colors and the maximum number of gingivitis patients in the distribution of normal numbers for gingivitis and periodontitis patients When the color tone between is the first boundary value, and the color tone intermediate between the maximum number of color tone of gingivitis patients and the maximum number of periodontitis patients is the second boundary value, the patient's saliva is the test paper A patient whose color tone exceeds a first boundary value after a certain period of time has been immersed in gingivitis, and a patient whose color tone exceeds a second boundary value is simply determined as periodontitis. .

  Here, there is gingivitis as a pre-stage of periodontitis, and gingivitis is caused by accumulation of plaque (plaque) and calculus between teeth and between teeth and gums (periodontal pocket), and inflammation of the gums. The condition is red and swollen. If this gingivitis is neglected, a large amount of plaque and calculus deposits in the periodontal pocket, resulting in mild periodontitis, and as the disease progresses, the periodontal pocket gradually deepens and plaque and calculus increase, As pus appears, it leads to moderate and severe periodontitis where the alveolar bone is absorbed.

Mild periodontitis has relatively little calculus and plaque deposits, and if you brush well and control plaque, it can recover from mild periodontitis to gingivitis and from gingivitis to normal. On the other hand, if it becomes severe periodontitis, it is necessary to remove the cause by surgery.

  By the way, for individual teeth in the oral cavity, whether or not periodontitis is mild, moderate or severe can be accurately determined from the inflammation of the gingiva and the depth of the periodontal pocket. However, saliva is generally related to the condition of the gingiva in the oral cavity, and it is not possible to judge the status of individual teeth from the saliva, and the oral cavity has mild periodontitis, moderate periodontitis, and severe teeth. It is practically difficult to determine whether peritonitis or not.

  On the other hand, when the LDH activity in the saliva of normal subjects, gingivitis patients and periodontitis patients was examined, even if normal, gingivitis, and periodontitis were found, there was a variation in LDH activity in saliva and the LDH activity It was found that there was an overlap.

  The feature of the present invention is that a normal person, a gingivitis patient, and a periodontitis patient obtain a distribution of the number of normal persons with respect to the color tone of the test paper, a distribution of the number of gingivitis patients and a periodontitis patient. The first boundary value is an intermediate color between the maximum number of colors and the maximum number of colors of gingivitis patients, and is between the maximum number of colors of gingivitis patients and the maximum number of colors of periodontitis patients. Patients with gingivitis and color tone exceeding the second boundary value are patients whose color tone exceeds the first boundary value after a certain period of time after the patient's saliva is immersed in the test paper. Is simply determined as periodontitis.

  Thereby, a person with normal oral cavity and a person with gingivitis or periodontitis in the oral cavity can be easily distinguished with high accuracy by coloring the test paper.

  In addition, by performing plaque control on a patient who has been subjected to simple determination, cleaning the tartar, and then performing simple determination again after a certain period of time, it is possible to easily determine whether the disease state has been improved or worsened.

  That is, according to the present invention, the normal person or the first boundary is obtained from a color tone in which the saliva of the patient who has performed the simple determination is immersed in the test paper and the coloration after the elapse of a predetermined time exceeds the first boundary value or the second boundary value. Gingivitis or periodontitis is improved when the color tone changes to a value, and it can be easily determined that gingivitis has deteriorated when the color tone exceeds the first boundary value and the color tone exceeds the second boundary value.

  The kit used for the simple determination is also novel. That is, the simple determination kit for gingivitis / periodontitis according to the present invention has a pH of 7 to 11 containing a lactate dehydrogenase substrate, lactate dehydrogenase coenzyme, diaphorase, tetrazolium salt, an enzyme stabilizer and a pH buffer. A test paper in which an aqueous solution is permeated into a water-absorbing substrate and dried, or a pH 7-11 containing a lactate dehydrogenase substrate, a lactate dehydrogenase coenzyme, a phenazinium methylsulfate derivative, a tetrazolium salt, an enzyme stabilizer and a pH buffer. A test paper in which an aqueous solution is permeated into a water-absorbing base material and dried, and saliva of normal subjects, gingivitis patients and periodontitis patients, lactate dehydrogenase substrate, lactate dehydrogenase coenzyme, diaphorase, tetrazolium salt, Test paper, or lactic acid, in which an aqueous solution of pH 7 to 11 containing an enzyme stabilizer and a pH buffer is permeated into a water-absorbing substrate and dried Constantly immersed in a test paper in which an aqueous solution of pH 7 to 11 containing a hydrogen enzyme substrate, lactate dehydrogenase coenzyme, a phenazinium methylsulfate derivative, a tetrazolium salt, an enzyme stabilizer and a pH buffer is permeated into a water-absorbing substrate and dried. The maximum number of normal and the maximum number of gingivitis patients in the distribution of the number of normal persons to the color tone of the test paper over time, the distribution of the number of patients of gingivitis patients and periodontitis patients When the intermediate color tone between the color tone is the first boundary value and the intermediate color tone between the maximum color tone of the gingivitis patient and the maximum number of periodontitis patients is the second boundary value, the patient The first boundary where a patient whose color tone exceeds a first boundary value after a certain period of time is immersed in test paper and a patient whose color tone exceeds the second boundary value is determined to be gingivitis, and a patient whose color tone exceeds the second boundary value is determined to be periodontitis Value color and second A color sample showing the color tone of Sakaichi, characterized by comprising a.

  The test paper is LDH substrate lactic acid or lactate, LDH coenzyme nicotinamide adenine dinucleotide (hereinafter referred to as NAD) or its salt, chromogenic enzyme diaphorase, diaphorase substrate tetrazolium salt, enzyme An aqueous solution having a pH of 7 to 11 in which a stabilizer and a pH buffer are dissolved, or the above aqueous solution using a phenazinylmethylsulfate (hereinafter referred to as PMS) derivative, which is an electron carrier instead of diaphorase, permeates a water-absorbing substrate and then dried. Can be manufactured.

  As a substrate for LDH, lactic acid, lithium lactate, sodium lactate, ammonium lactate, magnesium lactate, potassium lactate and the like are preferably used. The LDH substrate is preferably added so that the final concentration in the color developing solution for LDH activity test paper is 1.0 to 50 mg / mL.

  As the LDH coenzyme, NAD or its sodium salt or lithium salt is preferably used. The LDH coenzyme is preferably added so that the final concentration in the color developing solution for LDH activity test paper is 0.05 to 5.0 mg / mL.

  As the enzyme stabilizer, bovine serum albumin or the like is preferably used. The enzyme stabilizer is preferably added so that the final concentration in the color developing solution for LDH activity test paper is 0.05 to 20 mg / mL.

  The pH buffering agent has a buffering action at pH 7 to 11, such as Tris hydrochloric acid, phosphoric acid, boric acid, glycine, diethanolamine, sodium carbonate, sodium hydrogen carbonate, or HEPES, HEPPS, Tricine, Bicine, Glycylyglycine, TAPS, CAPS, etc. Good's buffer is preferably used. The pH buffering agent is preferably added so that the final concentration in the color developing solution for LDH activity test paper is 0.05 to 50 mg / mL.

Examples of the tetrazolium salt include 3,3 ′-[3,3′-Dimethoxy- (1,1′-biphenyl) -4,4′-diyl] -bis (2,5-diphenyl-2H-tetrazolimide), 3, 3 ′-[3,3′-Dimethyl- (1,1′-biphenyl) -4,4′-diyl] -bis [2- (4-nitrophenyl) -5-phenyl-2H-tetrazol chloride] (hereinafter, NBT) ), 2- (4-Iodophenyl) -3- (4
-Nitrophenyl) -5-phenyl-2H-tetrazole chloride (hereinafter referred to as INT), 3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazole bromide, 2- (4-Iodophenyl)- 3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt (hereinafter referred to as WST-1), 2- (4-Idophenyl) -3- (2,4-dinitrophenyl)- 5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt, etc. are preferably used. The tetrazolium salt is preferably added so that the final concentration in the color developing solution for LDH activity test paper is 0.05 to 20 mg / mL.

As the PMS derivative, 1-methoxy-5-methylphenylmethylsulfate (hereinafter referred to as 1-methoxyPMS) is particularly preferably used. The PMS derivative is desirably added so that the final concentration in the color developing solution for LDH activity test paper is 1.0 to 100 mg / mL.
For the water-absorbing substrate, ordinary filter paper, calligraphic blotting paper, polymer absorber, nonwoven fabric and the like are used.

  The test paper is a simple judgment of normal, gingivitis, and periodontitis after soaking saliva and comparing the coloration after standing at room temperature for 10 seconds to 10 minutes, preferably 1 minute to 5 minutes, with the color sample color. To do.

The color of the test paper varies depending on the tetrazolium salt used in the production. For example, the purple when using the NBT, red in the case of using the INT, Ru Teiirosu yellow when using the WST-1.

It is a figure which shows the relationship between the number of normal, gingivitis, periodontitis, and the color tone of test paper coloring. It is a figure which shows the example of a color sample.

  Hereinafter, the present invention will be described more specifically with reference to examples.

  A 12 mg / mL Tris-HCl buffer (pH 9.0, 25 ° C.) containing 40 mg / mL lithium lactate and 10 mg / mL bovine serum albumin was prepared (hereinafter referred to as buffer). NBT was added to distilled water so as to be 5.0 mg / mL and heated in hot water, and then completely dissolved by ultrasonic treatment (hereinafter referred to as NBT solution). NAD (lithium salt) (Nicotinamide adenindincuretide, Lithium salt; manufactured by Calbiochem) was dissolved in a buffer solution at a concentration of 3.347 mg / mL (hereinafter referred to as NAD solution). After diaphorase (Diaphorase; manufactured by Oriental Yeast Co., Ltd.) was suspended in distilled water at 500 U / mL, the supernatant obtained by centrifugation at 12,000 rpm × 5 minutes was used as a diaphorase solution. Buffer solution, NBT solution, NAD solution and diaphorase solution were mixed at a ratio of 4: 4: 1: 1 (ml), respectively, centrifuged again at 12,000 rpm × 5 minutes, and the resulting supernatant was treated with LDH activity. A coloring liquid for test paper was obtained. All of the above solutions were prepared in a brown bottle. The obtained color developing solution was permeated into a blotting paper (40 mm × 5 mm, manufactured by Sumikido Co., Ltd.) and then freeze-dried under light shielding to obtain a purple colored LDH test paper.

  A red color LDH test paper was obtained in the same manner as in Example 1 except that INT (manufactured by Dojindo Laboratories) was used instead of NBT.

  A yellow colored LDH test paper was obtained in the same manner as in Example 1 except that WST-1 (manufactured by Dojindo Laboratories) was used instead of NBT.

  A purple colored LDH test paper was obtained in the same manner as in Example 1 except that an aqueous solution of 67 mg / L1-methoxyPMS (manufactured by Dojindo Laboratories) was used instead of the diaphorase solution.

  A red-colored LDH test paper was obtained in the same manner as in Example 2 except that a 67 mg / L1-methoxyPMS aqueous solution was used instead of the diaphorase solution.

  A yellow colored LDH test paper was obtained in the same manner as in Example 3 except that a 67 mg / L1-methoxyPMS aqueous solution was used instead of the diaphorase solution.

Simple determination was performed using the test paper obtained in Example 1.
[Saliva test method]
Forty-one adult subjects were gargled with water, and after about 1 minute, about 100 μL of resting saliva was discharged onto the aluminum foil. The test paper was immersed in saliva and then shielded from light, and the color density after 1 minute was selected from the closest of 10 color samples. In addition, as for the darkness of the color, 10 is the darkest and 1 is the lightest.

[Evaluation of periodontal tissue]
Inspect all teeth of each subject using periodontal pocket probes at 6 locations for each tooth. Based on the presence of bleeding after probing and the depth of the periodontal pocket, normal, gingivitis, mild Evaluate periodontitis, moderate periodontitis and severe periodontitis, find the relationship between the color tone of test paper color and normal, gingivitis, mild periodontitis, moderate periodontitis and severe periodontitis, The relationship diagram shown in FIG. 1 was obtained.

  In FIG. 1, an intermediate color between the maximum number of normal colors and the maximum number of gingivitis patients is defined as a first boundary value A, and the maximum number of gingivitis patients and mild periodontitis patients. An intermediate color tone between the maximum number of color tones is set as the second boundary value B, the color sample shown in FIG. 2 is created, and the combination of the test paper and the color sample is used as a simple determination kit.

(Simple judgment)
Using the simple determination kit of this example, simple determination of normal, gingivitis, and periodontitis was possible. In addition, patients who performed simple judgment or those who removed calculus were instructed in brushing to perform plaque control. After 2 weeks, a simple judgment was made again using test paper. It was possible to easily determine that improvement was observed or that the medical condition was progressing.

Claims (3)

A test paper obtained by infiltrating a water-absorbing substrate and drying an aqueous solution having a pH of 7 to 11 containing a lactate dehydrogenase substrate, lactate dehydrogenase coenzyme, diaphorase, tetrazolium salt, enzyme stabilizer and pH buffer, or lactic acid Using a test paper in which an aqueous solution of pH 7-11 containing a dehydrogenase substrate, lactate dehydrogenase coenzyme, a phenazinium methylsulfate derivative, a tetrazolium salt, an enzyme stabilizer and a pH buffer is permeated into a water-absorbing substrate and dried,
Distribution of normal number of people for normal color, gingivitis patient and periodontitis patient's saliva in test paper and the color tone of test paper coloration after a certain time, gingivitis patient and periodontitis patient Find the distribution of numbers,
The intermediate color between the maximum number of normal subjects and the maximum number of gingivitis patients is the first boundary value, and the maximum number of gingivitis patients and the maximum number of periodontitis patients The middle color between the second boundary value,
Soaking a patient's saliva in a test paper and determining a patient whose color tone exceeds a first boundary value after a certain period of time as gingivitis, and a patient whose color tone exceeds a second boundary value is simply determined as periodontitis A simple determination assisting method for gingivitis and periodontitis characterized by the above. A test paper obtained by infiltrating a water-absorbing substrate and drying an aqueous solution having a pH of 7 to 11 containing a lactate dehydrogenase substrate, lactate dehydrogenase coenzyme, diaphorase, tetrazolium salt, enzyme stabilizer and pH buffer, or lactic acid Using a test paper in which an aqueous solution of pH 7-11 containing a dehydrogenase substrate, lactate dehydrogenase coenzyme, a phenazinium methylsulfate derivative, a tetrazolium salt, an enzyme stabilizer and a pH buffer is permeated into a water-absorbing substrate and dried,
Distribution of normal number of people for normal color, gingivitis patient and periodontitis patient's saliva in test paper and the color tone of test paper coloration after a certain time, gingivitis patient and periodontitis patient In the distribution of numbers, the first color is the middle color between the maximum number of colors of normal subjects and the maximum number of colors of gingivitis patients, and the maximum number of colors of gingivitis patients and the maximum of periodontitis patients When the intermediate color tone between a number of color tones is the second boundary value, gingivitis is defined as a patient whose color tone exceeds a first boundary value after a certain period of time by immersing the patient's saliva in a test paper, A simple determination assisting method for gingivitis and periodontitis, wherein a patient having a color tone exceeding the second boundary value is simply determined as periodontitis. When the saliva of the patient who performed the above simple determination is immersed in a test paper and the color change after a lapse of a certain time changes from a color tone exceeding the first boundary value or the second boundary value to a normal color or the color tone of the first boundary value The gingivitis according to claim 1 or 2, wherein the gingivitis or periodontitis is improved, and when the color tone exceeding the first boundary value is changed to the color tone exceeding the second boundary value, it is simply determined that the gingivitis has deteriorated.・ Simple determination assistance method for periodontitis.

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